Abstract
GDP-d-mannose pyrophosphorylase (GMPase) catalyzes the synthesis of GDP-d-mannose, which is a precursor for ascorbic acid (AsA) synthesis in plants. The rice genome encodes three GMPase homologs OsVTC1-1, OsVTC1-3 and OsVTC1-8, but their roles in AsA synthesis are unclear. The overexpression of OsVTC1-1 or OsVTC1-3 restored the AsA synthesis of vtc1-1 in Arabidopsis, while that of OsVTC1-8 did not, indicating that only OsVTC1-1 and OsVTC1-3 are involved in AsA synthesis in rice. Similar to Arabidopsis VTC1, the expression of OsVTC1-1 was high in leaves, induced by light, and inhibited by dark. Unlike OsVTC1-1, the expression level of OsVTC1-3 was high in roots and quickly induced by the dark, while the transcription level of OsVTC1-8 did not show obvious changes under constant light or dark treatments. In OsVTC1-1 RNAi plants, the AsA content of rice leaves decreased, and the AsA production induced by light was limited. In contrast, OsVTC1-3 RNAi lines altered AsA synthesis levels in rice roots, but not in the leaves or under the light/dark treatment. The enzyme activity showed that OsVTC1-1 and OsVTC1-3 had higher GMPase activities than OsVTC1-8 in vitro. Our data showed that, unlike in Arabidopsis, the rice GPMase homologous proteins illustrated a new model in AsA synthesis: OsVTC1-1 may be involved in the AsA synthesis, which takes place in leaves, while OsVTC1-3 may be responsible for AsA synthesis in roots. The different roles of rice GMPase homologous proteins in AsA synthesis may be due to their differences in transcript levels and enzyme activities.
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Author contribution
H.Q. measured the enzyme activities and the content of ascorbic acid, analyzed the expression of genes, analyzed the data, and wrote the article; Z.D. executed the measurement of enzyme activities and the expression of genes; C.Z.was involved in the generation of the transgenic plants; Y.W. was involving in measuring the ascorbic acid content of transgenic rice; J.W. was involved in measuring the ascorbic acid content of transgenic Arabidopsis. H.L. was involved in writing this article; Z.L.Z. was involved in data discussions; R.H. was involved in conceiving the project, designing the experiments and analyzing the data; and Z.J.Z. was involving in conceiving the project, designing the experiments, analyzing the data and writing the article. H.Q., Z.D. and C.Z. contributed equally to this work. R.H. and Z.J.Z. are the corresponding authors.
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Hua Qin, Zaian Deng and Chuanyu Zhang have contributed equally to this work.
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Fig. 1
The purified proteins of OsVTC1-1, OsVTC1-3, and OsVTC1-8 expressed in E. coli were identified by SDS-PAGE electrophoresis. –IPTG: the total protein from E. coli that was not induced by IPTG; +IPTG: the total protein from E. coli that was induced by IPTG; Purified: the purified recombinant protein. (JPEG 92 kb)
Fig. 2
The identification of transgenic Arabidopsis lines by western blotting. The total protein content was extracted from the leaves of 4-week-old transgenic lines, and the expression of exogenous proteins was analyzed by an anti-FLAG antibody. OE1-x, OE3-x, and OE8-x indicate the transgenic plants of OsVTC1-1, OsVTC1-3, and OsVTC1-8, respectively; “x” indicates the numbering of independent transgenic lines. Control indicates the transgenic lines transformed with the blank vector. (JPEG 78 kb)
Fig. 3
The expression of OsVTC1-1, OsVTC1-3, and OsVTC1-8 in OsVTC1-1 and OsVTC1-3 RI lines. The transcript levels of OsVTC1-1, OsVTC1-3, and OsVTC1-8 were determined using quantitative real-time PCR. The expression of Actin was used as the internal control. The transcript levels of OsVTC1-1, OsVTC1-3, and OsVTC1-8 in Zhonghua 17 were assigned as “1″ and the expression levels of OsVTC1-1, OsVTC1-3, and OsVTC1-8 in the RI lines were presented as the relative expression compared with those in Zhonghua 17. The assay was repeated three times. The bars represent the SE (±), and the asterisks indicate that results are significantly different from those in Zhonghua 17. The statistical significance was evaluated using the t test (** P < 0.01 and * P < 0.05). (JPEG 283 kb)
Table 1
Primers used in this paper. (DOCX 20 kb)
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Qin, H., Deng, Z., Zhang, C. et al. Rice GDP-mannose pyrophosphorylase OsVTC1-1 and OsVTC1-3 play different roles in ascorbic acid synthesis. Plant Mol Biol 90, 317–327 (2016). https://doi.org/10.1007/s11103-015-0420-0
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DOI: https://doi.org/10.1007/s11103-015-0420-0