Acid Biosynthesis In Plants Research Articles

Completion of the core β-oxidative pathway of benzoic acid biosynthesis in plants

Despite the importance of benzoic acid (BA) as a precursor for a wide array of primary and secondary metabolites, its biosynthesis in plants has not been fully elucidated. BA formation from phenylalanine requires shortening of the C(3) side chain by two carbon units, which can occur by a non-β-oxidative route and/or a β-oxidative pathway analogous to the catabolism of fatty acids. Enzymes responsible for the first and last reactions of the core BA β-oxidative pathway (cinnamic acid → cinnamoyl-CoA → 3-hydroxy-3-phenylpropanoyl-CoA → 3-oxo-3-phenylpropanoyl-CoA → BA-CoA) have previously been characterized in petunia, a plant with flowers rich in phenylpropanoid/benzenoid volatile compounds. Using a functional genomics approach, we have identified a petunia gene encoding cinnamoyl-CoA hydratase-dehydrogenase (PhCHD), a bifunctional peroxisomal enzyme responsible for two consecutively occurring unexplored intermediate steps in the core BA β-oxidative pathway. PhCHD spatially, developmentally, and temporally coexpresses with known genes in the BA β-oxidative pathway, and correlates with emission of benzenoid volatiles. Kinetic analysis of recombinant PhCHD revealed it most efficiently converts cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA, thus forming the substrate for the final step in the pathway. Down-regulation of PhCHD expression in petunia flowers resulted in reduced CHD enzyme activity, as well as decreased formation of BA-CoA, BA and their derived volatiles. Moreover, transgenic lines accumulated the PhCHD substrate cinnamoyl-CoA and the upstream pathway intermediate cinnamic acid. Discovery of PhCHD completes the elucidation of the core BA β-oxidative route in plants, and together with the previously characterized CoA-ligase and thiolase enzymes, provides evidence that the whole pathway occurs in peroxisomes.

The Shikimate Pathway and Aromatic Amino Acid Biosynthesis in Plants

L-tryptophan, L-phenylalanine, and L-tyrosine are aromatic amino acids (AAAs) that are used for the synthesis of proteins and that in plants also serve as precursors of numerous natural products, such as pigments, alkaloids, hormones, and cell wall components. All three AAAs are derived from the shikimate pathway, to which ≥30% of photosynthetically fixed carbon is directed in vascular plants. Because their biosynthetic pathways have been lost in animal lineages, the AAAs are essential components of the diets of humans, and the enzymes required for their synthesis have been targeted for the development of herbicides. This review highlights recent molecular identification of enzymes of the pathway and summarizes the pathway organization and the transcriptional/posttranscriptional regulation of the AAA biosynthetic network. It also identifies the current limited knowledge of the subcellular compartmentalization and the metabolite transport involved in the plant AAA pathways and discusses metabolic engineering efforts aimed at improving production of the AAA-derived plant natural products.

Targeting Acetyl-CoA Carboxylases: Small Molecular Inhibitors and their Therapeutic Potential

Acetyl-CoA carboxylases (ACCs) play a rate-limiting role in fatty acid biosynthesis in plants, microbes, mammals and humans. ACCs have the activity of both biotin carboxylase (BC) and carboxyltransferase (CT), catalyzing carboxylation of Acetyl-CoA to malonyl-CoA. In the past years, ACCs have been used as targets for herbicides in agriculture and for drug discovery and development of human diseases, such as microbial infections, diabetes, obesity and cancer. A great number of small molecule ACC inhibitors have been developed, including natural and non-natural (artificial) products. These chemicals target BC reaction, CT reaction or ACC phosphorylation. This article provides a comprehensive review and updates of ACC inhibitors, with a focus on their therapeutic application in metabolic syndromes and malignant diseases. The patent status of common ACC inhibitors is discussed.

Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over-expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE-TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map-based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant-containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed.

Molecular Mechanism of Enantioselective Inhibition of Acetolactate Synthase by Imazethapyr Enantiomers

Chiral compounds usually behave enantioselectively in phyto-biochemical processes. Imidazolinones are a class of chiral herbicides that are widely used. They inhibit branched-chain amino acid biosynthesis in plants by targeting acetolactate synthase (ALS). It has been reported that the imidazolinone enantiomers show different inhibiting activities to maize (Zea mays L.) seedlings and ALS. However, to date, the mechanism of enantioselective inhibition of imazethapyr (IM) on ALS activity has not been well studied. In this study, pure enantiomers of IM were used for characterizing their differences in activity to ALS. Computational molecular docking was performed to discover the molecular interaction between IM enantiomers and ALS at the first time. Results showed that the IM enantiomers enantioselectively suppressed the in vitro and in vivo ALS activity of maize leaves. R-(-)-IM was more active than S-(+)-IM. The in vivo ALS activity study showed only a 2-fold difference between R-(-)-IM and S-(+)-IM. Quite different from the in vivo study, the in vitro study showed that the difference in inhibition between the enantiomers fell sharply as concentration increased. At the lowest concentration of 40 microg L(-1), R-(-)-IM appeared 25 times more active than S-(+)-IM, but only 7 times at 200 microg L(-1). At the highest concentration of 25 mg L(-1), in vitro ALS activity was almost completely inhibited by S-(+)-, R-(-)-IM and (+/-)-IM, there was only 1.1 times differences between S-(+)- and R-(-)-IM. Molecular modeling results provide the rational structural basis to understand the mechanism of enantioselective inhibition of IM on ALS activity.

A new cotton SDR family gene encodes a polypeptide possessing aldehyde reductase and 3-ketoacyl-CoA reductase activities

To understand regulatory mechanisms of cotton fiber development, microarray analysis has been performed for upland cotton (Gossypium hirsutum). Based on this, a cDNA (GhKCR3) encoding a polypeptide belonging to short-chain alcohol dehydrogenase/reductase family was isolated and cloned. It contains an open reading frame of 987 bp encoding a polypeptide of 328 amino acid residues. Following its overexpression in bacterial cells, the purified recombinant protein specifically uses NADPH to reduce a variety of short-chain aldehydes. A fragment between Gly180 and Gly191 was found to be essential for its catalytic activity. Though the GhKCR3 gene shares low sequence similarities to the ortholog of Saccharomyces cerevisiae YBR159w that encodes 3-ketoacyl-CoA reductase (KCR) catalyzing the second step of fatty acid elongation, it was surprisingly able to complement the yeast ybr159wDelta mutant. Gas chromatography-mass spectrometry analysis showed that very long-chain fatty acids, especially C26:0, were produced in the ybr159wDelta mutant cells expressing GhKCR3. Applying palmitoyl-CoA and malonyl-CoA as substrates, GhKCR3 showed KCR activity in vitro. Quantitative real time-PCR analysis indicated GhKCR3 transcripts accumulated in rapidly elongating fibers, roots, and stems. Our results suggest that GhKCR3 is probably a novel KCR contributing to very long-chain fatty acid biosynthesis in plants.

Spatial distribution of cytosolic NADP+-isocitrate dehydrogenase in pine embryos and seedlings

Cytosolic NADP(+)-linked isocitrate dehydrogenase (NADP(+)-IDH) is considered the main enzyme catalyzing the production of 2-oxoglutarate for amino acid biosynthesis in plants. We characterized a full-length cDNA encoding the cytosolic NADP(+)-IDH in the gymnosperm Pinus pinaster Ait. The deduced amino acid sequence exhibited high homology with available sequences in angiosperms. Genomic analysis indicated that only one gene, or two genes with a high degree of homology, encodes the protein in P. pinaster. Cytosolic NADP(+)-IDH is up-regulated during embryo germination concomitantly with glutamine synthetase. Immunohistochemical analysis of germinating seeds and young seedlings showed a broad spatial pattern of NADP(+)-IDH expression. The protein was detected in vascular tissues of germinating embryo and seedling organs, as well as in other cellular types and tissues, including parenchyma and epidermal cells. The spatial pattern of NADP(+)-IDH expression in the embryo and seedling organs did not coincide with the reported spatial patterns for other key enzymes of nitrogen metabolism. Treatment of seedlings with phosphinothricin, an inhibitor of glutamine synthetase (GS), differentially affected GS and NADP-IDH in cotyledons. In response to herbicide treatment, GS was up-regulated in 0.5-cm-long cotyledons, whereas NADP(+)-IDH was up-regulated in 1.5-cm-long cotyledons, suggesting that 2-oxoglutarate is required to overcome the herbicide effect in tissues with a high demand for glutamate. The results indicated that cytosolic NADP(+)-IDH is a housekeeping enzyme that has not undergone functional specialization during evolution. Its spatial pattern in pine tissues suggests that it facilitates metabolism in different ways depending on the characteristics of the particular tissue and cellular type.

Effect of sulfur availability on the integrity of amino acid biosynthesis in plants

Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.

Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase

The sulfonylureas and imidazolinones are potent commercial herbicide families. They are among the most popular choices for farmers worldwide, because they are nontoxic to animals and highly selective. These herbicides inhibit branched-chain amino acid biosynthesis in plants by targeting acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This report describes the 3D structure of Arabidopsis thaliana AHAS in complex with five sulfonylureas (to 2.5 A resolution) and with the imidazolinone, imazaquin (IQ; 2.8 A). Neither class of molecule has a structure that mimics the substrates for the enzyme, but both inhibit by blocking a channel through which access to the active site is gained. The sulfonylureas approach within 5 A of the catalytic center, which is the C2 atom of the cofactor thiamin diphosphate, whereas IQ is at least 7 A from this atom. Ten of the amino acid residues that bind the sulfonylureas also bind IQ. Six additional residues interact only with the sulfonylureas, whereas there are two residues that bind IQ but not the sulfonylureas. Thus, the two classes of inhibitor occupy partially overlapping sites but adopt different modes of binding. The increasing emergence of resistant weeds due to the appearance of mutations that interfere with the inhibition of AHAS is now a worldwide problem. The structures described here provide a rational molecular basis for understanding these mutations, thus allowing more sophisticated AHAS inhibitors to be developed. There is no previously described structure for any plant protein in complex with a commercial herbicide.

Key Role of Conserved Histidines in Recombinant Mouse β-Carotene 15,15′-Monooxygenase-1 Activity

Alignment of sequences of vertebrate beta-carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V(max). The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.

ALBINO AND PALE GREEN 10 Encodes BBMII Isomerase Involved in Histidine Biosynthesis in Arabidopsis thaliana

We isolated an Arabidopsis albino and pale green 10 (apg10) mutant which exhibits pale green cotyledons and true leaves at the juvenile stage. We identified a valine to leucine change in BBMII (N'-[(5'-phosphoribosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) isomerase involved in histidine biosynthesis. The morphological abnormality of apg10 was recovered by histidine supplementation. The histidine limitation induced by apg10 mutation causes dynamic changes of the free amino acid profile, suggesting the existence of a cross-pathway regulatory mechanism of amino acid biosynthesis in plants. We also revealed that the APG10 knockout mutant exhibited embryo lethality, indicating the essential role of the Arabidopsis BBMII isomerase for plant growth.

Fine mapping of the rice low phytic acid (Lpa1) locus

Phytic acid is the primary storage form of phosphorus (P) in cereal grains. In addition to being essential for normal seedling growth and development, phytic acid plays an important role in human and animal nutrition. The rice low phytic acid mutation lpa1 results in a 45% reduction in seed phytic acid with a molar equivalent increase in inorganic P. The Lpa1 locus was previously mapped to the long arm of chromosome 2. Using microsatellite markers and a recombinant inbred line population, we fine mapped this locus between the markers RM3542 and RM482, which encompass a region of 135 kb. Additional markers were developed from the DNA sequence of this region. Two of these markers further delimited the locus to a 47-kb region containing eight putative open reading frames. Cloning and molecular characterization of the Lpa1 gene will provide insight into phytic acid biosynthesis in plants. The markers reported here should also be useful in introgressing the low phytic acid phenotype into other rice cultivars.

Crystal Structure of a Substrate Complex of Mycobacterium tuberculosis β-Ketoacyl-acyl Carrier Protein Synthase III (FabH) with Lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs.

Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme.

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis.

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.

Metabolic Engineering of Fatty Acid Biosynthesis in Plants

Fatty acids are the most abundant form of reduced carbon chains available from nature and have diverse uses ranging from food to industrial feedstocks. Plants represent a significant renewable source of fatty acids because many species accumulate them in the form of triacylglycerol as major storage components in seeds. With the advent of plant transformation technology, metabolic engineering of oilseed fatty acids has become possible and transgenic plant oils represent some of the first successes in design of modified plant products. Directed gene down-regulation strategies have enabled the specific tailoring of common fatty acids in several oilseed crops. In addition, transfer of novel fatty acid biosynthetic genes from noncommercial plants has allowed the production of novel oil compositions in oilseed crops. These and future endeavors aim to produce seeds higher in oil content as well as new oils that are more stable, are healthier for humans, and can serve as a renewable source of industrial commodities. Large-scale new industrial uses of engineered plant oils are on the horizon but will require a better understanding of factors that limit the accumulation of unusual fatty acid structures in seeds.

A mechanism of benzoic acid biosynthesis in plants and bacteria that mirrors fatty acid beta-oxidation.

Despite the simple structure and widespread occurrence of benzoic acid, its biosynthesis has resisted complete elucidation. However, recent studies reveal that the conversion of cinnamoyl-CoA into benzoyl-CoA (see scheme; R=Ph) can proceed by a route that resembles fatty acid β-oxidation. Feeding studies and sequence analyses show that both pathways not only proceed with the same stereochemistry, but are also mechanistically related.

Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants

Plants and most animals can synthesize their own L-ascorbic acid (vitamin C), but a mutation in the L-gulono-γ-lactone oxidase gene in the primate lineage makes it necessary for humans to acquire this vital compound from their diet. Despite the fact that plants and animals synthesize ascorbic acid via different pathways, transgenic tobacco and lettuce plants expressing a rat cDNA encoding L-gulono-γ-lactone oxidase accumulated up to seven times more ascorbic acid than untransformed plants. These results demonstrate that basal levels of ascorbic acid in plants can be significantly increased by expressing a single gene from the animal pathway.

Molecular biology and regulation of abscisic acid biosynthesis in plants

The phytohormone abscisic acid (ABA) is involved in seed dormancy and the response to various environmental stresses. Our understanding of the ABA biosynthetic pathway has been increased recently through the use of plant mutants and the cloning of many of the genes encoding for the enzymes involved. C 40 Xanthophylls are precursors of ABA and are now known to be derived from isopentenyl phosphate (IPP) synthesized in plastids via a mevalonate-independent pathway. Enzyme reactions downstream of zeaxanthin have recently been reported to be important for the precise regulation of ABA levels. Zeaxanthin epoxidase (ZEP) catalyses the conversion of zeaxanthin to violaxanthin. Changes in ZEP gene expression appear to regulate ABA biosynthesis in seeds and roots, but not in leaves which might be expected considering the important role of epoxy-carotenoids in photosynthesis and photoprotection. The isomerization of the resulting all- trans-violaxanthin to 9- cis-epoxy-carotenoids awaits elucidation. Although 9- cis-epoxy-carotenoid dioxygenase (NCED), which subsequently cleaves the resulting carotenoids could use the 9- cis isomers of both violaxanthin and neoxanthin as substrates in vitro, the in vivo substrates remain to be determined. NCEDs are apparently encoded by multigene families and identification of the various members is required to determine their relative contribution to the regulation of ABA levels. Studies on those already available indicate that their up-regulation upon water stress is compatible with a key role in the modulation of ABA levels. The genes encoding for the enzymes that convert the cleavage product xanthoxin to ABA are not yet known, although recently cloned aldehyde oxidases may act on ABA-aldehyde.