Understanding metabolism of arginine in biological systems via MALDI imaging.

Arginine is an important amino acid but has been barely studied in plants. The little research that has been done indicates that the pathways of synthesis are similar to those found in animals and procaryotes. However little is known about the cellular and tissue localization of the amino acid in plants. The research reported in this paper was designed to examine whether MALDI-MSI was sufficiently sensitive to examine the distribution of this amino acid in plant material, and whether the synthetic pathways were co-located. In wheat and orchid roots, the amount of arginine in tissues varies greatly and the pathways for its synthesis were not always detected with the amino acid.

Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1.

Transgenic potato (Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene (SnRK1) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%-167% compared with that in the wild-type. Glucose levels were decreased, at 17%-56% of the levels of the wild-type, and the starch content showed an increase of 23%-30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, alpha-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%-60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.

Characterization of a gene from chromosome 1B encoding the large subunit of ADPglucose pyrophosphorylase from wheat: evolutionary divergence and differential expression of Agp2 genes between leaves and developing endosperm.

A full-length genomic clone containing the gene encoding the large subunit of the ADPglucose pyrophosphorylase (Agp2), was isolated from a genomic library prepared from etiolated shoots of hexaploid wheat (Triticum aestivum L., cv, Chinese Spring). The coding region of this gene is identical to one of the cDNA clones previously isolated from a developing wheat grain cDNA library and is therefore an actively transcribed gene. The sequence represented by the cDNA spans 4.8 kb of the genomic clone and contains 15 introns. 2852 bp of DNA flanking the transcription start site of the gene was cloned upstream of the GUS (beta-glucuronidase) reporter gene. This Agp2::GUS construct and promoter deletions were used to study the pattern of reporter gene expression in both transgenic tobacco and wheat plants. Histochemical analysis of GUS expression in transgenic tobacco demonstrated that the reporter gene was expressed in guard cells of leaves and throughout the seed. In transgenic wheat, reporter gene expression was confined to the endosperm and aleurone with no expression in leaves. The cloned Agp2 gene was located to chromosome 1B by gene-specific PCR with nullisomic-tetrasomic lines. Northern analysis demonstrated that the Agp2 genes are differentially expressed in leaves and developing endosperm; while all three classes of Agp2 genes are transcribed in developing wheat grain endosperm, only one is transcribed in leaves. The differences between the Agp2 genes are discussed in relation to the evolution of hexaploid wheat.

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm.

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an alpha-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.

Modulation of fructokinase activity of potato (Solanum tuberosum) results in substantial shifts in tuber metabolism.

Potato plants (Solanum tuberosum L. cvs Desiree and Record) transformed with sense and antisense constructs of a cDNA encoding the potato fructokinase StFK1 exhibited altered transcription of this gene, altered amount of protein and altered enzyme activities. Measurement of the maximal catalytic activity of fructokinase revealed a 2-fold variation in leaf (from 90 to 180% of wild type activity) and either a 10- or 30-fold variation in tuber (from 10 or 30% to 300% in Record and Desiree, respectively) activity. The comparative effect of the antisense construct in leaf and tuber tissue suggests that this isoform is only a minor contributor to the total fructokinase activity in the leaf but the predominant isoform in the tuber. Antisense inhibition of the fructokinase resulted in a reduced tuber yield; however, its overexpression had no impact on this parameter. The modulation of fructokinase activity had few, consistent effects on carbohydrate levels, with the exception of a general increase in glucose content in the antisense lines, suggesting that this enzyme is not important for the control of starch synthesis. However, when metabolic fluxes were estimated, it became apparent that the transgenic lines display a marked shift in metabolism, with the rate of redistribution of radiolabel to sucrose markedly affected by the activity of fructokinase. These data suggest an important role for fructokinase, acting in concert with sucrose synthase, in maintaining a balance between sucrose synthesis and degradation by a mechanism independent of that controlled by the hexose phosphate-mediated activation of sucrose phosphate synthase.

Starch synthesis in potato tubers transformed with the wheat genes for ADPglucose pyrophosphorylase.

The aim of this work was to study the role of ADPglucose pyrophosphorylase (AGPase) in starch biosynthesis of non-photosynthetic organs. Agrobacterium tumefaciens was used to transform potato plants (Solanum tuberosum L. cv. Desireé) with the wheat AGPase genes (AGP-S and AGP-L, coding for the small and large subunits, respectively). Neither of these genes contains a recognisable plastid targeting sequence. Southern analysis and analysis of starch content identified four lines that contained both wheat sequences. Immunoblotting indicated that, in the tubers, three lines expressed the wheat small subunit (AGP-S), but AGP-L cross-reacting protein was not apparent. The fourth transgenic line had reduced AGPase activity. AGPase activity in the AGP-transgenic tubers ranged from 15 to 165% of that found in ß-glucuronidase (GUS) control lines.

Subcellular localization of ADPglucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform.

The intracellular location of ADPglucose pyrophosphorylase (AGPase) in wheat during endosperm development was investigated by analysis of the recovery of marker enzymes from amyloplast preparations. Amyloplast preparations contained 20-28% of the total endosperm activity of two plastidial marker enzymes and less than 0.8% of the total endosperm activity of two cytosolic marker enzymes. Amylo plasts prepared at various stages of development, from 8-30 d post anthesis, contained between 2% and 10% of the total AGPase activity; this implies that between 7% and 40% of the AGPase in wheat endosperm is plastidial during this period of development. Two proteins were recognized by antibodies to both the large and small subunits of wheat AGPase. The larger of the two AGPases was the major form of the enzyme in whole cell extracts, and the smaller, less abundant, form of AGPase was enriched in plastid preparations. The results are consistent with data from other graminaceous endosperms, suggesting that there are distinct plastidial and cytosolic isoforms of AGPase composed of different subunits. The plastidial isoform of AGPase from wheat endosperm is relatively insensitive to the allosteric regulators 3-phosphoglycerate and inorganic orthophos phate compared with plastidial AGPase from other species. Amyloplast AGPase showed no sensitivity to physiological concentrations of inorganic orthophosphate. 15 mM 3-phosphoglycerate caused no stimulation of the pyrophosphorolytic reaction, and only 2-fold stimulation of the ADPglucose synthesizing reaction.