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First published online December 24, 2008; 10.1104/pp.108.128314 Plant Physiology 149:1240-1250 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Small Family of Chloroplast Atypical Thioredoxins1,[C],[W],[OA]Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel
The reduction and the formation of regulatory disulfide bonds serve as a key signaling element in chloroplasts. Members of the thioredoxin (Trx) superfamily of oxidoreductases play a major role in these processes. We have characterized a small family of plant-specific Trxs in Arabidopsis (Arabidopsis thaliana) that are rich in cysteine and histidine residues and are typified by a variable noncanonical redox active site. We found that the redox midpoint potential of three selected family members is significantly less reducing than that of the classic Trxs. Assays of subcellular localization demonstrated that all proteins are localized to the chloroplast. Selected members showed high activity, contingent on a dithiol electron donor, toward the chloroplast 2-cysteine peroxiredoxin A and poor activity toward the chloroplast NADP-malate dehydrogenase. The expression profile of the family members suggests that they have distinct roles. The intermediate redox midpoint potential value of the atypical Trxs might imply adaptability to function in modulating the redox state of chloroplast proteins with regulatory disulfides.
Recent studies suggest that redox regulation is a major mechanism in cellular signal transduction and in control of gene expression in species as diverse as humans, plants, and bacteria (Danon, 2002  ; Toledano et al., 2004; Buchanan and Balmer, 2005; Holmgren et al., 2005). Members of the thioredoxin (Trx) superfamily of thiol-disulfide oxidoreductases play key roles in cellular redox regulation in almost all life forms (Debarbieux and Beckwith, 2000; Holmgren et al., 2005; Carvalho et al., 2006). Trxs share a similar fold (Trx-fold) and a common active site motif comprised of two vicinal Cys separated by two variable amino acids, the CXXC motif (Martin, 1995). Despite these common features, members of the Trx superfamily have diverse functions ranging from protein disulfide reductases to disulfide isomerases or to disulfide transferases. The versatility in function is reflected in part by the broad range of redox midpoint potentials measured for the Trxs. Redox midpoint potentials range from –300 mV for the reductive-type Trx-m (Hirasawa et al., 1999) to –120 mV for the oxidative-type Escherichia coli DsbA (Zapun et al., 1993; Aslund et al., 1997). Protein disulfide isomerases (PDIs), the bacterial DsbD, and glutaredoxins have intermediate redox midpoint potentials from –175 to –241 mV (Lundstrom and Holmgren, 1993; Aslund et al., 1997; Collet et al., 2002).
The observed differences in the redox midpoint potential between different Trxs reflect to some extent divergence in the stabilization of the negative charge of the N-terminal catalytic thiolate, and thus in the pKa, of the redox active site. One of the factors suggested to have an important role in determining the pKa of the catalytic Cys and the redox midpoint potential of the protein is the amino acid identity of the central dipeptide, located between the two Cys of the active site. The classic reductive-type Trxs share a canonical active site motif, the C(G/P)PC motif, while PDIs and the oxidative DsbA have the conserved CPHC and a CGHC motif, respectively. Mutating the central dipeptide was shown to change the redox midpoint potential of the protein, as well as its activity and its interactions with other proteins (Krause and Holmgren, 1991
In plants, the redox reactions of the photosynthetic machinery of the chloroplast need to adjust to both rapid and gradual environmental changes, such as photon flux, CO2 availability or water potential, and the associated free radical production. Four families of chloroplast-localized classic Trxs containing a canonical active site, Trx-f, Trx-m, Trx-x, and Trx-y, were characterized. The f-type and m-type Trxs were shown to be reduced by the ferredoxin-Trx system, which receives electrons from the light-capturing reactions of photosynthesis. The classic chloroplast Trxs are implicated in the light-dependent activation by reduction of different enzymes, including several metabolic enzymes such as NADP-malate dehydrogenase (MDH) and Fru-1,6-bisP, as well as in protection against oxidative stress (Schurmann and Buchanan, 2008 As studying atypical Trxs is expected to shed light on additional functions of Trxs in plants, we initiated a search in the Arabidopsis (Arabidopsis thaliana) genome for Trxs with a noncanonical active site. We characterized a small family of proteins, which we denoted AtACHTs (for atypical Cys His-rich Trxs). Notably, we found that the redox midpoint potential of three selected proteins, AtACHT1, AtACHT2a, and AtACHT4a, is significantly higher than that of the classic Arabidopsis Trxf1 (AtTrx-f1). Assays of the subcellular localization of the AtACHTs suggest that they are localized to the chloroplast. Examination of AtACHT1 and AtACHT4a showed that they are partitioned between the soluble stroma and the thylakoid membranes. AtACHT1, AtACHT2a, and AtACHT4a showed high preference in their activity toward the chloroplast 2-Cys peroxiredoxin A (2-Cys PrxA) relative to their activity toward the chloroplast NADP-MDH. The unique properties of the AtACHTs and their pattern of expression suggest distinct roles in the chloroplast.
Characteristics of the AtACHT Family of Proteins
To begin to look at Trxs with possible distinct activities from these of the well-characterized reductive-type chloroplast Trxs, we searched the Arabidopsis genome for small Trx-like genes with a noncanonical CXXC active site. The first protein with a noncanonical active site that emerged from a National Center for Biotechnology Information (NCBI) BLASTP search using a query of AtTrx-f1 sequence was AtACHT1. A follow-up analysis with AtACHT1 sequence identified a family of five genes denoted AtACHT1 to AtACHT5 (Fig. 1
). The proteins are homologs of the previously reported Lilium longiflorum Trx-like sequence (Meyer et al., 1999
To examine whether the proteins are active thiol-oxidoreductases, leaderless forms of three selected proteins, AtACHT1, AtACHT2a, and AtACHT4a, were expressed in E. coli, purified to homogeneity, and their catalytic activity was compared to that of the classic AtTrx-f1 in the standard insulin turbidity assay (Holmgren, 1979 ). We found that the catalytic activity of AtACHT1 was slightly higher, and that of AtACHT2a was slightly lower, than that of AtTrx-f1 (Fig. 2
). These results suggest that the AtACHT proteins preserve the catalytic activity that is common to members of the Trx oxidoreductase superfamily. Notably, the activity of the Trp-less AtACHT4a in parallel reactions was poor (Fig. 2).
The AtACHT Proteins Have a Higher Redox Midpoint Potential Than AtTrx-f1
It was shown that the central dipeptide sequence of the active site could influence the redox midpoint potential as well as the protein function (Krause and Holmgren, 1991
The AtACHTs Are Targeted to Chloroplasts
The presence of a leader peptide in the N-terminal sequence of all AtACHTs indicated that they might be localized to subcellular organelles. Thus, to study the localization of the AtACHTs, we compared the accumulation of each of the transiently expressed AtACHTs, fused with GFP at its C terminus (AtACHT1–5:GFP), to that of chloroplast-localized GFP and to the autofluorescence of chlorophyll, to mitochondrion-localized GFP, to an endoplasmic reticulum (ER)-localized GFP, and to cytoplasm-localized GFP in Arabidopsis protoplasts using confocal laser microscopy (Fig. 4A
). To avoid mislocalization due to overaccumulation of expressed proteins, we imaged only protoplasts displaying the earliest signal of GFP fluorescence, as previously done in our lab (Levitan et al., 2005
We chose two proteins, AtACHT1 and AtACHT4a, for further studies. First, to authenticate the protein localization, transgenic plants expressing AtACHT1 or AtACHT4a, each fused with yellow fluorescent protein (YFP) at their C terminus, were generated and imaged using confocal laser microscopy. The fluorescence images corroborated that both AtACHT1 and AtACHT4a are targeted to chloroplasts in planta and that they do not accumulate to detectible levels in other subcellular organelles (Fig. 4B). Second, to further study the subchloroplast localization of the proteins, we purified intact chloroplasts from transgenic plants, expressing the AtACHTs with HA3 affinity tag at their C terminus, and analyzed their soluble and membranal fractions by immunoblot analysis. The AtACHT1 and AtACHT4a proteins were found partitioned in both the stromal fraction, containing the large subunit of Rubisco, and the thylakoid membranes, containing the D1 protein (Fig. 4C), suggesting that they have a role in both compartments.
The plastidial localization of the AtACHT proteins prompted us to examine their activity toward known targets of chloroplast Trxs, such as 2-Cys Prx A and MDH. The 2-Cys Prxs detoxify peroxides by their reduction. Several Trx-like proteins were shown to reduce 2-Cys Prx (Dietz et al., 2006
The activation of MDH, which catalyzes the reduction of oxaloacetate into malate using NADPH as a cofactor, is dependent on the light-regulated reduction of two disulfide bonds by Trx (Schurmann and Buchanan, 2008 ). Though originally thought to be specifically light regulated through Trx-m, Trx-f was later shown to be the most efficient activator of sorghum (Sorghum bicolor) MDH (Collin et al., 2003, 2004). Activation assays were performed by incubating recombinant mature Arabidopsis NADP-MDH (AtMDH) with either AtTrx-f1 or selected AtACHTs in the presence of DTT and measuring the AtMDH enzymatic activity as oxidation of NADPH as described before (Jacquot and Buchanan, 1981). While 1 µM of AtTrx-f1 was sufficient to efficiently activate AtMDH under our experimental conditions, the same concentration of the AtACHTs did not appreciably activate AtMDH (Fig. 5C). By increasing the AtACHT concentration to 20 µM, a poor activation of AtMDH was observed (data not shown). Thus, the AtACHTs showed higher preference in their activity toward At-2-Cys PrxA than their activity toward AtMDH.
We investigated, using the MPSS (Meyers et al., 2004
The ACHT Family Is Unique to Plants
The plastid localization of AtACHTs suggested plant-specific function. Thus, to determine whether this family is unique to plants, a BLAST search was made against all available sequences in NCBI and in specific databases of selected organisms. We found homologs from the green algae Chlamydomonas reinhardtii and Ostreococcus tauri, the moss Physcomitrella patens, and the higher plants Oryza sativa and Zea mays. Notably, the genomes of nonplant species as well as cyanobacteria or other prokaryotes do not seem to contain ACHT homologs, suggesting that similarly to the chloroplast Trx-f (Schurmann and Buchanan, 2008
The phylogenetic analysis implies that the AtACHTs are subdivided into two classes (Fig. 7). Class 1 contains AtACHTs 1, 2a, and 2b, whereas Class 2 includes AtACHTs 3, 4a, 4b, and 5. All Class 2 AtACHTs and their homologs lack the conserved Trp preceding the redox active site and, except for one Oryza protein, have an identical active site motif, CGGC. All of them are typified by a large C-terminal extension. Class 1 AtACHTs have a C(G/A)SC active site motif and a shorter C terminus relative to Class 2 proteins. Both Class 1 and 2 proteins contain additional Cys, which are conserved in all proteins, and His, which are conserved within each class (Fig. 1). The relevance of the conserved Cys and His to possibly newly evolved activities of these proteins will have to be addressed in future studies. The high degree of conservation of this protein family along the plant evolution course, their absence from nonplant organisms, and their chloroplast localization imply that they have a preserved role in plants. Their unique sequence elements differentiate them from the classic Trxs, and their higher redox potential raises the possibility of specialized redox function in plant chloroplasts.
The AtACHTs constitute a small family of chloroplast Trx-like proteins that display a redox midpoint potential that is significantly less reducing compared to the classic Trxs (Fig. 3), a redox active site with a different central dipeptide than the canonical sequence, and several additional conserved Cys and His residues outside of the active site (Fig. 1). Previous experiments in which the central dipeptide sequence of one Trx family member was swapped with that of a second Trx resulted in a profound effect on the activity of the Trx. Often, a shift in the redox properties of the mutated Trx toward those of the second Trx was observed (Krause and Holmgren, 1991 ; Lundstrom et al., 1992; Chivers et al., 1996; Huber-Wunderlich and Glockshuber, 1998; Mossner et al., 1998; Quan et al., 2007). Notably, the two classes of AtACHTs differ in their central dipeptide; the Class 1 dipeptide is C(G/A)SC and the Class 2 is CGGC (Fig. 7). Yet, in spite of the difference in the central dipeptide, the redox midpoint potentials of selected members from both Class 1 and 2 AtACHTs were found to be almost identical (Fig. 3), suggesting that other residues in addition to the redox active site dipeptide might affect the redox midpoint potential as well. This assumption is further supported by the finding that the redox midpoint potential of different Trxs, containing the same canonical central dipeptide, may vary between –300 mV and –275 mV (Collin et al., 2003, 2004). Hence, the AtACHTs have evolved a redox active site with variable central dipeptide while maintaining a similar redox midpoint potential value that has been conceivably preserved by compensatory changes in other residues than the central dipeptide.
Both prokaryotic- and eukaryotic-type Trxs contain a highly conserved Trp residue preceding the active site. In Arabidopsis, 57 out of the 64 Trx-related gene products contain this Trp (Meyer et al., 2008 A plastidial function for the AtACHTs is implicated by their localization (Fig. 4) and by their phylogeny (Fig. 7), which is unique to the plastid-containing viridiplantae. What might be, then, the unique function of the AtACHTs among the 20 or so Trx-like proteins shown to reside in plastids? The first clue might be found in the expression pattern of the AtACHTs. AtACHT2a and AtACHT4a-4b express mainly in photosynthetic tissue (Fig. 6), suggesting that they might function in parallel to the classic Trxs, such as Trx-f and Trx-m. In contrast, AtACHT1 and AtACHT2b transcripts accumulate quite evenly in green and non-green tissues, suggesting a more generalized role that is not limited to the chloroplast (Fig. 6). The expression of the AtACHT5 and AtACHT3 transcripts appears to be unique. AtACHT5 transcript is enriched in floral tissues and accumulates to very high levels in the double mutant of regulators of floral meristem development (Sup/ap1; Fig. 6), whereas the AtACHT3 transcript is highly specific to pollen (Fig. 6), suggesting a more specialized function for these two proteins.
A second clue might be found in the activity in vitro of the AtACHTs, which, relative to AtTrx-f1, reacted with high efficiency with At-2-Cys PrxA and poorly with AtMDH (Fig. 5). The canonical site-containing AtTrx-x and AtTrx-y were also found to be inefficient activators of sorghum MDH and to react efficiently with peroxiredoxins in vitro (Collin et al., 2003
Another clue to the role of AtACHTs might be found in their redox midpoint potential, which is of an intermediate value (Fig. 3) between the two extremes of the oxidative DsbA (Zapun et al., 1993
Identification and Cloning of the AtACHT Proteins from Arabidopsis The protein sequence of AtTrx-f1 was used to search the Arabidopsis (Arabidopsis thaliana) database for Trx-like proteins containing a noncanonical active site using the BLASTP program at NCBI (http://www.ncbi.nlm.nih.gov). Total RNA was extracted using Tri-reagent (MRC). The RNA was subjected to RT using Superscript II (Invitrogen, Rhenium) and oligo(dT). PCR reactions were done with gene-specific primers.
Protein multiple alignments were generated using the ClustalX program (Thompson et al., 1997 To identify alternative splicing variants, the genome sequence of Arabidopsis was analyzed for gene structure positions (exons and introns borders). The borders between expressed sequences and introns were compared to the cDNA sequences of AtACHT2a and AtACHT2b, which were isolated by RT-PCR. The existence of the splice variants was predicted also by The Arabidopsis Information Resource (www.arabidopsis.org).
The cDNAs encoding AtACHTs, AtMDH (At5g58330), and At-2-Cys PrxA (At3g11630) without their putative transit peptide (primer sequences are listed in Supplemental Table S1), as predicted by TargetP (Emanuelsson et al., 2000
Recombinant proteins (5 µM) were used in the insulin reduction assay, as described previously (Holmgren, 1979
The redox potential was evaluated using the thiol-labeling reagent mBBr, as described previously (Hirasawa et al., 1999
Each AtACHT open reading frame (ORF) was ligated through SmaI sites into a puc18-GFP5-containing vector, yielding a fusion protein upstream and in frame to GFP5 ORF under the control of the cauliflower mosaic virus 35S promoter. Control fusion proteins were prepared as well and included the small subunit of Rubisco (kindly provided by Yoram Eyal, Agricultural Research Organization, Volcani Center), the chitinase ER marker (kindly provided by Jean-Marc Neuhaus, University of Neuchatel, Switzerland), the mitochondrial AtTrx-o1 (At2g35010), and the mature form of the GFP protein. The transient expression assays were performed as described before (Sheen, 2002
For transgenic plant generation, AtACHT1 and AtACHT4a were ligated upstream and in frame to HA3 or YFP tags. The fragments were cloned into pART7 vector under the control of the 35S promoter and 35S terminator. As a control, the YFP ORF was ligated as well. NotI-digested fragments were ligated into the binary vector pBART, which confer glufosinate (BASTA) resistance in plants. The binary plasmids were introduced into Agrobacterium tumefaciens through electroporation. Plant transformation was made by the floral dip method (Clough and Bent, 1998
Fluorescence images were obtained as described before (Levitan et al., 2005
Intact chloroplasts were purified from transgenic plants expressing AtACHT-HA3 fusion proteins as described before (Aronsson and Jarvis, 2002
At-2-Cys PrxA at a concentration of 5 µM was incubated with 2.5 µM of the different AtACHTs or 2.5 or 10 µM AtTrx-f1 in a reaction mixture containing 50 mM phosphate buffer, pH 7.4, and either 0.4 mM DTT or 0.8 mM GSH. The reaction was started by adding 100 µM hydrogen peroxide (H2O2). The concentration of H2O2 was determined at different time points using the PeroXOquant reagent (Pierce).
DTT-dependant activation of AtMDH by 1 µM of AtTrx-f1 or of the different AtACHTs was carried out with 1.5 µM AtMDH and 10 mM DTT in 100 mM Tris-HCl, pH 7.9. At fixed times, 20-µL aliquots of the activation mixture were used to determine the activity of the AtMDH by following the initial rate of consumption of NADPH at room temperature in a 1-mL assay mixture containing 140 µM NADPH and 750 µM oxaloacetate in 100 mM Tris-HCl, pH 7.9. The oxidation of NADPH was followed spectroscopically at 340 nm. One unit of MDH activity is defined as the amount oxidizing 1 µmol NADPH/min and corresponds to an absorbance change of 6.22.
The MPSS (http://mpss.udel.edu/at/java.html) and the Genevestigator database (www.genevestigator.ethz.ch/at) were used to evaluate differential gene expression. Validation of the main results was done using sqRT-PCR. Total RNA was extracted and equal amounts of RNA (2 µg) were subjected to RT using Superscript II (Invitrogen) and oligo(dT). PCR reactions were done with gene-specific primers (as listed in Supplemental Table S1). As a control, the actin and tubulin genes were amplified as well. Selected bands were excised, purified from the gel, and sequenced to authenticate their identity. Sequence data from this article can be found in the GenBank data libraries under AGI accession numbers At4g26160 (AtACHT1), At4g29670.1 (AtACHT2a), At4g29670.2 (AtACHT2b), At2g33270 (AtACHT3), At1g08570.1 (AtACHT4a), At1g08570.2 (AtACHT4b), and At5g61440 (AtACHT5).
The following materials are available in the online version of this article.
We thank Abdussalam Azem and his lab members at Tel Aviv University for helping us with the MDH activity assay. We are grateful for Vladimir Kiss for his assistance with the confocal microscopy. A.D. is incumbent of The Henry and Bertha Benson Chair, Weizmann Institute of Science. Received August 22, 2008; accepted December 22, 2008; published December 24, 2008.
1 This work was supported by the Israeli Science Foundation, by the Minerva Foundation, and by the Charles W. and Tillie K. Lubin Center for Plant Biotechnology at the Weizmann Institute of Science.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Avihai Danon (avihai.danon{at}weizmann.ac.il).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.128314 * Corresponding author; e-mail avihai.danon{at}weizmann.ac.il.
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TOP
ABSTRACT
RESULTS
-helices and arrows for β-sheet strands. The residues were color labeled: light blue (A, I, L, M, F, W, V, C), red (R, K), green (N, Q, S, T), magenta (D, E), orange (G), cyan (H, Y), and yellow (P). Conserved His are marked in red and conserved Cys are in blue. Arrows mark active site Cys. AGI accession numbers of the family members are: At4g26160 (AtACHT1), At4g29670.1 (AtACHT2a), At4g29670.2 (AtACHT2b), At2g33270 (AtACHT3), At1g08570.1 (AtACHT4a), At1g08570.2 (AtACHT4b), and At5g61440 (AtACHT5). [See online article for color version of this figure.]
-domain of the membrane-bound bacterial DsbD (Collet et al., 2002