全 文 ：植物学通报 2004, 21 (3): 352~359
Chinese Bulletin of Botany
①Foundation items: This work was supported by the grants of the key project on scientific and technological research
from the Ministry of Education (03098) and the‘Thousand-hundred-ten project’cultivating excellent person-
nel from the Department of Education of Guangdong province (02090).
②Author for correspondence.
Received: 2003-05-12 Accepted: 2003-09-13 Managing editor: SUN Dong-Hua
高等植物脱落酸生物合成途径及其酶调控①
万小荣 李 玲②
（华南师范大学生命科学学院 广州 510631）
摘要 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。
关键词 脱落酸, 生物合成, NCED基因, AO基因, 酶调控
Pathways and Related Enzymes of ABA Biosynthesis
in Higher Plants
WAN Xiao-Rong LI Ling②
(College of Life Science, South China Normal University, Guangzhou 510631)
Abstract Two pathways have been proposed for the biosynthesis of ABA: the ‘direct pathway’
and ‘indirect pathway’. In the direct pathway, which operates in some fungi, ABA is derived from
farnesyl diphosphate; in indirect pathway, ABA is produced from the oxidative cleavage of carotenoids.
The first committed step for ABA biosynthesis in plants is the oxidative cleavage of a 9-cis-
epoxycarotenoid(C40) to produce xanthoxin(C15) and a C25 by-product(Fig.2). Xanthoxin is then oxi-
dized to a ketone by an NAD-requiring enzyme. Consequently, there is a nonenzymatic desaturation
of the 2-3 bond and opening of the epoxide ring to form abscisic aldehyde. In the final step of the
pathway, abscisic aldehyde is oxidized to ABA. These endogenous enzymes such as zeaxanthin
epoxidase(ZEP), 9-cis-epoxycarotenoid dioxygenase(NCED) and aldehyde oxidase(AO) may play
key roles in the regulation of ABA biosynthesis in higher plants.
Key words Abscisic acid, Biosynthesis, NCED gene, AO gene, Enzymatic regulation
Abscisic acid (ABA) is a plant hormone involved in various physiological processes of plants.
The biosynthetic pathway of ABA remains a contentious issue to date. Nevertheless, the stress-
3532004 万小荣等：高等植物脱落酸生物合成途径及其酶调控
induced biosynthesis of ABA in plants is believed to occur as a result of dioxygenase-catalysed
cleavage of 9-Z-Xanthophylls(either neoxanthin or violaxanthin) to Xanthoxin(XAN) that is succes-
sively oxidized to ABA via abscisic aldehyde. The currently accepted pathway and the localization of
ABA biosynthesis in plants are depicted in Fig.1 (Seo and Koshiba, 2002). As highlighted in this
review, the recent glut of molecular, genetic and biochemical studies on ABA biosynthesis have
allowed remarkable breakthroughs towards understanding the regulatory mechanisms associated with
each gene or enzyme.
1 From IPP to Zeaxanthin
Two possible routes have been suggested for ABA biosynthesis, one direct and one indirect, in
which ABA is derived from the C15 compound farnesyl pyrophosphate and a C40 carotenoid,
respectively. Recent studies have shown that the indirect pathway is the main pathway in plants
(Fig.1). Substantial evidence has accumulated to support the existence of both mevalonate and non-
mevalonate pathways of isopentenyl pyrophosphate(IPP) synthesis in plants(Lichtenthaler, 1999).
IPP is synthesized from mevalonic acid in the cytosol, whereas in plastids, it is produced via 1-deoxy-
Fig. 1 The ABA biosynthetic pathway in plants
(a). Carotenoid precursor synthesis in the early steps of ABA biosynthesis; (b). Formation of epoxycarotenoid
and its cleavage in plastid; (c). Reactions in the cytosol for the formation of ABA (Seo and Koshiba, 2002)
354 21(3)
D-xylulose-5-phosphate(DXP) from pyruvate and glyceraldehyde-3-phosphate catalyzed by DXP
synthase(DXS) (Fig.1).IPP is converted to a C20 product, geranylgeranyl pyrophosphate(GGPP). Con-
version of GGPP to a C40 carotenoid phytoene, catalyzed by phytoene synthase(PSY), is the first
committed and rate-limiting step in carotenoid synthesis. Subsequently, phytoene is converted to ζ-
carotene, lycopene, b-carotene and then to a xanthophyll, zeaxanthin. Phytoene desaturase(PDS)
catalyzes the conversion of phytoene to ζ-carotene and is also one of the enzymes dedicated to
carotenoid synthesis (Hirschberg , 2001).
2 Epoxy-carotenoid Formation and its Cleavage in Plastids
The first step of the ABA-specific synthetic pathway is the conversion of zeaxanthin to all-trans-
violaxanthin by a two-step epoxidation (Fig. 1). The enzyme that catalyzes the reaction is zeaxanthin
epoxidase (ZEP) — the first enzyme to be identified as an ABA biosynthetic enzyme(Marin et al.,
1996). ABA-deficient mutants such as Arabidopsis thaliana aba1 and tobacco (Nicotiana
plumbaginifolia) aba2 are known to be impaired in ZEP (Duckham et al., 1991; Marin et al., 1996).
Because the level of epoxycarotenoids in green leaves is high relative to the amount of ABA synthesized,
it is considered unlikely that ZEP has a regulatory role in these tissues. The expression of ZEP tran-
scripts in green tissue did not increase in wild tobacco(Audran et al., 1998), tomato(Thompson et al.,
2000a) or cowpea (Iuchi et al., 2000) that were subjected to osmotic stress. In etiolated tissues, the
concentration of carotenoids is significantly lower and the increased expression of ZEP mRNA did
correlate with elevated ABA biosynthesis in roots and seeds(Audran et al., 1998; Borel et al., 2001). It
was reported that the expression of Arabidopsis AtZEP(encoding epoxidase) mRNA increased in
response to osmotic stress or ABA treatment in both roots and shoots(Xiong et al., 2002). The osmotic
induction of AtZEP transcript was impaired in ABA-deficient mutants and in the ABA-insensitive
mutant, abi1. Several additional genes necessary for the later steps in ABA biosynthesis were also
found to be induced by stress and ABA(Xiong et al., 2001; 2002). It was suggested that ABA biosyn-
thesis might be subjected to positive feedback regulation.
The enzyme(s) involved in the conversion of all-trans-violaxanthin to 9-cis-violaxanthin or 9-cis-
neoxanthin has not been isolated. Recently, a gene encoding the enzyme with the ability to convert all-
trans-violaxanthin to all-trans-neoxanthin was isolated from tomato(Lycopersicon esculentum)(Bouvier
et al., 2000) and potato(Solanum tuberosum)(Al-Babili et al., 2000) as a homolog of lycopene b-
cyclase. However, the gene from tomato is identical to a novel type of lycopene b-cyclase as revealed
by mutant analysis, indicating that the gene does not function in converting all-trans-violaxanthin to
all-trans-neoxanthin in vivo(Hirschberg, 2001). The xanthophylls cycle involves the light-dependent
and reversible conversion of violaxanthin to zeaxanthin (Fig. 2) ( Schwartz et al., 2003) and is thought
to be essential for the protection of the photosynthetic apparatus from photooxidative damage. Under
low or limiting light, zeaxanthin is converted into antheraxanthin and violaxanthin by ZEP. In excessive
light, de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin is catalyzed by violaxanthin
de-epoxidase(VDE). Whether the de-epoxidation leads to a reduction of violaxanthin pools in photo-
3552004 万小荣等：高等植物脱落酸生物合成途径及其酶调控
synthetic tissues, such that the availability of ABA precursors is limiting, remains to be investigated
(Liotenberg et al., 1999).
The committed step in ABA biosynthesis is the oxidative cleavage of xanthophylls, 9-cis-
violaxanthin or 9-cis-neoxanthin. Confirmation of the regulatory nature of the cleavage reaction was
provided by the characterization of a viviparous mutant of maize, vp14, that exhibited a defect in ABA
biosynthesis(Tan et al., 1997). The Vp14 gene was cloned and the derived protein sequence of Vp14
is related to lignostilbene dioxygenase, a bacterial enzyme that catalyzes a double-bond cleavage
reaction in ABA biosynthesis(Kamoda and Saburi, 1993). Recombinant VP14 protein catalyzes the
cleavage of 9-cis-xanthophylls to form xanthoxin and a C25 by-product (Fig. 2) in a reaction that
requires oxygen, ferrous iron, ascorbate and a detergent for activity in vitro (Schwartz et al., 1997b).
The characteristics of the cleavage reaction in its substrate specificity and the site of cleavage (11 and
12 position) were consistent with predictions. A 9-cis double bond in the carotenoid precursor was
necessary for activity. The product of this cleavage reaction is cis-xanthoxin, which is readily con-
verted to ABA [cis-(+)-S-ABA] by plants. Cleavage of an all trans-isomer would result in trans-
xanthoxin, which is converted to biologically inactive trans-ABA. Northern analysis of maize leaves
showed that Vp14 expression is induced during wilting in parallel with the increase in ABA levels(Tan
et al., 1997). In the time since the cloning of Vp14, a number of genes with sequence similarity to Vp14
have been reported. The nomenclature now used for designating genes that have homology to Vp14
is 9-cis-epoxycarotenoid dioxygenase genes(NCED). NCED cDNAs have been cloned from several
species, including maize, tomato(Burbidge et al., 1999), bean(Qin and Zeevaart, 1999), cowpea(Iuchi
et al., 2000), avocado (Chernys and Zeevaart, 2000) and Arabidopsis (Neill et al., 1998; Iuchi et al.,
2001). In all species examined, NCED comprises a gene family of several related genes. Although the
NCEDs display significant substrate plasticity in vitro, circumstantial evidence favors neoxanthin as
the primary precursor of ABA.Neoxanthin exits almost entirely as a 9-cis-isomer, whereas only a small
proportion of the violaxanthin is present as a 9-cis-isomer (Strand et al., 2000). Definitive evidence of
the endogenous substrate would require identification of the C25 by-product in plant. Previous efforts
to identify the C25 compounds in vegetative tissue have been unsuccessful.
It is suggested that there is a family of differentially regulated NCED genes that contributes to
environmental and developmental control of ABA biosynthesis in plants. A detailed study of NCED
expression during water stress using Phaseolus vulgaris(Qin and Zeevaart, 1999) and Nicotiana
plumbaginifolia(Qin and Zeevaart, 2002) showed a clear correlation between NCED(PvNCED1) mRNA
expression, NCED protein levels, and ABA levels in dehydrated leaves and roots, indicating the
regulatory role of NCED in ABA biosynthesis during water stress. The induction of NCED (LeNCED1/
NOTABILIS) expression in dehydrated leaves and roots has also been observed in tomato(Thompson
et al., 2000a). Furthermore, the induction of NCED expression after water stress in leaves has been
reported in cowpea(Iuchi et al., 2000) and avocado(Chernys and Zeevaart, 2000), although the expres-
sion of this gene in roots has not always been determined. Over-expression of LeNCED1 in tomato
plants results in the overproduction of ABA in leaves (Thompson et al., 2000b). These results strongly
356 21(3)
support the idea that NCED is a key regulatory enzyme in ABA biosynthesis, at least in leaves. Multi-
gene families often encode enzymes with related functions, but in cases where the function is the same,
differential regulation ensures that distinct genes are activated in response to different environmental
and other stimuli. Ultimately, the distinction between which gene is induced in response to a given
stimulus lies in the promoter region.
3 From Xanthoxin to ABA
After the cleavage of 9-cis-epoxycarotenoids, xanthoxin is converted to ABA in the cytosol.
Three possible pathways have been proposed via abscisic aldehyde(ABAld), xanthoxic acid or absci-
sic alcohol (Fig. 1 and Fig. 2).The first pathway via ABAld is the most probable to function in plants,
as shown by the characterization of Arabidopsis AO, which catalyzes the oxidation of ABAld. The
aba2 mutant in Arabidopsis was impaired in the conversion of xanthoxin to abscisic aldehyde(Schwartz
et al., 1997a). The gene corresponding to aba2 has recently been cloned and the gene product was
found to be similar to short-chain dehydrogenases/reductases(SDRs)( Cheng et al., 2002; Gonzalez-
Guzman et al., 2002). As expected , the ABA2 protein was able to catalyze the conversion of xanthoxin
to abscisic aldehyde utilizing NAD as a cofactor. It has not yet been reported whether the over-
expression of ABA2 would have any effect on ABA level. 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, and then an AO isoform(s) catalyzes the oxidation of abscisic aldehyde to
produce ABA. The second pathway via xanthoxic acid might also work. In this pathway, xanthoxin is
first oxidized to xanthoxic acid by AO and then xanthoxic acid is converted to ABA, presumably by
SDR. Pathway 3 via abscisic alcohol appears to be a shunt pathway but is important in mutants
impaired in the oxidization of ABAld (Seo and Koshiba, 2002).
4 Further Prospects
Significant progress has been achieved in recent years with the cloning of genes encoding for the
enzymes involved in the ABA biosynthetic pathway. The biochemical aspects of ABA synthesis,
such as the intermediates in the pathway and the sequence of reactions, have become well established.
The genes that encode most of the enzymes in the pathway have now been cloned (Fig.1 and Fig. 2).
This will contribute to a better understanding of the regulation of ABA levels and should make their
manipulation possible in transgenic plants.
However, there are several steps in ABA biosynthesis preceding the cleavage reaction that are
not well characterized. The epoxycarotenoid precursor must have a 9-cis configuration to be cleaved
by an NCED and for subsequent conversion to ABA [cis-(+)-S-ABA]. The formation of these 9-cis
isomers has not yet been established. An enzyme that catalyzes a similar reaction, the cis/trans
isomerization of prolycopene to lycopene, has recently been identified (Isaacson et al., 2002; Park et
al., 2002). This isomerase appears to be necessary only in non-photosynthetic tissue. In light-grown
tissue, photo-isomerization of lycopene is sufficient. It has not been established whether the 9-cis
3572004 万小荣等：高等植物脱落酸生物合成途径及其酶调控
isomerization of neoxanthin and violaxanthin is an enzymatic reaction. Alternatively, the 9-cis confor-
mations of some epoxycarotenoids could be stabilized by carotenoid-binding proteins. Although el-
evated expression in response to osmotic stress has been reported for several of these genes, the
significance of this up-regulation is still uncertain. Previous biochemical studies and the recent work
with transgenic plants clearly demonstrated that transcriptional regulation of the NCEDs is the major
control point in ABA synthesis(Qin and Zeevaart, 2002).The initial perception of stress and the signal
transduction pathway leading to elevated NCED expression remain to be elucidated.
The mechanisms of drought response have been investigated most extensively in a model plant,
Arabidopsis. On the contrary, the response of crops to drought stress has not been extensively studied.
Our program is focused on improving the drought tolerance of peanut by gene manipulation of NCED
and AO genes involved in ABA biosynthesis, and exploring the mechanisms of drought response in
crops. Molecular analysis of drought tolerance of peanut may be useful to elucidate the mechanism of
drought response, improving drought tolerance of crops by using transgenic plant technique.
Fig. 2 The pathway of ABA synthesis beginning with zeaxanthin
VDE. Violaxanthin de-epoxidase. The corresponding mutants and enzymes are indicated (Schwartz et al., 2003)
358 21(3)
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