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Regulation of the expression of the cytosolic O-acetylserine(thiol)lyase gene (Atcys-3A) from Arabidopsis thalianaunder heavy metal stress conditions has been investigated. Northern blot analysis of Atcys-3A expression shows a 7-fold induction after 18 h of cadmium treatment. Addition of 50 μm CdCl2 to the irrigation medium of matureArabidopsis plants induces a rapid accumulation of the mRNA throughout the leaf lamina, the root and stem cortex, and stem vascular tissues when compared with untreated plants, as observed byin situ hybridization. High pressure liquid chromatography analysis of GSH content shows a transient increase after 18 h of metal treatment. Our results are compatible with a high cysteine biosynthesis rate under heavy metal stress required for the synthesis of GSH and phytochelatins, which are involved in the plant detoxification mechanism.Arabidopsis-transformed plants overexpressing theAtcys-3A gene by up to 9-fold show increased tolerance to cadmium when grown in medium containing 250 μmCdCl2, suggesting that increased cysteine availability is responsible for cadmium tolerance. In agreement with these results, exogenous addition of cystine can, to some extent, also favor the growth of wild-type plants in cadmium-containing medium. Cadmium accumulates to higher levels in leaves of tolerant transformed lines than in wild-type plants. Regulation of the expression of the cytosolic O-acetylserine(thiol)lyase gene (Atcys-3A) from Arabidopsis thalianaunder heavy metal stress conditions has been investigated. Northern blot analysis of Atcys-3A expression shows a 7-fold induction after 18 h of cadmium treatment. Addition of 50 μm CdCl2 to the irrigation medium of matureArabidopsis plants induces a rapid accumulation of the mRNA throughout the leaf lamina, the root and stem cortex, and stem vascular tissues when compared with untreated plants, as observed byin situ hybridization. High pressure liquid chromatography analysis of GSH content shows a transient increase after 18 h of metal treatment. Our results are compatible with a high cysteine biosynthesis rate under heavy metal stress required for the synthesis of GSH and phytochelatins, which are involved in the plant detoxification mechanism.Arabidopsis-transformed plants overexpressing theAtcys-3A gene by up to 9-fold show increased tolerance to cadmium when grown in medium containing 250 μmCdCl2, suggesting that increased cysteine availability is responsible for cadmium tolerance. In agreement with these results, exogenous addition of cystine can, to some extent, also favor the growth of wild-type plants in cadmium-containing medium. Cadmium accumulates to higher levels in leaves of tolerant transformed lines than in wild-type plants. O-acetylserine(thiol)lyase phytochelatin high pressure liquid chromatography 2-(cyclohexylamino)ethanesulfonic acid In recent years, contamination of soils and waters by toxic heavy metals and organic pollutants represents a major environmental and human health problem. The possibility of the removal of these toxic heavy metals by phytoremediation technology has increased interest in the knowledge of the physiological and molecular mechanisms of plant adaptation to high levels of heavy metals. Several heavy metals such as cadmium and copper are tolerated to certain low levels through the synthesis of small peptides named phytochelatins, which form a tight complex with the metal ion to inactivate and store it in the vacuole (1Zenk M.H. Gene. 1996; 179: 21-30Crossref PubMed Scopus (879) Google Scholar). Phytochelatins are enzymatically synthesized from glutathione as a response of the plant to toxic levels of the heavy metals that produce a transient decrease of GSH content (2Noctor G. Arisi A.-C. Jouanin L. Kunert K.J. Rennenberg H. Foyer C.H. J. Exp. Bot. 1998; 49: 623-647Google Scholar). Glutathione is synthesized by a two-step ATP-dependent reaction catalyzed by the γ-glutamylcysteine synthetase and glutathione synthetase. These reactions seem to be highly regulated at the transcriptional and translational levels, as well as by feedback control of the γ-glutamylcysteine synthetase by GSH. InArabidopsis, a coordinated response of the glutathione biosynthesis genes to heavy metals has been reported (3Xiang C. Oliver D.J. Plant Cell. 1998; 10: 1539-1550Crossref PubMed Scopus (580) Google Scholar). One of the factors that also regulates GSH biosynthesis is cysteine availability, because exogenous addition of cysteine has been shown to increase the GSH content (4Farago S. Brunold C. J. Plant Physiol. 1994; 144: 433-437Crossref Scopus (22) Google Scholar). However, little work has been carried out on the regulation of cysteine biosynthesis under heavy metal stress or environmental conditions. The last step of cysteine biosynthesis is catalyzed by theO-acetylserine(thiol)lyase enzyme (OASTL)1 that incorporates sulfide into the O-acetyl-l-serine molecule. This molecule is produced by an acetylation reaction ofl-serine catalyzed by the serine acetyltransferase enzyme. Both serine acetyltransferase and OASTL activities have been demonstrated to be localized in the three cellular compartments involved in protein synthesis, cytosol, chloroplast, and mitochondrion (5Lunn J.E. Droux M. Martin J. Douce R. Plant Physiol. 1990; 94: 1345-1352Crossref PubMed Scopus (179) Google Scholar). However, the contribution of each OASTL and serine acetyltransferase isoform to a particular metabolic pathway is unknown (6Barroso C. Romero L.C. Vega J.M. Gotor C. Curr. Top. Phytochem. 1997; 1: 19-29Google Scholar, 7Leustek T. Physiol. Plant. 1996; 97: 411-419Crossref Scopus (65) Google Scholar). Recently, the Atcys-3A gene coding for the cytosolic OASTL isoform from Arabidopsis has been shown to be regulated by salt stress and the hormone abscisic acid (8Barroso C. Romero L.C. Cejudo F.J. Vega J.M. Gotor C. Plant Mol. Biol. 1999; 40: 729-736Crossref PubMed Scopus (68) Google Scholar). In this work, we describe the Cd induction of the cytosolic OASTL fromArabidopsis thaliana and the involvement of this enzyme in cadmium tolerance, by overexpression of the corresponding cDNA in transformed plants. Wild-typeA. thaliana (ecotype Columbia) plants were grown on moist vermiculite supplemented with Hoagland medium at 20 °C in the light and 18 °C in the dark, under a 16-h white light/8-h dark photoperiod. Cadmium chloride treatments were performed by addition to the Hoagland medium of CdCl2 to 50 μm final concentration, unless otherwise indicated. For the tolerance test, seeds were surface-sterilized and germinated on solid MS medium with and without CdCl2 at indicated concentrations, after cold treatment for 1 day to improve germination. The plants were grown in a growth chamber under the same conditions described above. For circadian experiments plants were grown on soil under a 16-h white light/8-h dark photoperiod for 3 weeks, before harvesting every 4 h for 36 h. After this treatment, plants were shifted to constant light and harvested every 4 h during 1 day. Seeds were germinated on solid MS medium for 5 days on vertical Petri dishes and transferred onto medium containing different concentrations of CdCl2. Plants were weighed after 6 days of further growth. Total seedling fresh weight was determined after gently drawing the root out of the agar medium. Cysteine and glutathione were extracted and subsequently quantified by reverse-phase HPLC after derivatization with monobromobimane (Molecular Probes) following the methods described by Rauser et al. (9Rauser W.E. Schupp R. Rennenberg H. Plant Physiol. 1991; 97: 128-138Crossref PubMed Scopus (70) Google Scholar). Plant tissues were homogenized in cold extraction buffer (4 ml/g fresh weight) containing 0.1 N HCl and 1 mm EDTA using a mortar and pestle with liquid nitrogen. Homogenates were centrifuged at 15,000 × g for 15 min in the cold. Thiols were reduced at 4 °C for 15 min by mixing 400 μl of extracted samples with 600 μl of 200 mm CHES (pH 9.2) and 100 μl of 250 mm NaBH4. A 330-μl aliquot was derivatized in the dark for 15 min by adding 20 μl of 15 mmmonobromobimane. The reaction was stopped by adding 250 μl of 0.25% (v/v) methanesulfonic acid at room temperature. Derivatized thiols were separated and quantified by reverse-phase HPLC (10Newton G.L. Dorian R. Fahey R.C. Anal. Biochem. 1981; 114: 383-387Crossref PubMed Scopus (417) Google Scholar).O-Acetylserine(thiol)lyase activity was measured in crude extracts from wild-type or transformed Arabidopsisplants following a previously described method (11Barroso C. Vega J.M. Gotor C. FEBS Lett. 1995; 363: 1-5Crossref PubMed Scopus (63) Google Scholar). Three-week-old plants were irrigated for 14 days with Hoagland medium supplemented with 250 μm CdCl2, final concentration. The leaves were collected and dried at 65 °C during 5 h. The dried material was wet-ashed in HNO3-HClO4(7:1) (v/v) at 65 °C until the volume of the solution was reduced from 20 to 2 ml. Finally, the samples were analyzed by inductively coupled plasma atomic emission spectrometry using a Fisons-ARL 3410 sequential multielement instrument. Total RNA was isolated from leaves, and Northern blot analysis was performed as previously described (11Barroso C. Vega J.M. Gotor C. FEBS Lett. 1995; 363: 1-5Crossref PubMed Scopus (63) Google Scholar). The blots were sequentially hybridized with a gene-specific Atcys-3A probe and then with maize 17 S DNA for detection of Arabidopsis 18 S rRNA (12Gotor C. Cejudo F.J. Barroso C. Vega J.M. Plant J. 1997; 11: 347-352Crossref PubMed Scopus (30) Google Scholar). Hybridization with CCA1 gene probe was used as a positive control of plant circadian oscillator (13Wang Z.-Y. Tobin E.M. Cell. 1998; 93: 1207-1217Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). For quantifying mRNA levels, radioactivity distributed on the blots was imaged and determined using an InstantImager electronic autoradiographer (Packard Instrument Co.). The level of Atcys-3A mRNA was normalized to the level of the 18 S rRNA obtained for each sample. Probes for in situhybridization were labeled with uridine 5′-α-thio35Striphosphate. The Atcys-3AcDNA insert (11Barroso C. Vega J.M. Gotor C. FEBS Lett. 1995; 363: 1-5Crossref PubMed Scopus (63) Google Scholar) subcloned into pBluescriptII KS was amplified by polymerase chain reaction using standard M13 reverse and forward primers. About 1 μg of the polymerase chain reaction product was used as template to synthesize 35S-labeled RNA using T7 RNA polymerase (antisense probe) or T3 RNA polymerase (sense probe). Mature Arabidopsis plants grown under standard conditions or treated with cadmium chloride (50 μm) for 18 h were used for in situ hybridization. Leaves, stems, and roots were cut into small pieces, fixed in 4% formaldehyde, embedded in wax, and sections were processed and hybridized under conditions as described previously (12Gotor C. Cejudo F.J. Barroso C. Vega J.M. Plant J. 1997; 11: 347-352Crossref PubMed Scopus (30) Google Scholar). For autoradiography, slides were coated with Amersham Pharmacia Biotech Hypercoat LM-1 nuclear emulsion and exposed for 11 days at 4 °C. The samples were then developed in Eastman Kodak Co. D19 developer prechilled at 14 °C and were fixed in 30% sodium thiosulfate for 5 min. After developing, the tissues were stained with 0.05% toluidine blue in water for 0.5 min, rinsed, dehydrated, and air-dried. For purposes of comparison, the tissue sections from control and stressed plants were fixed onto a single glass slide and hybridized with the same labeled probe. Tissue sections were observed in an Olympus BX50 microscope attached to a JVC TKC-1381 digital CCD color video camera for image capture. Images were processed and mounted by using the Olympus MicroImage analysis program and Adobe Photoshop software, respectively. For plant transformation, the β-glucuronidase gene in the binary vector pBI121 (CLONTECH), under the control of the cauliflower mosaic virus 35 S promoter, was replaced by the full-lengthAtcys-3A cDNA (11Barroso C. Vega J.M. Gotor C. FEBS Lett. 1995; 363: 1-5Crossref PubMed Scopus (63) Google Scholar). A 260-base pair fragment containing the polyadenylation signal from the nopaline synthase gene of theAgrobacterium Ti plasmid was placed downstream of theAtcys-3A gene. The resulting plasmid, named pBIOAS, was transformed into Agrobacterium tumefaciens strain CV50.A. thaliana (ecotype Columbia) was transformed by using the vacuum infiltration method described by Bechtold et al. (14Bechtold N. Ellis J. Pelletier G. C. R. Acad. Sci. ( Paris ). 1993; 316: 1194-1199Google Scholar). Transformed seeds were tested for kanamycin resistance on solid MS medium containing 50 mg l−1 kanamycin. Integration into the nuclear genome of the plant was analyzed by genomic Southern blot analysis. T5 or subsequent generations of each line were used for the experiments. Northern analysis showed a 7-fold induction ofAtcys-3A transcript level in leaves in response to cadmium chloride treatment when compared with nontreated plants (Fig.1, A and B). Although the maximum level of induction was observed 18 h after treatment, a significant increase in RNA accumulation after 1 h was observed. Concomitant with the increasing mRNA levels, OASTL enzyme activity increased by 2.5-fold after 18 h of exposure to metal (Fig. 1 C). The time course of changes inAtcys-3A transcript and OASTL enzyme activity levels could reflect a diurnal rhythm of this gene. To check this possibility, circadian experiments were performed on plants not exposed to Cd, using as a positive control the CCA1 (circadianclock-associated 1) gene (13Wang Z.-Y. Tobin E.M. Cell. 1998; 93: 1207-1217Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). We were unable to observe any fluctuation of Atcys-3A gene expression over a 60-h period, suggesting that the pattern of induction of Atcys-3A mRNA is specific to the heavy metal treatment (data not shown). To gain insight into this response, the effect of cadmium chloride onAtcys-3A expression was investigated at the tissue level byin situ hybridization. In roots, the transcript of theO-acetylserine(thiol)lyase Atcys-3A gene was localized in the cortex, but the amount of detected signal was higher in cadmium-treated plants as compared with nontreated plants (Fig.2, C and D). In stem sections, the level of expression of Atcys-3A was almost undetectable above background in control plants (Fig. 2,E and F). However, it was possible to detect signal in the cortex and the vascular tissue of cadmium-stressed plants (Fig. 2, G and H). In leaf, the basal level of expression observed in all cell types was increased throughout the leaf lamina after cadmium treatment (Fig. 2, L–O). Although thein situ hybridization technique is not a quantitative method, for purposes of comparison we hybridized the tissue sections on the same slide and therefore processed under the same conditions. The high level of Atcys-3A mRNA detected in of untreated plants (12Gotor C. Cejudo F.J. Barroso C. Vega J.M. Plant J. 1997; 11: 347-352Crossref PubMed Scopus (30) Google Scholar, G. Gotor C. M. Vega J.M. Romero L.C. Acad. Sci. S. 97: PubMed Scopus Google Scholar) was or increased in cadmium-treated Arabidopsis (data not shown). have demonstrated that cadmium induces the biosynthesis of phytochelatin peptides from GSH. To cadmium treatment induces the biosynthesis of the cysteine and we determined by HPLC analysis the content of in leaf tissues from was observed that the GSH content increased after 18 h of treatment with 50 μm cadmium and to the basal level h of treatment. However, the level of cysteine was not to the mRNA a rapid increase that after 1 h was also observed However, after exposure of higher concentrations of cadmium for a of the cysteine and GSH levels was observed. 100 μm Cd, the level of cysteine with to untreated plants, and a decrease of 30% was observed in the GSH in cysteine or GSH levels were detected at the of μm Cd used (data not shown). These results are in with the of the GSH content Cd treatment of Arabidopsis also observed by (3Xiang C. Oliver D.J. Plant Cell. 1998; 10: 1539-1550Crossref PubMed Scopus (580) Google Scholar). fluctuation of cysteine or GSH levels Cd was not observed (data not and glutathione content cadmium plants were with 50 for the indicated and the leaves were collected for cysteine and GSH as described under fresh weight of leaf, and and standard of at experiments are The significant from the at in a Arabidopsis plants were with 50 for the indicated and the leaves were collected for cysteine and GSH as described under fresh weight of leaf, and and standard of at experiments are The significant from the at We have plants overexpressing theAtcys-3A gene Atcys-3A cDNA was in to the cauliflower mosaic virus 35 S to expression of the gene. transformed tested by Southern were used for further analysis. Northern blot analysis of the transformed plants showed up to a 9-fold increase in Atcys-3A mRNA accumulation in the line compared with plants the pBI121 plasmid and The transformed plants also showed a increase in OASTL activity in leaf extracts (Fig. 3 B). We have tested the effect of the cadmium ion on the transformed plants by seeds on solid MS medium containing 250 The transformed lines the higher levels of overexpression of the Atcys-3A gene were to and on this medium (Fig. 3 C). or transformed control plants in the of the and plants that not produce leaves and after days (Fig. 3 C). The high cadmium resistance shown by the transformed lines overexpressing Atcys-3A could that cysteine availability is a for tolerance. In this we investigated exogenous addition of cysteine to pBI121 control is to the cadmium resistance observed in transformed To reaction in the medium the heavy metal and the of we used exogenous addition of which the Addition of 200 μm cystine to control plants was to growth in the of 250 μm not to the observed in the transformed lines (Fig. Cadmium analysis by atomic emission spectrometry of plants for 14 days on cadmium-containing showed an increase of cadmium accumulation in leaves compared with control plants Cadmium accumulation in different lines with mRNA and OASTL activity levels, with line the level of cadmium a higher cadmium than control in leaves of transformed Arabidopsis lines overexpressing the Arabidopsis plants were with 250 μm CdCl2 for 14 and leaves were then collected from different of the same plant line for Cd as described under are shown from The significant from the control plants. dried in a Mature Arabidopsis plants were with 250 μm CdCl2 for 14 and leaves were then collected from different of the same plant line for Cd as described under are shown from The significant from the control plants. dried different Cd resistance were In the test, transformed seeds were germinated in the of a of Cd concentrations and compared with Transformed seeds were to in the of CdCl2 up to 400 wild-type to or germinated but were unable to produce leaves, at 250 μm CdCl2 and white and at a cadmium of 400 μm In the test, we the of growth of wild-type and line Cd treatment. was to over μm concentration, wild-type showed a fresh weight of 2 mg (Fig. 5 B). The glutathione level was also determined in the transformed line and compared with the level in wild-type plants. In the of the concentrations of GSH in the transformed line and wild-type were However, treatment with 250 for 18 h produced in wild-type and the transformed a 30% decrease of the GSH content in and an increase of in the line metals such as and are highly with and a of is well that the the phytochelatins, an in the detoxification of cadmium and some heavy metals. in synthase is highly to cadmium ion R. Plant Physiol. 1995; PubMed Google Scholar). Arabidopsis plants treated with heavy metals such as cadmium and copper by increasing of genes involved in GSH synthesis and and (3Xiang C. Oliver D.J. Plant Cell. 1998; 10: 1539-1550Crossref PubMed Scopus (580) Google Scholar). increased levels of γ-glutamylcysteine synthetase and glutathione synthetase have also been observed in cadmium-treated such as maize Brunold C. Plant Physiol. PubMed Scopus Google and S. L. Bot. 1995; Scopus Google Scholar, J. J. Physiol. Plant. 1997; Google Scholar) T. Plant Mol. Biol. 1998; PubMed Scopus Google Scholar). phytochelatin synthesis a in GSH content (3Xiang C. Oliver D.J. Plant Cell. 1998; 10: 1539-1550Crossref PubMed Scopus (580) Google it is that the GSH has to be produced at higher to GSH biosynthesis under cadmium treatment. The reported show a transient increase of theO-acetylserine(thiol)lyase cadmium treatment, and this effect is observed at the transcriptional and Although the increase in OASTL activity is we have to out that these the of the three OASTL in the In situ hybridization also show that the transcript is in the leaf lamina and in the cortex and the vascular tissue of root and In changes of expression for have been observed in response to cadmium treatment T. Plant Mol. Biol. 1998; PubMed Scopus Google Scholar, S. T. Plant Mol. Biol. 1999; PubMed Scopus Google Scholar). these the that plants to cadmium by the genes required for phytochelatin synthesis, the genes involved in and in glutathione biosynthesis (3Xiang C. Oliver D.J. Plant Cell. 1998; 10: 1539-1550Crossref PubMed Scopus (580) Google Scholar). In because cadmium and heavy metals are with the reduced availability of cysteine for GSH biosynthesis and the amount of phytochelatin Addition of exogenous cystine plant growth in cadmium-containing and an for the cysteine molecule. In in transformed plants overexpressing the Atcys-3A the tolerance to cadmium ion also up to 400 μm CdCl2, in the of the line increasing the OASTL mRNA and enzyme the cysteine synthesis the transformed plants to be to the required for GSH synthesis and therefore for Although the level the of has been to be a step for cysteine we have not observed in metal tolerance by to the MS (data not shown). The effect of induction of cysteine biosynthesis in Cd resistance be observed in the transformed line high levels of OASTL mRNA and activity with a higher for Cd of this transformed line has shown an cadmium tolerance when compared with because of to to the Cd treatment with a increase of GSH The of O-acetylserine(thiol)lyase gene expression cysteine biosynthesis in transformed plants the of plants for toxic metal which be for phytoremediation of heavy of synthase in to increased tolerance to heavy but have been reported tolerance in plants S. J. 1999; PubMed Scopus Google Scholar, R. S. Plant Cell. 1999; 11: PubMed Scopus Google Scholar). be to of the synthase in Arabidopsis is to metal tolerance or is the step in the However, the step could be different in biosynthesis and not cysteine availability to be the an increase in OASTL expression is also observed on cadmium treatment T. Plant Mol. Biol. 1998; PubMed Scopus Google Scholar). of γ-glutamylcysteine synthetase in this cadmium tolerance to the same level Jouanin L. N. Plant Physiol. 1999; PubMed Scopus Google Scholar) as that reported To in cysteine availability to be of the for GSH for We L. of for of the and M. Romero of for the CCA1 gene. The of a complex a previously for of was from this The is PDF
Domínguez‐Solís et al. (Thu,) studied this question.