Glutathione-dependent root growth in Arabidopsis is linked to polar auxin transport (PAT). Arabidopsis mutants with reduced glutathione (GSH) levels also show reduced PAT.To gain an insight into the relationship between PAT and GSH level, we analysed tomato polycotyledon mutant, pct1-2, which has enhanced PAT. Microarray analysis of gene expression in pct1-2 mutant revealed underexpression of several genes related to glutamate and glutathione metabolism. In consonance with microarray analysis, enzymatic as well as in-vivo assay revealed higher glutathione level in the early phase of pct1-2 seedling growth than WT.The inhibition of auxin transport by 2,3,5-triiodobenzoic acid (TIBA) reduced both GSH level and PIN1 expression in pct1-2 root tips. The reduction of in vivo GSH accumulation in pct1-2 root tips by buthionine sulfoximine (BSO) stimulated elongation of the short root of pct1-2 mutant akin to TIBA. The rescue of short root phenotype of pct1-2 mutant was restricted to TIBA and BSO. The other auxin transport inhibitors 1-N-naphthylphthalamic acid (NPA), 2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid (BUM), 3-chloro-4-hydroxyphenylacetic acid (CHPAA), brefeldin and gravacin inhibited root elongation in both WT and pct1-2 mutant.Our results indicate a relationship between PAT and GSH level in tomato akin to Arabidopsis.Our work also highlights that TIBA rescues short root phenotype of the pct1-2 mutant by acting on a PAT component distinct from the site of action of other PAT inhibitors.
Tomato (Solanum lycopersicum), a model system for ripening of fleshy fruits, has ~40,000 genes predicted by in silico homology-based annotation. However, functional validation is lacking for most annotated tomato genes. Among the strategies for functional annotations, transposon-tagged mutagenesis is the most powerful approach. Transposon-tagged genes can be functionally validated by phenotyping and activation tagging. However, the lack of a robust in planta transformation system precludes large-scale transposon-mutagenesis of tomato. To overcome this limitation, we developed two sets of starter lines in tomato, each carrying maize transposon elements (Ds) and transposases (Ac), respectively. The Ds and Ac lines were crossed to allow the Ac-mediated transposition of the Ds in the F1 generation. In the F2 generation, the location of excised Ds at new sites in the tomato genome was monitored. The Ds transposition was interspersed on different chromosomes of the tomato, indicating unlinked transposition of the Ds. The analysis of DNA sequences flanking Ds showed random integration of Ds in intergenic regions, genes, and the promoter region of the genome. Our study paves the way for the generation of large-scale transposon-tagged tomato lines using Ac/Ds starter lines and provides a potential tool for the functional validation of genes in tomato.
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