The conformational stability and biophysical properties of the eukaryotic thioredoxins of Pisum sativum are not family-conserved

Thioredoxins (TRXs) are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1) the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas) of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations) belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common) for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core), are less so.     

A single glutaredoxin or thioredoxin gene is essential for viability in the yeast Saccharomyces cerevisiae

Glutaredoxins and thioredoxins are small heat-stable oxidoreductases that have been conserved throughout evolution. The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2). We report here that the quadruple trx1 trx2 grx1 grx2 mutant is inviable and that either a single glutaredoxin or a single thioredoxin (i.e. grx1 grx2 trx1, grx1 grx2 trx2, grx1 trx1 trx2, grx2 trx1 trx2) is essential for viability. Loss of both thioredoxins has been reported previously to lead to methionine auxotrophy consistent with thioredoxins being the sole reductants for 3'-phosphoadenosine 5'-phosphosulphate reductase (PAPS) in yeast. However, we present evidence for the existence of a novel yeast hydrogen donor for PAPS reductase, as strains lacking both thioredoxins assimilated sulphate under conditions that minimized the generation of reactive oxygen species (low aeration and absence of functional mitochondria). In addition, the assimilation of [35S]-sulphate was approximately 60-fold higher in the trx1 trx2 grx1 and trx1 trx2 grx2 mutants compared with the trx1 trx2 mutant. Furthermore, in contrast to the trx1 trx2 mutant, the trx1 trx2 grx2 mutant grew on minimal agar plates, and the trx1 trx2 grx1 mutant grew on minimal agar plates under anaerobic conditions. We propose a model in which the novel reductase activity normally functions in the repair of oxidant-mediated protein damage but, under conditions that minimize the generation of reactive oxygen species, it can serve as a hydrogen donor for PAPS reductase.     

Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.     

Thioredoxin and Redox Control within the New Concept of Oxidative Signaling

During the last decade, plant thioredoxins (TRX) h-type have been shown to be implicated in several new roles like the protection against the oxidative stress by their ability to reduce some antioxidant proteins as peroxiredoxins (PRX) or methionine-sulphoxide-reductases (MSR). However, the concept of the oxidative stress is changing and this fact raises the question of the TRX roles in this new context. In the January issue of Plant Physiology, we have presented two TRXsh from Pisum sativum differently involved in the control of the redox status. PsTRXh1 is an h-type TRX that acts by reducing classical antioxidant proteins. PsTRXh2 seems to be also involved in redox control, however it could act contrary to its counterpart h1. Both proteins may play antagonistic roles in pea in order to lead a better control of the redox status.Key Words: abiotic stress, oxidative signalling, thioredoxins, Pisum sativum, ROSHigh concentration of reactive oxygen species (ROS) in plant cells involve the activation of different antioxidant systems which reestablish the redox status leading to better physiological conditions. On the other hand, it has been well established that at certain levels, the ROS act as second messengers in signal transduction cascades in several processes in plant cells.¹ At the light of these events, it has been proposed the reevaluation of the concept of oxidative stress towards “oxidative signalling.”² This concept involves all the cellular mechanisms that let the plant cells sense and act in response to modified environmental conditions. Several cellular systems are involved in such role, and in these last years, plant TRXs have been shown to be involved in several number of metabolic pathways linked to the regulation of the redox imbalance,³ mainly for the case of the h-type cluster of the TRXs.^4–6In our last work,⁷ we have described two pea TRXs of the h-type cluster, PsTRXh1 and PsTRXh2 that are differentially, and even antagonistically, involved in the redoxregulation control, probably through their interaction with different target proteins. We proposed that PsTRXh1 might be involved in the control of the ROS levels in pea tissues due to its ability to interact with several antioxidant proteins in vivo. It is now very well known that some members of the TRX family reduce PRX,^8–10 an antioxidant enzyme involves in the direct deactivation of some oxidant agents or the MSR,^11,14,15 in charge of the recovery of the oxidized methionine, both in a very specific manner. Due to the increase of PsTRXh1 both at gene expression and protein levels in plant heterotrophic tissues in response to the H₂O₂ treatment, and because it is also capable of conferring resistance towards hydrogen peroxide when produced in a yeast trx1Δ trx2Δ strain,¹⁶ one function of this TRX member could be the reduction in vivo of some PRX and/or MSRA counterparts in Pea tissues in the context of the oxidative signalling.Interestingly, PsTRXh2 gene and its corresponding protein showed very different behaviours to that presented by its homologous h1, reinforcing the idea that some TRX isoforms in plants are capable of functional specificity in vivo. PsTRXh2 is expressed in all tissues assays, mainly in roots, but at an extremely low level compared with that of PsTRXh1. Its divergent functional behaviour was confirmed both in Pea plantlets and yeast. In fact, contrary to PsTRXh1, PsTRXh2 provides hypersensitivity in the yeast trx1Δ trx2Δ mutant. We explained the different behaviour by suggesting that PsTRXh2 might interact with some target(s) involved either directly or indirectly in hydrogen peroxide detoxification, either by compromising the target function in resistance to the ROS or by reinforcing the target function in producing sensitivity to H₂O₂. Most probably, PsTRXh1 and PsTRXh2 interact with very different partners, and the characterization of such targets may help in the deciphering of PsTRX isoforms. As short-term future experiments, using the TRX-specific two-hybrid system that was published recently,⁸ comparative efficiencies of PsTRX isoforms could be performed to reduce some putative target involved in H₂O₂ detoxification, including PsTRXh3 and PsTRXh4.¹⁷ Unravelling Pea TRX interactome should help in deciphering the function of each isoform.However, we have also considered the possibility that PsTRXh2 could interact with the same target that PsTRXh1, but producing an opposite effect. In the yeast context, the protein targeted by the heterologous plant TRXs responsible of this complementation is a type-II PRX.⁵ We think that PsTRXh2 could interact with this yeast PRX blocking it and producing the hypersensitivity. In fact, we have found a similar effect in other protein targeted in vitro by TRXs. In the (Fig. 1), we present the in vitro ability of PsTRXh1 and PsTRXh2 to reduce and activate the pea chloroplastic fructose-1,6-bisphosphatase (FBPase). In the presence of PsTRXh1, FBPase presents TRX-dependent activity but lower than that found when chloroplastic f and m1 isoforms are used.¹² On the opposite, PsTRXh2 presents no only FBPase activation capability but its presence induces the FBPase inhibition, as the enzymatic activity was lower than that exhibited by this enzyme without TRX.Open in a separate windowFigure 1TRX-dependent chloroplastic FBPase activity two-step procedure.¹³ , PsTRXf; ▴, PsTRXm1; •, PsTRXh1; , PsTRXh2. The negative control (no TRX) is represented by a symbol-less line. Numbers in each line represent maximum enzymatic velocity as OD₃₄₀/min.Considering all data, we think that the behaviour showed by both pea h-type TRXs is due to their interactions with several protein-targets, as the PRXs: when the ROS levels increase drastically, cells develop high redox imbalances or even undergo oxidative stress. In this situation, all antioxidant mechanisms must be activated, including the increase of PsTRXh1 expression and protein quantities, giving rise to a more efficient cell detoxification. Under nonimbalance conditions of the redox status, PsTRXh2 could act by interacting (activating or inhibiting) with some protein targets. However, the physiological target for PsTRXh2 are not yet described nor supposed. Our results suggest that its role in the redox control is by producing sensitivity to oxidant agents, maybe by allowing physiological ROS levels in cells.     

Implication of a pepper h-type thioredoxin in type I- and II-nonhost resistance to Xanthomonas axonopodis

Thioredoxins (TRXs) are distributed ubiquitously in prokaryotic and eukaryotic organisms. Plants have the most complex forms of TRXs. The functional roles of such TRXs have been studied in abiotic stress but their roles in plant defense responses against biotic stresses have been less well studied. Here, we identified an h-type TRX gene from pepper, CaTRXh1, and characterized its possible effect on Type II nonhost resistance, which entails localized programmed cell death in response to nonhost pathogens. Peptide sequences of CaTRXh1 showed a high degree of similarity with TRXhs from tobacco and Arabidopsis thaliana. Southern blot analyses revealed that CaTRXh1 was present as a single copy in the pepper genome. Intriguingly, leaf infiltration by Xanthomonas axonopodis pv. glycines 8ra, eliciting a visible type II nonhost hypersensitive response (HR), and its type III secretion-system null mutant 8–13, eliciting a type I nonhost non-HR, both induced CaTRXh1 at a level similar to that of pathogenesis-related protein 4, an HR marker gene in pepper. More surprisingly, expression of CaTRXh1 was significantly increased when X. axonopodis pv. vesicatoria race 3 infiltrated the leaf of a pepper cultivar containing a resistance gene, but not with infiltration of a susceptible pepper cultivar. Taken together, our study suggests that the expression of CaTRXh1 has a critical role in HR-mediated active defense responses in pepper. GenBank accession number: EF371503.     

The protein interaction of Saccharomyces cerevisiae cytoplasmic thiol peroxidase II with SFH2p and its in vivo function

Previously, we reported that the yeast cytoplasmic thiol peroxidase type II isoform (cTPx II), a member of the TSA/AhpC family, showed a very low peroxidase activity when compared with other cytoplasmic yeast isoforms, and that cTPx II mutant (cTPx II Delta) showed a severe growth retardation compared with that of the wild-type cells. To reveal the physiological function of cTPx II in yeast cell growth, we searched for proteins which react with cTPx II. In this study, we identified a novel interaction between cTPx II and CSR1p using the yeast two-hybrid system. CSR1p (SFH2p) has been known to be one member of Sec14 homologous (SFH2) proteins. SFH2p exhibits phosphatidylinositol transfer protein activity. Interestingly, we found that cTPx II selectively bound to SFH2p among the five types of SFH proteins and Sec14p. The interaction required the dimerization of cTPx II. In addition, SFH2p also specifically bound to cTPx II among the yeast thiol peroxidase isoforms. The selective interaction of the dimer form of cTPx II (the oxidized form) with SFH2p was also confirmed by glutathione S-transferase pull-down and immunoprecipitation assays. The growth retardation, clearly reflected by the length of the lag phase, of cTPx II Delta was rescued by deleting SFH2p in the cTPx II Delta strain. The SFH2 Delta strain did not show any growth retardation. In addition, the double mutant showed a higher susceptibility to oxidative stress. This finding provides the first in vivo demonstration of the specific interaction of cTPx II with SFH2p in an oxidative stress-sensitive manner and a novel physiological function of the complex of cTPx II and SFH2p.     

Plastidic phosphatidic acid phosphatases identified in a distinct subfamily of lipid phosphate phosphatases with prokaryotic origin

Plastidic phosphatidic acid phosphatase (PAP) dephosphorylates phosphatidic acid to yield diacylglycerol, which is a precursor for galactolipids, a primary and indispensable component of photosynthetic membranes. Despite its functional importance, the molecular characteristics and phylogenetic origin of plastidic PAP were unknown because no potential homologs have been found. Here, we report the isolation and characterization of plastidic PAPs in Arabidopsis that belong to a distinct lipid phosphate phosphatase (LPP) subfamily with prokaryotic origin. Because no homolog of mammalian LPP was found in cyanobacteria, we sought an LPP ortholog in a more primitive organism, Chlorobium tepidum, and its homologs in cyanobacteria. Arabidopsis had five homologs of cyanobacterial LPP, three of which (LPP gamma, LPP epsilon 1, and LPP epsilon 2) localized to chloroplasts. Complementation of yeast Delta dpp1 Delta lpp1 Delta pah1 by plastidic LPPs rescued the relevant phenotype in vitro and in vivo, suggesting that they function as PAPs. Of the three LPPs, LPP gamma activity best resembled the native activity. The three plastidic LPPs were differentially expressed both in green and nongreen tissues, with LPP gamma expressed the highest in shoots. A knock-out mutant for LPP gamma could not be obtained, although a lpp epsilon 1 lpp epsilon 2 double knock-out showed no significant changes in lipid composition. However, lpp gamma homozygous mutant was isolated only under ectopic overexpression of LPP gamma, suggesting that loss of LPP gamma may cause lethal effect on plant viability. Thus, in Arabidopsis, there are three isoforms of plastidic PAP that belong to a distinct subfamily of LPP, and LPP gamma may be the primary plastidic PAP.     

Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana

Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Delta12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.     

Three SAC1-like genes show overlapping patterns of expression in Arabidopsis but are remarkably silent during embryo development

In Saccharomyces cerevisiae, the SAC1 gene encodes a polyphosphoinositide phosphatase (PPIPase) that modulates the levels of phosphoinositides, which are key regulators of a number of signal transduction processes. SAC1p has been implicated in multiple cellular functions: actin cytoskeleton organization, secretory functions, inositol metabolism, ATP transport, and multiple-drug sensitivity. Here, we describe the characterization of three genes in Arabidopsis thaliana, AtSAC1a, AtSAC1b, and AtSAC1c, encoding proteins similar to those of yeast SAC1p. We demonstrated that the three AtSAC1 proteins are functional homologs of the yeast SAC1p because they can rescue the cold-sensitive and inositol auxotroph yeast sac1-null mutant strain. The fact that Arabidopsis and yeast SAC1 genes derived from a common ancestor suggests that this plant multigenic family is involved in the phosphoinositide pathway and in a range of cellular functions similar to those in yeast. Using GFP fusion experiments, we demonstrate that the three AtSAC1 proteins are targeted to the endoplasmic reticulum. Their expression patterns are overlapping, with at least two members expressed in each organ. Remarkably, AtSAC1 genes are not expressed during seed development, and therefore additional phosphatases are required to control phosphoinositide levels in seeds.     

An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase.

The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.     

GAMETOPHYTIC FACTOR 1, Involved in Pre-mRNA Splicing, Is Essential for Megagametogenesis and Embryogenesis in Arabidopsis

RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 (GFA1); a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homoiogs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogeneeis and embryogenesis in plant.     

In vivo characterization of a thioredoxin h target protein defines a new peroxiredoxin family.

Disruption of the two thioredoxin genes in yeast dramatically affects cell viability and growth. Expression of Arabidopsis thioredoxin AtTRX3 in the Saccharomyces thioredoxin Delta strain EMY63 restores a wild-type cell cycle, the ability to grow on methionine sulfoxide, and H2O2 tolerance. In order to isolate thioredoxin targets related to these phenotypes, we prepared a C35S (Escherichia coli numbering) thioredoxin mutant to stabilize the intermediate disulfide bridged complex and we added a polyhistidine N-terminal extension in order to purify the complex rapidly. Expression of this mutant thioredoxin in the wild-type yeast induces a reduced tolerance to H2O2, but only limited change in the cell cycle and no change in methionine sulfoxide utilization. Expression in the Delta thioredoxin strain EMY63 allowed us to isolate a complex of the thioredoxin with YLR109, an abundant yeast protein related to PMP20, a peroxisomal protein of Candida. No function has so far been attributed to this protein or to the other numerous homologues described in plants, animals, fungi, and prokaryotes. On the basis of the complementation and of low similarity with peroxiredoxins, we produced YLR109 and one of its Arabidopsis homologues in E. coli to test their peroxiredoxins activity. We demonstrate that both recombinant proteins present a thioredoxin-dependent peroxidase activity in vitro. The possible functions of this new peroxiredoxin family are discussed.     

Enhanced formaldehyde detoxification by overexpression of glutathione-dependent formaldehyde dehydrogenase from Arabidopsis

The ADH2 gene codes for the Arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism in eukaryotes. In the present work, we have investigated the potential role of FALDH in detoxification of exogenous formaldehyde. We have generated a yeast (Saccharomyces cerevisiae) mutant strain (sfa1Delta) by in vivo deletion of the SFA1 gene that codes for the endogenous FALDH. Overexpression of Arabidopsis FALDH in this mutant confers high resistance to formaldehyde added exogenously, which demonstrates the functional conservation of the enzyme through evolution and supports its essential role in formaldehyde metabolism. To investigate the role of the enzyme in plants, we have generated Arabidopsis transgenic lines with modified levels of FALDH. Plants overexpressing the enzyme show a 25% increase in their efficiency to take up exogenous formaldehyde, whereas plants with reduced levels of FALDH (due to either a cosuppression phenotype or to the expression of an antisense construct) show a marked slower rate and reduced ability for formaldehyde detoxification as compared with the wild-type Arabidopsis. These results show that the capacity to take up and detoxify high concentrations of formaldehyde is proportionally related to the FALDH activity in the plant, revealing the essential role of this enzyme in formaldehyde detoxification.     

Complementation of the fol2 deletion in Saccharomyces cerevisiae by human and Escherichia coli genes encoding GTP cyclohydrolase I

Saccharomyces cerevisiae is so far the only organism where a knock-out mutant in the gene encoding GTP cyclohydrolase I (FOL2) has been obtained. GTP cyclohydrolase I controls the de novo biosynthetic pathway of tetrahydrobiopterin and folic acid. Since deletion of yeast FOL2 leads to a recessive auxotrophy for folinic acid, we used a yeast fol2Delta mutant for an in vivo functional assay of heterologous GTP cyclohydrolases I. We show that the GTP cyclohydrolase I, encoded either by the E. coli folE gene or by the human cDNA, complements the yeast fol2Delta mutation by restoring folate prototrophy. Furthermore the folE-3x allele of the E. coli gene, carrying three base substitutions, failed to complement the yeast fol2Delta defect. This allele behaved as a negative semidominant to the wild type folE and, when overexpressed, completely abolished complementation of fol2Delta by folE. Thus, the yeast fol2 null mutant is a suitable system to characterize mutations in genes encoding GTP cyclohydrolase I.