Expression of Glycolate Oxidase in Rice Chloroplasts and Its Effects on Photosynthesis and Growth

doi:10.13560/j.cnki.biotech.bull.1985.2025-0550

Abstract

Abstract:

Objective Validate the hypothesis that plant chloroplasts possess the ability to metabolize glyoxylate into CO₂. Method In this study, the rice glycolate oxidase 1 (OsGLO1), glycolate oxidase 3 (OsGLO3) and catalase C (OsCATC) genes and the catalase (EcKAT) gene from Escherichia coli were cloned, and the chloroplast localization signal coding sequence RC2 was fused to their front ends; subsequently, GLO and CAT/KAT were combined in different combinations to construct different multi-gene expression vectors, which were directed its expression in rice chloroplasts, thereby catalyzing the production of glyoxylic acid from glycolic acid in chloroplasts. Result Results from transcript-level, protein-level, and enzyme activity analyses confirmed the high-efficiency expressions of target genes in rice and having normal catalytic function. However, all transgenic rice plants had phenotypes such as dwarfism, reduced tiller number, and decreased photosynthesis. Conclusion These findings indicate that only converting glycolate into glyoxylate in chloroplasts fails to enhance photosynthetic carbon fixation efficiency in rice, suggesting that rice chloroplasts may lack the capacity to catalyze glyoxylate into CO₂.

Key words: glycolate oxidase, catalase, glyoxylate, chloroplasts, photosynthesis, CO₂, rice, photorespiration

LI Bo-di, LI Zhi-chao, ZHU Guo-hui, PENG Xin-xiang, ZHANG Zhi-sheng. Expression of Glycolate Oxidase in Rice Chloroplasts and Its Effects on Photosynthesis and Growth[J]. Biotechnology Bulletin, 2025, 41(10): 87-97.

Figures/Tables 8

Table 1 Variation of column temperature during the determination of photorespiration-related metabolites by GCMS method

项目 Item

eating rate （℃/min）

升温速率 H

inal temperature （℃）

最终温度 F

保持时间 Holding time （min）

变化范围

Range of variation

Table 2 Retention times of photorespiration-related metabolites and their characteristic fragment ions

组分 Component	特征碎片离子大小 Characteristic fragment ion size	etention time （s）保留时间 R
乙醇酸 Glycollic acid	177	7.455
丙氨酸 Alanine	116	8.045
草酸 Oxalic acid	219	8.740
甘氨酸 Glycine	174	12.000
琥珀酸 Succinic acid	147	12.140
甘油酸 Glyceric acid	189	12.380
丝氨酸 Serine	204	12.870
苹果酸 Malic acid	117	14.743
α-酮戊二酸 α-ketoglutarate	198	15.910
核糖醇 Ribitol	73	17.760
柠檬酸 Citric acid	273	18.845

Fig. 1 Schematic diagram of GLO3-CAT pathway by the chloroplast photorespiratory metabolism in riceGLO: Glycolate oxidase 3. CAT: Catalase

Fig. 2 Acquirement and identification of transgenic rice plantsA: Transfomation procedure of rice (a1: screening of callus; a2: pre-differentiation; a3: differentiation; a4: rooting and strenghening). B: Western blot analysis of transgenic plants, GLO and CAT were detected by His monoclonal antibody in Western blot analysis. C-E: mRNA levels of transgenic plants (C: mRNA levels of OsGLO1; D: mRNA levels of OsGLO3; E: mRNA levels of CAT). F: GLO activity. G: CAT activity of G1K-OE and G3C-OE transgenic plants. H: DAB-staining of transgenic rice leaves. I: Relative expression of RbOH-b. G1-OE: Overexpressed line of chloroplast GLO1. G1K-OE: Over expressed line of chloroplast GLO1-KAT. G3-OE: Overexpressed line of chloroplast GLO3. G3C-OE: Overexpressed line of chloroplast GLO3-CAT. n=, *P＜0.05, **P＜0.01, ***P＜0.001, t-test. The same below

Fig. 3 Phenotypic observation of transgenic rice plantsA: Phenotypic observation of transgenic plants at seedling stage. B: Plant height. C: Maximum length of root. D: Transgenic rice plants of G1-OE and G1K-OE. E: Length of flag leaf. F: Width of flag leaf. G: Transgenic rice plants of G3-OE and G3C-OE. H: Plant height. I: Number of tillers. n=10

Fig. 4 Analysis of the photosynthetic-related parameters and carbohydrate content in the leaves of transigenic plants at tillering stage

Fig. 5 Content determination of photorespiration metabolites of GCGT tansgenic plants leaves at seedling stageTake the second leaf after hydroponic with Kimura B nutrient solution under natural light until the fourth leaf fully spreads, and the sampling time is 13:00‒15:00, n=3

Fig. 6 Analysis of mRNA levels of PLGG1 in transgenic rice plants (n=3)

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