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Cysteine synthesis and cysteine desulfuration in Arabidopsis plants at different developmental stages and light conditions
Plant Physiol. Biochem. 39 (2001) 861−870 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942801013055/FLA
Cysteine synthesis and cysteine desulfuration in Arabidopsis plants at different developmental stages and light conditions Petra Burandt, Ahlert Schmidt, Jutta Papenbrock* Institute for Botany, University of Hannover, Herrenhäuserstr. 2, 30419 Hannover, Germany
Received 9 March 2001; accepted 29 May 2001 Abstract – The last step in cysteine biosynthesis is catalysed by the pyridoxal 5’-phosphate-dependent enzyme O-acetyl-Lserine(thiol)lyase (OAS-TL). Several isoforms are localized in different compartments of the cell. OAS-TL and OAS-TL-like proteins also catalyse the formation of β-cyanoalanine and sulfide; the release of sulfide and of an unknown product was also observed. In Arabidopsis plants of different age, the OAS-TL activity decreased with increasing age whereas the enzymatical sulfide release from cysteine increased in older plants. The β-cyanoalanine synthase showed two activity peaks during the time course investigated. During a 12-h light/12-h dark rhythm, the OAS-TL and the sulfide-releasing activities ran parallel with a maximum in the light phase and a decrease of enzyme activity in the dark phase. β-Cyanoalanine synthase activities did not follow a special pattern. Three genes coding for the OAS-TL isoforms A, B, and C, which are presumably localized in the cytoplasm, in plastids, and in mitochondria, respectively, were differentially expressed. The mRNA steady-state levels of the three proteins varied at different developmental stages. The results are discussed with respect to the physiological relevance in the plant organism. © 2001 Éditions scientifiques et médicales Elsevier SAS Arabidopsis thaliana / β-cyanoalanine / cyanide / cysteine / diurnal rhythm / senescence / sulfide Bsas, β-substituted alanine synthase / CAS, β-cyanoalanine synthase / DES, L-cysteine desulfhydrase / OAS, O-acetyl-L-serine / OAS-TL, O-acetyl-L-serine(thiol)lyase / PLP, pyridoxal 5’-phosphate
1. INTRODUCTION The pyridoxal 5’-phosphate (PLP) containing enzyme O-acetyl-L-serine(thiol)lyase (OAS-TL, EC 4.2.99.8) catalyses the formation of cysteine from O-acetyl-L-serine (OAS) and sulfide. This reaction is responsible for the incorporation of inorganic sulfur into the amino acid cysteine which can be regarded as the primary organic compound containing reduced sulfur. The cysteine pool provides the basic molecule for several subsequent pathways such as methionine and glutathione biosynthesis and a number of secondary metabolites, and also for the release of the small molecule sulfide. Because the substrate OAS is derived from the carbon and nitrogen assimilation pathways, the cysteine synthase reaction links the sulfur and nitrogen assimilatory pathways together. Cysteine synthesis depends therefore on OAS-TL activity, sulfur *Correspondence and reprints: fax + 49 511 762 3992. E-mail address: [email protected] (J. Papenbrock).
availability and OAS supplied by serine acetyltransferase (reviewed in [18]). β-Cyanoalanine synthase (CAS) belongs to the same family as OAS-TL, the β-substituted alanine synthase (Bsas) gene family. CAS catalyses the formation of β-cyanoalanine from cyanide and cysteine, producing sulfide as a product. True OAS-TL enzymes can catalyse the CAS reaction at up to 36 % the rate of cysteine synthesis and CAS can catalyse the cysteine synthesis reaction at up to 7 % the rate of β-cyanoalanine synthesis ([10, 14]; cited in [34]). Originally, we were interested in sulfide-releasing enzymes in higher plants because sulfide may be involved in sulfur-induced resistance (SIR) against pathogens [4, 30]. L-Cysteine desulfhydrase proteins catalysing the degradation of L-cysteine to pyruvate, ammonium and sulfide or to alanine and sulfide are good candidates [7, 17, 24, 36]. Because of the nature of the PLP cofactor and the specific reaction mechanism of the OAS-TL proteins, it is assumed that OAS-TL isoforms could also evolve sulfide in a side reaction [32]. Early isotopic exchange experiments
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[28] and the demonstration that the recombinant plastid OAS-TL releases sulfide in a side activity support this assumption [4]. In our line of research, we were interested in the determination of enzyme activities of OAS-TL proteins and their respective side reactions at different physiological conditions to be able to differentiate between the sulfide released by true OAS-TL proteins, by OAS-TL-like proteins or by even another group of proteins. In addition, the expression of different OAS-TL isoforms was investigated at the RNA level to prove the putative correlation to the different types of enzyme activities at least at the expression level. So far OAS-TL enzyme activities and the expression of Bsas genes was studied in different plant organs, in response to varying sulfur and nitrogen supply and to certain light conditions [1, 11, 13, 26, 35]. For the experiments two different physiological conditions were chosen. The external factor light plays an essential role in plant development, growth and reproduction. The ability of all photosynthetically active organisms to adapt to periodic changes in the environment, such as diurnal or annual rhythms, improves their viability. The timely availability of the amino acid cysteine for protein biosynthesis and metabolic pathways in the different compartments of the plant cell could be a critical factor for optimal growth and development. It was shown before that the RNA expression and activity of one key enzyme in cysteine biosynthesis, the adenosine 5’-phosphosulfate reductase (APR), is diurnally regulated. Even the three isoforms (APR1–3) showed different time courses in
their diurnal fluctuations [15]. The purpose of this work was to analyse the light regulation of cysteine synthesis and of H2S release. Plant ageing is a naturally occurring process controlled by endogenous and exogenous factors. The activities of several enzymes that are likely to play a role in the breakdown and mobilization of nutrients have been shown to increase during senescence (reviewed in [23]). A few studies document that the activity of the sulfate assimilation pathway varies in plants during development and that isoforms are differentially expressed and/or differ in their specific activities. The highest activities of enzymes involved in cysteine biosynthesis occurred in the youngest plant leaves, and the activity declined as leaves mature [25, 33]. In this work it could be demonstrated that OAS-TL and sulfide-releasing activities showed different maxima and that the OAS-TL isoforms were differentially expressed during ageing.
2. RESULTS 2.1. Enzyme activity and expression levels of H2S-releasing proteins during plant development Plant parts above ground were harvested from Arabidopsis plants every week at different developmental stages. OAS-TL, L-cysteine desulfhydrase (DES), β-cyanoalanine synthase (CAS) activities were determined in crude plant extracts (figure 1). The
Figure 1. Enzyme activity determinations during different developmental stages of Arabidopsis plants. Crude extracts of soluble proteins were prepared from Arabidopsis plants grown for 6 weeks and harvested each week. OASTL, L-cysteine desulfhydrase (DES) and β-cyanoalanine synthase (CAS) activities were determined as described in Methods. Values indicated by an asterisk are different from the control (week 1) at α = 0.05.
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specific OAS-TL activity decreased drastically with increasing plant age up to about 50 % in the oldest 6-week-old plants in comparison to the youngest 1-week-old plants (figure 1, left graph). In contrast, the L-cysteine desulfhydrase activity increased with increasing plant age. About 170 nkat·mg–1 protein sulfide were formed by the youngest plants and about 270 nkat·mg–1 protein sulfide by the oldest plants harvested (figure 1, middle). The changes in the specific CAS activity were relatively small and did not follow a general pattern (figure 1, right). The highest level was reached in the 3-week-old plants, a second maximum was measured in the oldest plants. On an average molar basis, the amounts of synthesized cysteine were about ten times higher than the sulfide released from cysteine in the enzymatical conversion by DES and CAS. As mentioned above DES and CAS activities could be side activities of OAS-TL proteins. We asked the question whether the different OAS-TL isoforms contribute to a different extent to the total OAS-TL activity and also to the side activities. Thus the RNA expression levels of the three isozymes were analysed at different developmental stages. The organellar localization of the three proteins was unambiguously demonstrated before. OAS-TL A was localized in the cytoplasm [11] whereas OAS-TL B and OAS-TL C were imported into plastids and mitochondria, respectively [13]. Each of the OAS-TL proteins is encoded by a single copy β-substituted alanine synthase (Bsas) gene as was previously demonstrated [13, 14]. In agreement with these previous results, our Southern blot analysis revealed only one single band per probe using different non-gene cutting restriction enzymes (data not shown). The Southern blot patterns of all three Bsas genes investigated differed completely from each other indicating that the sequence-specific probes did not cross-hybridize with similar DNA sequences. RNA was extracted from the same plant material used for the enzyme activity measurements shown in figure 1 at different physiological stages and hybridized with isoform-specific probes. The DNA probe labelled with digoxigenin via PCR was not sensitive enough to detect RNA coding for the OAS-TL C protein. Therefore to prepare a probe recognizing the mitochondrial OAS-TL C form, RNA was digoxigeninlabelled because RNA-RNA hybridizations are supposed to be more sensitive. However, even the sensitivity of this probe was not sufficient to detect reasonable amounts. Therefore OAS-TL cDNA was also labelled radioactively. It was very difficult to extract sufficient amounts of intact high quality RNA from the plant
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samples harvested after 6 weeks. For this reason, only RNA samples extracted from 1- to 5-week-old plants were loaded on the gel (figure 2A). OAS-TL A RNA levels were quite similar during the time course of the experiment; both OAS-TL B and OAS-TL C RNA levels increased with increasing age (figure 2A). To summarize, the three OAS-TL isoforms investigated were differentially expressed under these particular physiological conditions. RNA expression patterns give only limited information on the in vivo situation because of regulation by translational and post-translational modifications. For this reason we next examined the protein steady-state levels of OAS-TL proteins by western blotting. It is not known which isoforms are recognized by the OAS-TL specific antibody. The same amounts of total protein extracts were loaded on an SDS gel (figure 2B, lower panel). The overall amount of OAS-TL proteins decreased continuously with increasing age (figure 2B, upper panel) in positive correlation with the OAS-TL enzyme activity determinations (figure 1, left graph). In the western blot analysis using the antibody specific for proteins with CAS activity, a decrease of the CAS protein levels could be demonstrated during the time course of the experiment (figure 2B, middle panel).
2.2. Enzyme activity and expression levels of H2S-releasing proteins during a diurnal light rhythm Next we examined the enzyme activities and expression levels during the influence of light/dark switches. The OAS-TL, DES and CAS activities were determined from plants which were grown in a diurnal 12-h light/12-h dark rhythm. The three enzyme activities did not differ massively in the plants harvested during the day/night cycle (figure 3). The tendency of slightly reduced OAS-TL in agreement with former results [1] and DES activities during the period of darkness could be observed. The CAS activities did not show a clear activity pattern. A maximum of CAS enzyme activity was determined 5 h after the onset of light (figure 3, lower graph). The same samples were used for northern blot analysis to determine the expression pattern of three true OAS-TL isoforms under investigation (figure 4A). The mRNA levels for OAS-TL A, OAS-TL B and OASTL C did not change significantly in correlation to a 12-h light/12-h dark cycle (figure 4A, upper panel). Equal amounts of total protein extracts were loaded on a SDS gel (figure 4B, lower panel). On the protein level, there were only slight changes during the diurnal rhythm for both immunoreactions using the OAS-TL
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Figure 2. Expression analysis at the RNA and protein levels of different OAS-TL isoforms. A, Arabidopsis plants were grown for 5 weeks in the greenhouse and the parts above ground were harvested each week and frozen in liquid nitrogen. Total RNA was extracted, separated in a denaturing agarose gel, blotted and hybridized with probes specific for sequences encoding for OAS-TL A, OAS-TL B and OAS-TL C labelled with digoxigenin or radioactively as described in Methods. Equal RNA loading was checked by staining with ethidium bromide (box at the bottom). B, Total protein extracts were prepared from the same plant material as described in A. Ten microgram protein were loaded in each lane. Antibodies specific for OAS-TL proteins and CAS proteins were used in the western blot analysis. To control an equal protein loading per lane the relevant section (between 20 and 70 kDa) from a Coomassie-stained gel is included (panel at the bottom). The RNA levels were quantified using the TINA program by comparing the intensity of the first band (first week corresponds to 100 %) with the intensities of the other bands.
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Figure 3. Enzyme activity determinations in Arabidopsis plant extracts during a diurnal light/dark rhythm. Arabidopsis plants were grown in a 12-h light/12-h dark rhythm and the parts above ground were harvested every 4 h and frozen in liquid nitrogen. Crude extracts of soluble proteins were prepared and used for OAS-TL, L-cysteine desulfhydrase (DES) and β-cyanoalanine synthase (CAS) activities as described in Methods. Values indicated by an asterisk are different from the control (1 h) at α = 0.1.
and the CAS antibody (figure 4B, upper panel). A small increase in the light phase was followed by a small decrease at the beginning of the dark phase.
3. DISCUSSION Three enzyme assays were used to determine the in vitro activity of presumably three separate enzymes at different plant ages or at different light conditions. All
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three enzyme activities varied under the conditions chosen for the experiments. The OAS-TL activity was about ten times higher than the DES and CAS activity calculated on a molar ratio with respect to the product formation of cysteine and sulfide, respectively. The OAS-TL activity correlated in plants with increasing age in a reverse proportional way to the DES activity measured in the same plant material. One could conclude that Arabidopsis thaliana contains a distinct L-cysteine desulfhydrase protein: its activity increases with increasing plant age and it contributes massively to the overall sulfide-evolution from higher plants. Maybe the release of cysteine increases in older plants, for example because of protein degradation. Cysteine desulfuration and evolution of gaseous sulfide could be a way to avoid the accumulation of toxic levels of cysteine in the cell. This protein would also be a good candidate for our research project concerning sulfurinduced resistance (SIR) by volatile sulfur-containing compounds [4, 30]. On the other hand, the reaction mechanisms of proteins containing PLP as a cofactor are rather complicated and under certain conditions by-products might be synthesized. The PLP-containing OAS-TL enzyme catalyses the substitution of acetate in the side chain of OAS by sulfide to give L-cysteine in a β-replacement reaction. The reaction can be divided into two. First, the amino acid substrate binds to the enzyme in a manner in which the PLP is in Schiff base linkage with the ε-amino group of an active-site lysine residue; transamination leads via several intermediates and the elimination of the β-substituent to α-aminoacrylate in a Schiff base linkage with PLP. Second half of the reaction is the reverse of the first half. Thus, OAS-TL proteins usually function in the organism in a ping-pong reaction mechanism (reviewed in [32]). However, under certain conditions, e.g. at higher pH-values, and also depending on the concentrations of the respective co-substrates or reaction products, the full reaction circle could arrest after the first half of the reaction. It was proposed before that after the evolution of sulfide from cysteine measured in the DES assay, another molecule of cysteine or another thiol might react with the C2-carbon of α-aminoacrylate leading finally to the formation of a thioester [32]. Again the physiological conditions in older leaves could promote the side reaction of OAS-TL proteins. However, the exact reaction mechanism of the side reaction of OAS-TL in plants needs to be elucidated in the future using analytical HPLC/MS methods and steady-state fluorescence studies. Preliminary results using OAS-TL antisense plants indi
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Figure 4. Expression analysis at the RNA and protein levels of different OAS-TL isoforms. A, Arabidopsis plants were grown in a 12-h light/12-h dark rhythm and the parts above ground were harvested every 4 h and frozen in liquid nitrogen. Total RNA was extracted, separated in a denaturing agarose gel, blotted and hybridized with probes specific for sequences encoding for OAS-TL A, OAS-TL B and OAS-TL C labelled with digoxigenin or radioactively as described in Methods. Equal RNA loading was checked by staining with ethidium bromide (box at the bottom). B, Total protein extracts were prepared from the same plant material as described in A. Ten microgram protein were loaded in each lane. Antibodies specific for OAS-TL proteins and CAS proteins were used in the western blot analysis. To control an equal protein loading per lane the relevant section (between about 15 and 65 kDa) from a Coomassie-stained gel is included (panel at the bottom). The RNA levels were quantified using the TINA program by comparing the intensity of the first band (1 h after illumination started) which corresponds to 100 % with the intensities of the other bands.
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cate that both the OAS-TL and the DES activities are reduced in these transgenic plants in comparison to wild-type plants (Burandt, Hesse, Papenbrock, unpubl. results). In general, CAS proteins are supposed to be involved in cyanide detoxification. High CAS activities were found in tissues of cyanogenic and non-cyanogenic species and were correlated with cyanide production and ethylene production [9]. This plant hormone is synthesized in a wide range of developmental processes in plants, such as growth, senescence and ripening [9]. In addition to the mitochondrial localized CAS, true OAS-TL proteins could also act as CAS proteins under certain conditions [10, 14, 34]. True OAS-TL proteins and OAS-TL-like/CAS proteins differ in their affinity for their respective substrates. CAS possesses a high affinity for cyanide at the expense of the affinity for OAS; as a side effect, the protein has a low affinity for cysteine. The reverse situation was demonstrated for true OAS-TL proteins whereas the affinity for sulfide is only mildly affected because of its different nature compared to cyanide [14]. Significant cysteine synthesis is thought to occur in mitochondria which account for about 14 % of the overall OAS-TL activity at least in spinach leaf tissue [19]. Therefore, cysteine could be available in concentrations high enough to allow the detoxification of incoming cyanide by CAS, despite the low affinity of this enzyme for cysteine. In our experiments, the overall changes between the specific CAS activities in younger and older plants were rather small. Obviously, relatively high levels of activity are constitutively available in plants to be able to detoxify evolving cyanide as fast as possible. It was shown before that in incubation experiments with cyanide, the levels of cyanide inside the cell increased about 10-fold whereas the CAS activity was only two times higher in comparison to control leaves [9]. In addition, other enzymes might be involved in cyanide detoxification. The compartmentalization in cells leads to specific physiological conditions inside the compartments. The chemical environment of organelles, next to a lot of other factors not mentioned, might influence isozyme activities and their side activities drastically. It is almost impossible to isolate pure intact mitochondria from older green Arabidopsis thaliana plants for measuring enzyme activity (Papenbrock, unpubl. results). For this reason, only the expression of OAS-TL isozymes was followed during ageing. We are aware that RNA and protein expression do not necessarily need to correlate with each other [22]. RNA levels
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coding for OAS-TL B and C increased in older plants whereas protein and activity levels decreased. Therefore, it was impossible to correlate the expression of at least one isoform with one of the three enzyme activities determined. In the diurnal rhythm experiments, the expression of the OAS-TL isoforms did not change significantly during the light and dark periods. In similar experiments published previously [13], the expression of the OAS-TL A isoform decreased when the plants were placed in the dark. However, the experimental design differed. To control the conditions chosen for our experiments, the expression of two genes, Lhc and Chl H, which are induced by light and repressed in the dark [22] was followed. They showed a typical diurnal expression pattern (data not shown). To check the expression of CAS, a cDNA probe specific for the CAS sequence should be chosen in a future experiment for a correlation with CAS enzyme activity. The differential expression of OAS-TL isoforms was investigated under several conditions in plant species. Differences in their expression, mainly of the cytosolic form, were demonstrated within the plant organs [1, 11], during sulfur starvation [1, 20], under stress conditions [35], under prolonged darkness [13, 20] or continuous light [11] to mention only a small number of experiments. One has to conclude that the Bsas genes are not mainly regulated at the transcriptional level. On the other hand in each of the expression experiments carried out, a maximum of three isoforms was investigated. High through-put analysis, like microarrays including all known Bsas genes, might help clarify the results. Also the amounts of proteins recognized by the antibodies did not correlate with enzyme activities. Obviously, post-translational modifications play an important role for the regulation of OAS-TL and CAS activities. One has to bear in mind that the cysteine synthesis reaction is also regulated by a complex formation of OAS-TL and serine acetyltransferase. Only the free OAS-TL is responsible for cysteine synthesis and functions as a regulatory subunit of serine acetyltransferase. In chloroplasts, the ratio between OAS-TL and serine acetyltransferase is about 300 to 1 and the majority of the OAS-TL is in the free form. The complex formation seems to be controlled by sulfide which promotes complex formation and OAS which disrupts it. Finally, the availability of OAS regulates cysteine synthesis. The enzymes of the complex are also localized in the cytosol and mitochondria, where their ratios differ markedly from that in chloroplasts [6] which makes the overall regulation even more
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difficult. Additionally, serine acetyltransferase isoforms should be included in microarray expression analysis. In the search for sulfide-releasing enzymes which might be involved in resistance against pathogen attack, true OAS-TL proteins might play an important role and their side reaction probably contributes significantly to the overall sulfide released from higher plants.
4. METHODS 4.1. Growth and harvest of plants Seeds of Arabidopsis thaliana, ecotype Columbia, were originally obtained from the Arabidopsis stock centre at the Ohio State University. Seeds were germinated on substrate TKS1; after 10 d, seedlings were transplanted in TKS2 substrate into pots (Floragard, Germany). After additional 10 d on soil for recovery, all plant parts above ground were harvested every week for 6 weeks (weeks 1–6) at 9:00 hours, 3 h after the additional light has been switched on, and were frozen in liquid nitrogen to follow the expression at different developmental stages. Conditions in the greenhouse were a 16-h light/8-h dark rhythm at temperatures of 23/21 °C. When necessary, additional light was switched on for 16 h per day to obtain a constant quantum fluence rate of 300 µmol·m–2·s–1 (sodium vapour lamps, SON-T Agro 400, Philips). To investigate the influence of light and darkness on expression and activity, 2-week-old plants were grown in a 12-h light/12-h dark rhythm in a growth chamber at a constant quantum fluence rate of 100 µmol·m–2·s–1. To follow one complete diurnal rhythm, plant parts above ground were harvested every 4 h for nearly one and a half day starting 1 hour after the onset of light and immediately frozen in liquid nitrogen.
4.2. RNA extraction and northern blotting Total RNA was extracted essentially as described previously [5]. Samples containing 15 µg RNA were separated on 1 % agarose-formaldehyde gels. Equal loading was controlled by staining the gels with ethidium bromide. After RNA transfer onto nylon membranes, filters were probed with cDNA probes labelled by PCR with digoxigenin using the PCR DIG probe synthesis kit (Roche). For the PCR amplification, the cDNA clones named pCS-A, pCS-B and pCS-C were used as templates [12, 13] with the following primer pairs: the cytosolic form (972 bp) with primer 44 (5’-GGATCCATGGCCTCGAGAATT-
3’) and primer 45 (5’-GTCGACTCAAGGCCTTGAAGG-3’), the plastid form (996 bp) with primer 17 (5’-GCGGATCCGCTGTATCTATCAA-3’) and primer 18 (5’-TATGTCGACTCAAAGCTCGGGCTG-3’) and the mitochondrial form (1 188 bp) with primer 36 (5’-GGATCCATGGCCGCCACATCTT-3’) and primer 37 (5’-GGTACCTCATACCTCAGGCTGA-3’). To prepare a more sensitive probe homologous to the mitochondrial form OAS-TL C, RNA was labelled using the DIG SP6/T7 RNA labelling kit (Roche). In addition, a radioactive labelled OAS-TL cDNA probe was prepared according to Papenbrock et al. [22] and used for hybridization. Hybridization was carried out as described previously [21].
4.3. SDS-PAGE and western blotting For the determination of OAS-TL protein steadystate levels in plants, 100 mg plant material was mortared to a fine powder in liquid nitrogen. Sample buffer (56 mM Na2CO3, 56 mM DTT, 2 % SDS, 12 % sucrose, 2 mM EDTA, 500 µL) was added, samples were incubated for 20 min at 95 °C and centrifuged. About 10 µg total protein supernatant was subjected to SDS/PAGE [16] and blotted [27]. Antibodies directed against purified spinach OAS-TL and purified spinach CAS [29] were used for the immunodetection. It is not known which isoforms were used for raising the antibodies because the N-terminal amino acid sequences of the purified proteins used for immunization were not determined. A colorimetric detection method using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3indolyl-phosphate (BCIP) was applied.
4.4. Enzyme activity measurements Enzyme activities were measured with crude plant extracts. Frozen plant material was mortared in 20 mM Tris-HCl (pH 8.0), in a ratio of 1:5 (w/w, 100 mg plant material plus 400 µL buffer). OAS-TL (EC 4.2.99.8) and L-cysteine desulfhydrase (EC 4.4.1.1 or EC 4.2.1.15) activities were measured as described [4, 29] using the method after Gaitonde [8] and Siegel [31], respectively. β-Cyanoalanine synthase (EC 4.4.1.9) activity was measured by the release of sulfide from cysteine in the presence of KCN (modified from Blumenthal et al. [2]). The assay contained in a total volume of 1 mL: 0.8 mM L-cysteine, 10 mM KCN, and 100 mM Tris-HCl (pH 9.0). The reaction was initiated by the addition of L-cysteine. After incubation for 15 min at 30 °C, the reaction was terminated by adding 100 µL 30 mM FeCl3 dissolved in 1.2 N HCl and 100 µL 20 mM N,N-dimethyl-p-phenylene
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diamine dihydrochloride dissolved in 7.2 N HCl [31]. The formation of methylene blue was determined at 670 nm in a spectrophotometer and quantified using a Na2S standard curve.
[7]
4.5. Miscellaneous Protein estimation was done according to Bradford [3] using bovine serum albumin as a protein standard. Both harvesting experiments were repeated twice independently. Each type of analysis was done in triplicate with the same frozen plant material. In addition each data point of enzyme activity determination was obtained by duplicates. For the enzyme activity graphs, the six replications were combined. Statistical analysis was performed using the Student method (SigmaPlot for Windows version 6.00). Representative northern and western blots are shown. The quantification of the RNA levels was done using TINA (version 2.09g).
Acknowledgments. The expert technical assistance of P. von Trzebiatowski and Julia Volker is gratefully acknowledged. Many thanks to our gardeners for caring for the plants. We would like to thank Dr H. Hesse for giving us pCS-A, pCS-B and pCS-C cDNA clones. This work was financially supported by the Deutsche Forschungsgemeinschaft (PA 764/2-1, SCHM 307/15-1).
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responses to sulfur, nitrogen and light, Gene 229 (1999) 155–161. Papenbrock J., Schmidt A., Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana, Eur. J. Biochem. 267 (2000) 5571–5579. Papenbrock J., Mock H.P., Kruse E., Grimm B., Expression studies in tetrapyrrole biosynthesis: inverse maxima of magnesium chelatase and ferrochelatase activity during cycling photoperiods, Planta 208 (1999) 264–273. Quirino B.F., Noh Y.S., Himelblau E., Amasino R.M., Molecular aspects of leaf senescence, Trends Plant Sci. 5 (2000) 278–282. Rennenberg H., Arabatzis N., Grundel I., Cysteine desulphydrase activity in higher plants: evidence for the action of L- and D-cysteine specific enzymes, Phytochemistry 26 (1987) 1583–1589. Rotte C., Leustek T., Differential subcellular localization and expression of ATP sulfurylase and 5’-adenylylsulfate reductase during ontogenesis of Arabidopsis leaves indicates that cytosolic and plastid forms of ATP sulfurylase may have specialized functions, Plant Physiol. 124 (2000) 715–724. Saito K., Tatsuguchi K., Takagi Y., Murakoshi I., Isolation and characterization of cDNA that encodes a putative mitochondrion-localizing isoform of cysteine synthase (O-acetylserine(thiol)-lyase) from Spinacia oleracea, J. Biol. Chem. 269 (1994) 28187–28192. Sambrook J., Fritsch E.F., Maniatis T., Molecular Cloning: A laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
[28] Schmidt A., Exchange of cysteine-bound sulfide with free sulfide and cysteine synthase activity in Chlorella pyrenoidosa, Z. Pflanzenphys. 84 (1977) 435–446. [29] Schmidt A., Sulphur metabolism. D. Cysteine synthase, in: Lea P.J. (Ed.), Methods in Plant Biochemistry, Academic Press, London, 1990, pp. 349–354. [30] Schnug E., Booth E., Haneklaus S., Walker K.C., Sulphur supply and stress resistance in oilseed rape, Proc. 9th Int. Rapeseed Congress, Cambridge, 1995, pp. 229–231. [31] Siegel M., A direct microdetermination for sulfide, Anal. Biochem. 11 (1965) 126–132. [32] Tai C.H., Cook P.F., O-Acetylserine sulfhydrylase, Adv. Enzymol. Relat. Areas Mol. Biol. 74 (2000) 185–234. [33] von Arb C., Brunold C., Enzymes of assimilatory sulfate reduction in leaves of Pisum sativum: changes during ontogeny and in vivo regulation by H2S and cysteine, Physiol. Plant. 67 (1986) 81–86. [34] Warrilow A.G., Hawkesford M.J., Separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach, J. Exp. Bot. 49 (1998) 1625–1636. [35] Yamaguchi Y., Nakamura T., Kusano T., Sano H., Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and β-cyanoalanine synthase, Plant Cell Physiol. 41 (2000) 465–476. [36] Zheng L., White R.H., Cash V.L., Jack R.F., Dean D.R., Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis, Proc. Natl. Acad. Sci. USA 90 (1993) 2754–2758.
Cysteine synthesis and cysteine desulfuration in Arabidopsis plants at different developmental stages and light conditions Petra Burandt, Ahlert Schmidt, Jutta Papenbrock* Institute for Botany, University of Hannover, Herrenhäuserstr. 2, 30419 Hannover, Germany
Received 9 March 2001; accepted 29 May 2001 Abstract – The last step in cysteine biosynthesis is catalysed by the pyridoxal 5’-phosphate-dependent enzyme O-acetyl-Lserine(thiol)lyase (OAS-TL). Several isoforms are localized in different compartments of the cell. OAS-TL and OAS-TL-like proteins also catalyse the formation of β-cyanoalanine and sulfide; the release of sulfide and of an unknown product was also observed. In Arabidopsis plants of different age, the OAS-TL activity decreased with increasing age whereas the enzymatical sulfide release from cysteine increased in older plants. The β-cyanoalanine synthase showed two activity peaks during the time course investigated. During a 12-h light/12-h dark rhythm, the OAS-TL and the sulfide-releasing activities ran parallel with a maximum in the light phase and a decrease of enzyme activity in the dark phase. β-Cyanoalanine synthase activities did not follow a special pattern. Three genes coding for the OAS-TL isoforms A, B, and C, which are presumably localized in the cytoplasm, in plastids, and in mitochondria, respectively, were differentially expressed. The mRNA steady-state levels of the three proteins varied at different developmental stages. The results are discussed with respect to the physiological relevance in the plant organism. © 2001 Éditions scientifiques et médicales Elsevier SAS Arabidopsis thaliana / β-cyanoalanine / cyanide / cysteine / diurnal rhythm / senescence / sulfide Bsas, β-substituted alanine synthase / CAS, β-cyanoalanine synthase / DES, L-cysteine desulfhydrase / OAS, O-acetyl-L-serine / OAS-TL, O-acetyl-L-serine(thiol)lyase / PLP, pyridoxal 5’-phosphate
1. INTRODUCTION The pyridoxal 5’-phosphate (PLP) containing enzyme O-acetyl-L-serine(thiol)lyase (OAS-TL, EC 4.2.99.8) catalyses the formation of cysteine from O-acetyl-L-serine (OAS) and sulfide. This reaction is responsible for the incorporation of inorganic sulfur into the amino acid cysteine which can be regarded as the primary organic compound containing reduced sulfur. The cysteine pool provides the basic molecule for several subsequent pathways such as methionine and glutathione biosynthesis and a number of secondary metabolites, and also for the release of the small molecule sulfide. Because the substrate OAS is derived from the carbon and nitrogen assimilation pathways, the cysteine synthase reaction links the sulfur and nitrogen assimilatory pathways together. Cysteine synthesis depends therefore on OAS-TL activity, sulfur *Correspondence and reprints: fax + 49 511 762 3992. E-mail address: [email protected] (J. Papenbrock).
availability and OAS supplied by serine acetyltransferase (reviewed in [18]). β-Cyanoalanine synthase (CAS) belongs to the same family as OAS-TL, the β-substituted alanine synthase (Bsas) gene family. CAS catalyses the formation of β-cyanoalanine from cyanide and cysteine, producing sulfide as a product. True OAS-TL enzymes can catalyse the CAS reaction at up to 36 % the rate of cysteine synthesis and CAS can catalyse the cysteine synthesis reaction at up to 7 % the rate of β-cyanoalanine synthesis ([10, 14]; cited in [34]). Originally, we were interested in sulfide-releasing enzymes in higher plants because sulfide may be involved in sulfur-induced resistance (SIR) against pathogens [4, 30]. L-Cysteine desulfhydrase proteins catalysing the degradation of L-cysteine to pyruvate, ammonium and sulfide or to alanine and sulfide are good candidates [7, 17, 24, 36]. Because of the nature of the PLP cofactor and the specific reaction mechanism of the OAS-TL proteins, it is assumed that OAS-TL isoforms could also evolve sulfide in a side reaction [32]. Early isotopic exchange experiments
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[28] and the demonstration that the recombinant plastid OAS-TL releases sulfide in a side activity support this assumption [4]. In our line of research, we were interested in the determination of enzyme activities of OAS-TL proteins and their respective side reactions at different physiological conditions to be able to differentiate between the sulfide released by true OAS-TL proteins, by OAS-TL-like proteins or by even another group of proteins. In addition, the expression of different OAS-TL isoforms was investigated at the RNA level to prove the putative correlation to the different types of enzyme activities at least at the expression level. So far OAS-TL enzyme activities and the expression of Bsas genes was studied in different plant organs, in response to varying sulfur and nitrogen supply and to certain light conditions [1, 11, 13, 26, 35]. For the experiments two different physiological conditions were chosen. The external factor light plays an essential role in plant development, growth and reproduction. The ability of all photosynthetically active organisms to adapt to periodic changes in the environment, such as diurnal or annual rhythms, improves their viability. The timely availability of the amino acid cysteine for protein biosynthesis and metabolic pathways in the different compartments of the plant cell could be a critical factor for optimal growth and development. It was shown before that the RNA expression and activity of one key enzyme in cysteine biosynthesis, the adenosine 5’-phosphosulfate reductase (APR), is diurnally regulated. Even the three isoforms (APR1–3) showed different time courses in
their diurnal fluctuations [15]. The purpose of this work was to analyse the light regulation of cysteine synthesis and of H2S release. Plant ageing is a naturally occurring process controlled by endogenous and exogenous factors. The activities of several enzymes that are likely to play a role in the breakdown and mobilization of nutrients have been shown to increase during senescence (reviewed in [23]). A few studies document that the activity of the sulfate assimilation pathway varies in plants during development and that isoforms are differentially expressed and/or differ in their specific activities. The highest activities of enzymes involved in cysteine biosynthesis occurred in the youngest plant leaves, and the activity declined as leaves mature [25, 33]. In this work it could be demonstrated that OAS-TL and sulfide-releasing activities showed different maxima and that the OAS-TL isoforms were differentially expressed during ageing.
2. RESULTS 2.1. Enzyme activity and expression levels of H2S-releasing proteins during plant development Plant parts above ground were harvested from Arabidopsis plants every week at different developmental stages. OAS-TL, L-cysteine desulfhydrase (DES), β-cyanoalanine synthase (CAS) activities were determined in crude plant extracts (figure 1). The
Figure 1. Enzyme activity determinations during different developmental stages of Arabidopsis plants. Crude extracts of soluble proteins were prepared from Arabidopsis plants grown for 6 weeks and harvested each week. OASTL, L-cysteine desulfhydrase (DES) and β-cyanoalanine synthase (CAS) activities were determined as described in Methods. Values indicated by an asterisk are different from the control (week 1) at α = 0.05.
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specific OAS-TL activity decreased drastically with increasing plant age up to about 50 % in the oldest 6-week-old plants in comparison to the youngest 1-week-old plants (figure 1, left graph). In contrast, the L-cysteine desulfhydrase activity increased with increasing plant age. About 170 nkat·mg–1 protein sulfide were formed by the youngest plants and about 270 nkat·mg–1 protein sulfide by the oldest plants harvested (figure 1, middle). The changes in the specific CAS activity were relatively small and did not follow a general pattern (figure 1, right). The highest level was reached in the 3-week-old plants, a second maximum was measured in the oldest plants. On an average molar basis, the amounts of synthesized cysteine were about ten times higher than the sulfide released from cysteine in the enzymatical conversion by DES and CAS. As mentioned above DES and CAS activities could be side activities of OAS-TL proteins. We asked the question whether the different OAS-TL isoforms contribute to a different extent to the total OAS-TL activity and also to the side activities. Thus the RNA expression levels of the three isozymes were analysed at different developmental stages. The organellar localization of the three proteins was unambiguously demonstrated before. OAS-TL A was localized in the cytoplasm [11] whereas OAS-TL B and OAS-TL C were imported into plastids and mitochondria, respectively [13]. Each of the OAS-TL proteins is encoded by a single copy β-substituted alanine synthase (Bsas) gene as was previously demonstrated [13, 14]. In agreement with these previous results, our Southern blot analysis revealed only one single band per probe using different non-gene cutting restriction enzymes (data not shown). The Southern blot patterns of all three Bsas genes investigated differed completely from each other indicating that the sequence-specific probes did not cross-hybridize with similar DNA sequences. RNA was extracted from the same plant material used for the enzyme activity measurements shown in figure 1 at different physiological stages and hybridized with isoform-specific probes. The DNA probe labelled with digoxigenin via PCR was not sensitive enough to detect RNA coding for the OAS-TL C protein. Therefore to prepare a probe recognizing the mitochondrial OAS-TL C form, RNA was digoxigeninlabelled because RNA-RNA hybridizations are supposed to be more sensitive. However, even the sensitivity of this probe was not sufficient to detect reasonable amounts. Therefore OAS-TL cDNA was also labelled radioactively. It was very difficult to extract sufficient amounts of intact high quality RNA from the plant
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samples harvested after 6 weeks. For this reason, only RNA samples extracted from 1- to 5-week-old plants were loaded on the gel (figure 2A). OAS-TL A RNA levels were quite similar during the time course of the experiment; both OAS-TL B and OAS-TL C RNA levels increased with increasing age (figure 2A). To summarize, the three OAS-TL isoforms investigated were differentially expressed under these particular physiological conditions. RNA expression patterns give only limited information on the in vivo situation because of regulation by translational and post-translational modifications. For this reason we next examined the protein steady-state levels of OAS-TL proteins by western blotting. It is not known which isoforms are recognized by the OAS-TL specific antibody. The same amounts of total protein extracts were loaded on an SDS gel (figure 2B, lower panel). The overall amount of OAS-TL proteins decreased continuously with increasing age (figure 2B, upper panel) in positive correlation with the OAS-TL enzyme activity determinations (figure 1, left graph). In the western blot analysis using the antibody specific for proteins with CAS activity, a decrease of the CAS protein levels could be demonstrated during the time course of the experiment (figure 2B, middle panel).
2.2. Enzyme activity and expression levels of H2S-releasing proteins during a diurnal light rhythm Next we examined the enzyme activities and expression levels during the influence of light/dark switches. The OAS-TL, DES and CAS activities were determined from plants which were grown in a diurnal 12-h light/12-h dark rhythm. The three enzyme activities did not differ massively in the plants harvested during the day/night cycle (figure 3). The tendency of slightly reduced OAS-TL in agreement with former results [1] and DES activities during the period of darkness could be observed. The CAS activities did not show a clear activity pattern. A maximum of CAS enzyme activity was determined 5 h after the onset of light (figure 3, lower graph). The same samples were used for northern blot analysis to determine the expression pattern of three true OAS-TL isoforms under investigation (figure 4A). The mRNA levels for OAS-TL A, OAS-TL B and OASTL C did not change significantly in correlation to a 12-h light/12-h dark cycle (figure 4A, upper panel). Equal amounts of total protein extracts were loaded on a SDS gel (figure 4B, lower panel). On the protein level, there were only slight changes during the diurnal rhythm for both immunoreactions using the OAS-TL
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Figure 2. Expression analysis at the RNA and protein levels of different OAS-TL isoforms. A, Arabidopsis plants were grown for 5 weeks in the greenhouse and the parts above ground were harvested each week and frozen in liquid nitrogen. Total RNA was extracted, separated in a denaturing agarose gel, blotted and hybridized with probes specific for sequences encoding for OAS-TL A, OAS-TL B and OAS-TL C labelled with digoxigenin or radioactively as described in Methods. Equal RNA loading was checked by staining with ethidium bromide (box at the bottom). B, Total protein extracts were prepared from the same plant material as described in A. Ten microgram protein were loaded in each lane. Antibodies specific for OAS-TL proteins and CAS proteins were used in the western blot analysis. To control an equal protein loading per lane the relevant section (between 20 and 70 kDa) from a Coomassie-stained gel is included (panel at the bottom). The RNA levels were quantified using the TINA program by comparing the intensity of the first band (first week corresponds to 100 %) with the intensities of the other bands.
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Figure 3. Enzyme activity determinations in Arabidopsis plant extracts during a diurnal light/dark rhythm. Arabidopsis plants were grown in a 12-h light/12-h dark rhythm and the parts above ground were harvested every 4 h and frozen in liquid nitrogen. Crude extracts of soluble proteins were prepared and used for OAS-TL, L-cysteine desulfhydrase (DES) and β-cyanoalanine synthase (CAS) activities as described in Methods. Values indicated by an asterisk are different from the control (1 h) at α = 0.1.
and the CAS antibody (figure 4B, upper panel). A small increase in the light phase was followed by a small decrease at the beginning of the dark phase.
3. DISCUSSION Three enzyme assays were used to determine the in vitro activity of presumably three separate enzymes at different plant ages or at different light conditions. All
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three enzyme activities varied under the conditions chosen for the experiments. The OAS-TL activity was about ten times higher than the DES and CAS activity calculated on a molar ratio with respect to the product formation of cysteine and sulfide, respectively. The OAS-TL activity correlated in plants with increasing age in a reverse proportional way to the DES activity measured in the same plant material. One could conclude that Arabidopsis thaliana contains a distinct L-cysteine desulfhydrase protein: its activity increases with increasing plant age and it contributes massively to the overall sulfide-evolution from higher plants. Maybe the release of cysteine increases in older plants, for example because of protein degradation. Cysteine desulfuration and evolution of gaseous sulfide could be a way to avoid the accumulation of toxic levels of cysteine in the cell. This protein would also be a good candidate for our research project concerning sulfurinduced resistance (SIR) by volatile sulfur-containing compounds [4, 30]. On the other hand, the reaction mechanisms of proteins containing PLP as a cofactor are rather complicated and under certain conditions by-products might be synthesized. The PLP-containing OAS-TL enzyme catalyses the substitution of acetate in the side chain of OAS by sulfide to give L-cysteine in a β-replacement reaction. The reaction can be divided into two. First, the amino acid substrate binds to the enzyme in a manner in which the PLP is in Schiff base linkage with the ε-amino group of an active-site lysine residue; transamination leads via several intermediates and the elimination of the β-substituent to α-aminoacrylate in a Schiff base linkage with PLP. Second half of the reaction is the reverse of the first half. Thus, OAS-TL proteins usually function in the organism in a ping-pong reaction mechanism (reviewed in [32]). However, under certain conditions, e.g. at higher pH-values, and also depending on the concentrations of the respective co-substrates or reaction products, the full reaction circle could arrest after the first half of the reaction. It was proposed before that after the evolution of sulfide from cysteine measured in the DES assay, another molecule of cysteine or another thiol might react with the C2-carbon of α-aminoacrylate leading finally to the formation of a thioester [32]. Again the physiological conditions in older leaves could promote the side reaction of OAS-TL proteins. However, the exact reaction mechanism of the side reaction of OAS-TL in plants needs to be elucidated in the future using analytical HPLC/MS methods and steady-state fluorescence studies. Preliminary results using OAS-TL antisense plants indi
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Figure 4. Expression analysis at the RNA and protein levels of different OAS-TL isoforms. A, Arabidopsis plants were grown in a 12-h light/12-h dark rhythm and the parts above ground were harvested every 4 h and frozen in liquid nitrogen. Total RNA was extracted, separated in a denaturing agarose gel, blotted and hybridized with probes specific for sequences encoding for OAS-TL A, OAS-TL B and OAS-TL C labelled with digoxigenin or radioactively as described in Methods. Equal RNA loading was checked by staining with ethidium bromide (box at the bottom). B, Total protein extracts were prepared from the same plant material as described in A. Ten microgram protein were loaded in each lane. Antibodies specific for OAS-TL proteins and CAS proteins were used in the western blot analysis. To control an equal protein loading per lane the relevant section (between about 15 and 65 kDa) from a Coomassie-stained gel is included (panel at the bottom). The RNA levels were quantified using the TINA program by comparing the intensity of the first band (1 h after illumination started) which corresponds to 100 % with the intensities of the other bands.
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cate that both the OAS-TL and the DES activities are reduced in these transgenic plants in comparison to wild-type plants (Burandt, Hesse, Papenbrock, unpubl. results). In general, CAS proteins are supposed to be involved in cyanide detoxification. High CAS activities were found in tissues of cyanogenic and non-cyanogenic species and were correlated with cyanide production and ethylene production [9]. This plant hormone is synthesized in a wide range of developmental processes in plants, such as growth, senescence and ripening [9]. In addition to the mitochondrial localized CAS, true OAS-TL proteins could also act as CAS proteins under certain conditions [10, 14, 34]. True OAS-TL proteins and OAS-TL-like/CAS proteins differ in their affinity for their respective substrates. CAS possesses a high affinity for cyanide at the expense of the affinity for OAS; as a side effect, the protein has a low affinity for cysteine. The reverse situation was demonstrated for true OAS-TL proteins whereas the affinity for sulfide is only mildly affected because of its different nature compared to cyanide [14]. Significant cysteine synthesis is thought to occur in mitochondria which account for about 14 % of the overall OAS-TL activity at least in spinach leaf tissue [19]. Therefore, cysteine could be available in concentrations high enough to allow the detoxification of incoming cyanide by CAS, despite the low affinity of this enzyme for cysteine. In our experiments, the overall changes between the specific CAS activities in younger and older plants were rather small. Obviously, relatively high levels of activity are constitutively available in plants to be able to detoxify evolving cyanide as fast as possible. It was shown before that in incubation experiments with cyanide, the levels of cyanide inside the cell increased about 10-fold whereas the CAS activity was only two times higher in comparison to control leaves [9]. In addition, other enzymes might be involved in cyanide detoxification. The compartmentalization in cells leads to specific physiological conditions inside the compartments. The chemical environment of organelles, next to a lot of other factors not mentioned, might influence isozyme activities and their side activities drastically. It is almost impossible to isolate pure intact mitochondria from older green Arabidopsis thaliana plants for measuring enzyme activity (Papenbrock, unpubl. results). For this reason, only the expression of OAS-TL isozymes was followed during ageing. We are aware that RNA and protein expression do not necessarily need to correlate with each other [22]. RNA levels
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coding for OAS-TL B and C increased in older plants whereas protein and activity levels decreased. Therefore, it was impossible to correlate the expression of at least one isoform with one of the three enzyme activities determined. In the diurnal rhythm experiments, the expression of the OAS-TL isoforms did not change significantly during the light and dark periods. In similar experiments published previously [13], the expression of the OAS-TL A isoform decreased when the plants were placed in the dark. However, the experimental design differed. To control the conditions chosen for our experiments, the expression of two genes, Lhc and Chl H, which are induced by light and repressed in the dark [22] was followed. They showed a typical diurnal expression pattern (data not shown). To check the expression of CAS, a cDNA probe specific for the CAS sequence should be chosen in a future experiment for a correlation with CAS enzyme activity. The differential expression of OAS-TL isoforms was investigated under several conditions in plant species. Differences in their expression, mainly of the cytosolic form, were demonstrated within the plant organs [1, 11], during sulfur starvation [1, 20], under stress conditions [35], under prolonged darkness [13, 20] or continuous light [11] to mention only a small number of experiments. One has to conclude that the Bsas genes are not mainly regulated at the transcriptional level. On the other hand in each of the expression experiments carried out, a maximum of three isoforms was investigated. High through-put analysis, like microarrays including all known Bsas genes, might help clarify the results. Also the amounts of proteins recognized by the antibodies did not correlate with enzyme activities. Obviously, post-translational modifications play an important role for the regulation of OAS-TL and CAS activities. One has to bear in mind that the cysteine synthesis reaction is also regulated by a complex formation of OAS-TL and serine acetyltransferase. Only the free OAS-TL is responsible for cysteine synthesis and functions as a regulatory subunit of serine acetyltransferase. In chloroplasts, the ratio between OAS-TL and serine acetyltransferase is about 300 to 1 and the majority of the OAS-TL is in the free form. The complex formation seems to be controlled by sulfide which promotes complex formation and OAS which disrupts it. Finally, the availability of OAS regulates cysteine synthesis. The enzymes of the complex are also localized in the cytosol and mitochondria, where their ratios differ markedly from that in chloroplasts [6] which makes the overall regulation even more
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difficult. Additionally, serine acetyltransferase isoforms should be included in microarray expression analysis. In the search for sulfide-releasing enzymes which might be involved in resistance against pathogen attack, true OAS-TL proteins might play an important role and their side reaction probably contributes significantly to the overall sulfide released from higher plants.
4. METHODS 4.1. Growth and harvest of plants Seeds of Arabidopsis thaliana, ecotype Columbia, were originally obtained from the Arabidopsis stock centre at the Ohio State University. Seeds were germinated on substrate TKS1; after 10 d, seedlings were transplanted in TKS2 substrate into pots (Floragard, Germany). After additional 10 d on soil for recovery, all plant parts above ground were harvested every week for 6 weeks (weeks 1–6) at 9:00 hours, 3 h after the additional light has been switched on, and were frozen in liquid nitrogen to follow the expression at different developmental stages. Conditions in the greenhouse were a 16-h light/8-h dark rhythm at temperatures of 23/21 °C. When necessary, additional light was switched on for 16 h per day to obtain a constant quantum fluence rate of 300 µmol·m–2·s–1 (sodium vapour lamps, SON-T Agro 400, Philips). To investigate the influence of light and darkness on expression and activity, 2-week-old plants were grown in a 12-h light/12-h dark rhythm in a growth chamber at a constant quantum fluence rate of 100 µmol·m–2·s–1. To follow one complete diurnal rhythm, plant parts above ground were harvested every 4 h for nearly one and a half day starting 1 hour after the onset of light and immediately frozen in liquid nitrogen.
4.2. RNA extraction and northern blotting Total RNA was extracted essentially as described previously [5]. Samples containing 15 µg RNA were separated on 1 % agarose-formaldehyde gels. Equal loading was controlled by staining the gels with ethidium bromide. After RNA transfer onto nylon membranes, filters were probed with cDNA probes labelled by PCR with digoxigenin using the PCR DIG probe synthesis kit (Roche). For the PCR amplification, the cDNA clones named pCS-A, pCS-B and pCS-C were used as templates [12, 13] with the following primer pairs: the cytosolic form (972 bp) with primer 44 (5’-GGATCCATGGCCTCGAGAATT-
3’) and primer 45 (5’-GTCGACTCAAGGCCTTGAAGG-3’), the plastid form (996 bp) with primer 17 (5’-GCGGATCCGCTGTATCTATCAA-3’) and primer 18 (5’-TATGTCGACTCAAAGCTCGGGCTG-3’) and the mitochondrial form (1 188 bp) with primer 36 (5’-GGATCCATGGCCGCCACATCTT-3’) and primer 37 (5’-GGTACCTCATACCTCAGGCTGA-3’). To prepare a more sensitive probe homologous to the mitochondrial form OAS-TL C, RNA was labelled using the DIG SP6/T7 RNA labelling kit (Roche). In addition, a radioactive labelled OAS-TL cDNA probe was prepared according to Papenbrock et al. [22] and used for hybridization. Hybridization was carried out as described previously [21].
4.3. SDS-PAGE and western blotting For the determination of OAS-TL protein steadystate levels in plants, 100 mg plant material was mortared to a fine powder in liquid nitrogen. Sample buffer (56 mM Na2CO3, 56 mM DTT, 2 % SDS, 12 % sucrose, 2 mM EDTA, 500 µL) was added, samples were incubated for 20 min at 95 °C and centrifuged. About 10 µg total protein supernatant was subjected to SDS/PAGE [16] and blotted [27]. Antibodies directed against purified spinach OAS-TL and purified spinach CAS [29] were used for the immunodetection. It is not known which isoforms were used for raising the antibodies because the N-terminal amino acid sequences of the purified proteins used for immunization were not determined. A colorimetric detection method using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3indolyl-phosphate (BCIP) was applied.
4.4. Enzyme activity measurements Enzyme activities were measured with crude plant extracts. Frozen plant material was mortared in 20 mM Tris-HCl (pH 8.0), in a ratio of 1:5 (w/w, 100 mg plant material plus 400 µL buffer). OAS-TL (EC 4.2.99.8) and L-cysteine desulfhydrase (EC 4.4.1.1 or EC 4.2.1.15) activities were measured as described [4, 29] using the method after Gaitonde [8] and Siegel [31], respectively. β-Cyanoalanine synthase (EC 4.4.1.9) activity was measured by the release of sulfide from cysteine in the presence of KCN (modified from Blumenthal et al. [2]). The assay contained in a total volume of 1 mL: 0.8 mM L-cysteine, 10 mM KCN, and 100 mM Tris-HCl (pH 9.0). The reaction was initiated by the addition of L-cysteine. After incubation for 15 min at 30 °C, the reaction was terminated by adding 100 µL 30 mM FeCl3 dissolved in 1.2 N HCl and 100 µL 20 mM N,N-dimethyl-p-phenylene
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diamine dihydrochloride dissolved in 7.2 N HCl [31]. The formation of methylene blue was determined at 670 nm in a spectrophotometer and quantified using a Na2S standard curve.
[7]
4.5. Miscellaneous Protein estimation was done according to Bradford [3] using bovine serum albumin as a protein standard. Both harvesting experiments were repeated twice independently. Each type of analysis was done in triplicate with the same frozen plant material. In addition each data point of enzyme activity determination was obtained by duplicates. For the enzyme activity graphs, the six replications were combined. Statistical analysis was performed using the Student method (SigmaPlot for Windows version 6.00). Representative northern and western blots are shown. The quantification of the RNA levels was done using TINA (version 2.09g).
Acknowledgments. The expert technical assistance of P. von Trzebiatowski and Julia Volker is gratefully acknowledged. Many thanks to our gardeners for caring for the plants. We would like to thank Dr H. Hesse for giving us pCS-A, pCS-B and pCS-C cDNA clones. This work was financially supported by the Deutsche Forschungsgemeinschaft (PA 764/2-1, SCHM 307/15-1).
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