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A Transcriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh
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Please cite this article as: Sun C., Deng L., Du M., Zhao J., Chen Q., Huang T., Jiang H., Li C.-B., and Li C. (2019). A Transcriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh. Mol. Plant. doi: https://doi.org/10.1016/j.molp.2019.10.010. # 7 ! 77 = 7 7 ! 7 7 < !< 7 = = 78= 7 77 7 ! 7 = < < 7 !< =7 = = =< 8 ! 7 7 ! < 7= 7 7 ! 7 7 9 8 7 < 7 $5"> ?": " < 7 4 $> 7 < 7 7 8 7 < < @ 7 7 78 A./*2
1
A Transcriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh
2 3
Chuanlong Sun1,2,6, Lei Deng1,6, Minmin Du3, Jiuhai Zhao4, Qian Chen4, Tingting
4
Huang5, Hongling Jiang1, Chang-Bao Li3,*, Chuanyou Li1,2,*
5
1
6
(Beijing), Institute of Genetics and Developmental Biology, Innovation Academy for
7
Seed Design, Chinese Academy of Sciences, Beijing 100101, China
8
2
University of Chinese Academy of Sciences, Beijing 100049, China
9
3
Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North
State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research
10
China), Ministry of Agriculture, Beijing Vegetable Research Center, Beijing Academy
11
of Agriculture and Forestry Sciences, Beijing 100097, China
12
4
13
University, Tai'an 271018, Shandong Province, China
14
5
15
Shandong Province, China
16
6
State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural
Institute of Vegetable, Qingdao Academy of Agricultural Sciences, Qingdao 266100,
These authors contributed equally to this article.
17 18
*
19
Chuanyou Li ([email protected])
20
Chang-Bao Li ([email protected])
Correspondence:
21 22
Running title: Anthocyanin Biosynthesis in Tomato Flesh
23
Short Summary: Through characterization of the purple tomato “Indigo Rose”,
24
which only shows anthocyanin pigmentation in the skin in a light-dependent manner,
25
we demonstrate that the R2R3-MYB protein SlAN2-like orchestrates a hierarchical
26
transcriptional network to promote anthocyanin accumulation. Expression of a
27
functional SlAN2-like could bypass light and activate anthocyanin biosynthesis in both
28
peel and flesh of tomato cultivars. 1
29
Abstract
30
Dietary anthocyanins are important health-promoting antioxidants that make a major
31
contribution to the quality of fruits. It is intriguing that most tomato cultivars do not
32
produce anthocyanins in fruit. However, the purple tomato variety Indigo Rose, which
33
combines the dominant Aft locus and the recessive atv locus from wild tomato
34
species, exhibits light-dependent anthocyanin accumulation in the skin. Here, we
35
report that whereas Aft encodes a functional allele of an anthocyanin activator named
36
SlAN2-like, atv encodes a non-functional allele of the anthocyanin repressor
37
SlMYBATV. The expression of SlAN2-like is responsive to light and a functional
38
SlAN2-like can activate both anthocyanin biosynthetic genes and their regulatory
39
genes, suggesting that SlAN2-like acts as a master regulator and plays a critical role
40
for the activation of anthocyanin biosynthesis. Our results reveal that cultivated
41
tomatoes contain a non-functional allele of this master regulator and therefore fail to
42
produce anthocyanins. Indeed, expression of a functional SlAN2-like in a tomato
43
cultivar led to the activation of the entire anthocyanin biosynthesis pathway and high
44
levels of anthocyanin accumulation in both peel and flesh. Our study exemplifies that
45
efficient engineering of complex metabolic pathways could be achieved through
46
tissue-specific expression of master transcriptional regulators.
47 48
Key words: anthocyanin biosynthesis, purple tomato, MBW complex, master
49
regulator, transcriptional regulation
50 51 52 53 54 55 56 2
57
Introduction
58
Anthocyanins are naturally occurring pigments responsible for red, purple, and blue
59
color of many plant organs, and play essential roles in multiple biological processes of
60
plants, such as attracting pollinators and dispersers, providing protection against biotic
61
and abiotic stresses (Gould, 2004; Zhang et al., 2013). Most importantly, growing
62
evidence reveals that anthocyanins are health-promoting compounds because of their
63
high antioxidant activity (Butelli et al., 2008). Dietary consumption of anthocyanins
64
reduces the risk of cardiovascular disease, age-related degenerative diseases and
65
certain cancers (He and Giusti, 2010; Li et al., 2017).
66 67
Anthocyanins are synthesized through the flavonoid branch of the general
68
phenylpropanoid pathway. It is well-recognized that anthocyanin biosynthesis is
69
tightly regulated at the transcriptional level by the evolutionarily conserved
70
MYB-bHLH-WD repeat (MBW) complex (Lloyd et al., 2017; Naing and Kim, 2018;
71
Xu et al., 2015). Many known high-anthocyanin accumulation traits in various crops,
72
such as purple-fleshed sweet potato (Mano et al., 2007), purple cauliflower (Chiu et
73
al., 2010), red-fleshed apple (Espley et al., 2009; Zhang et al., 2019), blood orange
74
(Butelli et al., 2012) and purple pummelo (Huang et al., 2018), are resulted from
75
elevated expression of MYB transcription factors, suggesting that MYB regulators
76
play an active and critical role in the activation of the MBW complex (Lloyd et al.,
77
2017; Xu et al., 2015).
78 79
Tomato (Solanum lycopersicum) is a favorite vegetable worldwide (Lin et al., 2014).
80
Whereas the most abundant antioxidant in tomato fruit is the lipophilic lycopene, the
81
levels of the water-soluble flavonoids (which include anthocyanins) are suboptimal
82
(Mes et al., 2008). Therefore, tomato is an ideal system to investigate the regulatory
83
mechanism of anthocyanin biosynthesis and provides an excellent candidate for
84
enhancement of anthocyanin content by genetic manipulations (Bovy et al., 2002; 3
85
Butelli et al., 2008; Jian et al., 2019; Mathews et al., 2003; Schreiber et al., 2012).
86 87
Although most cultivated tomatoes do not produce anthocyanins in fruit, some related
88
wild tomato species do produce little amount of anthocyanins in the fruit skin in a
89
light-dependent manner, and this trait has been transferred into tomato cultivars. For
90
example, introgression of the dominant Anthocyanin fruit (Aft) locus from Solanum
91
chilense or the Aubergine (Abg) locus from Solanum lycopersicoides into tomato
92
cultivars led to slight, light-dependent accumulation of anthocyanins in the fruit peel
93
(Mathews et al., 2003; Mes et al., 2008; Schreiber et al., 2012). Introgression of the
94
recessive atroviolacium (atv) gene from Solanum cheesmaniae into tomato cultivars
95
led to anthocyanin accumulation in vegetative organs but not in the fruit (Mes et al.,
96
2008). Notably, combining atv with Aft led to a dramatic increase of anthocyanin
97
pigmentation in the fruit peel (Mes et al., 2008), indicating that Aft genetically
98
interacts with atv in regulating anthocyanin biosynthesis. The popular purple tomato
99
variety Indigo Rose (InR) contains both Aft and atv and bears intense anthocyanin
100
pigmentation
in
the
fruit
peel
(Mes
et
al.,
2008)
101
(http://extension.oregonstate.edu/gardening/purple-tomato-debuts-indigo-rose).
102
should be noted that InR only produces anthocyanins in the peel that is exposed to
103
sunlight and no anthocyanin is accumulated in the shading peel and flesh (Qiu et al.,
104
2019), suggesting an essential role of light in regulating anthocyanin pigmentation.
It
105 106
Despite much efforts have been devoted to clone Aft and atv, the genes underlying
107
these loci have not been conclusively identified and/or verified (Cao et al., 2017;
108
Colanero et al., 2018; Kiferle et al., 2015; Schreiber et al., 2012). Thus, the molecular
109
mechanism by which InR only exhibits anthocyanin pigmentation in the fruit skin in a
110
light-dependent manner remains elusive.
111 112
Here, we employ a bulk population sequencing approach (Austin et al., 2014) to 4
113
identify the genes controlling anthocyanin accumulation in InR. We show that
114
whereas Aft encodes an R2R3-MYB protein SlAN2-like and positively regulates
115
anthocyanin biosynthesis, Atv encodes an R3-MYB protein SlMYBATV and
116
negatively regulates anthocyanin biosynthesis. In the light-exposing fruit peel of InR,
117
light activates SlAN2-like expression through SlHY5, a critical component of the light
118
signalling pathway (Lee et al., 2007). In turn, SlAN2-like triggers the expression of
119
both SlMYBATV and SlAN1 (Qiu et al., 2016), a bHLH transcription factor that
120
positively regulates anthocyanin biosynthesis. SlMYBATV competes SlAN2-like for
121
binding SlAN1 and thereby negatively regulating anthocyanin biosynthesis. Therefore,
122
SlAN2-like acts as a master regulator of anthocyanin biosynthesis and plays a critical
123
role in transmitting the light signal to the presumed MBW complex. These results led
124
us to a hypothesis that engineering the master regulator SlAN2-like could enhance
125
anthocyanin pigmentation in a light-independent manner. Indeed, expression of
126
SlAN2-like under the control of a fruit-specific promoter resulted in fruits with
127
enhanced anthocyanin accumulation in both peel and flesh. Our study uncovers the
128
transcriptional regulatory complex underlying anthocyanin biosynthesis in tomato
129
fruit and provides an excellent target for engineering high levels of anthocyanins and
130
other metabolites.
131 132
Results
133
A bulk population sequencing approach to map genes controlling anthocyanin
134
accumulation in InR
135
Since the original purple tomato InR was maintained in an undeclared background,
136
we introduced the anthocyanin accumulation trait into the background of the cultivar
137
Alisa Craig (AC) which does not show anthocyanin pigmentation in the fruit (Figure
138
1A and Supplemental Figure 1). Similar to InR, InR/AC accumulated large amounts
139
of anthocyanins in the light-exposing peel but not in the shading part of the peel
140
(Figure 1A and 1B). Notably, InR fruit does not accumulate anthocyanins in the flesh 5
141
(Figure 1A). These observations indicate that InR and InR/AC accumulate
142
anthocyanins in the fruit peel in a light-dependent manner.
143 144
To map the genes controlling anthocyanin pigmentation in InR, we crossed InR with
145
AC and generated an F2 population segregating the anthocyanin pigmentation trait.
146
Based on the anthocyanin pigmentation pattern of the fruit peel, F2 progeny can be
147
classified into InR-type (intense anthocyanin pigmentation), Aft-type (slight
148
anthocyanin
149
(Supplemental Figure 1). The segregation of InR-type:Aft-type:AC-type fits a ratio of
150
3:9:4 (108:302:151, P =0.48, chi-square test), confirming that the anthocyanin
151
pigmentation trait of InR is controlled by the dominant Aft locus and the recessive atv
152
locus (Mes et al., 2008). To map these loci using the same population, we performed
153
bulk population sequencing (Austin et al., 2014) on DNA pools of InR-type, Aft-type
154
and AC-type.
pigmentation)
and
AC-type
(no
anthocyanin
pigmentation)
155 156
SNP index comparison between DNA pools of the Aft-type and the AC-type revealed
157
that the Aft locus of InR was mapped to the bottom of chromosome 10 (Figure 1C),
158
which is consistent with previous mapping results using independent mapping
159
populations (De Jong et al., 2004). Similarly, SNP index comparison between DNA
160
pools of InR-type and Aft-type revealed that the atv locus of InR was mapped to
161
chromosome 7 (Figure 1D), which is also consistent with previous mapping results
162
(Cao et al., 2017; Colanero et al., 2018).
163 164
Aft encodes an R2R3-MYB and promotes anthocyanin accumulation
165
In the Aft mapping interval, we identified a gene cluster harbouring 4 R2R3-MYB
166
genes (Figure 1E), which have been designated as SlAN2 (De Jong et al., 2004; Jian et
167
al., 2019), SlANT1 (Mathews et al., 2003; Schreiber et al., 2012), SlANT1-like (Kiferle
168
et al., 2015) and SlAN2-like (Kiferle et al., 2015), respectively. Phylogenetic analysis 6
169
indicated that these four genes encode proteins that show high similarity to the known
170
R2R3-MYBs that play a regulatory role for anthocyanin biosynthesis (Supplemental
171
Figure 3). We then examined the expression of the 4 MYB genes in light-exposing and
172
shading peel of InR fruit and AC fruit. Reverse transcription-quantitative polymerase
173
chain reaction (RT-qPCR) analysis revealed that SlAN2-like is highly expressed in the
174
light-exposing peel (exhibits anthocyanin pigmentation) but not the shading peel (no
175
anthocyanin pigmentation) of InR fruit (Figure 2A). On the contrary, SlANT1, SlAN2
176
and SlANT1-like show negligible expression in both the light-exposing peel and the
177
shading peel of InR fruit (Figure 2A). Parallel experiments failed to detect significant
178
expression of the 4 MYB genes in the light-exposing and shading peel of AC fruit
179
(Figure 2A).
180 181
That the expression of SlAN2-like is co-related with the anthocyanin pigmentation
182
pattern of the InR fruit peel supports that SlAN2-like is a strong candidate for Aft.
183
Indeed, clustered regularly interspaced short palindromic repeats/CRISPR-associated
184
9 (CRISPR/Cas9)-mediated knock-out (Deng et al., 2017) mutations of SlAN2-like
185
(Supplemental Figure 2) largely abolished anthocyanin pigmentation of the InR fruit
186
peel (Figure 2B and 2C). On the contrary, CRISPR/Cas9-mediated editing of SlANT1,
187
SlAN2 and SlANT1-like (Supplemental Figure 2) showed negligible effects on
188
anthocyanin pigmentation of the InR fruit peel (Figure 2B and 2C). Together, these
189
results led us to the conclusion that Aft encodes the R2R3-MYB protein SlAN2-like.
190 191
Next, we examined the tissue-specific expression pattern of SlAN2-like in InR and
192
AC. Results showed that SlAN2-like was only highly expressed in the light-exposing
193
fruit peel of InR, but not in seedlings, leaves and stems of InR (Figure 2G). In
194
parallel, SlAN2-like expression was barely detectable in any tissue examined of AC
195
plants (Figure 2G).
196 7
197
To explore the function of SlAN2-like in regulating anthocyanin biosynthesis, the InR
198
allele of SlAN2-like (SlAN2-likeInR) was fused with green fluorescent protein (GFP)
199
and transferred to the AC background under the control of the 35S promoter to
200
generate SlAN2-likeInR-OE plants (Figure 2F). Similarly, the AC allele of SlAN2-like
201
(SlAN2-likeAC) was fused with GFP and transferred to the AC background under the
202
control of the 35S promoter to generate SlAN2-likeAC-OE plants. (Figure 2F). Notably,
203
SlAN2-likeInR-OE plants, but not SlAN2-likeAC-OE plants, showed anthocyanin
204
pigmentation in the fruit peel (Figure 2D and 2E) as well as in seedlings, roots, leaves
205
and flowers (Supplemental Figure 6), suggesting that SlAN2-likeInR is functional but
206
SlAN2-likeAC is not functional.
207 208
To address why SlAN2-like is non-functional in AC, we compared the genomic
209
sequence of SlAN2-like between InR and AC and found plenty variations
210
(Supplemental Figure 4C). For example, the nucleotide G653 of SlAN2-likeInR
211
genomic DNA, which locates in the conserved 5' splice site of the 2nd intron (Stamm
212
et al., 2005), changed to A657 in the genomic DNA of SlAN2-likeAC. Indeed, isolation
213
of the SlAN2-like cDNA from SlAN2-likeAC-OE plants revealed that SlAN2-likeAC
214
adopted an altered 5' splice site of the second intron, leading to a truncated protein
215
that lacks the R3 domain and the C-terminal (Figure1G, 1H and Supplemental Figure
216
4). RT-qPCR analysis using transcript-specific primers found that only the AC
217
version of the transcript, which encodes a truncated protein (Supplemental Figure 4B),
218
were detected in SlAN2-likeAC-OE plants, including in fruit peel and seedlings (Figure
219
2H). Additionally, we overexpressed the SlAN2-likeAC and SlAN2-likeInR genomic
220
sequence in Nicotiana benthamiana leaves under the 35S promoter. RT-qPCR
221
analysis with transcript-specific primers confirmed that SlAN2-likeAC only produced
222
the AC version of the transcript, while SlAN2-likeInR only produced the InR version of
223
the transcript (Supplemental Figure 7B).
224 8
225
Next, we made an A657G mutation in SlAN2-likeAC (SlAN2-likeAC-A657G)
226
(Supplemental Figure 7A) as well as a G653A mutation in SlAN2-likeInR
227
(SlAN2-likeInR-G653A) (Supplemental Figure 7A). These constructs were transformed
228
into N. benthamiana leaves to test their splicing pattern. As expected,
229
SlAN2-likeInR-G653A only produced the AC version of the transcript (Supplemental
230
Figure 7B). On the contrary, SlAN2-likeAC-A657G only produced the InR version of the
231
transcript (Supplemental Figure 7B). Taken together, these results demonstrate that
232
the G to A mutation at the splice site of the 2nd intron is the causal mutation that
233
determines the splicing pattern of SlAN2-like.
234 235
We then employed a dual-luciferase reporter (DLR) system (Hellens et al., 2005) to
236
examine whether genetic manipulation of SlAN2-like affects the expression of SlDFR,
237
which encodes a key enzyme of anthocyanin biosynthesis (Goldsbrough et al., 1994)
238
(Supplemental Figure 7C). Co-expression of a construct containing the SlAN2-likeInR
239
genomic sequence with the proSlDFR:LUC reporter led to high luciferase (LUC)
240
expression (Supplemental Figure 7D). In contrast, co-expression of a construct
241
containing the SlAN2-likeAC genomic sequence with the proSlDFR:LUC reporter
242
failed to activate LUC expression (Supplemental Figure 7D). However, co-expression
243
of SlAN2-likeAC-A657G with the reporter construct also led to high LUC expression
244
(Supplemental Figure 7D). Together, these results corroborate that SlAN2-likeInR, but
245
not SlAN2-likeAC, is functional to activate SlDFR expression. In addition, RT-qPCR
246
analysis showed that the expression of SlDFR was significantly increased in
247
SlAN2-likeInR-OE plants and decreased in SlAN2-likeInR-edited plants (slan2-like-c)
248
(Figure 2I and 2J), indicating that SlAN2-like positively regulates the expression of
249
SlDFR. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays using
250
the above-described SlAN2-likeInR-OE plants and anti-GFP antibody revealed an
251
enrichment of the SlAN2-likeInR-GFP recombinant protein on the SlDFR promoter
252
region containing a putative MYB binding site (MBS) (Zhu et al., 2015) (Figure 2K 9
253
and 2L). Collectively, these results support that SlAN2-likeInR promotes anthocyanin
254
accumulation through positively regulating anthocyanin biosynthetic genes.
255 256
Next, we sequenced SlAN2-like gene in some cultivars and wild tomatoes
257
(Supplemental Figure 5). Based on sequence alignment, most red-fruited tomatoes,
258
including cultivars and its ancestor Solanum pimpinellifolium (Lin et al., 2014),
259
contain the G to A mutation at the 5' splice site of the 2nd intron, indicating that
260
SlAN2-like are non-functional in these plants (Supplemental Figure 5). On the
261
contrary, several wild species showing anthocyanin accumulation including Solanum
262
chilense (Schreiber et al., 2012) and Solanum peruvianum (Kiferle et al., 2015)
263
contain the normal 5' splice site at the 2nd intron and encode a functional SlAN2-like
264
(Supplemental Figure 5). It has been reported that the cultivar LA1996 contains the
265
Aft locus introgressed from S. chilense (Jones et al., 2003), sequence analyses
266
indicated that this cultivar also contains a functional SlAN2-like (Supplemental Figure
267
5).
268 269
Together, our results reveal that, whereas most tomato cultivars contain a
270
loss-of-function allele of SlAN2-like (i.e., SlAN2-likeAC) (Supplemental Figure 5),
271
InR contains a functional allele of this anthocyanin activator (i.e., SlAN2-likeInR).
272 273
Atv encodes an R3-MYB and represses anthocyanin accumulation
274
In the atv mapping region of chromosome 7, an R3-MYB gene SlMYBATV was
275
proposed as a candidate gene underlying the atv mutation (Cao et al., 2017; Colanero
276
et al., 2018). In line with these observations, we found that, compare to its AC allele
277
counterpart (SlMYBATVAC), the InR allele of SlMYBATV (SlMYBATVInR) contains a
278
4-bp insertion in the second exon, which leads a truncated protein that lacks the R3
279
domain (Figure 1I and 1J).
280 10
281
To test whether this mutation is responsible for the effect of atv in the InR
282
background, we performed a genetic complementation assay. For this purpose,
283
SlMYBATVAC driven by its native promoter was transferred to the InR background
284
(proSlMYBATV:SlMYBATVAC). As expected, anthocyanin pigmentation of the
285
resulting transgenic plants was largely reduced compared to InR (Figure 3A and 3B),
286
supporting that the SlMYBATVInR mutation underlies the effect of the atv locus in InR.
287 288
Next, we generated CRISPR/Cas9-induced mutations of SlMYBATV in the cultivar
289
LA1996 (Jones et al., 2003), which contains the dominant Aft locus and exhibits a
290
typical “Aft-type” anthocyanin pigmentation pattern in the fruit peel (Supplemental
291
Figure 8A and 8B). The resulting SlMYBATV-edited plants (slmybatv-c) showed an
292
anthocyanin pigmentation pattern similar to that of InR (Figure 3C, 3D and 3E),
293
indicating that mutation of SlMYBATV in an Aft-containing background could
294
recapture the anthocyanin pigmentation phenotype of InR fruit.
295 296
The above results suggest that the cultivar allele of SlMYBATV (i.e., SlMYBATVAC)
297
negatively regulates anthocyanin accumulation and that SlMYBATVInR is a
298
loss-of-function allele. To prove this, we generated transgenic plants overexpressing
299
SlMYBATVAC in the background of InR (SlMYBATVAC-OE). Indeed, overexpression of
300
SlMYBATVAC abolished anthocyanin pigmentation as well as SlDFR expression of the
301
InR fruit peel (Figure 3F, 3G and 3H), suggesting a negative role of SlMYBATVAC in
302
anthocyanin biosynthesis.
303 304
It is believed that the recessive atv locus is derived from the wild tomato Solanum
305
cheesmaniae (Mes et al., 2008). Consistently, our sequence analyses indicated that
306
only InR and the S. cheesmaniae tomato LA0434 contain a loss-of-function allele of
307
SlMYBATV (Supplemental Figure 9), while most cultivars and wild species
11
308
(Solanum pimpinellifolium, Solanum chilense and Solanum peruvianum) are predicted
309
to encode a functional SlMYBATV (Supplemental Figure 9).
310 311
Taken together, our data support that Atv encodes the R3-MYB protein SlMYBATV
312
and that this protein acts as a repressor of anthocyanin biosynthesis. Whereas most
313
tomato cultivars like AC contain a functional allele of SlMYBATV (i.e.,
314
SlMYBATVAC) (Supplemental Figure 9), InR contains a non-functional allele of this
315
anthocyanin repressor (i.e., SlMYBATVInR).
316 317
SlAN1 promotes anthocyanin accumulation in the fruit peel
318
It has been shown that the bHLH transcription factor SlAN1 positively regulates
319
anthocyanin biosynthesis in tomato seedlings (Qiu et al., 2016). To test whether
320
SlAN1 contributes to the anthocyanin pigmentation of the InR fruit peel, we generated
321
SlAN1-edited plants (slan1-c) in the InR background. Sequence analyses indicated
322
that all SlAN1-edited plants carry mutations in the ORF, which lead to frame shift and
323
the generation of a premature stop codon (Supplemental Figure 10). As expected,
324
editing SlAN1 led to abolished anthocyanin pigmentation (Figure 4A and 4B) and
325
significantly reduced SlDFR expression in the InR fruit peel (Figure 4C), confirming
326
that SlAN1 plays a positive role for the anthocyanin pigmentation of InR fruit peel.
327 328
SlAN2-likeInR promotes the expression of SlAN1 and SlMYBATV
329
The above results demonstrate that both the positive effect of SlAN2-likeInR and SlAN1
330
and the negative effect of SlMYBATVAC contribute to the anthocyanin pigmentation
331
trait of the InR fruit. To test the possible inter-regulation of these genes, we examined
332
the expression of SlAN1 and SlMYBATV in the fruit peel of SlAN2-likeInR-edited plants
333
(slan2-like-c) and SlAN2-likeInR-OE plants. Results showed that whereas editing
334
SlAN2-likeInR led to reduced expression of SlAN1 and SlMYBATV (Figure 4D),
335
overexpression of SlAN2-likeInR led to increased expression of SlAN1 and SlMYBATV 12
336
(Figure 4E), indicating that SlAN2-likeInR positively regulates both the positive
337
regulator SlAN1 and the negative regulator SlMYBATV.
338 339
On the contrary, editing SlAN1 showed negligible effect on the expression of
340
SlAN2-likeInR (Figure 4F). Similarly, neither editing (Figure 4G) nor overexpression
341
of SlMYBATVAC (Figure 4H) significantly affected the expression of SlAN2-likeInR.
342
Together, these results support a scenario that SlAN2-likeInR acts upstream of SlAN1
343
and SlMYBATV and likely functions as a master regulator to orchestrate anthocyanin
344
biosynthesis.
345 346
SlMYBATVAC and SlAN2-likeInR compete for binding SlAN1
347
That the anthocyanin pigmentation phenotype of InR depends on the co-existence of
348
the dominant Aft locus and the recessive atv locus prompted us to explore the
349
biochemical basis of this genetic interaction. Yeast two-hybrid (Y2H) assays
350
indicated that the R3 domain of SlAN2-likeInR (SlAN2-likeR3) interacts with SlAN1
351
(Figure 5A). This interaction was verified by in vitro pull down assays showing that a
352
purified glutathione S-transferase (GST)-tagged SlAN2-likeInR (GST-SlAN2-likeInR)
353
could pull down the trigger factor (TF)-tagged SlAN1 fragment SlAN111-209
354
(TF-SlAN111-209) (Figure 5B). Domain mapping with Y2H assays revealed that
355
SlAN2-likeInR interacts the SlAN111-209 fragment of SlAN1 (Figure 5D), we therefore
356
designated SlAN111-209 as MYB-interacting region (MIR, SlAN1MIR).
357 358
In parallel experiments, we found that SlMYBATVAC also interacts with SlAN1 in
359
Y2H assays (Figure 5A), which was verified by in vitro pull down assays that
360
maltose-binding protein (MBP)-tagged SlMYBATVAC (MBP-SlMYBATVAC) could
361
pull down TF-SlAN1MIR (Figure 5C). Notably, domain mapping with Y2H assays
362
indicated that SlMYBATVAC also interacts the MIR of SlAN1 (Figure 5D).
363 13
364
SlAN2-likeInR and SlMYBATVAC interact with the same domain of SlAN1 raised the
365
possibility that the positive regulator SlAN2-likeInR and the negative regulator
366
SlMYBATVAC might compete with each other for binding SlAN1. Indeed, yeast
367
three-hybrid (Y3H) assays revealed SlAN2-likeInR–SlAN1 interaction on the synthetic
368
defined (SD) medium lacking Ade, His, Trp, and Leu (SD/-4); however, the induction
369
of SlMYBATVAC expression on SD medium lacking Ade, His, Trp, Leu, and Met
370
(SD/-5) led to a dramatic reduction in SlAN2-likeInR–SlAN1 interaction (Figure 5E),
371
suggesting that SlMYBATVAC interferes with SlAN2-likeInR–SlAN1 interaction. On
372
the other hand, SlMYBATVAC–SlAN1 interaction was detected on SD/-4 medium,
373
and this interaction was obviously reduced by inducing the expression of
374
SlAN2-likeInR on SD/-5 medium (Figure 5E), suggesting that SlAN2-likeInR also
375
impairs SlMYBATVAC–SlAN1 interaction.
376 377
To substantiate the above observations, we performed in vitro pull-down experiments
378
using a constant protein concentration of TF-SlAN1MIR and increasing protein
379
concentration of GST-SlAN2-likeInR or MBP-SlMYBATVAC. Results showed that the
380
ability of GST-SlAN2-likeInR to pull down TF-SlAN1MIR decreased as the amount of
381
MBP-SlMYBATVAC increased (Figure 5F). As a negative control, increasing the
382
amount of MBP failed to influence the ability of GST-SlAN2-likeInR to pull down
383
TF-SlAN1MIR (Figure 5F). Similarly, the ability of MBP-SlMYBATVAC to pull down
384
TF-SlAN1MIR decreased as the amount of GST-SlAN2-likeInR increased (Figure 5G),
385
and the increasing of GST has negligible effect on the ability of MBP-SlMYBATVAC
386
to pull down TF-SlAN1MIR (Figure 5G). These results corroborate that SlMYBATVAC
387
and SlAN2-likeInR compete with each other to interact with SlAN1.
388 389
SlHY5 plays a critical role in light-induced activation of SlAN2-likeInR
390
Next, we set out to identify the signalling components by which light activates
391
anthocyanin pigmentation in InR fruit. Considering that the bZIP transcription factor 14
392
SlHY5 plays a key role in light-induced anthocyanin accumulation in Arabidopsis
393
(Nguyen et al., 2015; Shin et al., 2013), we edited the tomato SlHY5 gene in the InR
394
background using the CRISPR/Cas9 gene editing system (Deng et al., 2017; Liu et al.,
395
2004) (Supplemental Figure 11A and 11B). In line with a recent report (Qiu et al.,
396
2019), editing SlHY5 led to abolished anthocyanin pigmentation (Figure 6Aand 6B)
397
and SlDFR expression (Figure 6C) in light-exposing fruit peel of InR. These results
398
indicated that SlHY5 plays a critical role in transmitting the light signal to the
399
anthocyanin biosynthesis machinery.
400 401
Not surprisingly, editing SlHY5 largely impaired light-induced expression of
402
SlAN2-likeInR in the InR fruit peel (Figure 6D), indicating that SlHY5 positively
403
regulates the expression of the master regulator SlAN2-likeInR. ChIP-qPCR assays with
404
transgenic SlHY5-GFP plants (Supplemental Figure 11C) and anti-GFP antibody
405
revealed an enrichment of the SlHY5-GFP fusion protein on a SlAN2-likeInR promoter
406
region containing an ACE-box (ACGT) (Figure 6E and 6F), a DNA element that can
407
be recognized by bZIP transcription factors (Lee et al., 2007).
408 409
We then examined the importance of the ACE-box for SlHY5-mediated activation of
410
SlAN2-likeInR expression using the above-described DLR assays. Considering that
411
SlHY5 itself lacks a transcriptional activation domain (Supplemental Figure 11E and
412
11G), we fused the VP16 activation domain (Stracke et al., 2010) to the C-terminal of
413
SlHY5 to generate the SlHY5-VP16 construct. Co-expression of SlHY5-VP16 with
414
proSlAN2-likeInR:LUC led to significantly increased LUC activity, but LUC signal
415
was significantly reduced when the ACE-box in the SlAN2-likeInR promoter was
416
mutated to “TTTT” (Supplemental Figure 12C). These results support that the
417
ACE-box in the SlAN2-likeInR promoter plays an important role for the SlHY5 activity
418
to activate SlAN2-likeInR expression.
419 15
420
Electrophoretic mobility shift assays (EMSAs) showed that the MBP-SlHY5
421
recombinant protein could bind a DNA probe containing the ACE-box but failed to
422
bind a DNA probe in which the ACE-box was mutated (Figure 6G). Collectively,
423
these results demonstrate that SlAN2-likeInR is a direct transcriptional target of SlHY5.
424 425
In summary, we proposed a model to explain the light-induced anthocyanin
426
pigmentation pattern of different genotypes (Figure 6H, 6I and Supplemental Figure
427
13). In the dominant Aft single mutant (i.e., LA1996), both the activator SlAN2-likeInR
428
and the repressor SlMYBATVAC are functional. In light-exposing peel of this
429
genotype, SlHY5 promotes the expression of SlAN2-likeInR, whose protein product in
430
turn activates both the activator SlAN1 and the repressor SlMYBATVAC. The
431
functional repressor SlMYBATVAC competes the binding of SlAN2-likeInR to SlAN1
432
and thereby reducing the formation of a functional SlAN2-likeInR–SlAN1 complex,
433
which determines the transcriptional output for anthocyanin biosynthesis (Figure 6H).
434
In InR which simultaneously harbors the dominant Aft locus and the recessive atv
435
locus, the activator SlAN2-likeInR is functional but the repressor SlMYBATVInR is
436
not-functional; the non-functional repressor SlMYBATVInR loses its ability to reduce
437
the transcriptional output for anthocyanin biosynthesis. Therefore, light-induced
438
anthocyanin accumulation in the fruit peel of InR is much higher than that of the Aft
439
single mutant (Figure 6I). In both AC (Supplemental Figure 13A) and the atv single
440
mutant (Supplemental Figure 13B), the master regulator SlAN2-likeAC is
441
not-functional and, as a consequence, light cannot activate the functional transcription
442
complex for anthocyanin biosynthesis. Our model highlights a central role of a
443
functional SlAN2-like (i.e., SlMYBATVInR) in linking the light signal to the
444
activation of anthocyanin biosynthesis in tomato fruit.
445 446
Engineering the expression of SlAN2-likeInR leads to anthocyanin accumulation
447
in both peel and flesh 16
448
If our model holds true, we predict that engineering the master regulator SlAN2-likeInR
449
could bypass light and activate anthocyanin biosynthesis in the fruit flesh of any
450
cultivar. To test this, SlAN2-likeInR under the control of the fruit-specific SlE8
451
promoter (Kneissl and Deikman, 1996) was transferred to the cultivar AC, which does
452
not accumulate anthocyanin in the peel and flesh. As expected, the resulting
453
proSlE8:SlAN2-likeInR plants exhibited anthocyanin accumulation in both peel and
454
flesh (Figure 7A). Anthocyanin pigmentation started at the breaker stage (42 days
455
post-anthesis, dpa) and intensified rapidly during the ripening process (Figure 7A),
456
which matched well with the expression pattern of the SlE8 gene (Kneissl and
457
Deikman, 1996). Whereas the anthocyanin content of the AC (wild type) fruits was
458
below the detection limit, the highest concentrations of transgenic fruits averaged
459
40.44 ± 4.39 (A535-A650)/g fresh weight, which equivalents to 2.22 ± 0.24 mg of
460
anthocyanin per g fresh weight (Figure 7B).
461 462
To evaluate the impact of the master regulator SlAN2-likeInR on gene expression in a
463
genome-wide scale, we performed RNA-sequencing (RNA-seq) experiments to
464
compare the transcriptome profiles between proSlE8:SlAN2-likeInR and AC fruits.
465
Fruits of each genotype were collected at the B+14 stage (56 dpa) for RNA extraction
466
and sequencing. Data quality assessment of three biological replications is shown in
467
Supplemental Figure 14. These analyses identified 2,074 differentially expressed
468
genes (DEGs) between transgenic and AC fruits (fold change ≥ 2, false discovery rate
469
[FDR]-adjusted P value < 0.05). Among them, 1,552 genes were up-regulated and
470
522 genes were down-regulated in proSlE8:SlAN2-likeInR fruits compared to that of
471
AC. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the
472
up-regulated
473
SlAN2-likeInR-up-regulated genes) are mainly enriched in pathways related to
474
phenylpropanoid biosynthesis, phenylalanine metabolism and flavonoid biosynthesis
475
(Figure 7C). Among the identified SlAN2-likeInR-up-regulated genes are most of the
genes
in
proSlE8:SlAN2-likeInR
17
fruits
(designated
as
476
known anthocyanin biosynthetic genes (Figure 7D) and several regulatory genes
477
including SlAN1 and SlMYBATV (Figure 7E), corroborating that SlAN2-likeInR
478
functions as a master regulator and orchestrates a hierarchical transcriptional
479
regulatory cascade for anthocyanin biosynthesis.
480 481
Discussion
482
Starting from the genetic analysis of the purple tomato InR, we identified the major
483
genes controlling anthocyanin pigmentation of tomato fruit and elucidated their action
484
mechanism. We demonstrated that whereas the dominant Aft locus encodes a
485
functional allele of the positive regulator SlAN2-like (i.e., SlAN2-likeInR), the
486
recessive atv locus encodes a non-functional allele of the negative regulator
487
SlMYBATV (i.e., SlMYBATVInR). We also demonstrated that the bHLH protein
488
SlAN1 is a positive regulator of anthocyanin pigmentation. Considering that the
489
MBW transcriptional regulators are generally conserved in eudicots (Lloyd et al.,
490
2017; Xu et al., 2015), it is reasonable to speculate that SlAN2-likeInR and SlAN1
491
might represent the MYB and the bHLH component, respectively, of the tomato
492
MBW regulatory complex. SlMYBATV, which negatively regulates anthocyanin
493
biosynthesis, might represent a new component of the tomato MBW complex. Our
494
results indicated that a functional allele of SlMYBATV (i.e., SlMYBATVAC)
495
competes the binding of SlAN2-likeInR to SlAN1 and thereby exerting a negative
496
effect on anthocyanin biosynthesis. Therefore, the anthocyanin pigmentation
497
phenotype of InR is resulted from the functional interaction between the functional
498
positive regulator SlAN2-likeInR and the non-functional negative regulator
499
SlMYBATVInR. On the contrary, most tomato cultivars (i.e., AC) contain the
500
non-functional positive regulator SlAN2-likeAC (Supplemental Figure 5) as well as the
501
functional negative regulator SlMYBATVAC (Supplemental Figure 9) and, as a
502
consequence, loss the capability to produce anthocyanins in fruits.
503 18
504
Furthermore, several lines of evidence suggest that the functional positive regulator
505
SlAN2-likeInR is an active and critical regulator of anthocyanin biosynthesis. First,
506
unlike SlAN2, SlANT1 and SlANT1-like, which barely express in tomato fruit,
507
SlAN2-likeInR is highly expressed in InR fruit peel under light-exposing. Second,
508
different with SlAN2-likeAC which is non-functional because of the G to A mutation
509
at the 5' splice site, SlAN2-likeInR is functional. Third, in addition to regulating the
510
expression of structural genes involved in anthocyanin biosynthesis, the functional
511
SlAN2-likeInR also regulates the expression of other regulatory genes including SlAN1
512
and SlMYBATV. Finally, the expression of SlAN2-likeInR itself is not affected by
513
SlAN1 and SlMYBATVAC. Together, these results suggest that genetic manipulation
514
of SlAN2-likeInR could activate anthocyanin biosynthesis in a light-independent
515
manner. Indeed, expression of SlAN2-likeInR under the control of the fruit-specific
516
SlE8 gene promoter led to high levels of anthocyanin accumulation in both peel and
517
flesh of the cultivar AC. Our study suggests two directions for future exploration.
518
First, to circumvent the concern of foreign DNA introgression, gene editing-based
519
technology (Voytas and Gao, 2014) could be exploited to replace the SlAN2-likeInR
520
promoter with the fruit-specific SlE8 promoter or insert the SlAN2-likeInR gene
521
downstream of the SlE8 promoter. Second, master transcriptional regulators such as
522
SlAN2-likeInR could provide effective tools for tissue-specific engineering high levels
523
of metabolites in other plants.
524 525
Notably, our results indicated that, while mutation of SlHY5 in InR abolished
526
SlAN2-likeInR expression and anthocyanin accumulation (Figure 6A and 6D),
527
overexpression
528
(Supplemental Figure 11D). In contrast, overexpression of SlHY5 failed to activate
529
SlAN2-likeAC expression in the AC background (Supplemental Figure 11I). The
530
differential effects of SlHY5 on the expression of SlAN2-likeInR and SlAN2-likeAC
531
could resulted from sequence variations of the SlAN2-likeInR and SlAN2-likeAC
of
SlHY5
in
InR
could
19
activate
SlAN2-likeInR expression
532
promoters. Indeed, our sequence comparison identified numerous variations between
533
the promoter regions of SlAN2-likeInR and SlAN2-likeAC (Supplemental Figure 12A).
534
Considering that SlHY5 itself does not possess activation capacity (Supplemental
535
Figure 11E and 11G) and that the putative SlHY5 binding site ACE-box does not
536
show difference between the promoter of SlAN2-likeInR and SlAN2-likeAC
537
(Supplemental Figure 12A), it is reasonable to speculate that there exist yet-to-be
538
identified sequence variations that determine the differential effects of SlHY5 on the
539
SlAN2-likeInR and SlAN2-likeAC promoters.
540 541
Methods
542
Plant materials and growth conditions
543
InR was used as the wild type (WT) for slant1-c, slan2-c, slant1-like-c, slan2-like-c,
544
proSlMYBATV:SlMYBATVAC, SlMYBATVAC-OE, slan1-c, slhy5-c and SlHY5-GFP.
545
AC was used as the WT for SlAN2-likeAC-OE, SlAN2-likeInR-OE, SlHY5-GFP/AC and
546
proSlE8:SlAN2-likeInR. LA1996 was used as the WT for slmybatv-c. InR/AC was
547
produced by introducing Aft and atv loci from InR into AC via backcrossing. Seeds of
548
AC, MicroTom, Moneymaker, VF36, M82, E6203, LA2750, LA0458, LA0462,
549
LA0448, LA1996, LA1406, LA0434, LA1610, LA2934, LA2398, LA0373 and
550
LA1279 were provided by the Tomato Genetics Resources Center at the University of
551
California, Davis. Tomato plants were grown side-by-side to ensure consistency of
552
the light condition.
553 554
Plasmid construction and plant transformation
555
DNA constructs for plant transformation were generated following standard
556
molecular biology protocols and using Gateway (Invitrogen) technology (Karimi et
557
al., 2002). To generate the SlAN2-like-OE construct, full-length SlAN2-like coding
558
sequence (CDS) from InR and the SlAN2-like genomic sequence from AC was cloned
559
(we failed to isolate SlAN2-like CDS from AC) into pENTR respectively, and then 20
560
recombined with the binary vector pK7WGF2 (Karimi et al., 2002) using a gateway
561
LR clonase enzyme mix kit. SlHY5-GFP and SlMYBATVAC-OE construct was
562
generated in a similar procedure, except that the CDS of SlHY5 and SlMYBATV were
563
isolated from AC. For proSlMYBATV:SlMYBATVAC construction, the 35S promoter of
564
pK7WGF2 was substituted by the 2752-bp SlMYBATV native promoter using the
565
unique enzymes Hind III and Spe I (Thermo Scientific), then the SlMYBATVAC CDS
566
was
567
proSlE8:SlAN2-likeInR construction, the 35S promoter of pK7WGF2 was substituted
568
by the 2187-bp SlE8 promoter using the unique enzymes Hind III and Spe I (Thermo
569
Scientific), then the full-length CDS of SlAN2-likeInR was cloned and recombined into
570
this modified binary vector. The above constructs were introduced into InR or AC
571
(except SlHY5-GFP which was transformed into both the InR and AC background) by
572
Agrobacterium tumefaciens (LBA4404)-mediated cotyledon explants transformation
573
as previously described (Deng et al., 2017). Transformants were selected based on
574
their resistance to kanamycin. T2 homozygous progeny were used for phenotypic and
575
molecular characterization.
cloned
and
recombined
into
this
modified
binary
vector.
For
576 577
Generation of gene editing plants using CRISPR/Cas9 technology
578
The generation of gene editing plants of SlANT1, SlAN2, SlANT1-like, SlAN2-like,
579
SlMYBATV, SlAN1 and SlHY5 was conducted as described previously (Deng et al.,
580
2017; Liu et al., 2019). Briefly, the two target sites of each gene were incorporated
581
into PCR forward and reverse primers, respectively. The PCR fragment was amplified
582
from pCBC-DT1T2_tomatoU6, and then purified and cloned into pTX041 at Bsa I
583
sites to generate pTX041_target gene. pTX041_SlMYBATV was introduced into
584
LA1996 and other constructs were introduced into InR by Agrobacterium tumefaciens
585
(LBA4404)-mediated cotyledon explants transformation. CRISPR/Cas9-induced
586
mutations were genotyped by PCR amplification and DNA sequencing. Primers used
587
for construction are listed in Supplemental Table 1. 21
588 589
Anthocyanin Measurement
590
Anthocyanin content was measured as previously described (Deikman and Hammer,
591
1995) with modification. Samples of fruit peel were ground to powder and about 0.1 g
592
powder was placed into 1 mL of extraction buffer (18% 1-propanol, 1% HCl, and
593
81% water) and incubated overnight at 4℃. Absorbance values (A535 and A650) of
594
the supernatant were measured using spectrophotometer after centrifugation for 20
595
min at 12,000 rpm. The anthocyanin content is presented as (A535-A650)/g fresh
596
weight. Anthocyanin content of proSlE8:SlAN2-likeInR flesh was further expressed as
597
mg/g fresh weight, based on an extinction coefficient of 17,000 and a molecular
598
weight of 934 (Butelli et al., 2008). Data from at least four independent biological
599
replicates were collected, and error bars represent the SD from the biological
600
replicates.
601 602
Bulk population sequencing
603
For bulk population sequencing, three DNA pools, InR-type pool, Aft-type pool and
604
AC-type pool, each with equal amount of DNA from 50 F2 individuals of InR x AC
605
cross were prepared. Three pooled libraries and the two parent libraries with insert
606
sizes of approximately 350-500 bp were prepared and sequenced by Oebiotech using
607
the Illunima HiSeq X-ten platform. To identify the mutation site, reads were mapped
608
on the tomato genome using BWA (Li and Durbin, 2009) with the default options.
609
SAMtools (Li et al., 2009) was used to convert alignment files to SAM/BAM format
610
and applied to SNP calling. SNP positions with SNP quality score < 20 and read
611
depth < 4 were excluded, as these positions may represent spurious SNPs called due
612
to sequencing and/or alignment errors. Homozygous SNPs between InR and AC but
613
different in the 3 pools were filtered, The proportion of reads harbouring InR
614
genotype in the total reads cover the SNP site is defined as the SNP-index and
615
candidate regions associated with anthocyanin were calculated according to ƸSNP 22
616
index between different pools as described before (Takagi et al., 2013).
617 618
RNA extraction and gene expression analysis
619
For reverse-transcription quantitative PCR (RT-qPCR) analysis, total RNA was
620
extracted from tomato fruit peel using the TRIzol Reagent (Invitrogen). 1μg of total
621
RNA was used to synthesize the first strand cDNA with the PrimeScript RT kit with
622
gDNA eraser (Takara). RT-qPCR was performed on Roche LightCycler 480 system
623
with the Kapa SYBR Fast qPCR kit (KAPA). The expression levels of target genes
624
were normalized to that of the tomato SlACTIN2 gene. Error bars represent the SD of
625
three biological replicates. Primer information is given in Supplemental Table 1.
626 627
Transient Expression Assays in Nicotiana benthamiana leaves
628
For the transient transcriptional activity assays of SlAN2-like to activate SlDFR
629
expression, the 1233-bp promoter sequence of SlDFR promoter was amplified from
630
genomic DNA from InR and cloned into the pGreenII 0800-LUC (Hellens et al., 2005)
631
vector to use as a reporter. The Renilla luciferase (REN) gene under the control of the
632
cauliflower 35S promoter in the pGreenII 0800-LUC vector was used as the internal
633
control. The genomic sequences of SlAN2-likeInR, SlAN2-likeAC and their derivatives
634
(SlAN2-likeInR-G653A and SlAN2-likeAC-A657G) were cloned into the pK7WGF2 (Karimi
635
et al., 2002) under the control of the 35S promoter and were used as effectors. Primers
636
used for generating these constructs are listed in Supplemental Table 1. Firefly LUC
637
and REN activities were measured using the Dual-Luciferase Reporter Assay System
638
(Promega) following the manufacturer’s instructions, and LUC/REN ratios were
639
calculated and presented. Data from three independent biological replicates were
640
collected, and error bars represent the standard deviation (SD) from three biological
641
replicates.
642 643
For the transient transcriptional activity assays of SlHY5-VP16 to activate SlAN2-like 23
644
expression, the 846-bp promoter sequence of SlAN2-likeInR promoter and its derivative
645
(SlAN2-likeInR promoter with a mutated ACE-box), as well as SlAN2-likeAC promoter
646
and its derivative (SlAN2-likeAC promoter with a mutated ACE-box) were amplified
647
from genomic DNA and cloned into the pGreenII 0800-LUC (Hellens et al., 2005).
648
VP16 activation domain fragment was amplified from pBT3-SUC (Dualsystems
649
Biotech.) and the merged sequences of SlHY5-VP16 were cloned into the pK7WGF2
650
(Karimi et al., 2002) under the control of the 35S promoter and were used as effectors.
651
Firefly LUC and REN activities were measured using the Dual-Luciferase Reporter
652
Assay System (Promega) following the manufacturer’s instructions, and the
653
experimental procedures were the same as those described above.
654 655
Expression profiling analysis
656
AC and proSlE8:SlAN2-likeInR fruits were harvested at the B+14 stage (56 dpa), and
657
flesh was collected for RNA isolation. Total RNA of each sample was extracted using
658
TRIzol Reagent (Invitrogen) and treated with DNase I. The quality of the total RNA
659
was assessed using a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer.
660
For each sample, 1 μg of total RNA was used to construct the Illumina sequencing
661
libraries according to the manufacturer’s instructions. The libraries were sequenced
662
by Biomarker Technologies using the Illunima HiSeq X-ten platform and
663
preliminarily analysed by using the BMKCloud platform (http://www.biocloud.net/).
664
Tomato reference genome (SL3.0) was downloaded from the SOL Genomics
665
Network (http://solgenomics.net/). Raw sequencing reads were first processed to
666
remove adaptors and low-quality bases, and then aligned to the genome sequences
667
using HISAT2 (Kim et al., 2015) with default parameters. Gene expression levels
668
were calculated in fragments per kilobase of transcript per million fragments mapped
669
(FPKMs) using StringTie (Pertea et al., 2015). Differentially expressed genes (DEGs)
670
between two groups were identified using the DEGSeq R package (Wang et al., 2010)
671
with fold change ≥ 2 and FDR-adjusted P value < 0.05. KEGG enrichment analysis 24
672
was implemented by using the KOBAS (Mao et al., 2005) with default parameters.
673 674
Yeast two-hybrid assay (Y2H)
675
Y2H assays were performed using the MATCHMAKER GAL4 Two-Hybrid System
676
(Clontech). To investigate the interactions of SlAN1 with SlAN2-likeInR and
677
SlMYBATVAC, full-length coding sequence of SlAN1 were cloned into pGADT7.
678
SlAN2-likeInR fragment spanning the R3 domain and the full-length CDS of
679
SlMYBATVAC were individually cloned into pGBKT7. To map the domains of SlAN1
680
involved in the SlAN1–SlAN2-likeInR interaction and SlAN1–SlMYBATVAC
681
interaction, derivatives of SlAN1 coding sequence were cloned into pGADT7.
682
Constructs used to test protein-protein interactions were co-transformed into yeast
683
(Saccharomyces cerevisiae) strain AH109 and co-transformation of the empty
684
pGADT7 vector was used as a negative control. Transformed yeast was grown on
685
medium lacking Leu and Trp (SD/-2) as transformation control or on medium lacking
686
Ade, His, Leu, and Trp (SD/-4) to test protein-protein interactions. The interactions
687
were observed after 3 d of incubation at 30°C. Primers used for vector construction
688
are listed in Supplemental Table 1.
689 690
Yeast three-hybrid assay (Y3H)
691
Y3H assays were performed using the MATCHMAKER GAL4 Two-Hybrid System
692
(Clontech). To construct pBridge-SlAN2-likeR3-SlMYBATVAC, the SlAN2-likeInR
693
fragment spanning the R3 domain was cloned into the MCS I (multiple cloning site I)
694
site of the pBridge vector (Clontech) fused to the GAL4 DNA-binding domain, and
695
the coding sequence of SlMYBATVAC was cloned into the MCS II site of the pBridge
696
vector expressed only in the absence of methionine. Constructs used for testing
697
protein-protein interactions were co-transformed into Saccharomyces cerevisiae strain
698
AH109. The presence of the transgenes was confirmed by growth on SD/-2 plates.
699
Transformed yeast cells were spread on SD/-4 medium to assess the SlAN1 and 25
700
SlAN2-likeInR interaction without the expression of SlMYBATVAC and on plates
701
containing SD/-Ade/-His/-Leu/-Trp/-Met (SD/-5) medium to induce SlMYBATVAC
702
expression. Interactions were observed after 3 days of incubation at 30°C. For the
703
construction of pBridge-SlMYBATVAC-SlAN2-likeR3, the SlMYBATVAC coding
704
sequence was cloned into the MCS I site and the SlAN2-likeInR fragment spanning the
705
R3 domain was cloned into the MCS II site of the pBridge vector and expressed only
706
in the absence of methionine. The experimental procedures were the same as those
707
described above.
708 709
In vitro pull-down assay
710
For pull-down assays to test the SlAN2-likeInR–SlAN1 and SlMYBATVAC–SlAN1
711
interactions, the full-length coding sequence of SlAN2-likeInR was PCR amplified and
712
cloned into the pGEX-4T-3 vector. To produce TF-SlAN1MIR fusion protein, a SlAN1
713
fragment spanning the MIR domain of SlAN1 was PCR amplified from InR and
714
cloned into pCold™ TF. Primers used for plasmid construction are listed in
715
Supplemental Table 1. The recombinant vectors were transformed into Escherichia
716
coli BL21 (DE3) cells. GST-SlAN2-likeInR, and TF-SlAN1MIR were purified using
717
GST-Bind Resin (Millipore) or Ni-NTA His-Bind Resin (Millipore), respectively. For
718
each reaction, 20 μL of agarose beads (Millipore) bound with 1 μg of
719
GST-SlAN2-likeInR was incubated with 1 μg of TF-SlAN1MIR in 1 mL of reaction
720
buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM DTT, and Roche protease
721
inhibitor cocktail) at 4°C for 2 h. Subsequently, beads were collected and washed
722
three times with washing buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1 mM
723
DTT). After washing, samples were denatured using sodium dodecyl sulfate (SDS)
724
loading buffer and separated using SDS-polyacrylamide gel electrophoresis
725
(SDS-PAGE). The TF-SlAN1MIR fusion protein was detected by immunoblotting with
726
anti-TF antibody. Purified GST was used as a negative control. 0.1μg of GST and
727
GST-SlAN2-likeInR fusion proteins were separated by SDS-PAGE, and then detected 26
728
by immunoblotting with anti-GST antibody (Abmart) as a loading control. For
729
pull-down assays to test SlMYBATVAC–SlAN1 interactions, the full-length coding
730
sequence of SlMYBATVAC was PCR amplified and cloned into the pMAL-c2X
731
vector. The recombinant vectors were transformed into E. coli BL21 (DE3) cells and
732
purified using amylose resin (NEB). The detection of SlMYBATVAC–SlAN1
733
interaction by in vitro pull-down was similar to those described above, except that
734
amylose resin (NEB) was used instead of GST-Bind Resin (Millipore).
735 736
For pull-down assays to confirm that SlMYBATVAC affects the SlAN2-likeInR–
737
SlAN1MIR interaction, 1 μg of purified GST-SlAN2-likeInR and TF-SlAN1MIR proteins
738
were added to each sample. Purified MBP-SlMYBATVAC and MBP protein was
739
added according to the concentration gradient. The GST-Bind Resin (Millipore) were
740
used to pull down proteins. Sequential procedures and buffers were the same as
741
described above. The effect of SlAN2-likeInR on SlMYBATVAC–SlAN1MIR
742
interaction was detected similarly.
743 744
Electrophoretic mobility shift assay
745
The full-length coding sequence of SlHY5 from InR was PCR amplified and cloned
746
into pMAL-c2X. The recombinant MBP-fusion proteins were expressed in E. coli
747
BL21 (DE3) cells and purified using amylase resin column (NEB). Oligonucleotide
748
probes were synthesized and labeled with biotin at the 5ʹ ends (Invitrogen).
749
Unlabelled probes with the same oligonucleotides were used as cold competitors.
750
EMSAs were performed as previously described (Du et al., 2017; Liu et al., 2019).
751
Briefly, biotin-labeled probes were incubated with MBP-fusion proteins at room
752
temperature for 20 min, and free and bound probes were separated via PAGE.
753
Mutated SlAN2-likeInR probes, in which the specific transcription factor-binding motif
754
5ʹ-ACGT-3ʹ was replaced by 5ʹ -TTTT-3ʹ was used as negative controls. Probes used
755
for EMSA are listed in Supplemental Table 1. 27
756 757
ChIP-qPCR Assay
758
The ChIP-qPCR assay was conducted as described previously (Du et al., 2017; Liu et
759
al., 2019). Briefly, 4 g peel of SlAN2-likeInR-OE, SlHY5-GFP mature green fruit (39
760
dpa) as well as the WT AC and InR were harvested and cross-lined in 1%
761
formaldehyde for 10 min, followed by neutralization with 0.125 M glycine. The
762
cross-linked samples were ground to powder in liquid nitrogen. Then the chromatin
763
complex was isolated, resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM
764
NaCl, 1 mM EDTA, 1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM
765
PMSF, and 1 × Roche protease inhibitor mixture), and sheared by sonication to
766
reduce the average DNA fragment size to around 500 bp. Then, 50 μL of sheared
767
chromatin was saved as input control. Anti-GFP antibody (Abcam) was incubated
768
with Dynabeads™ Protein G (Invitrogen) at 4°C for at least 6 h and added to the
769
remaining chromatin for overnight incubation at 4°C. The immunoprecipitated
770
chromatin–protein complex was then washed with low-salt washing buffer (20 mM
771
Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.2% SDS and 0.5% Triton X-100),
772
high salt washing buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA,
773
0.2% SDS and 0.5% Triton X-100), LiCl washing buffer (10mM Tris-HCl, pH 8.00,
774
25 mM LiCl, 1mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate), TE
775
buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA) sequentially, and then eluted with
776
elution buffer (1% SDS, 100mM NaHCO3) after washing. Reverse of protein-DNA
777
cross-linking was performed by incubating the immunoprecipitated complex at 65°C
778
overnight. DNA was recovered using the QIAquick PCR purification kit (Qiagen).
779
The ChIP signal was quantified as the percentage of total input DNA by qPCR. The
780
primers used in qPCR were designed at the promoters of the SlDFR and SlAN2-like
781
genes with or without MBS/ACE-box in the product fragments and listed in the
782
Supplemental Table 1. Each ChIP value was normalized to its respective input DNA
783
value and enrichment of DNA is shown as the percentage of input. 28
784 785
Data availability
786
The RNA-seq data have been deposited in the Genome Sequence Archive (GSA) at
787
the Beijing Institute of Genomics (BIG) Data Center, Chinese Academy of Sciences,
788
under the accession number CRA001936, and the bulk sequencing data have been
789
deposited in the GSA under the accession number CRA001937.
790 791
Accession Numbers
792
Sequence for genes described in this study can be found in the SOL Genomics
793
Network (http://solgenomics.net/) under the following accession numbers: SlANT1,
794
Solyc10g086250;
795
Solyc10g086270;
796
SlMYBATV, Solyc07g052490; SlDFR, Solyc02g085020; SlHY5, Solyc08g061130,
797
SlACTIN2, Solyc11g005330; SlE8, Solyc09g089580.
SlAN2 SlAN2-like,
(SlMYB75),
Solyc10g086060;
Solyc10g086290;
SlAN1,
SlANT1-like, Solyc09g065100;
798 799
Author contributions
800
C.L. and C-B.L. designed the project. C.S., L.D., M.D. and T.H. performed the
801
experiments, Q.C. and J.Z. performed the bioinformatics analysis. H.J. conducted
802
plant transformation. C.L., C.S., L.D. and M.D. wrote the manuscript. All authors
803
read and approved the final manuscript.
804 805
Acknowledgements
806
This work was supported by the Strategic Priority Research Program of the Chinese
807
Academy of Sciences (Precision Seed Design and Breeding), the National Key
808
Research and Development Program of China (2016YFD0100500), the Ministry of
809
Agriculture of China (2016ZX08009-003-001), and the Tai-Shan Scholar Program
810
from Shandong Province (No. tsxk20150901). No conflict of interest declared.
811 29
812
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979 33
980
Figure Legends
981
Figure 1. A bulk population sequencing approach to map genes controlling
982
anthocyanin accumulation in InR fruit.
983
(A) Photograph of Indigo Rose (InR, AftAft atvatv), Indigo Rose/AC (InR/AC, AftAft
984
atvatv) and AC (aftaft AtvAtv) fruits at the mature green stage (39 dpa). As shown on
985
the right, InR fruit does not accumulate anthocyanins in the flesh.
986
(B) Anthocyanin contents of light-exposing and shading fruit peel of InR, InR/AC and
987
AC. FW, fresh weight. Error bars represent the SD of four biological replicates.
988
(C) ƸSNP-index plot of chromosome 10 generated between DNA pools of the
989
Aft-type and the AC-type. ƸSNP-index between the two DNA pools is presented by
990
red lines, grey lines indicate 95% cutoff. (See also Figure S1.)
991
(D) ƸSNP-index plot of chromosome 7 generated between DNA pools of the
992
InR-type and Aft-type. ƸSNP-index between the two pools is presented by red lines,
993
grey lines indicate 95% cutoff. (See also Figure S1.)
994
(E) Candidate genes in Aft mapping region. The 4 R2R3-MYB genes are presented by
995
red arrows. Gene direction is indicated by the arrow.
996
(F) Candidate genes in atv mapping region. SlMYBATV is presented by blue arrow.
997
(G and I) Schematic diagram of the SlAN2-like gene (G) and the SlMYBATV gene (I)
998
in InR and AC. Black box and lines represent exons and introns, respectively. Dash
999
line indicates the corresponding position of AC splice site in the 2nd intron of
1000
SlAN2-like to InR. Red triangle represents the 4-bp ATAG insertion in the InR allele
1001
of SlMYBATV.
1002
(H and J) Schematic diagram of the SlAN2-like protein (H) and the SlMYBATV
1003
protein (J) in InR and AC. Orange box and green box represent the R2 domain and
1004
the R3 domain, respectively. Grey box represents sequences that are not identical to
1005
its functional counterpart.
1006 1007
Figure 2. SlAN2-like encodes a positive regulator of anthocyanin biosynthesis. 34
1008
(A) RT-qPCR results showing the expression levels of SlAN2, SlANT1, SlANT1-like
1009
and SlAN2-like, in light-exposing and shading fruit peel of InR and AC.
1010
(B) Photograph of InR, slan2-c, slant1-c, slant1-like-c and slan2-like-c fruits at the
1011
mature green stage (39 dpa). (See also Figure S2.)
1012
(C) Anthocyanin contents of light-exposing peel of InR, slan2-c, slant1-c,
1013
slant1-like-c and slan2-like-c fruits. FW, fresh weight.
1014
(D) Photograph of AC and SlAN2-like-OE fruits at the mature green stage (39 dpa).
1015
(See also Figure S6.)
1016
(E) Anthocyanin contents of light-exposing peel of AC, SlAN2-likeAC-OE and
1017
SlAN2-likeInR-OE fruits. FW, fresh weight.
1018
(F) RT-qPCR results showing the expression levels of SlAN2-like in light-exposing
1019
peel of AC, SlAN2-likeAC-OE and SlAN2-likeInR-OE fruits.
1020
(G) RT-qPCR results showing the expression levels of SlAN2-like in seedling, leaf,
1021
stem and light-exposing peel of AC and InR.
1022
(H) RT-qPCR results showing transcript-specific expression of SlAN2-like in InR, AC,
1023
SlAN2-likeAC-OE and SlAN2-likeInR-OE plants. (See also Figure S7.)
1024
(I and J) RT-qPCR results showing the expression levels of SlDFR in light-exposing
1025
peel of the indicated genotypes. AC was used as the WT for SlAN2-likeInR-OE plants
1026
(I), InR was used as the WT for slan2-like-c (J) plants.
1027
(K) Schematic diagram of the SlDFR promoter and the PCR amplicons (indicated as
1028
letters A and B) used for ChIP-qPCR. Red line indicates the Myb-binding site (MBS).
1029
The translational start site (ATG) is shown at position +1.
1030
(L) ChIP-qPCR showing the enrichment of SlAN2-likeInR on the chromatin of SlDFR.
1031
Chromatin of the WT and SlAN2-likeInR-OE plants was immunoprecipitated with an
1032
anti-GFP antibody. The immunoprecipitated DNA was used as a template for qPCR
1033
analysis, with primers targeting different regions of the SlDFR as shown in (K).
1034
SlACTIN2 was used as a non-specific target. ChIP signal was displayed as the
1035
percentage of total input DNA. 35
1036
For (C) and (E), error bars represent the SD of four biological replicates. For (A), (F),
1037
(G), (H), (I), (J) and (L), error bars represent the SD of three biological replicates.
1038
Asterisks indicate significant differences from the WT according to Student’s t-test at
1039
** P < 0.01. ns, not significant.
1040 1041
Figure 3. SlMYBATV encodes a negative regulator of anthocyanin biosynthesis.
1042
(A) Photograph of LA1996 (Aft), InR and proSlMYBATV:SlMYBATVAC fruits at the
1043
mature green stage (39 dpa). (See also Figure S8D.)
1044
(B)
1045
proSlMYBATV:SlMYBATVAC fruits. FW, fresh weight.
1046
(C) Photograph of LA1996 (Aft) and slmybatv-c fruits at the mature green stage (39
1047
dpa). (See also Figure S8A. and S8B.)
1048
(D) Anthocyanin contents of light-exposing peel of LA1996 and slmybatv-c fruits.
1049
FW, fresh weight.
1050
(E) RT-qPCR results showing the expression levels of SlDFR in light-exposing peel
1051
of LA1996 and slmybatv-c fruits.
1052
(F) Photograph of InR and SlMYBATVAC-OE fruits at the mature green stage (39 dpa).
1053
(See also Figure S8C.)
1054
(G) Anthocyanin contents of light-exposing peel of InR and SlMYBATVAC-OE fruits.
1055
FW, fresh weight.
1056
(H) RT-qPCR results showing the expression levels of SlDFR in light-exposing peel
1057
of InR and SlMYBATVAC-OE fruits.
1058
For (B), (D) and (G), error bars represent the SD of four biological replicates. For (E)
1059
and (H), error bars represent the SD of three biological replicates. Asterisks indicate
1060
significant differences from the WT according to Student’s t-test at ** P < 0.01.
Anthocyanin
contents
of
light-exposing
peel
of
LA1996,
1061 1062
Figure 4. SlAN2-like activates SlAN1 and SlMYBATV expression.
36
InR
and
1063
(A) Photograph of InR and slan1-c fruits at the mature green stage (39 dpa). (See also
1064
Figure S10.)
1065
(B) Anthocyanin contents of light-exposing peel of InR and slan1-c fruits. FW, fresh
1066
weight. Error bars represent the SD of four biological replicates.
1067
(C) RT-qPCR results showing the expression levels of SlDFR in light-exposing peel
1068
of InR and slan1-c fruits.
1069
(D and E) RT-qPCR results showing the expression levels of SlAN1 (left) and
1070
SlMYBATV (right) in the light-exposing fruit peel of the indicated genotypes. InR was
1071
used as the WT for SlAN2-like-c plants (D), and AC was used as the WT for
1072
SlAN2-likeInR-OE plants (E).
1073
(F and H) RT-qPCR results showing the expression levels of SlAN2-like in the
1074
light-exposing fruit peel of the indicated genotypes. InR was used as the WT for
1075
slan1-c (F) and SlMYBATVAC-OE plants (H), LA1996 was used as the WT for
1076
slmybatv-c plants (G).
1077
For (C-H), error bars represent the SD of three biological replicates. Asterisks
1078
indicate significant differences from the WT according to Student’s t-test at **P <
1079
0.01.
1080 1081
Figure 5. SlMYBATVAC and SlAN2-likeInR compete for binding SlAN1.
1082
(A) Yeast two-hybrid (Y2H) assay showing that both SlAN2-likeInR and
1083
SlMYBATVAC interact with SlAN1. The R3 domain of SlAN2-likeInR (SlAN2-likeR3)
1084
or full-length SlMYBATVAC were fused with the DNA binding domain (BD) in
1085
pGBKT7 and full length SlAN1 was fused with the activation domain (AD) in
1086
pGADT7, respectively. Transformed yeast was grown on selective SD/-2 media or
1087
SD/-4 media to test protein interaction. The empty pGADT7 vector was
1088
co-transformed with SlAN2-likeR3 or SlMYBATVAC in parallel as negative controls.
37
1089
(B) In vitro pull-down assay showing that SlAN2-likeInR interacts with SlAN1MIR.
1090
TF-SlAN1MIR was pulled down by GST-SlAN2-likeInR immobilized on GST-Bind
1091
resin. Protein bound to GST-Bind resin was eluted and analyzed by anti-TF antibody.
1092
(C) In vitro pull-down assay showing that SlMYBATVAC interacts with SlAN1MIR.
1093
TF-SlAN1MIR was pulled down by MBP-SlMYBATVAC immobilized on amylose
1094
resin. Protein bound to amylose resin was eluted and analyzed by anti-TF antibody.
1095
(D) Domains of SlAN1 involved in the SlAN2-likeInR–SlAN1 interaction and
1096
SlMYBATVAC–SlAN1 interaction were mapped using Y2H assays. Yeast cells
1097
co-transformed
1098
pGBKT7-SlAN2-likeR3 (bait), were dropped onto SD/-2 and SD/-4 media to assess
1099
interactions with SlAN2-likeInR (Left 2 panels). Yeast cells co-transformed with
1100
pGADT7-SlAN1 or its derivatives (preys) and pGBKT7-SlMYBATVAC (bait), were
1101
dropped onto SD/-2 and SD/-4 media to assess interactions with SlAN2-likeAC (Right
1102
2 panels).
1103
(E) Yeast three-hybrid (Y3H) assay showing that SlMYBATVAC and SlAN2-likeR3
1104
compete with each other to interact with SlAN1. (Top two panels) Yeast cells
1105
co-transformed with pGADT7-SlAN1 and pBridge-SlAN2-likeR3-SlMYBATVAC
1106
were dropped onto SD/-2 and SD/-4 media to assess the SlAN2-likeInR–SlAN1
1107
interaction. The co-transformed yeast cells were dropped onto SD/-5 medium to
1108
induce SlMYBATVAC. (Bottom two panels) Yeast cells co-transformed with
1109
pGADT7-SlAN1 and pBridge-SlMYBATVAC-SlAN2-likeR3 were dropped onto SD/-2
1110
and SD/-4 media to assess the SlMYBATVAC–SlAN1 interaction. The co-transformed
1111
yeast cells were dropped onto SD/-5 medium to induce SlAN2-likeR3.
1112
(F) Pull-down assay showing that SlMYBATVAC represses SlAN2-likeInR–SlAN1MIR
1113
interaction. For each sample, the amounts of TF-SlAN1MIR and GST-SlAN2-likeInR
1114
were equal, MBP-SlMYBATVAC and MBP were added according to the indicated
1115
gradient. TF-SlAN1MIR was pulled down by GST-SlAN2-likeInR immobilized on a
1116
GST-Bind resin. Proteins were eluted and analyzed using an anti-TF antibody.
with
pGADT7-SlAN1
38
or
its
derivatives
(preys)
and
1117
(G) Pull-down assay showing that SlAN2-likeInR represses SlMYBATVAC–SlAN1MIR
1118
interaction. For each sample, the amounts of TF-SlAN1MIR and MBP-SlMYBATVAC
1119
were equal, GST-SlAN2-likeInR and GST were added according to the indicated
1120
gradient. TF-SlAN1MIR was pulled down by MBP-SlMYBATVAC immobilized on an
1121
amylose resin. Proteins were eluted and analyzed using an anti-TF antibody.
1122 1123
Figure 6. SlHY5 activates SlAN2-likeInR expression in response to light.
1124
(A) Photograph of InR and slhy5-c fruits at the mature green stage (39 dpa). (See also
1125
Figure S11A. and S11B.)
1126
(B) Anthocyanin contents of light-exposing peel of InR and slhy5-c fruits. FW, fresh
1127
weight.
1128
(C and D) RT-qPCR results showing the expression levels of SlDFR (C) and
1129
SlAN2-likeInR (D) in light-exposing peel of InR and slhy5-c fruits.
1130
(E) Schematic diagram of the SlAN2-likeInR promoter. Green line represents the
1131
ACE-box motif. Short orange line indicates DNA probes used for EMSA. PCR
1132
amplicons used for ChIP-qPCR are indicated as A and B. The translational start site
1133
(ATG) is shown at position +1.
1134
(F) ChIP-qPCR showing the enrichment of SlHY5 on the chromatin of SlAN2-likeInR.
1135
Chromatin of the WT and SlHY5-GFP plants was immunoprecipitated with an
1136
anti-GFP antibody. The immunoprecipitated DNA was used as a template for qPCR
1137
analysis, with primers targeting different regions of the SlAN2-likeInR as shown in (E).
1138
SlACTIN2 was used as a non-specific target. ChIP signal was displayed as the
1139
percentage of total input DNA. Error bars represent the SD of three biological
1140
replicates. Asterisks indicate significant difference from the WT according to
1141
Student’s t-test at **P < 0.01.
1142
(G) EMSA showing that MBP-SlHY5 fusion protein directly binds to SlAN2-likeInR
1143
promoter in vitro. Biotin-labeled probes were incubated with MBP-SlHY5 protein,
1144
and free and bound DNA (arrows) was separated in an acrylamide gel. The MBP 39
1145
protein was incubated with the labeled probe in the first lane to serve as a negative
1146
control. Unlabeled probes were used as
1147
(Mu-competitor), mutated probe in which the ACE-box motif 5ʹ-ACGT-3ʹ was
1148
replaced with 5ʹ-TTTT-3ʹ.
1149
(H and I) Proposed working model for the light-induced anthocyanin biosynthesis in
1150
Aft single mutant (H) and InR (I). In the dominant Aft single mutant (i.e., LA1996),
1151
both SlAN2-likeInR and SlMYBATVAC are functional. In light-exposing fruit peel of
1152
this genotype, SlHY5 promotes the expression of SlAN2-likeInR, whose protein
1153
product in turn activates both the positive regulator SlAN1 and the negative regulator
1154
SlMYBATVAC. The functional negative regulator SlMYBATVAC competes the
1155
binding of SlAN2-likeInR to SlAN1 and thereby reducing the formation of a functional
1156
SlAN2-likeInR–SlAN1 complex, which determines the transcriptional output for
1157
anthocyanin biosynthesis (H). In InR which simultaneously harbors the dominant Aft
1158
locus and the recessive atv locus, the positive regulator SlAN2-likeInR is functional
1159
but the negative regulator SlMYBATVInR is not-functional; SlMYBATVInR loses the
1160
ability to reduce the transcriptional output for anthocyanin biosynthesis. Therefore,
1161
light-induced anthocyanin accumulation in the fruit peel of InR is much higher than
1162
that of the Aft single mutant (I). (See also Figure S13.)
competitors, as
indicated. Mu
1163 1164
Figure 7. Fruit-specific over-expression of SlAN2-likeInR leads to anthocyanin
1165
accumulation in fruit flesh.
1166
(A) Photograph of AC and proSlE8:SlAN2-likeInR fruits at the indicated stages. IMG,
1167
immature green; MG, mature green; B+n, n days after breaker.
1168
(B) Anthocyanin contents of AC and proSlE8:SlAN2-likeInR fruits harvested at the
1169
B+14 stage (56 dpa). FW, fresh weight. Error bars represent the SD of six biological
1170
replicates. Asterisks indicate significant differences from the WT (AC) according to
1171
Student’s t-test at ** P < 0.01.
40
1172
(C) KEGG pathway enrichment analysis of genes up-regulated (upper panel) or
1173
down-regulated (lower panel) by SlAN2-likeInR. The 1,552 genes up-regulated in
1174
proSlE8:SlAN2-likeInR (upper panel), and the 522 genes down-regulated in
1175
proSlE8:SlAN2-likeInR (lower panel) were used for analysis. The x axis shows the fold
1176
enrichment (ratio of genes in queried list/ratio of genes in the genome). The y axis
1177
shows the terms of metabolic pathway. The size of the plotted circle indicates the
1178
number of queried genes. The filled color is scaled to q value.
1179
(D and E) Expression of representative anthocyanin structural genes (D) and
1180
regulatory genes (E) in the RNA-seq experiments. log2(FPKM+1) of each gene from
1181
three biological replicates (1, 2 and 3) is shown. FPKM, fragments per kilobase of
1182
exon per million fragments mapped.
1183 1184
Supplemental Figure and Table Legends
1185
Figure S1. Genetic segregation and mapping of Aft and atv. Related to Figure 1.
1186
An F2 segregation population was generated by crossing InR (AftAft atvatv) with AC
1187
(aftaft AtvAtv). In the F2 population, fruit peel anthocyanin pigmentation pattern
1188
varied into 3 types: InR-type, Aft-type and AC-type, the predicted genotype was
1189
presented below the phenotype. The InR-type and Aft-type individuals were used to
1190
mapping atv locus. Aft-type and AC-type individuals were used to mapping Aft locus.
1191 1192
Figure S2. Generation of single mutants of the 4 R2R3-MYB genes. Related to Figure
1193
2.
1194
(A-D) CRISPR/Cas9-mediated editing of SlAN2 (A), SlANT1 (B), SlANT1-like (C)
1195
and SlAN2-like (D) in the InR background. Exons of the target genes are targeted by
1196
CRISPR/Cas9 using two single-guide RNAs (sgRNA; Target 1 and Target 2 (red
1197
arrows)). sgRNA targets and protospacer-adjacent motif (PAM) are indicated in red
1198
and bold font, respectively. Deletions and insertions are indicated by blue dashes and
1199
letters, respectively, and the numbers in parentheses indicate how many base pairs are 41
1200
involved (+, insertion; -, deletion).
1201
(E) RT-qPCR results showing the expression levels of SlAN2-like in light-exposing
1202
peel of InR and slan2-like-c fruits.
1203
(F) RT-qPCR results showing the expression levels of SlAN2 in light-exposing peel of
1204
InR and slan2-c fruits. It should be noted that the expression of slant1 and slant1-like
1205
in the fruit peel of InR and corresponding knock-out plants were below the detection
1206
limit.
1207
For (E) and (F), error bars represent the SD of three biological replicates. Asterisks
1208
indicate significant differences from the WT according to Student’s t-test at ** P <
1209
0.01.
1210 1211
Figure S3. Phylogenetic analysis of R2R3-MYB genes. Related to Figure 2.
1212
The evolutionary history was inferred using the Neighbor-Joining method in MEGA6.
1213
The tree is drawn to scale, with branch lengths in the same units as those of the
1214
evolutionary distances used to infer the phylogenetic tree. The percentage of replicate
1215
trees in which the groups clustered together in the bootstrap test (1000 replicates) are
1216
shown next to the branches. Solanum lycopersicum ANT1, SlANT1 (ABO26065.1); S.
1217
lycopersicum AN2, SlAN2 (AUG72357.1); S. lycopersicum ANT1-like, SlANT1-like
1218
(XP_004249668.1);
1219
Arabidopsis thaliana PAP1, AtPAP1 (AAG42001.1); A. thaliana MYB90, AtMYB90
1220
(NP_176813.1); Nicotiana tabacum AN2, NtAN2 (ACO52470.1); Antirrhinum majus
1221
ROSEA1, AmROS1 (ABB83826.1); A. majus ROSEA2, AmROS2 (ABB83827.1);
1222
Zea mays C1, ZmC1 (AAA33482.1); Z. mays Pl, ZmPl (AAA19819.1); Petunia
1223
hybrida AN2, PhAN2 (ABO21074.1); P. hybrida DPL, PhDPL (ADW94950.1); P.
1224
hybrida
1225
(AAX53089.1); Malus domestica MYB10, MdMYB10 (ABB84753); Citrus sinensis
1226
Ruby,
1227
(BAG68211.1).
PHZ,
CsRuby
S.
PhPHZ
lycopersicum
AN2-like,
(ADW94951.1);
(NP_001275818.1);
Solanum
Ipomoea
42
SlAN2-like
tuberosum
batatas
(AUG72358.1);
AN1,
MYB1,
StAN1
IbMYB1
1228 1229
Figure S4. Sequence alignment of SlAN2-like in InR and AC. Related to Figure 2.
1230
(A) Sequence alignment of SlAN2-like cDNA sequence. Sequence alignment was
1231
performed with Clustal X program. The stop codon is highlighted by blue square and
1232
AC harbors a premature stop codon.
1233
(B) Sequence alignment of SlAN2-like protein sequence. Sequence alignment was
1234
performed with Clustal X program. The R2 and R3 domain are represented in orange
1235
box and green box, respectively.
1236
(C) Sequence alignment of SlAN2-like genomic sequence. Sequence alignment was
1237
performed with Clustal X program. Exons in AC and InR were represented in red
1238
letters. The stop codon is highlighted by blue squares and AC harbors a premature
1239
stop codon. The causal SNP in the 5' splice site of the 2nd intron is indicated by arrow.
1240 1241
Figure S5. Sequence alignment of SlAN2-like genomic sequence in tomato cultivars
1242
and wild species. Related to Figure 2.
1243
Exons were represented in red letters. The causal SNP in the 5' splice site of the 2nd
1244
intron is indicated by arrow and performed with different colors, and the stop codon is
1245
highlighted by blue squares. Solanum lycopersicum (S. lycopersicum) represents
1246
cultivated tomatoes, Solanum chilense (S. chilense), Solanum peruvianum (S.
1247
peruvianum) and Solanum pimpinellifolium (S. pimpinellifolium) represent wild
1248
tomatoes. Sequence alignment was performed with Clustal X program.
1249 1250
Figure S6. Phenotype of 35S:SlAN2-likeInR plants. Related to Figure 2.
1251
Photograph of different plants of 35S:SlAN2-likeInR showing variation in anthocyanin
1252
accumulation in mature plants (A), seedlings (B), roots (C), leaves (D), stems (E) and
1253
flowers (F).
1254 1255
Figure S7. Alternative splicing of SlAN2-like. Related to Figure 2. 43
1256
(A) Schematic diagram of the SlAN2-like gene with different mutagenesis. InR
1257
features (exon, intron and SNP) in SlAN2-like were represented in orange and AC
1258
features (exon, intron and SNP) were represented in blue. SlAN2-likeInR-G653A
1259
represents a mutated SlAN2-likeInR with the “GCAAGT” mutated to “GCAAAT” in
1260
the 5' splice site of the 2nd intron. SlAN2-likeAC-A657G represents a mutated
1261
SlAN2-likeAC with the “GCAAAT” mutated to “GCAAGT” in the 5' splice site of the
1262
2nd intron.
1263
(B) RT-qPCR results showing transcript-specific expression of different forms of
1264
SlAN2-like indicated in (A) in the transient expression assays of (D).
1265
(C) A schematic diagram showing the constructs used in the transient expression
1266
assays of (D). Arrows, promoter regions; shaded boxes, coding sequence.
1267
(D) Transient expression assays in Nicotiana benthamiana showing the activation of
1268
the SlDFR promoter by different forms of SlAN2-like. The LUC/REN ratio represents
1269
the proSlDFR:LUC activity relative to the internal control (REN driven by the 35S
1270
promoter). EV, empty vector. Error bars represent the SD of four biological replicates.
1271
Asterisks indicate significant differences from the control according to Student’s t-test
1272
at ** P < 0.01. ns, not significant.
1273 SlMYBATVAC-OE
1274
Figure
1275
proSlMYBATV:SlMYBATVAC plants. Related to Figure 3.
1276
(A) CRISPR/Cas9-mediated editing of SlMYBATV. Exons of SlMYBATV was targeted
1277
by CRISPR/Cas9 using two single-guide RNAs (sgRNA; Target 1 and Target 2 (red
1278
arrows)). sgRNA targets and PAM are indicated in red and bold font, respectively.
1279
Deletions are indicated by blue dashes, and the numbers in parentheses indicate how
1280
many base pairs are involved.
1281
(B-D) RT-qPCR results showing the expression levels of SlMYBATV in
1282
light-exposing fruit peel of the indicated genotypes. LA1996 was used as the WT for
1283
slmybatv-c plants (B), and InR was used as the WT for SlMYBATVAC-OE and
S8.
Generation
of
slmybatv-c,
44
and
1284
proSlMYBATV:SlMYBATVAC plants (C and D).
1285
For (B-D), error bars represent the SD of three biological replicates. Asterisks indicate
1286
significant differences from the WT according to Student’s t-test at ** P < 0.01.
1287 1288
Figure S9. Sequence alignment of SlMYBATV genomic sequence in tomato cultivars
1289
and wild species. Related to Figure 3.
1290
Exons were represented in red letters. The “ATAG” insertion is indicated by arrow,
1291
and the stop codon is highlighted by blue squares. Solanum lycopersicum (S.
1292
lycopersicum) represents cultivated tomatoes, Solanum cheesmaniae (S. cheesmaniae),
1293
Solanum chilense (S. chilense), Solanum peruvianum (S. peruvianum) and Solanum
1294
pimpinellifolium (S. pimpinellifolium) represent wild tomatoes. Sequence alignment
1295
was performed with Clustal X program.
1296 1297
Figure S10. Generation of slan1-c knock-out mutants. Related to Figure 4.
1298
(A) CRISPR/Cas9-mediated editing of SlAN1. Exons of SlAN1 was targeted by
1299
CRISPR/Cas9 using two single-guide RNAs (sgRNA; Target 1 and Target 2 (red
1300
arrows)). sgRNA targets and PAM are indicated in red and bold font, respectively.
1301
Deletions and insertions are indicated by blue dashes and letters, respectively, and the
1302
numbers in parentheses indicate how many base pairs are involved (+, insertion; -,
1303
deletion).
1304
(B) RT-qPCR results showing the expression levels of SlAN1 in light-exposing peel of
1305
InR and slan1-c fruits. Error bars represent the SD of three biological replicates.
1306
Asterisks indicate significant differences from the WT according to Student’s t-test at
1307
** P < 0.01.
1308 1309
Figure S11. Generation of slhy5-c and SlHY5-GFP plants. Related to Figure 6.
1310
(A) CRISPR/Cas9-mediated editing of SlHY5. Exons of SlHY5 was targeted by
1311
CRISPR/Cas9 using two single-guide RNAs (sgRNA; Target 1 and Target 2 (red 45
1312
arrows)). sgRNA targets and PAM are indicated in red and bold font, respectively.
1313
Deletions are indicated by blue dashes and the numbers in parentheses indicate how
1314
many base pairs are involved.
1315
(B and C) RT-qPCR results showing the expression levels of SlHY5 in light-exposing
1316
fruit peel of the indicated genotypes.
1317
(D) RT-qPCR results showing the expression levels of SlAN2-like in light-exposing
1318
fruit peel of InR and SlHY5-GFP plants.
1319
(E) Yeast two-hybrid (Y2H) assay showing that SlHY5 lacks activation activity. The
1320
full-length coding sequence of SlHY5 was fused with the DNA binding domain (BD)
1321
in pGBKT7. Transformed yeast was grown on selective media lacking Ade, His, Leu,
1322
and Trp (SD/-4) to test activation activity. The SlAN2-likeInR-BD was co-transformed
1323
with pGADT7 as positive control and the empty pGBKT7 vector was co-transformed
1324
with pGADT7 as negative control.
1325
(F) A schematic diagram showing the constructs used in the transient expression
1326
assays of (G). Arrows, promoter regions; shaded boxes, coding sequence.
1327
(G) Transient expression assays in N. benthamiana showing the activation of the
1328
SlDFR
1329
proSlAN2-like:LUC activity relative to the internal control (REN driven by the 35S
1330
promoter).
1331
(H and I) RT-qPCR results showing the expression levels of SlHY5 and SlAN2-like in
1332
light-exposing fruit peel of AC and SlHY5-GFP/AC plants.
1333
For (B), (C), (D), (G), (H) and (I), error bars represent the SD of three biological
1334
replicates. Asterisks indicate significant differences from the WT (InR) according to
1335
Student’s t-test at ** P < 0.01.
promoter
by
SlHY5-VP16.
The
LUC/REN
ratio
represents
the
1336 1337
Figure S12. The ACE-box in SlAN2-like promoter plays an important role for SlHY5
1338
to activate SlAN2-likeInR expression. Related to Figure 6.
1339
(A) Sequence alignment of SlAN2-like promoter between InR and AC. The 46
1340
SlHY5-binding site (ACE-box) is highlighted in red. Sequence above the orange line
1341
represents probe used in the EMSA assay. Sequence alignment was performed with
1342
Clustal X program.
1343
(B) A Schematic diagram showing the constructs used in the transient expression
1344
assays of (C). Reporter 1 and 3 adopted wild type SlAN2-likeAC and SlAN2-likeInR
1345
promoter, respectively. The ACE-box was mutated to “TTTT” in Reptorter2 and 4.
1346
Arrows, promoter regions; shaded boxes, coding sequence.
1347
(C) Transient expression assays in N. benthamiana showing the activation of the
1348
SlAN2-like promoter by SlHY5-VP16. The LUC/REN ratio represents the
1349
proSlAN2-like:LUC activity relative to the internal control (REN driven by the 35S
1350
promoter). EV, empty vector.
1351 1352
Figure S13. Proposed working model for the light-induced anthocyanin biosynthesis
1353
in AC and atv single mutant. Related to Figure 6.
1354
In both AC (A) and the atv single mutant (B), the master regulator SlAN2-likeAC is
1355
not-functional and, as a consequence, light cannot activate the functional transcription
1356
complex for anthocyanin biosynthesis.
1357 1358
Figure S14. Summary of the RNA-seq analysis. Related to Figure 7.
1359
(A) Overview of RNA-seq data. Relative percentages of multiple mapped reads
1360
(multiple), unique mapped reads (uniq) and unmapped reads (unmapped) are shown.
1361
Three biological replicates were indicated by 1, 2 and 3.
1362
(B) Principal component analysis (PCA) showing the relatedness among the gene
1363
expression patterns of samples used for RNA-seq analysis. Red color represents AC
1364
samples and blue represents proSlE8:SlAN2-likeInR samples.
1365 1366
Table S1. Primers and probes used in this study.
47
B
120
Indigo Rose (InR)
Indigo Rose/AC (InR/AC)
AC
80 60 40 20
Indigo Rose transection
C
Light-exposing Shading
100
(A535-A650)g-1FW
A
0
InR
InR/AC
AC
D Aft
atv
1.0 △ SNP Index
△ SNP Index
1.0 0.5 0 -0.5
0.5 0 -0.5 -1.0
-1.0 10 Mb
20 Mb
30 Mb 40 Mb Chromosome 10
50 Mb
60 Mb
E
10 Mb
20 Mb
30 Mb 40 Mb Chromosome 7
50 Mb
60 Mb
F 60.98 Mbp Chr. 7
65.13 Mbp 65.14 Mbp 65.15 Mbp 65.16 Mbp 65.17 Mbp Chr. 10 SlAN2 SlANT1 SlANT1-like Heavy metal transport SlAN2-like
0 0 0 27 25 626 86 86 8 g0 g0 0g0 0 0 1 1 1 lyc lyc olyc So So S
60.99 Mbp
61.01 Mbp
SlMYBATV
0 0 28 29 86 86 g0 g0 0 0 1 1 lyc lyc So So
G
61.00 Mbp
0 0 49 50 52 52 g0 g0 7 7 0 0 lyc lyc So So
I SlMYBATV ATAG insertion
SlAN2-like InR ATG
InR
TAG
ATG TAA premature stop
AC ATG
AC
200 bp
H
200 bp ATG
TGA premature stop
TAA
J SlAN2-like
InR
R2
AC
R2
SlMYBATV R3
InR
AC
R3
61.02 Mbp
Relative expression levels
A
D
1.2 InR light-exposing InR shading AC light-exposing AC shading
1.0 0.8 0.6
1# 2# SlAN2-likeAC-OE
AC
0.4 0.2
E 12
0.0
SlANT1
**
SlANT1-like SlAN2-like (A535-A650)g FW
SlAN2
-1
B
10 8
**
6 4 2
ns InR
slan2-c 1#
slant1-c 2#
AC AC -OE 1# AC -OE 2# InR -OE 1# InR -OE 2# 2-like lAN2-like lAN2-like lAN2-like N S SlA S S
F 120 ns
100
ns
ns
80 60 40 **
20 0 InR
SlAN2-like
18000 16000 14000 12000 10000 8000 6000 4000 2000 0
slan2-c slant1-c slant1-like-c slan2-like-c 1# 2# 1# 1#
**
**
AC AC -OE 1# AC -OE 2# InR -OE 1# InR -OE 2# 2-like lAN2-like lAN2-like lAN2-like S S S
H 1.2
1000 Relative expression levels
AC InR
1.0 0.8 0.6 0.4 0.2 0.0
Seedling
Leaf
**
**
SlAN
G Relative expression levels
ns
0
slant1-like-c slan2-like-c 1# 1#
Relative expression levels
(A535-A650)g-1FW
C
1# 2# SlAN2-likeInR-OE
800
SlAN2-like AC version transcript InR version transcript
600 400 200 0 ng el edling peel dling ng eel eedli InR pe e s AC 2# seAe 2# p 2# seedli 2# peel InR AC s C -OE AC -OE InR -OE R -OE e In k li 2-like SlAN2- N2-like lAN2-like SlAN S SlA
Stem Light-exposing peel
MBS ATG
35
SlDFR
30
**
PCR amplicon
J **
25 20 15 10 5
L
1.0 0.8 0.6 0.4 0.2
**
AC
1#
2#
SlAN2-likeInR-OE
SlAN2-likeInR-OE AC
0.25 0.20 0.15 0.10 0.05
**
0.0
0
B
A
SlDFR
1.2 Relative expression levels
Relative expression levels
I
200 bp
+1
ChIP signal (%Input)
K
SlDFR
0.00 InR
1#
2#
slan2-like-c
A
B
SlACTIN2
A
C
InR
LA1996 (Aft)
1#
2#
B
D
4# slmybatv-c
E 80
SlDFR **
** (A535-A650)g-1FW
100 80 60 40
**
20
60
40
20
**
Relative expression levels
120
0
0 LA1 6 ( A t)
1#
InR p Sl
2# AT Sl
F
AT
AC
2#
SlMYBATVAC-OE
(A535-A650)g-1FW
3 2 1
2#
LA1 6 ( A t)
4#
sl
at -c
H 1.2
100
2#
4
slmybatv-c
120
1#
**
5
4#
G
InR
**
6
0 LA1 6 ( A t)
Relative expression levels
(A535-A650)g-1FW
2#
LA1996 (Aft)
proSlMYBATV:SlMYBATVAC
80 60 40 20
**
**
1#
2#
0
InR
Sl
AT
AC
-OE
SlDFR
1.0 0.8 0.6 0.4 0.2
**
0.0
**
1#
InR Sl
2# AT
AC
-OE
B
C
120
(A535-A650)g-1FW
100 80 60 40 20
1#
InR
sla
0.6
** ** ** **
0.0
ns
1.0 0.8 0.6 0.4 0.2 0.0
InR
1#
2# sla
2# -c
**
**
**
1#
InR
sla
A SlA SlA
-l -l
2# -c
R R
-OE -OE
**
30 20 10
**
**
SlA
G
-l ns
1.2
40
SlMYBATV
SlA
0.2
0
SlA
1.4
0.4
0.0
-c
SlA
1.2
ns
1.0
H
-l ns
Relative expression levels
0.2
0.6
50
0.8
0.4
0.8
E -c -c
1.0
Relative expression levels
Relative expression levels
sla
Relative expression levels
InR sla -l sla -l
1.0
**
1#
InR
1.2
Relative expression levels
0
-c
D
F
**
2#
SlDFR
1.2
Relative expression levels
A
0.8 0.6 0.4 0.2 0.0 LA1996 ( Aft)
2#
4#
slmybatv-c
SlMYBATV
1.2 1.0
SlA
-l
ns ns
0.8 0.6 0.4 0.2 0.0
1# 2# InR proSlMYBATV:SlMYBATVAC
A
B -2
re
ait
lA 1
lA 2-li e
A
lA 2-li eR3
lA 1
l
A
A
A
l
A
A
D
R3
-
- l
F- lA 1 IR A A
-
-
Anti- F
ll- o n
l
A
re
ait
IR
Anti- F
Inp t
- l
A
E
R3
A
ll- o n
- lA 2-li eInR
Inp t
R3
lA 2-li eR3
C F- lA 1 IR - lA 2-li eInR
-4
re
lA 2-li eR3 l
L
A
ait
-2
ri ge
lA 1
lA 2-li eR3
lA 1484-685
lA 1
lA 2-li eR3
lA 111-209
lA 1
l
A
A
-
lA 1210-483
lA 1
l
A
A
lA 2-li eR3
lA 1
-2
-4
F
-2
-4
-5
A
-
l
A
A
-4
G - l - lA 2-li eInR F- lA 1 - l
A
ll- o n
A IR
- lA 2-li eInR
IR A
A F- lA 1
-
-
1x
-
-
-
2x 4x -
-
-
-
-
1x
2x
4x
ll- o n Anti- F
-
-
1x
-
-
-
2x 5x -
-
-
-
-
1x
2x
5x Anti- F Anti-
AntiInp t Inp t
Anti-
Anti- F Anti-
Anti- F
A
A
E A SlA
-l
ox
A
R
1
R a pli on
A
500 p
InR
sl y -c
sl y -c
G F
o petitor
1.0
120 100 80 60 40 20
**
**
0 InR
4#
8#
I signal ( Inp t)
(A535-A650)g-1FW
B
ro e - l 5
Sl Y - F InR
**
0.8
-
- o petitor
- 10x 20x 50x - - - - - - - 10x 20x 50x -
o n pro e
0.6 0.4 0.2
sl y -c 0.0
Relative expression levels
C
A
lA
I 2
Free pro e
1.2 1.0 0.8
H
0.6 0.4 0.2
**
0.0 InR
4#
Lig t
Lig t
l
l
** 8#
5
5
sl y -c
Relative expression levels
D
R2R3 lA 2-li e
R2R3 lA 2-li e
1.2 1.0
lA 1
R3 l
IR
A
lA 1
IR
l
0.8
A
0.6 0.4
R2R3 lA 2-li e
0.2
**
0.0 InR
4#
** 8# sl y -c
lA 1
R2R3 lA 2-li e IR
IR
lA 1 SlDFR
SlDFR
A
B I
rea er
4
7
10
14
50
-1
(A535-A650)g FW
** 40
A
p SlE SlAN2-likeInR 2#
30
**
20 10 0
AC A
1#
2#
p SlE SlAN2-likeInR
D
Sl C
p-reg late genes (1552)
tatisti s o
l ol sis til enoi
l
at
a
nri
SlC S
oneogenesis
●
iar l eptanoi an gingerol ios nt esis
gene n 10 ● 20 ● 30
●
eta olis
●
en lalanine t rosine an tr ptop an ios nt esis en lalanine Flavonoi
ios nt esis
en lpropanoi
ios nt esis
ala tose
● ●
●
eta olis
SlF SlF SlDFR SlANS
val e 1.00 0.75 0.50 0.25 0.00
●
eta olis
SlC I
er
●
ios nt esis o a ino a i s ● l erolipi
ent
Sl T SlACC Sl ST Sl AT
●
1
2 Ri
SlF S
3 4 5 a tor
SlF SlRT
o n-reg late genes (522)
tatisti s o
Fatt a i
ios nt esis
at
a ●
nri ent gene n 10 ● 20 ● 30
E er
c g 2 c g c g c g 1 c1 g 1 c g 2 c g c g c g 1 1 c g c g 22 c g 1 2 c 2g c11g c 2g 2 c g c g 1 c12g 1 c 2g 1 c g 2 1 c g c 1g 1 c 1g1 c g 2 c g11 22 c11g 1 c11g 1
2 1
2
2 2 10
SlC
2#
1)
C
l l l l l l l l l l l l l l l l l l l l l l l l l l l
InR
Log2(F
Sl A
S S S S S S S S S S S S S S S S S S S S S S S S S S S
-l
2 1
0
AC proSlE :SlA 1 2 3 1 2 3
p SlE SlAN2-likeInR 2#
p SlE SlAN2-likeInR 2# A 1 2 3 1 2 3
iterpenoi
●
eta olis
●
ios nt esis
●
1
2 Ri
3 4 5 a tor
val e 1.00 0.75 0.50 0.25 0.00
SlAN2-like Sl Sl 2 Sl Sl AT Sl SlAN1 Sl AF1 Sl DR
S S S S S S S S S
l l l l l l l l l
c g 2 c g 2 c1 g 1 c 1g111 c g 2 c12g c g c g 11 c g
2 2 2
2 2 1
1)
Alanine aspartate an gl ta ate
●
Log2(F
one ios nt esis
0
a
10
●
erpenoi
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1
A Transcriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh
2 3
Chuanlong Sun1,2,6, Lei Deng1,6, Minmin Du3, Jiuhai Zhao4, Qian Chen4, Tingting
4
Huang5, Hongling Jiang1, Chang-Bao Li3,*, Chuanyou Li1,2,*
5
1
6
(Beijing), Institute of Genetics and Developmental Biology, Innovation Academy for
7
Seed Design, Chinese Academy of Sciences, Beijing 100101, China
8
2
University of Chinese Academy of Sciences, Beijing 100049, China
9
3
Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North
State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research
10
China), Ministry of Agriculture, Beijing Vegetable Research Center, Beijing Academy
11
of Agriculture and Forestry Sciences, Beijing 100097, China
12
4
13
University, Tai'an 271018, Shandong Province, China
14
5
15
Shandong Province, China
16
6
State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural
Institute of Vegetable, Qingdao Academy of Agricultural Sciences, Qingdao 266100,
These authors contributed equally to this article.
17 18
*
19
Chuanyou Li ([email protected])
20
Chang-Bao Li ([email protected])
Correspondence:
21 22
Running title: Anthocyanin Biosynthesis in Tomato Flesh
23
Short Summary: Through characterization of the purple tomato “Indigo Rose”,
24
which only shows anthocyanin pigmentation in the skin in a light-dependent manner,
25
we demonstrate that the R2R3-MYB protein SlAN2-like orchestrates a hierarchical
26
transcriptional network to promote anthocyanin accumulation. Expression of a
27
functional SlAN2-like could bypass light and activate anthocyanin biosynthesis in both
28
peel and flesh of tomato cultivars. 1
29
Abstract
30
Dietary anthocyanins are important health-promoting antioxidants that make a major
31
contribution to the quality of fruits. It is intriguing that most tomato cultivars do not
32
produce anthocyanins in fruit. However, the purple tomato variety Indigo Rose, which
33
combines the dominant Aft locus and the recessive atv locus from wild tomato
34
species, exhibits light-dependent anthocyanin accumulation in the skin. Here, we
35
report that whereas Aft encodes a functional allele of an anthocyanin activator named
36
SlAN2-like, atv encodes a non-functional allele of the anthocyanin repressor
37
SlMYBATV. The expression of SlAN2-like is responsive to light and a functional
38
SlAN2-like can activate both anthocyanin biosynthetic genes and their regulatory
39
genes, suggesting that SlAN2-like acts as a master regulator and plays a critical role
40
for the activation of anthocyanin biosynthesis. Our results reveal that cultivated
41
tomatoes contain a non-functional allele of this master regulator and therefore fail to
42
produce anthocyanins. Indeed, expression of a functional SlAN2-like in a tomato
43
cultivar led to the activation of the entire anthocyanin biosynthesis pathway and high
44
levels of anthocyanin accumulation in both peel and flesh. Our study exemplifies that
45
efficient engineering of complex metabolic pathways could be achieved through
46
tissue-specific expression of master transcriptional regulators.
47 48
Key words: anthocyanin biosynthesis, purple tomato, MBW complex, master
49
regulator, transcriptional regulation
50 51 52 53 54 55 56 2
57
Introduction
58
Anthocyanins are naturally occurring pigments responsible for red, purple, and blue
59
color of many plant organs, and play essential roles in multiple biological processes of
60
plants, such as attracting pollinators and dispersers, providing protection against biotic
61
and abiotic stresses (Gould, 2004; Zhang et al., 2013). Most importantly, growing
62
evidence reveals that anthocyanins are health-promoting compounds because of their
63
high antioxidant activity (Butelli et al., 2008). Dietary consumption of anthocyanins
64
reduces the risk of cardiovascular disease, age-related degenerative diseases and
65
certain cancers (He and Giusti, 2010; Li et al., 2017).
66 67
Anthocyanins are synthesized through the flavonoid branch of the general
68
phenylpropanoid pathway. It is well-recognized that anthocyanin biosynthesis is
69
tightly regulated at the transcriptional level by the evolutionarily conserved
70
MYB-bHLH-WD repeat (MBW) complex (Lloyd et al., 2017; Naing and Kim, 2018;
71
Xu et al., 2015). Many known high-anthocyanin accumulation traits in various crops,
72
such as purple-fleshed sweet potato (Mano et al., 2007), purple cauliflower (Chiu et
73
al., 2010), red-fleshed apple (Espley et al., 2009; Zhang et al., 2019), blood orange
74
(Butelli et al., 2012) and purple pummelo (Huang et al., 2018), are resulted from
75
elevated expression of MYB transcription factors, suggesting that MYB regulators
76
play an active and critical role in the activation of the MBW complex (Lloyd et al.,
77
2017; Xu et al., 2015).
78 79
Tomato (Solanum lycopersicum) is a favorite vegetable worldwide (Lin et al., 2014).
80
Whereas the most abundant antioxidant in tomato fruit is the lipophilic lycopene, the
81
levels of the water-soluble flavonoids (which include anthocyanins) are suboptimal
82
(Mes et al., 2008). Therefore, tomato is an ideal system to investigate the regulatory
83
mechanism of anthocyanin biosynthesis and provides an excellent candidate for
84
enhancement of anthocyanin content by genetic manipulations (Bovy et al., 2002; 3
85
Butelli et al., 2008; Jian et al., 2019; Mathews et al., 2003; Schreiber et al., 2012).
86 87
Although most cultivated tomatoes do not produce anthocyanins in fruit, some related
88
wild tomato species do produce little amount of anthocyanins in the fruit skin in a
89
light-dependent manner, and this trait has been transferred into tomato cultivars. For
90
example, introgression of the dominant Anthocyanin fruit (Aft) locus from Solanum
91
chilense or the Aubergine (Abg) locus from Solanum lycopersicoides into tomato
92
cultivars led to slight, light-dependent accumulation of anthocyanins in the fruit peel
93
(Mathews et al., 2003; Mes et al., 2008; Schreiber et al., 2012). Introgression of the
94
recessive atroviolacium (atv) gene from Solanum cheesmaniae into tomato cultivars
95
led to anthocyanin accumulation in vegetative organs but not in the fruit (Mes et al.,
96
2008). Notably, combining atv with Aft led to a dramatic increase of anthocyanin
97
pigmentation in the fruit peel (Mes et al., 2008), indicating that Aft genetically
98
interacts with atv in regulating anthocyanin biosynthesis. The popular purple tomato
99
variety Indigo Rose (InR) contains both Aft and atv and bears intense anthocyanin
100
pigmentation
in
the
fruit
peel
(Mes
et
al.,
2008)
101
(http://extension.oregonstate.edu/gardening/purple-tomato-debuts-indigo-rose).
102
should be noted that InR only produces anthocyanins in the peel that is exposed to
103
sunlight and no anthocyanin is accumulated in the shading peel and flesh (Qiu et al.,
104
2019), suggesting an essential role of light in regulating anthocyanin pigmentation.
It
105 106
Despite much efforts have been devoted to clone Aft and atv, the genes underlying
107
these loci have not been conclusively identified and/or verified (Cao et al., 2017;
108
Colanero et al., 2018; Kiferle et al., 2015; Schreiber et al., 2012). Thus, the molecular
109
mechanism by which InR only exhibits anthocyanin pigmentation in the fruit skin in a
110
light-dependent manner remains elusive.
111 112
Here, we employ a bulk population sequencing approach (Austin et al., 2014) to 4
113
identify the genes controlling anthocyanin accumulation in InR. We show that
114
whereas Aft encodes an R2R3-MYB protein SlAN2-like and positively regulates
115
anthocyanin biosynthesis, Atv encodes an R3-MYB protein SlMYBATV and
116
negatively regulates anthocyanin biosynthesis. In the light-exposing fruit peel of InR,
117
light activates SlAN2-like expression through SlHY5, a critical component of the light
118
signalling pathway (Lee et al., 2007). In turn, SlAN2-like triggers the expression of
119
both SlMYBATV and SlAN1 (Qiu et al., 2016), a bHLH transcription factor that
120
positively regulates anthocyanin biosynthesis. SlMYBATV competes SlAN2-like for
121
binding SlAN1 and thereby negatively regulating anthocyanin biosynthesis. Therefore,
122
SlAN2-like acts as a master regulator of anthocyanin biosynthesis and plays a critical
123
role in transmitting the light signal to the presumed MBW complex. These results led
124
us to a hypothesis that engineering the master regulator SlAN2-like could enhance
125
anthocyanin pigmentation in a light-independent manner. Indeed, expression of
126
SlAN2-like under the control of a fruit-specific promoter resulted in fruits with
127
enhanced anthocyanin accumulation in both peel and flesh. Our study uncovers the
128
transcriptional regulatory complex underlying anthocyanin biosynthesis in tomato
129
fruit and provides an excellent target for engineering high levels of anthocyanins and
130
other metabolites.
131 132
Results
133
A bulk population sequencing approach to map genes controlling anthocyanin
134
accumulation in InR
135
Since the original purple tomato InR was maintained in an undeclared background,
136
we introduced the anthocyanin accumulation trait into the background of the cultivar
137
Alisa Craig (AC) which does not show anthocyanin pigmentation in the fruit (Figure
138
1A and Supplemental Figure 1). Similar to InR, InR/AC accumulated large amounts
139
of anthocyanins in the light-exposing peel but not in the shading part of the peel
140
(Figure 1A and 1B). Notably, InR fruit does not accumulate anthocyanins in the flesh 5
141
(Figure 1A). These observations indicate that InR and InR/AC accumulate
142
anthocyanins in the fruit peel in a light-dependent manner.
143 144
To map the genes controlling anthocyanin pigmentation in InR, we crossed InR with
145
AC and generated an F2 population segregating the anthocyanin pigmentation trait.
146
Based on the anthocyanin pigmentation pattern of the fruit peel, F2 progeny can be
147
classified into InR-type (intense anthocyanin pigmentation), Aft-type (slight
148
anthocyanin
149
(Supplemental Figure 1). The segregation of InR-type:Aft-type:AC-type fits a ratio of
150
3:9:4 (108:302:151, P =0.48, chi-square test), confirming that the anthocyanin
151
pigmentation trait of InR is controlled by the dominant Aft locus and the recessive atv
152
locus (Mes et al., 2008). To map these loci using the same population, we performed
153
bulk population sequencing (Austin et al., 2014) on DNA pools of InR-type, Aft-type
154
and AC-type.
pigmentation)
and
AC-type
(no
anthocyanin
pigmentation)
155 156
SNP index comparison between DNA pools of the Aft-type and the AC-type revealed
157
that the Aft locus of InR was mapped to the bottom of chromosome 10 (Figure 1C),
158
which is consistent with previous mapping results using independent mapping
159
populations (De Jong et al., 2004). Similarly, SNP index comparison between DNA
160
pools of InR-type and Aft-type revealed that the atv locus of InR was mapped to
161
chromosome 7 (Figure 1D), which is also consistent with previous mapping results
162
(Cao et al., 2017; Colanero et al., 2018).
163 164
Aft encodes an R2R3-MYB and promotes anthocyanin accumulation
165
In the Aft mapping interval, we identified a gene cluster harbouring 4 R2R3-MYB
166
genes (Figure 1E), which have been designated as SlAN2 (De Jong et al., 2004; Jian et
167
al., 2019), SlANT1 (Mathews et al., 2003; Schreiber et al., 2012), SlANT1-like (Kiferle
168
et al., 2015) and SlAN2-like (Kiferle et al., 2015), respectively. Phylogenetic analysis 6
169
indicated that these four genes encode proteins that show high similarity to the known
170
R2R3-MYBs that play a regulatory role for anthocyanin biosynthesis (Supplemental
171
Figure 3). We then examined the expression of the 4 MYB genes in light-exposing and
172
shading peel of InR fruit and AC fruit. Reverse transcription-quantitative polymerase
173
chain reaction (RT-qPCR) analysis revealed that SlAN2-like is highly expressed in the
174
light-exposing peel (exhibits anthocyanin pigmentation) but not the shading peel (no
175
anthocyanin pigmentation) of InR fruit (Figure 2A). On the contrary, SlANT1, SlAN2
176
and SlANT1-like show negligible expression in both the light-exposing peel and the
177
shading peel of InR fruit (Figure 2A). Parallel experiments failed to detect significant
178
expression of the 4 MYB genes in the light-exposing and shading peel of AC fruit
179
(Figure 2A).
180 181
That the expression of SlAN2-like is co-related with the anthocyanin pigmentation
182
pattern of the InR fruit peel supports that SlAN2-like is a strong candidate for Aft.
183
Indeed, clustered regularly interspaced short palindromic repeats/CRISPR-associated
184
9 (CRISPR/Cas9)-mediated knock-out (Deng et al., 2017) mutations of SlAN2-like
185
(Supplemental Figure 2) largely abolished anthocyanin pigmentation of the InR fruit
186
peel (Figure 2B and 2C). On the contrary, CRISPR/Cas9-mediated editing of SlANT1,
187
SlAN2 and SlANT1-like (Supplemental Figure 2) showed negligible effects on
188
anthocyanin pigmentation of the InR fruit peel (Figure 2B and 2C). Together, these
189
results led us to the conclusion that Aft encodes the R2R3-MYB protein SlAN2-like.
190 191
Next, we examined the tissue-specific expression pattern of SlAN2-like in InR and
192
AC. Results showed that SlAN2-like was only highly expressed in the light-exposing
193
fruit peel of InR, but not in seedlings, leaves and stems of InR (Figure 2G). In
194
parallel, SlAN2-like expression was barely detectable in any tissue examined of AC
195
plants (Figure 2G).
196 7
197
To explore the function of SlAN2-like in regulating anthocyanin biosynthesis, the InR
198
allele of SlAN2-like (SlAN2-likeInR) was fused with green fluorescent protein (GFP)
199
and transferred to the AC background under the control of the 35S promoter to
200
generate SlAN2-likeInR-OE plants (Figure 2F). Similarly, the AC allele of SlAN2-like
201
(SlAN2-likeAC) was fused with GFP and transferred to the AC background under the
202
control of the 35S promoter to generate SlAN2-likeAC-OE plants. (Figure 2F). Notably,
203
SlAN2-likeInR-OE plants, but not SlAN2-likeAC-OE plants, showed anthocyanin
204
pigmentation in the fruit peel (Figure 2D and 2E) as well as in seedlings, roots, leaves
205
and flowers (Supplemental Figure 6), suggesting that SlAN2-likeInR is functional but
206
SlAN2-likeAC is not functional.
207 208
To address why SlAN2-like is non-functional in AC, we compared the genomic
209
sequence of SlAN2-like between InR and AC and found plenty variations
210
(Supplemental Figure 4C). For example, the nucleotide G653 of SlAN2-likeInR
211
genomic DNA, which locates in the conserved 5' splice site of the 2nd intron (Stamm
212
et al., 2005), changed to A657 in the genomic DNA of SlAN2-likeAC. Indeed, isolation
213
of the SlAN2-like cDNA from SlAN2-likeAC-OE plants revealed that SlAN2-likeAC
214
adopted an altered 5' splice site of the second intron, leading to a truncated protein
215
that lacks the R3 domain and the C-terminal (Figure1G, 1H and Supplemental Figure
216
4). RT-qPCR analysis using transcript-specific primers found that only the AC
217
version of the transcript, which encodes a truncated protein (Supplemental Figure 4B),
218
were detected in SlAN2-likeAC-OE plants, including in fruit peel and seedlings (Figure
219
2H). Additionally, we overexpressed the SlAN2-likeAC and SlAN2-likeInR genomic
220
sequence in Nicotiana benthamiana leaves under the 35S promoter. RT-qPCR
221
analysis with transcript-specific primers confirmed that SlAN2-likeAC only produced
222
the AC version of the transcript, while SlAN2-likeInR only produced the InR version of
223
the transcript (Supplemental Figure 7B).
224 8
225
Next, we made an A657G mutation in SlAN2-likeAC (SlAN2-likeAC-A657G)
226
(Supplemental Figure 7A) as well as a G653A mutation in SlAN2-likeInR
227
(SlAN2-likeInR-G653A) (Supplemental Figure 7A). These constructs were transformed
228
into N. benthamiana leaves to test their splicing pattern. As expected,
229
SlAN2-likeInR-G653A only produced the AC version of the transcript (Supplemental
230
Figure 7B). On the contrary, SlAN2-likeAC-A657G only produced the InR version of the
231
transcript (Supplemental Figure 7B). Taken together, these results demonstrate that
232
the G to A mutation at the splice site of the 2nd intron is the causal mutation that
233
determines the splicing pattern of SlAN2-like.
234 235
We then employed a dual-luciferase reporter (DLR) system (Hellens et al., 2005) to
236
examine whether genetic manipulation of SlAN2-like affects the expression of SlDFR,
237
which encodes a key enzyme of anthocyanin biosynthesis (Goldsbrough et al., 1994)
238
(Supplemental Figure 7C). Co-expression of a construct containing the SlAN2-likeInR
239
genomic sequence with the proSlDFR:LUC reporter led to high luciferase (LUC)
240
expression (Supplemental Figure 7D). In contrast, co-expression of a construct
241
containing the SlAN2-likeAC genomic sequence with the proSlDFR:LUC reporter
242
failed to activate LUC expression (Supplemental Figure 7D). However, co-expression
243
of SlAN2-likeAC-A657G with the reporter construct also led to high LUC expression
244
(Supplemental Figure 7D). Together, these results corroborate that SlAN2-likeInR, but
245
not SlAN2-likeAC, is functional to activate SlDFR expression. In addition, RT-qPCR
246
analysis showed that the expression of SlDFR was significantly increased in
247
SlAN2-likeInR-OE plants and decreased in SlAN2-likeInR-edited plants (slan2-like-c)
248
(Figure 2I and 2J), indicating that SlAN2-like positively regulates the expression of
249
SlDFR. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays using
250
the above-described SlAN2-likeInR-OE plants and anti-GFP antibody revealed an
251
enrichment of the SlAN2-likeInR-GFP recombinant protein on the SlDFR promoter
252
region containing a putative MYB binding site (MBS) (Zhu et al., 2015) (Figure 2K 9
253
and 2L). Collectively, these results support that SlAN2-likeInR promotes anthocyanin
254
accumulation through positively regulating anthocyanin biosynthetic genes.
255 256
Next, we sequenced SlAN2-like gene in some cultivars and wild tomatoes
257
(Supplemental Figure 5). Based on sequence alignment, most red-fruited tomatoes,
258
including cultivars and its ancestor Solanum pimpinellifolium (Lin et al., 2014),
259
contain the G to A mutation at the 5' splice site of the 2nd intron, indicating that
260
SlAN2-like are non-functional in these plants (Supplemental Figure 5). On the
261
contrary, several wild species showing anthocyanin accumulation including Solanum
262
chilense (Schreiber et al., 2012) and Solanum peruvianum (Kiferle et al., 2015)
263
contain the normal 5' splice site at the 2nd intron and encode a functional SlAN2-like
264
(Supplemental Figure 5). It has been reported that the cultivar LA1996 contains the
265
Aft locus introgressed from S. chilense (Jones et al., 2003), sequence analyses
266
indicated that this cultivar also contains a functional SlAN2-like (Supplemental Figure
267
5).
268 269
Together, our results reveal that, whereas most tomato cultivars contain a
270
loss-of-function allele of SlAN2-like (i.e., SlAN2-likeAC) (Supplemental Figure 5),
271
InR contains a functional allele of this anthocyanin activator (i.e., SlAN2-likeInR).
272 273
Atv encodes an R3-MYB and represses anthocyanin accumulation
274
In the atv mapping region of chromosome 7, an R3-MYB gene SlMYBATV was
275
proposed as a candidate gene underlying the atv mutation (Cao et al., 2017; Colanero
276
et al., 2018). In line with these observations, we found that, compare to its AC allele
277
counterpart (SlMYBATVAC), the InR allele of SlMYBATV (SlMYBATVInR) contains a
278
4-bp insertion in the second exon, which leads a truncated protein that lacks the R3
279
domain (Figure 1I and 1J).
280 10
281
To test whether this mutation is responsible for the effect of atv in the InR
282
background, we performed a genetic complementation assay. For this purpose,
283
SlMYBATVAC driven by its native promoter was transferred to the InR background
284
(proSlMYBATV:SlMYBATVAC). As expected, anthocyanin pigmentation of the
285
resulting transgenic plants was largely reduced compared to InR (Figure 3A and 3B),
286
supporting that the SlMYBATVInR mutation underlies the effect of the atv locus in InR.
287 288
Next, we generated CRISPR/Cas9-induced mutations of SlMYBATV in the cultivar
289
LA1996 (Jones et al., 2003), which contains the dominant Aft locus and exhibits a
290
typical “Aft-type” anthocyanin pigmentation pattern in the fruit peel (Supplemental
291
Figure 8A and 8B). The resulting SlMYBATV-edited plants (slmybatv-c) showed an
292
anthocyanin pigmentation pattern similar to that of InR (Figure 3C, 3D and 3E),
293
indicating that mutation of SlMYBATV in an Aft-containing background could
294
recapture the anthocyanin pigmentation phenotype of InR fruit.
295 296
The above results suggest that the cultivar allele of SlMYBATV (i.e., SlMYBATVAC)
297
negatively regulates anthocyanin accumulation and that SlMYBATVInR is a
298
loss-of-function allele. To prove this, we generated transgenic plants overexpressing
299
SlMYBATVAC in the background of InR (SlMYBATVAC-OE). Indeed, overexpression of
300
SlMYBATVAC abolished anthocyanin pigmentation as well as SlDFR expression of the
301
InR fruit peel (Figure 3F, 3G and 3H), suggesting a negative role of SlMYBATVAC in
302
anthocyanin biosynthesis.
303 304
It is believed that the recessive atv locus is derived from the wild tomato Solanum
305
cheesmaniae (Mes et al., 2008). Consistently, our sequence analyses indicated that
306
only InR and the S. cheesmaniae tomato LA0434 contain a loss-of-function allele of
307
SlMYBATV (Supplemental Figure 9), while most cultivars and wild species
11
308
(Solanum pimpinellifolium, Solanum chilense and Solanum peruvianum) are predicted
309
to encode a functional SlMYBATV (Supplemental Figure 9).
310 311
Taken together, our data support that Atv encodes the R3-MYB protein SlMYBATV
312
and that this protein acts as a repressor of anthocyanin biosynthesis. Whereas most
313
tomato cultivars like AC contain a functional allele of SlMYBATV (i.e.,
314
SlMYBATVAC) (Supplemental Figure 9), InR contains a non-functional allele of this
315
anthocyanin repressor (i.e., SlMYBATVInR).
316 317
SlAN1 promotes anthocyanin accumulation in the fruit peel
318
It has been shown that the bHLH transcription factor SlAN1 positively regulates
319
anthocyanin biosynthesis in tomato seedlings (Qiu et al., 2016). To test whether
320
SlAN1 contributes to the anthocyanin pigmentation of the InR fruit peel, we generated
321
SlAN1-edited plants (slan1-c) in the InR background. Sequence analyses indicated
322
that all SlAN1-edited plants carry mutations in the ORF, which lead to frame shift and
323
the generation of a premature stop codon (Supplemental Figure 10). As expected,
324
editing SlAN1 led to abolished anthocyanin pigmentation (Figure 4A and 4B) and
325
significantly reduced SlDFR expression in the InR fruit peel (Figure 4C), confirming
326
that SlAN1 plays a positive role for the anthocyanin pigmentation of InR fruit peel.
327 328
SlAN2-likeInR promotes the expression of SlAN1 and SlMYBATV
329
The above results demonstrate that both the positive effect of SlAN2-likeInR and SlAN1
330
and the negative effect of SlMYBATVAC contribute to the anthocyanin pigmentation
331
trait of the InR fruit. To test the possible inter-regulation of these genes, we examined
332
the expression of SlAN1 and SlMYBATV in the fruit peel of SlAN2-likeInR-edited plants
333
(slan2-like-c) and SlAN2-likeInR-OE plants. Results showed that whereas editing
334
SlAN2-likeInR led to reduced expression of SlAN1 and SlMYBATV (Figure 4D),
335
overexpression of SlAN2-likeInR led to increased expression of SlAN1 and SlMYBATV 12
336
(Figure 4E), indicating that SlAN2-likeInR positively regulates both the positive
337
regulator SlAN1 and the negative regulator SlMYBATV.
338 339
On the contrary, editing SlAN1 showed negligible effect on the expression of
340
SlAN2-likeInR (Figure 4F). Similarly, neither editing (Figure 4G) nor overexpression
341
of SlMYBATVAC (Figure 4H) significantly affected the expression of SlAN2-likeInR.
342
Together, these results support a scenario that SlAN2-likeInR acts upstream of SlAN1
343
and SlMYBATV and likely functions as a master regulator to orchestrate anthocyanin
344
biosynthesis.
345 346
SlMYBATVAC and SlAN2-likeInR compete for binding SlAN1
347
That the anthocyanin pigmentation phenotype of InR depends on the co-existence of
348
the dominant Aft locus and the recessive atv locus prompted us to explore the
349
biochemical basis of this genetic interaction. Yeast two-hybrid (Y2H) assays
350
indicated that the R3 domain of SlAN2-likeInR (SlAN2-likeR3) interacts with SlAN1
351
(Figure 5A). This interaction was verified by in vitro pull down assays showing that a
352
purified glutathione S-transferase (GST)-tagged SlAN2-likeInR (GST-SlAN2-likeInR)
353
could pull down the trigger factor (TF)-tagged SlAN1 fragment SlAN111-209
354
(TF-SlAN111-209) (Figure 5B). Domain mapping with Y2H assays revealed that
355
SlAN2-likeInR interacts the SlAN111-209 fragment of SlAN1 (Figure 5D), we therefore
356
designated SlAN111-209 as MYB-interacting region (MIR, SlAN1MIR).
357 358
In parallel experiments, we found that SlMYBATVAC also interacts with SlAN1 in
359
Y2H assays (Figure 5A), which was verified by in vitro pull down assays that
360
maltose-binding protein (MBP)-tagged SlMYBATVAC (MBP-SlMYBATVAC) could
361
pull down TF-SlAN1MIR (Figure 5C). Notably, domain mapping with Y2H assays
362
indicated that SlMYBATVAC also interacts the MIR of SlAN1 (Figure 5D).
363 13
364
SlAN2-likeInR and SlMYBATVAC interact with the same domain of SlAN1 raised the
365
possibility that the positive regulator SlAN2-likeInR and the negative regulator
366
SlMYBATVAC might compete with each other for binding SlAN1. Indeed, yeast
367
three-hybrid (Y3H) assays revealed SlAN2-likeInR–SlAN1 interaction on the synthetic
368
defined (SD) medium lacking Ade, His, Trp, and Leu (SD/-4); however, the induction
369
of SlMYBATVAC expression on SD medium lacking Ade, His, Trp, Leu, and Met
370
(SD/-5) led to a dramatic reduction in SlAN2-likeInR–SlAN1 interaction (Figure 5E),
371
suggesting that SlMYBATVAC interferes with SlAN2-likeInR–SlAN1 interaction. On
372
the other hand, SlMYBATVAC–SlAN1 interaction was detected on SD/-4 medium,
373
and this interaction was obviously reduced by inducing the expression of
374
SlAN2-likeInR on SD/-5 medium (Figure 5E), suggesting that SlAN2-likeInR also
375
impairs SlMYBATVAC–SlAN1 interaction.
376 377
To substantiate the above observations, we performed in vitro pull-down experiments
378
using a constant protein concentration of TF-SlAN1MIR and increasing protein
379
concentration of GST-SlAN2-likeInR or MBP-SlMYBATVAC. Results showed that the
380
ability of GST-SlAN2-likeInR to pull down TF-SlAN1MIR decreased as the amount of
381
MBP-SlMYBATVAC increased (Figure 5F). As a negative control, increasing the
382
amount of MBP failed to influence the ability of GST-SlAN2-likeInR to pull down
383
TF-SlAN1MIR (Figure 5F). Similarly, the ability of MBP-SlMYBATVAC to pull down
384
TF-SlAN1MIR decreased as the amount of GST-SlAN2-likeInR increased (Figure 5G),
385
and the increasing of GST has negligible effect on the ability of MBP-SlMYBATVAC
386
to pull down TF-SlAN1MIR (Figure 5G). These results corroborate that SlMYBATVAC
387
and SlAN2-likeInR compete with each other to interact with SlAN1.
388 389
SlHY5 plays a critical role in light-induced activation of SlAN2-likeInR
390
Next, we set out to identify the signalling components by which light activates
391
anthocyanin pigmentation in InR fruit. Considering that the bZIP transcription factor 14
392
SlHY5 plays a key role in light-induced anthocyanin accumulation in Arabidopsis
393
(Nguyen et al., 2015; Shin et al., 2013), we edited the tomato SlHY5 gene in the InR
394
background using the CRISPR/Cas9 gene editing system (Deng et al., 2017; Liu et al.,
395
2004) (Supplemental Figure 11A and 11B). In line with a recent report (Qiu et al.,
396
2019), editing SlHY5 led to abolished anthocyanin pigmentation (Figure 6Aand 6B)
397
and SlDFR expression (Figure 6C) in light-exposing fruit peel of InR. These results
398
indicated that SlHY5 plays a critical role in transmitting the light signal to the
399
anthocyanin biosynthesis machinery.
400 401
Not surprisingly, editing SlHY5 largely impaired light-induced expression of
402
SlAN2-likeInR in the InR fruit peel (Figure 6D), indicating that SlHY5 positively
403
regulates the expression of the master regulator SlAN2-likeInR. ChIP-qPCR assays with
404
transgenic SlHY5-GFP plants (Supplemental Figure 11C) and anti-GFP antibody
405
revealed an enrichment of the SlHY5-GFP fusion protein on a SlAN2-likeInR promoter
406
region containing an ACE-box (ACGT) (Figure 6E and 6F), a DNA element that can
407
be recognized by bZIP transcription factors (Lee et al., 2007).
408 409
We then examined the importance of the ACE-box for SlHY5-mediated activation of
410
SlAN2-likeInR expression using the above-described DLR assays. Considering that
411
SlHY5 itself lacks a transcriptional activation domain (Supplemental Figure 11E and
412
11G), we fused the VP16 activation domain (Stracke et al., 2010) to the C-terminal of
413
SlHY5 to generate the SlHY5-VP16 construct. Co-expression of SlHY5-VP16 with
414
proSlAN2-likeInR:LUC led to significantly increased LUC activity, but LUC signal
415
was significantly reduced when the ACE-box in the SlAN2-likeInR promoter was
416
mutated to “TTTT” (Supplemental Figure 12C). These results support that the
417
ACE-box in the SlAN2-likeInR promoter plays an important role for the SlHY5 activity
418
to activate SlAN2-likeInR expression.
419 15
420
Electrophoretic mobility shift assays (EMSAs) showed that the MBP-SlHY5
421
recombinant protein could bind a DNA probe containing the ACE-box but failed to
422
bind a DNA probe in which the ACE-box was mutated (Figure 6G). Collectively,
423
these results demonstrate that SlAN2-likeInR is a direct transcriptional target of SlHY5.
424 425
In summary, we proposed a model to explain the light-induced anthocyanin
426
pigmentation pattern of different genotypes (Figure 6H, 6I and Supplemental Figure
427
13). In the dominant Aft single mutant (i.e., LA1996), both the activator SlAN2-likeInR
428
and the repressor SlMYBATVAC are functional. In light-exposing peel of this
429
genotype, SlHY5 promotes the expression of SlAN2-likeInR, whose protein product in
430
turn activates both the activator SlAN1 and the repressor SlMYBATVAC. The
431
functional repressor SlMYBATVAC competes the binding of SlAN2-likeInR to SlAN1
432
and thereby reducing the formation of a functional SlAN2-likeInR–SlAN1 complex,
433
which determines the transcriptional output for anthocyanin biosynthesis (Figure 6H).
434
In InR which simultaneously harbors the dominant Aft locus and the recessive atv
435
locus, the activator SlAN2-likeInR is functional but the repressor SlMYBATVInR is
436
not-functional; the non-functional repressor SlMYBATVInR loses its ability to reduce
437
the transcriptional output for anthocyanin biosynthesis. Therefore, light-induced
438
anthocyanin accumulation in the fruit peel of InR is much higher than that of the Aft
439
single mutant (Figure 6I). In both AC (Supplemental Figure 13A) and the atv single
440
mutant (Supplemental Figure 13B), the master regulator SlAN2-likeAC is
441
not-functional and, as a consequence, light cannot activate the functional transcription
442
complex for anthocyanin biosynthesis. Our model highlights a central role of a
443
functional SlAN2-like (i.e., SlMYBATVInR) in linking the light signal to the
444
activation of anthocyanin biosynthesis in tomato fruit.
445 446
Engineering the expression of SlAN2-likeInR leads to anthocyanin accumulation
447
in both peel and flesh 16
448
If our model holds true, we predict that engineering the master regulator SlAN2-likeInR
449
could bypass light and activate anthocyanin biosynthesis in the fruit flesh of any
450
cultivar. To test this, SlAN2-likeInR under the control of the fruit-specific SlE8
451
promoter (Kneissl and Deikman, 1996) was transferred to the cultivar AC, which does
452
not accumulate anthocyanin in the peel and flesh. As expected, the resulting
453
proSlE8:SlAN2-likeInR plants exhibited anthocyanin accumulation in both peel and
454
flesh (Figure 7A). Anthocyanin pigmentation started at the breaker stage (42 days
455
post-anthesis, dpa) and intensified rapidly during the ripening process (Figure 7A),
456
which matched well with the expression pattern of the SlE8 gene (Kneissl and
457
Deikman, 1996). Whereas the anthocyanin content of the AC (wild type) fruits was
458
below the detection limit, the highest concentrations of transgenic fruits averaged
459
40.44 ± 4.39 (A535-A650)/g fresh weight, which equivalents to 2.22 ± 0.24 mg of
460
anthocyanin per g fresh weight (Figure 7B).
461 462
To evaluate the impact of the master regulator SlAN2-likeInR on gene expression in a
463
genome-wide scale, we performed RNA-sequencing (RNA-seq) experiments to
464
compare the transcriptome profiles between proSlE8:SlAN2-likeInR and AC fruits.
465
Fruits of each genotype were collected at the B+14 stage (56 dpa) for RNA extraction
466
and sequencing. Data quality assessment of three biological replications is shown in
467
Supplemental Figure 14. These analyses identified 2,074 differentially expressed
468
genes (DEGs) between transgenic and AC fruits (fold change ≥ 2, false discovery rate
469
[FDR]-adjusted P value < 0.05). Among them, 1,552 genes were up-regulated and
470
522 genes were down-regulated in proSlE8:SlAN2-likeInR fruits compared to that of
471
AC. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the
472
up-regulated
473
SlAN2-likeInR-up-regulated genes) are mainly enriched in pathways related to
474
phenylpropanoid biosynthesis, phenylalanine metabolism and flavonoid biosynthesis
475
(Figure 7C). Among the identified SlAN2-likeInR-up-regulated genes are most of the
genes
in
proSlE8:SlAN2-likeInR
17
fruits
(designated
as
476
known anthocyanin biosynthetic genes (Figure 7D) and several regulatory genes
477
including SlAN1 and SlMYBATV (Figure 7E), corroborating that SlAN2-likeInR
478
functions as a master regulator and orchestrates a hierarchical transcriptional
479
regulatory cascade for anthocyanin biosynthesis.
480 481
Discussion
482
Starting from the genetic analysis of the purple tomato InR, we identified the major
483
genes controlling anthocyanin pigmentation of tomato fruit and elucidated their action
484
mechanism. We demonstrated that whereas the dominant Aft locus encodes a
485
functional allele of the positive regulator SlAN2-like (i.e., SlAN2-likeInR), the
486
recessive atv locus encodes a non-functional allele of the negative regulator
487
SlMYBATV (i.e., SlMYBATVInR). We also demonstrated that the bHLH protein
488
SlAN1 is a positive regulator of anthocyanin pigmentation. Considering that the
489
MBW transcriptional regulators are generally conserved in eudicots (Lloyd et al.,
490
2017; Xu et al., 2015), it is reasonable to speculate that SlAN2-likeInR and SlAN1
491
might represent the MYB and the bHLH component, respectively, of the tomato
492
MBW regulatory complex. SlMYBATV, which negatively regulates anthocyanin
493
biosynthesis, might represent a new component of the tomato MBW complex. Our
494
results indicated that a functional allele of SlMYBATV (i.e., SlMYBATVAC)
495
competes the binding of SlAN2-likeInR to SlAN1 and thereby exerting a negative
496
effect on anthocyanin biosynthesis. Therefore, the anthocyanin pigmentation
497
phenotype of InR is resulted from the functional interaction between the functional
498
positive regulator SlAN2-likeInR and the non-functional negative regulator
499
SlMYBATVInR. On the contrary, most tomato cultivars (i.e., AC) contain the
500
non-functional positive regulator SlAN2-likeAC (Supplemental Figure 5) as well as the
501
functional negative regulator SlMYBATVAC (Supplemental Figure 9) and, as a
502
consequence, loss the capability to produce anthocyanins in fruits.
503 18
504
Furthermore, several lines of evidence suggest that the functional positive regulator
505
SlAN2-likeInR is an active and critical regulator of anthocyanin biosynthesis. First,
506
unlike SlAN2, SlANT1 and SlANT1-like, which barely express in tomato fruit,
507
SlAN2-likeInR is highly expressed in InR fruit peel under light-exposing. Second,
508
different with SlAN2-likeAC which is non-functional because of the G to A mutation
509
at the 5' splice site, SlAN2-likeInR is functional. Third, in addition to regulating the
510
expression of structural genes involved in anthocyanin biosynthesis, the functional
511
SlAN2-likeInR also regulates the expression of other regulatory genes including SlAN1
512
and SlMYBATV. Finally, the expression of SlAN2-likeInR itself is not affected by
513
SlAN1 and SlMYBATVAC. Together, these results suggest that genetic manipulation
514
of SlAN2-likeInR could activate anthocyanin biosynthesis in a light-independent
515
manner. Indeed, expression of SlAN2-likeInR under the control of the fruit-specific
516
SlE8 gene promoter led to high levels of anthocyanin accumulation in both peel and
517
flesh of the cultivar AC. Our study suggests two directions for future exploration.
518
First, to circumvent the concern of foreign DNA introgression, gene editing-based
519
technology (Voytas and Gao, 2014) could be exploited to replace the SlAN2-likeInR
520
promoter with the fruit-specific SlE8 promoter or insert the SlAN2-likeInR gene
521
downstream of the SlE8 promoter. Second, master transcriptional regulators such as
522
SlAN2-likeInR could provide effective tools for tissue-specific engineering high levels
523
of metabolites in other plants.
524 525
Notably, our results indicated that, while mutation of SlHY5 in InR abolished
526
SlAN2-likeInR expression and anthocyanin accumulation (Figure 6A and 6D),
527
overexpression
528
(Supplemental Figure 11D). In contrast, overexpression of SlHY5 failed to activate
529
SlAN2-likeAC expression in the AC background (Supplemental Figure 11I). The
530
differential effects of SlHY5 on the expression of SlAN2-likeInR and SlAN2-likeAC
531
could resulted from sequence variations of the SlAN2-likeInR and SlAN2-likeAC
of
SlHY5
in
InR
could
19
activate
SlAN2-likeInR expression
532
promoters. Indeed, our sequence comparison identified numerous variations between
533
the promoter regions of SlAN2-likeInR and SlAN2-likeAC (Supplemental Figure 12A).
534
Considering that SlHY5 itself does not possess activation capacity (Supplemental
535
Figure 11E and 11G) and that the putative SlHY5 binding site ACE-box does not
536
show difference between the promoter of SlAN2-likeInR and SlAN2-likeAC
537
(Supplemental Figure 12A), it is reasonable to speculate that there exist yet-to-be
538
identified sequence variations that determine the differential effects of SlHY5 on the
539
SlAN2-likeInR and SlAN2-likeAC promoters.
540 541
Methods
542
Plant materials and growth conditions
543
InR was used as the wild type (WT) for slant1-c, slan2-c, slant1-like-c, slan2-like-c,
544
proSlMYBATV:SlMYBATVAC, SlMYBATVAC-OE, slan1-c, slhy5-c and SlHY5-GFP.
545
AC was used as the WT for SlAN2-likeAC-OE, SlAN2-likeInR-OE, SlHY5-GFP/AC and
546
proSlE8:SlAN2-likeInR. LA1996 was used as the WT for slmybatv-c. InR/AC was
547
produced by introducing Aft and atv loci from InR into AC via backcrossing. Seeds of
548
AC, MicroTom, Moneymaker, VF36, M82, E6203, LA2750, LA0458, LA0462,
549
LA0448, LA1996, LA1406, LA0434, LA1610, LA2934, LA2398, LA0373 and
550
LA1279 were provided by the Tomato Genetics Resources Center at the University of
551
California, Davis. Tomato plants were grown side-by-side to ensure consistency of
552
the light condition.
553 554
Plasmid construction and plant transformation
555
DNA constructs for plant transformation were generated following standard
556
molecular biology protocols and using Gateway (Invitrogen) technology (Karimi et
557
al., 2002). To generate the SlAN2-like-OE construct, full-length SlAN2-like coding
558
sequence (CDS) from InR and the SlAN2-like genomic sequence from AC was cloned
559
(we failed to isolate SlAN2-like CDS from AC) into pENTR respectively, and then 20
560
recombined with the binary vector pK7WGF2 (Karimi et al., 2002) using a gateway
561
LR clonase enzyme mix kit. SlHY5-GFP and SlMYBATVAC-OE construct was
562
generated in a similar procedure, except that the CDS of SlHY5 and SlMYBATV were
563
isolated from AC. For proSlMYBATV:SlMYBATVAC construction, the 35S promoter of
564
pK7WGF2 was substituted by the 2752-bp SlMYBATV native promoter using the
565
unique enzymes Hind III and Spe I (Thermo Scientific), then the SlMYBATVAC CDS
566
was
567
proSlE8:SlAN2-likeInR construction, the 35S promoter of pK7WGF2 was substituted
568
by the 2187-bp SlE8 promoter using the unique enzymes Hind III and Spe I (Thermo
569
Scientific), then the full-length CDS of SlAN2-likeInR was cloned and recombined into
570
this modified binary vector. The above constructs were introduced into InR or AC
571
(except SlHY5-GFP which was transformed into both the InR and AC background) by
572
Agrobacterium tumefaciens (LBA4404)-mediated cotyledon explants transformation
573
as previously described (Deng et al., 2017). Transformants were selected based on
574
their resistance to kanamycin. T2 homozygous progeny were used for phenotypic and
575
molecular characterization.
cloned
and
recombined
into
this
modified
binary
vector.
For
576 577
Generation of gene editing plants using CRISPR/Cas9 technology
578
The generation of gene editing plants of SlANT1, SlAN2, SlANT1-like, SlAN2-like,
579
SlMYBATV, SlAN1 and SlHY5 was conducted as described previously (Deng et al.,
580
2017; Liu et al., 2019). Briefly, the two target sites of each gene were incorporated
581
into PCR forward and reverse primers, respectively. The PCR fragment was amplified
582
from pCBC-DT1T2_tomatoU6, and then purified and cloned into pTX041 at Bsa I
583
sites to generate pTX041_target gene. pTX041_SlMYBATV was introduced into
584
LA1996 and other constructs were introduced into InR by Agrobacterium tumefaciens
585
(LBA4404)-mediated cotyledon explants transformation. CRISPR/Cas9-induced
586
mutations were genotyped by PCR amplification and DNA sequencing. Primers used
587
for construction are listed in Supplemental Table 1. 21
588 589
Anthocyanin Measurement
590
Anthocyanin content was measured as previously described (Deikman and Hammer,
591
1995) with modification. Samples of fruit peel were ground to powder and about 0.1 g
592
powder was placed into 1 mL of extraction buffer (18% 1-propanol, 1% HCl, and
593
81% water) and incubated overnight at 4℃. Absorbance values (A535 and A650) of
594
the supernatant were measured using spectrophotometer after centrifugation for 20
595
min at 12,000 rpm. The anthocyanin content is presented as (A535-A650)/g fresh
596
weight. Anthocyanin content of proSlE8:SlAN2-likeInR flesh was further expressed as
597
mg/g fresh weight, based on an extinction coefficient of 17,000 and a molecular
598
weight of 934 (Butelli et al., 2008). Data from at least four independent biological
599
replicates were collected, and error bars represent the SD from the biological
600
replicates.
601 602
Bulk population sequencing
603
For bulk population sequencing, three DNA pools, InR-type pool, Aft-type pool and
604
AC-type pool, each with equal amount of DNA from 50 F2 individuals of InR x AC
605
cross were prepared. Three pooled libraries and the two parent libraries with insert
606
sizes of approximately 350-500 bp were prepared and sequenced by Oebiotech using
607
the Illunima HiSeq X-ten platform. To identify the mutation site, reads were mapped
608
on the tomato genome using BWA (Li and Durbin, 2009) with the default options.
609
SAMtools (Li et al., 2009) was used to convert alignment files to SAM/BAM format
610
and applied to SNP calling. SNP positions with SNP quality score < 20 and read
611
depth < 4 were excluded, as these positions may represent spurious SNPs called due
612
to sequencing and/or alignment errors. Homozygous SNPs between InR and AC but
613
different in the 3 pools were filtered, The proportion of reads harbouring InR
614
genotype in the total reads cover the SNP site is defined as the SNP-index and
615
candidate regions associated with anthocyanin were calculated according to ƸSNP 22
616
index between different pools as described before (Takagi et al., 2013).
617 618
RNA extraction and gene expression analysis
619
For reverse-transcription quantitative PCR (RT-qPCR) analysis, total RNA was
620
extracted from tomato fruit peel using the TRIzol Reagent (Invitrogen). 1μg of total
621
RNA was used to synthesize the first strand cDNA with the PrimeScript RT kit with
622
gDNA eraser (Takara). RT-qPCR was performed on Roche LightCycler 480 system
623
with the Kapa SYBR Fast qPCR kit (KAPA). The expression levels of target genes
624
were normalized to that of the tomato SlACTIN2 gene. Error bars represent the SD of
625
three biological replicates. Primer information is given in Supplemental Table 1.
626 627
Transient Expression Assays in Nicotiana benthamiana leaves
628
For the transient transcriptional activity assays of SlAN2-like to activate SlDFR
629
expression, the 1233-bp promoter sequence of SlDFR promoter was amplified from
630
genomic DNA from InR and cloned into the pGreenII 0800-LUC (Hellens et al., 2005)
631
vector to use as a reporter. The Renilla luciferase (REN) gene under the control of the
632
cauliflower 35S promoter in the pGreenII 0800-LUC vector was used as the internal
633
control. The genomic sequences of SlAN2-likeInR, SlAN2-likeAC and their derivatives
634
(SlAN2-likeInR-G653A and SlAN2-likeAC-A657G) were cloned into the pK7WGF2 (Karimi
635
et al., 2002) under the control of the 35S promoter and were used as effectors. Primers
636
used for generating these constructs are listed in Supplemental Table 1. Firefly LUC
637
and REN activities were measured using the Dual-Luciferase Reporter Assay System
638
(Promega) following the manufacturer’s instructions, and LUC/REN ratios were
639
calculated and presented. Data from three independent biological replicates were
640
collected, and error bars represent the standard deviation (SD) from three biological
641
replicates.
642 643
For the transient transcriptional activity assays of SlHY5-VP16 to activate SlAN2-like 23
644
expression, the 846-bp promoter sequence of SlAN2-likeInR promoter and its derivative
645
(SlAN2-likeInR promoter with a mutated ACE-box), as well as SlAN2-likeAC promoter
646
and its derivative (SlAN2-likeAC promoter with a mutated ACE-box) were amplified
647
from genomic DNA and cloned into the pGreenII 0800-LUC (Hellens et al., 2005).
648
VP16 activation domain fragment was amplified from pBT3-SUC (Dualsystems
649
Biotech.) and the merged sequences of SlHY5-VP16 were cloned into the pK7WGF2
650
(Karimi et al., 2002) under the control of the 35S promoter and were used as effectors.
651
Firefly LUC and REN activities were measured using the Dual-Luciferase Reporter
652
Assay System (Promega) following the manufacturer’s instructions, and the
653
experimental procedures were the same as those described above.
654 655
Expression profiling analysis
656
AC and proSlE8:SlAN2-likeInR fruits were harvested at the B+14 stage (56 dpa), and
657
flesh was collected for RNA isolation. Total RNA of each sample was extracted using
658
TRIzol Reagent (Invitrogen) and treated with DNase I. The quality of the total RNA
659
was assessed using a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer.
660
For each sample, 1 μg of total RNA was used to construct the Illumina sequencing
661
libraries according to the manufacturer’s instructions. The libraries were sequenced
662
by Biomarker Technologies using the Illunima HiSeq X-ten platform and
663
preliminarily analysed by using the BMKCloud platform (http://www.biocloud.net/).
664
Tomato reference genome (SL3.0) was downloaded from the SOL Genomics
665
Network (http://solgenomics.net/). Raw sequencing reads were first processed to
666
remove adaptors and low-quality bases, and then aligned to the genome sequences
667
using HISAT2 (Kim et al., 2015) with default parameters. Gene expression levels
668
were calculated in fragments per kilobase of transcript per million fragments mapped
669
(FPKMs) using StringTie (Pertea et al., 2015). Differentially expressed genes (DEGs)
670
between two groups were identified using the DEGSeq R package (Wang et al., 2010)
671
with fold change ≥ 2 and FDR-adjusted P value < 0.05. KEGG enrichment analysis 24
672
was implemented by using the KOBAS (Mao et al., 2005) with default parameters.
673 674
Yeast two-hybrid assay (Y2H)
675
Y2H assays were performed using the MATCHMAKER GAL4 Two-Hybrid System
676
(Clontech). To investigate the interactions of SlAN1 with SlAN2-likeInR and
677
SlMYBATVAC, full-length coding sequence of SlAN1 were cloned into pGADT7.
678
SlAN2-likeInR fragment spanning the R3 domain and the full-length CDS of
679
SlMYBATVAC were individually cloned into pGBKT7. To map the domains of SlAN1
680
involved in the SlAN1–SlAN2-likeInR interaction and SlAN1–SlMYBATVAC
681
interaction, derivatives of SlAN1 coding sequence were cloned into pGADT7.
682
Constructs used to test protein-protein interactions were co-transformed into yeast
683
(Saccharomyces cerevisiae) strain AH109 and co-transformation of the empty
684
pGADT7 vector was used as a negative control. Transformed yeast was grown on
685
medium lacking Leu and Trp (SD/-2) as transformation control or on medium lacking
686
Ade, His, Leu, and Trp (SD/-4) to test protein-protein interactions. The interactions
687
were observed after 3 d of incubation at 30°C. Primers used for vector construction
688
are listed in Supplemental Table 1.
689 690
Yeast three-hybrid assay (Y3H)
691
Y3H assays were performed using the MATCHMAKER GAL4 Two-Hybrid System
692
(Clontech). To construct pBridge-SlAN2-likeR3-SlMYBATVAC, the SlAN2-likeInR
693
fragment spanning the R3 domain was cloned into the MCS I (multiple cloning site I)
694
site of the pBridge vector (Clontech) fused to the GAL4 DNA-binding domain, and
695
the coding sequence of SlMYBATVAC was cloned into the MCS II site of the pBridge
696
vector expressed only in the absence of methionine. Constructs used for testing
697
protein-protein interactions were co-transformed into Saccharomyces cerevisiae strain
698
AH109. The presence of the transgenes was confirmed by growth on SD/-2 plates.
699
Transformed yeast cells were spread on SD/-4 medium to assess the SlAN1 and 25
700
SlAN2-likeInR interaction without the expression of SlMYBATVAC and on plates
701
containing SD/-Ade/-His/-Leu/-Trp/-Met (SD/-5) medium to induce SlMYBATVAC
702
expression. Interactions were observed after 3 days of incubation at 30°C. For the
703
construction of pBridge-SlMYBATVAC-SlAN2-likeR3, the SlMYBATVAC coding
704
sequence was cloned into the MCS I site and the SlAN2-likeInR fragment spanning the
705
R3 domain was cloned into the MCS II site of the pBridge vector and expressed only
706
in the absence of methionine. The experimental procedures were the same as those
707
described above.
708 709
In vitro pull-down assay
710
For pull-down assays to test the SlAN2-likeInR–SlAN1 and SlMYBATVAC–SlAN1
711
interactions, the full-length coding sequence of SlAN2-likeInR was PCR amplified and
712
cloned into the pGEX-4T-3 vector. To produce TF-SlAN1MIR fusion protein, a SlAN1
713
fragment spanning the MIR domain of SlAN1 was PCR amplified from InR and
714
cloned into pCold™ TF. Primers used for plasmid construction are listed in
715
Supplemental Table 1. The recombinant vectors were transformed into Escherichia
716
coli BL21 (DE3) cells. GST-SlAN2-likeInR, and TF-SlAN1MIR were purified using
717
GST-Bind Resin (Millipore) or Ni-NTA His-Bind Resin (Millipore), respectively. For
718
each reaction, 20 μL of agarose beads (Millipore) bound with 1 μg of
719
GST-SlAN2-likeInR was incubated with 1 μg of TF-SlAN1MIR in 1 mL of reaction
720
buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM DTT, and Roche protease
721
inhibitor cocktail) at 4°C for 2 h. Subsequently, beads were collected and washed
722
three times with washing buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1 mM
723
DTT). After washing, samples were denatured using sodium dodecyl sulfate (SDS)
724
loading buffer and separated using SDS-polyacrylamide gel electrophoresis
725
(SDS-PAGE). The TF-SlAN1MIR fusion protein was detected by immunoblotting with
726
anti-TF antibody. Purified GST was used as a negative control. 0.1μg of GST and
727
GST-SlAN2-likeInR fusion proteins were separated by SDS-PAGE, and then detected 26
728
by immunoblotting with anti-GST antibody (Abmart) as a loading control. For
729
pull-down assays to test SlMYBATVAC–SlAN1 interactions, the full-length coding
730
sequence of SlMYBATVAC was PCR amplified and cloned into the pMAL-c2X
731
vector. The recombinant vectors were transformed into E. coli BL21 (DE3) cells and
732
purified using amylose resin (NEB). The detection of SlMYBATVAC–SlAN1
733
interaction by in vitro pull-down was similar to those described above, except that
734
amylose resin (NEB) was used instead of GST-Bind Resin (Millipore).
735 736
For pull-down assays to confirm that SlMYBATVAC affects the SlAN2-likeInR–
737
SlAN1MIR interaction, 1 μg of purified GST-SlAN2-likeInR and TF-SlAN1MIR proteins
738
were added to each sample. Purified MBP-SlMYBATVAC and MBP protein was
739
added according to the concentration gradient. The GST-Bind Resin (Millipore) were
740
used to pull down proteins. Sequential procedures and buffers were the same as
741
described above. The effect of SlAN2-likeInR on SlMYBATVAC–SlAN1MIR
742
interaction was detected similarly.
743 744
Electrophoretic mobility shift assay
745
The full-length coding sequence of SlHY5 from InR was PCR amplified and cloned
746
into pMAL-c2X. The recombinant MBP-fusion proteins were expressed in E. coli
747
BL21 (DE3) cells and purified using amylase resin column (NEB). Oligonucleotide
748
probes were synthesized and labeled with biotin at the 5ʹ ends (Invitrogen).
749
Unlabelled probes with the same oligonucleotides were used as cold competitors.
750
EMSAs were performed as previously described (Du et al., 2017; Liu et al., 2019).
751
Briefly, biotin-labeled probes were incubated with MBP-fusion proteins at room
752
temperature for 20 min, and free and bound probes were separated via PAGE.
753
Mutated SlAN2-likeInR probes, in which the specific transcription factor-binding motif
754
5ʹ-ACGT-3ʹ was replaced by 5ʹ -TTTT-3ʹ was used as negative controls. Probes used
755
for EMSA are listed in Supplemental Table 1. 27
756 757
ChIP-qPCR Assay
758
The ChIP-qPCR assay was conducted as described previously (Du et al., 2017; Liu et
759
al., 2019). Briefly, 4 g peel of SlAN2-likeInR-OE, SlHY5-GFP mature green fruit (39
760
dpa) as well as the WT AC and InR were harvested and cross-lined in 1%
761
formaldehyde for 10 min, followed by neutralization with 0.125 M glycine. The
762
cross-linked samples were ground to powder in liquid nitrogen. Then the chromatin
763
complex was isolated, resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM
764
NaCl, 1 mM EDTA, 1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM
765
PMSF, and 1 × Roche protease inhibitor mixture), and sheared by sonication to
766
reduce the average DNA fragment size to around 500 bp. Then, 50 μL of sheared
767
chromatin was saved as input control. Anti-GFP antibody (Abcam) was incubated
768
with Dynabeads™ Protein G (Invitrogen) at 4°C for at least 6 h and added to the
769
remaining chromatin for overnight incubation at 4°C. The immunoprecipitated
770
chromatin–protein complex was then washed with low-salt washing buffer (20 mM
771
Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.2% SDS and 0.5% Triton X-100),
772
high salt washing buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA,
773
0.2% SDS and 0.5% Triton X-100), LiCl washing buffer (10mM Tris-HCl, pH 8.00,
774
25 mM LiCl, 1mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate), TE
775
buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA) sequentially, and then eluted with
776
elution buffer (1% SDS, 100mM NaHCO3) after washing. Reverse of protein-DNA
777
cross-linking was performed by incubating the immunoprecipitated complex at 65°C
778
overnight. DNA was recovered using the QIAquick PCR purification kit (Qiagen).
779
The ChIP signal was quantified as the percentage of total input DNA by qPCR. The
780
primers used in qPCR were designed at the promoters of the SlDFR and SlAN2-like
781
genes with or without MBS/ACE-box in the product fragments and listed in the
782
Supplemental Table 1. Each ChIP value was normalized to its respective input DNA
783
value and enrichment of DNA is shown as the percentage of input. 28
784 785
Data availability
786
The RNA-seq data have been deposited in the Genome Sequence Archive (GSA) at
787
the Beijing Institute of Genomics (BIG) Data Center, Chinese Academy of Sciences,
788
under the accession number CRA001936, and the bulk sequencing data have been
789
deposited in the GSA under the accession number CRA001937.
790 791
Accession Numbers
792
Sequence for genes described in this study can be found in the SOL Genomics
793
Network (http://solgenomics.net/) under the following accession numbers: SlANT1,
794
Solyc10g086250;
795
Solyc10g086270;
796
SlMYBATV, Solyc07g052490; SlDFR, Solyc02g085020; SlHY5, Solyc08g061130,
797
SlACTIN2, Solyc11g005330; SlE8, Solyc09g089580.
SlAN2 SlAN2-like,
(SlMYB75),
Solyc10g086060;
Solyc10g086290;
SlAN1,
SlANT1-like, Solyc09g065100;
798 799
Author contributions
800
C.L. and C-B.L. designed the project. C.S., L.D., M.D. and T.H. performed the
801
experiments, Q.C. and J.Z. performed the bioinformatics analysis. H.J. conducted
802
plant transformation. C.L., C.S., L.D. and M.D. wrote the manuscript. All authors
803
read and approved the final manuscript.
804 805
Acknowledgements
806
This work was supported by the Strategic Priority Research Program of the Chinese
807
Academy of Sciences (Precision Seed Design and Breeding), the National Key
808
Research and Development Program of China (2016YFD0100500), the Ministry of
809
Agriculture of China (2016ZX08009-003-001), and the Tai-Shan Scholar Program
810
from Shandong Province (No. tsxk20150901). No conflict of interest declared.
811 29
812
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Lin, T., Zhu, G., Zhang, J., Xu, X., Yu, Q., Zheng, Z., Zhang, Z., Lun, Y., Li, S., and Wang, X. (2014). Genomic analyses provide insights into the history of tomato breeding. Nat. Genet. 46:1220-1226. Liu, Y., Du, M., Deng, L., Shen, J., Fang, M., Chen, Q., Lu, Y., Wang, Q., Li, C., and Zhai, Q. (2019). MYC2 regulates the termination of jasmonate signaling via an autoregulatory negative feedback loop. Plant Cell 31:106-127. Liu, Y., Roof, S., Ye, Z., Barry, C.S., Van Tuinen, A., Vrebalov, J., Bowler, C., and Giovannoni, J.J. (2004). Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. Proc. Natl. Acad. Sci. U S A 101:9897-9902. Lloyd, A.M., Brockman, A., Aguirre, L., Campbell, A., Bean, A., Cantero, A., and Gonzalez, A. (2017). Advances in the MYB–bHLH–WD repeat (MBW) pigment regulatory model: Addition of a WRKY factor and co-option of an anthocyanin MYB for betalain regulation. Plant Cell Physiol. 58:1431-1441. Mano, H., Ogasawara, F., Sato, K., Higo, H., and Minobe, Y. (2007). Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato. Plant Physiol. 143:1252-1268. Mao, X., Cai, T., Olyarchuk, J.G., and Wei, L. (2005). Automated genome annotation and pathway identification using the kegg orthology (KO) as a controlled vocabulary. Bioinformatics 21:3787-3793. Mathews, H.V., Clendennen, S.K., Caldwell, C.G., Liu, X.L., Connors, K., Matheis, N., Schuster, D.K., Menasco, D.J., Wagoner, W., and Lightner, J. (2003). Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15:1689-1703. Mes, P.J., Boches, P., Myers, J.R., and Durst, R. (2008). Characterization of tomatoes expressing anthocyanin in the fruit. J. Am. Soc. Hortic. Sci. 133:262-269. Naing, A.H., and Kim, C.K. (2018). Roles of R2R3-MYB transcription factors in transcriptional regulation of anthocyanin biosynthesis in horticultural plants. Plant Molecular Biology 98:1-18. Nguyen, N.H., Jeong, C.Y., Kang, G.H., Yoo, S., Hong, S.W., and Lee, H. (2015). MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis. Plant J. 84:1192-1205. Pertea, M., Pertea, G., Antonescu, C., Chang, T.C., Mendell, J.T., and Salzberg, S.L. (2015). Stringtie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33:290-295. Qiu, Z., Wang, H., Li, D., Yu, B., Hui, Q., Yan, S., Huang, Z., Cui, X., and Cao, B. (2019). Identification of candidate HY5-dependent and -independent regulators of anthocyanin biosynthesis in tomato. Plant Cell Physiol. 60:643-656. Qiu, Z., Wang, X., Gao, J., Guo, Y., Huang, Z., and Du, Y. (2016). The tomato Hoffman's Anthocyaninless gene encodes a bHLH transcription factor 32
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involved in anthocyanin biosynthesis that is developmentally regulated and induced by low temperatures. PLoS One 11:e0151067. Schreiber, G., Reuveni, M., Evenor, D., Orenshamir, M., Ovadia, R., Sapirmir, M., Bootboolman, A., Nahon, S., Shlomo, H., and Chen, L. (2012). Anthocyanin1 from Solanum chilense is more efficient in accumulating anthocyanin metabolites than its Solanum lycopersicum counterpart in association with the anthocyanin fruit phenotype of tomato. Theor. Appl. Genet. 124:295-307. Shin, D.H., Choi, M., Kim, K., Bang, G., Cho, M., Choi, S., Choi, G., and Park, Y. (2013). HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis. FEBS Lett. 587:1543-1547. Stamm, S., Benari, S., Rafalska, I., Tang, Y., Zhang, Z., Toiber, D., Thanaraj, T.A., and Soreq, H. (2005). Function of alternative splicing. Gene 344:1-20. Stracke, R., Favory, J., Gruber, H., Bartelniewoehner, L., Bartels, S., Binkert, M., Funk, M., Weisshaar, B., and Ulm, R. (2010). The Arabidopsis bZIP transcription factor HY5 regulates expression of the PFG1/MYB12 gene in response to light and ultravioletϋB radiation. Plant Cell and Environment 33:88-103. Takagi, H., Abe, A., Yoshida, K., Kosugi, S., Natsume, S., Mitsuoka, C., Uemura, A., Utsushi, H., Tamiru, M., and Takuno, S. (2013). QTLϋseq: Rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J. 74:174-183. Voytas, D.F., and Gao, C. (2014). Precision genome engineering and agriculture: Opportunities and regulatory challenges. PLoS Biol. 12:1-6. Wang, L., Feng, Z., Wang, X., Wang, X., and Zhang, X. (2010). DEGseq: An r package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26:136-138. Xu, W., Dubos, C., and Lepiniec, L. (2015). Transcriptional control of flavonoid biosynthesis by MYB–bHLH–wdr complexes. Trends Plant Sci. 20:176-185. Zhang, L., Hu, J., Han, X., Li, J., Gao, Y., Richards, C.M., Zhang, C., Tian, Y., Liu, G., Gul, H., et al. (2019). A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour. Nat Commun 10:1494. Zhang, Y., Butelli, E., De Stefano, R., Schoonbeek, H., Magusin, A., Pagliarani, C., Wellner, N., Hill, L., Orzaez, D., and Granell, A. (2013). Anthocyanins double the shelf life of tomatoes by delaying overripening and reducing susceptibility to gray mold. Curr. Biol. 23:1094-1100. Zhu, Z., Wang, H., Wang, Y., Guan, S., Wang, F., Tang, J., Zhang, R., Xie, L., and Lu, Y. (2015). Characterization of the cis elements in the proximal promoter regions of the anthocyanin pathway genes reveals a common regulatory logic that governs pathway regulation. J. Exp. Bot. 66:3775-3789.
979 33
980
Figure Legends
981
Figure 1. A bulk population sequencing approach to map genes controlling
982
anthocyanin accumulation in InR fruit.
983
(A) Photograph of Indigo Rose (InR, AftAft atvatv), Indigo Rose/AC (InR/AC, AftAft
984
atvatv) and AC (aftaft AtvAtv) fruits at the mature green stage (39 dpa). As shown on
985
the right, InR fruit does not accumulate anthocyanins in the flesh.
986
(B) Anthocyanin contents of light-exposing and shading fruit peel of InR, InR/AC and
987
AC. FW, fresh weight. Error bars represent the SD of four biological replicates.
988
(C) ƸSNP-index plot of chromosome 10 generated between DNA pools of the
989
Aft-type and the AC-type. ƸSNP-index between the two DNA pools is presented by
990
red lines, grey lines indicate 95% cutoff. (See also Figure S1.)
991
(D) ƸSNP-index plot of chromosome 7 generated between DNA pools of the
992
InR-type and Aft-type. ƸSNP-index between the two pools is presented by red lines,
993
grey lines indicate 95% cutoff. (See also Figure S1.)
994
(E) Candidate genes in Aft mapping region. The 4 R2R3-MYB genes are presented by
995
red arrows. Gene direction is indicated by the arrow.
996
(F) Candidate genes in atv mapping region. SlMYBATV is presented by blue arrow.
997
(G and I) Schematic diagram of the SlAN2-like gene (G) and the SlMYBATV gene (I)
998
in InR and AC. Black box and lines represent exons and introns, respectively. Dash
999
line indicates the corresponding position of AC splice site in the 2nd intron of
1000
SlAN2-like to InR. Red triangle represents the 4-bp ATAG insertion in the InR allele
1001
of SlMYBATV.
1002
(H and J) Schematic diagram of the SlAN2-like protein (H) and the SlMYBATV
1003
protein (J) in InR and AC. Orange box and green box represent the R2 domain and
1004
the R3 domain, respectively. Grey box represents sequences that are not identical to
1005
its functional counterpart.
1006 1007
Figure 2. SlAN2-like encodes a positive regulator of anthocyanin biosynthesis. 34
1008
(A) RT-qPCR results showing the expression levels of SlAN2, SlANT1, SlANT1-like
1009
and SlAN2-like, in light-exposing and shading fruit peel of InR and AC.
1010
(B) Photograph of InR, slan2-c, slant1-c, slant1-like-c and slan2-like-c fruits at the
1011
mature green stage (39 dpa). (See also Figure S2.)
1012
(C) Anthocyanin contents of light-exposing peel of InR, slan2-c, slant1-c,
1013
slant1-like-c and slan2-like-c fruits. FW, fresh weight.
1014
(D) Photograph of AC and SlAN2-like-OE fruits at the mature green stage (39 dpa).
1015
(See also Figure S6.)
1016
(E) Anthocyanin contents of light-exposing peel of AC, SlAN2-likeAC-OE and
1017
SlAN2-likeInR-OE fruits. FW, fresh weight.
1018
(F) RT-qPCR results showing the expression levels of SlAN2-like in light-exposing
1019
peel of AC, SlAN2-likeAC-OE and SlAN2-likeInR-OE fruits.
1020
(G) RT-qPCR results showing the expression levels of SlAN2-like in seedling, leaf,
1021
stem and light-exposing peel of AC and InR.
1022
(H) RT-qPCR results showing transcript-specific expression of SlAN2-like in InR, AC,
1023
SlAN2-likeAC-OE and SlAN2-likeInR-OE plants. (See also Figure S7.)
1024
(I and J) RT-qPCR results showing the expression levels of SlDFR in light-exposing
1025
peel of the indicated genotypes. AC was used as the WT for SlAN2-likeInR-OE plants
1026
(I), InR was used as the WT for slan2-like-c (J) plants.
1027
(K) Schematic diagram of the SlDFR promoter and the PCR amplicons (indicated as
1028
letters A and B) used for ChIP-qPCR. Red line indicates the Myb-binding site (MBS).
1029
The translational start site (ATG) is shown at position +1.
1030
(L) ChIP-qPCR showing the enrichment of SlAN2-likeInR on the chromatin of SlDFR.
1031
Chromatin of the WT and SlAN2-likeInR-OE plants was immunoprecipitated with an
1032
anti-GFP antibody. The immunoprecipitated DNA was used as a template for qPCR
1033
analysis, with primers targeting different regions of the SlDFR as shown in (K).
1034
SlACTIN2 was used as a non-specific target. ChIP signal was displayed as the
1035
percentage of total input DNA. 35
1036
For (C) and (E), error bars represent the SD of four biological replicates. For (A), (F),
1037
(G), (H), (I), (J) and (L), error bars represent the SD of three biological replicates.
1038
Asterisks indicate significant differences from the WT according to Student’s t-test at
1039
** P < 0.01. ns, not significant.
1040 1041
Figure 3. SlMYBATV encodes a negative regulator of anthocyanin biosynthesis.
1042
(A) Photograph of LA1996 (Aft), InR and proSlMYBATV:SlMYBATVAC fruits at the
1043
mature green stage (39 dpa). (See also Figure S8D.)
1044
(B)
1045
proSlMYBATV:SlMYBATVAC fruits. FW, fresh weight.
1046
(C) Photograph of LA1996 (Aft) and slmybatv-c fruits at the mature green stage (39
1047
dpa). (See also Figure S8A. and S8B.)
1048
(D) Anthocyanin contents of light-exposing peel of LA1996 and slmybatv-c fruits.
1049
FW, fresh weight.
1050
(E) RT-qPCR results showing the expression levels of SlDFR in light-exposing peel
1051
of LA1996 and slmybatv-c fruits.
1052
(F) Photograph of InR and SlMYBATVAC-OE fruits at the mature green stage (39 dpa).
1053
(See also Figure S8C.)
1054
(G) Anthocyanin contents of light-exposing peel of InR and SlMYBATVAC-OE fruits.
1055
FW, fresh weight.
1056
(H) RT-qPCR results showing the expression levels of SlDFR in light-exposing peel
1057
of InR and SlMYBATVAC-OE fruits.
1058
For (B), (D) and (G), error bars represent the SD of four biological replicates. For (E)
1059
and (H), error bars represent the SD of three biological replicates. Asterisks indicate
1060
significant differences from the WT according to Student’s t-test at ** P < 0.01.
Anthocyanin
contents
of
light-exposing
peel
of
LA1996,
1061 1062
Figure 4. SlAN2-like activates SlAN1 and SlMYBATV expression.
36
InR
and
1063
(A) Photograph of InR and slan1-c fruits at the mature green stage (39 dpa). (See also
1064
Figure S10.)
1065
(B) Anthocyanin contents of light-exposing peel of InR and slan1-c fruits. FW, fresh
1066
weight. Error bars represent the SD of four biological replicates.
1067
(C) RT-qPCR results showing the expression levels of SlDFR in light-exposing peel
1068
of InR and slan1-c fruits.
1069
(D and E) RT-qPCR results showing the expression levels of SlAN1 (left) and
1070
SlMYBATV (right) in the light-exposing fruit peel of the indicated genotypes. InR was
1071
used as the WT for SlAN2-like-c plants (D), and AC was used as the WT for
1072
SlAN2-likeInR-OE plants (E).
1073
(F and H) RT-qPCR results showing the expression levels of SlAN2-like in the
1074
light-exposing fruit peel of the indicated genotypes. InR was used as the WT for
1075
slan1-c (F) and SlMYBATVAC-OE plants (H), LA1996 was used as the WT for
1076
slmybatv-c plants (G).
1077
For (C-H), error bars represent the SD of three biological replicates. Asterisks
1078
indicate significant differences from the WT according to Student’s t-test at **P <
1079
0.01.
1080 1081
Figure 5. SlMYBATVAC and SlAN2-likeInR compete for binding SlAN1.
1082
(A) Yeast two-hybrid (Y2H) assay showing that both SlAN2-likeInR and
1083
SlMYBATVAC interact with SlAN1. The R3 domain of SlAN2-likeInR (SlAN2-likeR3)
1084
or full-length SlMYBATVAC were fused with the DNA binding domain (BD) in
1085
pGBKT7 and full length SlAN1 was fused with the activation domain (AD) in
1086
pGADT7, respectively. Transformed yeast was grown on selective SD/-2 media or
1087
SD/-4 media to test protein interaction. The empty pGADT7 vector was
1088
co-transformed with SlAN2-likeR3 or SlMYBATVAC in parallel as negative controls.
37
1089
(B) In vitro pull-down assay showing that SlAN2-likeInR interacts with SlAN1MIR.
1090
TF-SlAN1MIR was pulled down by GST-SlAN2-likeInR immobilized on GST-Bind
1091
resin. Protein bound to GST-Bind resin was eluted and analyzed by anti-TF antibody.
1092
(C) In vitro pull-down assay showing that SlMYBATVAC interacts with SlAN1MIR.
1093
TF-SlAN1MIR was pulled down by MBP-SlMYBATVAC immobilized on amylose
1094
resin. Protein bound to amylose resin was eluted and analyzed by anti-TF antibody.
1095
(D) Domains of SlAN1 involved in the SlAN2-likeInR–SlAN1 interaction and
1096
SlMYBATVAC–SlAN1 interaction were mapped using Y2H assays. Yeast cells
1097
co-transformed
1098
pGBKT7-SlAN2-likeR3 (bait), were dropped onto SD/-2 and SD/-4 media to assess
1099
interactions with SlAN2-likeInR (Left 2 panels). Yeast cells co-transformed with
1100
pGADT7-SlAN1 or its derivatives (preys) and pGBKT7-SlMYBATVAC (bait), were
1101
dropped onto SD/-2 and SD/-4 media to assess interactions with SlAN2-likeAC (Right
1102
2 panels).
1103
(E) Yeast three-hybrid (Y3H) assay showing that SlMYBATVAC and SlAN2-likeR3
1104
compete with each other to interact with SlAN1. (Top two panels) Yeast cells
1105
co-transformed with pGADT7-SlAN1 and pBridge-SlAN2-likeR3-SlMYBATVAC
1106
were dropped onto SD/-2 and SD/-4 media to assess the SlAN2-likeInR–SlAN1
1107
interaction. The co-transformed yeast cells were dropped onto SD/-5 medium to
1108
induce SlMYBATVAC. (Bottom two panels) Yeast cells co-transformed with
1109
pGADT7-SlAN1 and pBridge-SlMYBATVAC-SlAN2-likeR3 were dropped onto SD/-2
1110
and SD/-4 media to assess the SlMYBATVAC–SlAN1 interaction. The co-transformed
1111
yeast cells were dropped onto SD/-5 medium to induce SlAN2-likeR3.
1112
(F) Pull-down assay showing that SlMYBATVAC represses SlAN2-likeInR–SlAN1MIR
1113
interaction. For each sample, the amounts of TF-SlAN1MIR and GST-SlAN2-likeInR
1114
were equal, MBP-SlMYBATVAC and MBP were added according to the indicated
1115
gradient. TF-SlAN1MIR was pulled down by GST-SlAN2-likeInR immobilized on a
1116
GST-Bind resin. Proteins were eluted and analyzed using an anti-TF antibody.
with
pGADT7-SlAN1
38
or
its
derivatives
(preys)
and
1117
(G) Pull-down assay showing that SlAN2-likeInR represses SlMYBATVAC–SlAN1MIR
1118
interaction. For each sample, the amounts of TF-SlAN1MIR and MBP-SlMYBATVAC
1119
were equal, GST-SlAN2-likeInR and GST were added according to the indicated
1120
gradient. TF-SlAN1MIR was pulled down by MBP-SlMYBATVAC immobilized on an
1121
amylose resin. Proteins were eluted and analyzed using an anti-TF antibody.
1122 1123
Figure 6. SlHY5 activates SlAN2-likeInR expression in response to light.
1124
(A) Photograph of InR and slhy5-c fruits at the mature green stage (39 dpa). (See also
1125
Figure S11A. and S11B.)
1126
(B) Anthocyanin contents of light-exposing peel of InR and slhy5-c fruits. FW, fresh
1127
weight.
1128
(C and D) RT-qPCR results showing the expression levels of SlDFR (C) and
1129
SlAN2-likeInR (D) in light-exposing peel of InR and slhy5-c fruits.
1130
(E) Schematic diagram of the SlAN2-likeInR promoter. Green line represents the
1131
ACE-box motif. Short orange line indicates DNA probes used for EMSA. PCR
1132
amplicons used for ChIP-qPCR are indicated as A and B. The translational start site
1133
(ATG) is shown at position +1.
1134
(F) ChIP-qPCR showing the enrichment of SlHY5 on the chromatin of SlAN2-likeInR.
1135
Chromatin of the WT and SlHY5-GFP plants was immunoprecipitated with an
1136
anti-GFP antibody. The immunoprecipitated DNA was used as a template for qPCR
1137
analysis, with primers targeting different regions of the SlAN2-likeInR as shown in (E).
1138
SlACTIN2 was used as a non-specific target. ChIP signal was displayed as the
1139
percentage of total input DNA. Error bars represent the SD of three biological
1140
replicates. Asterisks indicate significant difference from the WT according to
1141
Student’s t-test at **P < 0.01.
1142
(G) EMSA showing that MBP-SlHY5 fusion protein directly binds to SlAN2-likeInR
1143
promoter in vitro. Biotin-labeled probes were incubated with MBP-SlHY5 protein,
1144
and free and bound DNA (arrows) was separated in an acrylamide gel. The MBP 39
1145
protein was incubated with the labeled probe in the first lane to serve as a negative
1146
control. Unlabeled probes were used as
1147
(Mu-competitor), mutated probe in which the ACE-box motif 5ʹ-ACGT-3ʹ was
1148
replaced with 5ʹ-TTTT-3ʹ.
1149
(H and I) Proposed working model for the light-induced anthocyanin biosynthesis in
1150
Aft single mutant (H) and InR (I). In the dominant Aft single mutant (i.e., LA1996),
1151
both SlAN2-likeInR and SlMYBATVAC are functional. In light-exposing fruit peel of
1152
this genotype, SlHY5 promotes the expression of SlAN2-likeInR, whose protein
1153
product in turn activates both the positive regulator SlAN1 and the negative regulator
1154
SlMYBATVAC. The functional negative regulator SlMYBATVAC competes the
1155
binding of SlAN2-likeInR to SlAN1 and thereby reducing the formation of a functional
1156
SlAN2-likeInR–SlAN1 complex, which determines the transcriptional output for
1157
anthocyanin biosynthesis (H). In InR which simultaneously harbors the dominant Aft
1158
locus and the recessive atv locus, the positive regulator SlAN2-likeInR is functional
1159
but the negative regulator SlMYBATVInR is not-functional; SlMYBATVInR loses the
1160
ability to reduce the transcriptional output for anthocyanin biosynthesis. Therefore,
1161
light-induced anthocyanin accumulation in the fruit peel of InR is much higher than
1162
that of the Aft single mutant (I). (See also Figure S13.)
competitors, as
indicated. Mu
1163 1164
Figure 7. Fruit-specific over-expression of SlAN2-likeInR leads to anthocyanin
1165
accumulation in fruit flesh.
1166
(A) Photograph of AC and proSlE8:SlAN2-likeInR fruits at the indicated stages. IMG,
1167
immature green; MG, mature green; B+n, n days after breaker.
1168
(B) Anthocyanin contents of AC and proSlE8:SlAN2-likeInR fruits harvested at the
1169
B+14 stage (56 dpa). FW, fresh weight. Error bars represent the SD of six biological
1170
replicates. Asterisks indicate significant differences from the WT (AC) according to
1171
Student’s t-test at ** P < 0.01.
40
1172
(C) KEGG pathway enrichment analysis of genes up-regulated (upper panel) or
1173
down-regulated (lower panel) by SlAN2-likeInR. The 1,552 genes up-regulated in
1174
proSlE8:SlAN2-likeInR (upper panel), and the 522 genes down-regulated in
1175
proSlE8:SlAN2-likeInR (lower panel) were used for analysis. The x axis shows the fold
1176
enrichment (ratio of genes in queried list/ratio of genes in the genome). The y axis
1177
shows the terms of metabolic pathway. The size of the plotted circle indicates the
1178
number of queried genes. The filled color is scaled to q value.
1179
(D and E) Expression of representative anthocyanin structural genes (D) and
1180
regulatory genes (E) in the RNA-seq experiments. log2(FPKM+1) of each gene from
1181
three biological replicates (1, 2 and 3) is shown. FPKM, fragments per kilobase of
1182
exon per million fragments mapped.
1183 1184
Supplemental Figure and Table Legends
1185
Figure S1. Genetic segregation and mapping of Aft and atv. Related to Figure 1.
1186
An F2 segregation population was generated by crossing InR (AftAft atvatv) with AC
1187
(aftaft AtvAtv). In the F2 population, fruit peel anthocyanin pigmentation pattern
1188
varied into 3 types: InR-type, Aft-type and AC-type, the predicted genotype was
1189
presented below the phenotype. The InR-type and Aft-type individuals were used to
1190
mapping atv locus. Aft-type and AC-type individuals were used to mapping Aft locus.
1191 1192
Figure S2. Generation of single mutants of the 4 R2R3-MYB genes. Related to Figure
1193
2.
1194
(A-D) CRISPR/Cas9-mediated editing of SlAN2 (A), SlANT1 (B), SlANT1-like (C)
1195
and SlAN2-like (D) in the InR background. Exons of the target genes are targeted by
1196
CRISPR/Cas9 using two single-guide RNAs (sgRNA; Target 1 and Target 2 (red
1197
arrows)). sgRNA targets and protospacer-adjacent motif (PAM) are indicated in red
1198
and bold font, respectively. Deletions and insertions are indicated by blue dashes and
1199
letters, respectively, and the numbers in parentheses indicate how many base pairs are 41
1200
involved (+, insertion; -, deletion).
1201
(E) RT-qPCR results showing the expression levels of SlAN2-like in light-exposing
1202
peel of InR and slan2-like-c fruits.
1203
(F) RT-qPCR results showing the expression levels of SlAN2 in light-exposing peel of
1204
InR and slan2-c fruits. It should be noted that the expression of slant1 and slant1-like
1205
in the fruit peel of InR and corresponding knock-out plants were below the detection
1206
limit.
1207
For (E) and (F), error bars represent the SD of three biological replicates. Asterisks
1208
indicate significant differences from the WT according to Student’s t-test at ** P <
1209
0.01.
1210 1211
Figure S3. Phylogenetic analysis of R2R3-MYB genes. Related to Figure 2.
1212
The evolutionary history was inferred using the Neighbor-Joining method in MEGA6.
1213
The tree is drawn to scale, with branch lengths in the same units as those of the
1214
evolutionary distances used to infer the phylogenetic tree. The percentage of replicate
1215
trees in which the groups clustered together in the bootstrap test (1000 replicates) are
1216
shown next to the branches. Solanum lycopersicum ANT1, SlANT1 (ABO26065.1); S.
1217
lycopersicum AN2, SlAN2 (AUG72357.1); S. lycopersicum ANT1-like, SlANT1-like
1218
(XP_004249668.1);
1219
Arabidopsis thaliana PAP1, AtPAP1 (AAG42001.1); A. thaliana MYB90, AtMYB90
1220
(NP_176813.1); Nicotiana tabacum AN2, NtAN2 (ACO52470.1); Antirrhinum majus
1221
ROSEA1, AmROS1 (ABB83826.1); A. majus ROSEA2, AmROS2 (ABB83827.1);
1222
Zea mays C1, ZmC1 (AAA33482.1); Z. mays Pl, ZmPl (AAA19819.1); Petunia
1223
hybrida AN2, PhAN2 (ABO21074.1); P. hybrida DPL, PhDPL (ADW94950.1); P.
1224
hybrida
1225
(AAX53089.1); Malus domestica MYB10, MdMYB10 (ABB84753); Citrus sinensis
1226
Ruby,
1227
(BAG68211.1).
PHZ,
CsRuby
S.
PhPHZ
lycopersicum
AN2-like,
(ADW94951.1);
(NP_001275818.1);
Solanum
Ipomoea
42
SlAN2-like
tuberosum
batatas
(AUG72358.1);
AN1,
MYB1,
StAN1
IbMYB1
1228 1229
Figure S4. Sequence alignment of SlAN2-like in InR and AC. Related to Figure 2.
1230
(A) Sequence alignment of SlAN2-like cDNA sequence. Sequence alignment was
1231
performed with Clustal X program. The stop codon is highlighted by blue square and
1232
AC harbors a premature stop codon.
1233
(B) Sequence alignment of SlAN2-like protein sequence. Sequence alignment was
1234
performed with Clustal X program. The R2 and R3 domain are represented in orange
1235
box and green box, respectively.
1236
(C) Sequence alignment of SlAN2-like genomic sequence. Sequence alignment was
1237
performed with Clustal X program. Exons in AC and InR were represented in red
1238
letters. The stop codon is highlighted by blue squares and AC harbors a premature
1239
stop codon. The causal SNP in the 5' splice site of the 2nd intron is indicated by arrow.
1240 1241
Figure S5. Sequence alignment of SlAN2-like genomic sequence in tomato cultivars
1242
and wild species. Related to Figure 2.
1243
Exons were represented in red letters. The causal SNP in the 5' splice site of the 2nd
1244
intron is indicated by arrow and performed with different colors, and the stop codon is
1245
highlighted by blue squares. Solanum lycopersicum (S. lycopersicum) represents
1246
cultivated tomatoes, Solanum chilense (S. chilense), Solanum peruvianum (S.
1247
peruvianum) and Solanum pimpinellifolium (S. pimpinellifolium) represent wild
1248
tomatoes. Sequence alignment was performed with Clustal X program.
1249 1250
Figure S6. Phenotype of 35S:SlAN2-likeInR plants. Related to Figure 2.
1251
Photograph of different plants of 35S:SlAN2-likeInR showing variation in anthocyanin
1252
accumulation in mature plants (A), seedlings (B), roots (C), leaves (D), stems (E) and
1253
flowers (F).
1254 1255
Figure S7. Alternative splicing of SlAN2-like. Related to Figure 2. 43
1256
(A) Schematic diagram of the SlAN2-like gene with different mutagenesis. InR
1257
features (exon, intron and SNP) in SlAN2-like were represented in orange and AC
1258
features (exon, intron and SNP) were represented in blue. SlAN2-likeInR-G653A
1259
represents a mutated SlAN2-likeInR with the “GCAAGT” mutated to “GCAAAT” in
1260
the 5' splice site of the 2nd intron. SlAN2-likeAC-A657G represents a mutated
1261
SlAN2-likeAC with the “GCAAAT” mutated to “GCAAGT” in the 5' splice site of the
1262
2nd intron.
1263
(B) RT-qPCR results showing transcript-specific expression of different forms of
1264
SlAN2-like indicated in (A) in the transient expression assays of (D).
1265
(C) A schematic diagram showing the constructs used in the transient expression
1266
assays of (D). Arrows, promoter regions; shaded boxes, coding sequence.
1267
(D) Transient expression assays in Nicotiana benthamiana showing the activation of
1268
the SlDFR promoter by different forms of SlAN2-like. The LUC/REN ratio represents
1269
the proSlDFR:LUC activity relative to the internal control (REN driven by the 35S
1270
promoter). EV, empty vector. Error bars represent the SD of four biological replicates.
1271
Asterisks indicate significant differences from the control according to Student’s t-test
1272
at ** P < 0.01. ns, not significant.
1273 SlMYBATVAC-OE
1274
Figure
1275
proSlMYBATV:SlMYBATVAC plants. Related to Figure 3.
1276
(A) CRISPR/Cas9-mediated editing of SlMYBATV. Exons of SlMYBATV was targeted
1277
by CRISPR/Cas9 using two single-guide RNAs (sgRNA; Target 1 and Target 2 (red
1278
arrows)). sgRNA targets and PAM are indicated in red and bold font, respectively.
1279
Deletions are indicated by blue dashes, and the numbers in parentheses indicate how
1280
many base pairs are involved.
1281
(B-D) RT-qPCR results showing the expression levels of SlMYBATV in
1282
light-exposing fruit peel of the indicated genotypes. LA1996 was used as the WT for
1283
slmybatv-c plants (B), and InR was used as the WT for SlMYBATVAC-OE and
S8.
Generation
of
slmybatv-c,
44
and
1284
proSlMYBATV:SlMYBATVAC plants (C and D).
1285
For (B-D), error bars represent the SD of three biological replicates. Asterisks indicate
1286
significant differences from the WT according to Student’s t-test at ** P < 0.01.
1287 1288
Figure S9. Sequence alignment of SlMYBATV genomic sequence in tomato cultivars
1289
and wild species. Related to Figure 3.
1290
Exons were represented in red letters. The “ATAG” insertion is indicated by arrow,
1291
and the stop codon is highlighted by blue squares. Solanum lycopersicum (S.
1292
lycopersicum) represents cultivated tomatoes, Solanum cheesmaniae (S. cheesmaniae),
1293
Solanum chilense (S. chilense), Solanum peruvianum (S. peruvianum) and Solanum
1294
pimpinellifolium (S. pimpinellifolium) represent wild tomatoes. Sequence alignment
1295
was performed with Clustal X program.
1296 1297
Figure S10. Generation of slan1-c knock-out mutants. Related to Figure 4.
1298
(A) CRISPR/Cas9-mediated editing of SlAN1. Exons of SlAN1 was targeted by
1299
CRISPR/Cas9 using two single-guide RNAs (sgRNA; Target 1 and Target 2 (red
1300
arrows)). sgRNA targets and PAM are indicated in red and bold font, respectively.
1301
Deletions and insertions are indicated by blue dashes and letters, respectively, and the
1302
numbers in parentheses indicate how many base pairs are involved (+, insertion; -,
1303
deletion).
1304
(B) RT-qPCR results showing the expression levels of SlAN1 in light-exposing peel of
1305
InR and slan1-c fruits. Error bars represent the SD of three biological replicates.
1306
Asterisks indicate significant differences from the WT according to Student’s t-test at
1307
** P < 0.01.
1308 1309
Figure S11. Generation of slhy5-c and SlHY5-GFP plants. Related to Figure 6.
1310
(A) CRISPR/Cas9-mediated editing of SlHY5. Exons of SlHY5 was targeted by
1311
CRISPR/Cas9 using two single-guide RNAs (sgRNA; Target 1 and Target 2 (red 45
1312
arrows)). sgRNA targets and PAM are indicated in red and bold font, respectively.
1313
Deletions are indicated by blue dashes and the numbers in parentheses indicate how
1314
many base pairs are involved.
1315
(B and C) RT-qPCR results showing the expression levels of SlHY5 in light-exposing
1316
fruit peel of the indicated genotypes.
1317
(D) RT-qPCR results showing the expression levels of SlAN2-like in light-exposing
1318
fruit peel of InR and SlHY5-GFP plants.
1319
(E) Yeast two-hybrid (Y2H) assay showing that SlHY5 lacks activation activity. The
1320
full-length coding sequence of SlHY5 was fused with the DNA binding domain (BD)
1321
in pGBKT7. Transformed yeast was grown on selective media lacking Ade, His, Leu,
1322
and Trp (SD/-4) to test activation activity. The SlAN2-likeInR-BD was co-transformed
1323
with pGADT7 as positive control and the empty pGBKT7 vector was co-transformed
1324
with pGADT7 as negative control.
1325
(F) A schematic diagram showing the constructs used in the transient expression
1326
assays of (G). Arrows, promoter regions; shaded boxes, coding sequence.
1327
(G) Transient expression assays in N. benthamiana showing the activation of the
1328
SlDFR
1329
proSlAN2-like:LUC activity relative to the internal control (REN driven by the 35S
1330
promoter).
1331
(H and I) RT-qPCR results showing the expression levels of SlHY5 and SlAN2-like in
1332
light-exposing fruit peel of AC and SlHY5-GFP/AC plants.
1333
For (B), (C), (D), (G), (H) and (I), error bars represent the SD of three biological
1334
replicates. Asterisks indicate significant differences from the WT (InR) according to
1335
Student’s t-test at ** P < 0.01.
promoter
by
SlHY5-VP16.
The
LUC/REN
ratio
represents
the
1336 1337
Figure S12. The ACE-box in SlAN2-like promoter plays an important role for SlHY5
1338
to activate SlAN2-likeInR expression. Related to Figure 6.
1339
(A) Sequence alignment of SlAN2-like promoter between InR and AC. The 46
1340
SlHY5-binding site (ACE-box) is highlighted in red. Sequence above the orange line
1341
represents probe used in the EMSA assay. Sequence alignment was performed with
1342
Clustal X program.
1343
(B) A Schematic diagram showing the constructs used in the transient expression
1344
assays of (C). Reporter 1 and 3 adopted wild type SlAN2-likeAC and SlAN2-likeInR
1345
promoter, respectively. The ACE-box was mutated to “TTTT” in Reptorter2 and 4.
1346
Arrows, promoter regions; shaded boxes, coding sequence.
1347
(C) Transient expression assays in N. benthamiana showing the activation of the
1348
SlAN2-like promoter by SlHY5-VP16. The LUC/REN ratio represents the
1349
proSlAN2-like:LUC activity relative to the internal control (REN driven by the 35S
1350
promoter). EV, empty vector.
1351 1352
Figure S13. Proposed working model for the light-induced anthocyanin biosynthesis
1353
in AC and atv single mutant. Related to Figure 6.
1354
In both AC (A) and the atv single mutant (B), the master regulator SlAN2-likeAC is
1355
not-functional and, as a consequence, light cannot activate the functional transcription
1356
complex for anthocyanin biosynthesis.
1357 1358
Figure S14. Summary of the RNA-seq analysis. Related to Figure 7.
1359
(A) Overview of RNA-seq data. Relative percentages of multiple mapped reads
1360
(multiple), unique mapped reads (uniq) and unmapped reads (unmapped) are shown.
1361
Three biological replicates were indicated by 1, 2 and 3.
1362
(B) Principal component analysis (PCA) showing the relatedness among the gene
1363
expression patterns of samples used for RNA-seq analysis. Red color represents AC
1364
samples and blue represents proSlE8:SlAN2-likeInR samples.
1365 1366
Table S1. Primers and probes used in this study.
47
B
120
Indigo Rose (InR)
Indigo Rose/AC (InR/AC)
AC
80 60 40 20
Indigo Rose transection
C
Light-exposing Shading
100
(A535-A650)g-1FW
A
0
InR
InR/AC
AC
D Aft
atv
1.0 △ SNP Index
△ SNP Index
1.0 0.5 0 -0.5
0.5 0 -0.5 -1.0
-1.0 10 Mb
20 Mb
30 Mb 40 Mb Chromosome 10
50 Mb
60 Mb
E
10 Mb
20 Mb
30 Mb 40 Mb Chromosome 7
50 Mb
60 Mb
F 60.98 Mbp Chr. 7
65.13 Mbp 65.14 Mbp 65.15 Mbp 65.16 Mbp 65.17 Mbp Chr. 10 SlAN2 SlANT1 SlANT1-like Heavy metal transport SlAN2-like
0 0 0 27 25 626 86 86 8 g0 g0 0g0 0 0 1 1 1 lyc lyc olyc So So S
60.99 Mbp
61.01 Mbp
SlMYBATV
0 0 28 29 86 86 g0 g0 0 0 1 1 lyc lyc So So
G
61.00 Mbp
0 0 49 50 52 52 g0 g0 7 7 0 0 lyc lyc So So
I SlMYBATV ATAG insertion
SlAN2-like InR ATG
InR
TAG
ATG TAA premature stop
AC ATG
AC
200 bp
H
200 bp ATG
TGA premature stop
TAA
J SlAN2-like
InR
R2
AC
R2
SlMYBATV R3
InR
AC
R3
61.02 Mbp
Relative expression levels
A
D
1.2 InR light-exposing InR shading AC light-exposing AC shading
1.0 0.8 0.6
1# 2# SlAN2-likeAC-OE
AC
0.4 0.2
E 12
0.0
SlANT1
**
SlANT1-like SlAN2-like (A535-A650)g FW
SlAN2
-1
B
10 8
**
6 4 2
ns InR
slan2-c 1#
slant1-c 2#
AC AC -OE 1# AC -OE 2# InR -OE 1# InR -OE 2# 2-like lAN2-like lAN2-like lAN2-like N S SlA S S
F 120 ns
100
ns
ns
80 60 40 **
20 0 InR
SlAN2-like
18000 16000 14000 12000 10000 8000 6000 4000 2000 0
slan2-c slant1-c slant1-like-c slan2-like-c 1# 2# 1# 1#
**
**
AC AC -OE 1# AC -OE 2# InR -OE 1# InR -OE 2# 2-like lAN2-like lAN2-like lAN2-like S S S
H 1.2
1000 Relative expression levels
AC InR
1.0 0.8 0.6 0.4 0.2 0.0
Seedling
Leaf
**
**
SlAN
G Relative expression levels
ns
0
slant1-like-c slan2-like-c 1# 1#
Relative expression levels
(A535-A650)g-1FW
C
1# 2# SlAN2-likeInR-OE
800
SlAN2-like AC version transcript InR version transcript
600 400 200 0 ng el edling peel dling ng eel eedli InR pe e s AC 2# seAe 2# p 2# seedli 2# peel InR AC s C -OE AC -OE InR -OE R -OE e In k li 2-like SlAN2- N2-like lAN2-like SlAN S SlA
Stem Light-exposing peel
MBS ATG
35
SlDFR
30
**
PCR amplicon
J **
25 20 15 10 5
L
1.0 0.8 0.6 0.4 0.2
**
AC
1#
2#
SlAN2-likeInR-OE
SlAN2-likeInR-OE AC
0.25 0.20 0.15 0.10 0.05
**
0.0
0
B
A
SlDFR
1.2 Relative expression levels
Relative expression levels
I
200 bp
+1
ChIP signal (%Input)
K
SlDFR
0.00 InR
1#
2#
slan2-like-c
A
B
SlACTIN2
A
C
InR
LA1996 (Aft)
1#
2#
B
D
4# slmybatv-c
E 80
SlDFR **
** (A535-A650)g-1FW
100 80 60 40
**
20
60
40
20
**
Relative expression levels
120
0
0 LA1 6 ( A t)
1#
InR p Sl
2# AT Sl
F
AT
AC
2#
SlMYBATVAC-OE
(A535-A650)g-1FW
3 2 1
2#
LA1 6 ( A t)
4#
sl
at -c
H 1.2
100
2#
4
slmybatv-c
120
1#
**
5
4#
G
InR
**
6
0 LA1 6 ( A t)
Relative expression levels
(A535-A650)g-1FW
2#
LA1996 (Aft)
proSlMYBATV:SlMYBATVAC
80 60 40 20
**
**
1#
2#
0
InR
Sl
AT
AC
-OE
SlDFR
1.0 0.8 0.6 0.4 0.2
**
0.0
**
1#
InR Sl
2# AT
AC
-OE
B
C
120
(A535-A650)g-1FW
100 80 60 40 20
1#
InR
sla
0.6
** ** ** **
0.0
ns
1.0 0.8 0.6 0.4 0.2 0.0
InR
1#
2# sla
2# -c
**
**
**
1#
InR
sla
A SlA SlA
-l -l
2# -c
R R
-OE -OE
**
30 20 10
**
**
SlA
G
-l ns
1.2
40
SlMYBATV
SlA
0.2
0
SlA
1.4
0.4
0.0
-c
SlA
1.2
ns
1.0
H
-l ns
Relative expression levels
0.2
0.6
50
0.8
0.4
0.8
E -c -c
1.0
Relative expression levels
Relative expression levels
sla
Relative expression levels
InR sla -l sla -l
1.0
**
1#
InR
1.2
Relative expression levels
0
-c
D
F
**
2#
SlDFR
1.2
Relative expression levels
A
0.8 0.6 0.4 0.2 0.0 LA1996 ( Aft)
2#
4#
slmybatv-c
SlMYBATV
1.2 1.0
SlA
-l
ns ns
0.8 0.6 0.4 0.2 0.0
1# 2# InR proSlMYBATV:SlMYBATVAC
A
B -2
re
ait
lA 1
lA 2-li e
A
lA 2-li eR3
lA 1
l
A
A
A
l
A
A
D
R3
-
- l
F- lA 1 IR A A
-
-
Anti- F
ll- o n
l
A
re
ait
IR
Anti- F
Inp t
- l
A
E
R3
A
ll- o n
- lA 2-li eInR
Inp t
R3
lA 2-li eR3
C F- lA 1 IR - lA 2-li eInR
-4
re
lA 2-li eR3 l
L
A
ait
-2
ri ge
lA 1
lA 2-li eR3
lA 1484-685
lA 1
lA 2-li eR3
lA 111-209
lA 1
l
A
A
-
lA 1210-483
lA 1
l
A
A
lA 2-li eR3
lA 1
-2
-4
F
-2
-4
-5
A
-
l
A
A
-4
G - l - lA 2-li eInR F- lA 1 - l
A
ll- o n
A IR
- lA 2-li eInR
IR A
A F- lA 1
-
-
1x
-
-
-
2x 4x -
-
-
-
-
1x
2x
4x
ll- o n Anti- F
-
-
1x
-
-
-
2x 5x -
-
-
-
-
1x
2x
5x Anti- F Anti-
AntiInp t Inp t
Anti-
Anti- F Anti-
Anti- F
A
A
E A SlA
-l
ox
A
R
1
R a pli on
A
500 p
InR
sl y -c
sl y -c
G F
o petitor
1.0
120 100 80 60 40 20
**
**
0 InR
4#
8#
I signal ( Inp t)
(A535-A650)g-1FW
B
ro e - l 5
Sl Y - F InR
**
0.8
-
- o petitor
- 10x 20x 50x - - - - - - - 10x 20x 50x -
o n pro e
0.6 0.4 0.2
sl y -c 0.0
Relative expression levels
C
A
lA
I 2
Free pro e
1.2 1.0 0.8
H
0.6 0.4 0.2
**
0.0 InR
4#
Lig t
Lig t
l
l
** 8#
5
5
sl y -c
Relative expression levels
D
R2R3 lA 2-li e
R2R3 lA 2-li e
1.2 1.0
lA 1
R3 l
IR
A
lA 1
IR
l
0.8
A
0.6 0.4
R2R3 lA 2-li e
0.2
**
0.0 InR
4#
** 8# sl y -c
lA 1
R2R3 lA 2-li e IR
IR
lA 1 SlDFR
SlDFR
A
B I
rea er
4
7
10
14
50
-1
(A535-A650)g FW
** 40
A
p SlE SlAN2-likeInR 2#
30
**
20 10 0
AC A
1#
2#
p SlE SlAN2-likeInR
D
Sl C
p-reg late genes (1552)
tatisti s o
l ol sis til enoi
l
at
a
nri
SlC S
oneogenesis
●
iar l eptanoi an gingerol ios nt esis
gene n 10 ● 20 ● 30
●
eta olis
●
en lalanine t rosine an tr ptop an ios nt esis en lalanine Flavonoi
ios nt esis
en lpropanoi
ios nt esis
ala tose
● ●
●
eta olis
SlF SlF SlDFR SlANS
val e 1.00 0.75 0.50 0.25 0.00
●
eta olis
SlC I
er
●
ios nt esis o a ino a i s ● l erolipi
ent
Sl T SlACC Sl ST Sl AT
●
1
2 Ri
SlF S
3 4 5 a tor
SlF SlRT
o n-reg late genes (522)
tatisti s o
Fatt a i
ios nt esis
at
a ●
nri ent gene n 10 ● 20 ● 30
E er
c g 2 c g c g c g 1 c1 g 1 c g 2 c g c g c g 1 1 c g c g 22 c g 1 2 c 2g c11g c 2g 2 c g c g 1 c12g 1 c 2g 1 c g 2 1 c g c 1g 1 c 1g1 c g 2 c g11 22 c11g 1 c11g 1
2 1
2
2 2 10
SlC
2#
1)
C
l l l l l l l l l l l l l l l l l l l l l l l l l l l
InR
Log2(F
Sl A
S S S S S S S S S S S S S S S S S S S S S S S S S S S
-l
2 1
0
AC proSlE :SlA 1 2 3 1 2 3
p SlE SlAN2-likeInR 2#
p SlE SlAN2-likeInR 2# A 1 2 3 1 2 3
iterpenoi
●
eta olis
●
ios nt esis
●
1
2 Ri
3 4 5 a tor
val e 1.00 0.75 0.50 0.25 0.00
SlAN2-like Sl Sl 2 Sl Sl AT Sl SlAN1 Sl AF1 Sl DR
S S S S S S S S S
l l l l l l l l l
c g 2 c g 2 c1 g 1 c 1g111 c g 2 c12g c g c g 11 c g
2 2 2
2 2 1
1)
Alanine aspartate an gl ta ate
●
Log2(F
one ios nt esis
0
a
10
●
erpenoi