Abstract
GDP-mannose pyrophosphorylase (GMP) catalyzed the formation of GDP-mannose, which serves as a donor for the biosynthesis of mannose-containing polysaccharides. In this study, three GMP genes from Dendrobium officinale (i.e., DoGMPs) were cloned and analyzed. The putative 1000 bp upstream regulatory region of these DoGMPs was isolated and cis-elements were identified, which indicates their possible role in responses to abiotic stresses. The DoGMP1 protein was shown to be localized in the cytoplasm. To further study the function of the DoGMP1 gene, 35S:DoGMP1 transgenic A. thaliana plants with an enhanced expression level of DoGMP1 were generated. Transgenic plants were indistinguishable from wild-type (WT) plants in tissue culture or in soil. However, the mannose content of the extracted water-soluble polysaccharides increased 67%, 96% and 92% in transgenic lines #1, #2 and #3, respectively more than WT levels. Germination percentage of seeds from transgenic lines was higher than WT seeds and the growth of seedlings from transgenic lines was better than WT seedlings under salinity stress (150 mM NaCl). Our results provide genetic evidence for the involvement of GMP genes in the biosynthesis of mannose-containing polysaccharides and the mediation of GMP genes in the response to salt stress during seed germination and seedling growth.
Highlights
Previous studies demonstrated that mannan synthase isolated from plant species such as pea (Pisum sativum L.), fenugreek (Trigonella foenum-graecum L.) and guar (Cyamopsis tetragonoloba L.), used GDP-mannose as a substrate to synthesize a mannan backbone in vitro[13,14]
The complete DoGMP1 cDNA contains 1528 bp with an open reading frame (ORF) of 1086 bp encoding a protein of 361 amino acid residues with a calculated molecular mass of 39.373 kDa
DoGMP3 contains an ORF of 1086 bp encoding a protein of 361 amino acid residues with a calculated molecular mass of 39.604 kDa
Summary
Previous studies demonstrated that mannan synthase isolated from plant species such as pea (Pisum sativum L.), fenugreek (Trigonella foenum-graecum L.) and guar (Cyamopsis tetragonoloba L.), used GDP-mannose as a substrate to synthesize a mannan backbone in vitro[13,14]. Transformants of potato (Solanum tuberosum L.) with reduced GMP activity had 30–50% lower mannose content than WT plants[4]. The vast majority of the characterized GMPs from A. thaliana, rice or other higher plant species have been analyzed, it has been proposed that the involvement of GMPs in AsA synthesis and stress tolerance is conserved. It is not possible to predict gene functions of possible GMPs based only on nucleotide or amino acid sequence similarities. A probable GDP-mannose pyrophosphorylase (TAIR number: AT1G74910), predicted by amino acid sequence lacks GDP-mannose pyrophosphorylase activity[18]. We generated 35S:DoGMP1 A. thaliana transgenic lines, studied the relationship between DoGMP1 and mannose content of water-soluble polysaccharides, and assessed the tolerance of these lines to salinity stress. This work has implications for the development of abiotic stress-tolerant crops to overcome environmental stress limitations and improve production efficiency in the face of a burgeoning world population
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