Cloning and Expression Analysis of cDNAs Encoding ABA 8'-Hydroxylase in Peanut Plants in Response to Osmotic Stress

et al. (2014) Cloning and Expression Analysis of cDNAs Encoding ABA 8'-Hydroxylase in Peanut Plants in Response to Osmotic Stress. PLoS ONE 9(5): e97025. doi:10.1371/journal.pone.0097025 Cloning and Expression Analysis of cDNAs Encoding ABA 8'-Hydroxylase in Peanut Plants in Response to Osmotic Stress Shuai Liu 0 Yan Lv 0 Xiao-Rong Wan 0 Li-Mei Li 0 Bo Hu 0 Ling Li 0 Ji-Hong Liu, Key Laboratory of Horticultural Plant Biology (MOE), China 0 1 Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University , Guangzhou , China , 2 College of Life Sciences, Zhongkai University of Agriculture and Engineering , Guangzhou , China 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. - Funding: This work was jointly supported by the grants from the National Natural Science Foundation of China (approved Nos. 30800077 and 31171468 granted to XRW and LL, respectively) and Guangdong Natural Science Foundation (approved No. 10251063101000010 granted to LL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The plant hormone abscisic acid (ABA) regulates many important physiological and developmental processes in plants as well as adaptive responses to environmental stresses [1]. In theory, endogenous ABA content should be maintained by a balance between biosynthetic and catabolic activities. Thus, to further understand the molecular mechanism that controls the ABA contents in plants, the genes and enzymes in biosynthesis and catabolism of ABA must be investigated in detail. A number of enzymes for ABA biosynthesis have been identified by various genetic or biochemical approaches [2]. One of these, the 9-cisepoxycarotenoid dioxygenase (NCED), cleaves 11,12 double bonds of C40 carotenoids and produces the C15 precursor of ABA. This step is thought to be a critical reaction for de novo ABA biosynthesis in plants [35]. There are five members of the NCED family in the Arabidopsis genome. As the NCED family members exhibit various tissue specificities and expression patterns, it is suggested that each plays a distinct role [6]. Drought stress induced AtNCED3 predominantly among Arabidopsis NCED genes, therefore AtNCED3 is regarded as the most important enzyme for drought-inducible ABA biosynthesis [7]. We have characterized a peanut NCED gene, AhNCED1, and demonstrated that the expression of AhNCED1 gene plays an important role in the regulation of ABA level during water stress, and that waterstress tolerance of Arabidopsis plants can be improved by ectopic expression of the AhNCED1 gene causing accumulation of endogenous ABA [8,9]. Although much is known about ABA biosynthesis in plants, our knowledge about the catabolic pathway of ABA is still relatively limited [2]. ABA is catabolized into inactive forms by either oxidation or conjugation [2]. The oxidative pathways play a pivotal role in various physiological processes. The major oxidative pathway is triggered by the hydroxylation of C-8 to form 8hydroxy ABA (8OH-ABA), which is unstable and can be spontaneously isomerized to phaseic acid (PA), and finally reduced to dihydrophaseic acid (DPA) [2]. It has been predicted that ABA 8-hydroxylase belongs to the cytochrome P450 (CYP) monooxygenase superfamily and is named as CYP707A [2,10]. In recent years, considerable progress has been made in the identification and characterization of cDNAs encoding ABA 8hydroxylase, including four CYP707A genes in Arabidopsis [11,12], two in barley [13,14], two in rice [15], three in bean [16], five in maize [17], ten in soybean [18], and three in potato [19]. These investigations show that the expression of plant CYP707A genes is regulated developmentally and environmentally. In Arabidopsis, CYP707As are induced by exogenous ABA treatment, dehydration and rehydration [11,12]. The induction of CYP707As is likely to be important for the maintenance of endogenous ABA levels, especially when plants have to inactivate ABA promptly after release from dehydration [20]. Umezawa et al [21] further showed that CYP707A3 plays a prominent role in ABA catabolism during the dehydration and rehydration processes of Arabidopsis plants. In bean, PvCYP707A3 transcripts significantly increased in response to dehydration, no changes of mRNA levels of PvCYP707A1 and PvCYP707A2 were found, however, mRNA levels of PvCYP707A1 and PvCYP707A2 in dehydrated leaves rapidly increased in response to rehydration [16]. Transgenic Nicotiana sylvestris plants over-expressing PvCYP707As displayed a wilty phenotype with reduced ABA levels and increased PA levels, and it has been suggested that to increase ABA levels further by genetically repressing ABA 8-hydroxylase would be a more promising strategy than overexpressing NCEDs [16,22,23]. Strong induction of CYP707A1 and CYP707A4 tran (...truncated)