Conserved redox-dependent DNA binding of ROXY glutaredoxins with TGA transcription factors.
ABSTRACT: The Arabidopsis thaliana CC-type glutaredoxin (GRX) ROXY1 and the bZIP TGA transcription factor (TF) PERIANTHIA (PAN) interact in the nucleus and together regulate petal development. The CC-type GRXs exist exclusively in land plants, and in contrast to the ubiquitously occurring CPYC and CGFS GRX classes, only the CC-type GRXs expanded strongly during land plant evolution. Phylogenetic analyses show that TGA TFs evolved before the CC-type GRXs in charophycean algae. MpROXY1/2 and MpTGA were isolated from the liverwort Marchantia polymorpha to analyze regulatory ROXY/TGA interactions in a basal land plant. Homologous and heterologous protein interaction studies demonstrate that nuclear ROXY/TGA interactions are conserved since the occurrence of CC-type GRXs in bryophytes and mediated by a conserved ROXY C-terminus. Redox EMSA analyses show a redox-sensitive binding of MpTGA to the cis-regulatory as-1-like element. Furthermore, we demonstrate that MpTGA binds together with MpROXY1/2 to this motif under reducing conditions, whereas this interaction is not observed under oxidizing conditions. Remarkably, heterologous complementation studies reveal a strongly conserved land plant ROXY activity, suggesting an ancestral role for CC-type GRXs in modulating the activities of TGA TFs. Super-resolution microscopy experiments detected a strong colocalization of ROXY1 with the active form of the RNA polymerase II in the nucleus. Together, these data shed new light on the function of ROXYs and TGA TFs and the evolution of redox-sensitive transcription regulation processes, which likely contributed to adapt land plants to novel terrestrial habitats.
Project description:Glutaredoxins (GRXs) are ubiquitous glutathione-dependent oxidoreductase enzymes implicated in redox homeostasis, particularly oxidative stress response. Three major classes of GRX genes exist, the CPYC, CGFS classes are present in all pro- and eukaryote species, whereas the CC-type class GRXs are specific to land plants. In the basal land plant Physcomitrella patens, only two CC-type GRXs are present, compared with 21 in Arabidopsis. In contrast, sizes of the CPYC and CGFS classes remained rather similar throughout plant evolution, raising the interesting question as to when the CC-type GRXs first originated and how and why they expanded during land plant evolution. Recent evidence suggests that CC-type GRXs may have been recruited during evolution into diverse plant-specific functions of flower development (ROXY1, ROXY2) and pathogenesis response (ROXY19/GRX480). In the present study, GRX genes from the genomes of a range of green algae and evolutionarily diverse land plant species were identified; Ostreococcus, Micromonas, Volvox, Selaginella, Vitis, Sorghum, and Brachypodium. Previously identified sequences from Chlamydomonas, Physcomitrella, Oryza, Arabidopsis, and Populus were integrated to generate a more comprehensive understanding of the forces behind the evolution of various GRX classes. The analysis indicates that the CC-type GRXs probably arose by diversification from the CPYC class, at a time coinciding with colonization of land by plants. This strong differential expansion of the CC-type class occurred exclusively in the angiosperms, mainly through paleopolyploidy duplication events shortly after the monocot-eudicot split, and more recently through multiple tandem duplications that occurred independently in five investigated angiosperm lineages. The presented data suggest that following duplications, subfunctionalization, and subsequent neofunctionalization likely facilitated the sequestration of land plant-specific CC-type GRXs into novel functions such as development and pathogenesis response.
Project description:The Arabidopsis TGA transcription factor (TF) PERIANTHIA (PAN) regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG), which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly conserved regulatory AG cis-element, emphasizing the importance of redox-modifications in the regulation of flower developmental processes.
Project description:Glutaredoxins (Grxs) are a family of small multifunctional proteins involved in various cellular functions, including redox regulation and protection under oxidative stress. Despite the high number of Grx genes in plant genomes (48 Grxs in rice), the biological functions and physiological roles of most of them remain unknown. Here, the functional characterization of the two arsenic-responsive rice Grx family proteins, OsGrx_C7 and OsGrx_C2.1 are reported. Over-expression of OsGrx_C7 and OsGrx_C2.1 in transgenic Arabidopsis thaliana conferred arsenic (As) tolerance as reflected by germination, root growth assay, and whole plant growth. Also, the transgenic expression of OsGrxs displayed significantly reduced As accumulation in A. thaliana seeds and shoot tissues compared to WT plants during both AsIII and AsV stress. Thus, OsGrx_C7 and OsGrx_C2.1 seem to be an important determinant of As-stress response in plants. OsGrx_C7 and OsGrx_C2.1 transgenic showed to maintain intracellular GSH pool and involved in lowering AsIII accumulation either by extrusion or reducing uptake by altering the transcript of A. thaliana AtNIPs. Overall, OsGrx_C7 and OsGrx_C2.1 may represent a Grx family protein involved in As stress response and may allow a better understanding of the As induced stress pathways and the design of strategies for the improvement of stress tolerance as well as decreased As content in crops.
Project description:Glutaredoxins (GRXs) belong to the antioxidant and signalling network involved in the cellular response to oxidative stress in bacterial and eukaryotic cells. In spite of the high number of GRX genes in plant genomes, the biological functions and physiological roles of most of them remain unknown. Here the functional characterization of the Arabidopsis GRXS13 gene (At1g03850), that codes for two CC-type GRX isoforms, is reported. The transcript variant coding for the GRXS13.2 isoform is predominantly expressed under basal conditions and is the isoform that is induced by photooxidative stress. Transgenic lines where the GRXS13 gene has been knocked down show increased basal levels of superoxide radicals and reduced plant growth. These lines also display reduced tolerance to methyl viologen (MeV) and high light (HL) treatments, both conditions of photooxidative stress characterized by increased production of superoxide ions. Consistently, lines overexpressing the GRXS13.2 variant show reduced MeV- and HL-induced damage. Alterations in GRXS13 expression also affect superoxide levels and the ascorbate/dehydroascorbate ratio after HL-induced stress. These results indicate that GRXS13 gene expression is critical for limiting basal and photooxidative stress-induced reactive oxygen species (ROS) production. Together, these results place GRXS13.2 as a member of the ROS-scavenging/antioxidant network that shows a particularly low functional redundancy in the Arabidopsis GRX family.
Project description:Glutathione (GSH) was initially identified and characterized for its redox properties and later for its contributions to detoxification reactions. Over the past decade, however, the essential contributions of glutathione to cellular iron metabolism have come more and more into focus. GSH is indispensable in mitochondrial iron-sulfur (FeS) cluster biosynthesis, primarily by co-ligating FeS clusters as a cofactor of the CGFS-type (class II) glutaredoxins (Grxs). GSH is required for the export of the yet to be defined FeS precursor from the mitochondria to the cytosol. In the cytosol, it is an essential cofactor, again of the multi-domain CGFS-type Grxs, master players in cellular iron and FeS trafficking. In this review, we summarize the recent advances and progress in this field. The most urgent open questions are discussed, such as the role of GSH in the export of FeS precursors from mitochondria, the physiological roles of the CGFS-type Grx interactions with BolA-like proteins and the cluster transfer between Grxs and recipient proteins.
Project description:Despite their very close structural similarity, CxxC/S-type (class I) glutaredoxins (Grxs) act as oxidoreductases, while CGFS-type (class II) Grxs act as FeS cluster transferases. Here we show that the key determinant of Grx function is a distinct loop structure adjacent to the active site. Engineering of a CxxC/S-type Grx with a CGFS-type loop switched its function from oxidoreductase to FeS transferase. Engineering of a CGFS-type Grx with a CxxC/S-type loop abolished FeS transferase activity and activated the oxidative half reaction of the oxidoreductase. The reductive half-reaction, requiring the interaction with a second GSH molecule, was enabled by switching additional residues in the active site. We explain how subtle structural differences, mostly depending on the structure of one particular loop, act in concert to determine Grx function.
Project description:Glutaredoxins (GRXs) are glutathione-dependent oxidoreductase enzymes involved in a variety of cellular processes. In this study, our analysis revealed the presence of 48 genes encoding GRX proteins in the rice genome. GRX proteins could be classified into four classes, namely CC-, CGFS-, CPYC- and GRL-type, based on phylogenetic analysis. The classification was supported with organization of predicted conserved putative motifs in GRX proteins. We found that expansion of this gene family has occurred largely via whole genome duplication events in a species-specific manner. We explored rice oligonucleotide array data to gain insights into the function of GRX gene family members during various stages of development and in response to environmental stimuli. The comprehensive expression analysis suggested diverse roles of GRX genes during growth and development in rice. Some of the GRX genes were expressed in specific organs/developmental stages only. The expression of many of rice GRX genes was influenced by various phytohormones, abiotic and biotic stress conditions, suggesting an important role of GRX proteins in response to these stimuli. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provide a new avenue for in-depth characterization of selected genes of importance.
Project description:Glutaredoxins (GRXs) are small ubiquitous oxidoreductases involved in the regulation of the redox state in living cells. In an attempt to identify the full complement of GRXs in the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, three additional GRX homologs, besides the formerly characterized GintGRX1 (renamed here as RiGRX1), were identified. The three new GRXs (RiGRX4, RiGRX5 and RiGRX6) contain the CXXS domain of monothiol GRXs, but whereas RiGRX4 and RiGRX5 belong to class II GRXs, RiGRX6 belongs to class I together with RiGRX1. By using a yeast expression system, we observed that the newly identified homologs partially reverted sensitivity of the GRX deletion yeast strains to external oxidants. Furthermore, our results indicated that RiGRX4 and RiGRX5 play a role in iron homeostasis in yeast. Gene expression analyses revealed that RiGRX1 and RiGRX6 were more highly expressed in the intraradical (IRM) than in the extraradical mycelium (ERM). Exposure of the ERM to hydrogen peroxide induced up-regulation of RiGRX1, RiGRX4 and RiGRX5 gene expression. RiGRX4 expression was also up-regulated in the ERM when the fungus was grown in media supplemented with a high iron concentration. These data indicate the two monothiol class II GRXs, RiGRX4 and RiGRX5, might be involved in oxidative stress protection and in the regulation of fungal iron homeostasis. Increased expression of RiGRX1 and RiGRX6 in the IRM suggests that these GRXs should play a key role in oxidative stress protection of R. irregularis during its in planta phase.
Project description:Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The class III GRX gene family is unique to land plants, and Arabidopsis thaliana has 21 class III GRXs, which remain largely uncharacterized. About 80% of A. thaliana class III GRXs are transcriptionally regulated by nitrate, and several recent studies have suggested roles for these GRXs in nitrogen signaling. Our objective was to functionally characterize two nitrate-induced GRX genes, AtGRXS5 and AtGRXS8, defining their roles in signaling and development in the A. thaliana root. We demonstrated that AtGRXS5 and AtGRXS8 are primarily expressed in root and shoot vasculature (phloem), and that the corresponding GRX proteins display nucleo-cytosolic subcellular localization. Ectopic expression of AtGRXS8 in transgenic plants caused major alterations in root system architecture: Normal primary root development, but a near absence of lateral roots. RNA sequencing demonstrated that the roots of AtGRXS8-overexpressing plants show strongly reduced transcript abundance for many primary nitrate response genes, including the major high-affinity nitrate transporters. Correspondingly, high-affinity nitrate uptake and the transport of nitrate from roots to shoots are compromised in AtGRXS8-overexpressing plants. Finally, we demonstrated that the AtGRXS8 protein can physically interact with the TGA1 and TGA4 transcription factors, which are central regulators of early transcriptional responses to nitrate in A. thaliana roots. Overall, these results suggest that AtGRXS8 acts to quench both transcriptional and developmental aspects of primary nitrate response, potentially by interfering with the activity of the TGA1 and TGA4 transcription factors.
Project description:Glutaredoxins (Grxs) are a family of glutathione (GSH)-dependent thiol-disulfide oxidoreductases. They feature GSH-binding sites that directly connect the reversible redox chemistry of protein thiols to the abundant cellular nonprotein thiol pool GSSG/GSH. This work studied the pathways for oxidation of protein dithiols P(SH)2 and reduction of protein disulfides P(SS) catalyzed by Homo sapiens HsGrx1 and Escherichia coli EcGrx1. The metal-binding domain HMA4n(SH)2 was chosen as substrate as it contains a solvent-exposed CysCys motif. Quenching of the reactions with excess iodoacetamide followed by protein speciation analysis via ESI-MS allowed interception and characterization of both substrate and enzyme intermediates. The enzymes shuttle between three catalytically-competent forms (Grx(SH)(S-), Grx(SH)(SSG) and Grx(SS)) and employ conserved parallel monothiol and dithiol mechanisms. Experiments with dithiol and monothiol versions of both Grx enzymes demonstrate which monothiol (plus GSSG or GSH) or dithiol pathways dominate a specific oxidation or reduction reaction. Grxs are shown to be a class of versatile enzymes with diverse catalytic functions that are driven by specific interactions with GSSG/GSH.