Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant ( sre ) and salobreño ( sañ ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1 , sre1-2 , sañ3-1 , and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD-or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K m value for xanthoxin of 19 M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin → abscisic aldehyde → ABA transition as the last steps of the major ABA biosynthetic pathway.
The yeast trehalose-6-phosphate synthase gene (TPS1) was engineered under the control of the cauliflower mosaic virus regulatory sequences (CaMV35S) for expression in plants. Using Agrobacterium-mediated transfer, the gene was incorporated into the genomic DNA and constitutively expressed in Nicotiana tabacum L. plants. Trehalose was determined in the transformants, by anion-exchange chromatography coupled to pulsed amperometric detection. The non-reducing disaccharide accumulated up to 0.17 mg per g fresh weight in leaf extracts of transgenic plants. Trehaloseaccumulating plants exhibited multiple phenotypic alterations, including stunted growth, lancet-shaped leaves, reduced sucrose content and improved drought tolerance. These pleiotropic effects, and the fact that water loss from detached leaves was not significantly affected by trehalose accumulation, suggest that synthesis of this sugar, rather than leading to an osmoprotectant effect, had altered sugar metabolism and regulatory pathways affecting plant development and stress tolerance.
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