Impacts on the metabolome of down-regulating polyphenol oxidase in potato tubers.

Tubers of potato (Solanum tuberosum L. cv. Estima) genetically modified to reduce polyphenol oxidase (PPO) activity and enzymatic discolouration were assessed for changes in the metabolome using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS. Metabolome changes induced over a 48 hour (h) period by tuber wounding (sliced transverse sections) were also assessed using two PPO antisense lines (asPPO) and a wild-type (WT) control. Data were analysed using Principal Components Analysis and Analysis of Variance to assess differences between genotypes and temporal changes post-tuber wounding (by slicing). The levels of 15 metabolites (out of a total of 134 that were detected) differed between the WT and asPPO lines in mature tubers at harvest. A considerably higher number (63) of these metabolites changed significantly over a 48 h period following tuber wounding. For individual metabolites the magnitude of the differences between the WT and asPPO lines at harvest were small compared with the impacts of tuber wounding on metabolite levels. Some of the observed metabolite changes are explicable in terms of pathways known to be affected by wound responses. Whilst some statistically significant interactions (11 metabolites) were observed between line and time after wounding, very few profiles were consistent when comparing the WT with both asPPO lines, and the underlying metabolites appeared to be random in terms of the pathways they occupy. Overall, mechanical damage to tubers has a considerably greater impact on the metabolite profile than any potential unintended effects resulting from the down-regulation of PPO gene expression.

Characterizing the citrus cultivar Carrizo genome through 454 shotgun sequencing.

The citrus cultivar Carrizo is the single most important rootstock to the US citrus industry and has resistance or tolerance to a number of major citrus diseases, including citrus tristeza virus, foot rot, and Huanglongbing (HLB, citrus greening). A Carrizo genomic sequence database providing approximately 3.5×genome coverage (haploid genome size approximately 367 Mb) was populated through 454 GS FLX shotgun sequencing. Analysis of the repetitive DNA fraction indicated a total interspersed repeat fraction of 36.5%. Assembly and characterization of abundant citrus Ty3/gypsy elements revealed a novel type of element containing open reading frames encoding a viral RNA-silencing suppressor protein (RNA binding protein, rbp) and a plant cytokinin riboside 5'-monophosphate phosphoribohydrolase-related protein (LONELY GUY, log). Similar gypsy elements were identified in the Populus trichocarpa genome. Gene-coding region analysis indicated that 24.4% of the nonrepetitive reads contained genic regions. The depth of genome coverage was sufficient to allow accurate assembly of constituent genes, including a putative phloem-expressed gene. The development of the Carrizo database (http://citrus.pw.usda.gov/) will contribute to characterization of agronomically significant loci and provide a publicly available genomic resource to the citrus research community.

pBINPLUS/ARS: an improved plant transformation vector based on pBINPLUS.

Binary plant transformation vectors are widely used for introduction of transgenes into plants via Agrobacterium tumefaciens-mediated transformation. We report the construction of a binary plant vector pBINPLUS/ARS based on the pBINPLUS vector. Improvements introduced into pBINPLUS/ARS include the use of nonproprietary (ubiquitin-3 gene of Solanum tuberosum) promoter and terminator sequences for transcription of the NptII selectable marker and introduction of rare 8-bp restriction enzyme sites flanking both the NptII coding sequence (PmeI) and the entire selectable marker gene (FseI). This vector offers all of the advantages of its predecessor pBINPLUS and its helper plasmid pUCAP, which use the proprietary nopaline synthase promoter and terminator, while allowing for facile modification of selectable marker sequences in complex binary vector constructs. pBINPLUS/ARS has been used to introduce transgenes into potato and other crop species and is available to all researchers in academic, government, and industrial laboratories for proof-of-principle and commercial applications.

Potato glycosterol rhamnosyltransferase, the terminal step in triose side-chain biosynthesis.

Steroidal glycoalkaloids (SGAs) are potentially harmful specialty metabolites found in Solanaceous plants. Two tri-glycosylated alkaloids, alpha-chaconine and alpha-solanine accumulate in potato tubers. Expressed sequence tags (ESTs) were identified in the available database by searching for protein homology to the Sgt1 (SOLtu:Sgt1) steriodalalkaloid galactosyltransferase. The EST sequence data was used to isolate Sgt3 cDNA sequences by polymerase chain reaction (PCR) from a wounded potato tuber cDNA library. The resulting 1515bp open reading frame of Sgt3, encodes a predicted SGT3 amino acid sequence that is 18 residues longer than, 45% identical to, and 58% homologous to the SGT1 protein. The amino-terminal region of the Sgt3 cDNA was used to create an antisense transgene under control of the granule bound starch synthase, GBSS6, promoter and the ubiquitin, Ubi3, polyadenylation signal. Analysis of SGA metabolites in selected transgenic tubers revealed a dramatic decrease in the accumulation of alpha-chaconine and alpha-solanine. This decrease was compensated by an increase in beta-solanine and beta-chaconine with minor accumulation of alpha-SGAs. These results allowed the identification of the function for SGT3 as the beta-solanine/beta-chaconine rhamnosyl transferase, the terminal step in formation of the potato glycoalkaloid triose side chains.

The primary in vivo steroidal alkaloid glucosyltransferase from potato.

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase (Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to alpha-chaconine or alpha-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato (Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of alpha-solanine was increased and alpha-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.