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. 1998 Nov;118(3):1021–1028. doi: 10.1104/pp.118.3.1021

Expression Studies of the Zeaxanthin Epoxidase Gene in Nicotiana plumbaginifolia1

Corinne Audran 1, Charlotte Borel 2, Anne Frey 1, Bruno Sotta 3, Christian Meyer 1, Thierry Simonneau 2, Annie Marion-Poll 1,*
PMCID: PMC34775  PMID: 9808747

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.

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.

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] β-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-β-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.

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.

Figure 1.

Figure 1

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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.

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.

Figure 2.

Figure 2

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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.  

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.

Figure 3.

Figure 3

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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.

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).

Figure 4.

Figure 4

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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.  

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).

Figure 5.

Figure 5

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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.

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 g−1 dry weight, whereas roots contained, respectively, 0.8 ± 0.1, 0.9 ± 0.1, and 0.2 ± 0.0 nmol ABA g−1 dry weight. The variability of ABA content among these three experiments was likely due to small differences in culture conditions or plant age.

Figure 6.

Figure 6

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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.

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 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).

Figure 7.

Figure 7

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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.

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.

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.

Abbreviations:

DAP

days after pollination

RWC

relative water content

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).

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