ADP-glucose pyrophosphorylase large subunit 2 is essential for storage substance accumulation and subunit interactions in rice endosperm

Plant Science 249 (2016) 70–83

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ADP-glucose pyrophosphorylase large subunit 2 is essential for storage substance accumulation and subunit interactions in rice endosperm Xiao-Jie Tang a,1 , Cheng Peng a,1,2 , Jie Zhang a,1 , Yue Cai a , Xiao-Man You a , Fei Kong a , Hai-Gang Yan a , Guo-Xiang Wang a , Liang Wang a , Jie Jin a , Wei-Wei Chen a , Xin-Gang Chen a , Jing Ma a , Peng Wang a , Ling Jiang a , Wen-Wei Zhang a,∗∗ , Jian-Min Wan a,b,∗ a

State Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, 1 Weigang Road, Nanjing 210095, China Institute of Crop Science, The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing 100081, China b

a r t i c l e

i n f o

Article history: Received 10 March 2016 Received in revised form 4 May 2016 Accepted 13 May 2016 Available online 13 May 2016 Keywords: AGPase OsAGPL2 Starch synthesis Interaction

a b s t r a c t ADP-glucose pyrophosphorylase (AGPase) controls a rate-limiting step in the starch biosynthetic pathway in higher plants. Here we isolated a shrunken rice mutant w24. Map-based cloning identified OsAGPL2, a large subunit of the cytosolic AGPase in rice endosperm, as the gene responsible for the w24 mutation. In addition to severe inhibition of starch synthesis and significant accumulation of sugar, the w24 endosperm showed obvious defects in compound granule formation and storage protein synthesis. The defect in OsAGPL2 enhanced the expression levels of the AGPase family. Meanwhile, the elevated activities of starch phosphorylase 1 and sucrose synthase in the w24 endosperm might possibly partly account for the residual starch content in the mutant seeds. Moreover, the expression of OsAGPL2 and its counterpart, OsAGPS2b, was highly coordinated in rice endosperm. Yeast two-hybrid and BiFC assays verified direct interactions between OsAGPL2 and OsAGPS2b as well as OsAGPL1 and OsAGPS1, supporting the model for spatiotemporal complex formation of AGPase isoforms in rice endosperm. Besides, our data provided no evidence for the self-binding of OsAGPS2b, implying that OsAGPS2b might not interact to form higher molecular mass aggregates in the absence of OsAGPL2. Therefore, the molecular mechanism of rice AGPase assembly might differ from that of Arabidopsis. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27) carries out the first committed step in the synthesis of starch in both photosynthetic and non-photosynthetic plant tissues [1,2]. AGPase uses the substrates glucose-1-phosphate (G-1-P) and ATP to produce ADPglucose, the sugar nucleotide utilized by starch syn-

∗ Corresponding author at: State Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, 1 Weigang Road, Nanjing 210095, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (W.-W. Zhang), [email protected], [email protected] (J.-M. Wan). 1 These authors contributed equally to this work. 2 Present address: Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China. http://dx.doi.org/10.1016/j.plantsci.2016.05.010 0168-9452/© 2016 Elsevier Ireland Ltd. All rights reserved.

thase, and inorganic pyrophosphate (PPi). AGPase is an allosteric enzyme regulated by intermediates of the major pathway of carbon assimilation in the organism. AGPase activity is activated by 3phosphoglycerate (3-PGA) and inhibited by Pi in the leaves and sink tissues of most but not all organs of higher plants [1–3]. In addition, AGPase is subject to transcriptional regulation, with expression being increased by sugars [4–7] and decreased by nitrate [4] and phosphate [8]. The activity of the plastid-localized enzyme is also subject to fine regulation by redox control in response to changing light and sugar levels [9]. In higher plants, AGPase consists of two large subunits (LS) and two small subunits (SS) that interact and polymerize into the native heterotetrameric enzyme structure [10–12]. Increasing evidence has suggested that both subunits are essential for catalytic and allosteric regulatory properties of the enzyme [13–17]. In most organs, AGPase is entirely plastidial but in the cereal endosperm, there are both plastidial and cytosolic isoforms [18–22], with

X.-J. Tang et al. / Plant Science 249 (2016) 70–83

the latter the majority of AGPase activity during grain filling [2,23–25]. In rice, there are two genes encoding SSs, OsAGPS1 and OsAGPS2, and four encoding LSs, OsAGPL1, OsAGPL2, OsAGPL3, and OsAGPL4. OsAGPS1 and OsAGPL1 are plastid-targeted isoforms, both expressed at an early stage of endosperm development. OsAGPS2 can generate two transcripts via alternative splicing, designated as OsAGPS2a and OsAGPS2b, expressed in the chloroplast of leaf and the cytosol of endosperm, respectively [26]. OsAGPL2 encodes the cytosolic isoform in the endosperm while OsAGPL3 and OsAGPL4 encode plastid-targeted isoforms. OsAGPL3 is preferentially expressed in the leaf while OsAGPL4 is expressed at very low levels in seeds and leaves. Previous studies have shown that mutation in SS or LS genes leads to a severe depression of starch synthesis [2,23–25,27,28]. In a mutant lacking OsAGPL3, the AGPase activity and starch content in leaf blades were reduced to less than 1 and 5% of that in wild type blades, respectively [29]. In the absence of OsAGPL1, there was a reduction of 23% in the total AGPase activity in rice endosperm, whereas no difference in mature grain weight was found in the mutant. On the other hand, almost no starch accumulated in the culms of the mutant and the starch content was also reduced in the mutant embryos [30]. Therefore, OsAGPL1 is proposed to make a minor contribution to AGPase activity in the endosperm. In addition, the defective OsAGPS2 in the shrunken mutant EM22 reduces the total AGPase activity to about 20% of the wild type [31]. The reduced AGPase activity causes a decline in starch accumulation in the endosperm, leading to shrivelling of the seed at maturity [32]. Such shriveled seeds are also seen for OsAGPL2 null [shrunken1(shr1)] and missense [shrunken-1altered (shr1a)] mutants [33]. Of them, shr1a seeds exhibit a more severely shriveled phenotype yet with a higher AGPase activity (about 60% of that of the wild type) than that of shr1 seeds (about 40%). Nevertheless, the recombinant missense heterotetramer enzymes show lower affinity for the activator (3-PGA) but more sensitivity to the inhibitor (Pi) than the wild-type AGPase. Therefore, the sensitivity of the missense AGPases to Pi inhibition may lower their net catalytic activity to a level comparable with that of the shr1 seeds. Ohdan et al. and Lee et al. proposed a model for spatiotemporal complex formation of AGPase isoforms in the leaf and endosperm of rice [2,26]. OsAGPS2a combines with OsAGPL3 to function predominantly during transitory starch synthesis in rice leaves. At an early stage of the endosperm development, OsAGPS1 associates with OsAGPL1 to confer the dominant enzyme activity in starch synthesis. As the endosperm matures, the cytosolic OsAGPS2b/OsAGPL2 complex has the major function in starch accumulation. In maize, a direct interaction between Shrunken2 (SH2) and Brittle2 (BT2), i.e. endosperm-specific LS and SS, has been proven in yeast [34]. As either BT2 or SH2 shows no self-binding, the early steps in the assembly of maize AGPase were suggested to involve a heterodimer intermediate [34]. In contrast, Wang et al. proposed that the Arabidopsis AGPase assembly process may be initiated by homodimer formation of SSs, which interacts further with LSs or SSs [35]. The proposal was based on the fact that SSs can assemble into a homotetrameric quaternary structure in the absence of LSs [36], while in the absence of SSs, LSs become unstable in the chloroplast [37,38]. For rice AGPase, however, no valuable information is available so far about how subunits interact or polymerize into the heterotetrameric structure. In our present study, we characterized a shrunken rice mutant w24, containing a missense mutation in the cytosolic OsAGPL2 gene. Our data indicated a critical role of OsAGPL2 in starch synthesis and compound granule formation in the developing rice endosperm. In addition, direct interactions between OsAGPL2:OsAGPS2b as well as OsAGPL1:OsAGPS1 were verified through yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. N- or C-

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terminal domain of subunits necessary for assembly of rice AGPase was also defined. 2. Materials and methods 2.1. Plant materials and growth conditions In this study, we used a mutant line w24 that was generated by radiation-induced mutation of japonica rice (Oryza sativa L.) cv Koshihikari. Reciprocal crosses between w24 and Koshihikari were used for genetic analysis. An F2 population was produced from a cross between w24 and cv. Nanjing 11 (O. sativa L., indica) for mapping. All plants were grown during the summer under natural environmental conditions in an experimental field plot of Nanjing Agricultural University. The developing seeds at 5–25 d after flowering were used in biochemical and electron microscopic studies. 2.2. Microscopy Scanning electron microscopy was performed as described previously [39]. The procedure used for transmission electron microscopy (TEM) followed Wang et al. [40]. For the observation of compound granules, semi-thin sections were prepared according to the process described by Peng et al. [41]. 2.3. Determination of starch characters and soluble sugars Rice grains were processed using a dehuller and ground into fine flour with a miller. Amylose, starch and protein contents as well as the chain length distributions of amylopectin were determined according to a previous report [42]. Sucrose, glucose and fructose contents were measured with a glucose, fructose and saccharose assay kit (BioSenTec, Auzeville-Tolosane, France). Flag leaves harvested at the end of day at different filling stages were analyzed. Starch and soluble sugar contents in flag leaves were measured in the insoluble and soluble fractions of ethanol-water extracts and determined spectrophotometrically as described previously [43]. 2.4. SDS-PAGE and western-blot analysis Total protein extraction was performed as described by Takemoto et al. [44], and proteins were analyzed by SDS-PAGE. The immunoblot assay followed Wang et al. [45]. Synthetic peptide fragments of OsAGPL2, OsAGPS2b and Pho1 were chemically synthesized and used as antigen in rabbits to elicit antiserum. 2.5. Genetic mapping To identify markers linked to the w24 locus, we used both parents and 180 F2 plants with homozygous mutant alleles. More than 180 polymorphic simple sequence repeat (SSR) markers evenly distributed over the whole genome were selected. To fine-map the w24 locus, molecular markers were developed based on the nucleotide polymorphisms in the corresponding regions between Nipponbare and 93-11 (Supplementary Table 1). 2.6. Vector construction and rice transformation For complementation of the w24 mutant, the wild-type OsAGPL2 cDNA sequence was cloned into the binary vector pCUbi1390 under the control of the maize ubiquitin promoter to generate the transformation cassette p1390-Ubi-L2 (Table S2). For RNAi analysis of OsAGPL2, gene-specific fragments of OsAGPL2 were amplified by PCR (Table S2). The resulting DNA fragments were inserted in sense

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Fig. 1. Phenotype of the w24 mutant. (a) Grain filling of wild type and w24 at various stages of endosperm development. The weight of the grains indicates the dry weight of 100 brown grains. (b) Grain size of wild type and w24. The asterisk indicates statistical significance between the wild type and the mutant, as determined by a Student’s t-test (*P < 0.05). (c, d, e, f) Comparison of wild-type (c, e) and w24 seeds (d, f). Seeds were placed on a black surface (c, d) and on a light box (e, f). (g–j) Scanning electron microscopy analysis of the endosperm of the wild type (g, i) and w24 (h, j). Scale bars: 1 mm in (g) and (h); 20 ␮m in (i) and (j).

orientation between the KpnI/SacI sites and in antisense orientation between the BamHI/PstI sites in vector LH-FAD1390RNAi. These plasmids were introduced into Agrobacterium tumiefaciens strain EHA105, which was then used to transfect the w24 mutant and Koshihikari calli, respectively [46]. Hygromycin-resistant calli were regenerated and seedlings were grown in a greenhouse.

2.7. RNA extraction and real-time RT-PCR analysis Total RNA was extracted using an RNAprep pure Plant kit (TIANGEN Biotech, Beijing, China). A 1-␮g portion of total RNA was reverse-transcribed by priming with oligo (dT18 ) in a 20-␮l reaction volume based on the PrimeScript Reverse Transcriptase kit (Takara, Otsu, Japan). The primers used in this analysis were listed in Supplementary Table 2. The value of ActinI mRNA (accession number AK100267) was used as an internal control.

2.8. Yeast two-hybrid assay Pairs of recombinant plasmids were built in either the pGADT7 or pGBKT7 vectors (Clontech), including OsAGPL1, OsAGPL2, OsAGPS1, OsAGPS2b, and their truncations (Supplementary Table 2). Yeast transformation and screening procedures were performed according to the manufacturer’s instructions (Clontech).

2.9. BiFC assay The full-length cDNAs of OsAGPS1 and OsAGPS2b were cloned into the p2YN (eYFP) vector to construct the S1-eYFPN and S2beYFPN fusion proteins, respectively. OsAGPL1 and OsAGPL2 were cloned into the p2YC (eYFP) vector to produce L1-eYFPC and L2eYFPC fusion proteins, respectively (primer sequences are listed in Supplementary Table 2). The BiFC analyses were performed in tobacco, as described previously [41]. 2.10. Enzyme assays and native PAGE/activity staining A sample of 100 mg of developing endosperm harvested at 12 DAF was homogenized on ice in 1 ml of buffer: 50 mM HEPES NaOH (pH 7.4), 2 mM MgCl2 and 12.5% (v/v) glycerol. The homogenate was centrifuged at 20,000g for 10 min at 4 ◦ C and the supernatant was used for enzyme activity assays. AGPase activities were assayed in the pyrophosphorylase direction as described previously [47]. Sucrose synthase activities were determined following Doehlert et al. [48]. Debranching enzyme (DBE) and branching enzyme (BE) isozymes were separated by native PAGE and assayed according to the method described by Fujita et al. [49]. Starch synthase (SS) and phosphorylase (Pho) activities were measured in 7.5% polyacrylamide gels containing 0.8% (w/v) oyster glycogen (Sigma-Aldrich), according to the method described by Satoh et al. [50]. All electrophoretic separations were conducted at 4 ◦ C.

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73

15

**

60 45 30 15

*

9 6 3 0

0

0.350.35 0.30 0.30 0.25 0.25 0.20 0.150.20

w24 w24

Sucrose content (mg/g)

**

0.100.15 0.050.10 0.000.05

WT

0.25 0.20 0.15 0.10 0.05

W24 w24 w24 W24

2.50

**

2.00 1.50 1.00 0.50 0.00

WT

0.07

**

w24 w24

* *

0.06 0.05 0.04 0.03 0.02 0.01 0.00

0.00

WT WT

3.00

w24 w24

0.30

Glucose content (mg/g)

WT

Sucrose content (mg/g)

12

Fructose content (mg/g)

75

Amylose content (%)

Total starch content (%)

90

Crude protein content (mg/kernel)

a

WT

W24 w24

WT

W24 w24

b

8

w24-WT w24 - WT

∆ Molar (%)

6 4 2 0 6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52

-2 -4

Degree of Polymerization (DP)

Fig. 2. Important properties and amylopectin composition of wild type and the w24 grains. (a) Quality trait parameters and sugars of the wild type and w24 grains. Crude protein contents are expressed on the basis of the individual kernel. The other parameters except for amylose content are calculated based on dry weight. Values are expressed as means ± SDs (n = 3). The asterisks indicate statistical significance between the wild type and the mutant, as determined by a Student’s t-test (*P < 0.05; **P < 0.01). (b) Comparison of the amylopectin chain-length profiles of the wild type and the w24 mutant.

3. Results 3.1. The remarkable decline in starch accumulation in the w24 endosperm The w24 mutant did not exhibit any visibly abnormal phenotype at the vegetative stage of plant growth and development. After fertilization, the w24 mutant displayed a remarkably slower grainfilling rate compared with the wild type (Fig. 1a), which became apparent from approximately 15 days after flowering (DAF), and was maintained until seed maturation. Consistent with the low grain-filling rate, the mean weight of w24 seeds (g per 1000 grains) was 17.65 g, only approximately 72% of that of the wild-type (24.64 g). Moreover, the w24 mutant produced a shrunken and floury endosperm (Fig. 1d,f). Scanning electron microscopy analysis revealed that the w24 endosperm was filled with loosely packed, small, and round starch granules with large air spaces (Fig. 1h,j),

while the wild-type endosperm consisted of densely packed, polyhedral starch granules (Fig. 1g,i). Grain thickness of the w24 mutant was significantly lower than that of the wild type, but there were no statistically significant differences in grain width and grain length (Fig. 1b). The seeds of wild type had an average starch content of 82.71%, whereas those of the w24 mutant had a significantly decreased starch content of 64.45% (Fig. 2a). Correspondingly, the amylose content was also reduced in the w24 mutant. Structural changes in amylopectin were also noted, with short chains consisting of 6–10 degree of polymerization (DP) increasing and the intermediate chains with 11–36 DP decreasing (Fig. 2b). In addition, the w24 grain contained a significantly increased level of soluble sugars, specifically sucrose, glucose and fructose (Fig. 2a). Meanwhile, the crude protein content significantly decreased in the w24 mutant (Fig. 2a). Therefore, the mutation in the w24 mutant results in a pleiotropic defect in storage substance accumulation in the rice endosperm.

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Fig. 3. Abnormal compound granule formation in w24 seeds. (a–d) Semi-thin sections of wild-type (a,c) and w24 (b,d) endosperm at 9 DAF stained with I2 -KI. The center (a) and periphery (c) of wild-type endosperm cells. The center (b) and periphery (d) of w24 endosperm cells. Al, aleurone layers. White arrowheads indicate small, scattered starch granules in the cytosol in (b,d). The black arrowhead indicates the large empty space in w24 endosperm cells (b). Scale bars: 10 ␮m. (e,f) Semi-thin sections of wild-type (e) and w24 mutant (f) endosperm stained with Coomassie blue. Al, aleurone layers; Sl, subaleurone layers; En, starchy endosperm. Scale bars: 100 ␮m. (g,h) Transmission electron microscopy analysis of the endosperm of the wild type (g) and w24 (h). Scale bars: 5 ␮m. The white arrow indicates the unfilled protein storage vacuole. The black arrow indicates small and scattered starch granules.

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Fig. 4. Positional cloning and complementation of the w24 mutant. (a) Fine mapping of the W24 locus. The W24 locus was mapped to an 88.6-kb region by markers RM11383 and HY10 on chromosome 1 (Chr.1), which contained seven predicted genes. The number of recombinants is indicated below the map. (b) A single nucleotide substitution in OsAGPL2 led to Leu-155 replacement by Pro. NTP transferase, nucleotidyl transferase domain; LbH domain, C-terminal left-handed parallel beta helix (LbH) domain. (c,d) Complementation of w24 restored normal seed appearance. Complemented seeds (OsAGPL2-OX) became translucent (c), and the storage protein was restored to the normal level (d). In contrast, the endosperm of OsAGPL2 RNAi transgenic lines was shriveled (c). Arrows indicate glutelin precursors (d). (e) Immunoblot analysis of the OsAGPL2 protein in seeds of wild type and the w24 mutant, T1 vitreous grains of OsAGPL2-OX lines and shrunken grains of OsAGPL2 RNAi lines. AntiHSP82 antibodies were used as a loading control. The amount of protein loaded in (d) and (e) referred to grain number.

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3.2. The abnormal structures of compound starch granules and protein bodies in the w24 endosperm cells The structure of compound starch granules was observed using semi-thin sections prepared from developing endosperm at 9 DAF. Amyloplasts of wild-type endosperm contained compound granules (Fig. 3a), consisting of several dozen polyhedral, sharp-edged and easily separable granules. In contrast, the w24 endosperm contained a large cluster of small grains of amyloplasts (Fig. 3b). The abnormal amyloplasts were also observed in the peripheral endosperm cells (Fig. 3d). Moreover, compared to wild type, large empty spaces were often observed in the w24 endosperm cells (Fig. 3b, black arrowhead). Transmission electron microscopy analysis also indicated that small starch granules were scattered in the cytosol of the w24 endosperm cells, consistent with the result described above (Fig. 3h, black arrow). Coomassie blue staining of semi-thin sections showed that the number of protein bodies (PBs) was remarkably decreased in the subaleurone layer of the w24 endosperm (Fig. 3f), where the storage proteins were most abundant (Fig. 3e). SDS-PAGE analysis also revealed that there was an alteration of protein accumulation in the w24 mutant. In the w24 grains, reduced accumulation of glutelin, the major storage protein, was observed (Fig. 4d). Transmission electron microscopy images also showed that the glutelin-containing PBII in the w24 endosperm cells exhibited lower glutelin filling compared to those of wild type (Fig. 3g,h). In addition, some unfilled protein storage vacuoles were present in the w24 endosperm (Fig. 3h, white arrow). Taken together, these results suggested that the w24 mutation changed the production of storage substances in the rice endosperm.

3.3. Map-based cloning of the gene responsible for the w24 mutation For genetic analysis, w24 was crossed reciprocally with Koshihikari and the F2 population was analyzed for seed phenotype. Normal to shrunken seeds segregated at a ratio of approximately 3:1 (wild-type:mutant, n = 899, ␹2 = 0.964 < ␹2 0.05,1 = 3.84), indicating a single recessive inheritance. To genetically map the mutation, we developed an F2 population derived from a cross between w24 (japonica) and Nanjing 11 (indica). A total of 180 individuals showing the w24 phenotype were chosen and the mutated locus was first mapped to the long arm of chromosome 1 between the SSR markers RM5 and RM246. SSR and InDel analysis further indicated that the w24 locus was located within an 88.6-kb region, which includes seven open reading frames (ORFs) (Fig. 4a). Sequence analysis revealed a single nucleotide substitution of T-to-C in the fourth exon of Os01g0633100, which encodes a large subunit of rice AGPase, OsAGPL2 (Fig. 4b). This point mutation caused Leu-155 replacement by Pro. To verify that this locus was responsible for the w24 mutation, a vector bearing the OsAGPL2 coding region driven by the ubiquitin promoter was introduced into the w24 mutant to complement the phenotype. Among six positive OsAGPL2 transgenic lines, four were restored to vitreous seeds (Fig. 4c). The storage protein composition also reverted to wild type in these lines (Fig. 4d). In contrast, RNAi knockdown of OsAGPL2 in wild-type plants caused a shrunken phenotype (Fig. 4c). Western blot analysis verified the enhanced or decreased expression levels of OsAGPL2 in these transgenic lines (Fig. 4e). Therefore, OsAGPL2 indeed represents the gene responsible for the w24 mutation.

3.4. Effects of shrunken mutation on the expression of AGPase genes and the activities of major starch synthesis enzymes in the w24 endosperm To test whether the missense mutation in the OsAGPL2 gene has a severe effect on AGPase activity and, in turn, starch biosynthesis in the w24 mutant, we measured the expression levels and the catalytic activities of AGPase from the developing endosperms of the w24 mutant and the wild type. In the developing endosperm of the wild type, the transcript level of OsAGPL2 peaked at 5 DAF and remaining substantially high during the mid to late filling stages (Fig. 5a). In the w24 endosperm, the expression of OsAGPL2 was significantly higher than that of the wild type from 10 to 20 DAF and peak expression occurred at 15 DAF (Fig. 5a). Immunoblot analysis further confirmed the enhanced expression of OsAGPL2 in the w24 endosperm (Fig. 5b). However, the w24 endosperm retained only about 54% of the catalytic activity of the wild type endosperm AGPase (Fig. 5c), indicating that the Leu-155 residue of OsAGPL2 may play an important role in AGPase activity of rice endosperm. In addition, Giroux et al. found that mutation of either Sh2 or Bt2 gene could lead to an enhanced expression of the other gene [51]. Our Western blot analysis also verified that the OsAGPS2b protein, the counterpart of OsAGPL2 for the cytosolic AGPase, increased in the w24 endosperm (Fig. 5b), indicating the coordinated expression of OsAGPL2 and OsAGPS2b in rice endosperm. Moreover, real-time quantitative RT-PCR indicated that expression levels of the whole AGPase family were significantly increased in the mutant endosperm (Fig. 5d). Nevertheless, in the w24 leaves, the transcript levels of OsAGPS1 and OsAGPL1, but not OsAGPL3 and OsAGPS2b, the major isoforms of AGPase in the leaf, were dramatically increased (Fig. 5d), in agreement with the findings of Ohdan et al. in the OsAGPS2b null mutant EM22 [26]. To examine possible pleiotropic effects of the OsAGPL2 mutation on other starch synthesis enzymes, zymogram analyses were performed on the w24 mutant and the wild type. No significant differences in activities of isoamylases (ISA), pullulanase (Pul), Pho2, SSI and SSIIIa, or BE isoforms (BEI, BEIIa and BEIIb) were found between the w24 mutant and the wild type. But the activity of Pho1 appeared to be higher in the mutant than in the wild type. Real-time quantitative RT-PCR also showed the expression of Pho1 was increased about 1.9-fold in the mutant (data not shown). The result of Western blot confirmed the elevated expression of Pho1 in the w24 mutant (Fig. 5b). In addition, as sucrose synthase (SuSy) can provide an alternative source of ADPglucose [52], the activity of SuSy was determined in the w24 mutant and the wild type. A significantly increased activity of SuSy was observed in the w24 endosperm (Fig. 5c). 3.5. Interaction tests of AGPase subunits In yeast, a direct interaction between SH2 and BT2 has been shown [34]. Herein we also performed a yeast two-hybrid experiment to test whether OsAGPS2b and OsAGPL2 interact with each other. OsAGPS2b and OsAGPL2 were separately fused to both the activation domain (AD) and DNA-binding domain (BD) of the yeast transcriptional activator GAL4 (Table 1). As shown in Fig. 7a, positive interactions were observed in the reciprocal set of OsAGPS2b and OsAGPL2. Otherwise, in maize, SH2 or BT2 subunits show no self-binding in the yeast two-hybrid system [34]. Hence the OsAGPS2b or OsAGPL2 protein fused to both AD and BD was directly used to test the self-binding activity. No interaction was noted for the OsAGPS2b subunit whereas self-binding was evident for the OsAGPL2 subunit (Table 1, Fig. 7a). Terminal truncations of OsAGPL2 and OsAGPS2b were generated to determine whether sequences specific to either the C- or

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Fig. 6. Zymogram analysis of starch synthesizing enzymes. Native-PAGE/activity staining analysis was performed using enzymes extracted from the developing endosperms of wild type and the w24 mutant. (a) Activity bands for isoamylases (ISA), pullulanase (Pul) and starch phosphorylase 1 (Pho1). (b) Activity bands for Pho1 and Pho2. (c) Activity bands for starch synthases I and IIIa (SSI and SSIIIa). (d) Activity bands for branching enzyme (BE) isozymes, BEI, BEIIa and BEIIb.

Fig. 5. Expression, Western and activity analysis of AGPase in wild type and the w24 mutant. (a) Expression levels of OsAGPL2 during development of wild type and the w24 mutant seeds. Developing endosperms were harvested at 5, 10, 15, 20 and 25 DAF. The value of Actin I mRNA was used as an internal control for data normalization. Values are means ± SDs (n = 3). (b,c) Western blotting, AGPase and sucrose synthase activity analysis of wild type and the w24 mutant endosperm at 12 DAF. The OsAGPL2, OsAGPS2b and Pho1 gene products are 57, 53 and 106 kDa proteins, respectively. The amount of protein loaded referred to the fresh weight of endosperm. (d) Expression profiles of rice AGPase genes in developing endosperm and leaf of wild type and the w24 mutant at 12 DAF.

N-terminus are important for interaction. Two reciprocal sets of positive interactions were observed, specifically the C-terminus of OsAGPS2b with the full-length OsAGPL2 subunit, and the Cterminus of OsAGPL2 with the full-length OsAGPS2b (Table 1, Fig. 7a). The N-terminus of OsAGPL2 or OsAGPS2b displayed a positive but unidirectional interaction with the full-length counterpart subunit (Table 1, Fig. 7a). For self-binding of OsAGPL2, positive interactions were detected in the reciprocal set of the N-terminus of OsAGPL2 and the full-length OsAGPL2. The C-terminus of OsAGPL2 exhibited a positive but unidirectional interaction with the fulllength OsAGPL2 (Table 1, Fig. 7a). The L155P mutation in OsAGPL2 did not affect its interaction with OsAGPS2b as well as self-binding (Supplementary Fig.S1). For OsAGPS1 and OsAGPL1, a strong interaction occurred only with one of the two combinations: OsAGPS1 fused to BD and OsAGPL1 fused to AD. Another combination exhibited a weak interaction (Fig. 8a). Accordingly, terminal truncations of OsAGPS1 and OsAGPL1 were fused to BD and AD, respectively, to map the region

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Fig. 7. Analysis of OsAGPL2 and OsAGPS2b interactions. (a) Yeast two-hybrid analysis of OsAGPL2/S2b and their truncation interactions. (b) BiFC assay showing that L2-eYC and S2b-eYN interact to form a functional eYFP in tobacco leaf cytosol, as was the case with L2-eYC and L2-eYN. Scale bars: 20 ␮m. DIC, Differential Interference Contrast. Table 1 In vivo protein–protein interaction test results. Interacting polypeptides

A

B

C

L2/S2b L2/L2 S2b/S2b L2/S2b-N L2/S2b-C S2b/L2-N S2b/L2-C L2/L2-N L2/L2-C L1/S1 L1/L1 S1/S1 L1/S1-N L1/S1-C L1-N/S1 L1-C/S1

+ + – – + – + + – + – – – + + +

+ + – + + + + + + + – – nd nd nd nd

– – – – – – – – – – – – – – – –

“A” indicates that the first listed member of the binding pair was fused to GAL4AD, “B” indicates the second member was fused to GAL4-AD, and “C” indicates the interaction between either member and negative control plasmids lacking any rice sequence. nd, data for the indicated plasmid pair were not determined.

required for their interaction. As shown in Table 1 and Fig. 8a, the N-terminus of OsAGPS1 failed to interact with OsAGPL1 but the C-terminus of OsAGPS1 displayed a specific interaction with

OsAGPL1. In contrast, both the N- and C-terminuses of OsAGPL1 could bind to OsAGPS1. In addition, no self-binding was observed for both OsAGPS1 and OsAGPL1 (Table 1, Fig. 8a). A direct interaction was also observed between OsAGPS1 and OsAGPL2 (data not shown), which might result from the fact that the SSs of plant AGPases are much more conserved than the LSs [53]. In addition, no interactions were detected among OsAGPL2/OsAGPS2b and several cytosolic proteins involved in starch biosynthesis, including UDP-glucose pyrophosphorylase (Os09g0553200), phosphofructokinase ␤ subunit (Os06g0247500), and a putative plastidial ADP-glucose transporter (Os02g0202400), in the yeast two-hybrid system (data not shown). Finally, LSs (OsAGPL1 and OsAGPL2) and SSs (OsAGPS1 and OsAGPS2b) were fused to the enhanced yellow fluorescent protein (eYFP) C terminus (eYC) and N terminus (eYN), respectively, and were assayed for BiFC. eYFP fluorescence was reconstituted when the L2-eYC and S2b-eYN proteins were co-expressed in the leaf cells (Fig. 7b), verifying that they physically interacted with each other in the cytoplasm. Moreover, our BiFC assays demonstrated the interaction between OsAGPS1 and OsAGPL1 in the plastids (Fig. 8b). In addition, the OsAGPL2 protein was fused to both eYC and eYN, and the interaction between the OsAGPL2 subunits was evidenced by the detected eYFP fluorescence (Fig. 7b).

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Fig. 8. Interactions between OsAGPL1 and OsAGPS1. (a) Yeast two-hybrid analysis of OsAGPL1/S1 and their truncation interactions. (b) BiFC assay showing that L1-eYC and S1-eYN interact to form a functional eYFP in tobacco leaf plastids. Scale bars: 20 ␮m.

3.6. The mutation in OsAGPL2 affected the synthesis of transitory starch and soluble sugars in rice leaves Sink strength has been proposed to influence photosynthetic output [54]. That is, photosynthetic efficiency may be induced by increased sink strength, leading to greater biomass production [55]. However, though the starch synthesis was inhibited in the w24 seeds, no change was noted in photosynthesis rate and transpiration rate of the w24 plants (data not shown). Starch and soluble sugar levels were also determined in leaves of w24 and wild type plants (Fig. 9). The starch content in the w24 leaves was found to be severely reduced when compared with wild type leaves at 15 DAF (Fig. 9a). Moreover, the difference in the soluble sugar content of the leaves became apparent from 15 DAF, the same time when the grain-filling rate of the w24 seeds was distinct from that of the wild type seed (Fig. 1a). Therefore, the mutation in OsAGPL2 also affected the synthesis of transitory starch and soluble sugars in rice leaves. 4. Discussion 4.1. The aberrant storage substances in the w24 endosperm In this paper, we report the isolation and characterization of a rice shrunken mutant w24 that has a floury endosperm. The gene responsible for the w24 mutation encodes OsAGPL2, a large subunit of the cytosolic isoform of rice endosperm AGPase. The w24 mutant

had a T to C substitution in the fourth exon of OsAGPL2, resulting in Leu-155 (L155) replacement by Pro. This residue is just located in the nucleotidyl transferase domain (Fig. 4b). Sequence analysis showed that the L155 residue is conserved in the LS of diverse plant species (Supplementary Fig.S2), in addition to OsAGPL1. The missense (shr1a) mutants reported by Tuncel et al. contain mutations in the third or fourth exon of OsAGPL2, resulting in the replacement of T139I or A171V in the nucleotidyl transferase domain. The T139 and A171 residues, which are spatially very close to each other, are both part of loop structures and located close to the putative substrate and effector binding sites [33]. Interestingly, the L155 residue is just located in the middle of the T139 and A171 residues in the primary sequence of OsAGPL2. Our homology modeling of OsAGPL2 showed that L155 is also a part of loop structures near the putative substrate and effector binding sites (data not shown), as is the case with T139 and A171. Of the two residues, L155 is spatially closer to T139. Tuncel et al. suggested that both T139I and A171 V substitutions result in bulkier side chain that might cause a disorder of the local structure that alters the substrate and/or effector binding to OsAGPL2 [33]. In the same way, as the proline residue has exceptional conformational rigidity, the L155P substitution in the w24 mutant might have similar effects on the local loop structures as T139I and A171V substitutions. Moreover, the w24 seeds retained a relatively high AGPase activity (about 54% of that of the wild type), similar to the shr1a seeds (about 60%). We speculated that due to the high similarity of the T139, L155 and A171 residues

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a w24

WT

100 mg starch/g DW

* 80 60

4.2. Effects of OsAGPL2 mutation on starch synthesis pathway in the endosperm

40

* *

20 0 5

10

15

20

25

DAF

b

mg soluble sugar/g DW

w24

WT

30

* 25

* *

20 15

** 10 5 0 5

10

15

20

the endosperm [60]. Therefore, a complex and interlinked control might be present between the amount of major storage proteins and carbohydrate composition in storage sinks of higher plants [27]. In fact, RISBZ1/OsbZIP58, an OsbZIP transcription factor, has been proven to function as a key regulator of both storage protein and starch biosynthesis in rice endosperm [61–63].

25

DAF

Fig. 9. Starch and soluble sugar contents of wild type and the w24 mutant. Starch (a) and soluble sugar contents (b) in leaves.

in the spatial location and conformation, the L155 P substitution in the w24 mutant might impair the catalytic and regulatory properties of AGPase just like T139I and A171V substitutions in the shr1a mutants [33]. As the major AGPase activity responsible for starch accumulation in cereal endosperms is believed to be cytosolic [19], the missense mutation in cytosolic OsAGPL2 might account for significant decreases in both starch content and average weight of the w24 seeds. Moreover, large empty spaces were frequently observed in the w24 endosperm cells (Fig. 3b), consistent with the markedly slower grain-filling rate throughout the w24 endosperm development (Fig. 1a). Meanwhile, the decrease in starch content of the w24 endosperm was partly compensated for by an increase in soluble sugars, as was the case with many cytosolic AGPase mutants previously reported [31,56,57]. On the other hand, Kawagoe et al. suggested that the reduction of cytosolic AGPase activity in the endosperm of OsAGPS2b null mutant EM22 do not inhibit starch granule initiation but severely restrained the subsequent enlargement of granules [31]. Small grains of amyloplasts were also found in the w24 endosperm (Fig. 3b, d), indicating that AGPase may play a unique role in compound starch granule formation in rice endosperm. PBII with lower glutelin filling and unfilled protein storage vacuoles were found in the w24 endosperm at 9 DAF (Fig. 3h). SDSPAGE analysis also showed the reduction in the amount of glutelin in the w24 seeds (Fig. 4d). Müller-Röber et al. reported that the inhibition of starch biosynthesis resulted in a significant reduction in the expression levels of the major tuber proteins in potato [27]. In bt2 or sh2, a strongly reduced synthesis of the major storage proteins was observed in the endosperm [58,59]. Kawagoe et al. reported that the storage proteins in the EM22e ndosperm were undetectable at 8 DAF and remained in low levels at 21 DAF and maturity [31]. Besides, in the flo2 mutant, the production of storage starch and storage proteins was simultaneously decreased in

In this study, the missense mutation in OsAGPL2 was shown to lead to enhanced expressions of the OsAGPL2 gene itself and other AGPase genes in the w24 endosperm (Fig. 5a, d). The pronounced elevation in the OsAGPL2 and OsAGPS2b transcripts was associated with an increase in the cognate protein (Fig. 5b), indicating the possible existence of a positive feedback regulation of OsAGPL2 and OsAGPS2b in the w24 mutant. In the w24 endosperm, the reduction in AGPase activity caused a decrease in starch, which was accompanied by the accumulation of soluble sugars (Fig. 2a). Meanwhile, plant AGPase genes are believed to be induced by sugars [4–6,51,64–66]. High levels of sugars could enhance the expression of SH2 and BT2 [51]. Moreover, Akihiro et al. found that the expression levels of AGPase and starch contents in rice cultured cells are cooperatively regulated by sucrose and ABA [7]. Therefore, the dramatically enhanced expression of the AGPase genes might be related with the high levels of soluble sugars in the w24 endosperm. In addition, a strong correlation was observed between sugar levels and the redox-activation state of AGPase in potato tubers as well as pea, potato and Arabidopsis leaves [9,67,68]. The detached potato tuber discs fed with sucrose or glucose has increased levels of SS monomerization and thus elevated AGPase activity [67]. Though the cytosolic isoform of the cereal endosperm AGPase lacks the redox-regulatory Cys12 in its SS, it is proposed that the cytosolic isoform is also subject to redox control through modification of LS [69]. Therefore, further studies are necessary to consider whether sugar accumulation in the endosperm of cytosolic AGPase mutants might affect the redox status and then activity of AGPase. On the other hand, sucrose, or a metabolite of sucrose, has been suggested to be directly involved in inducing the expression of BE and Pho in detached potato leaves [70]. Likewise, the barley SUSIBA2 can bind to the sugar-responsive elements (SURE) in the iso1and sbeIIb promoters as a transcription activator [71]. Bioinformatics analysis also identifies putative SURE-elements in promoter regions of major enzymes controlling the starch biosynthesis in rice endosperm [72]. High expression of SUSIBA2 in rice has been shown to confer a shift of carbon flux and yield highstarch rice [73]. In this study, under a high sugar concentration in the w24 endosperm, no significant changes were observed in the activities of many major enzymes of starch biosynthesis except for Pho1 (Fig. 6). Both the transcript and protein levels of Pho1 were increased in the endosperm of w24 (Fig. 5b). Pho1 can use G-1-P as a substrate to add a glucose unit to the nonreducing end of the ␣-glucan chain with the release of Pi. Satoh et al. reported that the pho1 mutant exhibited more severe reduction in the starch level than the shr2 mutant harboring a defective OsAGPS2, indicating a crucial role for Pho1 activity for maximum starch biosynthesis [50]. In addition, Pho1 has a higher transcript level during the early stages of seed development than AGPase [26]. Due to an essential role of Pho1 in starch biosynthesis in rice endosperm, the elevated expression of Pho1 was assumed to enhance starch synthesis in the w24 endosperm. Furthermore, inactivation of AGPase leads to an increase of the level of its substrate G-1-P in both the cytosol and plastid in detached potato tubers [67]. Therefore, the concentration of G-1-P in the w24 endosperm cells might increase due to the declined AGPase activity. Likewise, based on the very high

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Km for G-1-P and the estimated low concentrations of this substrate in the plastid, Pho1 is suggested to have suboptimal activity in starch synthesis [74]. Hence an increase of G-1-P would favor the biosynthetic reaction carried out by Pho1 in the w24 endosperm. It is generally believed that the activity of plastidial AGPase and SuSy may partly account for the residual starch content in the shrunken mutant endosperm [33,52]. In this study, we proposed that Pho1 might also play an important role in the synthesis of residual starch in the w24 endosperm. In addition, SuSy is believed to be involved in the direct conversion of sucrose into ADPglucose in both leaf and heterotrophic organs [75,76]. In the w24 endosperm, the activity of SuSy was significantly increased, possibly implying a positive feedback regulation of SuSy in the mutant. However, the enhanced activity of SuSy was still unable to rescue the starch-deficient phenotype, thus unlikely to be a major determinant of ADPglucose formation in rice endosperm [33]. Besides, the starch and the soluble sugar contents were affected in the leaves of the w24 plants (Fig. 9). However, the expression levels of OsAGPL3 and OsAGPS2a did not dramatically change in the w24 leaves (Fig. 5d). Ohdan et al. also suggested that OsAGPL3 and OsAGPS2a transcript abundances were comparable in the leaves of the OsAGPS2b null mutant EM22 and the wild type [26]. Therefore, the impact of the OsAGPL2 mutation on the synthesis of transitory starch might need to be further investigated.

4.3. The interactions among AGPase subunits The recombinant SSs of the potato tuber, barley endosperm and Arabidopsis leaf AGPase are capable of forming a catalytically active homotetrameric enzyme [77–79], whereas the LS possesses catalytic and regulatory properties only when assembled with SS [80]. Therefore, in the absence of the LS, the AGPase mutant is highly likely to express a SS homotetramer in the cytosol [33]. However, the yeast two-hybrid results presented here provided no evidence for OsAGPS2b:OsAGPS2b homodimers. Likewise, our data showed that OsAGPL2 could exist as dimers. In addition, Greene and Hannah found that BT2 exists as a monomer in the absence of SH2, whereas the SH2 protein, in the absence of BT2, is found in a complex of 100 kD, consistent with a dimmer molecular mass [34]. This finding is in agreement with our results. Meanwhile, the primary sequence of OsAGPL2 shares higher identity with that of maize SH2 compared to wheat and barley [33]. The allosteric regulatory properties of the rice endosperm AGPases also aligns closer to the maize enzyme. Therefore, we proposed that SSs in maize and rice do not interact to form higher molecular mass aggregates in the absence of LSs, though SSs are quite evolutionarily conserved between different plant species [53]. Subunit interaction is believed to be critical for the overall stability of AGPase as well as the individual subunits [34]. The starch-deficient Arabidopsis mutant adg1, represents a mutation in the SS but it contains neither the LS nor SS proteins, suggesting that the presence of functional SSs is required for LS stability [35]. Similarly, although the SH2 protein was detected in the bt2 endosperm early in development, it greatly reduced later in development, indicating an increased lability when the formation of a SH2/BT2 polymer did not occur [28]. In addition, the level of the OsAGPL2 protein in the endosperm of the OsAGPS2 mutant osagps21 was also found to be reduced [2]. Likewise, there is also evidence that SSs are unstable in the absence of the LSs. In barley leaves, no SS protein was detected in the absence of LS [29]. In sh2 mutants, the turnover rate of the BT2 protein was identical with the SH2 protein in the bt2 mutants [28]. In the absence of OsAGPL1, the amount of OsAGPS1 protein is reduced in embryos and absent from culms [30]. In this study, the OsAGPS2b protein remarkably increased or

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reduced in OsAGPL2-OX lines or OsAGPL2 RNAi lines (Supplementary Fig.S3), in good coordination with OsAGPL2 in rice endosperm. In current study, direct interactions of OsAGPL2:OsAGPS2b and OsAGPL1:OsAGPS1 were verified in both the yeast two-hybrid and BiFC systems, supporting the model proposed by Ohdan et al. and Lee et al. [2,26]. Laughlin et al. found that the 19 amino acids located at the carboxy end of either LS or SS of potato AGPase were essential for proper folding and/or assembly of the subunits [81]. Our data also showed that protein motifs located in the C-terminal regions of OsAGPS2b and OsAGPL2 might be more essential for efficient interaction than their N-terminal regions (Table 1, Fig. 7a). The C-terminal region of AGPase contains the LbH domain which is involved in cooperative allosteric regulation and oligomerization while the catalytic domain is located in the N-terminus (http://www.ncbi.nlm.nih.gov/Structure, Fig. 7a). Similarly, the C-terminus of OsAGPS1 played an essential role in mediating the interaction between OsAGPL1 and OsAGPS1. On the other hand, both termini of OsAGPL1 displayed specific interactions with OsAGPS1, implying that each terminus of OsAGPL1 might initiate efficient interaction. In maize, the individual subunits of cytosolic AGPase, BT2 or SH2, do not interact when expressed in yeast. Accordingly, a heterodimer intermediate of BT2 and SH2 might be involved in the early steps in the polymerization of AGPase [34]. In this study, the subunits of plastidial AGPase in the rice endosperm, OsAGPS1 and OsAGPL1, also exhibited no self-binding (Table 1). We speculated that the assembly of OsAGPS1/OsAGPL1 heterotetramer may fit well to this model [34]. On the other hand, Wang et al. proposed that the AGPase assembly process may be initiated by homodimer formation of SSs, which interacts further with LSs or SSs [35]. Though there is no evidence for SS homodimers in rice and maize endosperm, it is still possible that OsAGPL2 may form homodimer (Fig. 7a) and subsequently interact with OsAGPS2b. Therefore, which of the two forms, OsAGPL2 homodimer or OsAGPL2/OsAGPS2b heterodimer, might possibly occur in the initial step of heterotetramer assembly? ISA, another major enzyme for starch biosynthesis, might provide useful reference for this problem. ISA can form two oligomers, the ISA1 homooligomer and the ISA1-ISA2 hetero-oligomer. Suppression of ISA2 gene expression caused the endosperm to have only the ISA1 homo-oligomer, while ISA2 overexpression led to endosperm having only the ISA1-ISA2 hetero-oligomer. Therefore, the relative amounts of the ISA1 and ISA2 proteins determine the ratio of the homo-oligomer to the hetero-oligomer [82]. Hence, we presumed that the formation of OsAGPL2 homodimer or OsAGPL2/OsAGPS2b heterodimer might also depend on the relative abundance of OsAGPS2b and OsAGPL2 in rice endosperm, as was the case with rice ISA oligomers. In summary, this work verifies the pivotal role of OsAGPL2 in starch synthesis and compound granule formation in the endosperm. The yeast two-hybrid and BiFC data reported here provide direct evidence for OsAGPL1:OsAGPS1 and OsAGPS2b:OsAGPL2 interactions. Still more in-depth studies are necessary to elucidate the precise mechanism by which these subunits polymerize into the native heterotetrameric structure.

Acknowledgments This work was financially supported by a project (No. 2014ZX08001-006) from Ministry of Agriculture of China for Transgenic Research, the National Natural Science Foundation of China (31501280), Jiangsu Science and Technology Development Program (BE2015363), Key Laboratory of Biology, Genetics and Breeding of Japonica Rice in Mid-lower Yangtze River, Ministry of Agriculture, P.R.China.

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