Cloning and characterization of a soluble acid invertase-encoding gene from muskmelon.
ABSTRACT Soluble acid invertase (S-AIV; EC 22.214.171.124) is thought to play a critical role in sucrose hydrolysis in muskmelon (Cucumis melo L.) fruit. A full-length cDNA clone encoding S-AIV was isolated from muskmelon by RT-PCR and RACE. The clone, designated as CmS-AIV1, contains 2178 nucleotides with an open reading frame of 1908 nucleotides. The deduced 636 amino acid sequence showed high identities with other plant soluble acid invertases. Northern blot analysis indicated that CmS-AIV1 was expressed in flowers and fruit, but was not detected in roots, stems or leaves. Moreover, the mRNA accumulation of CmS-AIV1 showed its maximum level at 10 days after pollination (DAP) and decreased gradually during fruit development until its minimum level at mature fruit. Interestingly, the sucrose content was very low in fruit before 20 DAP but increased dramatically between 20 and 30 DAP during fruit development. In contrast to sucrose content, the activities of S-AIV was very high in fruit before 20 DAP and decreased apparently between 20 and 30 DAP, suggesting that sucrose metabolism may be linked to the CmS-AIV1 transcript level in muskmelon fruit.
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ABSTRACT: Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyze sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn 184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.Biochimie 12/2013; DOI:10.1016/j.biochi.2013.12.013 · 3.14 Impact Factor
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ABSTRACT: Carbohydrates are the most important biochemical compounds in determining the quality of muskmelon fruit, and their accumulation in the muskmelon (Cucumis melo L.) fruits as substrate tub plants in solar greenhouse with different scion/rootstock graft combinations were studied. The results indicated that Zhongmi1/Shengzhen1 graft combination (GS) reduced all kinds of main sugars contents except galactinol and stachyose throughout the whole fruit development. However, the effects of Zhongmi1/RibenStrong graft combination (GR) on carbohydrates accumulation was less compared to GS, in which sucrose content was lower than that in self-rooted muskmelon (CK) only on 32 DAA, decreased raffinose content merely on 48 DAA, increased stachyose content only on 16 DAA and reduced total sugar accumulation dramatically. Grafting (GR and GS) enhanced starch content in the later fruit development. On the other hand, grafting GS significantly enhanced acid invertase or neutral invertase activity during the whole development, sucrose synthase activity only on 8 DAA and reduced sucrose phosphate synthase activity dramatically compared to CK during 16 to 48 DAA. Grafting GR increased acid invertase activity only on 24 and 40 DAA and sucrose phosphate synthase activity dramatically on 48 DAA. Grafting significantly increased alkaline �-galactosidase activity during 8 to 24 DAA; there were no significant differences between 32 to 48 DAA. Overall, considering the carbohydrate accumulation, Zhongmi1/RibenStrong graft combination was a superior graft combination, followed by Zhongmi1/Shengzhen1 graft combination.
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ABSTRACT: In plants, vacuolar invertase (β-fructofuranosidase, EC 126.96.36.199) is known to play as a key modulator for hexose accumulation and cell expansion. In this study, two cDNA clones (2,013 and 1,945 bp, with 99 % sequence identity) encoding vacuolar invertase isoforms were isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR. The corresponding predicted proteins consisted of 635 amino acids (designated as KC-VIN1, lacking a few amino acids at N-terminus) and 639 amino acids (designated as KC-VIN2), respectively. They showed 99 % identity, and found to vary at several locations with mostly non-conservative substitutions. Multiple sequence alignment of vacuolar invertase homologs covering four Solanaceae family members revealed some notable distinguishing sequence features (signature-type sequences). A consensus sequence was predicted using 45 vacuolar invertase sequences from 27 taxonomically different plant species, and a phylogenetic tree was generated to know the evolutionary relation between them. Hydrophobic characters were predicted, and compared in different plant species. All these data are presented in a comprehensive manner which were not documented in the earlier reports. As a preliminary study, vacuolar invertase expression patterns in the tubers of some Indian potato cultivars were analyzed by semi-quantitative RT-PCR and extractable enzyme assay. In all the potato cultivars, the overall expression level of invertase was found to be considerably higher after storage at low temperature as compared to the freshly harvested tubers.Acta Physiologiae Plantarum 07/2013; 35(7). DOI:10.1007/s11738-013-1240-y · 1.52 Impact Factor