Cloning and expression analysis of cDNAs encoding ABA 8'-hydroxylase in peanut plants in response to osmotic stress

Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic-stress responses. The major ABA catabolic pathway is triggered by ABA 8'-hydroxylation catalysed by ABA 8'-hydroxylase, the cytochrome P450 CYP707A family. In this study, the full-length cDNAs of AhCYP707A1 and AhCYP707A2 were cloned and characterized from peanut. Expression analyses showed that AhCYP707A1 and AhCYP707A2 were expressed ubiquitously in peanut roots, stems, and leaves with different transcript accumulation levels, including the higher expression of AhCYP707A1 in roots. The expression of AhCYP707A2 was significantly up-regulated by 20% PEG6000 or 250 mmol/L NaCl in peanut roots, stems, and leaves, whereas the up-regulation of AhCYP707A1 transcript level by PEG6000 or NaCl was observed only in roots instead of leaves and stems. Due to the osmotic and ionic stresses of high concentration of NaCl to plants simultaneously, low concentration of LiCl (30 mmol/L, at which concentration osmotic status of cells is not seriously affected, the toxicity of Li+ being higher than that of Na+) was used to examine whether the effect of NaCl might be related to osmotic or ionic stress. The results revealed visually the susceptibility to osmotic stress and the resistance to salt ions in peanut seedlings. The significant up-regulation of AhCYP707A1, AhCYP707A2 and AhNCED1 transcripts and endogenous ABA levels by PEG6000 or NaCl instead of LiCl, showed that the osmotic stress instead of ionic stress affected the expression of those genes and the biosynthesis of ABA in peanut. The functional expression of AhCYP707A1 cDNA in yeast showed that the microsomal fractions prepared from yeast cell expressing recombinant AhCYP707A1 protein exhibited the catalytic activity of ABA 8'-hydroxylase. These results demonstrate that the expressions of AhCYP707A1 and AhCYP707A2 play an important role in ABA catabolism in peanut, particularly in response to osmotic stress.