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Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.  相似文献   

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Insight into the role of sugars in bud burst under light in the rose   总被引:1,自引:0,他引:1  
Bud burst is a decisive process in plant architecture that requires light in Rosa sp. This light effect was correlated with stimulation of sugar transport and metabolism in favor of bud outgrowth. We investigated whether sugars could act as signaling entities in the light-mediated regulation of vacuolar invertases and bud burst. Full-length cDNAs encoding two vacuolar invertases (RhVI1 and RhVI2) were isolated from buds. Unlike RhVI2, RhVI1 was preferentially expressed in bursting buds, and was up-regulated in buds of beheaded plants exposed to light. To assess the importance of sugars in this process, the expression of RhVI1 and RhVI2 and the total vacuolar invertase activity were further characterized in buds cultured in vitro on 100 mM sucrose or mannitol under light or in darkness for 48 h. Unlike mannitol, sucrose promoted the stimulatory effect of light on both RhVI1 expression and vacuolar invertase activity. This up-regulation of RhVI1 was rapid (after 6 h incubation) and was induced by as little as 10 mM sucrose or fructose. No effect of glucose was found. Interestingly, both 30 mM palatinose (a non-metabolizable sucrose analog) and 5 mM psicose (a non-metabolizable fructose analog) promoted the light-induced expression of RhVI1 and total vacuolar invertase activity. Sucrose, fructose, palatinose and psicose all promoted bursting of in vitro cultured buds under light. These findings indicate that soluble sugars contribute to the light effect on bud burst and vacuolar invertases, and can function as signaling entities.  相似文献   

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By using immunolocalization and differential extraction methods we show that only apoplastic invertase, but not vacuolar invertase, was present in the mature, sucrose-accumulating L. hirsutum pericarp. In contrast, in the hexose-accumulating L. esculentum fruit, both the apoplastic and vacuolar invertase activities and protein content increase in the mature fruit. Quantitative expression studies of the soluble invertase gene (TIV1) and the apoplastic invertase genes (LINs) showed that only TIV1 gene expression could account for the species and developmental differences of both soluble and insoluble enzyme activity of the pericarp. The expression of the LIN genes encoding for apoplastic tomato invertases was unrelated to the differences in bound enzyme activity and could not account for the rise in bound invertase activity in the mature L. esculentum fruit. Evidence is presented that the bound invertase activity of tomato fruit is also the TIV1 gene product. The presence of apoplastic invertase in the mature sucrose-accumulating L. hirsutum fruit suggests a hydrolysis-resynthesis mechanism of sucrose uptake. In order to test this hypothesis, we studied short- and long-term uptakes of asymmetrically labelled 3H-fructosyl-sucrose accompanied by compartmental analysis of the sugars in attached whole fruits of L. hirsutum and L. esculentum. The results indicate that hydrolysis-resynthesis is slow in the sucrose-accumulating fruit but is not an integral part of an uptake and compartmentation mechanism.  相似文献   

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Plant protein inhibitors of invertases   总被引:12,自引:0,他引:12  
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Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp161 and Asn166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait.  相似文献   

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Although a lot of vacuolar invertase (EC 3.2.1.26) cDNAs are available from a diversity of plant species, up to now no sequence information is available on invertases from any dicot fructan-containing species. Therefore, we describe the cloning of vacuolar acid invertase cDNA from etiolated Belgian endive leaves ( Cichorium intybus L. var. foliosum cv. Flash), formed throughout the forcing process of the witloof chicory roots. Full-length cDNA was obtained by a combination of RT-PCR, PCR and 5'- and 3' RACE RT-PCR, starting with primers based on conserved amino acid sequences. The cloned chicory acid invertase groups together with vacuolar type invertases and fructan biosynthetic enzymes. A putative role for vacuolar type invertases in fructan synthesizing plants is discussed.  相似文献   

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This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall invertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl transferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar invertase, 1-FEH I might have evolved from a cell-wall invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.  相似文献   

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Different plant invertase isoenzymes are characterized by a single amino-acid difference in a conserved sequence, the WEC-P/V-D box. A proline residue is present in this sequence motif of extracellular invertase sequences, whereas a valine is found at the same position of vacuolar invertase sequences. The role of this distinct difference was studied by substituting the proline residue of extracellular invertase CIN1 from Chenopodium rubrum with a valine residue, by site-directed mutagenesis. The mutated gene was heterologously expressed in an invertase-deficient Saccharomyces cerevisiae strain. The single amino-acid difference was shown to be the molecular basis for two enzymatic properties of invertase isoenzymes, for both the pH optimum and the substrate specificity. A proline in the WEC-P/V-D box determines the more acidic pH optimum and the higher cleavage rate of raffinose of extracellular invertases, compared to vacuolar invertases that have a valine residue at this position.  相似文献   

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G Q Tang  M Lüscher    A Sturm 《The Plant cell》1999,11(2):177-189
To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.  相似文献   

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Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.  相似文献   

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