Metabolome and photochemical analysis of rice plants overexpressing Arabidopsis NAD kinase gene

Abstract

The chloroplastic NAD kinase (NADK2) is reported to stimulate carbon and nitrogen assimilation in Arabidopsis (Arabidopsis thaliana), which is vulnerable to high light. Since rice (Oryza sativa) is a monocotyledonous plant that can adapt to high light, we studied the effects of NADK2 expression in rice by developing transgenic rice plants that constitutively expressed the Arabidopsis chloroplastic NADK gene (NK2 lines). NK2 lines showed enhanced activity of NADK and accumulation of the NADP(H) pool, while intermediates of NAD derivatives were unchanged. Comprehensive analysis of the primary metabolites in leaves using capillary electrophoresis mass spectrometry revealed elevated levels of amino acids and several sugar phosphates including ribose-1,5-bisphosphate, but no significant change in the levels of the other metabolites. Studies of chlorophyll fluorescence and gas change analyses demonstrated greater electron transport and CO2 assimilation rates in NK2 lines, compared to those in the control. Analysis of oxidative stress response indicated enhanced tolerance to oxidative stress in these transformants. The results suggest that NADP content plays a critical role in determining the photosynthetic electron transport rate in rice and that its enhancement leads to stimulation of photosynthesis metabolism and tolerance of oxidative damages.

Figure 1.

Overexpression of Arabidopsis NADK2 in transgenic rice plants. A, Construction of the chimeric gene used for generation of Arabidopsis NADK2-overexpressing transgenic plants. The coding sequence of Arabidopsis NADK gene (AtNADK2) was cloned between the maize ubiquitin promoter and nos terminator. B, RT-PCR analysis of the AtNADK2 transgene expression. Total RNA was prepared from the vector control (C) or the NK2 lines. PCR primers specific to the NADK2 transgene or β-tubulin were used. C, Total NADK activity in leaf samples from three NK2 rice lines and vector control. Samples were taken from 1-month-old plants. Data are mean ± sem of measurements of five individual plants per line. *P < 0.05, compared with the vector control (by Student's t test).

Figure 2.

Pyridine nucleotide contents and ratio in transgenic rice plants. Values are mean ± sem of measurements of four plants per line. *P < 0.05, compared with the vector control. FW, Fresh weight.

Figure 3.

Summary of metabolite contents of leaves of transgenic rice plants. Quantitative comparison of amino acids (A), organic acids (B), and sugar phosphates (C). Values are the mean ± sem of measurements of four plants per line. *P < 0.05, compared with the vector control (by Student's t test). FW, Fresh weight; Mal, malate; Cit, citrate; Isocit, isocitrate; Succ, succinate; G6P, Glc-6-P; PGA, 3-phosphoglyceric acid; PEP, phosphoenolpyruvate.

Figure 4.

PCA of metabolites. A, Scores of PCA are presented based on a combination of two components (PC1 and PC2) and variances (26.8% for PC1 and 12.8% for PC2) of each component in the sample set. B and C, Loadings of metabolites with PC1 (B) and PC2 (C) components. The vertical axis represents the PC loading value.

Figure 5.

Metabolite profiling in transgenic rice plants. A, Hierarchical classification of metabolites. B, Heatmap corresponding to metabolites in relation to transgenic rice plants.

Figure 6.

Irradiance dependency of net CO₂ assimilation and chl fluorescence parameters in leaves of the vector control and NK2 lines. A, Light intensity dependence of ETR. B, Light intensity dependence of NPQ of chl fluorescence. C, Net CO₂ assimilation. Values are mean ± sd of determinations on five individual plants per line. The plants were kept in dark for 30 min before analysis.

Figure 7.

Ascorbate and GSH contents in rice plants. Values are mean ± sem of measurements of five plants per line. *P < 0.05, compared with the vector control (by Student's t test). C, Vector control; FW, fresh weight.

Figure 8.

Effects of MV on rice plants. Leaf discs of 10 mm diameter were floated on 10 μm MV and illuminated at 100 μmol photon m⁻² s⁻¹ for 3 and 6 h. A, F_v/F_m. B, MV-induced pigment degradation. C, MV-induced membrane damage. Ion leakage was estimated by measuring the increase in conductivity of the medium after MV treatment of leaf discs. D, Lipid peroxidation expressed as the MDA content in the vector control and NK2 lines. FW, Fresh weight. Values are the mean ± sem of measurements of five plants per line. *P < 0.05, compared with the vector control (by Student's t test).