Epoxycarotenoid cleavage by NCED5 fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with other NCED family members

Carotenoid cleavage, catalyzed by the 9-cis-epoxycarotenoid dioxygenase (NCED) constitutes a key step in the regulation of ABA biosynthesis. In Arabidopsis, this enzyme is encoded by five genes. NCED3 has been shown to play a major role in the regulation of ABA synthesis in response to water deficit, whereas NCED6 and NCED9 have been shown to be essential for the ABA production in the embryo and endosperm that imposes dormancy. Reporter gene analysis was carried out to determine the spatiotemporal pattern of NCED5 and NCED9 gene expression. GUS activity from the NCED5 promoter was detected in both the embryo and endosperm of developing seeds with maximal staining after mid-development. NCED9 expression was found at early stages in the testa outer integument layer 1, and after mid-development in epidermal cells of the embryo, but not in the endosperm. In accordance with its temporal- and tissue-specific expression, the phenotypic analysis of nced5 nced6 nced9 triple mutant showed the involvement of the NCED5 gene, together with NCED6 and NCED9, in the induction of seed dormancy. In contrast to nced6 and nced9, however, nced5 mutation did not affect the gibberellin required for germination. In vegetative tissues, combining nced5 and nced3 mutations reduced vegetative growth, increased water loss upon dehydration, and decreased ABA levels under both normal and stressed conditions, as compared with nced3. NCED5 thus contributes, together with NCED3, to ABA production affecting plant growth and water stress tolerance.     

The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis

Osmotic stress activates the biosynthesis of abscisic acid (ABA). One major step in ABA biosynthesis is the carotenoid cleavage catalyzed by a 9-cis epoxycarotenoid dioxygenase (NCED). To understand the mechanism for osmotic stress activation of ABA biosynthesis, we screened for Arabidopsis thaliana mutants that failed to induce the NCED3 gene expression in response to osmotic stress treatments. The ced1 (for 9-cis epoxycarotenoid dioxygenase defective 1) mutant isolated in this study showed markedly reduced expression of NCED3 in response to osmotic stress (polyethylene glycol) treatments compared with the wild type. Other ABA biosynthesis genes are also greatly reduced in ced1 under osmotic stress. ced1 mutant plants are very sensitive to even mild osmotic stress. Map-based cloning revealed unexpectedly that CED1 encodes a putative α/β hydrolase domain-containing protein and is allelic to the BODYGUARD gene that was recently shown to be essential for cuticle biogenesis. Further studies discovered that other cutin biosynthesis mutants are also impaired in osmotic stress induction of ABA biosynthesis genes and are sensitive to osmotic stress. Our work demonstrates that the cuticle functions not merely as a physical barrier to minimize water loss but also mediates osmotic stress signaling and tolerance by regulating ABA biosynthesis and signaling.     

Nucleotide Variation in the NCED3 Region of Arabidopsis thaliana and its Association Study with Abscisic Acid Content under Drought Stress

Drought tolerance is a comprehensive quantitative trait that is being understood further at the molecular genetic level. Abscisic acid (ABA) is the main drought-induced hormone that regulates the expression of many genes related to drought responses. 9-cis-epoxycarotenoid dioxygenase (NCED3) is thought to be a key enzyme in ABA biosynthesis. In this paper, we measured the ABA content increase under drought stress, and sequenced and compared the sequence of AtNCED3 among 22 Arabidopsis thaliana accessions. The results showed that the fold of ABA content increase under drought stress was highly variable among these accessions. High density single nucleotide polymorphism (SNP) and insertion/deletion (indel) were found in the AtNCED3 region, on average one SNP per 87.4 bp and one indel per 502 bp. Nucleotide diversity was significantly lower in the coding region than that in non-coding regions. The results of an association study with ANOVA analysis suggested that the 274th site (P←→S) and the 327th site (P←→R) amino acid variations might be the cause of ABA content increase of 163av accession under drought stress.     

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.     

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.     

Cloning and functional characterization of the <Emphasis Type="Italic">SmNCED3</Emphasis> in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

As one of the most important phytohormones, the abscisic acid (ABA) is often used to breed stress-tolerant crop lines with both higher yields and active ingredient contents. In higher plants, the 9-cis-epoxycarotenoid dioxygenase (NCED) has been found to be a regulatory enzyme involved in ABA biosynthesis. In research, the novel gene SmNCED3 was isolated from S. miltiorrhiza. The open reading frame of SmNCED3 was 1725-bp, and it was encoding 574 amino acids with a calculated molecular mass of 63,822 kDa, which was verified by the expression of SmNCED3 in E. coli. The deduced SmNCED3 amino acid sequence had high sequence homology with NCED sequences from other plants and contained a putative chloroplast transit targeting signal peptide at its N terminus. Phylogenetic analysis demonstrated that SmNCED3 had a closer affinity to NCED3 in Arabidopsis thaliana (AtNCED3). The 1732-bp 5′ flanking sequence of SmNCED3 was also cloned. It contained several phytohormone response elements, biotic or abiotic stress-related elements, and plant development-related elements. Real-time PCR revealed that SmNCED3 was highly expressed in leaves, and was strongly induced by exogenous ABA. A subcellular localization experiment indicated that SmNCED3 was located in chloroplast stroma, chloroplast membranes, and thylakoid membranes. The overexpression of SmNCED3 promoted ABA accumulation. These results indicated that SmNCED3 might be a rate-limiting gene regulating ABA biosynthesis, and improving abiotic stresses tolerance and active ingredient contents in plants.     

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.     

Localization and targeting of the VP14 epoxy-carotenoid dioxygenase to chloroplast membranes

Abscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges. ABA is synthesized via an oxidative cleavage of 9-cis epoxy-carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway. Vp14 of maize encodes an epoxy-carotenoid dioxygenase that is soluble when expressed in E. coli. An important goal has been to determine how the soluble VP14 protein is targeted to epoxy-carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids. Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma-targeting domain. The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes. Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N-terminal sequence. A putative amphipathic alpha-helix within this region is essential, but not sufficient, for the membrane targeting. Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S-transferase failed to associate with the thylakoid membrane. The membrane-bound VP14 was partially resistant to chaotropic washes such as 0.1 M Na2CO3 (pH 11.5) and 6 M urea. Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane.     

Effect of hydroxylated carotenoid deficiency on ABA accumulation in Arabidopsis

Abscisic acid (ABA) is a sesquiterpene compound (C₁₅) derived from C₄₀ carotenoids. The immediate carotenoid precursors for ABA biosynthesis, 9- cis -violaxanthin and 9'- cis -neoxanthin, are produced from β -carotene by a series of hydroxylation, epoxidation, and isomerization reactions. Carotenoid hydroxylase deficient mutants contain severely reduced levels of violaxanthin and neoxanthin ( < 20% of wild type level) and provide a unique system to correlate carotenoid substrate availability and ABA production in photosynthetic tissues under non-stressed conditions. Quantitative measurements indicated that ABA levels in the carotenoid hydroxylase mutants are reduced nearly 50% compared to the wild type plants under non-stressed conditions. When drought-stressed, wild type plants showed up to a 17-fold increase in ABA levels, while ABA levels in the carotenoid hydroxylase mutants were only increased 6- to 7-fold (25% of wild type drought-stressed ABA levels). Expression of AtNCED3 ( Arabidopsis thaliana nine- cis -epoxycarotenoid dioxygenase 3, the rate-limiting activity for ABA biosynthesis) was induced in the carotenoid hydroxylase mutants, but to a lesser extent than the 40-fold increase in wild type plants. Therefore, the reduced ABA accumulation in response to drought-stress is at least partially due to the attenuated increase in AtNCED3 gene expression in the carotenoid hydroxylase mutants. The remaining violaxanthin and neoxanthin in the carotenoid hydroxylase mutants can not be converted into ABA, indicating that there is probably a separate pool of violaxanthin and neoxanthin that is not accessible to the cleavage enzymes, because it is sequestered in the light-harvesting complexes.     

Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes

The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48 h after wounding and remained elevated through 96 h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8′-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3 h of wounding and remained elevated through 96 h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.     

Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid

The tomato mutant notabilis has a wilty phenotype as a result of abscisic acid (ABA) deficiency. The wild-type allele of notabilis, LeNCED1, encodes a putative 9-cis-epoxycarotenoid dioxygenase (NCED) with a potential regulatory role in ABA biosynthesis. We have created transgenic tobacco plants in which expression of the LeNCED1 coding region is under tetracycline-inducible control. When leaf explants from these plants were treated with tetracycline, NCED mRNA was induced and bulk leaf ABA content increased by up to 10-fold. Transgenic tomato plants were also produced containing the LeNCED1 coding region under the control of one of two strong constitutive promoters, either the doubly enhanced CaMV 35S promoter or the chimaeric 'Super-Promoter'. Many of these plants were wilty, suggesting co-suppression of endogenous gene activity; however three transformants displayed a common, heritable phenotype that could be due to enhanced ABA biosynthesis, showing increased guttation and seed dormancy. Progeny from two of these transformants were further characterized, and it was shown that they also exhibited reduced stomatal conductance, increased NCED mRNA and elevated seed ABA content. Progeny of one transformant had significantly higher bulk leaf ABA content compared to the wild type. The increased seed dormancy was reversed by addition of the carotenoid biosynthesis inhibitor norflurazon. These data provide strong evidence that NCED is indeed a key regulatory enzyme in ABA biosynthesis in leaves, and demonstrate for the first time that plant ABA content can be increased through manipulating NCED.