ci21A/Asr1 expression influences glucose accumulation in potato tubers.

Asr genes are exclusively found in the genomes of higher plants. In many species, this gene family is expressed under abiotic stress conditions and during fruit ripening. The encoded proteins have nuclear localisation and consequently a transcription factor function has been suggested. Interestingly, yeast-one-hybrid experiments revealed that a grape ASR binds to the promoter of a hexose transporter gene (VvHT1). However, the role of these proteins in planta is still elusive. By using a reverse genetics approach in potato we found that modification of Asr1 expression has no incidence on the aerial phenotype of the plant but exerts a dramatic effect in tuber. Asr1 antisense potatoes displayed decreased tuber fresh weight whereas Asr1 overexpressors had a diminished number of tubers. Moreover, overexpression lines showed lower transcript levels of a plasma membrane hexose transporter and a concomitant decrease in glucose content in parenchyma cells of potato tubers. On the same hand glucose uptake rate was also reduced in one of the overexpressing lines. It thus seems likely that Asr1 is involved in the control of hexose uptake in heterotrophic organs. In addition, the transgenic plants were characterized by several other changes in steady state metabolite levels. Results presented here support a role for ci21A/Asr1 in glucose metabolism of potato tuber.

Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes.

Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-(13)C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and well-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism.

Constitutive expression of the beta-ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants.

Polyhydroxybutyrate (PHB) is a member of a class of thermoelastic polymers called polyhydroxyalkanoates that serve many bacteria as intracellular storage molecules for carbon and energy. Transgenic plants provide a potential means of producing this polymer cost-effectively. To date, however, few reports of the successful production of this polymer have been published, with the exception of work with transgenic Arabidopsis. Using a variety of chimeric constructs, we have determined that the constitutive, chloroplast-localized expression of one of the genes involved in PHB production-the beta-ketothiolase (phbA) gene-is detrimental to the efficient production of transgenic PHB. The alternate use of either inducible or somatically activated promoters allowed the construction of transgenic PHB-producing potato (Solanum tuberosum) and tobacco (Nicotiana tabacum) plants, although the amount of PHB formed was still rather low. Taking advantage of an inducible promoter, the maximal amount of PHB produced in transgenic potato was 0.09 mg g(-1) dry weight. In transgenic tobacco using a somatically activated promoter, up to 3.2 mg g(-1) dry weight was accumulated. In Arabidopsis, the formation of high levels of PHB had previously been shown to be accompanied by severe negative effects on growth and development of the plant. Phasins are proteins known from PHB-producing bacteria speculated to serve as protectants against the highly hydrophobic surface of the PHB granules in the bacterial intracellular milieu. Co-expression of the phasin gene in parallel with the PHB synthesis genes, however, did not lead to reduced symptom development.