Yet another plant thioredoxin

Yet another plant thioredoxin

Research Update TRENDS in Plant Science Vol.7 No.5 May 2002 191 Yet another plant thioredoxin Yves Balmer and Bob B. Buchanan Thioredoxins are wide...

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Research Update

TRENDS in Plant Science Vol.7 No.5 May 2002

191

Yet another plant thioredoxin Yves Balmer and Bob B. Buchanan Thioredoxins are widely distributed proteins that function in a broad spectrum of cellular reactions. Plant cells have well characterized chloroplast and cytosolic thioredoxin systems, but, unlike animals and yeast, a mitochondrial counterpart has not been clearly defined. Recently, a complete thioredoxin system has been described in plant mitochondria, opening a new door for the study of thioredoxins as well as mitochondria.

not to NADP, but to ferredoxin by the novel iron–sulfur enzyme, ferredoxin–thioredoxin reductase (FTR), whose mechanism has been elucidated recently. Ferredoxin is reduced photochemically from H2O via the photosynthetic electron transport chain. In both cases, thioredoxin either reduces specific disulfide bridges on target proteins, thereby altering their activity, or serves as a substrate reductant in an enzymatic reaction (reviewed in Refs [3–5]).

Published online: 4 April 2002 Bacteria, yeast and animals

In a 1995 perspective on redox regulation in chloroplasts, Charles Levings and James Siedow [1] raised the question of the role of thioredoxin in plant mitochondria. At that time, thioredoxin had been reported to occur in these organelles, but evidence for an enzyme catalyzing its reduction was not available. In a recent article, Christophe Laloi and colleagues [2] have begun to fill this gap. The authors report the occurrence not only of an NADP-linked thioredoxin reductase (NTR), but also of a thioredoxin that appears to be unrelated to known prokaryotic or eukaryotic types. Some background

Thioredoxins are small (12 kDa) globular proteins present in all types of living organisms. They function in a growing number of regulatory processes, including enzyme regulation, response to oxidative stress, transcription and translation. In addition, as a hydrogen donor, they participate in several reductive reactions. Thioredoxins are characterized by a redox active site made up of two neighboring cyst(e)ines in a conserved motif, WCGPC, or in some cases, WCPPC. The oxidized form of the protein is reduced by one of two mechanisms (Fig. 1). The more widely distributed mechanism is the NADP linked-system, present in all types of cells, in which thioredoxin obtains electrons from NADPH via the flavin enzyme NTR. NADP can be reduced by several routes, including carbohydrate breakdown or light plus an electron donor (other than H2O). In chloroplasts and oxygenic photosynthetic prokaryotes, the reduction of thioredoxin is linked http://plants.trends.com

Bacteria, typified by E. coli, present a simple thioredoxin system composed of one or two thioredoxins and an NTR that is a 37 kDa homodimer. The yeast cytosolic thioredoxin system is similar to that of E. coli. By contrast, the animal cytosolic system has an NTR that is a 55 kDa homodimer, containing an essential selenocysteine near the C-terminus. In addition, unlike its bacterial and yeast counterparts, the animal enzyme is characterized by a lack of thioredoxin specificity. Thus, whereas E. coli and yeast NTR are specific for indigenous thioredoxins, the animal counterpart is active with a wide range of substrates, including other types of thioredoxins and chemical oxidants, such as lipid hydroxyperoxides and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) [6]. In spite of these differences, the bacterial, yeast and animal systems have similar functions. For example, each acts as a hydrogen donor in DNA synthesis (ribonucleotide reductase) and protein repair (methionine-sulfoxide reductase). Each also plays an important role in sensing oxidative stress. Finally, in certain cases, these thioredoxins function in the regulation of apoptosis, protein folding (cytochrome c maturation) and gene expression (for example, via yeast Yap1p and animal NFκB transcription factors) [7–9]. In yeast and animal cells, the thioredoxin profile is more extensive than in bacteria owing to a second NADP-dependent thioredoxin system present in mitochondria [10,11]. Because the thioredoxin system has been characterized only recently, the function of mitochondrial thioredoxin

(a) Target protein

(b) Target protein 2H

2H Thioredoxin

Thioredoxin

2H+ –

H+ NTR e– H NADP

2e FTR Ferredoxin e–

e– H

Light + H2O

Substrate

Ferredoxin linked

NADP linked TRENDS in Plant Science

Fig. 1. Mechanisms of thioredoxin reduction. Thioredoxins are reduced by either (a) reduced ferredoxin (via the iron–sulfur enzyme, ferredoxin–thioredoxin reductase, FTR) or (b) NADPH (via the flavin enzyme, NADP-dependent thioredoxin reductase, NTR). The ferredoxin-linked system is found in chloroplasts and oxygenic photosynthetic prokaryotes. NADPH is used as the reductant for thioredoxin by a broad spectrum of cells: those that grow on organic compounds (heterotrophs) and anaerobes that require light (photosynthetic bacteria that are unable to evolve oxygen), heterotrophic eukaryotes and by explastidic compartments of oxygenic photosynthetic eukaryotes. Reduced NADP is generated metabolically except for the anoxygenic photosynthetic bacteria that reduce the coenzyme photochemically. To our knowledge, thioredoxins have not been studied in chemoautotrophs, aerobic bacteria that grow on CO2 by oxidizing inorganic compounds. Not shown is the ferredoxin-linked reduction of thioredoxin via the flavoprotein pathway described for Clostridium pasteurianum [22].

systems is not as well documented as their cytosolic counterparts. At this stage, one common reaction that has been described for mitochondrial thioredoxin is the removal of reactive oxygen species (H2O2) via a thioredoxin-dependent peroxidase (peroxiredoxin) [11,12]. Plants

With representatives in chloroplasts, cytosol and mitochondria, plants present a complex profile of thioredoxins. The cytosolic system consists of thioredoxin h and an NTR similar to its yeast and prokaryotic counterparts. Thioredoxin h functions are beginning to be uncovered: in reduction of H2O2 via a peroxidase, self-incompatibility and seed germination [4,13–15]. At least eight different genes encode for thioredoxin h in the Arabidopsis thaliana genome [2].

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Research Update

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TRENDS in Plant Science Vol.7 No.5 May 2002

Bacteria and photosynthetic m-type

Spinach Porphyra Synechococcus Anabaena Pea

Plant cytosol, h

At h1 Tobacco

E. coli

At h3

At m1

Yeast

Corynebacterium

Cytosol 2 Cytosol 1 Mitochondria

Bovine Human

Spinach Rat

Pea

Animal mitochondria

At o1 Wheat At o2

Bovine

Rat Human

Animal cytosol

At f2 At f1

Plant chloroplast, f

Plant mitochondria, o TRENDS in Plant Science

Fig. 2. Patterns of sequence similarity showing the relationship of mitochondrial thioredoxins in comparison to other types. Included are representatives from bacteria, algae and yeast, animals and plants. The phylogenetic tree was generated by the Clustal method and PhyloDraw software using amino acid sequences of the indicated thioredoxins, including bacterial and photosynthetic f and m-types, plant h and o-types, yeast and animal cytosolic and mitochondrial counterparts. Clockwise, beginning with bacteria and photosynthetic m-type thioredoxins: Corynebacterium nephridii (P00275), At m1, Arabidopsis thaliana m1 (O48737), E. coli, Escherichia coli (P00274), pea, Pisum sativum m (P48384), spinach, Spinacia oleracea m (P07591), Porphyra purpurea m (P51225), Synechococcus sp. m (P12243), Anabaena sp. m (P06544), At h1, A. thaliana h1 (P29448), tobacco, Nicotiana tabacum h (P29449), At h3, A. thaliana h3 (Q42403), cytosol 2, Saccharomyces cerevisiae 2 (P22217), cytosol 1, S. cerevisiae 1 (P22803), mitochondrial S. cerevisiae 3 (P25372), spinach, S. oleracea f (P09856), pea, P. sativum f (P29450), At f 2, A. thaliana f 2 (Q9XFH9), At f 1 A. thaliana f 1 (Q9XFH8), rat, Rattus norvegicus (P11232), human, Homo sapiens (P10599), bovine, Bos taurus (O97680), wheat, Triticum aestivum (GenBank BF478722), At o2, A. thaliana o2 (GenBank AAK83918), At o1, A. thaliana o1 (GenBank AAC12840), rat, R. norvegicus (P97615), human, H. sapiens (Q99757), bovine, B. taurus (Q95108).

The chloroplast contains two major types of thioredoxins, m and f, both reduced by ferredoxin and FTR via the photosynthetic electron transport chain. Both chloroplast thioredoxins are represented by multiple genes in Arabidopsis: two and at least four genes for thioredoxin f and m, respectively [16]. Thioredoxin f regulates several chloroplast enzymes, including members of the Calvin cycle [fructose1,6-bisphosphatase, sedoheptulo1,7-bisphosphatase, phosphoribulokinase, glycerhaldehyde-3-phosphate dehydrogenase and Rubisco (via Rubisco activase)], ATP synthesis (CF1 ATP synthase) and fatty acid synthesis (acetyl-CoA carboxylase) [3,4]. Less is known about the function of thioredoxin m, which is involved in the deactivation of the carbohydratedegrading enzyme, glucose-6-phosphate http://plants.trends.com

dehydrogenase. Thioredoxins m as well as f activate NADP-dependent malate dehydrogenase, an enzyme functional in CO2 and hydrogen transport [17,18]. Thioredoxin specificity in the activation of the translation of a thylakoid protein (D1) has not been determined [19]. New development

For more than a decade, the evidence that plant mitochondria contain a thioredoxin rested on activity and western blot measurements [20,21]. One study indicated the presence of thioredoxin h in these organelles [20]. Recent work on Arabidopsis has revealed a new dimension – a complete thioredoxin system composed of a new type of thioredoxin (designated ‘o’) and an NTR similar to the plant cytosolic counterpart [2]. Both the thioredoxin o and NTR genes encode an mRNA with a putative

mitochondrial targeting sequence. In addition, the processed proteins are located in the mitochondrial matrix as confirmed by western blot analysis and in vitro import experiments. Thioredoxin o appears to be unrelated to known thioredoxins. As for yeast and mammals, the role of thioredoxin in plant mitochondria needs further study. Possible functions include the detoxification of reactive oxygen via a peroxiredoxin, as seen with other systems, and, as proposed earlier [1], the regulation of an alternative oxidase – an enzyme characteristic of mitochondria from many plant species. ‘…the T-DNA destination vector p*WG allows the rapid cloning of any gene or sequence to be transformed into plants.’

The presence of yet another type of thioredoxin in plants raises the question of its relationship to counterparts in other organelles. Except in certain algae in which the m-type is found in the chloroplast chromosome, plant thioredoxins are encoded in the nucleus [4]. Extensive phylogenetic analysis suggests that thioredoxin m is of prokaryotic origin, whereas thioredoxins f and h are of the eukaryotic type. In their analysis, Laloi et al. [2] compared the newly found thioredoxin o with counterparts in other compartments of plant cells and found that the thioredoxins clustered in four main phylogenetic groups: o, m, h and f, with the f-type related to h. A fifth group was formed by a single member, thioredoxin x [16]. We have constructed a tree based on the amino acid sequences of representative thioredoxins to compare thioredoxin o with counterparts from other sources and found a clustering of seven major groups: (1) bacterial and photosynthetic m-types, (2) plant cytosolic h, (3) yeast cytosolic and mitochondrial thioredoxins, (4) chloroplast f, (5) animal cytosolic, (6) plant mitochondrial thioredoxin o and (7) animal mitochondrial thioredoxin (Fig. 2). As expected, bacterial and m-type, f-type, h-type and animal and yeast cytosolic thioredoxins formed separate clusters. Interestingly, yeast mitochondrial thioredoxin appeared in the cluster with cytosolic members from the same source, whereas plant and animal mitochondrial thioredoxins formed independent groups, suggesting that the different mitochondrial thioredoxins are unrelated.

Research Update

TRENDS in Plant Science Vol.7 No.5 May 2002

What’s next?

Although, the functional question raised by Levings and Siedow [1] remains unanswered, knowledge of thioredoxin o and the associated NTR enriches the field and opens the door to the identification of new regulatory events in plant mitochondria. Acknowledgement

Y.B. gratefully acknowledges the support of a fellowship from the Swiss National Science Foundation. References 1 Levings, C.S. and Siedow, J.N. (1995) Regulation by redox poise in chloroplast. Science 268, 695–696 2 Laloi, C. et al. (2001) Identification and characterization of a mitochondrial thioredoxin system in plant. Proc. Natl. Acad. Sci. U. S. A. 98, 14144–14149 3 Buchanan, B.B. (1980) Role of light in the regulation of chloroplast enzymes. Annu. Rev. Plant Physiol. 31, 341–374 4 Schürmann, P. and Jacquot, J-P. (2000) Plant thioredoxin system revisited. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 371–400 5 Buchanan, B.B. et al. The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond. Photosynth. Res. (in press) 6 Williams, C.H., Jr et al. (2000) Thioredoxin reductase: two modes of catalysis have evolved. Eur. J. Biochem. 267, 6110–6117

7 Arner, E.S.J. and Holmgren, A. (2000) Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267, 6102–6109 8 Fabianeck, R.A. et al. (1998) The active-site cysteines of the periplasmic thioredoxin-like protein CcmG of Escherichia coli are important but not essential for cytochrome c maturation in vivo. J. Bacteriol. 180, 1947–1950 9 Carmel-Harel, O. and Storz, G. (2000) Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae response to oxidative stress. Annu. Rev. Microbiol. 54, 439–461 10 Pedrajas, J.R. et al. (1999) Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae. J. Biol. Chem. 274, 6366–6373 11 Miranda-Vizuete, A. et al. (2000) The mitochondrial thioredoxin system. Antioxid. Redox Signal. 2, 801–810 12 Rabilloud, T. et al. (2001) The mitochondrial antioxidant defense system and its response to oxidative stress. Proteomics 1, 1105–1110 13 Rouhier, N. et al. (2001) Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as proton donor. Plant Physiol. 127, 1299–1309 14 Cabrillac, D. et al. (2001) The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins. Nature 410, 220–223 15 Besse, I. and Buchanan, B.B. (1996) Thiocalsin: a thioredoxin-linked substrate-specific protease dependent on calcium. Proc. Natl. Acad. Sci. U. S. A. 93, 3169–3175

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16 Mestres-Ortega, D. and Meyer, Y. (1999) The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin. Gene 240, 307–331 17 Scheibe, R. (1991) Redox modulation of chloroplast enzymes. A common principle for individual control. Plant Physiol. 96, 1–2 18 Ruelland, E. and Miginiac-Maslow, M. (1999) Regulation of chloroplast enzyme activities by thioredoxins: activation or relief of inhibition? Trends Plant Sci. 4, 136–141 19 Trebitsh, T. and Danon, A. (2001) Translation of psbA mRNA is regulated by signals initiated by both photosystems II and I. Proc. Natl. Acad. Sci. U. S. A. 98, 12289–12294 20 Bodenstein-Lang, J. et al. (1989) Animal and plant mitochondria contain specific thioredoxins. FEBS Lett. 258, 22–26 21 Marcus, F. et al. (1991) Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments. Arch. Biochem. Biophys. 287, 195–198 22 Hammel, K.E. et al. (1983) Ferredoxin/flavoprotein-linked pathway for the reduction of thioredoxin. Proc. Natl. Acad. Sci. U. S. A. 80, 3681–3689

Yves Balmer Bob B. Buchanan* Dept of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. *e-mail: [email protected]

Techniques & Applications

 vectors for Agrobacterium-mediated plant GATEWAY transformation Mansour Karimi, Dirk Inzé and Ann Depicker Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species. Commonly, the genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can replicate both in E. coli and Agrobacterium. Because these vectors are generally large, cloning can be time-consuming and laborious. Recently,  conversion technology the GATEWAY has provided a fast and reliable alternative to the cloning of sequences into large acceptor plasmids. Published online: 11 April 2002

The GATEWAY conversion technology (Invitrogen, Gaithersburg, MD, USA) is based on the site-specific recombination http://plants.trends.com

reaction mediated by phage λ. DNA fragments flanked by recombination sites (att) can be transferred into vectors that contain compatible recombination sites (attB × attP or attL × attR) in a reaction mediated by the GATEWAY BP Clonase or LR Clonase Enzyme Mix (Invitrogen). The entry clones, which can be considered general donor plasmids, are made by recombining the DNA fragment of interest with the flanking attB sites into the attP site pDONR201 mediated by the GATEWAY BP Clonase Enzyme Mix. Subsequently, the fragment in the entry clone can be transferred to any destination vector that contains the attR sites by mixing both plasmids and by using the GATEWAY LR Clonase Enzyme Mix.

Here we describe a set of GATEWAYcompatible binary T-DNA destination vectors for a wide range of different applications (Fig. 1). Details can be found on the web site (http://www.plantgenetics. rug.ac.be/gateway/), which provides the complete DNA sequence, a map, and a Vector NTI view of all constructs. The web site will be updated regularly by adding new constructs and relevant information. -compatible vectors Backbone of GATEWAY

The backbone of all described GATEWAY-compatible vectors is the plasmid pPZP200 [1]. This plasmid is relatively small (6.7 kb), contains an origin for replication in E. coli (ColE1) and in Agrobacterium (pVS1), has a pBR322 bom site for mobilization from E. coli to

1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(02)02251-3