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AtGRX4, an Arabidopsis chloroplastic monothiol glutaredoxin, is able to suppress yeast grx5 mutant phenotypes and respond to oxidative stress
FEBS Letters 582 (2008) 848–854
AtGRX4, an Arabidopsis chloroplastic monothiol glutaredoxin, is able to suppress yeast grx5 mutant phenotypes and respond to oxidative stress Ning-Hui Cheng* Plant Physiology Group, USDA/ARS ChildrenÕs Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030, USA Received 12 November 2007; revised 1 February 2008; accepted 5 February 2008 Available online 12 February 2008 Edited by Julian Schroeder
Abstract Arabidopsis monothiol glutaredoxin (Grx), AtGRX4, was targeted to chloroplasts/plastids and had high similarity to yeast Grx5. In yeast expression assays, AtGRX4 localized to the mitochondria and suppressed the sensitivity of grx5 cells to oxidants. In addition, AtGRX4 reduced iron accumulation and rescued the lysine auxotrophy of grx5 cells. In planta, AtGRX4 RNA transcripts accumulated in growing tissues. Furthermore, AtGRX4expression was altered under various stresses. Genetic analysis revealed that seedlings of atgrx4 mutants were sensitive to oxidants. Taken together, these results suggest that AtGRX4 may have important functions in plant growth and development under extreme environments. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Glutaredoxin; Oxidative stress; Redox signaling; Chloroplast; Plastid; Arabidopsis thaliana
1. Introduction Reactive oxygen species (ROS) generated through activation of various oxidases and peroxidases can act as secondary messengers to trigger numerous stress-related signal cascades in response to internally developmental cues and/or externally environmental stimuli [1]. However, due to their cytotoxic nature and often excess accumulation as by-products during photosynthesis and carbon metabolism, ROS also cause wide-ranging damage to macromolecules [2]. Plants must orchestrate an elaborate antioxidant network to overcome such oxidative damage and control signaling events [1]. Of those antioxidant systems, the physiological roles of glutaredoxins (Grxs) have not been fully defined [3,4]. Grxs are disulfide oxidoreductases (thioltransferase) and conserved in both prokaryotes and eukaryotes [3,4]. Biochemical analyses of Grxs (dithiol Grx) from various organisms reveals that this group of proteins can catalyze the reduction of protein disulfides and of glutathione (GSH)-protein mixed disulfides via a dithiol or monothiol mechanism [5]. As a result, Grxs appear to be involved in many cellular processes and regulation of redox homeostasis [6,7]. Recently, a new group of Grxs has been identified [8]. These Grxs contain a single cysteine residue in the putative * Fax: +1 713 798 7078. E-mail address: [email protected]
motif, ‘‘CGFS’’, thus are termed monothiol Grxs [8]. Monothiol Grxs were initially identified in yeast (Grx3, 4, and 5), then found in all types of living cells [9]. There is a growing body of evidence that monothiol Grxs may have multiple functions in biogenesis of mitochondrial iron–sulfur clusters, detoxification of cytotoxin, cell growth and proliferation, regulation of iron homeostasis, Ca2+ signaling, protein kinase C (PKC) mediated stress responses, and protection of protein oxidation [9]. Furthermore, biochemical studies indicate that monothiol Grxs consist of unique structural features, implicating that those Grxs have distinct functions [10–12]. Photosynthetic organisms, including higher plants, have a large Grx gene family; however, until recently, only a few plant Grxs have been studied [4]. In dicots, Arabidopsis and poplar genomes contain at least 36 and 31 ORFs coding for putative Grxs, respectively, while in the rice genome, there are 27 putative Grxs identified [13,14]. In contrast to other species, plant Grxs can be divided into three major classes: dithiol, monothiol, and a unique CC-type Grxs [13,14]. Most recently, structure-function analyses indicate that poplar Grx C1, like human Grx2, contains a [2Fe–2S] cluster in the holo form, suggesting a potential role of this Grx in sensing oxidative stress and iron– sulfur biosynthesis [15]. Previously, we isolated and characterized the first plant monothiol Grx, AtGRXcp, which was targeting to chloroplasts and played a critical role in redox regulation in chloroplasts [16]. In the present work, we identify and isolate another member of monothiol Grxs from Arabidopsis thaliana, termed AtGRX4, and examine the subcellular location of AtGRX4. We characterize the function of AtGRX4 using yeast expression assays and determine AtGRX4 expression and altered responses to stresses. We further isolate AtGRX4 knockout mutants and describe the phenotypes of these plants. Our results show a conserved function among those Arabidopsis monothiol Grxs.
2. Materials and methods 2.1. Isolation of AtGRX4 null alleles To isolate atgrx4 alleles, two T-DNA insertional mutant lines were obtained from the SALK and SAIL T-DNA collection [17,18]. Homozygous plants from each T3 generation were obtained by PCR screening using AtGRX4-specific and T-DNA border primers (Table S1). The location of the T-DNA insertion was determined by sequencing the PCR product. Both atgrx4 alleles were backcrossed to their respective parental plants to remove any potential unlinked mutations.
0014-5793/$34.00 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.02.006
N.-H. Cheng / FEBS Letters 582 (2008) 848–854 2.2. Plant growth conditions Arabidopsis wild-type (ecotype Columbia, Col-0) and atgrx4 mutant seeds were surface-sterilized, germinated and grown on one-half strength Murashige and Skoog (MS) medium solidified with 0.8% agar and the same media supplemented with various concentrations of H2O2 and diamide. Diamide is a sulfhydryl reagent which oxidizes sulfhydryl groups of proteins to the disulfide form. Iron-sufficient and -deficient media were made following the published protocol [16]. 2.3. DNA constructs and yeast growth assays The full-length cDNA of AtGRX4was amplified by using a gene specific primer (Table S1). The fidelity of all clones was confirmed by sequencing. In order to express AtGRX4 in yeast cells, the full-length cDNA of AtGRX4 was subcloned into yeast expression vector, piUGpd [16]. Yeast strains used in this study and growth assays were followed the published protocol [16]. 2.4. Localization of AtGRX4-GFP fusions in yeast and plant cells Full-length AtGRX4 was fused to the N-terminus of green fluorescent protein (GFP) using a procedure described previously [16]. The AtGRX4-GFP fusion was then subcloned into yeast and plant expression vectors as described previously [16]. In yeast, AtGRX4-GFP was imaged in comparison with AtGRXcp-GFP and the mitochondrial marker, COX6a-DsRed fusion. Cytochrome c Oxidase subunit VI (COX6a) encoded by an Arabidopsis gene (At4g37830) is a mitochondria-resident protein [19]. In plant cells, the subcellular localization of the fused protein was imaged in comparison with labeled organelle markers-chloroplasts, mitochondria, Golgi, and peroxisome-as described previously [16].
849 2.5. AtGRX4::GUS transgenic plants and histochemical analysis A 505 bp DNA sequence upstream of the ATG of AtGRX4 ORF was amplified from genomic DNA by using gene promoter forward and reverse primers (Table S1). The PCR fragment was cloned into pBI121 to replace the 35S promoter, resulting in plasmid pAtGRX4::GUS. Agrobacterium transformation of Arabidopsis plants was performed as described previously [16]. Histochemical analysis was performed following the published protocol [16]. 2.6. RNA isolation and northern blotting analysis RNA isolation and northern blotting analysis was conducted according to the protocol described previously [16].
3. Results 3.1. AtGRX4 is a member of monothiol glutaredoxins and suppresses the sensitivity of yeast grx5 cells to H2O2 Searching for a new member of the monothiol Grx family in the Arabidopsis genome database revealed that there exists another ORF coding a polypeptide similar to AtGRXcp (Fig. 1). This new Arabidopsis gene was termed AtGRX4 (At3G15660). AtGRX4 cDNA was cloned by a RT-PCR approach using gene specific primers. Sequence analysis indicated that AtGRX4 had 24.5% identity to yeast Grx5 at the amino acid
Fig. 1. Sequence analyses of monothiol Grxs. Alignment of monothiol Grx sequences was performed with ClustalW software (http://www.ebi.ac.uk/ clustalw/). The conserved putative motif ‘‘CGFS’’ and cysteine residue are labeled with asterisk and arrowhead, respectively. AtGRXcp (At3g54900; AY157988), AtGRX4 (At3g15660; BAB02297), EcGrx4 (P37010), DrGrx5 (AAZ30729), HsGrx5 (AAH47680), MmGrx5 (Q80Y14), PfGLP-1 (CAB38997), and ScGrx5 (Q02784). At, Arabidopsis thaliana; Dr, Danio rerio; Ec, Escherichia coli; Hs, Homo sapiens; Mm, Mus musculus; Pf, Plasmodium falciparum; Sc, Saccharomyces cerevisiae.
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level and contained one Grx-domain at its C-terminus with the conserved putative motif ‘‘CGFS’’ (Fig. 1). To determine if AtGRX4 could complement Grx5 function and suppress the sensitivity of grx5 cells to oxidant, such as H2O2, we expressed vector control, yeast Grx5, and AtGRX4 in grx5 cells. All yeast strains grew normally in YPD liquid media (rich media) after 24 h growth (Fig. 2A). While vector-expressing grx5 cells were growth impaired in the medium containing 1 mM H2O2, both AtGRX4-, and Grx5-expressing grx5 cells grew in a similar manner to wild-type cells (Fig. 2A). These observations suggest that AtGRX4 is able to suppress the sensitivity of grx5 to oxidative stress. Given that yeast Grx5 is a mitochondrial Grx [20], the suppression of grx5 phenotypes suggests that subcellular localization of AtGRX4 in yeast cells could be similar to yeast Grx5. To investigate this, AtGRX4-GFP was expressed in yeast cells.
N.-H. Cheng / FEBS Letters 582 (2008) 848–854
Yeast growth assays revealed that AtGRX4-GFP could also suppress the sensitivity of grx5 cells to H2O2 (Fig. 2A). A previous study demonstrated that AtGRXcp was targeting to mitochondria when expressed in yeast cells (Fig. 2B; [16]). Similarly, the AtGRX4-GFP is co-localized with COX6a-DsRed in mitochondria (Fig. 2C). 3.2. AtGRX4 is able to rescue the lysine auxotrophy of a yeast grx5 mutant and suppress iron accumulation in grx5 cells Yeast Grx5 deletion mutants are defective in lysine synthesis due to disruption of the maturation of a mitochondrial Fe–S cluster-containing enzyme, homoaconitase [20]. As a result, grx5 cells failed to grow on lysine deficient media, while wild-type cells grew very well on the same media (Fig. 3; [20]). Expression of AtGRX4 and AtGRX4-GFP rescued the lysine auxotrophy of grx5 cells in a similar manner to
Fig. 2. AtGRX4 is able to suppress the sensitivity of grx5 cells to H2O2. (A) Vector-, AtGRX4-, AtGRX4-GFP, and Grx5-expressing grx5 cells were grown in YPD liquid media and the same media supplemented with 1.0 mM H2O2. Cell density was measured at A600 after 24 h growth at 30 C. Shown is one representative experiment from three independent experiments conducted. The bars indicate the standard error (n = 6) (***P < 0.001). (B and C) Co-localization of AtGRXcp-GFP (B) and AtGRX4-GFP (C) with mitochondrial markers (Cox6a-DsRed fusion) in yeast cells. BF: bright field. Scale bars = 10 lm.
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Fig. 3. AtGRX4 rescues the lysine auxotrophy of grx5 mutant. Wild-type expressing vector, grx5 cells expressing vector, Grx5, AtGRX4, and AtGRX4-GFP were assayed on SC medium with or without lysine. The photographs were taken after 3 day growth at 30 C.
Grx5-expressing grx5 cells (Fig. 3). This result indicates that AtGRX4 can restore Grx5 function in yeast. Deletion of Grx5 disrupts iron–sulfur cluster assembly and as a consequence, increases the level of intracellular iron [20]. This iron accumulation results in the production of toxic radicals through the iron-mediated Fenton reaction [21]. We have demonstrated that AtGRX4 could protect yeast cells against oxidative damage (Figs. 2 and 3). To further delineate the potential function of AtGRX4, we assayed the iron content in wild-type and grx5 cells expressing vector, Grx5, and AtGRX4. As shown in Fig. 4, AtGRX4- and Grx5-expressing cells were both able to suppress the iron accumulation (as measured at the intracellular iron level). 3.3. AtGRX4 is a chloroplast/plastid-localized monothiol Grx Sequence analysis using ChloroP program (version 1.1) predicted that AtGRX4 contained a chloroplastic/plastidic signal peptide [4,13]. In order to experimentally verify this, AtGRX4 was fused with the green fluorescent protein (GFP) at its C-terminus and the fusion was transiently expressed in tobacco leaf cells. Indeed, AtGRX4-GFP, like AtGRXcp-GFP, predominately localized to chloroplasts, rather than mitochondria,
Golgi, or peroxisomes in epidermal cells (Fig. 5; data not shown). Because AtGRX4 is not highly expressed in green tissues, like leaves, it will be expected that AtGRX4-GFP targets to plastids as well. 3.4. AtGRX4 expression is induced by various stresses AtGRX4 is ubiquitously expressed in all tissues (Fig. 1S; data not shown). In order to understand the function of AtGRX4 under stress conditions, AtGRX4 expression was analyzed upon various stress treatments. When 10-day-old seedlings were exposed to different sources of lights, AtGRX4 expression was down-regulated in response to light treatments (Fig. 6A). Interestingly, AtGRX4 expression was differentially regulated with various temperature treatments. In detail, AtGRX4 was induced in the cold (4 C) treatment, but down-regulated upon heat shocks (37 and 45 C; Fig. 6B). As shown in Fig. 6C, AtGRX4 was constantly induced by water stress (submerging the plant). Furthermore, AtGRX4 expression was altered by metal–ion stresses (Fig. 6D). For example, AtGRX4 was induced by mixed nutrient solution (MS), but down-regulated by NaCl and ZnCl2 (Fig. 6D).
Fig. 4. AtGRX4 suppresses free iron accumulation in grx5 cells. Intracellular iron content was examined with a QuantiChron Iron Assay Kit (BioAssay Systems, Hayward, CA, USA). All results shown here are the means of three independent experiments, and the bars indicate the standard errors (**P < 0.01; ***P < 0.001).
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N.-H. Cheng / FEBS Letters 582 (2008) 848–854
Fig. 5. Subcellular localization of AtGRX4-GFP in plant cells. AtGRX4-GFP is localized to chloroplasts in tobacco cells. Left panels display the transient expression of AtGRX4-GFP in tobacco epidermal cells; central panels display fluorescing chloroplasts; right panels show the merged images. Scale bars = 25 lm.
Fig. 6. AtGRX4 expression in response to various stress stimuli. (A) AtGRX4 was expressed in response to various light sources. Total RNA samples were extracted from 10-day-old Col-0 seedlings (lane1) and seedlings grown in greenhouse (natural sunlight) for 3 h (lane 2), UV light for 15 min (lane 3) and 30 min (lane 4). (B) AtGRX4 was expressed in response to temperature stresses. 10-day-old seedlings grown at 22 C (lane1), 4 C for 2 days (lane2), grown at 28 C (lane3), 37 C for 1.5 h (lane 4), and 45 C for 1 h (lane 5). (C) AtGRX4 was expressed in response to submerging treatments. 10-day-old seedlings were subjected to submerging treatment for 0 day (lane 1), 1 day (lane 2), 2 days (lane3), 3 days (lane 4), and 4 days (lane 5). (D) AtGRX4 expression in response to metal-ion treatments. 3-week-old seedlings were previously treated for 12 h with the following solutions: water (as a control, lane 1), Murashige and Skoog (MS) nutrient media (lane 2), 80 mM NaCl (lane 3), 1 mM ZnCl2 (lane 4), 2 mM MnCl2 (lane 5), 0.1 mM NiSO4 (lane 6), and 50 mM CaCl2 (lane 7). Twenty micrograms of total RNA were hybridized with gene-specific probes for AtGRX4. Ethidium bromide (EtBr) stained rRNA are shown as a loading control.
3.5. Analysis of atgrx4 mutants To gain insight into the function of AtGRX4 in planta, we analyzed two T-DNA insertional lines of AtGRX4 (Fig. 7A). Sequence analysis of the T-DNA flanking regions revealed that in atgrx4-1(Salk_112767) the T-DNA was located in the 5Õ-
UTR at position 331nt upstream of the ATG of AtGRX4, and in atgrx4-2 (Sail_431_H03) the T-DNA was inserted at position Lys-24 (Fig. 7A). AtGRX4 expression was reduced in atgrx4-1 allele, and not detectable in atgrx4-2 allele as determined by RNA blot analysis (Fig. 2S). Both atgrx4-1 and atgrx4-2 did not display visible defects in seedling growth, flowering, and seed production when grown in soil under normal condition in comparison with wild-type (data not shown). When wild-type and atgrx4 seeds were grown on 1/2 MS media, atgrx4 seedlings grew slightly shorter in comparison to wild-type. The primary root length of atgrx4 seedlings were 88% (atgrx4-1) and 79% (atgrx4-2) of that of wild-type (Fig. 7B and C). However, when grown on the same media containing 3 mM H2O2, atgrx4-1 and atgrx4-2 had 45% and 57% reduction in primary root growth compared to wildtype (Fig. 7B and C). Apparently, root growth of wild-type and atgrx4 seedlings was inhibited by H2O2 if compared to that on 1/2 MS media. However, the inhibition of atgrx4 seedlings is significant greater than that of wild-type (78% in atgrx4-1 and 80% in atgrx4-2 compared to 65% in wild-type, P < 0.0001). When tested on the media containing 0.4 mM diamide, both alleles displayed 23% and 25% reduction of primary root growth, but this reduction caused by diamide is not significantly different from that of controls (data not shown). In addition, no difference was seen between wild-type and atgrx4 seedlings tested on iron-sufficient or iron-deficient media (data not shown).
4. Discussion In the present study, our findings reveal that AtGRX4 is a functional monothiol Grx localized to chloroplasts/plastids and able to complement yeast Grx5 function (Figs. 2–5). We also demonstrate that AtGRX4 expression is regulated under various environmental stimuli and loss of AtGRX4 in Arabidopsis leads
N.-H. Cheng / FEBS Letters 582 (2008) 848–854
Fig. 7. AtGRX4 knockout mutants are sensitive to external oxidants. (A) Genomic structure of AtGRX4 showing the sites of T-DNA insertions. (B) Wild-type (Col-0) and atgrx4 allele seeds were germinated and grown on 1/2 MS and the same media supplemented with 3 mM H2O2 for ten days. Shown is one representative experiment of three independent experiments. (C) Root growth measurement. The length of primary roots of seedlings was measured after 10 days of growth on the medium with or without H2O2. Error bars represent the standard error (n P 15; StudentÕs t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
to seedlings sensitive to oxidants (Figs. 6 and 7), suggesting a critical role of AtGRX4 in regulating redox state in plant growth and development. AtGRX4, like AtGRXcp, targets to mitochondria when expressed in yeast and also suppresses yeast grx5 mutant phenotypes (Figs. 2–4). It has been shown that all Grx5 homologues tested from various species are able to complement yeast Grx5 function [9]. These findings demonstrate the functional interchangeability among those monothiol Grxs [9]. Interestingly, entire AtGRX4 has only one cysteine residue within the ‘‘CGFS’’ motif, while AtGRXcp has four cysteine residues (Fig. 1). The ability of AtGRX4 to restore Grx5 function suggests that Cys91 is sufficient for AtGRX4 function. This finding is consistent with the previous report where Cys60, but not
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Cys171, is essential for yeast Grx5 function [10]. However, subsequent analysis indicates that Cys171 also has an important role in the formation of an intramolecular disulfide bond between Cys60 and Cys171 during catalysis [11]. In the present study, the biochemical property of AtGRX4 has been initially analyzed, but like AtGRXcp, there are no enzymatic activities in the insulin reduction and the b-hydroxyethyl disulfide (HED) assays defined in AtGRX4 (data not shown) [16]. Given the functional similarity in yeast complementation assay and the structural diversity of cysteine residues, AtGRX4 and AtGRXcp provide a unique platform to further delineate the structure-function relationship in monothiol Grxs. Both AtGRX4 and AtGRXcp are localized to chloroplasts/ plastids, implicating a critical role these Grxs may play in these organelles. The ability of AtGRX4 and AtGRXcp to partially rescue the Fe–S enzyme activities and suppress iron accumulation in yeast grx5 cells suggests that these AtGRXs may be required for biogenesis of Fe–S clusters in chloroplasts/plastids. Although the direct evidence for proving this function is pending, several recent studies suggest a connection between Grxmediated redox regulation and Fe–S cluster biosynthesis in chloroplasts/plastids [9]. In addition, different, but partially overlapping biosynthesis pathways for mitochondrial and plastidic Fe–S clusters (the Isc and Suf systems, respectively) could explain why AtGRXs only partially restored Grx5 function in vivo (Fig. 3) [9,16]. It is also possible that AtGRX4 (AtGRXcp) suppression of yeast grx5 mutant phenotypes is due to general activation of the ROS scavenging system in yeast (Figs. 2–4). In support of this hypothesis, recent studies indicate that yeast Grx3, a nuclear-localized monothiol Grx, complements Grx5 function when targeting to mitochondria [22]. However, rather than functioning as Grx5 in Fe–S biosynthesis, Grx3 globally regulates iron homeostasis through interacting with a transcriptional factor, Aft1 [23,24]. Future work will be necessary to determine the target(s) of AtGRXs and understand the complexity of ROS regulation in planta. Recent reports have shown that Arabidopsis has high steady-state levels of protein oxidation during vegetative growth and under stress conditions [25,26]. Altered expression of AtGRX4 in response to various environmental stimuli suggests that AtGRX4 may play an important role in growth and development. In agreement with this, atgrx4 roots are more sensitive to H2O2 (Fig. 7). Although atgrx4 plants did not display any altered sensitivity to iron imbalance (data not shown), this may be due to tight regulation of redox state at the whole plant level or compensation by functional redundancy among Grxs [4,13,14]. The interplay among Grxs and various antioxidant systems will be addressed in future studies. Acknowledgements: We are very grateful to Dr. Elison Blancaflor for helping in confocal imaging, Drs. Toshiro Shigaki and Jon Pittman for critical reading of the manuscript. This work is supported by United State Department of Agriculture/Agricultural Research Service under Cooperation Agreement 6250-21520-042-02.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet. 2008.02.006.
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AtGRX4, an Arabidopsis chloroplastic monothiol glutaredoxin, is able to suppress yeast grx5 mutant phenotypes and respond to oxidative stress Ning-Hui Cheng* Plant Physiology Group, USDA/ARS ChildrenÕs Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030, USA Received 12 November 2007; revised 1 February 2008; accepted 5 February 2008 Available online 12 February 2008 Edited by Julian Schroeder
Abstract Arabidopsis monothiol glutaredoxin (Grx), AtGRX4, was targeted to chloroplasts/plastids and had high similarity to yeast Grx5. In yeast expression assays, AtGRX4 localized to the mitochondria and suppressed the sensitivity of grx5 cells to oxidants. In addition, AtGRX4 reduced iron accumulation and rescued the lysine auxotrophy of grx5 cells. In planta, AtGRX4 RNA transcripts accumulated in growing tissues. Furthermore, AtGRX4expression was altered under various stresses. Genetic analysis revealed that seedlings of atgrx4 mutants were sensitive to oxidants. Taken together, these results suggest that AtGRX4 may have important functions in plant growth and development under extreme environments. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Glutaredoxin; Oxidative stress; Redox signaling; Chloroplast; Plastid; Arabidopsis thaliana
1. Introduction Reactive oxygen species (ROS) generated through activation of various oxidases and peroxidases can act as secondary messengers to trigger numerous stress-related signal cascades in response to internally developmental cues and/or externally environmental stimuli [1]. However, due to their cytotoxic nature and often excess accumulation as by-products during photosynthesis and carbon metabolism, ROS also cause wide-ranging damage to macromolecules [2]. Plants must orchestrate an elaborate antioxidant network to overcome such oxidative damage and control signaling events [1]. Of those antioxidant systems, the physiological roles of glutaredoxins (Grxs) have not been fully defined [3,4]. Grxs are disulfide oxidoreductases (thioltransferase) and conserved in both prokaryotes and eukaryotes [3,4]. Biochemical analyses of Grxs (dithiol Grx) from various organisms reveals that this group of proteins can catalyze the reduction of protein disulfides and of glutathione (GSH)-protein mixed disulfides via a dithiol or monothiol mechanism [5]. As a result, Grxs appear to be involved in many cellular processes and regulation of redox homeostasis [6,7]. Recently, a new group of Grxs has been identified [8]. These Grxs contain a single cysteine residue in the putative * Fax: +1 713 798 7078. E-mail address: [email protected]
motif, ‘‘CGFS’’, thus are termed monothiol Grxs [8]. Monothiol Grxs were initially identified in yeast (Grx3, 4, and 5), then found in all types of living cells [9]. There is a growing body of evidence that monothiol Grxs may have multiple functions in biogenesis of mitochondrial iron–sulfur clusters, detoxification of cytotoxin, cell growth and proliferation, regulation of iron homeostasis, Ca2+ signaling, protein kinase C (PKC) mediated stress responses, and protection of protein oxidation [9]. Furthermore, biochemical studies indicate that monothiol Grxs consist of unique structural features, implicating that those Grxs have distinct functions [10–12]. Photosynthetic organisms, including higher plants, have a large Grx gene family; however, until recently, only a few plant Grxs have been studied [4]. In dicots, Arabidopsis and poplar genomes contain at least 36 and 31 ORFs coding for putative Grxs, respectively, while in the rice genome, there are 27 putative Grxs identified [13,14]. In contrast to other species, plant Grxs can be divided into three major classes: dithiol, monothiol, and a unique CC-type Grxs [13,14]. Most recently, structure-function analyses indicate that poplar Grx C1, like human Grx2, contains a [2Fe–2S] cluster in the holo form, suggesting a potential role of this Grx in sensing oxidative stress and iron– sulfur biosynthesis [15]. Previously, we isolated and characterized the first plant monothiol Grx, AtGRXcp, which was targeting to chloroplasts and played a critical role in redox regulation in chloroplasts [16]. In the present work, we identify and isolate another member of monothiol Grxs from Arabidopsis thaliana, termed AtGRX4, and examine the subcellular location of AtGRX4. We characterize the function of AtGRX4 using yeast expression assays and determine AtGRX4 expression and altered responses to stresses. We further isolate AtGRX4 knockout mutants and describe the phenotypes of these plants. Our results show a conserved function among those Arabidopsis monothiol Grxs.
2. Materials and methods 2.1. Isolation of AtGRX4 null alleles To isolate atgrx4 alleles, two T-DNA insertional mutant lines were obtained from the SALK and SAIL T-DNA collection [17,18]. Homozygous plants from each T3 generation were obtained by PCR screening using AtGRX4-specific and T-DNA border primers (Table S1). The location of the T-DNA insertion was determined by sequencing the PCR product. Both atgrx4 alleles were backcrossed to their respective parental plants to remove any potential unlinked mutations.
0014-5793/$34.00 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.02.006
N.-H. Cheng / FEBS Letters 582 (2008) 848–854 2.2. Plant growth conditions Arabidopsis wild-type (ecotype Columbia, Col-0) and atgrx4 mutant seeds were surface-sterilized, germinated and grown on one-half strength Murashige and Skoog (MS) medium solidified with 0.8% agar and the same media supplemented with various concentrations of H2O2 and diamide. Diamide is a sulfhydryl reagent which oxidizes sulfhydryl groups of proteins to the disulfide form. Iron-sufficient and -deficient media were made following the published protocol [16]. 2.3. DNA constructs and yeast growth assays The full-length cDNA of AtGRX4was amplified by using a gene specific primer (Table S1). The fidelity of all clones was confirmed by sequencing. In order to express AtGRX4 in yeast cells, the full-length cDNA of AtGRX4 was subcloned into yeast expression vector, piUGpd [16]. Yeast strains used in this study and growth assays were followed the published protocol [16]. 2.4. Localization of AtGRX4-GFP fusions in yeast and plant cells Full-length AtGRX4 was fused to the N-terminus of green fluorescent protein (GFP) using a procedure described previously [16]. The AtGRX4-GFP fusion was then subcloned into yeast and plant expression vectors as described previously [16]. In yeast, AtGRX4-GFP was imaged in comparison with AtGRXcp-GFP and the mitochondrial marker, COX6a-DsRed fusion. Cytochrome c Oxidase subunit VI (COX6a) encoded by an Arabidopsis gene (At4g37830) is a mitochondria-resident protein [19]. In plant cells, the subcellular localization of the fused protein was imaged in comparison with labeled organelle markers-chloroplasts, mitochondria, Golgi, and peroxisome-as described previously [16].
849 2.5. AtGRX4::GUS transgenic plants and histochemical analysis A 505 bp DNA sequence upstream of the ATG of AtGRX4 ORF was amplified from genomic DNA by using gene promoter forward and reverse primers (Table S1). The PCR fragment was cloned into pBI121 to replace the 35S promoter, resulting in plasmid pAtGRX4::GUS. Agrobacterium transformation of Arabidopsis plants was performed as described previously [16]. Histochemical analysis was performed following the published protocol [16]. 2.6. RNA isolation and northern blotting analysis RNA isolation and northern blotting analysis was conducted according to the protocol described previously [16].
3. Results 3.1. AtGRX4 is a member of monothiol glutaredoxins and suppresses the sensitivity of yeast grx5 cells to H2O2 Searching for a new member of the monothiol Grx family in the Arabidopsis genome database revealed that there exists another ORF coding a polypeptide similar to AtGRXcp (Fig. 1). This new Arabidopsis gene was termed AtGRX4 (At3G15660). AtGRX4 cDNA was cloned by a RT-PCR approach using gene specific primers. Sequence analysis indicated that AtGRX4 had 24.5% identity to yeast Grx5 at the amino acid
Fig. 1. Sequence analyses of monothiol Grxs. Alignment of monothiol Grx sequences was performed with ClustalW software (http://www.ebi.ac.uk/ clustalw/). The conserved putative motif ‘‘CGFS’’ and cysteine residue are labeled with asterisk and arrowhead, respectively. AtGRXcp (At3g54900; AY157988), AtGRX4 (At3g15660; BAB02297), EcGrx4 (P37010), DrGrx5 (AAZ30729), HsGrx5 (AAH47680), MmGrx5 (Q80Y14), PfGLP-1 (CAB38997), and ScGrx5 (Q02784). At, Arabidopsis thaliana; Dr, Danio rerio; Ec, Escherichia coli; Hs, Homo sapiens; Mm, Mus musculus; Pf, Plasmodium falciparum; Sc, Saccharomyces cerevisiae.
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level and contained one Grx-domain at its C-terminus with the conserved putative motif ‘‘CGFS’’ (Fig. 1). To determine if AtGRX4 could complement Grx5 function and suppress the sensitivity of grx5 cells to oxidant, such as H2O2, we expressed vector control, yeast Grx5, and AtGRX4 in grx5 cells. All yeast strains grew normally in YPD liquid media (rich media) after 24 h growth (Fig. 2A). While vector-expressing grx5 cells were growth impaired in the medium containing 1 mM H2O2, both AtGRX4-, and Grx5-expressing grx5 cells grew in a similar manner to wild-type cells (Fig. 2A). These observations suggest that AtGRX4 is able to suppress the sensitivity of grx5 to oxidative stress. Given that yeast Grx5 is a mitochondrial Grx [20], the suppression of grx5 phenotypes suggests that subcellular localization of AtGRX4 in yeast cells could be similar to yeast Grx5. To investigate this, AtGRX4-GFP was expressed in yeast cells.
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Yeast growth assays revealed that AtGRX4-GFP could also suppress the sensitivity of grx5 cells to H2O2 (Fig. 2A). A previous study demonstrated that AtGRXcp was targeting to mitochondria when expressed in yeast cells (Fig. 2B; [16]). Similarly, the AtGRX4-GFP is co-localized with COX6a-DsRed in mitochondria (Fig. 2C). 3.2. AtGRX4 is able to rescue the lysine auxotrophy of a yeast grx5 mutant and suppress iron accumulation in grx5 cells Yeast Grx5 deletion mutants are defective in lysine synthesis due to disruption of the maturation of a mitochondrial Fe–S cluster-containing enzyme, homoaconitase [20]. As a result, grx5 cells failed to grow on lysine deficient media, while wild-type cells grew very well on the same media (Fig. 3; [20]). Expression of AtGRX4 and AtGRX4-GFP rescued the lysine auxotrophy of grx5 cells in a similar manner to
Fig. 2. AtGRX4 is able to suppress the sensitivity of grx5 cells to H2O2. (A) Vector-, AtGRX4-, AtGRX4-GFP, and Grx5-expressing grx5 cells were grown in YPD liquid media and the same media supplemented with 1.0 mM H2O2. Cell density was measured at A600 after 24 h growth at 30 C. Shown is one representative experiment from three independent experiments conducted. The bars indicate the standard error (n = 6) (***P < 0.001). (B and C) Co-localization of AtGRXcp-GFP (B) and AtGRX4-GFP (C) with mitochondrial markers (Cox6a-DsRed fusion) in yeast cells. BF: bright field. Scale bars = 10 lm.
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Fig. 3. AtGRX4 rescues the lysine auxotrophy of grx5 mutant. Wild-type expressing vector, grx5 cells expressing vector, Grx5, AtGRX4, and AtGRX4-GFP were assayed on SC medium with or without lysine. The photographs were taken after 3 day growth at 30 C.
Grx5-expressing grx5 cells (Fig. 3). This result indicates that AtGRX4 can restore Grx5 function in yeast. Deletion of Grx5 disrupts iron–sulfur cluster assembly and as a consequence, increases the level of intracellular iron [20]. This iron accumulation results in the production of toxic radicals through the iron-mediated Fenton reaction [21]. We have demonstrated that AtGRX4 could protect yeast cells against oxidative damage (Figs. 2 and 3). To further delineate the potential function of AtGRX4, we assayed the iron content in wild-type and grx5 cells expressing vector, Grx5, and AtGRX4. As shown in Fig. 4, AtGRX4- and Grx5-expressing cells were both able to suppress the iron accumulation (as measured at the intracellular iron level). 3.3. AtGRX4 is a chloroplast/plastid-localized monothiol Grx Sequence analysis using ChloroP program (version 1.1) predicted that AtGRX4 contained a chloroplastic/plastidic signal peptide [4,13]. In order to experimentally verify this, AtGRX4 was fused with the green fluorescent protein (GFP) at its C-terminus and the fusion was transiently expressed in tobacco leaf cells. Indeed, AtGRX4-GFP, like AtGRXcp-GFP, predominately localized to chloroplasts, rather than mitochondria,
Golgi, or peroxisomes in epidermal cells (Fig. 5; data not shown). Because AtGRX4 is not highly expressed in green tissues, like leaves, it will be expected that AtGRX4-GFP targets to plastids as well. 3.4. AtGRX4 expression is induced by various stresses AtGRX4 is ubiquitously expressed in all tissues (Fig. 1S; data not shown). In order to understand the function of AtGRX4 under stress conditions, AtGRX4 expression was analyzed upon various stress treatments. When 10-day-old seedlings were exposed to different sources of lights, AtGRX4 expression was down-regulated in response to light treatments (Fig. 6A). Interestingly, AtGRX4 expression was differentially regulated with various temperature treatments. In detail, AtGRX4 was induced in the cold (4 C) treatment, but down-regulated upon heat shocks (37 and 45 C; Fig. 6B). As shown in Fig. 6C, AtGRX4 was constantly induced by water stress (submerging the plant). Furthermore, AtGRX4 expression was altered by metal–ion stresses (Fig. 6D). For example, AtGRX4 was induced by mixed nutrient solution (MS), but down-regulated by NaCl and ZnCl2 (Fig. 6D).
Fig. 4. AtGRX4 suppresses free iron accumulation in grx5 cells. Intracellular iron content was examined with a QuantiChron Iron Assay Kit (BioAssay Systems, Hayward, CA, USA). All results shown here are the means of three independent experiments, and the bars indicate the standard errors (**P < 0.01; ***P < 0.001).
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Fig. 5. Subcellular localization of AtGRX4-GFP in plant cells. AtGRX4-GFP is localized to chloroplasts in tobacco cells. Left panels display the transient expression of AtGRX4-GFP in tobacco epidermal cells; central panels display fluorescing chloroplasts; right panels show the merged images. Scale bars = 25 lm.
Fig. 6. AtGRX4 expression in response to various stress stimuli. (A) AtGRX4 was expressed in response to various light sources. Total RNA samples were extracted from 10-day-old Col-0 seedlings (lane1) and seedlings grown in greenhouse (natural sunlight) for 3 h (lane 2), UV light for 15 min (lane 3) and 30 min (lane 4). (B) AtGRX4 was expressed in response to temperature stresses. 10-day-old seedlings grown at 22 C (lane1), 4 C for 2 days (lane2), grown at 28 C (lane3), 37 C for 1.5 h (lane 4), and 45 C for 1 h (lane 5). (C) AtGRX4 was expressed in response to submerging treatments. 10-day-old seedlings were subjected to submerging treatment for 0 day (lane 1), 1 day (lane 2), 2 days (lane3), 3 days (lane 4), and 4 days (lane 5). (D) AtGRX4 expression in response to metal-ion treatments. 3-week-old seedlings were previously treated for 12 h with the following solutions: water (as a control, lane 1), Murashige and Skoog (MS) nutrient media (lane 2), 80 mM NaCl (lane 3), 1 mM ZnCl2 (lane 4), 2 mM MnCl2 (lane 5), 0.1 mM NiSO4 (lane 6), and 50 mM CaCl2 (lane 7). Twenty micrograms of total RNA were hybridized with gene-specific probes for AtGRX4. Ethidium bromide (EtBr) stained rRNA are shown as a loading control.
3.5. Analysis of atgrx4 mutants To gain insight into the function of AtGRX4 in planta, we analyzed two T-DNA insertional lines of AtGRX4 (Fig. 7A). Sequence analysis of the T-DNA flanking regions revealed that in atgrx4-1(Salk_112767) the T-DNA was located in the 5Õ-
UTR at position 331nt upstream of the ATG of AtGRX4, and in atgrx4-2 (Sail_431_H03) the T-DNA was inserted at position Lys-24 (Fig. 7A). AtGRX4 expression was reduced in atgrx4-1 allele, and not detectable in atgrx4-2 allele as determined by RNA blot analysis (Fig. 2S). Both atgrx4-1 and atgrx4-2 did not display visible defects in seedling growth, flowering, and seed production when grown in soil under normal condition in comparison with wild-type (data not shown). When wild-type and atgrx4 seeds were grown on 1/2 MS media, atgrx4 seedlings grew slightly shorter in comparison to wild-type. The primary root length of atgrx4 seedlings were 88% (atgrx4-1) and 79% (atgrx4-2) of that of wild-type (Fig. 7B and C). However, when grown on the same media containing 3 mM H2O2, atgrx4-1 and atgrx4-2 had 45% and 57% reduction in primary root growth compared to wildtype (Fig. 7B and C). Apparently, root growth of wild-type and atgrx4 seedlings was inhibited by H2O2 if compared to that on 1/2 MS media. However, the inhibition of atgrx4 seedlings is significant greater than that of wild-type (78% in atgrx4-1 and 80% in atgrx4-2 compared to 65% in wild-type, P < 0.0001). When tested on the media containing 0.4 mM diamide, both alleles displayed 23% and 25% reduction of primary root growth, but this reduction caused by diamide is not significantly different from that of controls (data not shown). In addition, no difference was seen between wild-type and atgrx4 seedlings tested on iron-sufficient or iron-deficient media (data not shown).
4. Discussion In the present study, our findings reveal that AtGRX4 is a functional monothiol Grx localized to chloroplasts/plastids and able to complement yeast Grx5 function (Figs. 2–5). We also demonstrate that AtGRX4 expression is regulated under various environmental stimuli and loss of AtGRX4 in Arabidopsis leads
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Fig. 7. AtGRX4 knockout mutants are sensitive to external oxidants. (A) Genomic structure of AtGRX4 showing the sites of T-DNA insertions. (B) Wild-type (Col-0) and atgrx4 allele seeds were germinated and grown on 1/2 MS and the same media supplemented with 3 mM H2O2 for ten days. Shown is one representative experiment of three independent experiments. (C) Root growth measurement. The length of primary roots of seedlings was measured after 10 days of growth on the medium with or without H2O2. Error bars represent the standard error (n P 15; StudentÕs t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
to seedlings sensitive to oxidants (Figs. 6 and 7), suggesting a critical role of AtGRX4 in regulating redox state in plant growth and development. AtGRX4, like AtGRXcp, targets to mitochondria when expressed in yeast and also suppresses yeast grx5 mutant phenotypes (Figs. 2–4). It has been shown that all Grx5 homologues tested from various species are able to complement yeast Grx5 function [9]. These findings demonstrate the functional interchangeability among those monothiol Grxs [9]. Interestingly, entire AtGRX4 has only one cysteine residue within the ‘‘CGFS’’ motif, while AtGRXcp has four cysteine residues (Fig. 1). The ability of AtGRX4 to restore Grx5 function suggests that Cys91 is sufficient for AtGRX4 function. This finding is consistent with the previous report where Cys60, but not
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Cys171, is essential for yeast Grx5 function [10]. However, subsequent analysis indicates that Cys171 also has an important role in the formation of an intramolecular disulfide bond between Cys60 and Cys171 during catalysis [11]. In the present study, the biochemical property of AtGRX4 has been initially analyzed, but like AtGRXcp, there are no enzymatic activities in the insulin reduction and the b-hydroxyethyl disulfide (HED) assays defined in AtGRX4 (data not shown) [16]. Given the functional similarity in yeast complementation assay and the structural diversity of cysteine residues, AtGRX4 and AtGRXcp provide a unique platform to further delineate the structure-function relationship in monothiol Grxs. Both AtGRX4 and AtGRXcp are localized to chloroplasts/ plastids, implicating a critical role these Grxs may play in these organelles. The ability of AtGRX4 and AtGRXcp to partially rescue the Fe–S enzyme activities and suppress iron accumulation in yeast grx5 cells suggests that these AtGRXs may be required for biogenesis of Fe–S clusters in chloroplasts/plastids. Although the direct evidence for proving this function is pending, several recent studies suggest a connection between Grxmediated redox regulation and Fe–S cluster biosynthesis in chloroplasts/plastids [9]. In addition, different, but partially overlapping biosynthesis pathways for mitochondrial and plastidic Fe–S clusters (the Isc and Suf systems, respectively) could explain why AtGRXs only partially restored Grx5 function in vivo (Fig. 3) [9,16]. It is also possible that AtGRX4 (AtGRXcp) suppression of yeast grx5 mutant phenotypes is due to general activation of the ROS scavenging system in yeast (Figs. 2–4). In support of this hypothesis, recent studies indicate that yeast Grx3, a nuclear-localized monothiol Grx, complements Grx5 function when targeting to mitochondria [22]. However, rather than functioning as Grx5 in Fe–S biosynthesis, Grx3 globally regulates iron homeostasis through interacting with a transcriptional factor, Aft1 [23,24]. Future work will be necessary to determine the target(s) of AtGRXs and understand the complexity of ROS regulation in planta. Recent reports have shown that Arabidopsis has high steady-state levels of protein oxidation during vegetative growth and under stress conditions [25,26]. Altered expression of AtGRX4 in response to various environmental stimuli suggests that AtGRX4 may play an important role in growth and development. In agreement with this, atgrx4 roots are more sensitive to H2O2 (Fig. 7). Although atgrx4 plants did not display any altered sensitivity to iron imbalance (data not shown), this may be due to tight regulation of redox state at the whole plant level or compensation by functional redundancy among Grxs [4,13,14]. The interplay among Grxs and various antioxidant systems will be addressed in future studies. Acknowledgements: We are very grateful to Dr. Elison Blancaflor for helping in confocal imaging, Drs. Toshiro Shigaki and Jon Pittman for critical reading of the manuscript. This work is supported by United State Department of Agriculture/Agricultural Research Service under Cooperation Agreement 6250-21520-042-02.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet. 2008.02.006.
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