RESUMO
Many plants accumulate proline in response to salt stress. Δ-pyrroline-5-carboxylate synthetase (P5CS) is the rate-limiting enzyme in proline biosynthesis in plants. Plasmid DNA (pCHF3-PvP5CS1 and pCHF3-PvP5CS2) containing the selectable neomycin phosphotransferase gene for kanamycin resistance and Phaseolus vulgaris P5CS (PvP5CS1 and PvP5CS2) cDNA was introduced into Arabidopsis plants using Agrobacterium-mediated gene transfer. Southern blot, northern blot and RT-PCR analyses demonstrated that the foreign genes were integrated into Arabidopsis chromosomal DNA and expressed. Single-gene transformants were analysed in this study. Transgenic plants expressed higher levels of PvP5CS1 and PvP5CS2 transcripts under salt stress conditions than under normal conditions. When treated with 0, 100 and 200 mM NaCl, the average proline content in leaves of transgenic plants was significantly higher (P < 0.01) than control plants. The average relative electrical conductivity (REC) of transgenic lines was significantly lower (P < 0.01) than control plants under salt stress condition. Biomass production of transgenic lines was significantly higher (P < 0.05) than control plants under 200 mM NaCl stress treatment. These results indicated that introducing PvP5CS1 and PvP5CS2 cDNA into transgenic Arabidopsis caused proline overproduction, increasing salt tolerance. Although the expression of PvP5CS1 in L4 lines and PvP5CS2 in S4 lines was the same under salt stress condition, the S4 lines accumulated 1.6 and 1.9 times more proline than the L4 lines under 100 and 200 mM NaCl treatments, respectively. The REC of S4 plants was 0.5 (100 mM NaCl) and 0.6 times (200 mM NaCl) that of L4 plants. The biomass production of S4 plants was 1.6 times (200 mM NaCl) more than in L4 plants. Total P5CS enzyme activity of S4 was significantly higher than that of L4. These results implied that the PvP5CS2 protein had stronger capacity to catalyze proline synthesis than PvP5CS1 under salt stress condition.
Assuntos
Arabidopsis/fisiologia , Phaseolus/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/genética , Condutividade Elétrica , Engenharia Genética , Glutamato-5-Semialdeído Desidrogenase/genética , Glutamato-5-Semialdeído Desidrogenase/metabolismo , Ornitina-Oxo-Ácido Transaminase/genética , Ornitina-Oxo-Ácido Transaminase/metabolismo , Phaseolus/enzimologia , Proteínas de Plantas/metabolismo , Prolina/biossíntese , Plantas Tolerantes a Sal/enzimologia , Plantas Tolerantes a Sal/genética , Plântula/genética , Plântula/crescimento & desenvolvimento , Estresse FisiológicoRESUMO
A full-length cDNA denominated PvP5CS for Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), an enzyme involved in the biosynthesis of proline, was cloned from common bean using a candidate gene approach. PvP5CS contains an open reading frame encoding a 716 amino acid polypeptide. Sequence analysis showed that PvP5CS shares 95.1% homology in nucleotide sequence and 93.2% identity in amino acid sequence with the mothbean (Vigna aconitifolia) P5CS. The expression patterns of PvP5CS in common bean treated with drought, cold (4 degrees C), and salt (200 mM NaCl) stresses were examined using real-time quantitative PCR. These abiotic stresses caused significant up-regulation of the expression of PvP5CS in leaves. The PvP5CS mRNA transcript increased to 2.5 times the control level after 4d drought stress. A rapid up-regulation of PvP5CS, to about 16.3 times the control at 2h post-treatment was observed under salt stress. A significant increase in PvP5CS expression (11.7-fold) was detected after 2h of cold stress. The peaks of proline accumulation appeared at 8d for drought, 24h for cold and 9h for salt stress, somewhat later than the peaks of PvP5CS expression. These results suggest that PvP5CS was a stress-inducible gene regulating the accumulation of proline in plants subjected to stress. Finally, subcellular localization assays showed that the PvP5CS protein was present in the nucleus and at the plasmalemma.
