Vacuolar Invertase Research Articles

Sugars take a central position in plant growth, development and, stress responses. A focus on apical dominance.

Sugars take a central position in plant growth, development and, stress responses. A focus on apical dominance.

Exaggerated root respiration accounts for growth retardation in a starchless mutant of Arabidopsis thaliana

The knock-out mutation of plastidial phosphoglucomutase (pgm) causes a starchless phenotype in Arabidopsis thaliana, and results in a severe growth reduction of plants cultivated under diurnal conditions. It has been speculated that high soluble sugar levels accumulating during the light phase in leaf mesophyll might cause a reduction of photosynthetic activity or that shortage of reduced carbon during the night is the reason for the slow biomass gain of pgm. Separate simultaneous measurements of leaf net photosynthesis and root respiration demonstrate that photosynthetic activity per unit fresh weight is not reduced in pgm, whereas root respiration is strongly elevated. Comparison with a mutant defective in the dominating vacuolar invertase (AtβFruct4) revealed that high sucrose concentration in the cytosol, but not in the vacuole, of leaf cells is responsible for elevated assimilate transport to the root. Increased sugar supply to the root, as observed in pgm mutants, forces substantial respiratory losses. Because root respiration accounts for 80% of total plant respiration under long-day conditions, this gives rise to retarded biomass formation. In contrast, reduced vacuolar invertase activity leads to reduced net photosynthesis in the shoot and lowered root respiration, and affords an increased root/shoot ratio. The results demonstrate that roots have very limited capacity for carbon storage but exert rigid control of supply for their maintenance metabolism.

Cloning, 3D modeling and expression analysis of three vacuolar invertase genes from cassava (Manihot Esculenta Crantz).

Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (β-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed β-propeller module at N-terminus domain, and forms a β-sandwich module at the C-terminus domain. The active site of the protein is situated in the β-propeller module. MeVINVs are classified in two subfamilies, α and β groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while β group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development.

Silencing the vacuolar invertase gene GhVIN1 blocks cotton fiber initiation from the ovule epidermis, probably by suppressing a cohort of regulatory genes via sugar signaling

Cotton fibers, the most important source of cellulose for the global textile industry, are single-celled trichomes derived from the ovule epidermis at or just prior to anthesis. Despite progress in understanding cotton fiber elongation and cell-wall biosynthesis, knowledge regarding the molecular basis of fiber cell initiation, the first step of fiber development determining the fiber yield potential, remains elusive. Here, we provide evidence that expression of a vacuolar invertase (VIN) is an early event that is essential for cotton fiber initiation. RNAi-mediated suppression of GhVIN1, a major VIN gene that is highly expressed in wild-type fiber initials, resulted in significant reduction of VIN activity and consequently a fiberless seed phenotype in a dosage dependent manner. The absence of a negative effect on seed development in these fiberless seeds indicates that the phenotype is unlikely to be due to lack of carbon nutrient. Gene expression analyses coupled with in vitro ovule culture experiments revealed that GhVIN1-derived hexose signaling may play an indispensable role in cotton fiber initiation, probably by regulating the transcription of several MYB transcription factors and auxin signaling components that were previously identified as required for fiber initiation. Together, the data represent a significant advance in understanding the mechanisms of cotton fiber initiation, and provide the first indication that VIN-mediated hexose signaling may act as an early event modulating the expression of regulatory genes and hence cell differentiation from the ovule epidermis.

Vacuolar Invertase Gene Silencing in Potato (Solanum tuberosum L.) Improves Processing Quality by Decreasing the Frequency of Sugar-End Defects

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.

Cloning and Functional Analysis of GhVacInc2a Encoding Vacuolar Invertase in Cotton

Cloning and Functional Analysis of <i>GhVacInc2a</i> Encoding Vacuolar Invertase in Cotton

Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse 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 Asn184), 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.

Differential responses of root growth, acid invertase activity and transcript level to copper stress in two contrasting populations of Elsholtzia haichowensis

The present study aimed to test a hypothesis that acid invertases in root of metallophytes might play important roles in root growth under heavy metal stress. Plants of two contrasting populations, one from an ancient Cu mine (MP) and the other from a non-contaminated site (NMP), of metallophyte Elsholtzia haichowensis were treated with Cu in controlled experiments. The results showed that MP was Cu tolerant under 10 μM Cu2+ treatment. Cu treatment resulted in a higher root/shoot biomass ratio in MP compared to NMP. Scaling exponent in root/shoot allometric function in MP was lower than NMP. More complicated root architecture was observed in MP under Cu stress. Four full-length cDNAs (EhNcwINV, EhCcwINV, EhNvINV and EhCvINV) encoding cell wall and vacuolar invertases were cloned. Both of the transcript level and activity of the acid invertase in MP elevated under Cu treatment. There were positive correlations between root acid invertase transcript level, activity and root/shoot biomass ratio. The results indicated important roles of acid invertase in governing root growth under Cu stress. It also suggested that there was a possible interrelation between acid invertases and Cu tolerance mechanisms in MP of E. haichowensis.

Interaction proteins of invertase and invertase inhibitor in cold-stored potato tubers suggested a protein complex underlying post-translational regulation of invertase

The activity of vacuolar invertase (VI) is vital to potato cold-induced sweetening (CIS). A post-translational regulation of VI activity has been proposed which involves invertase inhibitor (VIH), but the mechanism for the interaction between VI and VIH has not been fully understood. To identify the potential partners of VI and VIH, two cDNA libraries were respectively constructed from CIS-resistant wild potato species Solanum berthaultii and CIS-sensitive potato cultivar AC035-01 for the yeast two-hybrid analysis. The StvacINV1 (one of the potato VIs) and StInvInh2B (one of the potato VIHs), previously identified to be associated with potato CIS, were used as baits to screen the two libraries. Through positive selection and sequencing, 27 potential target proteins of StvacINV1 and eight of StInvInh2B were clarified. The Kunitz-type protein inhibitors were captured by StvacINV1 in both libraries and the interaction between them was confirmed by bimolecular fluorescence complementation assay in tobacco cells, reinforcing a fundamental interaction between VI and VIH. Notably, a sucrose non-fermenting-1-related protein kinase 1 was captured by both the baits, suggesting that a protein complex could be necessary for fine turning of the invertase activity. The target proteins clarified in present research provide a route to elucidate the mechanism by which the VI activity can be subtly modulated.

The role of sucrose-metabolizing enzymes in pear fruit that differ in sucrose accumulation

In the paper, the soluble sugar composition and activities of enzymes metabolizing sucrose: invertase (β-fructosidase, EC 3.2.1.26), sucrose synthase (SS; EC 2.4.1.13) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) were investigated during fruit development of two pear species: Pyrus bretschneideri Rehd. cv. ‘Yali’ and P. pyrifolia Nakai cv. ‘Aikansui’, characterized as low and high sucrose types, respectively. It was found that, at the end of fruit development of ‘Aikansui’, the level of sucrose was five times higher than in ‘Yali’ in the same period. It was coincident with the significantly higher activities of SS (synthesis) and SPS and lower activities of invertase (vacuolar and cell wall-bound acid invertase and neutral invertase). The high correlation was found between sucrose level and SS (synthesis) and SPS activities in ‘Aikansui’ pears.

Effects of Copper Stress on Sucrose Metabolism in Seed of Two &lt;i&gt;Rumex dentatus L.&lt;/i&gt; Populations

Two Rumex dentatus populations, one from copper (Cu) mine and the other from uncontaminated site, were studied for sucrose metabolism of seed under Cu stress. Cu treatment significantly inhibited vacuolar invertase activity in non-metallicolous population (NMP), while it in metallicolous population (MP) was markedly higher than control. At control, cell wall invertase activity in MP showed little difference from NMP. However, after Cu treatment, cell wall invertase activity in MP was significantly higher than NMP. Activity of N/A-Inv was not affected by population or Cu treatment. Compared with control, sucrose and reducing sugars contents in MP were increased by Cu treatment, but those in NMP was decreased. Cu concentration in seed of NMP was higher than MP under Cu stress. These results suggested less Cu transported to seed and efficient sucrose metabolism in seed of MP might be partly reasons why R. dentatus can successful reproduction at Cu enriched soils.

Effects of auxin and methyl jasmonate on cut rose petal growth through activation of acid invertase

Petal growth associated with flower opening depends on cell expansion caused by water influx. To understand the mechanism of flower opening and to develop a method for improving cut flower quality, we investigated the changes in the amount of soluble carbohydrates and invertase activities in rose (Rosa ‘Meivildo’) petals using attached and cut flowers, and the effects of α-napthylacetic acid (NAA) and methyl jasmonate (MeJA) on cut flowers. Cut rose flowers were harvested at the tight bud stage (TB), or mature bud stage (MB), which is the commercial harvest stage, and 2 days after TB. Cut flowers were immediately treated with deionized water or 1% (w/v) glucose. Fresh weight (FW) and the levels of soluble carbohydrates in petals in cut flowers were lower under postharvest conditions than those of attached flowers during flower opening. Although invertase activities in petals of attached flowers increased drastically during TB and MB, those in the petals harvested at TB did not increase under postharvest conditions. These results suggested that sucrose metabolism, including invertase activity, could be an important factor in cut rose flower opening to a greater extent after harvest. In addition, we tried to control flower opening by affecting invertase activities in petals of cut rose flowers. Cut flowers treated with NAA opened faster and those treated with MeJA opened later than in controls. Levels of soluble carbohydrates and invertase activity in petals were also changed by these treatments. In flowers treated with NAA, activities of both vacuolar and cell wall invertases increased 1 day after treatment and then decreased through to the end of the treatment, although activities in control flowers never increased after harvested. By contrast, cell wall invertase activity in MeJA treated flowers increased 1 day later than with the NAA treatment and remained at a relatively high level until 4 days after treatment compared to the controls. Our results suggest that inducing invertase activity in postharvest conditions may important for the quality of cut roses.

Trafficking of Plant Vacuolar Invertases: From a Membrane-Anchored to a Soluble Status. Understanding Sorting Information in Their Complex N-Terminal Motifs

Vacuolar invertases (VIs) are highly expressed in young tissues and organs. They may have a substantial regulatory influence on whole-plant metabolism as well as on photosynthetic efficiency. Therefore, they are emerging as potentially interesting biotechnological targets to increase plant biomass production, especially under stress. On the one hand, VIs are well known as soluble and extractable proteins. On the other hand, they contain complex N-terminal propeptide (NTPP) regions with a basic region (BR) and a transmembrane domain (TMD). Here we analyzed in depth the Arabidopsis thaliana VI2 (AtVI2) NTPP by mutagenesis. It was found that correct sorting to the lytic vacuole (LV) depends on the presence of intact dileucine (SSDALLPIS), BR (RRRR) and TMD motifs. AtVI2 remains inserted into membranes on its way to the LV, and the classical sorting pathway (endoplasmic reticulum→Golgi→LV) is followed. However, our data suggest that VIs might follow an alternative, adaptor protein 3 (AP3)-dependent route as well. Membrane-anchored transport and a direct recognition of the dileucine motif in the NTPP of VIs might have evolved as a simple and more efficient sorting mechanism as compared with the vacuolar sorting receptor 1/binding protein of 80 kDa (VSR1/BP80)-dependent sorting mechanism followed by those proteins that travel to the vacuole as soluble proteins.

Profiling of StvacINV1 Expression in Relation to Acid Invertase Activity and Sugar Accumulation in Potato Cold-Stored Tubers

Reducing sugars (RS) accumulation in potato (Solanum tuberosum L.) tubers stored at low temperature, a phenomenon known as cold-induced sweetening, is a serious postharvest problem for the potato processing industry. RS accumulation is prevented by suppression of a gene encoding potato vacuolar acid invertase (StvacINV1), which catalyzes irreversible hydrolysis of sucrose into glucose and fructose. Six potato genotypes were selected for dynamic analysis of sugar accumulation and invertase expression pattern in tubers during 1 month of low-temperature storage. The significant increase of sucrose content was found to occur earlier than the remarkable rise of RS content in all the genotypes tested. RS accumulation continued from 15 to 30 days of storage while sucrose content was almost steady in this period. The transcripts of StvacINV1 increased rapidly in tubers exposed to cold treatment and the activity of soluble acid invertase increased accompanied with sugar accumulation. Correlation analysis across eight genotypes revealed that invertase activity was positively correlated with RS content while the correlation between soluble acid invertase activity and the level of StcacINV1 transcripts was weak. Our results suggest that sucrose cleavage is causal for RS accumulation in cold-stored tubers and the acid invertase activity is possibly modulated by post-translational mechanisms.

Characterisation of genes encoding key enzymes involved in sugar metabolism of apple fruit in controlled atmosphere storage

Sugars are essential contributors to fruit flavour. Controlled atmosphere (CA) storage has been proved to be beneficial for maintaining harvested fruit quality. To explore regulatory mechanism of sugar metabolism in fruit stored in CA condition, we cloned several genes, encoding key enzymes, involved in sugar metabolism in apple fruit, and analyzed sugar contents, along with gene expression and enzyme activities in fruits stored in air and CA. The results indicated that CA could maintain higher contents of sugars, including sucrose, fructose and glucose. Expression levels of key genes, such as sucrose synthase (SS), sucrose phosphate synthase (SPS), fructokinase (FK) and hexokinase (HK), were shown to be correlated with the corresponding enzyme activities. We found that activities of neutral invertase (NI), vacuolar invertase (VI), FK and HK were inhibited, but SPS activity was promoted in apple fruit stored in CA, suggesting that CA storage could enhance sucrose synthesis and delay hydrolysis of sucrose and hexose. These findings provided molecular evidence to explain why higher sugar levels in harvested fruit are maintained under CA storage.

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10 % of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24 h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentose phosphate pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-013-1907-z) contains supplementary material, which is available to authorized users.

Promoter regions of potato vacuolar invertase gene in response to sugars and hormones

Potato vacuolar acid invertase (StvacINV1) (β-fructofuranosidase; EC 3.2.1.26) has been confirmed to play an important role in cold-induced sweetening of potato tubers. However, the transcriptional regulation mechanisms of StvacINV1 are largely unknown. In this study, the 5'-flanking sequence of StvacINV1 was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINV1 promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINV1 promoter was suppressed by sucrose, glucose, fructose, and cold, while enhanced by indole-3-acetic acid (IAA), and gibberellic acid (GA3). Further deletion analysis clarified that the promoter regions from-118 to-551,-551 to-1021, and-1021 to-1521 were required for responding to sucrose/glucose, GA3, and IAA, respectively. These findings provide essential information regarding transcriptional regulation mechanisms of StvacINV1.

Differential Expression of Genes Encoding Acid Invertases in Multiple Shoots of Bamboo in Response to Various Phytohormones and Environmental Factors

The promoter regions of two cell wall invertase genes, Boβfruct1 and Boβfruct2, and a vacuolar invertase gene, Boβfruct3, in Bambusa oldhamii were cloned, and putative regulatory cis-elements were identified. The expression of these three genes in multiple shoots of bamboo that were cultured in vitro under different conditions was analyzed by real-time PCR. The two cell wall invertase genes were upregulated by indole-3-acetic acid and cytokinins but responded differently to other phytohormones and different temperatures. Boβfruct1 was also upregulated by sucrose and glucose. In contrast, the Boβfruct2 expression was induced by the depletion of sucrose, and this induction could be suppressed by glucose and sucrose. The expression of Boβfruct3 was light-dependent; however, abscisic acid (ABA) could induce its expression in the dark. ABA and light exhibited an additive effect on the expression of Boβfruct3. Our results suggest that these three Boβfruct genes have individual roles in the adaption of the plant to environmental changes. Boβfruct2 might also have an essential role in the immediate response of cells to sucrose availability and in the maintenance of sink activity. Moreover, Boβfruct3 might be one of the interacting nodes of the light and ABA signaling pathways.

A VIN1 GUS::GFP fusion reveals activated sucrose metabolism programming occurring in interspersed cells during tomato fruit ripening

The tomato is a model for fleshy fruit development and ripening. Here we report on the identification of a novel unique cell autonomous/cellular pattern of expression that was detected in fruits of transgenic tomato lines carrying a GFP GUS driven by the fruit specific vacuolar invertase promoter VIN1. The VIN1 promoter sequence faithfully reproduced the global endogenous VIN expression by conferring a biphasic pattern of expression with a second phase clearly associated to fruit ripening. A closer view revealed a salt and pepper pattern of expression characterized by individual cells exhibiting a range of expression levels (from high to low) surrounded by cells with no expression. This type of pattern was detected across different fruit tissues and cell types with some preferences for vascular, sub-epidermal layer and the inner part of the fruit. Cell ability to show promoter activity was neither directly associated with overall ripening – as we find VIN+ and – VIN− cells at all stages of ripening, nor with cell size. Nevertheless the number of cells with active VIN-driven expression increased with ripening and the activity of the VIN promoter seems to be inversely correlated with cell size in VIN+ cells. Gene expression analysis of FACS-sorted VIN+ cells revealed a transcriptionally distinct subpopulation of cells defined by increased expression of genes related to sucrose metabolism, and decreased activity in protein synthesis and chromatin remodeling. This finding suggests that local micro heterogeneity may underlie some aspects (i.e. the futile cycles involving sucrose metabolism) of an otherwise more uniform looking ripening program.

A synthetic tuber-specific and cold-induced promoter is applicable in controlling potato cold-induced sweetening

Cold-induced sweetening (CIS) in potato seriously hinders the potato processing industry. It could be of great value for genetic improvement of potato CIS to have a target gene specifically expressed in cold stored tubers. In this study, we used a synthetic promoter, pCL, in potato transformation to drive an antisense expression of StvacINV1, the acid vacuolar invertase gene from Solanum tuberosum. The measurements of expression and enzyme activity of target gene showed that pCL promoter could efficiently govern target gene to express specifically and remarkably regulate the activity of acid vacuolar invertase in potato tubers at low temperature, furthermore, it had almost no effect in other tissues or the tubers under room temperature. The transgenic tubers showed decrease in reducing sugar content during storage atlow temperature and acceptable chip color without significant changes observed in plant morphology and tuberization between the nontransgenic and transgenic lines. This tuber-specific and cold-induced feature could maximally reduce the background expression of the target gene which might bring about potential negative or detrimental effects to plant development. The synthetic promoter confirmed here would be optimal for gene function research in potato tubers in response to low temperature.