Gene co-expression reveals the modularity and integration of C4 and CAM in Portulaca

Abstract

C4 photosynthesis and Crassulacean acid metabolism (CAM) have been considered as largely independent adaptations despite sharing key biochemical modules. Portulaca is a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C4 but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C4 + CAM. It has been hypothesized that C4 + CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly of Portulaca amilis and sampled mRNA from P. amilis and Portulaca oleracea during CAM induction. Gene co-expression network analyses identified C4 and CAM gene modules shared and unique to both Portulaca species. A conserved CAM module linked phosphoenolpyruvate carboxylase to starch turnover during the day-night transition and was enriched in circadian clock regulatory motifs in the P. amilis genome. Preservation of this co-expression module regardless of water status suggests that Portulaca constitutively operate a weak CAM cycle that is transcriptionally and posttranscriptionally upregulated during drought. C4 and CAM mostly used mutually exclusive genes for primary carbon fixation, and it is likely that nocturnal CAM malate stores are shuttled into diurnal C4 decarboxylation pathways, but we found evidence that metabolite cycling may occur at low levels. C4 likely evolved in Portulaca through co-option of redundant genes and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C4 evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.

Figure 1

Overview of CCMs in the Portullugo. Simplified C₄ (A) and CAM (B) pathways showing shared carboxylation and decarboxylation pathways. ASP, aspartateaminotransferase; BCA, beta carbonic anhydrase; CBC, Calvin–Benson cycle; MDH, malate dehydrogenase; ME, malic enzyme; PEPC, PEP carboxylase. Cartoon dendrogram of the Portullugo (Portulacineae + Molluginaceae), highlighting major Portulaca clades (C), which are colored by primary C₄ biochemical pathway. Mean age estimates for focal Portulaca clades are from Ocampo and Columbus (2012). Boxplots of night-day differences in titratable acidity (n = 6) of experimental P. amilis (orange) and P. oleracea plants (purple) (D); boxplots show median and interquartile range; whiskers show 1.5 × interquartile range. WW, well-watered; D, drought. Asterisks connecting lines indicate significant differences between well-watered and drought treatments (independent t test; “***”, P < 0.001; “NS”, P = 0.121). Images of P. amilis when well-watered and after 8 days without water (E); images were digitally extracted for comparison.

Figure 2

Genome and genomic content of P. amilis. Idiogram of the nine primary scaffolds of the P. amilis genome with key C₄ and CAM genes highlighted (A). Breakdown of repetitive elements (B); the horizontal bar chart shows the fractions of major classes of repetitive elements and the repeat landscape (histogram) shows the relative abundances of repetitive elements versus the Kimura substitution level from the consensus. Scatterplot of number of gene models versus the log10-transformed genome size for 107 angiosperm genomes (C) with notable benchmarking and CCM genomes highlighted; data are from Zhao and Schranz (2019). Line in (C) shows results of ordinary least squares regression; one sample t_slope (106) > 0, p_slope = 0.0017.

Figure 3

PGNs. Well-watered and drought PGNs of P. amilis (A and B), and P. oleracea (E and F) colored by WGCNA co-expression module identity. Each node in the graph represents one gene and node sizes represent their degrees. Each module’s size and functional composition are shown in the histogram, and the z-score normalized expression of each module’s constituent genes is shown along the horizontal axis. An example of the z-score normalized expression for module paWW1 is enlarged in (A) to show individual gene expression with the median highlighted in bold. TPM normalized expression profiles of two focal PPC transcripts (PaPPC-1E1a’ and PaPPC-1E1c) are shown in (C) and (D), respectively; points show median of six biological replicates and error bars show interquartile range; black and red lines represent well-watered and drought samples, respectively.

Figure 4

TPM normalized abundance of selected P. amilis genes hypothesized to be involved in CAM. Gene model and name are shown above each plot, and black and red lines show median well-watered and drought abundance of all biological replicates (n = 6), respectively; error bars show interquartile range. Module assignments are listed for well-watered and drought networks in black and red, respectively. (A) GWD, glucan water dikinase; (B) LSF, phosphoglucan phosphatase; (C) PHS; (D) ISA, isoamylase; (E) DPE, 4-alpha-glucanotransferase; (F) AMY; (G) BAM, beta-amylase; (H) BCA; (I) ALMT, aluminum-activated malate transporter; (J) PPCK, PEPC kinase; (K) NAD-MDH; (L) TDT; (M) PUMP, mitochondrial uncoupling protein/dicarboxylate carrier; (N) NAD-ME, NAD-dependent ME; (O) NADP-ME, NADP-dependent ME. Significant differences in abundance across each time series between well-watered and drought conditions are shown in the upper right corner of each plot. P-values for unique transcripts were aggregated for each gene model and transformed into q-values by adjusting for false discovery rate (see “Materials and methods”); “NS”  = nonsignificant, “*” = q < 0.05, “**”  = q < 0.01, “***” = q < 0.001.

Figure 5

PPC-1E1c PGN preservation and subnetworks of common genes. Stacked bar charts showing the number and functional composition of genes that were exclusive to the well-watered or drought PPC-1E1c PGNs, or preserved across treatments in P. amilis (A) and P. oleracea (B). Subnetworks induced by genes common to both P. amilis (C) and P. oleracea (D) drought PPC-1E1c PGNs; drought PGNs include preserved and drought exclusive genes. Orange edges represent correlations among genes shared between species, while black edges are unique to each respective species. Metabolic pathways of the shared PPC-1E1c subnetworks (E), showing PEPC, sugar transport, and starch metabolism. PEP, phosphoenolpyruvate; OAA, oxaloacetate; ADO, Adagio; APRR,Arabidopsis pseudo-response regulator; DPE, 4-alpha-glucanotransferase; ELF, EARLY FLOWERING;GI GIGANTEA; GWD, glucan water dikinase; ISA, isoamylase; LSF, phosphoglucanphosphatase; LUX, LUX ARRHYTHMO; PHS, alpha-glucan phosphorylase; PEPC, PEP carboxylase;SBE, starch branching enzyme; RVE, REVEILLE. Colors of bar plot units (A and B) and gene boxes (C–E) indicate functional categories as in Figure 3.

Figure 6

Hypothesized photosynthetic carbon fluxes of C₄ + CAM in P. amilis. Major carbon fluxes when well-watered (A) and droughted (B). Red, blue, and purple lines indicate NAD-specific, NADP specific, and shared reactions, respectively, and gray lines show possible pathways for malate that are unique to C₄ + CAM photosynthesis. Line thicknesses are indicative of relative fluxes through pathways. ALAAT, alanine aminotransferase; ASP, aspartate aminotransferase; BCA, beta carbonic anhydrase; CBC, Calvin-Benson cycle; NAD-MDH, NAD-dependent malate dehydrogenase; NAD-ME, NAD-dependent malic enzyme; NADP-MDH, NADP-dependent malate dehydrogenase; NADP-ME, NADP-dependent malic enzyme; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; PPCK, PEPC kinase; PPDK, pyruvate, phosphate dikinase.