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TRENDS in Plant Science Vol.7 No.1 January 2002
28 Stitt, M. and Sonnewald, U. (1995) Regulation of metabolism in transgenic plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 341–368 29 Neuhaus, H.E. and Emes, M.J. (2000) Non-photosynthetic metabolism in plastids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 111–140 30 Givan, C.V. (1999) Evolving concepts in plant glycolysis: two centuries of progress. Biol. Rev. Cambridge Phil. Soc. 74, 277–309 31 Hatzfeld, W.D. and Stitt, M. (1990) A study of the rate of recycling of triose phosphates in heterotrophic Chenpodium rubrum cells, potato tubers and maize endosperm. Planta 180, 198–204 32 Entwistle, G. and ap Rees, T. (1990) Lack of fructose-1,6-bisphosphatase in a range of higher plants that store starch. Biochem. J. 271, 467–472 33 Naeem, M. et al. (1997) Starch synthesis in amyloplasts purified from developing potato tubers. Plant J. 11, 1095–1103 34 Schott, K. et al. (1995) Transport of inorganic phosphates across the envelope membranes of potato tuber amyloplasts. Planta 196, 647–652 35 Kammerer, B. et al. (1998) Molecular characterization of a carbon transporter in plastids from heterotrophic tissues: the glucose 6-phosphate phosphate antiporter. Plant Cell 10, 105–117 36 Beckles, D. et al. (2001) A cytosolic ADPglucose pyrophosphorylase is a feature of graminaceous endosperms, but not of other starch-storing organs. Plant Physiol. 125, 818–827 37 Farré, E.M. et al. (2001) Analysis of the compartmentation of glycolytic intermediates,
nucleotides, sugars, organic acids, amino acids and sugar alcohols in potato tubers using a non-aqueous fractionation method. Plant Physiol. 127, 685–700 Stark, D.M. et al. (1991) Regulation of the amount of starch in plant tissues. Science 258, 287–292 Fernie, A.R. et al. (2001) Contribution of plastidial phosphoglucomutase to the control of starch synthesis in potato tubers. Planta 213, 418–426 Smith, A.M. et al. (1997) The synthesis of the starch granule. Annu. Rev. Plant. Physiol. Plant Mol. Biol. 48, 65–87 Kossmann, J. and Lloyd, J. (2000) Understanding and influencing starch biochemistry. Crit. Rev. Plant Sci. 19, 171–226 Denyer, K. et al. (2001) The control of amylose synthesis. J. Plant Physiol. 479–487 Sehnke, P.C. et al. (2001) Regulation of starch accumulation by granule associated plant 14-3-3 proteins. Proc. Natl. Acad. Sci. U. S. A. 98, 765–770 Rodriguez-Lopez, M. et al. (2000) Adenosine diphosphate glucose pyrophosphatase: a plastidial phosphodiesterase that prevents starch biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 97, 8705–8710 Geigenberger, P. et al. (1998) High-temperature perturbation of starch synthesis by decreased levels of glycerate-3-phosphate in growing potato tubers. Plant Physiol. 117, 1307–1316 Geigenberger, P. et al. (1999) Contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis is decreased by water stress in growing potato tubers. Planta 209, 338–345 Tjaden, J. et al. (1998) Altered plastidic ATP/ADP-transporter activity influences potato
(Solanum tuberosum L.) tuber morphology, yield and composition of tuber starch. Plant J. 16, 531–540 Loef, I. et al. (1999) Feeding orotate leads to a specific increase in uridine nucleotide levels, resulting in a stimulation of sucrose degradation and starch synthesis in discs of growing potato tubers. Planta 209, 314–323 Schafer-Pregl, R. et al. (1998) Analysis of quantitative trait loci (QTLs) and quantitative trait alleles (QTAs) for potato tuber yield and starch content. Theor. Appl. Genet. 97, 834–846 Sweetlove, L.J. et al. (1996) Starch metabolism in tubers of transgenic potato (Solanum tuberosum) with increased ADPglucose pyrophosphorylase. Biochem. J. 320, 493–498 Bachem, C. et al. (2000) Functional genomic analysis of potato tuber life-cycle. Potato Res. 43, 297–312 Roessner, U. et al. (2000) Simultaneous analysis of metabolites in potato tuber by gas chromatography – mass spectrometry. Plant J. 23, 131–142 Fiehn, O. et al. (2000) Metabolic profiling for plant functional genomics. Nat. Biotechnol. 18, 1157–1161 Roessner, U. et al. (2001) Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems. Plant Cell 13, 11–29 Roessner, U. et al. (2001) High-resolution metabolic phenotyping of genetically and environmentally diverse potato tuber systems – identification of phenocopies. Plant Physiol. 127, 749–764 Kehr, J. (2001) High-resolution spatial analysis of plant systems. Curr. Opin. Plant Biol. 4, 197–201
Complex regulation of ABA biosynthesis in plants
Mitsunori Seo and Tomokazu Koshiba
Abscisic acid (ABA) is a plant hormone that plays important roles during many phases of the plant life cycle, including seed development and dormancy, and in plant responses to various environmental stresses. Because many of these physiological processes are correlated with endogenous ABA levels, the regulation of ABA biosynthesis is a key element facilitating the elucidation of these physiological characteristics. Recent studies on the identification of genes encoding enzymes involved in ABA biosynthesis have revealed details of the main ABA biosynthetic pathway. At the same time, the presence of gene families and their respective organ-specific expression are indicative of the complex mechanisms governing the regulation of ABA biosynthesis in response to plant organ and/or environmental conditions. There have been recent advances in the study of ABA biosynthesis and new insights into the regulation of ABA biosynthesis in relation to physiological phenomena.
Abscisic acid (ABA) is a plant hormone involved in various physiological processes of plants . In developing seeds, ABA is necessary for inducing the synthesis of reserve proteins and lipids as well as for the onset of seed dormancy and the acquisition
of desiccation tolerance. Endogenous ABA levels peak during seed maturation and the onset of primary dormancy. ABA also plays important roles in vegetative development in response to various environmental stresses such as drought and highsalinity conditions. Moreover, ABA is known to control the expression of many genes related to these phenomena. Thus, an understanding of the mechanisms underlying the regulation of plant ABA levels is a crucial part of determining ABA action in plant growth and physiological responses, which is correlated to endogenous ABA levels. Because the rates of synthesis and the rates of breakdown determine ABA levels in situ, both mechanisms should be studied in detail, although the rate of breakdown remains largely unknown . The recent glut of molecular, genetic and biochemical studies on ABA biosynthesis have allowed remarkable breakthroughs towards
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DXP synthase. are synthesized from the C5 precursor. ζ-carotene. DXS.com Two possible routes have been suggested for ABA biosynthesis. as shown by the characterization of Arabidopsis AO. (b) Formation of epoxycarotenoid and its cleavage in plastid. phytoene desaturase. catalyzed by phytoene synthase (PSY).3]. NCED catalyzes the oxidative cleavage of a 9-cis isomer of epoxycarotenoid such as 9-cis-violaxanthin and 9′-cis-neoxanthin to form xanthoxin. whereas in plastids where carotenoid synthesis takes place. The second pathway via xanthoxic acid (2) might also work. IPP is synthesized via DXP from glyceraldehyde-3phosphate and pyruvate. Hachioji-shi. (c) Reactions in the cytosol for the formation of ABA. *e-mail: seo@ hoshoku-mac2. http://plants. one direct and one indirect. Abbreviations: ABA. short-chain dehydrogenase/reductase. lycopene and β-carotene). Subsequently. isopentenyl pyrophosphate (IPP). 1-deoxy-Dxylulose-5-phosphate. GGPP . AO. IPP is synthesized from mevalonic acid in the cytosol. zeaxanthin epoxidase. . The ABA biosynthetic pathway in plants. 1). respectively [1. A member of SDR such as ABA2 in Arabidopsis converts xanthoxin to ABAld.trends. aldehyde oxidase. IPP . The first step of this specific ABA synthetic pathway is the two-step epoxidation of zeaxanthin to form all-transviolaxanthin catalyzed by ZEP . This review focuses on more recent studies of ABA biosynthetic genes and enzymes. zeaxanthin. which catalyzes the oxidation of ABAld. and discusses the complex regulatory systems of ABA synthesis and their respective physiological aspects. abscisic acid. abscisic aldehyde. including the characterization of ABAdeficient mutants and the isolation of the relevant mutated genes. is the first committed and rate-limiting step in carotenoid synthesis. The first pathway via ABAld (1) is the most probable to function in plants. like other isoprenoids. PDS. ZEP .biol. DXP . Recent studies. In plastids. have shown that the indirect pathway is the main pathway (Fig. Biosynthetic pathways and related enzymes Early steps: synthesis of the carotenoid precursor in plastids understanding the regulatory mechanisms associated with each gene or enzyme [2–6].8]). The enzyme(s) catalyzing the conversion of all-trans-violaxanthin to 9-cis-violaxanthin or 9′-cisneoxanthin has not been identified. geranylgeranyl pyrophosphate. In this pathway. Carotenoids are synthesized from a C5 compound. Three possible pathways are proposed. ABA is synthesized from C40 carotenoids (phytoene. geranylgeranyl pyrophosphate (GGPP). it is produced via 1-deoxy-D-xylulose-5-phosphate (DXP) from pyruvate and glyceraldehyde-3-phosphate. in which ABA is derived from the C15 compound farnesyl pyrophosphate and a C40 carotenoid. NCED. ABAld.jp Fig. IPP is converted to a C20 product. 9-cisepoxycarotenoid dioxygenase. Enzymes (red) and compound names (black) discussed in the text are shown in the figure.42 Review TRENDS in Plant Science Vol. xanthoxin is first oxidized to xanthoxic acid by AO and then xanthoxic acid is converted to ABA. Tokyo 192-0397. Pathway (3) via abscisic alcohol appears to be a shunt pathway but is important in mutants impaired in the oxidation of ABAld. The biosynthetic pathway of carotenoids is well defined (reviewed in [9. Carotenoids. Tokyo Metropolitan University. SDR.ac. presumably by SDR. Phytoene desaturase (PDS) catalyzes the conversion of phytoene to ζ-carotene and is also one of the enzymes dedicated to carotenoid synthesis. Japan. β-carotene and then to a xanthophyll. IPP . DXP synthase (DXS) is the enzyme that catalyzes the first step of the non-mevalonic acid IPP synthesis pathway (reviewed in [7.10]).7 No. isopentenyl pyrophosphate. metro-u. phytoene synthase. PSY. 1. lycopene.1 January 2002 (a) Glyceraldehyde-3-phosphate + pyruvate DXS DXP (b) OH Zeaxanthin HO O ZEP ZEP O OH Antheraxanthin IPP (C5) Farnesyl pyrophosphate (C15) GGPP (C20) PSY Phytoene (C40) PDS ζ-Carotene Lycopene β-Carotene NCED HO OH O all-trans-Violaxanthin HO HO O O HO HO O HO OH all-trans-Neoxanthin 9-cis-Violaxanthin O 9′-cis-Neoxanthin HO (c) Xanthoxin (2) Xanthoxic acid HO O O OH CHO NCED OH Plastid Cytosol (3) AO? HO SDR OH CHO O OH CH2OH ABAld COOH O SDR? ABA O (1) AO OH COOH Abscisic alcohol TRENDS in Plant Science Mitsunori Seo* Tomokazu Koshiba Dept of Biological Sciences. phytoene is converted to ζ-carotene. (a) Carotenoid precursor synthesis in the early steps of ABA biosynthesis. Conversion of GGPP to a C40 carotenoid phytoene.
The first step of the ABA-specific synthetic pathway is the conversion of zeaxanthin to all-trans-violaxanthin by a two-step epoxidation (Fig. suggesting that these isoforms might act to convert xanthoxin to xanthoxic acid. and then an AO isoform(s) catalyzes the oxidation of abscisic aldehyde to produce ABA. In ripening avocado fruits.38]. Seo and T. with a low Km value of 0. a potent inhibitor of the molybdo-enzymes in plants. among them. Arabidopsis aba3. The second pathway of ABA synthesis via xanthoxic acid might also be operative [6. Recombinant ABA2 protein has been shown to catalyze the conversion of xanthoxin to abscisic aldehyde in vitro (A. Recently. respectively.. although the corresponding gene has not been identified . The sitiens tomato mutant is thought to contain a mutation in an AO gene specific for ABA biosynthesis . exogenously supplied abscisic aldehyde is converted to abscisic alcohol. consistent with its proposed enzymatic reaction between xanthoxin and abscisic aldehyde .7 No. a tomato ABA-deficient mutant.com family. Endo et al. The third and last pathway. is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) (Fig. The shunt pathway appears to be a minor source of ABA in wild-type plants but might play a significant . cowpea (Vigna unguiculata) . Later steps in the cytosol: xanthoxin to ABA After the cleavage of 9-cis-epoxycarotenoids. avocado (Persea americana)  and Arabidopsis [23. Four members of the Arabidopsis aldehyde oxidase genes (AAO1-4) have been cloned. xanthoxin is converted to ABA in the cytosol. Recently. ABA-deficient mutants such as Arabidopsis thaliana aba1 and tobacco (Nicotiana plumbaginifolia) aba2 are known to be impaired in ZEP [11–13]. unpublished). inhibition of AO activity by tungstate.33] and recombinant ABA3 protein was shown to have Moco sulfurase activity . In all the species examined. 1). The recombinant VP14 protein specifically cleaves 9-cis isomers of epoxy-xanthophylls such as 9-cis-violaxanthin and 9′-cis-neoxanthin . 1).30]. the oxidative cleavage of xanthophylls. AOδ. indicating that the gene does not function in converting all-trans-violaxanthin to all-trans-neoxanthin in vivo . AAO3. 1). The Arabidopsis aba2 mutant lacks the activity to synthesize ABA from xanthoxin. In flacca and sitiens tomato mutants. a gene encoding the enzyme with the ability to convert all-trans-violaxanthin to all-trans-neoxanthin was isolated from tomato (Lycopersicon esculentum)  and potato (Solanum tuberosum)  as a homolog of capsanthin capsorubin synthase or lycopene β-cyclase. unpublished). which is required for their activity [25. might be activated in some mutants. the gene from tomato is identical to a novel type of lycopene β-cyclase as revealed by mutant analysis . However tomato flacca retains considerable residual activity in roots. The Arabidopsis ABA3 gene was cloned [32. including bean (Phaseolus vulgaris) . supporting the biochemical characterization of the mutants. However. Furthermore. and tomato flacca and sitiens .Review TRENDS in Plant Science Vol. results in the accumulation of xanthoxin. The gene encoding NCED was first identified during the characterization of the maize (Zea mays) viviparous14 (vp14) mutant . The next step.51 µM for abscisic aldehyde [35. Subsequently notabilis. 9-cis-violaxanthin and/or 9′-cisneoxanthin to produce xanthoxin. via abscisic alcohol. the ABA2 gene was isolated and shown to encode an enzyme related to a short-chain dehydrogenase/reductase (SDR) http://plants. The enzyme(s) involved in the conversion of all-trans-violaxanthin to 9-cis-violaxanthin or 9′-cis-neoxanthin has not been isolated. an Arabidopsis aao3 mutant with a defect in the AAO3 gene exhibits a wilty phenotype and ABA biosynthesis is impaired . The enzyme that catalyzes the reaction is zeaxanthin epoxidase (ZEP) – the first enzyme to be identified as an ABA biosynthetic enzyme . NCED cDNAs have been cloned from several species.trends. indicating organ-specific regulation of Moco sulfuration . Further analysis of the substrate specificity of the Arabidopsis ABA2-related enzyme and the AO isoforms for xanthoxic acid and xanthoxin. tobacco aba1 and tomato flacca lack the activity of aldehyde oxidase (AO) isoforms because of a defect in generating the sulfurylated form of the molybdenum cofactor (Moco). was also shown to be defective in the Vp14-related gene (LeNCED1) . tobacco aba1 . All these studies support the pathway whereby xanthoxin is first converted to abscisic aldehyde by an enzyme of the type encoded by the Arabidopsis ABA2 gene. Three possible pathways have been proposed via abscisic aldehyde. would aid in establishing the feasibility of the second pathway. The defects in the mutants result in their incapacity to convert abscisic aldehyde to ABA.24]. showing that abscisic aldehyde is reduced to abscisic alcohol and then oxidized to ABA via a shunt pathway . Some of the Arabidopsis AO isoforms can oxidize xanthoxin in activity gel staining after native gel electrophoresis (M. Koshiba. suggesting that xanthoxin is a substrate of AO . NCED comprises a gene family of several related genes.1 January 2002 43 Specific ABA biosynthetic pathway: xanthophyll formation and its cleavage in plastids Mutants containing a defect in carotenoid precursor synthesis exhibit a pleiotropic phenotype in addition to impaired ABA biosynthesis. One might speculate on the possibility of the two ABA biosynthetic pathways operating in an organ and/or developmental-stage-dependent manner. Typical mutants impaired in the later steps of ABA biosynthesis in the cytosol are Arabidopsis aba2 and aba3 .26.29.36]. xanthoxic acid or abscisic alcohol (Fig. which encodes an AO isoform. but can oxidize abscisic aldehyde to form ABA by in vitro enzyme preparations .
However. towards the regulation of ABA biosynthesis. When DXS is over. had not previously been considered to be a regulatory step in ABA biosynthesis because the bulk enzymatic activity is constitutive and the rate of this activity does not change during water stress . tocopherols or carotenoids. Some data indicate that the rate of carotenoid synthesis does not affect the rate of ABA biosynthesis  because the expression of PDS mRNA and its product in developing maize seeds does not correlate with endogenous ABA levels . It has been shown that the expression of tobacco (ABA2/NpZEP) and tomato (LeZEP1) ZEP genes in leaves is not correlated to endogenous ABA levels during water stress [45. from xanthoxin to ABA. These results strongly support the idea that NCED is a key regulatory enzyme in ABA biosynthesis. the expression of ZEP in roots is induced by dehydration [45. but wilts readily after dehydration with no significant increase in ABA levels in the dehydrated leaves . suggesting a regulatory role for ZEP in seeds . The expression of AAO3 mRNA in the leaves of wildtype plants increases rapidly in water-stressed leaves . the level of protein or its activity in leaf extracts does not significantly change during the stress. expression levels of DXS change according to endogenous isoprenoid content in the form of chlorophylls. and accumulation of ABA is not observed. endogenous ABA levels are also influenced by DXS expression. resulting in enhanced dormancy. The last two reactions in the cytosol. and over-expression of the PSY gene (PSY1) in tomato does not result in increased ABA levels . The expression of NpZEP in the transgenic plants under water-stress conditions remains constant. It is possible that these different regulatory systems modulating AO activity at the transcriptional and posttranscriptional levels are coordinated at tissue levels during water-stress responses. A detailed study of NCED expression during water stress using Phaseolus vulgaris showed a clear correlation between NCED (PvNCED1) mRNA expression. The induction of NCED (LeNCED1/NOTABILIS) expression in dehydrated leaves and roots has also been observed in tomato . the aao3 mutant exhibits a relatively normal phenotype in well watered conditions. NCED protein levels. In contrast to the effect of dehydration on leaves observed in Arabidopsis.trends. This is supported by the observation that the expression of Arabidopsis ABA3.46]. xylem sap and leaves of drought-stressed plants has been widely reported . A tobacco aba2 mutant complemented by NpZEP under the control of a constitutive promoter produces normal ABA levels under well watered conditions. which peaks during the middle of seed development . Seed maturation and dormancy ABA plays a crucial role in seed maturation. This suggests that early ABA synthesis steps. and ABA levels in dehydrated leaves and roots. Expression of ZEP in leaves seems to be influenced by diurnal functions. The expression of NpZEP correlates well with endogenous ABA levels during seed development. the expression of AAO3 in roots is not induced by dehydration . the induction of NCED expression after water stress in leaves has been reported in http://plants. However. Because AO requires Moco for activity. ABA has been suggested to induce a dormant state during the late phase of seed maturation.or under-expressed in Arabidopsis. particularly the formation of DXP. and might be responsible for the insignificant effects observed on bulk AO activity under these conditions. However. Transgenic plants over-expressing NpZEP cDNA accumulate ABA in seeds. However. Surprisingly.33]. Cheng et al. unpublished). the availability of the cofactor would contribute towards the regulation of its activity levels. although the expression of this gene in roots has not always been determined.46]. It has been reported that the expression of an Arabidopsis NCED mRNA (AtNCED3) in whole plants is induced by dehydration and that the over-expression of AtNCED3 results in the accumulation of ABA . is induced by dehydration treatment of shoots [32. encoding Moco sulfurase.44 Review TRENDS in Plant Science Vol. indicating the regulatory role of NCED in ABA biosynthesis during water stress . Furthermore.1 January 2002 role in mutants impaired in their capacity to oxidize abscisic aldehyde to ABA directly. recent studies on an Arabidopsis aao3 mutant indicate that AAO3 plays an important role in plant dehydration tolerance. The increase in the ABA content of root. and in the establishment and maintenance of dormancy.7 No.. Regulation during the early stages of ABA biosynthesis The contribution of carotenoid synthesis towards the regulation of ABA biosynthesis has not been extensively discussed. might contribute. at least in leaves. The expression of other genes involved in . Over-expression of LeNCED1 in tomato plants results in the over-production of ABA in leaves . at least in part.22]. It is possible that post-transcriptional regulation is involved in controlling the activity of AAO3. indicating the importance of drought-induced NpZEP expression in controlling ABA synthesis in roots .com cowpea and avocado [21. recent studies suggest a possible link between the early and later stages of ABA synthesis. suggesting the possible role of DXS in the regulation of isoprenoids biosynthesis . Regulation of ABA biosynthesis in relation to physiological processes Plant responses to water stress One of the most striking aspects of ABA biosynthesis is the drastic increase in ABA levels during dehydration. although the expression is up-regulated by sugar treatment (W-H. Regulation of Arabidopsis ABA2 gene expression by dehydration is not observed.
where endogenous ABA transiently accumulates. which result in different diurnal rhythms. ZEP expression fluctuates according to a diurnal rhythm during a time lapse consisting of light and dark periods [45.Review TRENDS in Plant Science Vol. water loss occurs faster in vp14 leaves compared with wild-type leaves. It is surprising that diurnal fluctuations of LeNCED1 expression have been observed in tomato leaves . because NCED is thought to be a key regulatory enzyme in leaves. suggesting that regulation of ABA synthesis plays an important role in plant sugar responses . the expression of ZEP is affected by a factor.58]. In tobacco and tomato. ZEP converts zeaxanthin to violaxanthin through a two-step epoxidation. expressed in ripening fruits. ABA levels in the seeds decrease and hence the seeds exhibit enhanced ability to germinate due to weaker dormancy. The peak of NCED expression occurs at the end of the light period.7 No. Although it remains uncertain whether all the gene products in these families are involved in ABA biosynthesis. Violaxanthin is converted to zeaxanthin by the enzyme violaxanthin de-epoxidase under conditions of excessive light. ABA levels in embryos of vp14 mutants are significantly lower than in the wild type. but imbibition at high temperature prevents significant changes in ABA levels. In Arabidopsis. Only four out of six gene products exhibit NCED activity when tested. independent of ABA synthesis. leaf and root development. Because leaves contain substantial amounts of xanthophylls. ABA levels increase when dormant but not non-dormant tobacco seeds imbibe.com Gene families encode NCED and AO. This is consistent with the observation that most of the ABA-deficient mutants screened based on sugar responsiveness were identified as aba2 alleles. The influence of the diurnal oscillation of NCED on the rate of ABA synthesis needs to be examined further. The expression of ABA2/GIN1 is induced by the treatment of sugar but not by water stress (W-H. Under low or limiting light. whereas PaNCED3 is not detected in leaves . Cheng et al. Different genes of a given gene family contribute to ABA synthesis in different physiological processes Xanthophylls are involved in protecting the photosynthetic apparatus from photo-oxidative damage . Maize Vp14 (ZmNCED1) is highly expressed in embryos and roots.46]. In ripening avocado fruits. These results indicate the presence of NCED gene(s) other than Vp14 that are responsible for ABA synthesis in at least non-stressed leaves. High-temperature treatment inhibits germination of lettuce seeds .1 January 2002 45 ABA biosynthesis during seed development has not been examined in detail. http://plants. there are nine NCED-related genes (AtNCEDs) on the genome database. ABA levels are increased by sugar treatment of Arabidopsis seedlings. When the seeds are imbibed at high temperature in the presence of fluridone. Sugar signals are known to modulate many important processes in higher plants such as germination. The levels of both PaNCED1 and PaNCED3.54–56]. light intensity. Three NCED-related cDNAs (PaNCED1. PaNCED2 and PaNCED3) have been isolated from avocado. Overexpression of LeNCED1 in tomato results in increased seed dormancy . The gin5 mutant is also likely to be an ABA-deficient mutant because the glucose-insensitive phenotype of the mutant is rescued by the application of ABA. indicating the existence of different regulatory systems for each gene. but no significant difference is observed in ABA content in non-stressed leaves between the wild type and vp14. In this case. The diurnal oscillation of ZEP expression in leaves of tobacco and tomato are probably related to its role in the xanthophyll cycle. The gin1. The relevance of ABA synthesis in imbibed seeds for maintaining seed dormancy has been reported recently. although specific genes have yet to be assigned to this function. unpublished). in conjunction with the expression of the relevant respective genes [57. some might function in ABA synthesis. . indicating that the expression of ABA2 is important in sugar responses. but less in leaves. a carotenoid synthesis inhibitor . correlated with similar daily fluctuations of endogenous ABA levels . The ABA content in lettuce seeds decreases rapidly during normal germination conditions. These findings point to a specific regulatory system of ABA biosynthesis that maintains seed dormancy in imbibed seeds. and treatment with ABA increases sugar sensitivity. and senescence. whereas the expression of ZEP peaks in the middle of the light period.trends. the increase in ABA content is blocked by fluridone. The physiological role of sugar-regulated ABA synthesis has been discussed in relation to sugar-induced starch biosynthesis during early seed development . PaNCED1 is highly expressed in leaves and its expression is induced by dehydration. seedling growth. However. the fluctuation of ZEP expression and the quantities of each xanthophyll might not be limiting for ABA synthesis. Diurnal functions Sugar response Interesting findings on novel ABA function have been reported from a study in which several ABA-deficient Arabidopsis mutants were isolated during the screening of sugar-response mutants [28. two of which (PaNCED1 and PaNCED3) have the ability to convert 9-cis-violaxanthin and 9′-cis-neoxanthin to xanthoxin. and stressed leaves of vp14 accumulated lower ABA levels compared with the wild type . These results indicate that NCED might also play an important role in ABA synthesis during seed development.. the expression of the two NCED mRNAs (PaNCED1 and PaNCED3) fluctuates in a similar way to that observed for endogenous ABA levels . sis4 and isi4 mutants are allelic to aba2.
a.and hetero-dimers by three AAO gene products.p.33] [27. stems.) Induced by dehydration in leaves (Z. Phaseolus vulgaris. Because hydrophobic carotenoids exist exclusively in the membranes of plastids.p.30. are subjected to different post-transcriptional regulatory mechanisms. ZEP .t. maize  and tomato ..v. at least four AO isoforms are generated as homo. This indicates that AOδ has a role in leaf-specific ABA synthesis. It is possible that different NCEDs in a given plant species. V.p.m.36. which..e.) notabilis (L. genes and enzymes involved in the process. Sports and Culture. AOγ (AAO2-AAO2) and AOδ (AAO3-AAO3). but their deduced amino acid sequence share <50% homology with ABA2.a.65].S. Vigna unguiculata. V. AtNCED3. with distinct physiological roles. L. NCED. exhibits a wilty phenotype in leaves but is not affected in seed dormancy.) Developing seeds (N. in turn. fDefective in the synthesis of molybdenum cofactor.a.u.v.) Induced by sugar in seedlings. Arabidopsis thaliana. The localization of VP14 inside the plastids might be under regulatory control. depending upon the expression of the corresponding genes [36. is involved in ABA biosynthesis in vivo .)f flacca (L. In Arabidopsis.)f aba1 (N.t. N..) aba3 (A. the gene product probably does not function in ABA biosynthesis.e. L.. At present..com similar subunits.e. some of these AOs probably act within the ABA biosynthetic pathway. P .. this implies that there is another AO isoform(s) responsible for ABA biosynthesis in other organs. at least two types of NCED exist. a soluble stromal form and an insoluble thylakoid membrane-bound form in plastids . is supported in part by a Grant-in Aid for Scientific Research (B) 13490024 from the Ministry of Education. Bean PvNCED1 exists mainly in the insoluble fractions of chloroplasts such as the thylakoid and envelope membranes.. lacks the targeting signal peptide to plastids and in most instances the recombinant protein cleaves a variety of carotenoids symmetrically to produce a C14-dialdehyde and two C13 products . flowers and seeds.) Gene family Noc Mutants Refs (plant speciesb) aba2 (N. and another. is supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. dW-H. L.K. roots (P .32. one. The isoforms exhibit different substrate preferences.t. whereas maize VP14 exists in two distinct fractions. Cheng et al.) aba1 (A. In Arabidopsis. Arabidopsis ABA3 has been shown to encode Moco sulfurase. Acknowledgements We are grateful to S.31] [26. Table 1 represents the characteristics of ABA synthetic enzymes and the expression of the respective genes in relation to the physiological processes. might contribute towards the regulation of ABA biosynthesis.)d Induced by dehydration in leaves but not in roots (A.m..v.e. but not by dehydration of leaves (A. Plant AO consists of two http://plants.trends.1 January 2002 Table 1. Among them. Further prospects Recent advances in ABA biosynthesis research have yielded substantial information on the pathways. which localizes only in the thylakoid membrane. which exits as two distinct forms .t. particularly in the roots based on the dominant expression of these genes in this organ [36. P . The data indicate that ABA synthesis is not merely a group of reactions but . such as roots.30] [25.p.e. Nicotiana plumbaginifolia.. and the gene family has been found in Arabidopsis . eThere are several related genes in the Arabidopsis database.28]  [27.) vp14 (Z.e.) Diurnal fluctuation in leaves (L. 9-cis-epoxycarotenoid dioxygenase.)f [25.e. P .t.7 No. L.46 Review TRENDS in Plant Science Vol. cThe existence of a homologous gene in tomato has been proposed .e. which is consistent with the high expression of AAO3 in leaves . V. At the same time. T. L.. such as PvNCED1. unpublished.. detailed characterization and organ-specific expression profiles of each AtNCED gene..)     NCED Yes SDR AO Oxidation of xanthoxin (Oxidation of xanthoxic acid?) Oxidation of abscisic aldehyde (Oxidation of xanthoxin?) Noe Yes aba2 (A. Z.e. NCED localizes at the membrane of plastids to interact with its substrate. The presence of AO isoforms has been reported in several plant species [31.) Diurnal fluctuation in leaves (N. as well as on the regulation of each step. such as VP14..) Ripening fruits (P . aldehyde oxidase. AOβ and AOγ remains unclear.61–63]. Lycopersicon esculentum. M.t.29] aAO. as well as the physiological roles of their respective gene products remain to be determined. P .. Characteristics of ABA synthetic enzymes Enzymea Enzyme reaction ZEP Conversion of zeaxanthin to violaxanthin Oxidative cleavage of 9-cisepoxycarotenoid Gene expression (plant speciesb) Induced by dehydration in roots but not in leaves (N.m. Although the role of AOα.) or whole plants (A. and are distributed differently in the organs or tissues.t. SDR.p.. whose expression is up-regulated by dehydration.u. AAO4 is a possible candidate for the AO involved in ABA production in immature seeds because AAO4 mRNA is expressed mainly in siliques and the expression in leaves is up-regulated by dehydration . bA. zeaxanthin epoxidase. impaired in the AAO3 gene.p. the first NCED identified in Arabidopsis.u. Herman Lips at Ben-Gurion University and Eiji Nambara at RIKEN for their critical reading of the manuscript. Although the expression is induced by dehydration .65]. short-chain dehydrogenase/reductase. AtNCED1. AOα (AAO1-AAO1). The Arabidopsis aao3 mutant. Science. Although the enzymatic nature of AAO4 has not been characterized.)..) aao3 (A.t. Persea americana. AOβ (AAO1-AAO2). Zea mays.) sitiens (L. Japan.
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