Post‐translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold‐induced sweetening of potato tubers

Cold‐induced sweetening (CIS) is a serious post‐harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark‐coloured and bitter‐tasting product and generating the probable carcinogen acrylamide as a by‐product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS‐susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS‐resistant line increased susceptibility to CIS. The results show that post‐translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.     

Association mapping of cold‐induced sweetening in potato using historical phenotypic data

In this study, historical phenotypic data from a potato breeding programme were used with an association mapping approach to identify alleles of candidate genes associated with cold‐induced sweetening of potato. Molecular marker analysis was used to determine allelic variation of candidate genes potentially involved in cold‐induced sweetening. Variations in the UDP‐glucose pyrophosphorylase (UGPase, EC 2.7.7.9) and apoplastic invertase genes (EC 3.2.1.26) were significantly associated with cold‐induced sweetening, and a possible interaction of apoplastic invertase and apoplastic invertase inhibitor was identified. This demonstrates that breeding programme phenotypic data collected over multiple years and environments can be used successfully with pedigree information for association mapping. It also confirms that the UGPase and apoplastic invertase markers are transferable across breeding programmes with distinct germplasm.     

Vacuolar invertases in potato (Solanum tuberosum L.): molecular cloning, characterization, sequence comparison, and analysis of gene expression in the cultivars

In plants, vacuolar invertase (β-fructofuranosidase, EC 3.2.1.26) 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.     

Cloning and functional analysis of chicory root fructan1‐exohydrolase I (1‐FEH I): a vacuolar enzyme derivedfrom a cell‐wall invertase ancestor? Mass fingerprint of the 1‐FEH I enzyme

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.     

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.     

Effect of sugars on the development of oxidative stress induced by hypothermia in potato plants expressing yeast invertase gene

The influence of sugars on the development of oxidative stress induced by hypothermia was investigated in the leaves of two genotypes of potato (Solanum tuberosum L.) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. We used wild-type plants of potato, cv. Désirée, and potato plants expressing a yeast invertase gene under the control of the B33 class I patatin promoter and carrying a sequence of proteinase inhibitor II leader peptide for the apoplastic enzyme localization. At temperature of 22°C optimal for growth, expression of the yeast invertase gene in the leaves of transformed plants brought about a modification in the carbohydrate metabolism manifested in the activation of acid forms of invertase and accumulation of intracellular sugars (predominantly of sucrose because of its resynthesis). The exposure of plants to light under prolonged hypothermia (5°C, 6 days) activated all the forms of invertase (predominantly of acid invertase) and induced accumulation of sugars. In the leaves of potato expressing the yeast invertase gene, these processes were more intense. Under chilling, superoxide dismutase activity and the rate of lipid peroxidation in the leaves of investigated potato genotypes depended on the level of accumulated intracellular sugars. It was concluded that sugars play an important role as stabilizers of cellular membranes and scavengers of reactive oxygen species decelerating the processes of free radical oxidation of biomolecules upon the development of oxidative stress induced by hypothermia.     

Sucrose uptake,invertase localization and gene expression in developing fruit of Lycopersicon esculentum and the sucrose-accumulating Lycopersicon hirsutum

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.     

Cloning of a vacuolar invertase from Belgian endive leaves (Cichorium intybus)

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.     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

Nucleotide diversity and molecular evolution of the WAG-2 gene in common wheat (Triticum aestivum L) and its relatives

In this work, we examined the genetic diversity and evolution of the WAG-2 gene based on new WAG-2 alleles isolated from wheat and its relatives. Only single nucleotide polymorphisms (SNP) and no insertions and deletions (indels) were found in exon sequences of WAG-2 from different species. More SNPs and indels occurred in introns than in exons. For exons, exons+introns and introns, the nucleotide polymorphism π decreased from diploid and tetraploid genotypes to hexaploid genotypes. This finding indicated that the diversity of WAG-2 in diploids was greater than in hexaploids because of the strong selection pressure on the latter. All dn/ds ratios were < 1.0, indicating that WAG-2 belongs to a conserved gene affected by negative selection. Thirty-nine of the 57 particular SNPs and eight of the 10 indels were detected in diploid species. The degree of divergence in intron length among WAG-2 clones and phylogenetic tree topology suggested the existence of three homoeologs in the A, B or D genome of common wheat. Wheat AG-like genes were divided into WAG-1 and WAG-2 clades. The latter clade contained WAG-2, OsMADS3 and ZMM2 genes, indicating functional homoeology among them.     

马铃薯转化酶抑制子St-inh cDNA克隆及在大肠杆菌中的表达和功能测定

用RT-PCR结合5’RACE方法从马铃薯(Solarium tuberosum L．)栽培种JH块茎中克隆了转化酶抑制子St-inh全长cDNA。序列分析表明,St-inh基因编码区全长663bp,编码221个氨基酸。将含St-inh基因cDNA的DNA片段克隆到pET28a( )上,转化大肠杆菌BL21(DE3)后成功实现了表达。基因表达产物与马铃薯栽培品种(系)E1、JH试管块茎以及番茄果实的转化酶提取物共孵育结果显示,转化酶活性分别下降了34．3％、21％和33．8％,说明St-inh的翻译产物具有转化酶抑制子功能。BLAST基因序列分析表明,St-inh与Kunitz-type C类基因序列同源性达95％以上,T-COFFEE氨基酸序列对比分析显示,该基因编码的蛋白质具有典型的Kunitz-type结构域[L,I,V,M]-X-D-X-[E,D,N,T,Y]-[D,G]-[R,K,H,D,E,N,Q]-X-[L,I,V,M]-X(5)-Y-X-[L,I,V,M],因此,St-inh基因可能为Kunitz-type家族成员。     

INVINH1 Gene Alleliс Polymorphism in Maize Accessions from VIR Collection

Russian Journal of Genetics - The sequences of the apoplastic invertase inhibitor gene INVINH1 were identified in 71 maize accessions from the collection of the Vavilov All-Russian Institute of...     

Expression of biotin-binding proteins,avidin and streptavidin,in plant tissues using plant vacuolar targeting sequences

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotinmetabolism.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.