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Plant Physiol, April 2003, Vol. 131, pp. 1591-1601
UPDATE ON ABSCISIC ACID BIOSYNTHESIS
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INTRODUCTION |
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 TOP
 INTRODUCTION
 ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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Abscisic acid (ABA) was discovered independently by several groups in the early 1960s. Originally believed to be involved in the abscission of fruit and dormancy of woody plants, the role of ABA in these processes is still not clear. ABA is, however, necessary for seed development, adaptation to several abiotic stresses, and sugar sensing. The regulation of these processes is in large part mediated by changes in de novo synthesis of ABA.
Two pathways have been proposed for the synthesis of ABA. In the
"direct pathway," which operates in some fungi, ABA is derived from
farnesyl diphosphate (Hirai et al., 2000
). Because of
structural similarities, an "indirect pathway" in which ABA is
produced from the cleavage of carotenoids also had been proposed
(Taylor and Smith, 1967). The first committed step for
ABA synthesis in plants is the oxidative cleavage of a
9-cis-epoxycarotenoid (C40) to produce xanthoxin
(C15) and a C25 by-product
(Fig. 1). The 4'-hydroxyl of xanthoxin is
oxidized to a ketone by an NAD-requiring enzyme. As a consequence,
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.
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Evidence for the indirect pathway in plants had initially come from a variety of biochemical studies, 18O2-labeling experiments, and the characterization of ABA-deficient mutants. In recent years, the genes encoding enzymes for many steps in the pathway have been identified. Much of the recent work in characterizing these genes has confirmed previous biochemical studies. Advances in the elucidation of the ABA biosynthetic pathway and its regulation also have allowed the manipulation of ABA levels in transgenic plants. Of particular interest is the cloning and characterization of the nine-cis-epoxycarotenoid dioxygenases (NCEDs) that catalyze the rate-limiting step in ABA synthesis. The identification of the NCEDs also has had an impact beyond plant biology. Similar proteins are present in a diverse array of organisms. Their enzymatic activities are responsible for the synthesis of a variety of compounds from carotenoids, including vitamin A in animals.
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ABA-DEFICIENT MUTANTS |
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TOP
INTRODUCTION
ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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Our understanding of the functions and synthesis of ABA has been greatly enhanced by the identification and characterization of ABA-deficient mutants (Table I). The ABA-deficient mutants have been identified by the following phenotypes: precocious germination, susceptibility to wilting, an increase in stomatal conductance, and an ability to germinate and grow on media containing a high concentration of Suc or salt. In recent years, these mutants have also been very useful in cloning the genes that encode ABA biosynthetic enzymes.
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The pathway of ABA synthesis can be traced back to the early steps of
isoprenoid synthesis in plastids
(Rodríguez-Concepción and Boronat, 2002).
Isoprenoids are an extremely diverse class of natural products that
serve a variety of functions in plants. Although it is not a committed
step in ABA synthesis, the epoxidation of zeaxanthin seems to be a
reasonable place to begin a review on ABA synthesis in plants (Fig. 1).
Mutants impaired in the epoxidation of zeaxanthin were first identified
by an ABA-deficient phenotype. In addition, the ZEP appears to have a
role in the regulation of ABA synthesis in non-chlorophyllous organs.
The aba1 mutant in Arabidopsis (Karssen et al.,
1983; Duckham et al., 1991; Rock and
Zeevaart, 1991a) and the aba2 mutant in N. plumbaginifolia (Marin et al., 1996) both contain
lesions in the enzyme that catalyzes the epoxidation of zeaxanthin to antheraxanthin and violaxanthin (ZEP in Fig. 1). To avoid confusion, the genes corresponding to these mutants will subsequently be referred
to as AtZEP and NpZEP, respectively.
The atzep mutant provided definitive evidence that ABA is
derived from an epoxycarotenoid precursor (Duckham et al.,
1991; Rock and Zeevaart, 1991a). This mutant
also has been used extensively to characterize the role of
epoxycarotenoids in the xanthophyll cycle and as components of the
light-harvesting complexes (Lokstein et al.,
2002).
An epoxidase mutant in N. plumbaginifolia, npzep,
has been identified and the corresponding gene cloned (Marin et
al., 1996). The NpZEP protein, which is similar to some
bacterial monooxygenases, was able to catalyze the epoxidation of
zeaxanthin to antheraxanthin and violaxanthin. For this activity, it
was necessary to add an additional component from chloroplasts. It was
later determined that reduced ferredoxin is a necessary cofactor
(Bouvier 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 transcripts in green tissue does not increase in wild
tobacco (Nicotiana plumbaginifolia; Audran et
al., 1998), tomato (Thompson et al., 2000a), or
cowpea (Vigna unguiculata; Iuchi et al.,
2000) that are subjected to osmotic stress. In etiolated tissues, the concentration of carotenoids is significantly lower and
the increased expression of ZEP mRNA does correlate with
elevated ABA synthesis in roots and seeds (Audran et al.,
1998; Borel et al., 2001). The overexpression of
ZEP in transgenic tobacco resulted in increased seed
dormancy (Frey et al., 1999), thus providing further
evidence that the level of epoxycarotenoids limits ABA synthesis in
some tissues.
The Arabidopsis epoxidase, AtZEP, was cloned by sequence
similarity to the tobacco ZEP (Audran et al.,
2001; Xiong et al., 2002). Contrasting reports
on the expression of AtZEP mRNA have appeared in the
literature. In one study, it was found that the level of
AtZEP mRNA was induced by drought stress in root tissues (Audran et al., 2001). The expression of the
AtZEP transcript was unaffected in several ABA-deficient and
-insensitive mutants (Audran et al., 2001). In another
study, it was reported that the expression of AtZEP 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 synthesis also were found to
be induced by stress and ABA (Xiong et al., 2001,
2002). The authors suggested that ABA synthesis might be
subject to positive feedback regulation.
The significance of ZEP up-regulation in green leaves is
uncertain. The
18O2-labeling experiments,
which were instrumental in establishing the indirect pathway of ABA
synthesis, also provide some indication of flux through the pathway.
The 1'-hydroxyl in ABA is derived from the epoxide in the carotenoid
precursor. In 18O2-labeling
experiments, there is little incorporation of 18O
at this position for time points less than 8 h (Zeevaart et al., 1989). Therefore, de novo synthesis of epoxycarotenoids
appears to be unnecessary for ABA synthesis in leaves.
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THE CLEAVAGE REACTION |
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TOP
INTRODUCTION
ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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The first committed step in ABA synthesis is the oxidative
cleavage of a 9-cis-epoxycarotenoid. For many years, the pathway of ABA
synthesis had been a point of contention because of difficulties in
demonstrating this activity in vitro. This problem was eventually resolved by the identification and characterization of an ABA-deficient mutant in maize, vp14 (viviparous 14).
Biochemical characterization of vp14 indicated that there
was no lesion in carotenoid synthesis or in the later steps of ABA
synthesis (Tan et al., 1997). By the process of
elimination, it appeared that vp14 was impaired in the
cleavage reaction.
The vp14 mutant resulted from a transposon
insertion, which allowed the corresponding gene to be cloned
(Tan et al., 1997). At the time the Vp14 gene
was identified, the deduced amino acid sequence was most similar to
lignostilbene dioxygenases (LSDs) from Pseudomonas
(Sphingomonas) paucimobilis. The LSDs catalyze a
double-bond cleavage reaction (Kamoda and Samejima,
1991) that is very similar to the cleavage reaction in ABA
synthesis. The recombinant VP14 protein was able to cleave
9-cis-neoxanthin and 9-cis-violaxanthin to form xanthoxin and a
C25 by-product (Schwartz et al.,
1997b). The characteristics of the cleavage reaction in its
substrate specificity and the site of cleavage (11-12 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 converted 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.
Additional ABA synthetic cleavage enzymes have
been identified and characterized in a variety of plant species
(Qin and Zeevaart, 1999; Chernys and Zeevaart,
2000; Iuchi et al., 2000, 2001).
The recombinant enzymes from these species display the same substrate specificity as VP14. The nomenclature that has been adopted for these
enzymes is NCEDs, which is consistent with either 9-cis-violaxanthin or
9-cis-neoxanthin as a substrate.
Although the NCEDs display significant substrate plasticity in vitro,
circumstantial evidence favors neoxanthin as the primary precursor of
ABA. Neoxanthin exists 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). In addition, the
Km for the recombinant PvNCED1 and VP14 is
lower with neoxanthin as substrate relative to 9-cis-violaxanthin
(Qin and Zeevaart, 1999; Schwartz et al.,
2003). Definitive evidence of the endogenous substrate would
require identification of the C25 by-product in
planta. Previous efforts to identify the C25
compounds in vegetative tissue have been unsuccessful. It has been
suggested that these compounds are rapidly degraded after the cleavage
reaction (Parry and Horgan, 1991). The
C25-epoxy-apocarotenal and related compounds have
been identified in fruits that produce high levels of ABA during
ripening (Molnár and Szabolics, 1980; Gross
and Eckhardt, 1981; see also Parry and Horgan,
1991).
Carotenoids in plants are synthesized within
plastids and are associated with the thylakoid and envelope membranes.
Therefore, it was expected that the cleavage reaction would also occur
in chloroplasts. The PvNCED1 from bean (Phaseolus vulgaris)
was imported into pea (Pisum sativum) chloroplasts,
where it was found to associate exclusively with the thylakoid membrane
(Qin and Zeevaart, 1999). An N-terminal targeting
sequence from a cowpea enzyme, VuNCED1, was capable of targeting the
green fluorescent protein to chloroplasts (Iuchi et al.,
2000). After in vitro import assays, the VP14 protein was found
in the stroma and on the thylakoid membrane exposed to the stroma
(Tan et al., 2001). In this study, deletion or
disruption of a putative amphipathic-helix in the N terminus of VP14
interfered with the association of VP14 with thylakoids. The binding of
VP14 to the thylakoid was saturable, suggesting that it associates with
specific components in the thylakoid membrane that have not yet been identified.
The not mutant in tomato also is impaired in the cleavage
step (Burbidge et al., 1999). Both the vp14
and not mutants have weak phenotypes relative to other
ABA-deficient mutants. The vp14 null mutant shows only a
35% reduction of ABA levels in stressed leaves and a 70% reduction in
developing embryos (Tan et al., 1997). This indicates
that there are multiple NCEDs involved in ABA synthesis. In avocado
(Persea americana), PaNCED1 and
PaNCED3 were both shown to encode ABA biosynthetic enzymes
(Chernys and Zeevaart, 2000).
In the Arabidopsis genome, there are nine hypothetical proteins that
share sequence similarity to NCEDs (Fig.
2). In a phylogenetic analysis of the
NCEDs and other similar proteins, five of the Arabidopsis proteins are
clustered with previously characterized NCEDs. It has been reported
that AtNCED2, 3, 6, and 9 are able to catalyze the cleavage
reaction in ABA synthesis (Iuchi et al., 2001). Based
upon sequence similarity, it is expected that AtNCED5 is also an ABA
synthetic enzyme. However, the biochemical role of AtNCED5 has not been
verified experimentally.
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Presumably, the different NCEDs in Arabidopsis are
expressed in different tissues and at different developmental stages.
The AtNCED3 transcript is induced by water stress and
reduced expression results in a wilty phenotype (Iuchi et al.,
2001), indicating that this gene is an important regulator of
ABA levels during water stress. The expression of AtNCED9
also is elevated slightly in response to water stress (Iuchi et
al., 2001). The expression pattern and physiological role of
the other AtNCED genes has not been reported yet. One or
more of these genes is expected to display elevated expression during
seed development when ABA begins to accumulate.
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SYNTHESIS OF APOCAROTENOIDS |
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TOP
INTRODUCTION
ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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A variety of natural products like ABA are derived from the
oxidative cleavage of carotenoids. Collectively referred to as apocarotenoids, these compounds serve important functions in a range of
organisms. Retinal is a chromophore used for phototaxis in green algae
(Nagel et al., 2002; Sineshchekov et al.,
2002) and for light-driven proton pumping in
Halobacterium NRC-1. (Kolbe et al.,
2000).
In animals, vitamin A is necessary for normal vision and development.
Based on sequence similarity to NCEDs, a vitamin A biosynthetic enzyme
was identified in fruitfly (von Lintig and Vogt, 2000) and subsequently in other species (Wyss et al., 2000;
Redmond et al., 2001; Lindqvist and Andersson,
2002). Hypothetical proteins that are similar to NCEDs are also
present in a variety of prokaryotes (Fig. 2). The biochemical and
biological roles of the putative cleavage enzymes in prokaryotes have
not been reported.
All of the proteins in the alignment (Fig.
3) have been shown to catalyze a
double-bond cleavage reaction. With the exception of SpLSD, the
substrates are carotenoids. The N terminus of the ZmVP14, which
does not align well with the other proteins, contains a
chloroplast-targeting sequence of approximately 45 amino acids (Tan et al., 2001). The AtCCD7 protein also contains a
predicted chloroplast-targeting sequence. The specific functions for
conserved residues have not been yet been defined. There are several
highly conserved His and acidic residues that may be necessary for
coordinating iron in the active site.
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There are four hypothetical proteins in Arabidopsis that share some degree of sequence similarity with the NCEDs, but are not thought to be involved in ABA synthesis (Fig. 2). Recent findings demonstrate that at least two of these proteins are able to catalyze carotenoid cleavage reactions. To distinguish these enzymes from the NCEDs, the nomenclature of CCDs has been adopted.
The AtCCD1 protein and a likely ortholog in bean catalyze a symmetric
9-10 (9'-10') cleavage with several different carotenoids (Schwartz et al., 2001). A variety of volatile and
semivolatile compounds, such as the ionones and 
-damascenone, are
derived from 9-10 cleavage reactions. These compounds, which are often produced in flowers and fruits, are believed to serve as attractants for pollination and seed dispersal. Several products derived from 9-10, (9'-10') cleavage reactions also accumulate in the roots of plants
inoculated with arbuscular mycorrhizal fungi (Walter et al.,
2000). The function of these compounds in mycorrhizae has not
been determined yet. Recently, a 7-8 (7'-8') CCD has been cloned from
Crocus sativus, which specifically catalyzes the synthesis of crocetin dialdehyde (C20) and
hydroxy--cyclocitral (C10) from zeaxanthin
(Bouvier et al., 2003). Crocetin dialdehyde is a
precursor of crocin, the primary pigment in saffron.
The most disparate members of this enzyme family in Arabidopsis are
AtCCD7 and AtCCD8. The recombinant AtCCD7 protein is able to cleave
,-carotene at the 9-10 position (S.H. Schwartz and J.A.D.
Zeevaart, unpublished data). The AtCCD8 gene corresponds to the max4 mutant in Arabidopsis, which exhibits extensive
lateral shoot growth. The CCD8/MAX4 gene in Arabidopsis is
orthologous to the RMS1 gene in pea (O. Leyser, personal
communication). Grafting experiments indicate that the rms1
mutant is impaired in the synthesis of a signal molecule that
originates in the wild-type rootstock and inhibits outgrowth of lateral
buds on the mutant scion (Beveridge et al., 2000). There
is also evidence for a similar signaling molecule in Arabidopsis
(Turnbull et al., 2002). The biochemical function and
role of AtCCD8 in this process have not been determined yet.
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THE LATER STEPS. CONVERSION OF XANTHOXIN TO ABA |
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TOP
INTRODUCTION
ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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In contrast to the cleavage reaction, the later steps in ABA
synthesis have been characterized extensively by feeding potential intermediates to intact plants and cell-free extracts (Sindhu and Walton, 1987). By this approach, the sequence of reactions subsequent to cleavage has been determined. The first steps are the
oxidation of the 4'-hydroxyl to a ketone followed by the nonenzymatic desaturation of 2'-3' bond and opening of the epoxide ring. The final
step in the pathway is the oxidation of abscisic aldehyde to ABA.
The aba2 mutant in Arabidopsis was first identified by
screening for the ability to germinate in the presence of the GA
biosynthetic inhibitor, paclobutrazol (Léon-Kloosterziel
et al., 1996). By feeding potential intermediates to extracts
of the aba2 mutant, it was determined that this mutant was
impaired in the conversion of xanthoxin to abscisic aldehyde
(Schwartz et al., 1997a). The aba2 is the
only mutant identified to date that is blocked at this step in the
pathway. Additional alleles of aba2 have since been
identified in screens for a sugar-insensitive phenotype (Laby et
al., 2000; Cheng et al., 2002; see Table I),
altered stomatal conductance (Merlot et al., 2002), and
germination and growth on a medium containing a high NaCl concentration
(González-Guzmán et al., 2002). The gene
corresponding to aba2 has recently been cloned and the gene
product was found to be similar to short chain dehydrogenases/reductases (Cheng et al., 2002;
González-Guzmán et al., 2002). As expected,
the ABA2 protein was able to catalyze the conversion of xanthoxin to
abscisic aldehyde utilizing NAD as a cofactor. The ABA2
transcript level was not affected by stress (González-Guzmán et al., 2002) but was
induced by Glc (Cheng et al., 2002). It has not yet been
reported whether the overexpression of ABA2 would have any
effect on ABA levels.
Mutants impaired in the final step of ABA synthesis, the oxidation of
abscisic aldehyde to ABA, have been identified in a variety of plants.
A loss of this abscisic aldehyde oxidase activity may result from a
mutation in the aldehyde oxidase apoprotein or a lesion in the
synthesis of a MoCo that the enzyme requires for activity. A lesion in
an early step of MoCo synthesis would affect a number of activities.
For example, the nar2a mutant in barley lacks aldehyde
oxidase, xanthine dehydrogenase, and nitrate reductase activities
(Walker-Simmons et al., 1989). The aba3
mutant in Arabidopsis and the flacca mutant in tomato lack
aldehyde oxidase activity, but the activity of nitrate reductase is
unaffected. This phenotype results from a defect in the formation of a
desulfo moiety of the MoCo that is specifically required by certain
hydroxylases (Schwartz et al., 1997a; Akaba et
al., 1998; Sagi et al., 1999). The gene
corresponding to aba3 has been cloned and the N terminus of
the deduced protein is similar to the NifS sulfurase (Bittner et
al., 2001; Xiong et al., 2001). Using Cys as a
sulfur donor, the recombinant protein was able to activate aldehyde
oxidase activity (Bittner et al., 2001). The
flacca mutant also results from a mutation in this sulfurase
(Sagi et al., 1999, 2002). It was found
that ABA3 expression increased in response to osmotic stress
or ABA (Bittner et al., 2001; Xiong et al.,
2001).
Four abscisic aldehyde oxidase (AAO) genes have been identified in
Arabidopsis (AAO1 through 4). Of the aldehyde
oxidases characterized so far, only AAO3 uses abscisic aldehyde
efficiently as a substrate (Seo et al., 2000a). A wilty,
ABA-deficient mutant with a lesion in AAO3 has been
identified (Seo et al., 2000b), demonstrating that AAO3
is responsible for ABA synthesis in vegetative tissues. In contrast to
aba3, the aao3 mutants are not subject to
precocious germination. Therefore, another aldehyde oxidase appears to
be necessary for ABA synthesis in some tissues. In plants subjected to
dehydration, AAO3 mRNA expression was elevated. However, the
level of the corresponding protein was unaffected by water stress
(Seo et al., 2000a). In addition, feeding experiments and assays with cell-free preparations indicate that the conversion of
xanthoxin to ABA is unaffected by stress (Sindhu and Walton, 1987; Schwartz et al., 1997a).
Several variations in the later steps of the pathway may be responsible
for a small portion of ABA synthesis. It has been suggested that
oxidation of the aldehyde may occur subsequent to cleavage and before
the ring modifications (Cowan, 2000), indicating that
xanthoxic acid would be an intermediate in the pathway. However, the
conversion of xanthoxic acid is very low in cell-free extracts (Sindhu and Walton, 1987). Also, the ABA2 protein is
unable to convert xanthoxic acid to ABA (Cheng et al.,
2002). Both the flacca and sitiens
mutants in tomato are blocked in the final step of the pathway, the
oxidation of abscisic aldehyde to ABA, and accumulate 2-trans-ABA
alcohol (Linforth et al., 1987). These mutants are able
to synthesize some ABA by a shunt pathway in which abscisic alcohol is
oxidized to ABA (Rock et al., 1991b).
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TRANSGENIC PLANTS WITH ELEVATED ABA LEVELS |
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TOP
INTRODUCTION
ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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Inhibitors of transcription and translation block stress-induced
ABA accumulation (Quarrie and Lister, 1984;
Guerrero and Mullet, 1986), indicating gene expression
is up-regulated for one or more steps in ABA synthesis. The genes
encoding most of the enzymes in the ABA biosynthetic pathway have now
been identified. Based upon elevated expression during stress, a
regulatory role has been proposed for several of the genes
(Audran et al., 1998, 2001; Seo et
al., 2000b; Bittner et al., 2001; Xiong
et al., 2001, 2002).
In etiolated tissues, the levels of epoxycarotenoids are low and it
appears that elevated expression of ZEP may be important for ABA
synthesis (Frey et al., 1999). In green tissue, however, most of the biochemical evidence indicates that the NCED-catalyzed cleavage reaction is the primary regulatory step in ABA synthesis. In
all instances studied to date, stress-induced ABA accumulation correlates well with increased expression of NCED mRNA
(Tan et al., 1997; Qin and Zeevaart,
1999; Chernys and Zeevaart, 2000; Iuchi et al., 2000, 2001; Thompson
et al., 2000a) and also with NCED protein levels (Qin
and Zeevaart, 1999). The expression of PaNCED1 and
PaNCED3 also increased before the accumulation of high ABA
levels during fruit ripening in avocado (Chernys and Zeevaart,
2000).
The overexpression of NCEDs is sufficient for elevated
ABA synthesis. Overexpression of the LeNCED1 in tomato
(Thompson et al., 2000b), PvNCED1 in
tobacco (Qin and Zeevaart, 2002), and AtNCED3 in Arabidopsis (Iuchi et al., 2001)
resulted in increased ABA levels. In N. plumbaginifolia,
induced expression of PvNCED1 resulted in decreased stomatal
conductance and increased stress tolerance (Fig.
4). Similar results were obtained with
the other species listed above. For the overexpression of
LeNCED1 (Thompson et al., 2000b) and
PvNCED1 (Qin and Zeevaart, 2002), increased seed dormancy was also reported.
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CATABOLISM OF ABA |
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TOP
INTRODUCTION
ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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The level of ABA in plants is controlled not only by its
synthesis, but also through its catabolism. One of the primary
catabolites of ABA is phaseic acid (PA). The conversion of ABA to PA
begins with the hydroxylation of the 8' position by ABA
8'-hydroxylase. The 8' hydroxyl appears to be an unstable
intermediate that spontaneously rearranges to form PA. The ABA
8'-hydroxylase is a cytochrome P450 (Krochko et al.,
1998), which may be induced by ABA (Windsor and
Zeevaart, 1997). This negative feedback regulation is
consistent with time course measurements of ABA and PA accumulation in
stressed plants (Zeevaart, 1980) and recent work with
NCED overexpression in plants (Qin and Zeevaart,
2002). ABA may also be inactivated by the formation of ABA Glc
ester in some tissues. An ABA glucosyltransferase gene from adzuki bean
(Vigna angularis) has been cloned recently (Xu
et al., 2002). Interestingly, this gene is also up-regulated by
ABA. The physiological significance of ABA Glc ester formation and the
potential for engineering ABA levels by decreased glucosylation may now
be investigated.
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FUTURE DIRECTIONS |
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TOP
INTRODUCTION
ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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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 isomerization of neoxanthin and violaxanthin is an enzymatic
reaction. Alternatively, the 9-cis conformations of some
epoxycarotenoids could be stabilized by carotenoid-binding proteins.
In most plant tissues, neoxanthin is the predominant carotenoid with a
9-cis conformation and is considered the most likely precursor of ABA
(Strand et al., 2000). Neoxanthin is derived through the
opening of an epoxy ring in violaxanthin followed by an intramolecular
rearrangement to form an allenic bond. Allenic carotenoids, such as
neoxanthin, are among the most abundant carotenoids in nature.
Therefore, an understanding of their synthesis and functions in
photosynthetic organisms is of considerable interest. Two genes that
encode neoxanthin synthases (NSY) have been identified in potato
(Solanum tuberosum) and tomato (Al-Babili et
al., 2000; Bouvier et al., 2000). The NSY gene
products are similar to lycopene cyclases from various plants and a
capsanthin-capsorubin synthase from pepper (Capsicum
annuum). Transient expression in tobacco and in vitro assays both
demonstrated that the tomato NSY was capable of converting violaxanthin
to neoxanthin (Bouvier et al., 2000). No lycopene
cyclase activity was found by co-expression in a lycopene-accumulating
strain of Escherichia coli (Bouvier et al.,
2000). However, the NSY gene corresponds to the
old-gold mutant in tomato, which accumulates higher levels
of lycopene due to the loss of a fruit-specific lycopene -cyclase,
CYC-B (Ronen et al., 2000; Hirschberg,
2001). It has been suggested that the NSY is a bifunctional
enzyme capable of converting lycopene to ,-carotene or
violaxanthin to neoxanthin (Hirschberg, 2001). Presumably, there is an additional gene responsible for neoxanthin synthesis in plants, because the old-gold mutant is able to
produce neoxanthin. Moreover, no ortholog of the NSY gene is
apparent in the Arabidopsis genome.
The oxidative cleavage products of carotenoids serve important roles in
both plants and animals. Based upon sequence similarity to NCEDs,
putative cleavage enzymes have been identified in a number of plants
and prokaryotes. The characterization of CCDs in plants suggests that
apocarotenoids have various roles in growth and development. The
synthesis of apocarotenoids is well documented in cyanobacteria and a
carotenoid cleavage activity has been described in the cyanobacterium
Microcystis PCC7806 (Jüttner
and Höflacher, 1985). However, the biological functions
of these compounds in cyanobacteria and other prokaryotes have not yet
been determined.
Despite the important roles that apocarotenoids serve in various
organisms and the growing number of putative cleavage enzymes appearing
in the sequence databases, little is known about the mechanism by which
these enzymes catalyze reactions. In an isotopic labeling experiment
with ,-carotene 15, 15'-dioxygenase from chicken
(Gallus gallus), approximately 50% of the cleavage products contained oxygen derived from O2
(Leuenberger et al., 2001). In this experiment, the
second oxygen was derived from water, and the authors proposed a
monooxygenase mechanism. However, no reducing equivalents are required
for assays with any of the recombinant enzymes that have been
characterized. In addition,
18O2-labeling experiments
with plants indicate that the initial cleavage product in ABA
synthesis, xanthoxin, results entirely from O2
(Zeevaart et al., 1989). At this point, the mechanism by
which these enzymes catalyze reactions is still uncertain.
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. Although elevated 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 demonstrate that transcriptional regulation of the NCEDs is the major control point in ABA synthesis. The initial perception of stress and the signal transduction pathway leading to elevated NCED expression remain to be elucidated.
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ACKNOWLEDGMENTS |
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We thank Dr. Nam-Hai Chua (Rockefeller University, New York) for providing the pTA7002 plasmid and Dr. Ottoline Leyser (University of York, UK) for sharing her unpublished results with the atccd8/max4 mutant.
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FOOTNOTES |
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Received November 19, 2002; returned for revision December 9, 2002; accepted January 13, 2003.
1 This work was supported by the National Science Foundation (grant no. IBN-9982758) and by the U.S. Department of Energy (grant no. DE-FG02-91ER20021).
* Corresponding author; e-mail zeevaart{at}msu.edu; fax 517-353-9168.
www.plantphysiol.org/cgi/doi/10.1104/pp.102.017921.
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LITERATURE CITED |
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TOP
INTRODUCTION
ABA-DEFICIENT MUTANTS
THE CLEAVAGE REACTION
SYNTHESIS OF APOCAROTENOIDS
THE LATER STEPS. CONVERSION...
TRANSGENIC PLANTS WITH ELEVATED...
CATABOLISM OF ABA
FUTURE DIRECTIONS
LITERATURE CITED
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-cyclohexenyl carotenoid epoxidase from pepper (Capsicum annuum).
J Biol Chem
271: 28861-28867-carotene and xanthophylls in plants.
Plant Cell
14: 333-342-carotene 7,8 (7',8') oxygenase (-cyclocitral, crocetindial generating) in Microcystis.
Arch Microbiol
141: 337-343[CrossRef]
,-dioxygenase gene from Pseudomonas paucimobilis TMY1009.
Agric Biol Chem
55: 1411-1412[Medline]-carotene to retinal.
Angew Chem Int Ed
40: 2614-2617-carotene 15,15'-monooxygenase.
J Biol Chem
277: 23942-23948-Citraurin epoxide, a new carotenoid from Valencia orange peel.
Phytochemistry
19: 633-637[CrossRef]-carotene 15,15'-dioxygenase.
J Biol Chem
276: 6560-6565-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato.
Proc Natl Acad Sci USA
97: 11102-11107
