Molecular cloning and characterization of a dehydration-inducible cDNA encoding a putative 9-cis-epoxycarotenoid dioxygenase in Arachis hygogaea L.

A rate-limiting step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). Here we present the cloning, characterization of a cDNA from dehydrated peanut (Arachis hygogaea L.) leaves that encodes a putative NCED. The 2486-bp full-length cDNA (designated as AhNCED1), obtained by rapid amplification of cDNA ends (RACE), has an open reading frame of 601 amino acid residues and encodes a protein with a calculated molecular weight of 66.86 kDa and an isoelectric point of 8.39. Sequence analysis shows that the deduced amino acid sequence of AhNCED1 shares high identity with the reported NCED protein sequences. There is a 30-amino-acid chloroplast-targeting peptide at the N-terminus of the AhNCED1 protein predicted by iPSORT algorithm. Semi-quantification by duplex RT-PCR reveals that the expression of AhNCED1 is up-regulated by dehydration and that rehydration represses its expression. The organ specific expression pattern of AhNCED1 has been examined, which indicates its dominant expression in leaves and stems. Molecular analysis of the drought-inducible gene of peanut may be useful to investigate the response of agricultural crops to drought stress.     

Identification of different ABA biosynthesis sites at seedling and fruiting stages in Arachis hypogaea L. following water stress

Abscisic acid (ABA) is one of the most important phytohormones involved in abiotic stress responses. ABA transport in plants is important in determining endogenous ABA levels and their resulting physiological responses. However, the regulation of ABA transport remains unclear. In this study, we compared the ABA concentrations and AhNCED1 levels at seedling and fruiting stages in peanut (Arachis hypogaea L.), in response to water stress. At the seedling stage, ABA initially accumulated in roots (1 h), followed by the lower stem (2 h) and finally in the upper stem (4 h). The expression/activity of an ABA biosynthesis rate-limiting enzyme, AhNCED1, showed the same accumulation patterns. In contrast, during the fruiting stage, ABA and AhNCED1 increases were initially detected in the first apical leaf of main stem, followed by the stem, and finally in the root. These results imply that biosynthesis of ABA in peanut plants subject to water deficiency could be dependent on developmental stage with the roots being the initial site of ABA biosynthesis during the seedling stage, whereas during the fruiting stage ABA biosynthesis occurs initially in the leaf. The distribution patterns of ABA in seedling stage peanuts in response to water stress were: root-stem-leaf, while in fruiting stage peanuts the distribution patterns of ABA were: leaf-stem-root. These findings will help to understand plant regulatory water deficit resistance mechanisms at seedling and fruiting stages and to advance our total understanding of the regulation of ABA transport.     

Drought induction of Arabidopsis 9-cis-epoxycarotenoid dioxygenase occurs in vascular parenchyma cells

The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.     

A Distal ABA Responsive Element in AtNCED3 Promoter Is Required for Positive Feedback Regulation of ABA Biosynthesis in Arabidopsis

The plant hormone abscisic acid (ABA) plays a crucial role in plant development and responses to abiotic stresses. Recent studies indicate that a positive feedback regulation by ABA exists in ABA biosynthesis in plants under dehydration stress. To understand the molecular basis of this regulation, we analyzed the cis-elements of the AtNCED3 promoter in Arabidopsis. AtNCED3 encodes the first committed and highly regulated dioxygenase in the ABA biosynthetic pathway. Through delineated and mutagenesis analyses in stable-transformed Arabidopsis, we revealed that a distal ABA responsive element (ABRE: GGCACGTG, -2372 to -2364 bp) is required for ABA-induced AtNCED3 expression. By analyzing the AtNCED3 expression in ABRE binding protein ABF3 over-expression transgenic plants and knock-out mutants, we provide evidence that the ABA feedback regulation of AtNCED3 expression is not mediated by ABF3.     

Regulatory function of AMP1 in ABA biosynthesis and drought resistance in arabidopsis

In this study we reported the isolation of a mutant in which the reporter pVP14-LUC was highly expressed in Arabidopsis. The gene expression of maize VP14 is closely correlated with the endogenous ABA levels, and the Arabidopsis homolog of VP14, AtNCED1, encoding an enzyme of ABA biosynthesis, was up-regulated, and high ABA level was detected in the mutant. Map-based cloning revealed that the mutated gene is a novel allele of the AMP1 (Altered Meristem Program 1) which encodes a glutamate carboxypeptidase that plays an important role in shoot apical meristem development and phytohormone homeostasis. We found that the mutant displayed obvious drought tolerance, being with more lateral roots, high seed germination under mannitol, increased ABA accumulation, and highly induced gene expression of RD29A. Using the approaches of artificial microRNA gene silencing in transgenic plants, three AMP1 down-regulated lines were obtained. The AMP1 down-regulated plants exhibited a low rate of water loss, decreased stomatal aperture, and enhanced drought tolerance. These results provide evidence demonstrating the regulatory function of AMP1 in plant drought tolerance and stress responsive gene expression.     

不同花生品种响应干旱胁迫后叶片内ABA与AhNCEDl的分布

以粤油7号和汕优523两个不同抗旱性品种为材料,研究响应干旱胁迫后叶片ABA（abscisicacid,脱落酸）和AhNCEDl（Arachishypogaeanine-cis-epoxycarotenoiddioxygenase）的分布以及含量变化。结果表明,两种花生品种响应干旱胁迫后叶片的维管组织中ABA分布增强且含量增加,AhNCEDl蛋白分布也增强;且在水分胁迫初期粤油7号花生AhNCEDl蛋白分布强于汕优523,其体内ABA分布水平也高于汕优523;经ABA生物合成抑制剂N印roxen处理后,两种花生叶片ABA分布减弱,但粤油7号叶片维管组织ABA分布水平仍高于汕优523。结果表明维管组织是干旱胁迫下花生叶片中ABA和AhNCEDl分布的主要区域,且粤油7号花生抗旱性强可能与其体内AhNCEDl和ABA的分布量较高有关。     

Abscisic acid (ABA) inhibition of lateral root formation involves endogenous ABA biosynthesis in Arachis hypogaea L.

ABA has been found to play a significant role in post-embryonic developmental in peanut seedlings. The results from the current study indicate that in the presence of exogenous 10 μmol l⁻¹ ABA, lateral roots (LRs) number decreased and seedling development was delayed. This effect was eliminated by 25 μmol l⁻¹ naproxen, an inhibitor of ABA biosynthesis. The Arabidopsis mutant deficient in ABA biosynthesis, nced3, displays a phenotype with more and longer LRs. We found that ABA decreased root-branching in peanut in a dose-dependent way. ABA-treated seedlings showed higher endogenous ABA levels than the control and naproxen-treated seedlings. RT-PCR results indicated that the expression of AhNCED1, a key gene in the ABA biosynthetic pathway, was significantly up-regulated by exogenous ABA in peanut. The mRNA levels of AhNCED1 began to increase 2 days after ABA treatment. The results from the current study show that ABA inhibits peanut LR development by increasing endogenous ABA contents.     

CYP707A3, a major ABA 8'-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana

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 the cytochrome P450 CYP707A family. Among four members of Arabidopsis CYP707As, the expression of CYP707A3 was most highly induced in response to both dehydration and subsequent rehydration. A T-DNA insertional cyp707a3-1 mutant contained higher ABA levels in turgid plants, which showed a reduced transpiration rate and hypersensitivity to exogenous ABA during early seedling growth. On dehydration, the cyp707a3-1 mutant accumulated a higher amount of stress-induced ABA than the wild type, an event that occurred relatively later and was coincident with slow drought induction of CYP707A3. The cyp707a3 mutant plants exhibited both exaggerated ABA-inducible gene expression and enhanced drought tolerance. Conversely, constitutive expression of CYP707A3 relieved growth retardation by ABA, increased transpiration, and a reduction of endogenous ABA in both turgid and dehydrated plants. Taken together, our results indicate that CYP707A3 plays an important role in determining threshold levels of ABA during dehydration and after rehydration.