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Manipulation of the rice L-galactose pathway: evaluation of the effects of transgene overexpression on ascorbate accumulation and abiotic stress tolerance.

Zhang GY, Liu RR, Zhang CQ, Tang KX, Sun MF, Yan GH, Liu QQ - PLoS ONE (2015)

Bottom Line: The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated.The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants.Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China; Agricultural Science Institute of Coastal Region of Jiangsu, Yancheng 224002, Jiangsu, China.

ABSTRACT
Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3',5'-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress.

No MeSH data available.


Expression of ascorbate biosynthetic genes in rice roots, endosperm, stems, sheaths, old and young leaves.All tissues except young leaves were taken from DAF20 (20 days after flowering) plants. The young leaves were taken from 30 day-old plants. Data obtained by real-time RT-PCR were normalized using actin mRNA expression. Data shown are the average of three measurements with standard errors. Different letters indicate statistically significant differences at P<0.05 (ANOVA, Tukey post-host). The GeneBank accession numbers for OsGMP2, OsGME1, OsGME2, OsGGP, OsGPP, OsGDH, and OsGalLDH1 are AK122126, AK069385, AK102348, AK071188, AK071149, AK067039, and AK241565, respectively.
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pone.0125870.g003: Expression of ascorbate biosynthetic genes in rice roots, endosperm, stems, sheaths, old and young leaves.All tissues except young leaves were taken from DAF20 (20 days after flowering) plants. The young leaves were taken from 30 day-old plants. Data obtained by real-time RT-PCR were normalized using actin mRNA expression. Data shown are the average of three measurements with standard errors. Different letters indicate statistically significant differences at P<0.05 (ANOVA, Tukey post-host). The GeneBank accession numbers for OsGMP2, OsGME1, OsGME2, OsGGP, OsGPP, OsGDH, and OsGalLDH1 are AK122126, AK069385, AK102348, AK071188, AK071149, AK067039, and AK241565, respectively.

Mentions: The rice genome contains only one copy each of the OsPMM, OsGGP and OsGPP genes, while there are two copies of the OsGME genes (OsGME1 and OsGME2) and OsGalLDH (OsGalLDH1 and OsGalLDH2) and three copies of OsGMP genes (OsGMP1, OsGMP2 and OsGMP3) [37]. To increase our understanding of rice AsA biosynthesis, we first performed expression profiling of the genes involved in the final six L-galactose pathway steps in various tissues. As there is high identity (more than 90%) among the coding sequences of three OsGMP genes, and also very high identity (98%) between OsGalLDH1 and OsGalLDH2, the expression analysis was only carried out by using the primer according to the OsGMP2 or OsGalLDH1, which might represent the whole of three OsGMP genes or two OsGalLDH genes, respectively. As shown in Fig 3, this analysis demonstrated expression of all tested genes in all tissues; however, their transcription levels were quite low in root, stem and developing endosperm samples compared with their levels in leaves. Moreover, their expression in young leaves from 4-week-old seedlings were much greater than in old leaves from plants 20 days after flowering, implying that the expression of L-galactose pathway was enriched in young rice leaves.


Manipulation of the rice L-galactose pathway: evaluation of the effects of transgene overexpression on ascorbate accumulation and abiotic stress tolerance.

Zhang GY, Liu RR, Zhang CQ, Tang KX, Sun MF, Yan GH, Liu QQ - PLoS ONE (2015)

Expression of ascorbate biosynthetic genes in rice roots, endosperm, stems, sheaths, old and young leaves.All tissues except young leaves were taken from DAF20 (20 days after flowering) plants. The young leaves were taken from 30 day-old plants. Data obtained by real-time RT-PCR were normalized using actin mRNA expression. Data shown are the average of three measurements with standard errors. Different letters indicate statistically significant differences at P<0.05 (ANOVA, Tukey post-host). The GeneBank accession numbers for OsGMP2, OsGME1, OsGME2, OsGGP, OsGPP, OsGDH, and OsGalLDH1 are AK122126, AK069385, AK102348, AK071188, AK071149, AK067039, and AK241565, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418601&req=5

pone.0125870.g003: Expression of ascorbate biosynthetic genes in rice roots, endosperm, stems, sheaths, old and young leaves.All tissues except young leaves were taken from DAF20 (20 days after flowering) plants. The young leaves were taken from 30 day-old plants. Data obtained by real-time RT-PCR were normalized using actin mRNA expression. Data shown are the average of three measurements with standard errors. Different letters indicate statistically significant differences at P<0.05 (ANOVA, Tukey post-host). The GeneBank accession numbers for OsGMP2, OsGME1, OsGME2, OsGGP, OsGPP, OsGDH, and OsGalLDH1 are AK122126, AK069385, AK102348, AK071188, AK071149, AK067039, and AK241565, respectively.
Mentions: The rice genome contains only one copy each of the OsPMM, OsGGP and OsGPP genes, while there are two copies of the OsGME genes (OsGME1 and OsGME2) and OsGalLDH (OsGalLDH1 and OsGalLDH2) and three copies of OsGMP genes (OsGMP1, OsGMP2 and OsGMP3) [37]. To increase our understanding of rice AsA biosynthesis, we first performed expression profiling of the genes involved in the final six L-galactose pathway steps in various tissues. As there is high identity (more than 90%) among the coding sequences of three OsGMP genes, and also very high identity (98%) between OsGalLDH1 and OsGalLDH2, the expression analysis was only carried out by using the primer according to the OsGMP2 or OsGalLDH1, which might represent the whole of three OsGMP genes or two OsGalLDH genes, respectively. As shown in Fig 3, this analysis demonstrated expression of all tested genes in all tissues; however, their transcription levels were quite low in root, stem and developing endosperm samples compared with their levels in leaves. Moreover, their expression in young leaves from 4-week-old seedlings were much greater than in old leaves from plants 20 days after flowering, implying that the expression of L-galactose pathway was enriched in young rice leaves.

Bottom Line: The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated.The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants.Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China; Agricultural Science Institute of Coastal Region of Jiangsu, Yancheng 224002, Jiangsu, China.

ABSTRACT
Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3',5'-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress.

No MeSH data available.