Identification and characterization of intraspecific variability of the sucrose synthase gene <Emphasis Type="Italic">Sus4</Emphasis> of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

Nucleotide and amino acid variability of fragments of the Sus4 gene encoding the sucrose synthase enzyme was studied in 24 potato cultivars bred in Russia and other countries and differing in starch content in tubers. Both SNPs and indels were detected in a chosen Sus4 gene fragment including the sequence from exon 3 to exon 6 and corresponding to the main part of the sucrose synthase domain. Four types of Sus4 sequences were revealed depending on the presence of an insertion in introns 4 and 5 and of the mononucleotide octamer (T)₈ in intron 5. Differentiation of these sequences was confirmed by statistical methods. Sixteen amino acid substitutions were identified in the translated sequence, of which eleven were nonsynonymous. Specific cultivar-specific nucleotide and amino acid substitutions were also revealed, which can be used in future for identifying potato cultivars/genotypes. No direct associations between the mutational changes and the starch content were found in the potato cultivars studied.     

Intraspecific polymorphism of the sucrose synthase genes in Russian and Kazakh potato cultivars

In 12 different Russian and Kazakh potato cultivars, the polymorphism of the glucosyltransferase domain of the sucrose synthase gene was first examined, as well as the polymorphism of the sucrose synthase domain fragment of the same gene in the potato cultivars of Kazakh breed. It was demonstrated that the examined sequences contained point mutations, as well as insertions and deletions, including those not described earlier. Amino acid substitutions specific to heat- and drought-tolerant varieties were also identified and could be associated with the development of abiotic stress resistance.     

High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5' and 3' flanking sequences and the leader intron.

The 3.6 kb of 5' flanking sequence, leader intron, and 0.7 kb of 3' sequence from the potato sucrose synthase gene Sus4-16 are sufficient to direct high-level expression in developing tubers, in basal tissues of axillary buds and shoots, and in meristems and caps of roots, and to confer sucrose inducibility in leaves. By examining a series of deletion and substitution constructs in transgenic potato plants, we found that this pattern of expression requires 5' flanking sequences both upstream and downstream of position -1500 and that sequences between positions -1500 and -267 are essential for sucrose induction. Replacement of the native 3' sequence with the nopaline synthase 3' sequence resulted in the loss of sucrose inducibility and of expression in basal tissues of axillary buds. A general decrease in expression in other tissues was also observed. Removal of the 1612-bp leader intron also had a dramatic effect on both the pattern and level of expression.     

A potato Sus3 sucrose synthase gene contains a context-dependent 3' element and a leader intron with both positive and negative tissue-specific effects.

To examine which sequences are involved in regulating the potato sucrose synthase gene Sus3-65, we examined a series of deletion and substitution constructs in transgenic potato and tobacco plants. In a construct containing 3.9 kb of 5' flanking region, substitution of the native 3' sequence with the nopaline synthase 3' sequence and deletion of the leader intron did not significantly affect expression in vegetative tissues. However, in a construct containing only 320 bp of 5' flanking region, these changes had marked effects. Replacing the native 3' sequences with nopaline synthase 3' sequences caused a six- to 20-fold increase in expression in vascular tissue, and removing the leader intron almost completely abolished expression in potato plants. Surprisingly, removal of the leader intron from either the full-length construct or a construct containing only 320 bp of 5' flanking sequence reduced expression in vascular tissue of tobacco anthers at later stages of development but increased expression in pollen by more than 100-fold.     

Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice (Oryza sativa L.)

The waxy gene, which encodes the granule bound starch synthase enzyme, is one of the key genes influencing starch synthesis in the rice endosperm. To investigate functional differences between GBSS alleles, we cloned and sequenced GBSS cDNA from a series of cultivars that differed substantially in apparent amylose content and starch viscosity characteristics. We found two single nucleotide polymorphisms in exons 6 and 10 that resulted in amino acid substitutions. These substitutions are associated with differences in apparent amylose content and viscosity characteristics. Subsequent sequencing of these regions from additional cultivars confirmed their association with particular rice quality characteristics. These point mutations could prove useful as molecular markers in the production of cultivars with superior eating, cooking and processing quality, and contribute to our understanding of the various structural and functional differences among granule bound starch synthase alleles.     

小麦面粉黄色素相关基因研究

小麦八氢番茄红素合成酶(PSY)基因和脂肪氧化酶(LOx)基因可能影响面粉黄色素含量。根据玉米P5y 基因序列设计引物,扩增出小麦PSY基因的部分片段,序列比较表明小麦和玉米PSY基因外显子DNA序列长度一致,但存在单核苷酸多态性(SNP)位点,序列一致性为90％;蛋白质氨基酸序列比较发现其序列一致性为97％, 说明一些SNP并未导致氨基酸的改变,该基因在玉米和小麦中应具有类似的功能活性。利用非整倍体材料将小麦 PSY基因初步定位到1D染色体。用同样方法,发现小麦的LOX基因与大麦、水稻、玉米的L0X基因具有很高的一致性,并将其初步定位到4BS染色体。     

Molecular cloning and sequence comparison of a cDNA encoding α-glucan, water dikinase (GWD) from potato (Solanum tuberosum L.), and analysis of gene expression

Starch phosphorylation is an important biochemical aspect of plant starch metabolism as it influences the overall structure of the starch granule, and a prerequisite for its degradation. There is a growing interest on the isolation and characterization of α-glucan/glucan-like, water dikinases (GWDs) from plants, particularly agriculturally important crops, because GWD is known to catalyze starch phosphorylation both in leaves and different plant storage organs. In the present study, a 4,789-bp full-length cDNA encoding a GWD isoform was isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. The predicted protein consisted of 1,463 amino acids having N-terminal 77-amino acid transit peptide, and 1,386-amino acid mature protein shorter by one amino acid as compared to the other mature GWDs from potato and tomato. The mature GWD showed 98 % sequence identity with the GWD isolated earlier from the potato cv. Desiree. Variations were found at 25 locations representing mostly non-conservative substitutions. The GWD represents a distinct isoform from potato, as revealed by sequence and phylogenetic analyses. Amino acid composition, segment-wise hydrophobic characters, predicted secondary structures were also analyzed and documented in this report. Broadly, the level of GWD expression as analyzed by semi-quantitative RT-PCR approach was found to be nearly uniform both in the mature tubers and leaves from most of the potato cultivars. By immunodetection technique, a band corresponding to ~155 kDa protein was detected only in the tuber protein extracts. The tuber starch-bound phosphorus content data showed minor variations between the potato cultivars.     

Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum)

Tuberising stolon tips of potato ( Solanum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2 cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present) to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum, ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase. fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation.     

Salinity Induces Accumulation of Soluble Sugars and Alters the Activity of Sugar Metabolising Enzymes in Rice Plants

Changes in the starch and sucrose contents, and the sucrose phosphate synthase, acid invertase, and starch phosphorylase activities were studied in the seedlings of salt sensitive and salt tolerant rice cultivars growing under two NaCl concentrations (7 and 14 dS m^-1) for 20 d. Under salinity, the starch content in roots declined more in salt sensitive cvs. Ratna and Jaya than in salt tolerant cvs. CSR-1 and CSR-3 and was unchanged in shoots. The contents of reducing and non-reducing sugars, and the activity of sucrose phosphate synthase was increased more in the sensitive than in the tolerant cultivars. Acid invertase activity decreased in shoots of the salt tolerant cultivars, whereas increased in salt sensitive cultivars. Starch phosphorylase activity decreased in all cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

In the potato, Kunitz-type enzyme inhibitors are abundant and highly polymorphic small proteins found in tubers. DNA sequence analysis of 1596 unselected ESTs (expressed sequence tags) from mature tubers of the cultivars Provita and Saturna resulted in the identification of 55 different DNA sequences with high sequence similarity to Kunitz-type enzyme inhibitors. The frequency of Kunitz-type inhibitor ESTs in Provita was four times higher than in Saturna tubers, and none of the Provita ESTs was identical to any of the Saturna ESTs. A phenogram constructed from the deduced amino acid sequences of the inhibitors revealed three major homology groups-A, B and C. Group A inhibitors were all derived from Provita ESTs. Inhibitor groups A and B were more similar to each other than to group C inhibitors, and for most members within-group similarity was at least 90%. Non-conservative amino acid substitutions and insertion/deletion polymorphisms suggest functional differentiation between members of the gene family. A minimum of 21 genes for Kunitz-type enzyme inhibitors (six for group A, nine for group B and six for group C) was estimated to exist in the potato genome. Genetic mapping and the identification of BAC (bacterial artificial chromosome) clones containing more than one member of the gene family indicated that most inhibitor genes of groups A, B and C are organized in a cluster that maps to a single region on potato chromosome III.     

Multiple, distinct isoforms of sucrose synthase in pea

Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum). The genes have distinct patterns of expression in different organs of the plant, and during organ development. Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties. Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms. Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1. One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95%. The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected. The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele

Summary The genomic sequence of the potato gene for starch granule-bound starch synthase (GBSS; waxy protein) has been determined for the wild-type allele of a monoploid genotype from which an amylose-free (amf) mutant was derived, and for the mutant part of the amf allele. Comparison of the wild-type sequence with a cDNA sequence from the literature and a newly isolated cDNA revealed the presence of 13 introns, the first of which is located in the untranslated leader. The promoter contains a G-box-like sequence. The deduced amino acid sequence of the precursor of GBSS shows a high degree of identity with monocot waxy protein sequences in the region corresponding to the mature form of the enzyme. The transit peptide of 77 amino acids, required for routing of the precursor to the plastids, shows much less identity with the transit peptides of the other waxy preproteins, but resembles the hydropathic distributions of these peptides. Alignment of the amino acid sequences of the four mature starch synthases with the Escherichia coli glgA gene product revealed the presence of at least three conserved boxes; there is no homology with previously proposed starch binding domains of other enzymes involved in starch metabolism. We report the use of chimeric constructs with wild-type and amf sequences to localize, via complementation experiments, the region of the amf allele in which the mutation resides. Direct sequencing of polymerase chain reaction products confirmed that the amf mutation is a deletion of a single AT basepair in the region coding for the transit peptide. Premature termination of translation as a result of this frameshift mutation results in a small peptide. However, a protein reacting with anti-GBSS serum, slightly larger than the wild-type mature GBSS, can be detected in a membrane fraction from amylose-free tubers. A possible explanation for this phenomenon will be discussed.     

A human metallothionein pseudogene containing AG/CT repetitive elements

Summary A lambda phage recombinant clone, 25 S, which contains a 15.5-kb EcoRI human genomic DNA fragment, has been characterized. Restriction mapping and Southern blot hybridization indicated a 3.0-kb HindIII fragment containing metallothionein (MT)-like sequences. Several interesting features were found upon comparison of this nucleotide sequence with that of other human MT genes: (1) sequences representing the 5 regulatory region, the 5 untranslated region, and the first exon are not contained in the 3.0-kb HindIII fragment; (2) the coding sequence of the second exon (amino acids 10–31 encoding a portion of the -domain of the MT protein) has 11 amino acid changes out of a total of 21, whereas, the third exon (amino acids 32–61, representing the complete -domain of the MT protein) has only 4 amino acid substitutions; however, all cysteine residues are conserved; (3) this MT-like gene retains intron sequences and processing signals; (4) Southern blot analysis of human genomic DNA indicated this MT-like gene is located on a 10.5-kb EcoRI genomic DNA fragment; and (5) unusual AG/CT-rich repetitive elements are located within the second intron and upstream of the second exon of this MT-like gene. This gene is not expressed in response to metal induction in two human cell lines, as shown by northern blot analyses. Based on these observations, this MT-like gene represents a unique nonprocessed pseudogene of the human MT multigene family.