Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado

Avocado (Persea americana Mill. cv Lula) is a climacteric fruit that exhibits a rise in ethylene as the fruit ripens. This rise in ethylene is followed by an increase in abscisic acid (ABA), with the highest level occurring just after the peak in ethylene production. ABA is synthesized from the cleavage of carotenoid precursors. The cleavage of carotenoid precursors produces xanthoxin, which can subsequently be converted into ABA via ABA-aldehyde. Indirect evidence indicates that the cleavage reaction, catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED), is the regulatory step in ABA synthesis. Three genes encoding NCED cleavage-like enzymes were cloned from avocado fruit. Two genes, PaNCED1 and PaNCED3, were strongly induced as the fruit ripened. The other gene, PaNCED2, was constitutively expressed during fruit ripening, as well as in leaves. This gene lacks a predicted chloroplast transit peptide. It is therefore unlikely to be involved in ABA biosynthesis. PaNCED1 was induced by water stress, but expression of PaNCED3 was not detectable in dehydrated leaves. Recombinant PaNCED1 and PaNCED3 were capable of in vitro cleavage of 9-cis-xanthophylls into xanthoxin and C(25)-apocarotenoids, but PaNCED2 was not. Taken together, the results indicate that ABA biosynthesis in avocado is regulated at the level of carotenoid cleavage.     

A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions

Glc has hormone-like functions and controls many vital processes through mostly unknown mechanisms in plants. We report here on the molecular cloning of GLUCOSE INSENSITIVE1 (GIN1) and ABSCISIC ACID DEFICIENT2 (ABA2) which encodes a unique Arabidopsis short-chain dehydrogenase/reductase (SDR1) that functions as a molecular link between nutrient signaling and plant hormone biosynthesis. SDR1 is related to SDR superfamily members involved in retinoid and steroid hormone biosynthesis in mammals and sex determination in maize. Glc antagonizes ethylene signaling by activating ABA2/GIN1 and other abscisic acid (ABA) biosynthesis and signaling genes, which requires Glc and ABA synergistically. Analyses of aba2/gin1 null mutants define dual functions of endogenous ABA in inhibiting the postgermination developmental switch modulated by distinct Glc and osmotic signals and in promoting organ and body size and fertility in the absence of severe stress. SDR1 is sufficient for the multistep conversion of plastid- and carotenoid-derived xanthoxin to abscisic aldehyde in the cytosol. The surprisingly restricted spatial and temporal expression of SDR1 suggests the dynamic mobilization of ABA precursors and/or ABA.     

高等植物脱落酸生物合成途径及其酶调控

脱落酸(ABA)生物合成一般有两条途径:C15直接途径和C40间接途径,前者经C15法呢焦磷酸(FPP)直接形成ABA;后者经由类胡萝卜素的氧化裂解间接形成ABA,是高等植物ABA生物合成的主要途径.9-顺式环氧类胡萝卜素氧化裂解为黄质醛是植物ABA生物合成的关键步骤,然后黄质醛被氧化形成一种酮,该过程需NAD为辅因子,酮再转变形成ABA-醛,ABA-醛氧化最终形成ABA.在该途径中,玉米黄质环氧化酶(ZEP)、9-顺式环氧类胡萝卜素双加氧酶(NCED)和醛氧化酶(AO)可能起重要作用.     

高等植物脱落酸生物合成途径及其酶调控

脱落酸（ABA）生物合成一般有两条途径：C15直接途径和C40间接途径, 前者经C15法呢焦磷酸（FPP）直接形成ABA;后者经由类胡萝卜素的氧化裂解间接形成ABA, 是高等植物ABA生物合成的主要途径。9-顺式环氧类胡萝卜素氧化裂解为黄质醛是植物ABA生物合成的关键步骤, 然后黄质醛被氧化形成一种酮, 该过程需NAD为辅因子, 酮再转变形成ABA-醛, ABA-醛氧化最终形成ABA。在该途径中,玉米黄质环氧化酶（ZEP）、9-顺式环氧类胡萝卜素双加氧酶（NCED）和醛氧化酶（AO）可能起重要作用。     

Abscisic acid promotes lectin biosynthesis in developing and germinating rice embryos

Immature rice (Oryza sativa, L) embryos isolated about 12 days post anthesis are fully able to develop into young seedlings when cultured in vitro. Concomitantly, they rapidly loose their lectin synthesis activity. Abscisic acid added to the nutrient medium prevents precocious germination of the immature embryos and simultaneously strongly promotes lectin biosynthesis activity. Similarly, abscisic acid keeps mature embryos grown in a nutrient medium in a dormant state and maintains their lectin synthesis activity, whereas control embryos rapidly germinate but also quickly loose their lectin synthesis activity. It appears, therefore, that rice lectin is typically synthesized in embryos which are kept in a dornant state.Abbreviations ABA abscisic acid - GA₃ gibberellic acid - WGA wheat germ agglutinin - DPA days post anthesis     

Regulation of hormone metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism

In a wide range of plant species, seed germination is regulated antagonistically by two plant hormones, abscisic acid (ABA) and gibberellin (GA). In the present study, we have revealed that ABA metabolism (both biosynthesis and inactivation) was phytochrome-regulated in an opposite fashion to GA metabolism during photoreversible seed germination in Arabidopsis. Endogenous ABA levels were decreased by irradiation with a red (R) light pulse in dark-imbibed seeds pre-treated with a far-red (FR) light pulse, and the reduction in ABA levels in response to R light was inhibited in a phytochrome B (PHYB)-deficient mutant. Expression of an ABA biosynthesis gene, AtNCED6, and the inactivation gene, CYP707A2, was regulated in a photoreversible manner, suggesting a key role for the genes in PHYB-mediated regulation of ABA metabolism. Abscisic acid-deficient mutants such as nced6-1, aba2-2 and aao3-4 exhibited an enhanced ability to germinate relative to wild type when imbibed in the dark after irradiation with an FR light pulse. In addition, the ability to synthesize GA was improved in the aba2-2 mutant compared with wild type during dark-imbibition after an FR light pulse. Activation of GA biosynthesis in the aba2-2 mutant was also observed during seed development. These data indicate that ABA is involved in the suppression of GA biosynthesis in both imbibed and developing seeds. Spatial expression patterns of the AtABA2 and AAO3 genes, responsible for last two steps of ABA biosynthesis, were distinct from that of the GA biosynthesis gene, AtGA3ox2, in both imbibed and developing seeds, suggesting that biosynthesis of ABA and GA in seeds occurs in different cell types.     

Formation and breakdown of ABA

The phytohormone, abscisic acid (ABA) is found in all photosynthetic organisms. The amount of ABA present is determined by the dynamic balance between biosynthesis and degradation: these two processes are influenced by development, environmental factors such as light and water stress, and other growth regulators. ABA is synthesized from a C₄₀ carotenoid precursor and the first enzyme committed specifically to ABA synthesis is a plastid- localized 9-cis-epoxycarotenoid dioxygenase, which cleaves an epoxycarotenoid precursor to form xanthoxin. Subsequently, xanthoxin is converted to ABA by two cytosolic enzymes via abscisic aldehyde, but there appears to be at least one minor alternative pathway. The major catabolic route leads to 8′-hydroxy ABA and phaseic acid formation, catalyzed by the cytochrome P450 enzyme ABA 8′-hydroxylase. In addition, there are alternate catabolic pathways via conjugation, 4′-reduction and 7′-hydroxylation. As a consequence of recent developments, the mechanism by which the concentration of hormonally active ABA is controlled at the cellular, tissue and whole plant level can now be analyzed in detail.     

Carotenogenic and abscisic acid biosynthesizing activity in a cell-free system

Abscisic acid is considered an apocarotenoid formed by cleavage of a C-40 precursor and subsequent oxidation of xanthoxin and abscisic aldehyde. Confirmation of this reaction sequence is still awaited, and might best be achieved using a cell-free system capable of both carotenoid and abscisic acid biosynthesis. An abscisic acid biosynthesizing cell-free system, prepared from flavedo of mature orange fruits, was used to demonstrate conversion of farnesyl pyrophosphate, geranylgeranyl pyrophosphate and all-trans-β-carotene into a range of β,β-xanthophylls, xanthoxin, xanthoxin acid, 1′,4′-trans-abscisic acid diol and abscisic acid. Identification of product carotenoids was achieved by high-performance liquid chromatography and on-line spectral analysis of individual components together with co-chromatography. Putative C-15 intermediates and product abscisic acid were identified by combined capillary gas chroma-tography-mass spectrometry. Kinetic studies revealed that β-carotene, formed from either famesyl pyrophosphate or geranylgeranyl pyrophosphate, reached a maximum within 30 min of initiation of the reaction. Thereafter, β-carotene levels declined exponentially. Catabolism of substrate β-carotene into xanthophylls, putative abscisic acid precursors and product abscisic acid was restricted to the all-trans-isomer. However, when a combination of all-trans- and 9-cis-β-carotene in the ratio 1:1 was used as substrate, formation of abscisic acid and related metabolites was enhanced. Biosyn-thetically prepared [¹⁴C]-all-trans-violaxanthin, [¹⁴C]-all-trans-neoxanthin and [¹⁴C]-9′-cis-neoxanthin were used as substrates to confirm the metabolic interrelationship between carotenoids and abscisic acid. The results are consistent with 9′-cis-neoxan-thin being the immediate carotenoid precursor to ABA, which is oxidatively cleaved to produce xanthoxin. Formation of abscisic aldehyde was not observed. Rather, xanthoxin appeared to be converted to abscisic acid via xanthoxin acid and 1′,4′-trans-abscisic acid diol. An alternative pathway for abscisic acid biosynthesis is therefore proposed.     

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.     

In Situ Abscisic Acid Synthesis : A Requirement for Induction of Embryo Dormancy in Helianthus annuus

When applied to young nondormant embryos of sunflower (Hellanthus annus) (7-10 day[s] after pollination [DAP]), abscisic acid (ABA) inhibited germination as long as it was present. However, whatever the dose used and the duration of its application, ABA was unable to induce dormancy because after transfer of treated embryos to control (without ABA) medium, germination occurred. Thereafter, exogenous ABA became effective and allowed the dormancy to develop in 13 and 17 DAP embryos, i.e. in embryos which after isolation were still able to germinate in high percentage. After embryo dormancy was well established (21 DAP), application of fluridone allowed the germination to occur very quickly on control medium. Isolated dormant axes were also induced to germinate by an application of fluridone. Radioimmunological analysis showed that 24 hours after these treatments, endogenous ABA levels were drastically reduced in the axes. When these fluridone-treated embryos were cultured on ABA medium, germination was again inhibited as long as exogenous ABA was present but germination occurred as soon as embryos were transferred to control medium. Such behavior suggested that in situ ABA synthesis is necessary to impose and maintain the embryo dormancy.     

Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. ¹⁸O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.     

Abscisic acid,xanthoxin and violaxanthin in the caps of gravistimulated maize roots

The occurrence and distribution of abscisic acid (ABA), xanthoxin (Xa) and the carotenoid violaxanthin (Va) were investigated in root tips of maize (Zea mays L. cv. Merit). In roots grown in the dark, Va and ABA were present in relatively high amounts in the root cap and in low amounts in the adjacent terminal 1.5 mm of the root. Xanthoxin was present in equal concentrations in both regions. In roots exposed to light, the ABA distribution was reversed, with relatively low levels in the root cap and high levels in the adjacent 1.5-mm segment. Light also caused a decrease in Va in both regions of the root and an increase in Xa, especially in the cap. In the maize cultivar used for this work, light is necessary for gravitropic curving. This response occurs within the same time frame as the light-induced ABA redistribution as well as the changes in the levels of Va and Xa. These data are consistent with a role for ABA in root gravitropism and support the proposal that Xa may arise from the turnover of Va.Abbreviations ABA abscisic acid - GC gas chromatography - HPLC high-performance liquid chromatography - GC-MS gas chromatography-mass spectroscopy - V_a violaxanthin - Xa xanthoxin     

Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation

In response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2-1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1-1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.     

Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.     

Changes in Abscisic Acid Biosynthesis and Catabolism during Dormancy Breaking in Fagus sylvatica Embryo

At harvest, embryos of Fagus sylvatica are dormant. A cold pretreatment without medium at 30% moisture content allowed them to germinate. A comparison of the abscisic acid (ABA) content before and after the pretreatment has no significant relevance since dormancy is expressed during the culture at 23°C. During this culture, both de novo biosynthesis and conjugate hydrolysis contributed to maintain a high level of ABA in the dormant axis. The level of conjugates and the rate of hydrolysis were not modified substantially by the cold pretreatment. In contrast, the dormancy release was associated with a strong decrease in the capacity for ABA synthesis. Moreover, feeding (+)-[³H]ABA to untreated and pretreated embryos proved that the cold treatment also induced a hastening of ABA catabolism. Received August 15, 1996; accepted December 6, 1996     

Control of abscisic acid synthesis

The abscisic acid (ABA) biosynthetic pathway involves the formation of a 9-cis-epoxycarotenoid precursor. Oxidative cleavage then results in the formation of xanthoxin, which is subsequently converted to ABA. A number of steps in the pathway may control ABA synthesis, but particular attention has been given to the enzyme involved in the oxidative cleavage reaction, i.e. 9-cis-epoxycarotenoid dioxygenase (NCED). Cloning of a gene encoding this enzyme in maize was first reported in 1997. Mapping and DNA sequencing studies indicated that a wilty tomato mutant was due to a deletion in the gene encoding an enzyme with a very similar amino acid sequence to this maize NCED. The potential use of this gene in altering ABA content will be discussed together with other genes encoding ABA biosynthetic enzymes.