Low abundance does not mean less importance in cysteine metabolism

The cysteine molecule plays an essential role in cells because it is part of proteins and because it functions as a reduced sulfur donor molecule. In addition, the cysteine molecule may also play a role in the redox signaling of different stress processes. Even though the synthesis of cysteine by the most abundant of the isoforms of O-acetylserine(thiol) lyase in the chloroplast, the mitochondria and the cytosol is relatively well-understood, the role of the other less common isoforms homologous to O-acetylserine(thiol)lyase is unknown. Several studies on two of these isoforms, one located in the cytosol and the other one in the chloroplast, have shown that while one isoform operates with a desulfhydrase activity and is essential to regulate the homeostasis of cysteine in the cytosol, the other, located in the chloroplast, synthesizes S-sulfocysteine. This metabolite appears to be essential for the redox regulation of the chloroplast under certain lighting conditions.Key words: cysteine, S-sulfocysteine, desulfhydrase, sulfur metabolism, redox regulation, ArabidopsisCysteine occupies a central position in the plant primary and secondary metabolism due to its biochemical functions. Cysteine is the first organic compound with reduced sulfur synthesized by the plant in the photosynthetic primary sulfate assimilation. The importance of cysteine for plants derives from its role as an amino acid in proteins but also because of its functions as a precursor for a huge number of essential bio-molecules, such as many plant defense compounds formed in response to different environmental adverse conditions.^1,2 All of these bio-molecules contain sulfur moieties that act as functional groups and are derived from cysteine, and therefore, are intimately linked via their biosynthetic pathways.In addition to the final destination of the reduced sulfur atom in the primary and secondary metabolism of cells, the thiol residue of the cysteine molecule is a functional group that translates the physico-chemical signal (redox) of ROS and RNS into a functional signal, altering the properties of small molecules such as GSH or proteins whose enzymatic or functional properties depend on the redox state of its cysteine residues.³Sulfate is the major sulfur form available to plants. Sulfate is taken up to plant cells through specific sulfate transporters and is activated to adenosine 5′-phosphosulfate (APS). The reduction of the activated sulfate form, APS, is linked to plastids and the photosynthetic activity; therefore, APS is reduced to sulfite by the APS reductase using two GSH molecules as donors of the two electrons required in this step. Sulfite is further reduced to sulfide by the sulfite reductase that uses photosynthetically reduced ferredoxine (Fd) as an electron donor of the six required electrons. The biosynthesis of cysteine is further accomplished by the sequential reaction of two enzymes: First, the serine acetyltransferase (SAT) synthesizes the intermediary product, O-acetylserine (OAS), from acetyl-CoA and serine; and second, the O-acetylserine(thiol)lyase (OASTL) incorporates the sulfide to OAS producing the cysteine. Recently, much progress has been made toward understanding the action of the O-acetylserine(thiol)lyase (OASTL) enzyme, one of the enzymes responsible for the biosynthesis of cysteine, using as a model system the plant Arabidopsis thaliana. The focus of the research has been mainly placed on the most abundant enzymes based on their involvement in the primary sulfate assimilation pathway. Biochemical and molecular analysis of the major OASTL knockout mutants in Arabidopsis thaliana revealed that part of the produced sulfide is incorporated to O-acetylserine to form cysteine in the chloroplast with the assistance of the OAS-B isoform. However, most of the chloroplastic sulfide overflows and escapes into the cytosol and the mitochondria, where it is also assimilated into cysteine by the OAS-A1 and OAS-C isoforms, respectively.^4–6The three major OASTL isoforms seem to be redundant under normal growth conditions. However, our investigations on the major cytosolic isoform, the OAS-A1, revealed new insights on the function of this enzyme as a determinant of the antioxidative capacity of the cytosol.⁷ The OASTL homolog, CYS-C1, exhibits OASTL activity, but in fact, it is a β-cyanoalanine synthase enzyme that uses cysteine to detoxify cyanide within the mitochondria.⁸ Furthermore, Arabidopsis cells contain four additional O-acetylserine(thiol)lyase isoforms encoded by the CYS-D1 (At3g04940), CYS-D2 (At5g28020), CS26 (At3g03630) and CS-LIKE (At5g28030) genes with unknown function. Are these four isoforms authentic OASTL and are, therefore, redundant enzymes or do they have different activities and, therefore, different functions?Our recent research on the less-common isoforms, CS-like and CS26, shed light on this issue, and we are decoding two important aspects of the sulfur metabolism in plants.^9,10 The CS-LIKE protein was identified by sequence homology upon the completion of the sequencing of the Arabidopsis genome. Because of its cytosolic localization, it is thought to have an auxiliary function with respect to the major cytosolic isoform, the OAS-A1. The characterization of the purified recombinant protein has shown that the CS-LIKE isoform catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate; thus, CS-LIKE is a novel L-cysteine desulfhydrase (EC 4.4.1.1), and it is designated as DES1 (Fig. 1). This enzyme is important for maintaining the homeostasis of cysteine in the cell, and the loss of function of this protein in knockout mutant plants results in higher levels of cysteine and glutathione. This increased level of soluble thiols results also in a higher antioxidant capacity of the plant, which, in turn, becomes more resistant to abiotic stress phenomena such as the presence of heavy metals or hydrogen peroxide. This observation may indicate that the regulation of this enzyme may be a key component of the plant physiological processes that involve redox reactions. Cytosolic cysteine degrading enzymes with desulfhydrase activity has been found in plants, but the protein responsible for this activity remained unisolated until now that it is revealed with our investigation on DES1.¹¹ From the standpoint of biotechnology, plants with this modified enzyme may result in abiotic stress-resistant lines that deserve to be studied.Open in a separate windowFigure 1Biosynthesis of cysteine and S-sulfocysteine in the chloroplast and cytosol of Arabidopsis and subcellular localization of the responsible enzymes. The cytosolic and plastidial O-acetylserine(thiol)lyase, L-cysteine desulfhydrase and S-sulfocysteine synthase are shown in red. A single representative of a grana thylakoid is shown as a grey oval compartment.The other less common enzyme studied, called CS26 and localized in the chloroplast, has proved to be an enzyme with S-sulfocysteine synthase activity.¹⁰ This enzyme synthesizes the incorporation of thiosulfate to O-acetylserine to form S-sulfocysteine (RSSO₃⁻). This activity, discovered for the first time in plants, was previously reported in bacteria where the biosynthesis of cysteine can be accomplished by two enzymes encoded by the cysK and cysM genes.^12,13 This enzyme activity is essential for the chloroplast function under long-day growing conditions but seems to be superfluous under short-day conditions. Morphologic and biochemical phenotype comparisons of the knockout oas-b and cs26 highlight the importance of the metabolite S-sulfocysteine and not the cysteine in the redox control of the chloroplast. Under long-day growth conditions, the cs26 mutants exhibit a reduction in size and show leaf paleness, have reductions in the chlorophyll content and photosynthetic activity, and are not able to properly detoxify reactive oxygen species, which are accumulated to high levels. None of these changes are observed in the oas-b mutant.Although we do not know the function of the S-sulfocysteine molecule in the chloroplast, two aspects are important to note. On the one hand, the enzyme CS26 can be located in the chloroplast''s lumen in opposition to the enzyme OAS-B, which is located in the stroma. The second aspect is the difference in chemical reactivity of S-sulfocysteine and cysteine. The S-sulfocysteine has two sulfur atoms with different degrees of oxidation, −1 and +5; therefore, it may act as an oxidant molecule by reacting with reduced thiols forming a disulfide bridge and releasing sulfite.¹⁴ We have suggested that a putative target of S-sulfocysteine can be the STN7 kinase, which contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved cysteines that are critical for its activity. A disulfide bridge between these two cysteines is required for the kinase activity, but how the redox states of these two cysteines are regulated in the lumen remains an open question.¹⁵ In general, how the thiol oxidation of proteins located in the thylakoid lumen takes place is still unclear because no sulfhydryl oxidases have been identified in this compartment. In fact, this process is highly important because the chaperones and peptidyl-prolyl cis-trans isomerases, such as the AtFKBP13, need to be oxidized in order to be functional in the lumen and to regulate the folding of the Rieske protein.^16–18     

The role of PGR5 in the redox poising of photosynthetic electron transport

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO₂ concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.     

Multi‐level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ‐subunit through the ferredoxin–thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild‐type levels as irradiance was increased. This effect was caused by an altered redox state of the γ‐subunit under low, but not high, light. The low light‐specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non‐photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin–thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.     

A Monogalactosyldiacylglycerol Synthase Found in the Green Sulfur Bacterium Chlorobaculum tepidum Reveals Important Roles for Galactolipids in Photosynthesis

Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll c aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants.     

The Thylakoid Membrane Protein CGL160 Supports CF1CF0 ATP Synthase Accumulation in Arabidopsis thaliana

The biogenesis of the major thylakoid protein complexes of the photosynthetic apparatus requires auxiliary proteins supporting individual assembly steps. Here, we identify a plant lineage specific gene, CGL160, whose homolog, atp1, co-occurs with ATP synthase subunits in an operon-like arrangement in many cyanobacteria. Arabidopsis thaliana T-DNA insertion mutants, which no longer accumulate the nucleus-encoded CGL160 protein, accumulate less than 25% of wild-type levels of the chloroplast ATP synthase. Severe cosmetic or growth phenotypes result under either short day or fluctuating light growth conditions, respectively, but this is ameliorated under long day constant light growth conditions where the growth, ATP synthase activity and photosynthetic electron transport of the mutants are less affected. Accumulation of other photosynthetic complexes is largely unaffected in cgl160 mutants, suggesting that CGL160 is a specific assembly or stability factor for the CF₁CF₀ complex. CGL160 is not found in the mature assembled complex but it does interact specifically with subunits of ATP synthase, predominantly those in the extrinsic CF₁ sub-complex. We suggest therefore that it may facilitate the assembly of CF₁ into the holocomplex.     

A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa

Galactosylglycerolipids (GGLs) and chlorophyll are characteristic components of chloroplast in photosynthetic organisms. Although chlorophyll is anchored to the thylakoid membrane by phytol (tetramethylhexadecenol), this isoprenoid alcohol has never been found as a constituent of GGLs. We here described a novel GGL, in which phytol was linked to the glycerol backbone via an ether linkage. This unique GGL was identified as an Alkaline-resistant and Endogalactosylceramidase (EGALC)-sensitive GlycoLipid (AEGL) in the marine green alga, Ulva pertusa. EGALC is an enzyme that is specific to the R-Galα/β1-6Galβ1-structure of galactolipids. The structure of U. pertusa AEGL was determined following its purification to 1-O-phytyl-3-O-Galα1-6Galβ1-sn-glycerol by mass spectrometric and nuclear magnetic resonance analyses. AEGLs were ubiquitously distributed in not only green, but also red and brown marine algae; however, they were rarely detected in terrestrial plants, eukaryotic phytoplankton, or cyanobacteria.     

Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in Chlamydomonas

Mitochondrial F₁F_O ATP synthase (Complex V) catalyses ATP synthesis from ADP and inorganic phosphate using the proton-motive force generated by the substrate-driven electron transfer chain. In this work, we investigated the impact of the loss of activity of the mitochondrial enzyme in a photosynthetic organism. In this purpose, we inactivated by RNA interference the expression of the ATP2 gene, coding for the catalytic subunit β, in the green alga Chlamydomonas reinhardtii. We demonstrate that in the absence of β subunit, complex V is not assembled, respiratory rate is decreased by half and ATP synthesis coupled to the respiratory activity is fully impaired. Lack of ATP synthase also affects the morphology of mitochondria which are deprived of cristae. We also show that mutants are obligate phototrophs and that rearrangements of the photosynthetic apparatus occur in the chloroplast as a response to ATP synthase deficiency in mitochondria. Altogether, our results contribute to the understanding of the yet poorly studied bioenergetic interactions between organelles in photosynthetic organisms.     

Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF₁-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF₁-γ. Previous studies showed that the light-dependent reduction of CF₁-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF₁-γ, especially under physiological conditions. We therefore performed direct assessment of the CF₁-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF₁-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF₁-γ. The results showed that CF₁-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.     

Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine synthase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.     

Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.     

Photosynthetic adaptation to length of day is dependent on s-sulfocysteine synthase activity in the thylakoid lumen

Arabidopsis (Arabidopsis thaliana) chloroplasts contain two O-acetyl-serine(thiol)lyase (OASTL) homologs, OAS-B, which is an authentic OASTL, and CS26, which has S-sulfocysteine synthase activity. In contrast with OAS-B, the loss of CS26 function resulted in dramatic phenotypic changes, which were dependent on the light treatment. We have performed a detailed characterization of the photosynthetic and chlorophyll fluorescence parameters in cs26 plants compared with those of wild-type plants under short-day growth conditions (SD) and long-day growth conditions (LD). Under LD, the photosynthetic characterization, which was based on substomatal CO(2) concentrations and CO(2) concentration in the chloroplast curves, revealed significant reductions in most of the photosynthetic parameters for cs26, which were unchanged under SD. These parameters included net CO(2) assimilation rate, mesophyll conductance, and mitochondrial respiration at darkness. The analysis also showed that cs26 under LD required more absorbed quanta per driven electron flux and fixed CO(2). The nonphotochemical quenching values suggested that in cs26 plants, the excess electrons that are not used in photochemical reactions may form reactive oxygen species. A photoinhibitory effect was confirmed by the background fluorescence signal values under LD and SD, which were higher in young leaves compared with mature ones under SD. To hypothesize the role of CS26 in relation to the photosynthetic machinery, we addressed its location inside of the chloroplast. The activity determination and localization analyses that were performed using immunoblotting indicated the presence of an active CS26 enzyme exclusively in the thylakoid lumen. This finding was reinforced by the observation of marked alterations in many lumenal proteins in the cs26 mutant compared with the wild type.     

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.     

Expression by Chlamydomonas reinhardtii of a chloroplast ATP synthase with polyhistidine-tagged beta subunits

The green alga Chlamydomonas reinhardtii is a model organism for the study of photosynthesis. The chloroplast ATP synthase is responsible for the synthesis of ATP during photosynthesis. Using genetic engineering and biolistic transformation, a string of eight histidine residues has been inserted into the amino-terminal end of the β subunit of this enzyme in C. reinhardtii. The incorporation of these amino acids did not impact the function of the ATP synthase either in vivo or in vitro and the resulting strain of C. reinhardtii showed normal growth. The addition of these amino acids can be seen through altered gel mobility of the β subunit and the binding of a polyhistidine-specific dye to the subunit. The purified his-tagged CF1 has normal Mg²⁺-ATPase activity, which can be stimulated by alcohol and detergents and the enzyme remains active while bound to a nickel-coated surface. Potential uses for this tagged enzyme as a biochemical tool are discussed.     

The stoichiometry of the chloroplast ATP synthase oligomer III in Chlamydomonas reinhardtii is not affected by the metabolic state

The chloroplast H⁺-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO₂ concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H⁺-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.