Spatial resolution of an integrated C4+CAM photosynthetic metabolism

Abstract

C₄ and CAM photosynthesis have repeatedly evolved in plants over the past 30 million years. Because both repurpose the same set of enzymes but differ in their spatial and temporal deployment, they have long been considered as distinct and incompatible adaptations. Portulaca contains multiple C₄ species that perform CAM when droughted. Spatially explicit analyses of gene expression reveal that C₄ and CAM systems are completely integrated in Portulaca oleracea, with CAM and C₄ carbon fixation occurring in the same cells and CAM-generated metabolites likely incorporated directly into the C₄ cycle. Flux balance analysis corroborates the gene expression findings and predicts an integrated C₄+CAM system under drought. This first spatially explicit description of a C₄+CAM photosynthetic metabolism presents a potential new blueprint for crop improvement.

Fig. 1.. Drought induction of CAM and laser capture microdissection.

(A) Diel fluctuation of titratable acidity from whole leaves of well-watered plants and after a 7-day drought treatment. NS, not significant. (B) P. oleracea, illustrating the orientation of paradermal sections (blue box). (C) Fresh, flash-frozen paradermal leaf section in cryosection block. The red square indicates an area used for tissue dissection. (D) Microphotograph of a 12-μm-thick leaf paradermal section indicating bundle sheath (BS) and mesophyll (M) tissues before (D) and after (E) BS cell capture. Orange arrows in (D) indicate calcium oxalate crystals in M cells. The red line in (E) indicates a laser-cut area of BS tissue, and the orange line illustrates an area of M tissue for laser capture. (F) RNA profile by electrophoresis for quality control. CP, chloroplast RNAs; RFU, relative fluorescence units.

Fig. 2.. Transcriptome-wide gene expression patterns across M and BS samples.

(A) All laser microdissection (LMD) samples projected into the first two dimensions of a transcriptome-wide multidimensional scaling analysis of log-transformed expression. Treatments: D, drought; WW, well-watered. (B) Venn diagram with number of DE genes across variables. (C) Heatmap with the log of gene expression of the 500 most variable genes across samples. Dendrograms cluster mRNA libraries (on the top) and genes (on the left) on the basis of gene expression similarities.

Fig. 3.. Differential gene expression of M and BS across experimental conditions.

(A) Schematic of C₄, CAM, and accessory biochemical pathways. Solid and dotted lines indicate C₄ and CAM routes of carbon concentration, respectively; the gray box contains night reactions, and key substrates are shown (PEP). Intracellular compartments are indicated within red boxes (V, vacuole; C, chloroplast). (B) Differential transcript abundance (measured in log₂FC) of selected genes in M relative to BS tissue (left) and at 7 hours relative to 23 hours (middle ) across LMD samples. Gene color backgrounds reflect pathways in (A). In the third panel, white triangles indicate log₂FC of 7-hour M samples in D relative to 7-hour M samples WW. Black triangles indicate log₂FC of 23-hour M samples in drought compared to 23-hour M samples WW. A triangle in the WW region (negative log₂FC, left to the red line) indicates higher expression during WW, while triangles within the D region (right to the red line) indicate higher expression in D. In all panels, asterisks indicate DE significance (P_adj < 0.05).

Fig. 4.. Transcription abundance of selected CCM-related genes.

Median of transcripts per million (TPM; y axis) across time points (7 and 23 hours, x axis) in LMD mRNA libraries. Black and red lines indicate M or BS samples, respectively. Plain lines indicate watered, and dotted lines indicate drought. Error bars represent the interquartile range of expression.

Fig. 5.. Visium spatial gene expression.

(A) Microphotograph of a leaf paradermal section; K-means clustering of total gene expression; and abundance of the main C₄, CAM, and Calvin cycle carboxylases using the 10x Genomics Visium platform. K-means clustering of sampling spots corresponds to BS (dark blue), M (light blue), and water storage (WS; orange) tissues; abundances of PPC-1E1a′, PPC-1E1c, and RBCS are shown relative to their observed unique molecule index (UMI) ranges. (B) Violin plots of transcript abundance in UMI across sample spots classified by tissue type in 2300-hour samples.

Fig. 6.. Correlations between predicted enzymatic fluxes and estimated gene expression in M and BS samples.

Pearson correlation for individual enzymes between predicted reaction fluxes in the most efficient C₄+CAM flux balance model (FBA) and the expression (EXP) of their encoding genes as the mean of transcript abundance in the LMD samples (TPM). Each data point corresponds to an experimental group of samples composed of D or wet conditions (W), M or BS tissue, and daytime (7 h) or nighttime (23 h). Time labels are shown next to each point. Expression and flux balance results are normalized by maximal values for each enzyme/gene. Regression lines are shown in blue, and SEs are shown in gray.

Fig. 7.. Biochemical flux results in the FBA model of an integrated C₄+CAM photosynthesis.

Schematic of major metabolic fluxes related to C₄ and CAM in the M and BS; orange and blue arrows indicate daytime and nighttime reactions, respectively. Black numbers indicate fluxes under WW conditions (no specific CO₂ uptake constraint; stomata open responding to WW condition); red numbers indicate fluxes under drought conditions (daytime CO₂ uptake constraint as 45% stomata closure compared to WW condition). Minor fluxes are not shown for simplicity, and therefore, not all shown input and output fluxes are equal. The unit for the flux values is mmol gDW⁻¹ day⁻¹.