A Partial C4 Photosynthetic Biochemical Pathway in Rice

Abstract

Introduction of a C₄ photosynthetic pathway into C₃ rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO₂ at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously accumulates four of the core C₄ photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME), and pyruvate phosphate dikinase (ZmPPDK). This led to enhanced enzyme activity and mild phenotypic perturbations but was largely neutral in its effects on photosynthetic rate. Measurements of the flux of ¹³CO₂ through photosynthetic metabolism revealed a significant increase in the incorporation of ¹³C into malate, consistent with increased fixation of ¹³CO₂ via PEP carboxylase in lines expressing the maize PEPC enzyme. However, there was no significant differences in labeling of 3-phosphoglycerate (3PGA) indicating that there was no carbon flux through NADP-ME into the Calvin-Benson cycle. There was also no significant difference in labeling of phosphoenolpyruvate (PEP) indicating that there was no carbon flux through PPDK. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labeling of malate, aspartate and citrate than in the quintuple line. There was evidence of ¹³C labeling of aspartate indicating ¹³CO₂ fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C₄ cycle, these results demonstrate for the first-time a partial flux through the carboxylation phase of NADP-ME C₄ metabolism in transgenic rice containing two of the key metabolic steps in the C₄ pathway.

Keywords: 13C labeling; C4 photosynthesis; C4 rice; NADP-malic enzyme; Oryza sativa (rice); malate; metabolic engineering; transgenic rice.

FIGURE 1

Soluble leaf protein. Immunoblots for (A) Quadruple and (B) Quintuple crosses. Maize (Zm), wild-type rice (WT), and plants of F₂ crosses (numbers). Protein was extracted from the youngest fully expanded leaf at mid-tillering stage. Samples were loaded on an equal leaf area basis (0.2364 mm² for ZmPEPC and ZmPPDK, and 2.364 mm² for ZmNADP-MDH, ZmNADP-ME, and OsGDCH).

FIGURE 2

(A) Leaf Chlorophyll content, (B) tiller number, and (C) plant height of wild-type (WT), Quadruple, and Quintuple lines. Chlorophyll SPAD values, tiller number, and plant height are means ± SE of eight individual F₂ plants and eight WT plants, 90 days post germination. Different letters within groups indicated those values that are statistically different based on a one-way ANOVA with a Tukey multiple comparison test for post hoc pairwise comparison, P-value < 0.05. ns indicates non-significant.

FIGURE 3

(A,B) Net CO₂ assimilation rate (A) in response to intercellular pCO₂ (Ci) and (C,D) photosynthetic photon flux density (PPFD). Measurements (A,B) were made at 2000 μmol photons m^–2 s^–1 under 21% O₂ (A,B) and 2% O₂ (B). Measurements (C,D) were made at a pCO₂ (C_a) of 400 μmol CO₂ mol air^–1. Values are means ± SE of three F₂ cross plants and three wild-type (WT) plants for quadruple comparisons and seven F₂ cross plants and four WT plants for quintuple comparisons. A Student’s t-test was performed for (C,D). Significant differences between WT and quintuple within PARi level are indicated by *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001.

FIGURE 4

¹³CO₂ pulse-labeling of wild-type, quadruple and quintuple rice cross lines. Fully expanded leaves at mid-tillering stage were pulse-labeled with ¹³CO₂ (300 ppm) for 60 s under steady state photosynthetic conditions. Isotopomers of malate, aspartate, 3PGA, PEP, and citrate + isocitrate were measured in extracts from pulse-labeled (60 s) and non-labeled (0 s) leaves by LC-MS/MS, and ¹³C enrichment (%) was calculated after correction for natural abundance. Values are means ± SE (n = 2–4). The original data are presented in Supplementary Dataset A. Different letters within groups indicated those values that are statistically different based on a one-way ANOVA, P-value < 0.05.