RESUMO
Phytochelatins (PCs) are major chelators of toxic elements including inorganic arsenic (As) in plant cells. Their synthesis confers tolerance and influences within-plant mobility. Previous studies had shown that various metal/metalloid ions differentially activate PC synthesis. Here we identified C-terminal parts involved in arsenite- [As(III)] dependent activation of AtPCS1, the primary Arabidopsis PC synthase. The T-DNA insertion in the AtPCS1 mutant cad1-6 causes a truncation in the C-terminal regulatory domain that differentially affects activation by cadmium (Cd) and zinc (Zn). Comparisons of cad1-6 with the AtPCS1 null mutant cad1-3 and the double mutant of tonoplast PC transporters abcc1/2 revealed As(III) hypersensitivity of cad1-6 equal to that of cad1-3. Both cad1-6 and cad1-3 showed increased As distribution to shoots compared with Col-0, whereas Zn accumulation in shoots was equally lower in cad1-6 and cad1-3. Supporting these phenotypes of cad1-6, PC accumulation in the As(III)-exposed plants were at trace level in both cad1-6 and cad1-3, suggesting that the truncated AtPCS1 of cad1-6 is defective in PCS activity in response to As(III). Analysis of a C-terminal deletion series of AtPCS1 using the PCS-deficient mutant of fission yeast suggested important regions within the C-terminal domain for As(III)-dependent PC synthesis, which were different from the regions previously suggested for Cd- or Zn-dependent activation. Interestingly, we identified a truncated variant more strongly activated than the wild-type protein. This variant could potentially be used as a tool to better restrict As mobility in plants.
Assuntos
Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arsenitos/farmacologia , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Cisteína/metabolismo , Glutationa/metabolismo , Minerais/metabolismo , Mutação/genética , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
The AR2/ERF transcription factor genes ERF71/HRE2 and ERF73/HRE1 were induced at hypoxic conditions in Arabidopsis thaliana roots. ERF73/HRE1 but not its related gene ERF71/HRE2 was furthermore regulated by ethylene. Treatment with 1 ppm ethylene promoted ERF73/HRE1 expression fivefold. This induction did not occur in the presence of the ethylene receptor inhibitor 1-methylcyclopropene. ERF73/HRE1 expression positively regulated alcohol dehydrogenase (ADH) activity, which was analyzed as a marker enzyme for metabolic adaptation to hypoxic stress. The knock out lines erf73/hre1-1 and erf73/hre1-2 showed lowered ADH activity; the overexpressing lines ERF73/HRE1-ox1 and ERF73/HRE1-ox5 displayed elevated ADH activity. Treatment of wild-type Arabidopsis with 5% O2 and 1 ppm ethylene resulted in higher induction of ADH activity than that observed with 5% O2 or 1 ppm ethylene alone. ERF73/HRE1-ox1 and ERF73/HRE1-ox5 plants that were exposed to 5% O2 did not show enhanced ADH activity after treatment with ethylene, indicating that the ethylene response with respect to ADH activity was saturated in the ERF73/HRE1ox lines. In contrast, erf73/hre1-1 and erf73/hre1-2 lines displayed ethylene-dependent ADH activity pointing to redundant factor(s) that can mediate ethylene regulation of ADH activity in the Arabidopsis root. Our data show that ethylene regulates metabolic adaptation to low oxygen stress in the Arabidopsis root through ERF73/ HRE1.