Installation of C4 photosynthetic pathway enzymes in rice using a single construct

Abstract

Introduction of a C₄ photosynthetic mechanism into C₃ crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C₄ rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. ¹³ CO₂ labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C₄ proteins introduced thus far, a functional C₄ pathway is achievable in rice.

Keywords: C4 photosynthesis; metabolic engineering; rice.

Figure 1

Expression of C₄ enzymes in O. sativa. (a) Transcript abundance (in transcripts per million) of Z. mays transgenes and orthologous to them endogenous genes in wild‐type (WT) and three transgenic rice lines. CA, carbonic anhydrase; MDH, NADP‐malate dehydrogenase; NADP‐ME, NADP‐dependent malic enzyme; PEPC, PEP carboxylase; PPDK, pyruvate orthophosphate dikinase. Mean ± SD, n = 3 biological replicates. (b) Immunodetection of proteins in leaf extracts loaded on leaf area basis. Z. mays leaf extract dilution series was used for relative quantification; three plants from each transgenic line were analysed. Signal from RbcL (the large subunit of ribulose bisphosphate carboxylase oxygenase) was used as loading control. (c) Confocal micrographs of protein localization on leaf cross‐sections. Fluorescence signals are pseudo‐coloured: green ‐ protein of interest labelled with secondary antibodies conjugated with Alexa Fluor 488; magenta ‐ chlorophyll autofluorescence; blue ‐ calcofluor white‐stained cell walls. BS, bundle sheath; M, mesophyll. Scale bars = 20 µm. Localization of C₄ enzymes in transgenic lines 1 and B6 is presented in Figure S1 and the summary of localization is presented in Table S1.

Figure 2

¹³C enrichment (%) during ¹³CO₂‐pulse labelling of wild‐type (WT) rice and three transgenic lines expressing enzymes of the C₄ metabolic pathway. 3PGA, 3‐phosphoglycerate; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; RuBP, ribulose‐1,5‐bisphosphate; 2PG, 2‐phosphoglycolate. The x‐axes show the pulse labelling time on a log scale. Values from the 30 s time point are mean ± SD, n = 3‐4 biological replicates. Values at all other time points are from individual samples or means of two biological replicates. Abundances of individual isotopomers are shown in Figure 3 and Figure S9. The original data are presented in Data S1.

Figure 3

Isotopomer distribution (%) of malate and aspartate during ¹³CO₂‐pulse labelling of wild‐type (WT) rice and three transgenic lines expressing enzymes of the C₄ metabolic pathway. The relative abundance of each isotopomer (m_n ) for a given metabolite is represented, where n is the number of ¹³C atoms incorporated. The x‐axes show the pulse labelling time on a log scale. Values from the 30 s time point are mean ± SD, n = 3‐4 biological replicates. Values at all other time points are from individual samples or means of two biological replicates. Isotopomer abundances for further metabolites are shown in Figure S9. The original data are presented in Data S1.

Figure 4

Gas‐exchange and fluorescence analysis of wild‐type (WT) O. sativa and three transgenic lines expressing enzymes of the C₄ metabolic pathway. (a) A, net CO₂ assimilation rate; g_{s_H2O}, leaf stomatal conductance to water vapour; ETR/4, light‐driven electron transport rate through PHotosystem II divided to four, measured at different intercellular pCO₂ (C_i), PPFD of 1500 µmol m^‐2 s^‐1 and ambient pO₂ (20 kPa). Parameters determined by fitting the A‐C_i response curves and statistical analysis are provided in Table 2. (b) Leaf CO₂ compensation point (Г). Mean ± SE, n = 5 biological replicates for WT, n = 4 otherwise. Statistical analysis was performed using one‐way ANOVA and Tukey’s post hoc test, letters indicate significant differences between the groups (α > 0.05) .

Figure 5

Gas‐exchange and fluorescence analysis of wild‐type (WT) O. sativa and three transgenic lines expressing enzymes of the C₄ metabolic pathway grown at lower irradiance of about 200 µmol m^‐2 s^‐1 during the first 20 min after dark‐to‐light transition. Measurements were done on 40‐min dark‐adapted leaves at PPFD of 500 µmol m^‐2 s^‐1 and ambient pO₂ (20 kPa). A, net CO₂ assimilation rate; g_{s_H2O}, leaf stomatal conductance to water vapour; C_i/C_a, ratio between intercellular and ambient pCO₂; Φ_PSII, quantum efficiency of Photosystem II; NPQ, non‐photochemical quenching. Mean ± SE, n = 5 biological replicates for WT, n = 4 otherwise (α> 0.05). Statistical analysis was performed at 5, 10, 15 and 20 min after the beginning of illumination using one‐way ANOVA and Tukey’s post hoc test, letters indicate significant differences between the groups (α > 0.05); ns, not significant.