Step In Abscisic Acid Biosynthesis Research Articles

Genome-wide identification of the NCED gene family and functional characterization of PavNCED5 related to bud dormancy in sweet cherry

Abscisic acid (ABA) is involved in responding to various stresses and regulating the dormancy of flower buds in fruit trees. The cleavage of carotenoids is catalyzed by 9-cis epoxy carotenoid dioxygenase (NCED), and is a key step in ABA biosynthesis. Although NCED genes have been identified in a variety of plants, the characteristics of these genes in sweet cherry (Prunus avium L.) trees have been seldom reported. In the present study, 10 PavNCED genes were identified in the sweet cherry genome and distributed on three chromosomes. Cis-element analysis of PavNCEDs indicated that they might be related to hormone regulation, stress responses, and plant development. Through co-expression network analysis, the transcription factors were identified, such as ERF, bHLH and TCP, which might be involved in the regulation of PavNCEDs. Expression pattern analysis of the PavNCED family genes showed that PavNCED1–5 genes were highly expressed during the flower bud dormancy. Importantly, the expression of PavNCED5 was decreased with the decline of ABA content in the sweet cherry. Additionally, the overexpression of PavNCED5 in Arabidopsis showed dormancy deficiency of seeds. This implies that PavNCED5 may play an important role during the dormancy stage of sweet cherry. In general, we characterized the NCED family in sweet cherry and revealed the essential role of PavNCED5 in flower bud dormancy. These findings open up future research directions and provide valuable information for the further functional study of PavNCEDs in sweet cherry.

Disruption of the ABA1 encoding zeaxanthin epoxidase caused defective suberin layers in Arabidopsis seed coats.

Suberin, a complex polyester deposited in the seed coat outer integument, acts as a hydrophobic barrier to control the movement of water, ions, and gas. However, relatively little is known about the signal transduction involved in suberin layer formation during seed coat development. In this study, the effect of the plant hormone abscisic acid (ABA) on suberin layer formation in seed coats was investigated by characterizing mutations in Arabidopsis related to ABA biosynthesis and signaling. Seed coat permeability to tetrazolium salt was noticeably elevated in aba1-1 and abi1-1 mutants, but not significantly altered in snrk2.2/3/6, abi3-8, abi5-7, and pyr1pyl1pyl2pyl4 quadruple mutants compared with that in the wild-type (WT). ABA1 encodes a zeaxanthin epoxidase that functions in the first step of ABA biosynthesis. aba1-1 and aba1-8 mutant seed coats showed reduced autofluorescence under UV light and increased tetrazolium salt permeability relative to WT levels. ABA1 disruption resulted in decreased total seed coat polyester levels by approximately 3%, with a remarkable reduction in levels of C24:0 ω-hydroxy fatty acids and C24:0 dicarboxylic acids, which are the most abundant aliphatic compounds in seed coat suberin. Consistent with suberin polyester chemical analysis, RT-qPCR analysis showed a significant reduction in transcript levels of KCS17, FAR1, FAR4, FAR5, CYP86A1, CYP86B1, ASFT, GPAT5, LTPG1, LTPG15, ABCG2, ABCG6, ABCG20, ABCG23, MYB9, and MYB107, which are involved in suberin accumulation and regulation in developing aba1-1 and aba1-8 siliques, as compared with WT levels. Together, seed coat suberization is mediated by ABA and partially processed through canonical ABA signaling.

Abscisic acid modulates neighbor proximity-induced leaf hyponasty in Arabidopsis.

Leaves of shade-avoiding plants such as Arabidopsis (Arabidopsis thaliana) change their growth pattern and position in response to low red to far-red ratios (LRFRs) encountered in dense plant communities. Under LRFR, transcription factors of the phytochrome-interacting factor (PIF) family are derepressed. PIFs induce auxin production, which is required for promoting leaf hyponasty, thereby favoring access to unfiltered sunlight. Abscisic acid (ABA) has also been implicated in the control of leaf hyponasty, with gene expression patterns suggesting that LRFR regulates the ABA response. Here, we show that LRFR leads to a rapid increase in ABA levels in leaves. Changes in ABA levels depend on PIFs, which regulate the expression of genes encoding isoforms of the enzyme catalyzing a rate-limiting step in ABA biosynthesis. Interestingly, ABA biosynthesis and signaling mutants have more erect leaves than wild-type Arabidopsis under white light but respond less to LRFR. Consistent with this, ABA application decreases leaf angle under white light; however, this response is inhibited under LRFR. Tissue-specific interference with ABA signaling indicates that an ABA response is required in different cell types for LRFR-induced hyponasty. Collectively, our data indicate that LRFR triggers rapid PIF-mediated ABA production. ABA plays a different role in controlling hyponasty under white light than under LRFR. Moreover, ABA exerts its activity in multiple cell types to control leaf position.

Interlinked regulatory loops of ABA catabolism and biosynthesis coordinate fruit growth and ripening in woodland strawberry

Fruit growth and ripening are controlled by multiple phytohormones. How these hormones coordinate and interact with each other to control these processes at the molecular level is unclear. We found in the early stages of Fragaria vesca (woodland strawberry) fruit development, auxin increases both widths and lengths of fruits, while gibberellin [gibberellic acid (GA)] mainly promotes their longitudinal elongation. Auxin promoted GA biosynthesis and signaling by activating GA biosynthetic and signaling genes, suggesting auxin function is partially dependent on GA function. To prevent the repressive effect of abscisic acid (ABA) on fruit growth, auxin and GA suppressed ABA accumulation during early fruit development by activating the expression of FveCYP707A4a encoding cytochrome P450 monooxygenase that catalyzes ABA catabolism. At the onset of fruit ripening, both auxin and GA levels decreased, leading to a steep increase in the endogenous level of ABA that drives fruit ripening. ABA repressed the expression of FveCYP707A4a but promoted that of FveNCED, a rate-limiting step in ABA biosynthesis. Accordingly, altering FveCYP707A4a expression changed the endogenous ABA levels and affected FveNCED expression. Hence, ABA catabolism and biosynthesis are tightly linked by feedback and feedforward loops to limit ABA contents for fruit growth and to quickly increase ABA contents for the onset of fruit ripening. These results indicate that FveCYP707A4a not only regulates ABA accumulation but also provides a hub to coordinate fruit size and ripening times by relaying auxin, GA, and ABA signals.

RNA interference of NtNCED3 reduces drought tolerance and impairs plant growth through feedback regulation of isoprenoids in Nicotiana tabacum

Abscisic acid (ABA) regulates plant growth and development and responses to various abiotic stresses. The enzyme 9-cis-epoxycarotenoid dioxygenase (NCED) catalyses the crucial rate-limiting step in ABA biosynthesis. Here, we isolated two variants of the NCED3 gene (NtNCED3-1 and NtNCED3-2) from tobacco. Our study suggested that NtNCED3 is mainly expressed in the roots, stems, and young upper leaves, and is rapidly induced by drought and heavy metal stress. The promoter of NtNCED3-2 contains a number of abiotic stress-responsive cis-acting elements. RNA interference-based silencing of NtNCED3-2 reduced the ABA content and drought tolerance of tobacco. Suppression of NtNCED3-2 also inhibited primary root development and root hair growth, led to a stunted phenotype, and reduced the number of leaf glandular trichomes. Compared with wild type plant, NtNCED3-2 knockdown plants had reduced diterpenoids, photosynthetic pigments, plant hormone contents, and lower expression of related genes in the isoprenoid metabolism pathway, which resulted in lower photosynthetic ability. Taken together, our results indicate that NtNCED3-2 is involved in the response to drought stress and the regulation of plant growth and development via feedback regulation of the isoprenoid metabolism pathway flux.

Promotion of Germination Using Hydroxamic Acid Inhibitors of 9-cis-Epoxycarotenoid Dioxygenase.

Abscisic acid (ABA) inhibits seed germination and the regulation of ABA biosynthesis has a role in maintenance of seed dormancy. The key rate-limiting step in ABA biosynthesis is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). Two hydroxamic acid inhibitors of carotenoid cleavage dioxygenase (CCD), D4 and D7, previously found to inhibit CCD and NCED in vitro, are shown to have the novel property of decreasing mean germination time of tomato (Solanum lycopersicum L.) seeds constitutively overexpressing LeNCED1. Post-germination, D4 exhibited no negative effects on tomato seedling growth in terms of height, dry weight, and fresh weight. Tobacco (Nicotiana tabacum L.) seeds containing a tetracycline-inducible LeNCED1 transgene were used to show that germination could be negatively and positively controlled through the chemical induction of gene expression and the chemical inhibition of the NCED protein: application of tetracycline increased mean germination time and delayed hypocotyl emergence in a similar manner to that observed when exogenous ABA was applied and this was reversed by D4 when NCED expression was induced at intermediate levels. D4 also improved germination in lettuce (Lactuca sativa L.) seeds under thermoinhibitory temperatures and in tomato seeds imbibed in high osmolarity solutions of polyethylene glycol. D4 reduced ABA and dihydrophaseic acid accumulation in tomato seeds overexpressing LeNCED1 and reduced ABA accumulation in wild type tomato seeds imbibed on polyethylene glycol. The evidence supports a mode of action of D4 through NCED inhibition, and this molecule provides a lead compound for the design of NCED inhibitors with greater specificity and potency.

Molecular characterization of a mutation affecting abscisic acid biosynthesis and consequently stomatal responses to humidity in an agriculturally important species.

Mutants deficient in the phytohormone abscisic acid (ABA) have been instrumental in determining not only the biosynthetic pathway for this hormone, but also its physiological role in land plants. The wilty mutant of Pisum sativum is one of the classical, well-studied ABA-deficient mutants; however, this mutant remains uncharacterized at a molecular level. Using a candidate gene approach, we show that the wilty mutation affects the xanthoxin dehydrogenase step in ABA biosynthesis. To date, this step has only been represented by mutants in the ABA2 gene of Arabidopsis thaliana. Functional ABA biosynthesis appears to be critical for normal stomatal responses to changes in humidity in angiosperms, with wilty mutant plants having no increase in foliar ABA levels in response to a doubling in vapour pressure deficit, and no closure of stomata. Phylogenetic analysis of the ABA2 gene family from diverse land plants indicates that an ABA-biosynthesis-specific short-chain dehydrogenase (ABA2) evolved in the earliest angiosperms. The relatively recent origin of specificity in this step has important implications for both the evolution of ABA biosynthesis and action in land plants.

Molecular cloning and characterization of SoNCED, a novel gene encoding 9-cis-epoxycarotenoid dioxygenase from sugarcane (Saccharum officinarum L.)

Abscisic acid (ABA) plays important roles in adaptive responses to various environmental stresses. The rate-limiting step in ABA biosynthesis is the oxidative cleavage of cis-epoxycarotenoids, which is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). In this experiment, a full-length cDNA encoding NCED gene was cloned by RT-PCR and RACE from sugarcane (Saccharum officinarum L.). The full-length of SoNCED is 2,521 bp with 1,827 bp open reading frame, encoding a peptide of 608 amino acids. The calculated molecular weight of protein was 65.9 kDa with isoelectric point of 6.04. Conserved domains prediction indicated a chloroplast-targeting peptide located at N-terminus of SoNCED. Phylogenetic tree, constructed by Neighbor-Joining method indicated that SoNCED shared high identity with the NCEDs reported from other plant species. Sequence alignment revealed that the basic secondary structure including α-helices, β-strands, β-propeller and His residues coordinating catalytic sites of SoNCED were highly conserved as in the NCEDs from other plants. Tissue specific expression analysis using quantitative real-time PCR showed a significant increase in SoNCED mRNA level and its correlation with O2 – production rate and ABA accumulation in leaves and roots of sugarcane variety GT21 when exposed to water stress. Further, the stimulation of SoNCED mRNA level, O2 – production rate and ABA content after exogenous application of ABA (100 μMol l−1) proved its involvement in pathways providing tolerance to drought stress.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

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.

A viviparous mutant of maize exhibiting permanent water stress symptoms

In maize vivipary, the precocious germination of the seed while it is still attached to the ear is a reliable phenotype for the identification of mutants impaired in the biosynthesis or response to abscisic acid (ABA). Here we present the characterization of a new allele of vp10, a gene encoding for a cofactor (MoCo) required for the last step of ABA biosynthesis. The lesion in this gene leads to a reduction in the endogenous ABA level. Embryonic messenger RNAs of the ABA inducible genes glb1, lea3, and rab17 are barely detectable, although their level increases when stimulated by exogenous ABA administration. These findings confirm that the mutant can be ascribed to a defect in ABA biosynthesis. In the absence of water stress, mutant plants grow like wild-type siblings; however when mutant tissues are exposed to air they differ from non-mutant ones by showing a higher rate of water loss, of transpiration and of stomatal conductance. These events are restored to almost normal values by adding exogenous ABA. All these defects are ascribable to an impairment in the regulation of stomatal opening since, in contrast to wild-type, some of the mutant stomata exhibit partially or totally open rims. The defect in ABA biosynthesis is also associated with loss of regulation of the expression of rab17 and rab28, two genes expressed in vegetative tissues under abiotic stress. These genes are constitutively expressed in the mutant plant tissues independently of the water regime applied. Thus this mutant may provide a tool for the study of molecular mechanisms underlying drought-stress responses in crop plants.

Sesquiterpene-like inhibitors of a 9- cis-epoxycarotenoid dioxygenase regulating abscisic acid biosynthesis in higher plants

Abscisic acid (ABA) is a carotenoid-derived plant hormone known to regulate critical functions in growth, development and responses to environmental stress. The key enzyme which carries out the first committed step in ABA biosynthesis is the carotenoid cleavage 9- cis-epoxycarotenoid dioxygenase (NCED). We have developed a series of sulfur and nitrogen-containing compounds as potential ABA biosynthesis inhibitors of the NCED, based on modification of the sesquiterpenoid segment of the 9 -cis-xanthophyll substrates and product. In in vitro assays, three sesquiterpene-like carotenoid cleavage dioxygenase (SLCCD) inhibitor compounds 13, 17 and 18 were found to act as inhibitors of Arabidopsis thaliana NCED 3 (AtNCED3) with K i′s of 93, 57 and 87 μM, respectively. Computational docking to a model of AtNCED3 supports a mechanism of inhibition through coordination of the heteroatom with the non-heme iron in the enzyme active site. In pilot studies, pretreatment of osmotically stressed Arabidopsis plants with compound 13 resulted lower levels of ABA and catabolite accumulation compared to levels in mannitol-stressed plant controls. This same inhibitor moderated known ABA-induced gene regulation effects and was only weakly active in inhibition of seed germination. Interestingly, all three inhibitors led to moderation of the stress-induced transcription of AtNCED3 itself, which could further contribute to lowering ABA biosynthesis in planta. Overall, these sesquiterpenoid-like inhibitors present new tools for controlling and investigating ABA biosynthesis and regulation.

MOLYBDENUM COFACTOR BIOSYNTHESIS AND MOLYBDENUM ENZYMES

The molybdenum cofactor (Moco) forms the active site of all eukaryotic molybdenum (Mo) enzymes. Moco consists of molybdenum covalently bound to two sulfur atoms of a unique tricyclic pterin moiety referred to as molybdopterin. Moco is synthesized from GTP by an ancient and conserved biosynthetic pathway that can be divided into four steps involving the biosynthetic intermediates cyclic pyranopterin monophosphate, molybdopterin, and adenylated molybdopterin. In a fifth step, sulfuration or bond formation between Mo and a protein cysteine result in two different catalytic Mo centers. There are four Mo enzymes in plants: (1) nitrate reductase catalyzes the first and rate-limiting step in nitrate assimilation and is structurally similar to the recently identified, (2) peroxisomal sulfite oxidase that detoxifies excessive sulfite. (3) Aldehyde oxidase catalyzes the last step of abscisic acid biosynthesis, and (4) xanthine dehydrogenase is essential for purine degradation and stress response.

The Biochemical Characterization of Two Carotenoid Cleavage Enzymes from Arabidopsis Indicates That a Carotenoid-derived Compound Inhibits Lateral Branching

Enzymes that are able to oxidatively cleave carotenoids at specific positions have been identified in animals and plants. The first such enzyme to be identified was a nine-cis-epoxy carotenoid dioxygenase from maize, which catalyzes the rate-limiting step of abscisic acid biosynthesis. Similar enzymes are necessary for the synthesis of vitamin A in animals and other carotenoid-derived molecules in plants. In the model plant, Arabidopsis, there are nine hypothetical proteins that share some degree of sequence similarity to the nine-cis-epoxy carotenoid dioxygenases. Five of these proteins appear to be involved in abscisic acid biosynthesis. The remaining four proteins are expected to catalyze other carotenoid cleavage reactions and have been named carotenoid cleavage dioxygenases (CCDs). The hypothetical proteins, AtCCD7 and AtCCD8, are the most disparate members of this protein family in Arabidopsis. The max3 and max4 mutants in Arabidopsis result from lesions in AtCCD7 and AtCCD8. Both mutants display a dramatic increase in lateral branching and are believed to be impaired in the synthesis of an unidentified compound that inhibits axillary meristem development. To determine the biochemical function of AtCCD7, the protein was expressed in carotenoid-accumulating strains of Escherichia coli. The activity of AtCCD7 was also tested in vitro with several of the most common plant carotenoids. It was shown that the recombinant AtCCD7 protein catalyzes a specific 9-10 cleavage of beta-carotene to produce the 10 black triangle down-apo-beta-carotenal (C27) and beta-ionone (C13). When AtCCD7 and AtCCD8 were co-expressed in a beta-carotene-producing strain of E. coli, the 13-apo-beta-carotenone (C18) was produced. The C18 product appears to result from a secondary cleavage of the AtCCD7-derived C27 product. The sequential cleavages of beta-carotene by AtCCD7 and AtCCD8 are likely the initial steps in the synthesis of a carotenoid-derived signaling molecule that is necessary for the regulation lateral branching.

Characterization of the expression of Phaseolus vulgaris OCT1, a dehydration-regulated gene that encodes a new type of phloem transporter.

A cDNA coding for a putative organic cation transporter (OCT) was isolated from Phaseolus vulgaris roots by differential display RT-PCR and the corresponding full-length cDNA (named PvOCT1) was subsequently obtained by RACE-PCR. Hydropathy profiles of the deduced amino acid sequence (547 residues) predicted the existence of twelve membrane-spanning domains, which are highly conserved in the major facilitator superfamily (MFS). Three specific domains, which characterize organic ion transporters in animals, can also be observed in the predicted protein. In the non-stressed plants, northern analysis showed that PvOCT1 is strongly expressed in roots and stems, while in situ hybridization revealed the presence of PvOCT1 transcripts in phloem cells. In roots PvOCT1 transcript levels transitorily increased after one hour of dehydration and then dramatically decreased. This decrease was associated with enhanced abundance of PvNeED1 mRNA encoding the enzyme thought to catalyze the limiting step of abscisic acid biosynthesis.

The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-responsive gene expression.

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.

The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves.

Abscisic acid (ABA) is a plant hormone involved in seed development and germination and in responses to various environmental stresses. The last step of ABA biosynthesis involves oxidation of abscisic aldehyde, and aldehyde oxidase (EC ) is thought to catalyze this reaction. An aldehyde oxidase isoform, AOdelta, encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3, and AAO4), is the most likely candidate for the enzyme, because it can efficiently catalyze the oxidation of abscisic aldehyde to ABA. Here, we report the isolation and characterization of an ABA-deficient Arabidopsis mutant that maps at the AAO3 locus. The mutant exhibits a wilty phenotype in rosette leaves, but seed dormancy is not affected. ABA levels were significantly reduced in the mutant leaves, explaining the wilty phenotype in rosettes, whereas the level in the mutant seeds was less reduced. No AOdelta activity could be detected in the rosette leaves of the mutant. Sequence data showed that the mutant contains a G to A substitution in the AAO3 gene. The mutation causes incorrect splicing of the ninth intron of AAO3 mRNA. We thus conclude that the ABA-deficient mutant is impaired in the AAO3 gene and that the gene product, AOdelta, is an aldehyde oxidase that catalyzes the last step of ABA biosynthesis in Arabidopsis, specifically in rosette leaves. Other aldehyde oxidases may be involved in ABA biosynthesis in other organs.

Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana.

Abscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses. Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 1.2.3.1). We have reported the occurrence of three isoforms of aldehyde oxidase, AOalpha, AObeta and AOgamma, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde. Here we report a new isoform, AOdelta, found in rosette leaf extracts, which efficiently oxidizes abscisic aldehyde. AO delta was specifically recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4). Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOdelta. These results indicate that AOdelta is encoded by AAO3. AOdelta produced in P. pastoris exhibited a very low Km value for abscisic aldehyde (0.51 microM), and the oxidation product was determined by gas chromatography-mass spectrometry to be ABA. Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves. When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment. These results suggest that AOdelta, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves.

Characterization of the ABA-deficient tomato mutant notabilis and its relationship with maize Vp14.

The notabilis (not) mutant of tomato has a wilty phenotype due to a deficiency in the levels of the plant hormone abscisic acid (ABA). The mutant appears to have a defect in a key control step in ABA biosynthesis--the oxidative cleavage of a 9-cis xanthophyll precursor to form the C15 intermediate, xanthoxin. A maize mutant, viviparous 14 (vp14) was recently obtained by transposon mutagenesis. This maize genetic lesion also affects the oxidative cleavage step in ABA synthesis. Degenerate primers for PCR, based on the VP14 predicted amino acid sequence, have been used to provide probes for screening a wilt-related tomato cDNA library. A full-length cDNA clone was identified which is specific to the not gene locus. The ORFs of the tomato cDNA and maize Vp14 are very similar, apart from parts of their N-terminal sequences. The not mutation has been characterized at the DNA level. A specific A/T base pair deletion of the coding sequence has resulted in a frameshift mutation, indicating that not is a null mutant. This observation is discussed in connection with the relatively mild phenotype exhibited by not mutant homozygotes.

Carotenoids and abscisic acid (ABA) biosynthesis in higher plants

Recent research has revealed that abscisic acid (ABA), synthesised in response to water stress, is an apo‐carotenoid. Two potential carotenoid precursors, 9′‐cis‐neoxanthin and 9‐cis‐violaxanthin, have been identified in light‐grown and etiolated leaves, and in roots of a variety of species. Experiments utilizing etiolated Phaseolus vulgaris leaves and deuterium oxide strongly suggest that 9′‐cis‐neoxanthin, synthesised from all‐trans‐violaxanthin, is the immediate pre‐cleavage precursor of ABA. The cleavage of 9′‐cis‐neoxanthin, performed by an inducible and specific dioxygenase, is likely to be the rate‐limiting step in ABA biosynthesis. Any apocarotenoids formed as by‐products of cleavage are probably rapidly degraded by lipoxygenase or related enzymes. After cleavage xanthoxin is converted via ABA‐aldehyde to ABA by constitutive enzymes in the cytosol.