A Fructan Exohydrolase from Maize Degrades Both Inulin and Levan and Co-Exists with 1-Kestotriose in Maize.

Enzymes with fructan exohydrolase (FEH) activity are present not only in fructan-synthesizing species but also in non-fructan plants. This has led to speculation about their functions in non-fructan species. Here, a cell wall invertase-related Zm-6&1-FEH2 with no "classical" invertase motif was identified in maize. Following heterologous expression in Pichia pastoris and in Nicotiana benthamiana leaves, the enzyme activity of recombinant Zm-6&1-FEH2 displays substrate specificity with respect to inulin and levan. Subcellular localization showed Zm-6&1-FEH2 exclusively localized in the apoplast, and its expression profile was strongly dependent on plant development and in response to drought and abscisic acid. Furthermore, formation of 1-kestotriose, an oligofructan, was detected in vivo and in vitro and could be hydrolyzed by Zm-6&1-FEH2. In summary, these results support that Zm-6&1-FEH2 enzyme from maize can degrade both inulin-type and levan-type fructans, and the implications of the co-existence of Zm-6&1-FEH2 and 1-kestotriose are discussed.

Apoplastic maize fructan exohydrolase Zm-6-FEH displays substrate specificity for levan and is induced by exposure to levan-producing bacteria.

Fructan exohydrolases (FEHs) are structurally related to cell wall invertases. While the latter are ubiquitous in higher plants, the role of FEHs in non-fructan species has remained enigmatic. To explore possible roles of FEHs in maize, a full length putative Zm-6-FEH-encoding cDNA was cloned displaying high sequence similarity with cell wall invertases. For functional characterization, Zm-6-FEH protein was expressed in Picha pastoris and in Nicotiana benthamiana leaves. Enzyme activity of recombinant Zm-6-FEH protein showed a strong preference for levan as substrate. Expression profiling in maize seedlings revealed higher transcript amounts in the more mature leaf parts as compared to the growth zone at the base of the leaf, in good correlation with FEH enzyme activities. Subcellular localization analysis indicated Zm-6-FEH location in the apoplast. Noteworthy, incubation of leaf discs with levan and co-incubation with high levan-producing bacteria selectively up-regulated transcript levels of Zm-6-FEH, accompanied by an increase of 6-FEH enzyme activity. In summary, the results indicate that Zm-6-FEH, a novel fructan exohydrolase of a non-fructan species, may have a role in plant defense against levan-producing bacteria.

A 6&1-FEH Encodes an Enzyme for Fructan Degradation and Interact with Invertase Inhibitor Protein in Maize (Zea mays L.).

About 15% of higher plants have acquired the ability to convert sucrose into fructans. Fructan degradation is catalyzed by fructan exohydrolases (FEHs), which are structurally related to cell wall invertases (CWI). However, the biological function(s) of FEH enzymes in non-fructan species have remained largely enigmatic. In the present study, one maize CWI-related enzyme named Zm-6&1-FEH1, displaying FEH activity, was explored with respect to its substrate specificities, its expression during plant development, and its possible interaction with CWI inhibitor protein. Following heterologous expression in Pichia pastoris and in N. benthamiana leaves, recombinant Zm-6&1-FEH1 revealed substrate specificities of levan and inulin, and also displayed partially invertase activity. Expression of Zm-6&1-FEH1 as monitored by qPCR was strongly dependent on plant development and was further modulated by abiotic stress. To explore whether maize FEH can interact with invertase inhibitor protein, Zm-6&1-FEH1 and maize invertase inhibitor Zm-INVINH1 were co-expressed in N. benthamiana leaves. Bimolecular fluorescence complementation (BiFC) analysis and in vitro enzyme inhibition assays indicated productive complex formation. In summary, the results provide support to the hypothesis that in non-fructan species FEH enzymes may modulate the regulation of CWIs.

Plant glutathione biosynthesis revisited: redox-mediated activation of glutamylcysteine ligase does not require homo-dimerization.

Plant γ-glutamylcysteine ligase (GCL), catalyzing the first and tightly regulated step of glutathione (GSH) biosynthesis, is redox-activated via formation of an intramolecular disulfide bond. In vitro, redox-activation of recombinant GCL protein causes formation of homo-dimers. Here, we have investigated whether dimerization occurs in vivo and if so whether it contributes to redox-activation. FPLC analysis indicated that recombinant redox-activated WT (wild type) AtGCL dissociates into monomers at concentrations below 10-6 M, i.e. below the endogenous AtGCL concentration in plastids, which was estimated to be in the micromolar range. Thus, dimerization of redox-activated GCL is expected to occur in vivo To determine the possible impact of dimerization on redox-activation, AtGCL mutants were generated in which salt bridges or hydrophobic interactions at the dimer interface were interrupted. WT AtGCL and mutant proteins were analyzed by non-reducing SDS-PAGE to address their redox state and probed by FPLC for dimerization status. Furthermore, their substrate kinetics (KM, Vmax) were compared. The results indicate that dimer formation is not required for redox-mediated enzyme activation. Also, crystal structure analysis confirmed that dimer formation does not affect binding of GSH as competitive inhibitor. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated.

Suppression of extracellular invertase inhibitor gene expression improves seed weight in soybean (Glycine max).

Cell wall invertase (CWI) and vacuolar invertase (VI) play multiple functions in plant growth. As well as depending on transcriptional and post-transcriptional regulation, there is growing evidence that CWI and VI are also subject to post-translational control by small inhibitory proteins. Despite the significance of this, genes encoding inhibitors, their molecular and biochemical properties, and their potential roles in regulating seed production have not been well documented in soybean (Glycine max). In this study, two invertase inhibitor isoforms, GmCIF1 and GmC/VIF2, were characterized to possess inhibitory activities in vitro via heterologous expression. Transcript analyses showed that they were predominantly expressed in developing seeds and in response to ABA. In accordance with this, surveys of primary targets showed subcellular localizations to the apoplast in tobacco epidermis after expressing YFP-fusion constructs. Investigations using RNAi transgenic plants demonstrated marked elevations of CWI activities and improvements in seed weight in conjunction with higher accumulations of hexoses, starch, and protein in mature seeds. Further co-expression analyses of GmCIF1 with several putative CWI genes corroborated the notion that GmCIF1 modulation of CWI that affects seed weight is mainly contingent on post-translational mechanisms. Overall, the results suggest that post-translational elevation of CWI by silencing of GmCIF1 expression orchestrates the process of seed maturation through fine-tuning sucrose metabolism and sink strength.

Regulation of secondary cell wall biosynthesis by a NAC transcription factor from Miscanthus.

Cell wall recalcitrance is a major limitation for the sustainable exploitation of lignocellulosic biomass as a renewable resource. Species and hybrids of the genus Miscanthus have emerged as candidate crops for the production of lignocellulosic feedstock in temperate climates, and dedicated efforts are underway to improve biomass yield. However, nothing is known about the molecular players involved in Miscanthus cell wall biosynthesis to facilitate breeding efforts towards tailored biomass. Here, we identify a Miscanthus sinensis transcription factor related to SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1), which acts as a master switch for the regulation of secondary cell wall formation and lignin biosynthesis. MsSND1 is expressed in growth stages associated with secondary cell wall formation, together with its potential targets. Consistent with this observation, MsSND1 was able to complement the secondary cell wall defects of the Arabidopsis snd1 nst1 double mutant, and ectopic expression of MsSND1 in tobacco leaves was sufficient to trigger patterned deposition of cellulose, hemicellulose, and lignin reminiscent of xylem elements. Transgenic studies in Arabidopsis thaliana plants revealed that MsSND1 regulates, directly and indirectly, the expression of a broad range of genes involved in secondary cell wall formation, including MYB transcription factors which regulate only a subset of the SCW differentiation program. Together, our findings suggest that MsSND1 is a transcriptional master regulator orchestrating secondary cell wall biosynthesis in Miscanthus.

Reassessment of an Arabidopsis cell wall invertase inhibitor AtCIF1 reveals its role in seed germination and early seedling growth.

In higher plants, cell wall invertase (CWI) and vacuolar invertase (VI) are recognized as essential players in sugar metabolism and sugar signaling, thereby affecting source-sink interactions, plant development and responses to environmental cues. CWI and VI expression levels are transcriptionally controlled; however, both enzymes are also subject to posttranslational control by invertase inhibitor proteins. The physiological significances of inhibitor proteins during seed germination and early seedling development are not yet fully understood. Here, we demonstrate that the inhibitor isoform AtCIF1 impacted on seed germination and early seedling growth in Arabidopsis. The primary target of AtCIF1 was shown to be localized to the apoplast after expressing an AtCIF1 YFP-fusion construct in tobacco epidermis and transgenic Arabidopsis root. The analysis of expression patterns showed that AtCWI1 was co-expressed spatiotemporally with AtCIF1 within the early germinating seeds. Seed germination was observed to be accelerated independently of exogenous abscisic acid (ABA) in the AtCIF1 loss-of-function mutant cif1-1. This effect coincided with a drastic increase of CWI activity in cif1-1 mutant seeds by 24 h after the onset of germination, both in vitro and in planta. Accordingly, quantification of sugar content showed that hexose levels were significantly boosted in germinating seeds of the cif1-1 mutant. Further investigation of AtCIF1 overexpressors in Arabidopsis revealed a markedly suppressed CWI activity as well as delayed seed germination. Thus, we conclude that the posttranslational modulation of CWI activity by AtCIF1 helps to orchestrate seed germination and early seedling growth via fine-tuning sucrose hydrolysis and, possibly, sugar signaling.

Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae.

The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed.

The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera.

Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB-type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.

Generation of Se-fortified broccoli as functional food: impact of Se fertilization on S metabolism.

Selenium (Se)-fortified broccoli (Brassica oleracea var. italica) has been proposed as a functional food for cancer prevention, based on its high glucosinolate (GSL) content and capacity for Se accumulation. However, as selenate and sulphate share the initial assimilation route, Se fertilization could interfere with sulphur metabolism and plant growth. Consequently, GSL accumulation could be compromised. To evaluate these potentially adverse effects of Se fertilization, we performed a comprehensive study on sand-grown young broccoli plants (weekly selenate applications of 0.8 µmol plant(-1) via the root) and field-grown adult broccoli plants during head formation (single foliar selenate application: 25.3 or 253 µmol plant(-1) ). The results show that under these conditions, Se application does not affect plant growth, contents of cysteine, glutathione, total GSL, glucoraphanin (major aliphatic GSL) or the expression of BoMYB28 (encoding a functionally confirmed master regulator for aliphatic GSL biosynthesis). Conversely, due to the changed expression of sulphate transporters (BoSULTR1;1, 1;2, 2;1, and 2;2), sulphate and total S contents increased in the shoot of young plants while decreasing in the root. We conclude that broccoli can be fertilized with Se without reduction in GSL content, even with Se accumulation exceeding the level recommended for human consumption.

Enhanced formation of methane in plant cell cultures by inhibition of cytochrome c oxidase.

The claim of methane (CH4) formation in plants has caused much controversy and debate within the scientific community over the past 4 years. Here, using both stable isotope and concentration measurements, we demonstrate that CH4 formation occurs in plant cell cultures that were grown in the dark under sterile conditions. Under non-stress conditions the plant cell cultures produced trace amounts [0.3-0.6 ng g⁻¹ dry weight (DW) h⁻¹] of CH4 but these could be increased by one to two orders of magnitude (up to 12 ng g⁻¹ DW h⁻¹) when sodium azide, a compound known to disrupt electron transport flow at the cytochrome c oxidase (complex IV) in plant mitochondria, was added to the cell cultures. The addition of other electron transport chain (ETC) inhibitors did not result in significant CH4 formation indicating that a site-specific disturbance of the ETC at complex IV causes CH4 formation in plant cells. Our study is an important first step in providing more information on non-microbial CH4 formation from living plants particularly under abiotic stress conditions that might affect the electron transport flow at the cytochrome c oxidase in plant mitochondria.

Exploiting plants for glutathione (GSH) production: Uncoupling GSH synthesis from cellular controls results in unprecedented GSH accumulation.

Glutathione (GSH) is a key factor for cellular redox homeostasis and tolerance against abiotic and biotic stress (May et al., 1998; Noctor et al., 1998a). Previous attempts to increase GSH content in plants have met with moderate success (Rennenberg et al., 2007), largely because of tight and multilevel control of its biosynthesis (Rausch et al., 2007). Here, we report the in planta expression of the bifunctional gamma-glutamylcysteine ligase-glutathione synthetase enzyme from Streptococcus thermophilus (StGCL-GS), which is shown to be neither redox-regulated nor sensitive to feedback inhibition by GSH. Transgenic tobacco plants expressing StGCL-GS under control of a constitutive promoter reveal an extreme accumulation of GSH in their leaves (up to 12 micromol GSH/gFW, depending on the developmental stage), which is more than 20- to 30-fold above the levels observed in wild-type (wt) plants and which can be even further increased by additional sulphate fertilization. Surprisingly, this dramatically increased GSH production has no impact on plant growth while enhancing plant tolerance to abiotic stress. Furthermore, StGCL-GS-expressing plants are a novel, cost-saving source for GSH production, being competitive with current yeast-based systems (Li et al., 2004).

Buckwheat (Fagopyrum esculentum Moench) FeMT3 gene in heavy metal stress: protective role of the protein and inducibility of the promoter region under Cu(2+) and Cd(2+) treatments.

The protective role in vivo of buckwheat metallothionein type 3 (FeMT3) during metal stress and the responsiveness of its promoter to metal ions were examined. Increased tolerance to heavy metals of FeMT3 producing Escherichia coli and cup1(Delta) yeast cells was detected. The defensive ability of buckwheat MT3 during Cd and Cu stresses was also demonstrated in Nicotiana debneyii leaves transiently expressing FeMT3. In contrast to phytochelatins, the cytoplasmatic localization of FeMT3 was not altered under heavy metal stress. Functional analysis of the corresponding promoter region revealed extremely high inducibility upon Cu(2+) and Cd(2+) treatments. The confirmed defense ability of FeMT3 protein in vivo and the great responsiveness of its promoter during heavy metal exposure make this gene a suitable candidate for biotechnological applications.

The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus.

The pectin matrix of the cell wall, a complex and dynamic network, impacts on cell growth, cell shape and signaling processes. A hallmark of pectin structure is the methylesterification status of its major component, homogalacturonan (HGA), which affects the biophysical properties and enzymatic turnover of pectin. The pectin methylesterases (PMEs), responsible for de-esterification, encompass a protein family of more than 60 isoforms in the Arabidopsis genome. The pivotal role of PME in the regulation of pectin properties also requires tight control at the post-translational level. Type-I PMEs are characterized by an N-terminal pro region, which exhibits homology with pectin methylesterase inhibitors (PMEIs). Here, we demonstrate that the proteolytic removal of the N-terminal pro region depends on conserved basic tetrad motifs, occurs in the early secretory pathway, and is required for the subsequent export of the PME core domain to the cell wall. In addition, we demonstrate the involvement of AtS1P, a subtilisin-like protease, in Arabidopsis PME processing. Our results indicate that the pro region operates as an effective retention mechanism, keeping unprocessed PME in the Golgi apparatus. Consequently, pro-protein processing could constitute a post-translational mechanism regulating PME activity.

Inhibitors of plant invertases do not affect the structurally related enzymes of fructan metabolism.

Plant fructan active enzymes (FAZYs), including the enzymes involved in inulin metabolism, namely sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) and fructan 1-exohydrolase (1-FEH; EC 3.2.1.153), are evolutionarily related to acid invertases (AIs), that is, plant cell wall invertase (CWI) and vacuolar invertase (VI). Acid invertases are post-translationally controlled by proteinaceous inhibitors. Whether FAZYs are subject to similar controls is not known. To probe their possible interactions with invertase inhibitors, we transiently expressed chicory (Cichorium intybus) FAZYs, as well as several previously characterized invertase inhibitors from nonfructan species, and the C. intybus cell wall/vacuolar inhibitor of fructosidase (CiC/VIF), a putative invertase inhibitor of a fructan-accumulating plant, in leaves of Nicotiana benthamiana. Leaf extracts containing recombinant, enzymatically active FAZYs were used to explore the interaction with invertase inhibitors. Neither heterologous inhibitors nor CiC/VIF affected FAZY activities. CiC/VIF was confirmed as an AI inhibitor with a stronger effect on CWI than on VI. Its expression in planta was developmentally regulated (high in taproots, and undetectable in leaves and flowers). In agreement with its target specificities, CiC/VIF was associated with the cell wall. It is concluded that subtle structural differences between AIs and FAZYs result in pronounced selectivity of inhibitor action.

The redox switch of gamma-glutamylcysteine ligase via a reversible monomer-dimer transition is a mechanism unique to plants.

In plants, the first committed enzyme for glutathione biosynthesis, gamma-glutamylcysteine ligase (GCL), is under multiple controls. The recent elucidation of GCL structure from Brassica juncea (BjGCL) has revealed the presence of two intramolecular disulfide bridges (CC1, CC2), which both strongly impact on GCL activity in vitro. Here we demonstrate that cysteines of CC1 are confined to plant species from the Rosids clade, and are absent in other plant families. Conversely, cysteines of CC2 involved in the monomer-dimer transition in BjGCL are not only conserved in the plant kingdom, but are also conserved in the evolutionarily related alpha- (and some gamma-) proteobacterial GCLs. Focusing on the role of CC2 for GCL redox regulation, we have extended our analysis to all available plant (31; including moss and algal) and related proteobacterial GCL (46) protein sequences. Amino acids contributing to the homodimer interface in BjGCL are highly conserved among plant GCLs, but are not conserved in related proteobacterial GCLs. To probe the significance of this distinction, recombinant GCLs from Nicotiana tabacum (NtGCL), Agrobacterium tumefaciens (AtuGCL, alpha-proteobacteria) and Xanthomonas campestris (XcaGCL, gamma-proteobacteria) were analyzed for their redox response. As expected, NtGCL forms a homodimer under oxidizing conditions, and is activated more than threefold. Conversely, proteobacterial GCLs remain monomeric under oxidizing and reducing conditions, and their activities are not inhibited by DTT, despite the presence of CC2. We conclude that although plant GCLs are evolutionarily related to proteobacterial GCLs, redox regulation of their GCLs via CC2-dependent dimerization has been acquired later in evolution, possibly as a consequence of compartmentation in the redox-modulated plastid environment.

Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins.

In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca(2+) ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.

Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings.

The protective function of a plant type-2 metallothionein was analysed after expression in Escherichia coli and in Arabidopsis thaliana seedlings. BjMT2 from Brassica juncea was expressed in E. coli as a TrxABjMT2 fusion protein. After affinity chromatography and cleavage from the TrxA domain, pure BjMT2 protein was obtained which strongly reacted with the thiol reagent monobromobimane. Escherichia coli cells expressing the TrxABjMT2 fusion were more tolerant to Cu2+ and Cd2+ exposure than control strains. Likewise, when BjMT2 cDNA was expressed in A. thaliana under the regulation of the 35S promoter, seedlings exhibited an increased tolerance against Cu2+ and Cd2+ based on shoot growth and chlorophyll content. Analysis of transiently transformed cells of A. thaliana and tobacco leaves by confocal laser scanning microscopy (CLSM) revealed exclusive cytosolic localization of a BjMT2::EGFP (enhanced green fluorescent protein) fusion protein in control and heavy metal-exposed plant cells. Remarkably, ectopic expression of BjMT2 reduced root growth in the absence of heavy metal exposure, whereas in the presence of 50 or 100 microM Cu2+ root growth in control and transgenic lines was identical. The results indicate that in A. thaliana, root and shoot development are differentially affected by ectopic expression of BjMT2.

Structural basis for the redox control of plant glutamate cysteine ligase.

Glutathione (GSH) plays a crucial role in plant metabolism and stress response. The rate-limiting step in the biosynthesis of GSH is catalyzed by glutamate cysteine ligase (GCL) the activity of which is tightly regulated. The regulation of plant GCLs is poorly understood. The crystal structure of substrate-bound GCL from Brassica juncea at 2.1-A resolution reveals a plant-unique regulatory mechanism based on two intramolecular redox-sensitive disulfide bonds. Reduction of one disulfide bond allows a beta-hairpin motif to shield the active site of B. juncea GCL, thereby preventing the access of substrates. Reduction of the second disulfide bond reversibly controls dimer to monomer transition of B. juncea GCL that is associated with a significant inactivation of the enzyme. These regulatory events provide a molecular link between high GSH levels in the plant cell and associated down-regulation of its biosynthesis. Furthermore, known mutations in the Arabidopsis GCL gene affect residues in the close proximity of the active site and thus explain the decreased GSH levels in mutant plants. In particular, the mutation in rax1-1 plants causes impaired binding of cysteine.

Glutathione, photosynthesis and the redox regulation of stress-responsive gene expression.

The ubiquitous antioxidant thiol tripeptide glutathione is present in millimolar concentrations in plant tissues and is regarded as one of the major determinants of cellular redox homeostasis. Recent research has highlighted a regulatory role for glutathione in influencing the expression of many genes important in plants' responses to both abiotic and biotic stress. Therefore, it becomes important to consider how glutathione levels and its redox state are influenced by environmental factors, how glutathione is integrated into primary metabolism and precisely how it can influence the functioning of signal transduction pathways by modulating cellular redox state. This review draws on a number of recent important observations and papers to present a unified view of how the responsiveness of glutathione to changes in photosynthesis may be one means of linking changes in nuclear gene expression to changes in the plant's external environment.