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Plant Physiol. (1998) 118: 1021-1028
Expression Studies of the Zeaxanthin Epoxidase Gene in
Nicotiana plumbaginifolia1
Corinne Audran,
Charlotte Borel,
Anne Frey,
Bruno Sotta,
Christian Meyer,
Thierry Simonneau, and
Annie Marion-Poll*
Laboratoire de Biologie Cellulaire, Institut National de la
Recherche Agronomique, Route de St Cyr, 78026 Versailles cedex, France
(C.A., A.F., C.M., A.M.-P.); Laboratoire d'Ecophysiologie des Plantes
sous Stress Environnementaux, Institut National de la Recherche
Agronomique, 2 place Viala, 34060 Montpellier cedex 2, France (C.B.,
T.S.); and Laboratoire de Physiologie du Développement des
Plantes, Unité, Mixte de Recherche de Physiologie Cellulaire et
Moléculaire des Plantes, Université Pierre et Marie Curie,
tour 53 (E5, casier 156), 4 place Jussieu, 75252 Paris cedex 05, France (B.S.)
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ABSTRACT |
Abscisic acid (ABA) is a plant
hormone involved in the control of a wide range of physiological
processes, including adaptation to environmental stress and seed
development. In higher plants ABA is a breakdown product of xanthophyll
carotenoids (C40) via the C15 intermediate
xanthoxin. The ABA2 gene of Nicotiana
plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes
the conversion of zeaxanthin to violaxanthin. In this study we analyzed
steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in
all plant organs, but transcript levels were found to be higher in
aerial parts (stems and leaves) than in roots and seeds. In leaves
ABA2 mRNA accumulation displayed a day/night cycle;
however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the
ABA2 mRNA level peaked around the middle of development,
when ABA content has been shown to increase in many species. In
conditions of drought stress, ABA levels increased in both leaves and
roots. A concomitant accumulation of ABA2 mRNA was
observed in roots but not in leaves. These results are discussed in
relation to the role of zeaxanthin epoxidase both in the xanthophyll
cycle and in the synthesis of ABA precursors.
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INTRODUCTION |
ABA is ubiquitous in higher plants and is also produced by some
algae and several phytopathogenic fungi. It modulates the growth and
development of plants, particularly during seed formation and in
response to environmental stresses (Zeevaart and Creelman, 1988 ;
Giraudat et al., 1994). During seed development endogenous ABA content
fluctuates in a number of species (Black, 1991) and has been implicated
in the control of many events during seed formation, including the
accumulation of nutritive reserves, the acquisition of desiccation
tolerance, and the onset and maintenance of dormancy (McCarty, 1995;
Ingram and Bartels, 1996). In vegetative tissues ABA levels increase in
various stress conditions, and application of ABA creates effects
similar to plant-stress responses. It has been shown that increased ABA
levels limit water loss through transpiration by reducing stomatal
aperture (Leung and Giraudat, 1998). Moreover, the responses to ABA
result in long-term physiological changes that require modifications of
gene expression at the transcriptional level (Bray, 1993, 1997;
Chandler and Robertson, 1994; Shinozaki and Yamaguchi-Shinozaki, 1996).
It is now clear that ABA is a breakdown product of xanthophyll
carotenoids (C40) via the
C15 intermediate xanthoxin (Walton and Li, 1995).
Studies of mutants defective in ABA synthesis have contributed to the
clarification of the biosynthetic pathway and to the analysis of the
physiological role of endogenous ABA (Zeevaart and Creelman, 1988;
Taylor, 1991). Such mutant plants show a reduced seed dormancy and have
a strong tendency to wilt, a condition that can be reversed by applying
ABA. Moreover, ABA-deficient mutants have been shown to be impaired in
cold and drought adaptation and in the stress regulation of various
genes (Leung and Giraudat, 1998).
Mutants impaired in several steps of the ABA biosynthesis pathway have
been reported. Mutants blocked in the early steps of carotenoid
synthesis, for example, some viviparous mutants of maize
(vp2, vp5, vp7, or vp9),
lack carotenoids essential for photosystem protection and therefore
exhibit photobleaching and ABA deficiency (Neill et al., 1986). In
contrast, mutants impaired in the downstream steps of carotenoid
biosynthesis do not show photobleaching. The aba1 mutant of
Arabidopsis and the aba2 mutant of
Nicotiana plumbaginifolia are impaired in the epoxidation of zeaxanthin and have been shown to be either slightly or not at all
affected in PSII photochemical efficiency (Rock and Zeevaart, 1991;
Rock et al., 1992; Marin et al., 1996; Tardy and Havaux, 1996; Hurry et
al., 1997). Zeaxanthin was able to replace the missing
epoxy-carotenoids antheraxanthin, violaxanthin, and neoxanthin as a
stabilizing component of the light-harvesting complex II in the
aba1 mutant of Arabidopsis.
The N. plumbaginifolia ABA2 cDNA has been cloned and shown
to encode zeaxanthin epoxidase, a chloroplast-imported protein of 72.5 kD (Marin et al., 1996). Homologous zeaxanthin epoxidase cDNAs were
subsequently cloned in pepper (Bouvier et al., 1996) and tomato
(Burbidge et al., 1997b). ABA2 protein catalyzes the conversion
of zeaxanthin into antheraxanthin and, subsequently, violaxanthin via
reduced Fd (Bouvier et al., 1996). Recently, a new
viviparous mutant of maize, vp14, was isolated
and the corresponding gene cloned (Schwartz et al., 1997b; Tan
et al., 1997). The protein VP14 catalyzes the oxidative cleavage of
9-cis-xanthophylls to xanthoxin, which is the first
C15 precursor of ABA. Vp14
likely belongs to a multigene family (four to six related genes in
maize). The homolog of Vp14 has been cloned in tomato
(Burbidge et al., 1997a). Indirect evidence suggests that this cleavage
might be the key regulatory step in the ABA biosynthetic pathway
(Walton and Li, 1995).
Other mutants affected in the later steps of the ABA biosynthetic
pathway are known in a variety of plant species (Giraudat et al.,
1994). Recently, two new Arabidopsis mutants have been described. The
aba2 mutant is impaired in the conversion of xanthoxin to
ABA-aldehyde and the aba3 mutant is affected in the
biosynthesis of the molybdenum cofactor necessary for ABA-aldehyde
oxidase activity (Léon-Kloosterziel et al., 1996; Schwartz et
al., 1997a). As is the case for the Arabidopsis mutant aba3,
the N. plumbaginifolia mutant aba1 lacks
ABA-aldehyde oxidase activity because of a molybdenum cofactor
deficiency (Leydecker et al., 1995).
The recent isolation of genes involved in the downstream steps of the
ABA biosynthetic pathway provides the opportunity to study their
regulation and to investigate their contribution to the regulation of
ABA biosynthesis. In this report we show that accumulation of
ABA2 mRNA and ABA2 protein are higher in leaves than in
roots. We also demonstrate that ABA2 transcript levels in
roots are up-regulated by drought stress and that this increase in
transcript level is correlated with an increase in ABA accumulation. We
also show that ABA2 mRNA steady-state levels peak during
seed development.
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
The mutant aba2-s1 of Nicotiana
plumbaginifolia var Viviani was obtained as previously
described (Marin et al., 1996). Wild-type and mutant seeds were
surface-sterilized and sown on agar-solidified B medium (Bourgin et
al., 1979). Seeds were first incubated for 2 to 3 weeks under short
days (8 h of light/16 h of dark) with a substantial day/night
temperature difference (25°C/17°C) for uniform germination. Young
plants were then transferred to a growth chamber (25°C; 16 h of
light/8 h of dark). Finally, 6-week-old plants were transferred to soil
or to sand. Wild-type and mutant plants were grown in a growth chamber
with 80% RH, 16 h of light at 25°C, and 8 h of dark at
17°C. Some experiments were performed on aba2-s1
plants grafted onto wild-type tobacco stocks in greenhouse conditions.
Northern Analysis
Unless otherwise stated, tissue samples were collected 3 to 4 h after the beginning of the light period. Total RNA was obtained from
0.5 g of plant tissue after it was ground in liquid nitrogen and
extracted with phenol as previously described (Verwoerd et al.,
1989), except that the extraction buffer was 100 mM
Tris-HCl, pH 8.0, 100 mM LiCl, 10 mM EDTA, pH
8.0, and 1% SDS. Total RNA from seeds was extracted after the outer
and inner portions of the plant capsules were removed as previously
described (Dean et al., 1985). Six to eight micrograms of RNA was
fractionated on a 1.2% (w/v) agarose gel containing formaldehyde in
Mops buffer (Sambrook et al., 1989) and then transferred onto
GeneScreen (DuPont) or Hybond-N (Amersham) membranes following the
manufacturer's instructions.
Probes were labeled with [32P]dCTP using a
random-primer kit (Pharmacia). Blots were hybridized with a 1.4-kb
HindIII internal fragment of the ABA2 cDNA (Marin
et al., 1996). To check for equal loading, blots were either
rehybridized with a 0.5-kb cDNA fragment of 25S rRNA (Unfried and
Gruendler, 1990) or a reverse picture of the ethidium bromide-stained
gel was used. Washes were carried out under high stringency (Sambrook
et al., 1989). Quantification of mRNA was performed using a phosphor
imager (BAS-1500, Fuji, Tokyo, Japan) or by scanning of autoradiograms
(Power Look II scanner, UMAX) with subsequent analysis using the
program Mac Bas version 2.2 (Fuji) and normalized with respect to the
25S RNA reference. Each blot was repeated several times with mRNA from
independent experiments.
Western Analysis
Plant tissue (0.5 g) was ground in liquid nitrogen and incubated
in 1 mL of denaturation buffer (125 mM Tris-HCl, pH 6.8, 10% [v/v]  -mercaptoethanol, 4% [w/v] SDS, 20% [v/v]
glycerol, and 25 mg L 1 bromphenol blue) at
100°C for 5 min. After high-speed centrifugation, total proteins were
run in a 10% acrylamide gel under denaturing conditions. SDS-PAGE and
immunoblotting were performed as previously described
(Crété et al., 1997). Antibodies were prepared against N. plumbaginifolia zeaxanthin epoxidase protein expressed in
Escherichia coli. A PvuII internal fragment of
0.87 kb was cloned into the SmaI site of pQE32 (Qiagen,
Chatsworth, CA). The resulting plasmid was used to transform E. coli M15 (pREP4). Bacteria were grown at 37°C up to an
A600 of 0.7 before
isopropylthio--galactoside was added to a final concentration of 2 mM. After 5 h, E. coli cells were
centrifuged and resuspended in 0.1 M Tris-HCl, pH 8.0, 8 M urea, and 0.1 M sodium phosphate. After the
sample was centrifuged the supernatant was loaded onto a
Ni-nitrilotriacetic acid column (Qiagen). The column was then
washed with the same buffer at pH 6.3 before elution of recombinant
protein at pH 4.6. Urea concentration was then reduced to 4 M by dialysis. Rabbit antiserum was collected after two
injections of 1 mg of purified protein.
ABA Measurements
Plant material was frozen in liquid nitrogen and lyophilized prior
to being ground into a powder. Extraction, purification, quantification
by ELISA, and identification of immunoreactive molecules has been
previously described (Kraepiel et al., 1994). We used a monoclonal
anti-ABA antibody (LPDP 229, Jussieu, France) labeled with
peroxidase-conjugated goat antibody to mouse immunoglobulins (Sigma).
Hormonal content was determined five times for each sample.
Dehydration Experiments
Dehydration experiments were performed on whole plants and on
detached roots. In the first experiment N. plumbaginifolia
plants were cultivated in the growth chamber in sand for 6 weeks.
Rosette-stage plants were removed from the sand and roots were rapidly
washed and blotted dry on tissue paper. Entire plants were placed under a laminar flow hood. Leaves and roots of three plants were collected separately after 0, 4, and 8 h for ABA measurements and RNA and protein extractions.
For the second set of experiments N. plumbaginifolia plants
were cultivated in a hydroponic device placed in a growth chamber. Roots of adult plants were cut, blotted dry on tissue paper, and divided into two groups. The control group (100% RWC) was sampled immediately, and the other group was rapidly dehydrated in a stream of
dry air until a RWC of 60% was reached. The water content was calculated as the difference between the fresh weight and the dry
weight after lyophilization. RWC was determined on additional samples
as the ratio of the water content of dehydrated roots to the water
content of roots before dehydration. For both groups subsamples were
put into 10-mL tubes and incubated for durations ranging from 30 min to
4 h. Tubes were placed in a moist atmosphere at 24°C in darkness
to prevent water loss. After incubation samples were frozen and stored
at  80°C. RNA extraction was performed as previously described.
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RESULTS |
Tissue-Specific Expression of the ABA2
Gene
To get an insight into the regulation of the ABA2
gene, we first examined its expression pattern in various organs by
northern analysis (Fig. 1).
ABA2 cDNA was previously shown to hybridize to a single mRNA
band of approximately 2.5 kb in leaves (Marin et al., 1996).
Accordingly, the ABA2 probe detected a single band
corresponding to the same RNA size in all of the organs tested.
Steady-state levels of mRNA were high in leaves and stems. Lower
amounts of mRNA were detected in roots and flowers. In flowers the
level of the transcript was almost constant during development, except
in 50-mm-long flowers, in which a higher mRNA accumulation was
observed.
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| Figure 1.
ABA2 mRNA accumulation in N. plumbaginifolia plants. Total RNA from roots (R), stems (St),
rosette leaves (rL), cauline leaves (cL), and flowers (a, 3 mm; b, 5 mm; c, 8 mm; d, 15 mm; e, 20 mm; and f, 50 mm in length) were
hybridized with an ABA2 probe and then with a 25S rRNA
probe as a loading control.
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It has been shown that ABA content changes during seed development,
with a significant increase at about one-third to one-half of the time
from seed initiation to maturity (Black, 1991; Rock and Quatrano,
1995). The accumulation kinetics of ABA2 mRNA were analyzed
during seed development in wild-type N. plumbaginifolia (Fig. 2). Seed RNA was extracted from
capsules from 3 to 22 DAP, when seed capsules were opening. At early
stages of development, the ABA2 mRNA level was low.
Abundance of the transcript increased between 5 and 10 DAP and then
decreased to very low levels 18 DAP. These data show that
ABA2 mRNA levels change during seed development, with a
maximum between one-third and one-half of the time from seed initiation
to maturity.
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| Figure 2.
Steady-state ABA2 mRNA levels in
developing seeds of wild-type plants. RNA was extracted from seeds
harvested from 3 to 22 DAP when seed capsules were opening. Ethidium
bromide staining of 25S RNA is shown as a control.
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ABA, ABA2 Protein, and ABA2 mRNA Levels in Wild-Type
and Mutant Leaves
The mutant aba2-s1 results from an imprecise excision
of a maize Ac element from the ABA2 gene and was
shown to contain a stop codon in the 5 region of the coding sequence
(Marin et al., 1996).
ABA levels in the mutant aba2-s1 were lower than in the wild
type but were unexpectedly quite variable (Marin et al., 1996). Because
previous experiments were performed on detached leaves of unstressed
plants grown in humid conditions, where ABA synthesis is low, we chose
to measure endogenous ABA levels in stressed leaves of the wild type
and in the mutant aba2-s1. ABA levels were measured in
detached leaves before and after 1 h of dehydration under a
laminar flow hood (Fig. 3). When leaves
were not stressed, the ABA content in the aba2-s1 mutant
varied from 10% to 15% of the wild-type content. After 1 h of
dehydration, a 2-fold increase in ABA content was observed in the
wild-type leaves, whereas it remained unchanged in mutant leaves.
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| Figure 3.
ABA levels in response to dehydration in leaves of
the wild type (WT) and of the mutant aba2-s1. ABA was
quantified before (not stressed, NS) and after (stressed, S) detached
leaves were placed for 1 h under a laminar flow hood. Similar
results were obtained in four independent experiments. Only one is
presented here. DW, Dry weight.
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Polyclonal antibodies were raised against the ABA2-purified protein
produced in E. coli and were shown to cross-react with the
purified protein in western analysis and in ELISA (data not shown). In
wild-type leaves we could detect only one specific band that was not
present in mutant leaves (Fig. 4).
Nonspecific cross-reacting bands were observed in all extracts. The
size of the major band was approximately 67 kD, which is in accordance with the expected size of ABA2 protein deduced from the cDNA sequence. The ABA2 cDNA encodes a 72-kD protein including a
chloroplast transit peptide that is subsequently cleaved (Marin et al.,
1996). We can conclude that, in the mutant aba2-s1, the
presence of a stop codon at the beginning of the ABA2-coding
sequence prevents ABA2 protein synthesis and probably also reduces mRNA
stability, since northern analysis showed that ABA2
transcript levels were very low in leaves of the mutant
aba2-s1 (data not shown).
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| Figure 4.
Immunoblot of leaf extracts from the mutant
aba2-s1 and from the wild type (WT). Nine wild-type
extracts were tested during a period of 24 h. Because the mutant
aba2-s1 produced no detectable ABA2 protein, only one
extract was loaded. Mutant leaves were collected 3.5 h after the
beginning of the light period. Proteins were visualized with an
antiserum against the ABA2 protein.
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Diurnal Oscillations of Steady-State mRNA and Protein Levels
We analyzed steady-state mRNA and protein levels in plants at the
rosette stage during a 24-h period to monitor possible diurnal mRNA
and/or protein fluctuations. Plants were cultivated in sand in a growth
chamber (16 h of light/8 h of dark). Leaves and roots were collected
during a period of 24 h. More samples were collected at the
beginning of the day, when rapid modification of mRNA accumulation was
observed.
We detected significant mRNA fluctuations in leaves using northern
analysis (Fig. 5). Steady-state levels of
mRNA increased at the beginning of the light period and reached a
maximum after 3 to 5 h of light. The mRNA level then decreased to
very low levels during the dark period. However, a small increase was
observed 30 min before the beginning of the light period. In contrast, a low and constant accumulation of ABA2 mRNA was observed in
roots (data not shown).
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| Figure 5.
Diurnal rhythm in ABA2 mRNA
abundance in N. plumbaginifolia leaves. Leaf total RNA
was extracted at different intervals during 24 h and hybridized
with an ABA2 probe. Ethidium bromide staining of 25S RNA
is shown as a control.
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In leaves we did not observe significant variations of ABA2 protein
(Fig. 4), which was confirmed by scanning densitometry. Therefore, in
spite of a clear diurnal rhythm in ABA2 mRNA abundance, the
ABA2 protein level remained constant in leaves. In roots no ABA2
protein was detected by the antiserum (data not shown). Because a
reduced level of mRNA was observed in roots compared with leaves (Fig.
1), the ABA2 protein level in root extracts was likely to be too low to
be detected.
Regulation of ABA2 Gene Expression by
Dehydration
In a wide range of species an increase in endogenous ABA
concentration in plant roots and leaves has been observed after an imposed water deficit (Zeevaart and Creelman, 1988). We therefore analyzed the accumulation of ABA2 mRNA in response to
dehydration treatments.
In the first set of experiments we studied the levels of ABA and
ABA2 mRNA in roots and leaves of wild-type plants after
dehydration under a laminar flow hood for several hours. Plants were
grown in sand in growth-chamber conditions. After the sand was removed, entire young plants were placed under a laminar flow hood. Experiments were started (time 0) when the ABA2 mRNA level in leaves was
maximum, after 4 h of light. The relative abundance of endogenous
ABA was measured in leaves (Fig. 6A) and
in roots (Fig. 6B) after 0, 4, and 8 h of dehydration. Three
independent experiments are presented. Before dehydration leaves
contained, respectively, 2.6 ± 0.2, 2.7 ± 0.4, and
2.1 ± 0.4 nmol ABA g1 dry weight, whereas
roots contained, respectively, 0.8 ± 0.1, 0.9 ± 0.1, and
0.2 ± 0.0 nmol ABA g1 dry weight. The
variability of ABA content among these three experiments was likely due
to small differences in culture conditions or plant age.
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| Figure 6.
ABA accumulation and ABA2
transcript levels in dehydrated leaves and roots. Entire N. plumbaginifolia plants were dehydrated under a laminar flow
hood for 0 h (white bars), 4 h (checkered bars), or 8 h
(black bars). Three independent experiments are presented here (1, 2, and 3). Relative abundance of ABA was measured in leaves (A) and in
roots (B). Hormonal content was determined five times for each sample.
Total RNA was extracted from leaves and roots and used for northern
analysis using ABA2 and 25S rRNA probes. Relative
ABA2 mRNA expression levels were determined using 25S
rRNA as a standard in leaves (C) and in roots (D). Relative abundance
of ABA was calculated by giving the value 1 to the ABA level observed
at 0 h of dehydration, and relative abundance of
ABA2 mRNA was calculated by giving the value 1 to the
mRNA level observed at the same time in roots as in leaves, even if
their absolute values were different.
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In our experiments ABA content was always lower in roots than in
leaves. A similar increase in ABA level was observed both in leaves and
roots. In leaves endogenous ABA content increased 2- to 7-fold after
4 h of dehydration and 3- to 12-fold after 8 h. In roots the
ABA level increased 3- to 5-fold after 4 h of dehydration and 7- to 15-fold after 8 h. We analyzed the relative abundance of
ABA2 mRNA in leaves (Fig. 6C) and in roots (Fig. 6D). In
leaves the ABA2 mRNA level decreased 2- to 3-fold after 4 h of dehydration and 1.5- to 5-fold after 8 h of
dehydration. In roots the level of ABA2 mRNA increased 2.5- to 5-fold after 4 h of dehydration and 3- to 7-fold after 8 h
of dehydration. This increased accumulation of ABA2 mRNA in
roots was maintained after 24 h of dehydration (data not shown);
therefore, although a similar increase in ABA levels was observed in
roots and leaves in response to drought stress, the relative abundance
of ABA2 mRNA increased in roots but decreased in leaves.
The decrease of ABA2 mRNA in leaves paralleled the diurnal
oscillations of steady-state ABA2 mRNA levels previously
observed (Fig. 5). The times 0, 4, and 8 h of dehydration
corresponded, respectively, to 4, 8, and 12 h after the beginning
of the light period. The relative abundance of ABA2 mRNA in
leaves of control (nondehydrated) plants decreased similarly, as
presented in Figure 5 (data not shown). The ABA2 protein level in
leaves was constant during dehydration, as was previously observed in
nondehydrated leaves (Fig. 4), but no ABA2 protein was detected in
roots by the antiserum either before or after dehydration (data not
shown). The level of this protein was probably too low to be detected even if there was an increased level of ABA2 mRNA in roots.
In the second set of experiments we studied the abundance of
ABA2 mRNA in roots that were rapidly dehydrated and then
maintained at a constant water content for several hours. In contrast
to the first experiment, in which the water loss increased with time, this method consisted of imposing a constant stress throughout the
experiment. Wild-type plants were cultivated in a hydroponic device and
roots were cut just before the beginning of the stress (see
``Materials and Methods''). ABA2 mRNA levels were
quantified every 30 min for 4 h in control roots (100% RWC) and
in dehydrated roots, which were rapidly dehydrated to 60% RWC. The
relative abundance of ABA2 mRNA increased 2- to 4-fold
in dehydrated roots compared with control roots 2 to 4 h after
dehydration (Fig. 7).
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| Figure 7.
ABA2 transcript levels in roots after an imposed
dehydration. N. plumbaginifolia plants were cultivated
in a hydroponic device. Roots were cut and divided into two batches.
One was immediately sampled (100% RWC) and another was rapidly
dehydrated in a stream of dry air until its weight was at 60% RWC.
Samples were incubated from 30 min to 4 h. Total RNA from roots
was extracted, and relative ABA2 mRNA expression levels
were determined using 25S rRNA as a standard.
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DISCUSSION |
Carotenoids are synthesized in plant chloroplasts, and
violaxanthin is one of the most abundant carotenoids in the thylakoid membrane. Its synthesis from zeaxanthin by zeaxanthin epoxidase and its
reconversion to zeaxanthin by violaxanthin de-epoxidase constitute
enzymatic reactions called the xanthophyll cycle. The xanthophyll cycle
is thought to be essential for protection of the photosynthetic
apparatus from photo-oxidation, and zeaxanthin, antheraxanthin, and
violaxanthin are associated with the light-harvesting complexes
(Demmig-Adams and Adams, 1996). In accordance with the role of
zeaxanthin and violaxanthin in photosynthesis, our experiments demonstrate that ABA2 mRNA is more abundant in
photosynthetic than in nonphotosynthetic tissues (Fig. 1). Furthermore,
in leaves mRNA content fluctuates periodically, indicating that a
diurnal rhythm is likely involved in the regulation of the expression of the ABA2 gene (Fig. 5).
The ABA2 mRNA level increases at the beginning of the light
period to reach a maximum after 3 to 5 h of light and then
decreases gradually to be undetectable during the dark period. A small
but reproducible increase in ABA2 mRNA accumulation was also
noticed 30 min before the night/day transition. This endogenous rhythm of ABA2 mRNA levels is similar to oscillations of transcript
levels of genes encoding proteins of the light-harvesting complex of PSI and PSII (Piechulla, 1993), providing further evidence for the
existence of a coordinated regulation of genes involved in the assembly
and function of the photosynthetic apparatus. However, western analysis
showed that oscillations of steady-state ABA2 mRNA levels do
not result in any fluctuation in ABA2 protein level (Fig. 4). This
observation might reflect the stability of the ABA2 protein, but the
physiological significance of variations affecting only mRNA levels is
unknown. Because we did not observe any clear daily fluctuations of ABA
levels in leaves in our experimental conditions (data not shown), we
can assume that the diurnal rhythm of ABA2 mRNA accumulation
does not reflect a general regulation of the ABA synthesis
pathway.
ABA may be involved in water-stress responses. Many reports have shown
an increase in ABA concentration in roots, xylem sap, and leaves of
drought-stressed plants (Davies and Zhang, 1991). In N. plumbaginifolia plants an increased ABA level was observed in
roots upon dehydration, and ABA2 gene expression was induced by drought stress in roots of intact plants or in detached roots (Figs.
6 and 7). Our data suggest that drought-induced ABA in the roots likely
arises from de novo synthesis, in accordance with previous studies. In
a number of species, both phloem blocking and experiments with excised
roots indicate that the root ABA level increase does not require the
transport of ABA, an ABA precursor, or other factors from the shoot and
may instead be due to de novo synthesis (Walton et al., 1976; Cornish
and Zeevaart, 1985). Furthermore, the increase in ABA levels in tomato
roots has been shown to correlate with a decrease in the levels of
specific xanthophylls that are ABA precursors (Parry et al., 1992).
Stress-induced ABA production by roots might have considerable
importance, modifying the plant water balance before the leaves have
even detected a water stress. Stomatal closure allows reduced leaf
transpiration, and analyses of the control of stomatal conductance strongly suggest that roots are the primary sensor of water deficit (Tardieu, 1996). Moreover, changes in gene expression in the shoot during water deficit might also be induced by signals originating in
the roots (Griffiths and Bray, 1996). Although other factors may play a
role, it is clear that ABA acts as a positive signal from drying roots
to promote stomatal closure and to slow down leaf expansion (Jackson,
1997). ABA has also been shown to be accumulated in intact or detached
N. plumbaginifolia leaves upon dehydration (Figs. 3 and 6).
In leaves the lack of correlation between ABA2 transcript
levels and the increase in ABA concentration probably indicates that
the de novo ABA synthesis does not require an increase in ABA2 mRNA accumulation. Indeed, in several plant species,
including N. plumbaginifolia, xanthophylls have been shown
to be abundant in leaves compared with roots (Parry and Horgan,
1992). Migration of ABA synthesized in roots may contribute to
its accumulation in leaves; however, we have shown that ABA pools were
significantly lower in roots than in leaves (Fig. 6). From our data we
conclude that accumulation of zeaxanthin epoxidase mRNA is controlled
by drought stress in roots. In leaves, where the level of
ABA2 mRNA is high compared with roots, availability of
xanthophyll precursors might not be limiting for ABA synthesis, even
under water stress. An increased gene expression would therefore not be
needed or would be restricted to certain tissues and might not be
detected.
We cannot exclude that the regulation of the ABA2 gene
expression might be controlled at different levels or by different factors in leaves compared with roots because of the contribution of
zeaxanthin epoxidase to the xanthophyll cycle. The different steps of
the ABA biosynthesis pathway appear to be differentially regulated,
since the expression of genes involved in the oxidative cleavage of
9-cis-epoxy-carotenoids has been shown to be controlled by
water stress in leaves. Accumulation of Vp14 mRNA increased in detached maize leaves upon dehydration (Tan et al., 1997), and an
increase in mRNA levels of its homolog in tomato was also observed in
leaves of unwatered plants (Burbidge et al., 1997a).
We demonstrated here that the ABA2 mRNA level changes during
N. plumbaginifolia seed development, with a maximum between
one-third to one-half of the time from seed initiation to maturity. It
has been widely documented that ABA content increases to high levels at
the same time in a number of species such as Arabidopsis (Karssen et
al., 1983), Brassica napus (Juricic et al., 1995), alfalfa (Xu and Bewley, 1995), and Nicotiana tabacum (Jiang et al.,
1996). In this study ABA2 mRNA accumulation was measured in
whole seeds; in developing seeds ABA has two origins, maternal and
embryonic. These two ABA sources may have different contributions to
the regulation of seed development. In Arabidopsis genetic analysis has
shown that dormancy induction is exclusively a function of ABA
synthesized in the embryo (Karssen et al., 1983). The use of in situ
hybridization techniques would be necessary to further investigate the
tissue specificity of ABA2 gene expression in seeds.
Nevertheless, the kinetics of ABA2 mRNA accumulation in N. plumbaginifolia are similar to those of ABA accumulation
in developing seeds of various species. Our data indicate that the regulation of steady-state levels of zeaxanthin epoxidase mRNA might
contribute to the regulation of ABA synthesis in seeds. We also showed
that an overexpression of the ABA2 gene in transgenic seeds
results in an increased seed dormancy, indicating that ABA2 gene expression may limit ABA biosynthesis in the seed embryo (A. Frey,
unpublished data).
In conclusion, in nonphotosynthetic tissues such as roots and seeds,
ABA2 mRNA levels appear to be coordinately regulated with
changes in ABA levels. Studies of other biosynthetic genes will provide
further information about the existence of a common regulation of the
ABA biosynthetic pathway. In contrast, the regulation of
ABA2 transcript levels in leaves appears to be more related to the regulation of photosynthetic genes, confirming its potential role in photosystem function.
| |
FOOTNOTES |
1
This work was supported by the Ministère
de l'Education Nationale et de la Recherche Scientifique et Technique
(grant no. 95282 to C.A.) and by the European Community BIOTECH program
(grant no. BIO4-CT-960062).
*
Corresponding author; e-mail poll{at}versailles.inra.fr; fax
33-1-30-83-30-99.
Received April 16, 1998;
accepted August 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DAP, days after pollination.
RWC, relative water
content.
| |
ACKNOWLEDGMENTS |
We thank J. Goujaud and J. Talbotec for care of the plants. We
are grateful to H. McKhann and S. Filleur for critical reading of the
manuscript.
| |
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Top
Abstract
Introduction