The Kalanchoë genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism

Abstract

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C₃ photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO₂ fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C₃ photosynthesis engineering to enhance water-use efficiency in crops.

Fig. 1

A species tree reconstructed from 210 single-copy genes using a summary method. a Diploid plant of Kalanchoë fedtschenkoi. b Individual maximum-likelihood gene trees were reconstructed from the CDS alignments for each of the 210 single-copy-gene ortholog groups using RAxML, and the species tree was summarized from the gene trees using ASTRAL-II. Pie graphs on nodes represent the proportion of gene trees that support the various quartets at every node, with red for the main topology shown in this tree, blue for the first alternative, and green for the second alternative, respectively. Quartet frequencies displayed in pie graphs and the posterior-probability at each node are calculated by ASTRAL-II

Fig. 2

Genome duplication in Kalanchoë fedtschenkoi. a Syntenic depth of the K. fedtschenkoi genome for each grape gene. Syntenic depth refers to the number of times a genomic region is covered by synteny blocks against another genome. b Typical micro-colinearity patterns between genomic regions from grape and K. fedtschenkoi. Rectangles show predicted gene models with colors showing relative orientations (blue: same strand, black: opposite strand). Matching gene pairs are displayed as connecting shades. Three orthologous gene groups that were maximally retained as four copies in K. fedtschenkoi were highlighted with phylogenetic trees on the right suggesting two rounds of genome duplications in the Kalanchoë lineage. c Four-fold transversion substitution rate (4dtv) in K. fedtschenkoi and six other eudicot plant species

Fig. 3

An overview of CAM pathway in Kalanchoë fedtschenkoi. a The CAM pathway map in K. fedtschenkoi. Orange colors indicate the key enzymes involved in the CAM pathway. The numbers in parenthesis are the four-fold transversion substitution rate (4dtv) values. b Diel expression profiles of duplicated genes in CAM-related gene families. ALMT tonoplast aluminum-activated malate transporter, β-CA β type carbonic anhydrase, ME malic enzyme, MDH malate dehydrogenase, PEP phosphoenolpyruvate, PEPC PEP carboxylase, PPCK PEPC kinase, PPDK pyruvate phosphate dikinase, TDT tonoplast dicarboxylate transporter. White and black bars indicate daytime (12-h) and nighttime (12-h), respectively

Fig. 4

Examples of convergent change in diel transcript expression pattern in CAM species. a The four time-windows for comparative analysis of temporal changes in transcript expression, which were represented by 12 time points: 2, 4, …, 24 h after the beginning of the light period. b and c Comparison of diel transcript expression pattern of phosphoenolpyruvate carboxylase kinase 1 (PPCK1) and phototropin 2 (PHOT2), respectively, between CAM species (Kalanchoë fedtschenkoi and pineapple) and C₃ species (Arabidopsis). Left panels show the diel transcript expression profiles. Right panels show enrichment triangle networks, in which a K. fedtschenkoi gene and a pineapple ortholog had significantly enriched expression in the same time-window, whereas an Arabidopsis ortholog had significantly enriched expression in the opposite time-window. The numbers are the time shifts in diel transcript expression pattern between genes connected by each edge. White and black bars indicate daytime (12-h) and nighttime (12-h), respectively. X-axis represents the time after the beginning of the light period

Fig. 5

Two phosphoenolpyruvate carboxylase (PEPC) genes with relative high transcript abundance in Kalanchoë fedtschenkoi. a Regulation of PEPC1 activity. b Diel expression profiles of PEPC1 (Kaladp0095s0055.1) and PEPC2 (Kaladp0048s0578.1) transcripts in K. fedtschenkoi, shown in the left and right Y-axis, respectively. OAA Oxaloacetate, PEP phosphoenolpyruvate, PEPC PEP carboxylase, PPCK PEPC kinase, PP2A protein phosphatase 2 A. White and black bars indicate daytime (12-h) and nighttime (12-h), respectively

Fig. 6

A convergent change in phosphoenolpyruvate carboxylase (PEPC) protein sequences in CAM species. a convergent- vs. divergent-substitutions in PEPC2 protein sequences between species listed in Supplementary Table 9. The arrow head indicates the comparison of K. fedtschenkoi vs. P. equestris. b Probability of convergent changes in PEPC2 protein sequence between K. fedtschenkoi and orchid. Red arrow indicates the protein sequence alignment site of convergent change (highlighted in red font at the alignment in panel c). c A convergent amino-acid change (from R/K/H to D) in PEPC2 shared by diverse species (highlighted in red font) at the alignment position indicated by the red arrow. d In vitro activity of PEPC isoforms in the absence of phosphorylation by PPCK. KfPEPC1: Kaladp0095s0055; KfPEPC1^R515D: KfPEPC1 with mutation at residue 515 from arginine (R) to aspartic acid (D); KfPEPC2: Kaladp0048s0578.1; KfPEPC2^D509K: KfPEPC2 with mutation at residue 509 from D to lysine (K); PqPEPC2: P. equestris PEPC gene PEQU07008; PqPEPC2^D504K: PqPEPC2 with mutation at residue 504 from D to K. “*” indicates significant difference between wild-type and mutant of PEPC1 or PEPC2 (Student’s t-test; P < 0.01). The error bars indicate standard deviation (SD) calculated from three replicates

Fig. 7

Protein structure model of phosphoenolpyruvate carboxylase 2 (PEPC2) in Kalanchoë fedtschenkoi. a PEPC2 (Kaladp0048s0578.1) structural model with a glucose-6-phosphate (G6P) substrate (orange spheres) bound at the β-barrel active site (yellow). D509 (red spheres) is located at an α-helix (red) in adjacent to the β-barrel and far from the hallmark serine residue (S8, green spheres) that is the phosphorylation target of PPCK1. b PEPC tetramer structure. The phosphorylation site (S8, green) is located at the interphase of the tetramer and D509 (spheres) is located at the peripheral of the tetramer. The β-barrel active site is shown in red, and no G6P activator may be required for activation of the PEPC activity following the competitive activating model of PEPC

Fig. 8

Convergent changes in diel transcript expression of heat-shock proteins (HSPs) in CAM species in comparison with C₃ species. a Schematic representation of the possible roles of HSP40, HSP60, and HSP70 in leaf heat tolerance. b Comparison of diel transcript expression pattern of HSP70 between CAM species (Kalanchoë fedtschenkoi and pineapple) and C₃ species (Arabidopsis). Left panel shows the diel transcript expression patterns. Right panel shows enrichment triangle network, in which a K. fedtschenkoi gene and a pineapple ortholog had significantly enriched expression in the same time-window, whereas an Arabidopsis ortholog had significantly enriched expression in the opposite time-window. The numbers are the time shifts in diel transcript expression pattern between genes connected by each edge. White and black bars indicate daytime (12-h) and nighttime (12-h), respectively. X-axis represents the time after the beginning of the light period. RuBisCO: Ribulose-1,5-bisphosphate carboxylase/oxygenase; RCA: rubisco activase; RuBP: ribulose-1,5-bisphosphate; PGA: 3-phosphoglycerate

Fig. 9

A convergent change in elongated hypocotyl 5 (HY5) protein sequences in CAM species. a An overview of the signaling pathway involved in circadian rhythm in plants. b Convergent change in HY5 protein sequences in diverse species (highlighted in red font). The black line indicates the protein sequence alignment position (located within the bZIP domain) where the mutation (E-to-R) occurred. CCA1 circadian clock associated 1, COP1 constitutive photomorphogenic 1, CRY cryptochrome, EC evening complex, ELF 3/4 early flowering 3/4, GI gigantea, LHY late elongated hypocotyl, LUX lux arrhythmo, PRR5/7/9 pinoresinol reductase 5/7/9, PHYs phytochromes, RVEs reveilles, TOC1 timing of cab expression 1