- Email: [email protected]
Pineapple Genome: A Reference for Monocots and CAM Photosynthesis
TIGS 1307 No. of Pages 7
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have originated in lowland South America, perhaps in the Southwest Brazil Paraná– Paraguay basin [1]. It performs obligate CAM photosynthesis that fixes CO2 at night, indicating that it originated in arid or semi-arid regions. It is perennial, living as long as 50 years. Pineapple is propagated vegetatively and its main propagule, the crown, can endure months without Ray Ming,1,2,* 2 water. Although pineapple can survive in Ching Man Wai, and 3 areas with a total annual rainfall of as little Romain Guyot as 500 mm (a condition likely to represent that in its region of origin), it can also Pineapple occupies an important tolerate annual levels as high as phylogenetic position and its refer5500 mm [2]. It is now grown in 85 ence genome expedites genomic countries.
Pineapple Genome: A Reference for Monocots and CAM Photosynthesis
research within the family Bromeliaceae and more widely among monocots. One such research focus is the evolution of crassulacean acid metabolism (CAM) photosynthesis. Acquiring circadian clock cis-regulatory elements in CAM-related genes might be a critical step in the evolution of this form of photosynthesis. Followup studies will clarify the processes and evolutionary forces leading to the multiple independent origins of CAM photosynthesis within the family Bromeliaceae and in over 400 genera across 36 families. A Genome Domesticated through Somatic Mutations
The discovery of pineapple [Ananas comosus (L.) Merr.] by Christopher Columbus during his second voyage to the New World is a key event in the subsequent distribution of pineapple across tropical and subtropical regions worldwide. The fact that Columbus encountered pineapple in a Carib village rather than in its native lowland South America shows that pineapple had been domesticated by Tupi-Guarani Indians long before Columbus arrived and was already distributed throughout the Americas and Caribbean islands [1]. Pineapple is a member of the Bromeliaceae family and is thought to
rearrangements after two ancient wholegenome duplication (WGD) events shared by all monocots, likely due to its vegetative propagation and, thus, reduced meiosis. Through structural comparisons of the pineapple genome to itself, homologous intragenomic blocks and genes were identified to reconstruct the ancient seven chromosomes before the two rounds of WGD. After the most ancient t WGD, the seven chromosomes doubled to 14. Two chromosomal fusion events reduced the chromosome number to 12 before the second s WGD. After the s WGD, the 12 chromosomes doubled to 24, two chromosomal fusion events reduced the chromosome number to 22, and two Pineapple fruit is the primary product of the chromosome fission events produced pineapple plant. The edible part of the fruit three additional chromosomes, resulting is developed from expended carpels, the in the current 25 chromosomes. bases of sepals, and bracts of more than 200 fused flowers, and the surface of the The well-conserved pineapple karyotype fruit is covered by sepals, bracts, and the makes its genome a reference for comapices of ovaries [3]. Cultivated pineapple parative analysis among monocot is self-incompatible with the gametophytic genomes. Its pivotal phylogenetic position control of pollen phenotype, while all wild at the base of the order Poales resulted in relatives are self-fertile [4]. Seedlessness is revised dating of the pan-cereal genome a desirable trait because it increases the duplication event r to 95–115 million years edible portion of the fruit. It has been ago (MYA); this duplication event is lacking hypothesized that self-incompatibility was in pineapple [6]. The earlier s WGD was selected during the domestication pro- revised to 100–120 MYA. The application cess, and seedless mutants could be mul- of the pineapple genome as a reference tiplied quickly through vegetative for monocots was also demonstrated by propagation [5]. Regardless of the origin showing the sorghum lineage-specific of self-incompatibility, most pineapple cul- translocation of gene families of type I tivars were selected as a result of somatic MADS-box genes and GDSL-like lipase/ mutation and the remainder from the acyl hydrolase genes using pineapple hybridization of distantly related cultivars genome as a reference. or wild relatives following the breakdown of self-incompatibility. Both methods Circadian Regulation of CAM resulted in highly heterozygous genomes Photosynthesis that are challenging to assemble. Photosynthesis is the process of converting light energy to chemical energy through conversion of carbon dioxide A Reference Genome for (CO2) into organic compound in plants, Monocots Recently, an international consortium algae, and certain bacteria. There are overcame the challenges of sequencing three photosynthetic pathways in terresa heterozygous genome and assembled trial plants: C3, C4, and CAM. The first a reference genome for pineapple [6]. The photosynthetic product is a three-carbon consortium found that the chromosome molecule in C3 plants and a four-carbon karyotype of pineapple is surprisingly well molecule in C4 and CAM plants. The conserved, with limited chromosomal difference between C4 and CAM
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photosynthesis is that the separation of the auxiliary CO2 from the Calvin cycle occurs spatially in two cell types (mesophyll and bundle sheath) in C4 plants but temporally in the same mesophyll cell in CAM plants. C3 photosynthesis is the ancestral pathway and occurs in all taxonomic plant groups. It was proposed that C4 and CAM photosynthesis evolved through reorganization of the gene network in C3 plants [7]. The hallmark of CAM photosynthesis is the fixation of CO2 at night, whereas C3 and C4 plants fix CO2 during the day. The circadian rhythm of CO2 fixation was observed and has been extensively studied in CAM plants, and this pattern persists in detached leaves or under constant environmental conditions [8]. Pineapple is the most important crop exhibiting CAM photosynthesis. CAM plants consume one-sixth the amount of water used by C3 plants and a quarter of the amount of water used by C4 plants [9], and this high water-use efficiency is the result of stomatal closing during the day and opening at night to enable such plants to survive in hot and arid regions. However, how CAM photosynthesis evolved independently in 36 families has been a longstanding question [9]. Candidate genes for CAM photosynthesis were identified based on genes homologous to those encoding enzymes of the C3 and C4 pathways. Analyzing contrasting expression patterns in photosynthetic green[4_TD$IF] leaf tip and nonphotosynthetic white base leaf tissues separated the gene family members involved in carbon fixation from those involved in other processes [6]. Genome analysis revealed that WGD and tandem duplication did not contribute new genes in CAM photosynthesis, at least in the pineapple lineage [6]. Pineapple genes involved in carbon fixation have a diel expression pattern in photosynthetic tissues with low or no expression in nonphotosynthetic tissue [6]. These CAMrelated genes are enriched in known circadian clock cis-regulatory elements,
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including the morning element (CCACAC), evening element (AAAATATC), G-box (CACGTG), and Circadian Clock-Associated 1 (CCA1) (AAAAATCT). Carbonic anhydrase (CA) catalyzes the conversion of CO2 into bicarbonate and is the first step in CO2 fixation in CAM photosynthesis. Among the three CA families (/, b, and g) in pineapple, only bCA is expressed at night and during the early morning in photosynthetic tissue, indicating that bCA catalyzes carbon fixation. Promoters of all three bCA genes contain CCA1-binding sites (AAAAATCT). Among all the bCA genes in C3 rice, and C4 maize and sorghum, only one bCA gene in sorghum contains a CCA1-binding site at its promoter and this gene has no known photosynthetic function [10]. Acquiring these circadian clock cis-elements in CAMrelated genes might be critical for the evolution of CAM photosynthesis.
C3 and C4 plants (Table 1). Circadian clock cis-regulatory elements were present in all nine genes of all species with variation in their frequency. These nine genes carry out similar functions in C3, C4, and CAM species, and are under circadian regulation with different diel patterns. These cis-elements are six or eight base pairs (bp), and appear in the genome randomly at expected frequencies of 1/ 4096 bp and 1/65536 bp, respectively The expected and actual frequencies of these elements was examined in 2-kb promoter regions of all genes and photosynthetic genes in two CAM, two C3 and one C4 species (Table 2A,B). For the 6-bp morning element and G-box, the morning element appeared at higher than expected frequencies, whereas the Gbox appeared at lower than expected frequencies across all five species. The frequency of the morning element was about twice that of the G-box element. Similar discrepancies were also observed for the 8-bp evening element and the CCA1binding site, with a higher than expected frequency for the CCA1-binding site and a lower than expected frequency for evening elements across all five species. The frequency of the CCA1-binding site was about three times that of the evening element. These elements are not randomly distributed in the genome and selection has likely acted on them, causing the biased distribution frequency in promoter regions genome wide.
The second step is the conversion of phosphoenolpyruvate to oxaloacetate (OAA) by phosphoenolpyruvate carboxylase (PEPC) and the third step is the conversion of OAA to malate by the enzyme malate dehydrogenase (MDH). Genomic sequences of pineapple CA, PEPC, and MDH revealed the presence of CCA1 elements within their promoters (Table 1). PEPC becomes activated at night via phosphorylation by PEPC kinase [11]. The promoter region of PEPC kinase contains a G-box. The circadian rhythm of CAM photosynthesis is likely a concerted genome-wide adaptation with a selection of circadian clock cis-elements as a crucial Analysis of circadian clock cis-elements in if not essential component of this evolu- CAM-related genes revealed enrichment of tionary process. more than 10% of such cis-elements in CAM species of orchid for all four elements and in pineapple for three elements comThe Origin of Circadian Clock pared with the genome-wide frequencies Cis-Elements in CAM-Related of these elements in all genes (Table 2B). Genes of Pineapple The pineapple genome offered an oppor- Such enrichment was also observed for tunity to investigate the distribution and two elements in the C3 genera Arabidopsis frequency of these circadian clock cis-ele- and rice, and for one element in the C4 ments in CAM-related genes. Two-kb pro- genus Sorghum (Table 2B). As a control, moter regions of the nine most important the frequencies of these elements were genes in CAM photosynthesis were exam- analyzed in promoter regions of isoleucine ined in CAM species and their orthologs in and valine biosynthesis genes that are not
Arabidopsis
beta-CA
PEPC
MDH
Orchid
Pineapple
Rice
Sorghum
Gene ID
TF binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
AT1G23720
–
PEQU_27755
ME
Aco002732
CCA1, G-box
LOC_Os01g45274
ME
Sobic.002G230100
G-box, ME
AT1G58180
–
PEQU_37822
–
Aco006181
CCA1, ME
LOC_0s09g28910
–
Sobic.003G234200
G-box
AT1G70410
ME
PEQU_31387
–
Aco005402
CCA1
Sobic.003G234400
–
AT3G01500
ME
Sobic.003G234500
CCA1
AT4G33580
ME
Sobic.003G234600
–
AT5G14740
–
AT1G53310
G-box, CCA1
PEQU_07008
EE
Aco010025
EE, ME, CCA1, PBX
LOC_Os01g55350
G-box
Sobic.002G167000
G-box
AT2G42600
–
PEQU_14315
CCA1
Aco018093
ME, CCA1
LOC_Os02g14770
CCA1
Sobic.004G106900
ME
AT3G14940
ME, CCA1
Aco016429
G-box, PBX, CCA1
LOC_Os08g27840
CCA1
Sobic.007G106500
ME, G-box
AT1G68750
–
LOC_Os09g14670
–
Sobic.003G301800
ME
LOC_Os01g02050
ME, G-box, PBX
Sobic.010G160700
ME
LOC_Os01g11054
CCA1
Sobic.003G100600
ME
LOC_Os08g33720
ME, EE
Sobic.009G240700
CCA1
PEQU_05708
–
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AT1G53240
ME, CCA1
Aco006122
CCA1
AT3G15020
ME
PEQU_17024
EE, CCA1
Aco007734
CCA1
LOC_Os05g49880
ME
Sobic.003G238500
G-box
AT5G43330
ME, EE
PEQU_19417
–
Aco013935
ME
LOC_Os03g56280
ME, PBX, G-box
Sobic.001G219300
ME, G-box, CCA1
AT1G04410
–
PEQU_08514
ME
Aco002885
ME, G-box
LOC_Os01g46070
–
Sobic.007G137600
ME
AT3G47520
ME
PEQU_20254
PBX, CCA1
Aco004349
ME, G-box, PBX
LOC_Os01g61380
ME
Sobic.004G004500
PBX
AT4G17260
ME, PBX
PEQU_35914
G-box, PBX, CCA1
Aco014690
G-box, PBX
LOC_Os04g46560
–
Sobic.004G004400
ME
AT5G58330
ME, CCA1
Aco017525
CCA1
LOC_Os10g33800
ME, PBX, G-box
Sobic.006G170800
-
AT2G22780
EE
Aco017526
PBX
LOC_Os02g01510
G-box
Sobic.007G166300
ME, CCA1
AT5G09660
ME, CCA1
LOC_Os08g44810
CCA1
Aco017527
CCA1
Sobic.007G166200
ME, G-box
Aco017528
CCA1
Sobic.001G073900
EE
Aco019631
CCA1
Sobic.008G186200
ME, PBX, CCA1
Aco010232
ME, G-box
Aco004996
ME, PBX
Aco008626
PBX
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Table 1. Cis-Elements Annotated at Promoter Sequences of Selected Photosynthetic Genes across Five Speciesa
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Arabidopsis
PEPC kinase
PEPC-related kinase
NAD-ME
NADP-ME
PEPCK
PPDK
PPDK regulatory protein
a
Orchid
Pineapple
Rice
Sorghum
Gene ID
TF binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
AT1G08650
ME
PEQU_38595
ME
Aco010095
G-box
LOC_Os02g56310
EE, PBX, CCA1
Sobic.004G338000
G-box, PBX
AT3G04530
–
PEQU_29948
ME, G-box, CCA1
Aco013938
G-box
LOC_Os02g41580
ME, PBX, G-box
Sobic.006G148300
ME, EE
PEQU_12226
ME, G-box
Sobic.004G219900
ME, G-box
PEQU_15364
ME, CCA1 Sobic.010G016900
ME, G-box, PBX
AT1G12580
G-box
PEQU_06553
ME, G-box
Aco022525
EE, ME, G-box, CCA1
LOC_Os06g03682
ME, PBX, CCA1
AT1G12680
–
PEQU_09615
ME, G-box
Aco001261
EE, ME, PBX
LOC_Os09g29170
ME, G-box, CCA1
Sobic.002G231300
G-box, CCA1
Sobic.007G160600
ME
AT2G13560
EE, G-box
PEQU_05938
ME
Aco016569
–
LOC_Os07g31380
ME, PBX
Sobic.002G309400
CCA1
AT4G00570
G-box
PEQU_22911
ME, EE, G-box
Aco007622
G-box, PBX, CCA1
LOC_Os10g35960
CCA1
Sobic.001G201700
ME, CCA1
AT5G25880
G-box
PEQU_29209
EE, CCA1
Aco009967
G-box, PBX, ME
LOC_Os05g09440
ME, G-box
Sobic.001G201700
ME, CCA1
AT5G11670
CCA1
PEQU_01237
G-box, PBX
Aco005631
CCA1
LOC_Os01g09320
ME, EE
AT1G79750
ME
PEQU_08427
G-box
Aco005989
G-box
LOC_Os01g52500
G-box
AT2G19900
–
LOC_Os01g54030
–
AT4G37870
G-box, ME, CCA1
PEQU_14885
–
Aco017762
ME, EE, G-box, CCA1
LOC_Os03g15050
–
Sobic.001G432800
ME
AT5G65690
ME, EE
PEQU_25034
ME, EE, CCA1
LOC_Os10g13700
ME, G-box, PBX
LOC_Os04g43710
ME, G-box
AT4G15530
ME, CCA1
PEQU_01453
G-box, PBX
LOC_Os03g31750
ME
Sobic.009G132900
-
LOC_Os05g33570
-
Sobic.001G326900
ME
LOC_Os06g02200
-
AT3G14330
ME, EE, G-box
PEQU_07618
ME
Aco024818
Aco014488
EE, PBX, G-box, CCA1
-
Abbreviations: beta-CA, beta-carbonic anhydrase; ME, malic enzyme; PPDK, pyruvate orthophosphate dikinase.
Sobic.002G324400
–
Sobic.002G324500
G-box, CCA1
Sobic.002G324700
CCA1
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Table 1. (continued)
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Table 2. Frequency of Circadian Motifs (per kb) in Promoter Regions of all Genes (A), Photosynthetic Genes (B), and Nonphotosynthetic Isoleucine and Valine Biosynthesis Genes (C) across Five Speciesa[1_TD$IF] Circadian mof
Expected frequencyb
Arabidopsis
Orchid
Pineapple
Rice
Sorghum
(A) All Genes Morning element (6 bp) Evening element (8 bp) CCA1-binding site (8 bp) G-box element (6 bp)
0.250 0.105 0.105 0.250
0.288 0.063 0.212 0.155
0.291 0.050 0.166 0.085
0.302 0.064 0.210 0.183
0.460 0.042 0.132 0.213
0.478 0.040 0.148 0.180
(B) Photosynthec Genes Morning element (6 bp) Evening element (8 bp) CCA1-binding site (8 bp) G-box element (6 bp)
0.250 0.105 0.105 0.250
0.315 0.130 0.296 0.019
0.370 0.130 0.217 0.196
0.328 0.121 0.328 0.293
0.419 0.048 0.145 0.274
0.438 0.047 0.156 0.156
0.350 0.100 0.300 0.150
0.350 0.200 0.050 0.200
0.250 0.200 0.100 0.100
0.692 0.115 0.154 0.308
0.450 0.050 0.350 0.200
(C) Nonphotosynthec Isoleucine and Valine Biosynthesis Genes Morning element (6 bp) 0.250 Evening element (8 bp) 0.105 CCA1-binding site (8 bp) 0.105 G-box element (6 bp) 0.250 a
Circadian motfis with 10% or higher frequency occurrence in photosynthetic genes than genome wide frequency is highlighted in red. Expected frequency of the motif occurrence (per kb) in pineapple genome was calculated based on the assumption of randomization and both forward and reverse strands were included. Given that the pineapple genome GC content is 38%, the occurrence probabilities of G/C and A/T are 0.19 and 0.31, respectively.
*b
under circadian clock regulation. These binding site from one element to another elements were enriched the most in rice in the same promoter region that alters the diel pattern of gene expression (Table 1). and the least in pineapple (Table 2C). Nevertheless, selection most likely acted Acquiring these circadian clock cis-ele- on CAM-related genes, leading to the ments in CAM-related genes might not enrichment of these cis-elements in probe as spectacular [5_TD$IF]and innovation as initially moter regions. appeared, based on genome-wide analysis of their distribution and frequencies. Future Research Opportunities The morning elements regulate gene The presence of circadian clock cis-eleexpression at dawn, and the evening ele- ments in orthologs of CAM-related genes ments at dusk. The G-box is regulated by in C3 species partially solved the mystery of light and the phytohormone abscisic acid where these cis-elements arose. Enrich[12], and by PEPC kinase in pineapple. ment of these cis-elements in promoter CCA1 regulates the reactive oxygen spe- regions of CAM-related genes in pineapple cies response as part of light harvesting for suggested that natural selection of these photosynthesis, as well as for responses to cis-elements contributed to the indepennitrogen and abiotic stresses [13,14] and, dent origin of CAM photosynthesis in 36 as recently revealed, for CO2 fixation in families. The large family Bromeliaceae has CAM photosynthesis in pineapple. None around 3170 species, native mainly to of these cis-elements are CAM specific. tropical Americas, and CAM photosyntheCircadian regulation occurs for 89% of sis evolved in this family a minimum of five genes in Arabidopsis [15], but it does times [17]. C3 photosynthesis was ancesnot imply that 89% of genes have circadian tral in the subfamilies Tillandsioideae, Pitclock cis-elements, because as few as 5% cairnioideae, and Puyoideae, with 28% of of transcription factors could generate 792 species, 28% of 331 species, and proper circadian rhythms [16]. Acquiring 21% of 132 species tested performing these cis-elements in CAM-related genes CAM photosynthesis, respectively (Figcould happen as easily as shifting the ure 1). By contrast, CAM photosynthesis
was ancestral in the subfamilies Bromelioideae, with 90% of the 499 species tested performing CAM photosynthesis, including pineapple, and Hechtioideae with all 25 species performing CAM photosynthesis. No CAM photosynthesis has evolved in the subfamilies Brocchinioideae, Lindmanioideae, and Navioideae. C3 and CAM photosynthesis coexist in five subfamilies, indicating that both gain and loss of CAM photosynthesis occurred during the evolution of the Bromeliaceae, providing an opportunity to study the evolution of CAM photosynthesis. CAM photosynthesis in CAM species from the Puyoideae evolved more recently than those in Bromelioideae, and more intermediate types are likely present in former. Comparative genomic analysis of CAM-related genes in C3, intermediate types, and CAM species in Puyoideae would reveal progressive changes towards CAM photosynthesis, whereas such analysis of CAM and C3 species in Bromelioideae would reveal genes with critical changes resulting in the loss of CAM photosynthesis. Genome-wide ChIP-seq analysis of circadian clock regulatory gene-binding sites in selected C3 and CAM species within each subfamily
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Subfamily
C3
CAM
Bromelioideae
49
450
Puyoideae
104
28
Pitcairnioideae
238
93
Navioideae
70
0
Hechoideae
0
25
Tillandsioideae
569
223
CAM
C3 C3
C3
CAM
C3
photosynthesis in pineapple include: (i) the evolution and control of stomata movement and their reverse pattern of closing during the day and opening at night, another essential component of the evolution and reversion of CAM photosynthesis; (ii) gene interaction networks in CAM photosynthesis; (iii) the molecular mechanism of self-incompatibility; (iv) the molecular basis of spineless leaves, a major trait of domestication; (v) the regulation of protease bromelain production; (vi) gene and regulatory elements underlying the composition and nutritional value of the pineapple fruit; and (vii) genes and gene interaction networks controlling the partitioning of photosynthetic products into the growing fruit. Acknowledgments This work was supported by the Fujian Provincial Key Lab of Applied Plant Systems Biology and a start-up
C3
fund from Fujian Agriculture and Forestry University to
Lindmanioideae C3
Brocchinioideae
28
16
0
0
Figure 1. Phylogenetic Distribution of C3 and Crassulacean Acid Metabolism (CAM) photosynthesis Species among Eight Subfamilies in Bromeliaceae. The ancestral photosynthetic pathway of each
R.M. 1 FAFU and UIUC-SIB Joint Center for Genomics and Biotechnology,[3_TD$IF] Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China 2 Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 3 IRD, UMR IPME, COFFEEADAPT, BP 64501, 34394 Montpellier Cedex 5, France
subfamily is labeled on the left and the number of C3 and CAM species in each subfamily is on the right. Based on [17].
*Correspondence: [email protected] (R. Ming).
will reveal gains and losses of target cis- analysis. Given that CAM repeatedly elements, providing evidence for their roles evolved in over 400 genera in 36 families, in acquiring or losing CAM photosynthesis. it appears most likely that CAM-related genes evolved from C3 photosynthesis Genome editing using the CRISPR/CAS9 genes, because no CAM-specific genes system opens another dimension for have yet been identified in pineapple. If studying the evolution of CAM photosyn- acquiring circadian clock cis-elements is thesis. Altering selected circadian clock proven to be the major cause of the shift cis-elements of major genes in the CAM from C3 to CAM photosynthesis, engiphotosynthesis pathway would validate neering C3 crops to become CAM phototheir role and pleiotropic effects of such synthesizers would be expedited by changes, complementing genome-wide editing short cis-elements rather than genomic, transcriptomic, and ChIP-Seq transferring a set of new genes into a analyses. After the validation of the role of crop, which would be slow and have to circadian clock cis-elements in CAM go through lengthy deregulation prophotosynthesis, CAM-related genes cesses at high cost. and regulatory elements in other CAM species could be analyzed through com- Additional research opportunities on parative genomic and gene expression genome evolution and CAM
References 1. Collins, J.L. (1960) The Pineapple, Botany, Utilisation, Cultivation, Leonard Hill
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2. Pickersgill, B. (1976) Pineapple. In Evolution of Crop Plants (Simmonds, N.W., ed.), pp. 14–17, Longman 3. Okimoto, M.C. (1948) Anatomy and histology of the pineapple inflorescence and fruit. Bot. Gaz. 110, 217–231 4. Brewbaker, J.L. and Gorrez, D.D. (1967) Genetics of selfincompatibility in the monocot genera, Ananas (pineapple) and Gasteria. Am. J. Bot. 54, 611–616 5. Coppens d’Eeckenbrugge, G. et al. (1992) Fertility and selfincompatibility in the genus Ananas. In International Pineapple Symposium (Bartholomew, D.P. and Rohrbach, K. G., eds), pp. 45–52, International Society for Horticultural Science (ISHS) 6. Ming, R. et al. (2015) The pineapple genome and the evolution of CAM photosynthesis. Nat. Genet. 47, 1435– 1442 7. West-Eberhard, M.J. et al. (2011) Photosynthesis, reorganized. Science 332, 311–312 8. Wilkins, M.B. (1992) Circadian rhythms: their origin and control. New Phytol. 121, 347–375 9. Yang, X. et al. (2015) A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world. New Phytol. 207, 491–504
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10. Wang, X. et al. (2009) Comparative genomic analysis of C4 photosynthetic pathway evolution in grasses. Genome Biol. 10, R68 11. Carter, P.J. et al. (1991) Circadian rhythms in the activity of a plant protein kinase. EMBO J. 10, 2063 12. Filichkin, S.A. et al. (2011) Global profiling of rice and poplar transcriptomes highlights key conserved circadian-controlled pathways and cis-regulatory modules. PLoS ONE 6, e16907
13. Gutiérrez, R.A. (2012) Systems biology for enhanced plant nitrogen nutrition. Science 336, 1673–1675 14. Lai, A.G. et al. (2012) CIRCADIAN CLOCK-ASSOCIATED 1 regulates ROS homeostasis and oxidative stress responses. Proc. Natl. Acad. Sci. U.S.A. 109, 17129–17134 15. Michael, T.P. et al. (2008) Network discovery pipeline elucidates conserved time-of-day-specific cis-regulatory modules. PLoS Genet. 4, e14
16. Hanano, S. et al. (2008) A systematic survey in Arabidopsis thaliana of transcription factors that modulate circadian parameters. BMC Genomics 9, 182 17. Crayn, D.M. et al. (2015) Photosynthetic pathways in Bromeliaceae: phylogenetic and ecological significance of CAM and C3 based on carbon isotope ratios for 1893 species. Bot. J. Lin. Soc. 178, 169–221
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have originated in lowland South America, perhaps in the Southwest Brazil Paraná– Paraguay basin [1]. It performs obligate CAM photosynthesis that fixes CO2 at night, indicating that it originated in arid or semi-arid regions. It is perennial, living as long as 50 years. Pineapple is propagated vegetatively and its main propagule, the crown, can endure months without Ray Ming,1,2,* 2 water. Although pineapple can survive in Ching Man Wai, and 3 areas with a total annual rainfall of as little Romain Guyot as 500 mm (a condition likely to represent that in its region of origin), it can also Pineapple occupies an important tolerate annual levels as high as phylogenetic position and its refer5500 mm [2]. It is now grown in 85 ence genome expedites genomic countries.
Pineapple Genome: A Reference for Monocots and CAM Photosynthesis
research within the family Bromeliaceae and more widely among monocots. One such research focus is the evolution of crassulacean acid metabolism (CAM) photosynthesis. Acquiring circadian clock cis-regulatory elements in CAM-related genes might be a critical step in the evolution of this form of photosynthesis. Followup studies will clarify the processes and evolutionary forces leading to the multiple independent origins of CAM photosynthesis within the family Bromeliaceae and in over 400 genera across 36 families. A Genome Domesticated through Somatic Mutations
The discovery of pineapple [Ananas comosus (L.) Merr.] by Christopher Columbus during his second voyage to the New World is a key event in the subsequent distribution of pineapple across tropical and subtropical regions worldwide. The fact that Columbus encountered pineapple in a Carib village rather than in its native lowland South America shows that pineapple had been domesticated by Tupi-Guarani Indians long before Columbus arrived and was already distributed throughout the Americas and Caribbean islands [1]. Pineapple is a member of the Bromeliaceae family and is thought to
rearrangements after two ancient wholegenome duplication (WGD) events shared by all monocots, likely due to its vegetative propagation and, thus, reduced meiosis. Through structural comparisons of the pineapple genome to itself, homologous intragenomic blocks and genes were identified to reconstruct the ancient seven chromosomes before the two rounds of WGD. After the most ancient t WGD, the seven chromosomes doubled to 14. Two chromosomal fusion events reduced the chromosome number to 12 before the second s WGD. After the s WGD, the 12 chromosomes doubled to 24, two chromosomal fusion events reduced the chromosome number to 22, and two Pineapple fruit is the primary product of the chromosome fission events produced pineapple plant. The edible part of the fruit three additional chromosomes, resulting is developed from expended carpels, the in the current 25 chromosomes. bases of sepals, and bracts of more than 200 fused flowers, and the surface of the The well-conserved pineapple karyotype fruit is covered by sepals, bracts, and the makes its genome a reference for comapices of ovaries [3]. Cultivated pineapple parative analysis among monocot is self-incompatible with the gametophytic genomes. Its pivotal phylogenetic position control of pollen phenotype, while all wild at the base of the order Poales resulted in relatives are self-fertile [4]. Seedlessness is revised dating of the pan-cereal genome a desirable trait because it increases the duplication event r to 95–115 million years edible portion of the fruit. It has been ago (MYA); this duplication event is lacking hypothesized that self-incompatibility was in pineapple [6]. The earlier s WGD was selected during the domestication pro- revised to 100–120 MYA. The application cess, and seedless mutants could be mul- of the pineapple genome as a reference tiplied quickly through vegetative for monocots was also demonstrated by propagation [5]. Regardless of the origin showing the sorghum lineage-specific of self-incompatibility, most pineapple cul- translocation of gene families of type I tivars were selected as a result of somatic MADS-box genes and GDSL-like lipase/ mutation and the remainder from the acyl hydrolase genes using pineapple hybridization of distantly related cultivars genome as a reference. or wild relatives following the breakdown of self-incompatibility. Both methods Circadian Regulation of CAM resulted in highly heterozygous genomes Photosynthesis that are challenging to assemble. Photosynthesis is the process of converting light energy to chemical energy through conversion of carbon dioxide A Reference Genome for (CO2) into organic compound in plants, Monocots Recently, an international consortium algae, and certain bacteria. There are overcame the challenges of sequencing three photosynthetic pathways in terresa heterozygous genome and assembled trial plants: C3, C4, and CAM. The first a reference genome for pineapple [6]. The photosynthetic product is a three-carbon consortium found that the chromosome molecule in C3 plants and a four-carbon karyotype of pineapple is surprisingly well molecule in C4 and CAM plants. The conserved, with limited chromosomal difference between C4 and CAM
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TIGS 1307 No. of Pages 7
photosynthesis is that the separation of the auxiliary CO2 from the Calvin cycle occurs spatially in two cell types (mesophyll and bundle sheath) in C4 plants but temporally in the same mesophyll cell in CAM plants. C3 photosynthesis is the ancestral pathway and occurs in all taxonomic plant groups. It was proposed that C4 and CAM photosynthesis evolved through reorganization of the gene network in C3 plants [7]. The hallmark of CAM photosynthesis is the fixation of CO2 at night, whereas C3 and C4 plants fix CO2 during the day. The circadian rhythm of CO2 fixation was observed and has been extensively studied in CAM plants, and this pattern persists in detached leaves or under constant environmental conditions [8]. Pineapple is the most important crop exhibiting CAM photosynthesis. CAM plants consume one-sixth the amount of water used by C3 plants and a quarter of the amount of water used by C4 plants [9], and this high water-use efficiency is the result of stomatal closing during the day and opening at night to enable such plants to survive in hot and arid regions. However, how CAM photosynthesis evolved independently in 36 families has been a longstanding question [9]. Candidate genes for CAM photosynthesis were identified based on genes homologous to those encoding enzymes of the C3 and C4 pathways. Analyzing contrasting expression patterns in photosynthetic green[4_TD$IF] leaf tip and nonphotosynthetic white base leaf tissues separated the gene family members involved in carbon fixation from those involved in other processes [6]. Genome analysis revealed that WGD and tandem duplication did not contribute new genes in CAM photosynthesis, at least in the pineapple lineage [6]. Pineapple genes involved in carbon fixation have a diel expression pattern in photosynthetic tissues with low or no expression in nonphotosynthetic tissue [6]. These CAMrelated genes are enriched in known circadian clock cis-regulatory elements,
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including the morning element (CCACAC), evening element (AAAATATC), G-box (CACGTG), and Circadian Clock-Associated 1 (CCA1) (AAAAATCT). Carbonic anhydrase (CA) catalyzes the conversion of CO2 into bicarbonate and is the first step in CO2 fixation in CAM photosynthesis. Among the three CA families (/, b, and g) in pineapple, only bCA is expressed at night and during the early morning in photosynthetic tissue, indicating that bCA catalyzes carbon fixation. Promoters of all three bCA genes contain CCA1-binding sites (AAAAATCT). Among all the bCA genes in C3 rice, and C4 maize and sorghum, only one bCA gene in sorghum contains a CCA1-binding site at its promoter and this gene has no known photosynthetic function [10]. Acquiring these circadian clock cis-elements in CAMrelated genes might be critical for the evolution of CAM photosynthesis.
C3 and C4 plants (Table 1). Circadian clock cis-regulatory elements were present in all nine genes of all species with variation in their frequency. These nine genes carry out similar functions in C3, C4, and CAM species, and are under circadian regulation with different diel patterns. These cis-elements are six or eight base pairs (bp), and appear in the genome randomly at expected frequencies of 1/ 4096 bp and 1/65536 bp, respectively The expected and actual frequencies of these elements was examined in 2-kb promoter regions of all genes and photosynthetic genes in two CAM, two C3 and one C4 species (Table 2A,B). For the 6-bp morning element and G-box, the morning element appeared at higher than expected frequencies, whereas the Gbox appeared at lower than expected frequencies across all five species. The frequency of the morning element was about twice that of the G-box element. Similar discrepancies were also observed for the 8-bp evening element and the CCA1binding site, with a higher than expected frequency for the CCA1-binding site and a lower than expected frequency for evening elements across all five species. The frequency of the CCA1-binding site was about three times that of the evening element. These elements are not randomly distributed in the genome and selection has likely acted on them, causing the biased distribution frequency in promoter regions genome wide.
The second step is the conversion of phosphoenolpyruvate to oxaloacetate (OAA) by phosphoenolpyruvate carboxylase (PEPC) and the third step is the conversion of OAA to malate by the enzyme malate dehydrogenase (MDH). Genomic sequences of pineapple CA, PEPC, and MDH revealed the presence of CCA1 elements within their promoters (Table 1). PEPC becomes activated at night via phosphorylation by PEPC kinase [11]. The promoter region of PEPC kinase contains a G-box. The circadian rhythm of CAM photosynthesis is likely a concerted genome-wide adaptation with a selection of circadian clock cis-elements as a crucial Analysis of circadian clock cis-elements in if not essential component of this evolu- CAM-related genes revealed enrichment of tionary process. more than 10% of such cis-elements in CAM species of orchid for all four elements and in pineapple for three elements comThe Origin of Circadian Clock pared with the genome-wide frequencies Cis-Elements in CAM-Related of these elements in all genes (Table 2B). Genes of Pineapple The pineapple genome offered an oppor- Such enrichment was also observed for tunity to investigate the distribution and two elements in the C3 genera Arabidopsis frequency of these circadian clock cis-ele- and rice, and for one element in the C4 ments in CAM-related genes. Two-kb pro- genus Sorghum (Table 2B). As a control, moter regions of the nine most important the frequencies of these elements were genes in CAM photosynthesis were exam- analyzed in promoter regions of isoleucine ined in CAM species and their orthologs in and valine biosynthesis genes that are not
Arabidopsis
beta-CA
PEPC
MDH
Orchid
Pineapple
Rice
Sorghum
Gene ID
TF binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
AT1G23720
–
PEQU_27755
ME
Aco002732
CCA1, G-box
LOC_Os01g45274
ME
Sobic.002G230100
G-box, ME
AT1G58180
–
PEQU_37822
–
Aco006181
CCA1, ME
LOC_0s09g28910
–
Sobic.003G234200
G-box
AT1G70410
ME
PEQU_31387
–
Aco005402
CCA1
Sobic.003G234400
–
AT3G01500
ME
Sobic.003G234500
CCA1
AT4G33580
ME
Sobic.003G234600
–
AT5G14740
–
AT1G53310
G-box, CCA1
PEQU_07008
EE
Aco010025
EE, ME, CCA1, PBX
LOC_Os01g55350
G-box
Sobic.002G167000
G-box
AT2G42600
–
PEQU_14315
CCA1
Aco018093
ME, CCA1
LOC_Os02g14770
CCA1
Sobic.004G106900
ME
AT3G14940
ME, CCA1
Aco016429
G-box, PBX, CCA1
LOC_Os08g27840
CCA1
Sobic.007G106500
ME, G-box
AT1G68750
–
LOC_Os09g14670
–
Sobic.003G301800
ME
LOC_Os01g02050
ME, G-box, PBX
Sobic.010G160700
ME
LOC_Os01g11054
CCA1
Sobic.003G100600
ME
LOC_Os08g33720
ME, EE
Sobic.009G240700
CCA1
PEQU_05708
–
Trends in Genetics, Month Year, Vol. xx, No. yy
AT1G53240
ME, CCA1
Aco006122
CCA1
AT3G15020
ME
PEQU_17024
EE, CCA1
Aco007734
CCA1
LOC_Os05g49880
ME
Sobic.003G238500
G-box
AT5G43330
ME, EE
PEQU_19417
–
Aco013935
ME
LOC_Os03g56280
ME, PBX, G-box
Sobic.001G219300
ME, G-box, CCA1
AT1G04410
–
PEQU_08514
ME
Aco002885
ME, G-box
LOC_Os01g46070
–
Sobic.007G137600
ME
AT3G47520
ME
PEQU_20254
PBX, CCA1
Aco004349
ME, G-box, PBX
LOC_Os01g61380
ME
Sobic.004G004500
PBX
AT4G17260
ME, PBX
PEQU_35914
G-box, PBX, CCA1
Aco014690
G-box, PBX
LOC_Os04g46560
–
Sobic.004G004400
ME
AT5G58330
ME, CCA1
Aco017525
CCA1
LOC_Os10g33800
ME, PBX, G-box
Sobic.006G170800
-
AT2G22780
EE
Aco017526
PBX
LOC_Os02g01510
G-box
Sobic.007G166300
ME, CCA1
AT5G09660
ME, CCA1
LOC_Os08g44810
CCA1
Aco017527
CCA1
Sobic.007G166200
ME, G-box
Aco017528
CCA1
Sobic.001G073900
EE
Aco019631
CCA1
Sobic.008G186200
ME, PBX, CCA1
Aco010232
ME, G-box
Aco004996
ME, PBX
Aco008626
PBX
TIGS 1307 No. of Pages 7
Table 1. Cis-Elements Annotated at Promoter Sequences of Selected Photosynthetic Genes across Five Speciesa
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Arabidopsis
PEPC kinase
PEPC-related kinase
NAD-ME
NADP-ME
PEPCK
PPDK
PPDK regulatory protein
a
Orchid
Pineapple
Rice
Sorghum
Gene ID
TF binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
Gene ID
TF [1_TD$IF]binding [2_TD$IF]motif
AT1G08650
ME
PEQU_38595
ME
Aco010095
G-box
LOC_Os02g56310
EE, PBX, CCA1
Sobic.004G338000
G-box, PBX
AT3G04530
–
PEQU_29948
ME, G-box, CCA1
Aco013938
G-box
LOC_Os02g41580
ME, PBX, G-box
Sobic.006G148300
ME, EE
PEQU_12226
ME, G-box
Sobic.004G219900
ME, G-box
PEQU_15364
ME, CCA1 Sobic.010G016900
ME, G-box, PBX
AT1G12580
G-box
PEQU_06553
ME, G-box
Aco022525
EE, ME, G-box, CCA1
LOC_Os06g03682
ME, PBX, CCA1
AT1G12680
–
PEQU_09615
ME, G-box
Aco001261
EE, ME, PBX
LOC_Os09g29170
ME, G-box, CCA1
Sobic.002G231300
G-box, CCA1
Sobic.007G160600
ME
AT2G13560
EE, G-box
PEQU_05938
ME
Aco016569
–
LOC_Os07g31380
ME, PBX
Sobic.002G309400
CCA1
AT4G00570
G-box
PEQU_22911
ME, EE, G-box
Aco007622
G-box, PBX, CCA1
LOC_Os10g35960
CCA1
Sobic.001G201700
ME, CCA1
AT5G25880
G-box
PEQU_29209
EE, CCA1
Aco009967
G-box, PBX, ME
LOC_Os05g09440
ME, G-box
Sobic.001G201700
ME, CCA1
AT5G11670
CCA1
PEQU_01237
G-box, PBX
Aco005631
CCA1
LOC_Os01g09320
ME, EE
AT1G79750
ME
PEQU_08427
G-box
Aco005989
G-box
LOC_Os01g52500
G-box
AT2G19900
–
LOC_Os01g54030
–
AT4G37870
G-box, ME, CCA1
PEQU_14885
–
Aco017762
ME, EE, G-box, CCA1
LOC_Os03g15050
–
Sobic.001G432800
ME
AT5G65690
ME, EE
PEQU_25034
ME, EE, CCA1
LOC_Os10g13700
ME, G-box, PBX
LOC_Os04g43710
ME, G-box
AT4G15530
ME, CCA1
PEQU_01453
G-box, PBX
LOC_Os03g31750
ME
Sobic.009G132900
-
LOC_Os05g33570
-
Sobic.001G326900
ME
LOC_Os06g02200
-
AT3G14330
ME, EE, G-box
PEQU_07618
ME
Aco024818
Aco014488
EE, PBX, G-box, CCA1
-
Abbreviations: beta-CA, beta-carbonic anhydrase; ME, malic enzyme; PPDK, pyruvate orthophosphate dikinase.
Sobic.002G324400
–
Sobic.002G324500
G-box, CCA1
Sobic.002G324700
CCA1
TIGS 1307 No. of Pages 7
Table 1. (continued)
TIGS 1307 No. of Pages 7
Table 2. Frequency of Circadian Motifs (per kb) in Promoter Regions of all Genes (A), Photosynthetic Genes (B), and Nonphotosynthetic Isoleucine and Valine Biosynthesis Genes (C) across Five Speciesa[1_TD$IF] Circadian mof
Expected frequencyb
Arabidopsis
Orchid
Pineapple
Rice
Sorghum
(A) All Genes Morning element (6 bp) Evening element (8 bp) CCA1-binding site (8 bp) G-box element (6 bp)
0.250 0.105 0.105 0.250
0.288 0.063 0.212 0.155
0.291 0.050 0.166 0.085
0.302 0.064 0.210 0.183
0.460 0.042 0.132 0.213
0.478 0.040 0.148 0.180
(B) Photosynthec Genes Morning element (6 bp) Evening element (8 bp) CCA1-binding site (8 bp) G-box element (6 bp)
0.250 0.105 0.105 0.250
0.315 0.130 0.296 0.019
0.370 0.130 0.217 0.196
0.328 0.121 0.328 0.293
0.419 0.048 0.145 0.274
0.438 0.047 0.156 0.156
0.350 0.100 0.300 0.150
0.350 0.200 0.050 0.200
0.250 0.200 0.100 0.100
0.692 0.115 0.154 0.308
0.450 0.050 0.350 0.200
(C) Nonphotosynthec Isoleucine and Valine Biosynthesis Genes Morning element (6 bp) 0.250 Evening element (8 bp) 0.105 CCA1-binding site (8 bp) 0.105 G-box element (6 bp) 0.250 a
Circadian motfis with 10% or higher frequency occurrence in photosynthetic genes than genome wide frequency is highlighted in red. Expected frequency of the motif occurrence (per kb) in pineapple genome was calculated based on the assumption of randomization and both forward and reverse strands were included. Given that the pineapple genome GC content is 38%, the occurrence probabilities of G/C and A/T are 0.19 and 0.31, respectively.
*b
under circadian clock regulation. These binding site from one element to another elements were enriched the most in rice in the same promoter region that alters the diel pattern of gene expression (Table 1). and the least in pineapple (Table 2C). Nevertheless, selection most likely acted Acquiring these circadian clock cis-ele- on CAM-related genes, leading to the ments in CAM-related genes might not enrichment of these cis-elements in probe as spectacular [5_TD$IF]and innovation as initially moter regions. appeared, based on genome-wide analysis of their distribution and frequencies. Future Research Opportunities The morning elements regulate gene The presence of circadian clock cis-eleexpression at dawn, and the evening ele- ments in orthologs of CAM-related genes ments at dusk. The G-box is regulated by in C3 species partially solved the mystery of light and the phytohormone abscisic acid where these cis-elements arose. Enrich[12], and by PEPC kinase in pineapple. ment of these cis-elements in promoter CCA1 regulates the reactive oxygen spe- regions of CAM-related genes in pineapple cies response as part of light harvesting for suggested that natural selection of these photosynthesis, as well as for responses to cis-elements contributed to the indepennitrogen and abiotic stresses [13,14] and, dent origin of CAM photosynthesis in 36 as recently revealed, for CO2 fixation in families. The large family Bromeliaceae has CAM photosynthesis in pineapple. None around 3170 species, native mainly to of these cis-elements are CAM specific. tropical Americas, and CAM photosyntheCircadian regulation occurs for 89% of sis evolved in this family a minimum of five genes in Arabidopsis [15], but it does times [17]. C3 photosynthesis was ancesnot imply that 89% of genes have circadian tral in the subfamilies Tillandsioideae, Pitclock cis-elements, because as few as 5% cairnioideae, and Puyoideae, with 28% of of transcription factors could generate 792 species, 28% of 331 species, and proper circadian rhythms [16]. Acquiring 21% of 132 species tested performing these cis-elements in CAM-related genes CAM photosynthesis, respectively (Figcould happen as easily as shifting the ure 1). By contrast, CAM photosynthesis
was ancestral in the subfamilies Bromelioideae, with 90% of the 499 species tested performing CAM photosynthesis, including pineapple, and Hechtioideae with all 25 species performing CAM photosynthesis. No CAM photosynthesis has evolved in the subfamilies Brocchinioideae, Lindmanioideae, and Navioideae. C3 and CAM photosynthesis coexist in five subfamilies, indicating that both gain and loss of CAM photosynthesis occurred during the evolution of the Bromeliaceae, providing an opportunity to study the evolution of CAM photosynthesis. CAM photosynthesis in CAM species from the Puyoideae evolved more recently than those in Bromelioideae, and more intermediate types are likely present in former. Comparative genomic analysis of CAM-related genes in C3, intermediate types, and CAM species in Puyoideae would reveal progressive changes towards CAM photosynthesis, whereas such analysis of CAM and C3 species in Bromelioideae would reveal genes with critical changes resulting in the loss of CAM photosynthesis. Genome-wide ChIP-seq analysis of circadian clock regulatory gene-binding sites in selected C3 and CAM species within each subfamily
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TIGS 1307 No. of Pages 7
Subfamily
C3
CAM
Bromelioideae
49
450
Puyoideae
104
28
Pitcairnioideae
238
93
Navioideae
70
0
Hechoideae
0
25
Tillandsioideae
569
223
CAM
C3 C3
C3
CAM
C3
photosynthesis in pineapple include: (i) the evolution and control of stomata movement and their reverse pattern of closing during the day and opening at night, another essential component of the evolution and reversion of CAM photosynthesis; (ii) gene interaction networks in CAM photosynthesis; (iii) the molecular mechanism of self-incompatibility; (iv) the molecular basis of spineless leaves, a major trait of domestication; (v) the regulation of protease bromelain production; (vi) gene and regulatory elements underlying the composition and nutritional value of the pineapple fruit; and (vii) genes and gene interaction networks controlling the partitioning of photosynthetic products into the growing fruit. Acknowledgments This work was supported by the Fujian Provincial Key Lab of Applied Plant Systems Biology and a start-up
C3
fund from Fujian Agriculture and Forestry University to
Lindmanioideae C3
Brocchinioideae
28
16
0
0
Figure 1. Phylogenetic Distribution of C3 and Crassulacean Acid Metabolism (CAM) photosynthesis Species among Eight Subfamilies in Bromeliaceae. The ancestral photosynthetic pathway of each
R.M. 1 FAFU and UIUC-SIB Joint Center for Genomics and Biotechnology,[3_TD$IF] Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China 2 Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 3 IRD, UMR IPME, COFFEEADAPT, BP 64501, 34394 Montpellier Cedex 5, France
subfamily is labeled on the left and the number of C3 and CAM species in each subfamily is on the right. Based on [17].
*Correspondence: [email protected] (R. Ming).
will reveal gains and losses of target cis- analysis. Given that CAM repeatedly elements, providing evidence for their roles evolved in over 400 genera in 36 families, in acquiring or losing CAM photosynthesis. it appears most likely that CAM-related genes evolved from C3 photosynthesis Genome editing using the CRISPR/CAS9 genes, because no CAM-specific genes system opens another dimension for have yet been identified in pineapple. If studying the evolution of CAM photosyn- acquiring circadian clock cis-elements is thesis. Altering selected circadian clock proven to be the major cause of the shift cis-elements of major genes in the CAM from C3 to CAM photosynthesis, engiphotosynthesis pathway would validate neering C3 crops to become CAM phototheir role and pleiotropic effects of such synthesizers would be expedited by changes, complementing genome-wide editing short cis-elements rather than genomic, transcriptomic, and ChIP-Seq transferring a set of new genes into a analyses. After the validation of the role of crop, which would be slow and have to circadian clock cis-elements in CAM go through lengthy deregulation prophotosynthesis, CAM-related genes cesses at high cost. and regulatory elements in other CAM species could be analyzed through com- Additional research opportunities on parative genomic and gene expression genome evolution and CAM
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