Annals of Forest Science

, Volume 73, Issue 1, pp 119–134 | Cite as

Structural and functional characterization of tree proteins involved in redox regulation: a new frontier in forest science

  • Jean-Pierre Jacquot
  • Jérémy Couturier
  • Claude Didierjean
  • Eric Gelhaye
  • Mélanie Morel-Rouhier
  • Arnaud Hecker
  • Christophe Plomion
  • Desirée D. Gütle
  • Nicolas Rouhier
Review Paper

Abstract

Key message

This paper describes how the combination of genomics, genetic engineering, and 3D structural characterization has helped clarify the redox regulatory networks in poplar with consequences not only in system biology in plants but also in bacteria and mammalian systems.

Context

Tree genomes are increasingly available with a large number of orphan genes coding for proteins, the function of which is still unknown.

Aims and methods

Modern techniques of genome analysis coupled with recombinant protein technology and massive 3D structural determination of tree proteins should help elucidate the function of many of the proteins encoded by orphan genes. X-ray crystallography and NMR will be the methods of choice for protein structure determination.

Results

In this review, we provide examples illustrating how the above-mentioned techniques improved our understanding of redox regulatory circuits in poplar, the first forest tree species sequenced. We showed that poplar peroxiredoxins use either thioredoxin or glutaredoxin as electron donors to reduce hydrogen peroxide. That glutaredoxin could be a reductant was unknown at the time of this discovery even in other biological organisms and was later confirmed notably by the observation that the two genes are fused in some bacteria and by the resolution of the structure of the bacterial hybrid protein. Similarly, genome analysis coupled to in vitro analysis of enzymatic properties led to the discovery that some plant methionine sulfoxide reductases can also use both thioredoxins and glutaredoxins as electron donors. Besides their disulfide reductase activity, it has been demonstrated that some poplar glutaredoxins are also involved in iron-sulfur center biogenesis and assembly. The original 3D structure determination has been made with poplar glutaredoxin C1 and then confirmed in a variety of other biological organisms including human. Our work also showed that in plants, so-called glutathione peroxidases use thioredoxins and not glutathione as electron donors. This is true for all non-selenocysteine-containing glutathione peroxidases. Finally, connections between the thioredoxin and glutaredoxin systems have been elucidated through the study of atypical poplar thioredoxins.

Conclusion

Altogether, these data illustrate how the combination of genetic engineering and structural biology improves our understanding of biological processes and helps fuel systems biology for trees and other biological species.

Keywords

3D protein structure Genome sequence Glutaredoxin Redox Thioredoxin Poplar 

1 Introduction

There are currently more than 50 plant genomes sequenced and published (Michael and Jackson 2013; see also: http://en.wikipedia.org/wiki/List_of_sequenced_plant_genomes), and of these, more than ten concern tree species. The majority of those are constituted by fruit trees (peach, plum, pear, sweet orange, clementine, apple, coffee, papaya, and cocoa tree). The first forest tree genome published is Populus trichocarpa (Tuskan et al. 2006) followed by Picea abies (Nystedt et al. 2013), Hevea brasiliensis (Rahman et al. 2013), Picea glauca (Birol et al. 2013), Pinus taeda (Neale et al. 2014), and Eucalyptus grandis (Myburg et al. 2014).

The analysis of the first tree genomes has helped in identifying the total number of genes present in these genomes and also a number of orphan genes that have no counterpart in other biological organisms. A recent estimate of the number of orphan genes in poplar is 44 %, a value much higher than the 14 % estimation in Arabidopsis (Guo et al. 2007; Feldmann and Goff 2013). The mechanisms leading to orphan gene emergence have been discussed in a number of biological systems (Wissler et al. 2013). In order to understand the functions and interaction properties of the proteins encoded by these genes (if any), one possibility is to produce the corresponding recombinant proteins. The elucidation of their 3D structures together with molecular docking is one possibility that can be used to reach that understanding. Other techniques as the yeast two-hybrid system screen and co-immunoprecipitation can also help in identifying the potential ligands either metabolites or macromolecules as other proteins, carbohydrates, or lipids. Together with the results of transcriptomic and proteomic experiments, these approaches will help decipher metabolic and regulation networks. We illustrate here the use of genetic engineering and 3D structure determination to describe the redox interaction networks in trees, concentrating on the glutathione/glutaredoxin- (Grx) and thioredoxin- (Trx) dependent pathways in poplar. Most of the enzymes presented are members of the thioredoxin fold class. They share a small thioredoxin domain (consisting of a four-stranded beta sheet surrounded by three alpha helices) to fulfill many molecular functions.

2 Redox regulation background

When biological organisms including trees are submitted to stress either abiotic or biotic, a major response is the establishment of an oxidative burst which has to be controlled in order to avoid excessive accumulation of reactive oxygen species and subsequent subcellular damage. A number of antioxidant systems can be found in trees including the thioredoxin and glutaredoxin systems and structural derivatives such as thiol peroxidases (peroxiredoxins (Prx) and glutathione peroxidases (Gpx)) or methionine sulfoxide reductases (Msr). Notably, the thioredoxin-linked systems control the rate and hence yield of photosynthesis but they are also a good defense line against excess oxidants such as H2O2 (Schürmann and Jacquot 2000; Rouhier et al. 2008). The general positioning of the thioredoxin- or glutaredoxin-linked redox networks in plants is shown in Fig. 1. In this review, we provide a number of examples detailing how poplar has served as a model for understanding redox regulation not only in plants but also in other systems including animal and bacterial cells. We will also indicate how poplar has become a model species for establishing the 3D structures of plant proteins, a field still in infancy.
Fig. 1

Positioning and major roles of the glutaredoxin- and thioredoxin-linked systems in plants. This figure emphasizes the central position of the glutaredoxin and thioredoxin systems for the cellular redox control in photosynthetic organisms. Abbreviations used are as follows: GR glutathione reductase, GSH glutathione, TR thioredoxin reductases, Grx glutaredoxin, Trx Thioredoxin

3 Poplar thiol peroxidases: peroxiredoxins and “glutathione” peroxidases

The studies that have been initiated in poplar were initially a follow-up of the previous characterization of a peroxide-detoxifying enzyme called peroxiredoxin or thioredoxin peroxidase (Verdoucq et al. 1999). Peroxiredoxins are very abundant enzymes which rely on a catalytic cysteine residue to attack various organic peroxides (including hydrogen peroxide) with the release of an alcohol-like molecule and water according to the equation:
$$ \mathrm{RO}\mathrm{O}\mathrm{H}\to \mathrm{R}\mathrm{O}\mathrm{H}+{\mathrm{H}}_2\mathrm{O} $$
In the process, the catalytic cysteine residue becomes oxidized into a sulfenic acid which has to be regenerated, the best known regenerating system being the thioredoxin system (NADPH or photoreduced ferredoxin relayed by a thioredoxin reductase and thioredoxin itself). Including glutathione peroxidases, there are five classes of thioredoxin peroxidases in plants based on their amino acid sequence and oligomerization state (Rouhier and Jacquot 2005). Several of them are solely dependent on thioredoxin for their regeneration (Rouhier et al. 2004b; Navrot et al. 2006), but surprisingly our initial data which concerned a specific class called atypical type II peroxiredoxin (later referred to as poplar PrxIIB) indicated that they could be regenerated equally well by thioredoxin and glutaredoxin (Rouhier et al. 2001, 2002a). That glutaredoxins could be an alternate reducing system to atypical type II peroxiredoxins was unknown at that time in other biological organisms. We could in subsequent experiments determine that poplar type II peroxiredoxins are present in several cellular compartments and in particular in the cytosol, mitochondria, and plastids (Gama et al. 2007, 2008). The 3D structure of the cytosolic poplar Prx IIB has been solved both by X-ray crystallography and NMR revealing molecular aspects of its organization, catalytic and regeneration mechanisms (Echalier et al. 2005; Noguera-Mazon et al. 2006). A nice confirmation that glutaredoxin could indeed be a physiological reductant for type II Prx was the detection of hybrid genes in several bacteria. As for thioredoxin reductase-thioredoxin gene fusions observed in some mycobacteria as Mycobacterium leprae (Jacquot et al. 2009), Prx and Grx genes can be associated in operons and even fused in some species as Haemophilus influenzae and Neisseria meningitidis resulting in the generation of a hybrid enzyme (Kim et al. 2003; Rouhier and Jacquot 2003). Based on the sequence of the linker peptide between the two domains (Grx and Prx), we were then able in “fun experiments” to construct functional Prx-Grx and Prx-Trx fusion proteins made with poplar sequences (Rouhier et al. 2006b). We have also studied deeply another variety of peroxiredoxin from poplar called peroxiredoxin Q (Prx Q in plants or BCP in bacteria) (Rouhier et al. 2004b). Prx Q is exclusively chloroplastic and the poplar enzyme is brought back to the reduced form by the thioredoxin system but not via the glutaredoxin system unlike type II Prx. Prx Q expression is modified when poplar leaves are infected by the rust fungus Melampsora larici-populina. We could not get a 3D structure of poplar Prx Q but we have been later able to isolate and characterize structurally a hybrid enzyme from Thermotoga maritima which contains a Prx Q-like module linked to a nitroreductase domain (Couturier et al. 2013b). Interestingly in T. maritima, the hybrid enzyme is reduced by an unusual reduction system composed of thioredoxin reductase and glutaredoxin-like enzymes, indicating that glutaredoxins can be donors in other biological systems than poplar. In non-photosynthetic organisms, another line of defense against excessive peroxide generation is constituted by Gpxs. Those enzymes employ similar catalytic mechanisms and sulfenic acid chemistry as Prxs. There are several classes of Gpxs in mammals including selenocysteine-containing enzymes which are present only in algae but not in land plants, the Gpxs of which contain cysteines instead of selenocysteines. For the selenoenzymes, the selenenic acid is normally brought back to the thiol state via reduced glutathione. Interestingly, we have observed using the poplar model that plant Gpxs are present in different subcellular compartments but also that they differ from their mammalian selenoenzyme counterparts in using thioredoxin but not glutathione for their regeneration. Whereas it is in principle possible for GSH to reduce sulfenic acids, the presence of a resolving cysteine in plant Gpxs leads to the formation on an intramolecular disulfide which can only be reduced by Trxs. In this respect, cysteine-containing Gpxs can be considered as thioredoxin peroxidases or peroxiredoxins. Indeed, the structural determination of poplar Gpx5 indicated that it has a fold similar to peroxiredoxins, an observation that was not clear from the examination of amino acid sequences (Navrot et al. 2006; Koh et al. 2007). Figure 2 illustrates this structural convergence between type II Prx and so-called plant Gpx (panels a, b). The monomer organization is similar, based on the thioredoxin model plus a helix-beta strand insertion between the second helix and the second β-strand of the Trx fold and a β-hairpin N-terminal extension. Interestingly, plant Gpx can reduce not only hydroperoxides but also peroxynitrites (Selles et al. 2012). In conclusion, this set of experiments has allowed us to determine that unlike their mammalian counterparts, plant Gpxs do not rely on glutathione but rather on the thioredoxin system for their functioning and regeneration. The peroxiredoxin and Gpx “stories” have also provided one of the examples of the interchangeability between the thioredoxin and glutaredoxin pathways with regard to peroxide detoxification.
Fig. 2

Selected structures of poplar enzymes. In all these structures, β-strands are shown in blue, α-helices are in red, and connecting loops are in purple. a Cartoon representation of the overall fold of the dimeric Prx IIB (PDB number, 1TP9). b Cartoon representation of the overall fold of the dimeric Gpx5 in the reduced form (PDB number, 2P5Q). Note that, in the oxidized form, the formation of an intramolecular disulfide bond between Cys44 and Cys92 induces a large conformational rearrangement leading in particular to the complete unwinding of the α1 helix. By comparing both structures, you could note the similar architecture of the monomers and the characteristic double β-strand external to the central pleated β-sheet. c Cartoon representation of the overall fold of the plastidial methionine sulfoxide reductase A (PDB number, 2J89). The structure of the monomer in the reduced form is shown here. The positions of the sulfur atoms of the important cysteine residues are indicated by yellow spheres. Site-directed mutagenesis experiments coupled to activity assays indicated that the catalytic Cys46 forms a disulfide with Cys202 before its subsequent reduction by Cys196. It is clear that considerable conformational rearrangements are needed to bring the sulfur atoms close enough to form these disulfides. This may explain why we have been unable to get the 3D structure of oxidized forms. d Cartoon representation of the overall fold of the monomeric thioredoxin h4 under two different oxidized forms (PDB numbers 3D21, 3D22). The sulfur atoms of the cysteine residues of the disulfide bonds are shown in yellow spheres. Left panel, disulfide bond between Cys58 and Cys61; right panel, disulfide bond between Cys4 and Cys58. Lacking residues (7–15) are shown in an imaginary gray dashed loop for a better overview of the right panel structure. e, f Structural comparison of Grx C1 and Grx C4 dimers. In the Grx C1 structure, there is one homodimer containing a [2Fe-2S] ISC plus two additional monomers symmetrically positioned but bearing no ISC. GSH indicates an external glutathione molecule (shown in stick) and GS glutathione molecule covalently linked to the Fe2S2 center. Sulfur and iron atoms are represented, respectively, as yellow and red spheres. Grx C4 makes a homodimer with no ISC. The PDB number of Grx C1 is 2E7P, whereas no coordinates were deposited for Grx C4

4 Poplar methionine sulfoxide reductases

Another example of a repair enzyme using sulfenic acid chemistry is the methionine sulfoxide reductase. Oxidative stress can indeed induce the oxidation of the side chain of methionine residues resulting in methionine sulfoxide generation. Depending on the stereochemistry, two types of Msrs can repair this oxidative modification: MsrA and MsrB. MsrA has been characterized in poplar and its 3D structure deciphered. Unlike Prx whose structure is based on the thioredoxin fold, MsrA exhibits a unique alpha/beta architecture. Figure 2c shows the structure of the subunit of MsrA together with the position of the cysteines involved in catalysis. For catalysis, the two poplar MsrAs characterized rely on a three cysteine mechanism, the order of the cysteine involvement being different from the Escherichia coli enzyme (Rouhier et al. 2007a). Initially for poplar MsrA, the most efficient reductant system was found to be the thioredoxin system. However, it was found later that, in Gracilaria gracilis, MsrA and Grx are linked in a Grx-Grx-MsrA hybrid enzyme, a situation reminiscent of the earlier identification of Prx-Grx fusions. Additionally, subsequent experiments have shown that Msrs of the two types (A and B) including poplar MsrA are also efficiently regenerated by the glutaredoxin system (Vieira dos Santos et al. 2007; Tarrago et al. 2009; Couturier et al. 2012). Overall, these data provide further evidence concerning the interchangeability of the two reduction systems.

5 Poplar thioredoxins

Additional data connecting the thioredoxin and glutaredoxin systems were provided by the thorough study of poplar thioredoxins. We first set out to describe the first structure of an active site variant (CPPC instead of CGPC) in poplar Trxh1 (Coudevylle et al. 2004, 2005). The NMR structural analysis revealed that changing the glycine residue of the active site into a proline has very moderate effect on the structure (Menchise et al. 2001), although based on yeast complementation, CPPC containing thioredoxins are less active in sulfate reduction but more efficient for protection against H2O2 than their CGPC counterparts (Bréheĺin et al. 2000). Further characterization of poplar thioredoxins included the study of poplar Trxh3 (Gelhaye et al. 2003b) and also showed that the Trxh2 isoform is located in mitochondria where it serves to regulate alternative oxidase and the mitochondrial electron flow (Gelhaye et al. 2004). Incidentally, this was the first clear demonstration of the presence of thioredoxin h in plant mitochondria. A very interesting piece of information arose when studying poplar Trx h4. This protein possesses a classical active site (CGPC) but has the unique characteristic to contain a conserved cysteine near the N-terminus at position 4 (Gelhaye et al. 2003a). Through site-directed mutagenesis and structural determination, we have shown that this was related to an unusual three cysteine mechanism where two successive and different disulfides are formed upon catalysis, one of them involving Cys4. Remarkably, the regeneration system of Trx h4 is the glutaredoxin system and not the NAPDH-thioredoxin reductase system (Koh et al. 2008). Figure 2d shows the structure of poplar Trx h4 with its characteristic pleated β-sheet surrounded by α helices found in all thioredoxins. Unlike in non-photosynthetic organisms, there are in fact a large number of thioredoxin genes in plants and the situation is similar in poplar (Gelhaye et al. 2005; Chibani et al. 2009). One of the newly discovered thioredoxins, Trx z, is located in plastids and reduced by the ferredoxin-thioredoxin reductase (FTR) (Chibani et al. 2011). Several of the thioredoxin genes code for proteins with highly unusual active sites. We showed quite recently that in contrast with their classification based on amino acid sequence similarities, poplar Trx-like2.1 and Trx-lilium2.2 are regenerated in vitro either by the NADPH-thioredoxin reductase system or by glutathione but not the FTR system (Chibani et al. 2012). As a consequence, since these two thioredoxins are plastidial, their likely in vivo reducing system is glutathione, thus resembling glutaredoxins. Consistently, Trx-like2.1 was found to be effective in MsrB1 and PrxIIE regeneration, two known Grx target enzymes. Altogether, these results further exemplify the need to back up phylogenetic studies with “wet lab” biochemistry. A similar need was apparent from the wrong classification of plant “glutathione” peroxidases which turned out to be thioredoxin peroxidases as detailed previously. Concerning our own redox research, these data also reinforce the notion of thioredoxin-glutaredoxin cross talk already previously mentioned. To conclude on the past studies on poplar thioredoxins on a more physiological note, we have very recently obtained evidence that thioredoxin h1 is involved in the gravitropism response being itself localized in the amyloplasts of the endoderm, a localization of high interest for gravitropism signal perception (Azri et al. 2013). Overall, the international recognition of these studies on poplar in a competitive field (including research groups in medicine and other areas of biology) is apparent in a paper by Perez-Jimenez et al. (2009) where two poplar thioredoxin h were selected in a study detailing thioredoxin catalysis at the single molecule and atomic level.

6 Poplar glutaredoxins

Another section of the redox field where we have made significant contributions is the study of glutaredoxins, the initial breakthrough papers having been made with poplar Grxs. As observed for Trxs, Grxs make an extended phylogenetic family in photosynthetic organisms including poplar but conversely the number of Grx genes is much reduced in bacteria or mammals (Rouhier et al. 2004a, 2006a; Couturier et al. 2009a, 2013a). Overall, there are three major groups in land plants with active sites in the form of CxxC (class I), CGFS (class II), and CCxC/S (class III). Class III Grxs are present solely in land plants and play a role in flower development (Li et al. 2009). From genomic and phylogenetic analyses, a fourth Grx class is also present in land plants but whether they exhibit glutathione-dependent reductase activity awaits confirmation (Couturier et al. 2009a). Additional classes formed by fusion enzymes are found in lower eukaryotes and prokaryotes. The Grx studies were initiated with poplar Grx C4, a class I member with a “classical” CYPC active site. The corresponding protein was produced in a soluble form by genetic engineering but only after optimization of its DNA sequence and its biochemical properties studied. It did not turn out to be different from other classical Grxs (d’Ambrosio et al. 2003; Rouhier et al. 2002b, 2003). On the other hand, the characterization of poplar Grx C1 (class I, CGYC active site) led to surprising results as the recombinant protein partitioned between an apo monomeric form and a holo dimeric form. Very interestingly, the holo form was shown to contain an iron sulfur center (ISC) of the Fe2S2 type (Feng et al. 2005, 2006; Rouhier et al. 2007b). NMR-linked structural determination of Grx C4 which does not normally assemble such holo structures indicated nevertheless that the protein makes homodimers in solution with the active sites facing one another (Noguera et al. 2005). Figures 2e, f show dimers of Grx C1 and Grx C4, with the ISC present in the former but not the latter. A thorough comparison of the structures of both Grxs revealed that the Pro residue present between the two Cys of the disulfide is probably too bulky to allow the formation of the ISC in Grx C4. In agreement with this observation, the P to G mutation in Grx C4 allows the protein to incorporate an ISC into a homodimer, and the reverse mutation in Grx C1 abolishes that property (Rouhier et al. 2007b). A crucial observation in Grx C1 is that two of the Fe-S ligands are external glutathione molecules, a situation also encountered in a number of non-photosynthetic homologs (Rouhier et al. 2008). Incidentally, these data complexify the bioinformatics prediction of sequences able to bind an ISC. Normally, they should contain four cysteines or a similar number of cysteine and histidine ligand residues, but in the permissive glutaredoxins, only one cysteine per monomer is sufficient (Selles et al. 2009). The structural study of class I poplar Grx S12 and also of Arabidopsis thaliana Grx C5 has added more to our understanding concerning the sequence requirements permissive for ISC binding (Couturier et al. 2009b, 2011). The observation that Grxs from class II can bind ISC has shed new light on a completely different research area, the biosynthesis of ISC. Indeed, it was known that in yeast the mitochondrial Grx5 (class II, CGFS active site) is required for Fe-S cluster biogenesis but it was not known why (Rodríguez-Manzaneque et al. 2002). Work performed with CGFS Grxs from poplar or Arabidopsis has demonstrated that they also ligate ISC. In addition, they can very efficiently transfer these centers to acceptor proteins as apo ferredoxin (Bandyopadhyay et al. 2008; Rouhier et al. 2010). Overall, these very original contributions made with poplar proteins support an additional role for glutaredoxins in Fe-S protein assembly, separate from the classical reductase activity.

While progress has been remarkable on class I and II members, we know little about the biochemical and structural properties of class III Grxs. Indeed, they are notoriously difficult to work with owing to insolubility problems perhaps linked to the very hydrophobic C-terminus of the protein. Nevertheless, we have made a number of constructs using poplar Grx C1 and Grx C4 as templates in which we have introduced active site signatures (CCMC and CCMS) present in class III Grxs. In agreement with the structural predictions made from earlier work on Grx C1 and C4, the engineered Grxs bind Fe-S centers and possess reductase activity in the monomeric form (Couturier et al. 2010). Whether these data reflect specific physiological functions awaits further characterization.

7 Biochemical and structural studies of redox enzymes in progress

Having isolated to homogeneity a number of poplar thioredoxins and glutaredoxins has allowed us to trap their potential targets. This was performed by genetic engineering and modifying the second cysteine of the active sites into serines which in effect prevents the backup reaction and allows trapping the target disulfide-linked to the redoxin. Using this technology, we have identified ca 100 Grx targets suggesting that redox post-translational modifications control a large number of cellular processes. A carbonic anhydrase was further characterized (Rouhier et al. 2005). Likewise, the use of a mutated poplar thioredoxin h led to the identification of more than 50 Trx-linked proteins in the mitochondria (Balmer et al. 2004). The structural and regulatory properties of those potential targets are still largely unknown even though redox regulation in mitochondria was reported for 2-oxoaciddehydrogenase complexes (Bunik et al. 1999).

There are a large number of glutathione-S-transferases (GST) genes in photosynthetic organisms and especially in poplar (Lan et al. 2009; Lallement et al. 2014a). These proteins are involved mainly in cellular detoxification. Some of these proteins possess a catalytic cysteine and they are functionally similar to Grxs. Three GSTs of the lambda type (GSTL1 to L3) have been recently characterized until the molecular level. The proteins are monomeric and they bind glutathionylated substrates for deglutathionylation reactions (Lallement et al. 2014b). Overall, this study strengthened the proposal that GSTLs are involved in the management of secondary metabolites via their redox properties. Additional poplar GSTs from different classes are currently being studied.

We have mentioned earlier the existence of thioredoxin-like proteins in poplar (like2- and lilium-types especially), indicating that these proteins functionally behave as glutaredoxins. From this observation, it becomes of high interest to elucidate what in their structure makes them glutaredoxin-like from a catalytic point of view. As a matter of fact, several of these structures are being solved presently and we should be able to answer this question soon.

Another area where we are currently active concerns the regulatory enzymes of the Benson-Calvin cycle and especially the phosphatases, fructose-1,6-bisphosphatase (FBPase), and sedoheptulose-1,7-bisphosphatase (SBPase). It is known that both enzymes are redox-regulated but the relationship between them is still unclear at the molecular level. Although we know precisely the structure and regulatory properties of pea FBPase, little is known about the structure-function relationship of SBPase (Jacquot et al. 1995; Chiadmi et al. 1999). In a first series of experiments, we have produced the corresponding recombinant proteins from poplar and Chlamydomonas reinhardtii but the enzymes turned out not to be sufficiently stable and rather rapidly denatured. We have then turned to another model Physcomitrella patens (see companion paper by Müller et al. this issue) from where we could produce stable FBPase and SBPase as recombinant proteins. The biochemical and structural properties of both proteins are currently under investigation. The Physcomitrella patens model is also currently being used to create mutants of the ferredoxin-thioredoxin system using site-directed mutagenesis and gene replacement for the catalytic subunit of FTR.

Another area of current excitement is the involvement of class II Grxs in ISC biosynthesis. In three recent papers, the structures of poplar Grx S14 were solved and its Fe-S cluster transfer capacity described (Wang et al. 2011, 2012, 2014). Current emphasis is made on the interactions between class II Grxs and BolA proteins or similar components such as SufE1 protein (Couturier et al. 2014; Roret et al. 2014). A last domain of interest that is being investigated concerns the link between redox and biotic stress. Indeed when poplar leaves are infected by rust there is a very active exchange of information including the release of small secreted proteins (SSP) by both interaction partners. These SSP very often possess unusually high cysteine contents with the possibility to make several disulfides. We are currently studying the properties of some corresponding recombinant proteins up to the 3D molecular level. Interestingly, many of these SSPs would qualify as orphan gene products.

8 Census of tree protein 3D structures

As explained above, the availability of gene models from fully sequenced genomes is extremely helpful and even required for the development of high throughput structural biology with the aim of deciphering tree protein structures. In the pdb database, the structures are listed with a link to their botanical classification and unfortunately the item tree is not present. So making a census of the present tree protein structures is not an easy task. Also the definition of a tree is not so clear; for example, we chose not to define banana (overall nine protein structures available of three different proteins) and coconut (one structure in pdb) as trees as various botanical considerations indicate they should not be labeled as such. Another example is Vitis vinifera (overall 20 protein structures nearly all of them describing oxidoreductases of the secondary metabolism) which we chose not to include as a tree although it is an organism which contains lignin but it does not have the stance of a tree. Tables 1 and 2 provide the current data concerning protein structures from forest and non-forest trees, respectively. Using a very empirical approach (looking systematically for most well-known tree species), we have found 141 protein structures belonging to forest species including H. brasiliensis and 33 structures to non-forest species (including fruit trees but also plantation trees as coffee). The non-forest tree protein structures describe ca 14 different proteins from five different botanical families. The known forest tree protein structures belong to seven genera (Betulaceae, Cesalpinaceae, Euphorbiaceae, Fabaceae, Salicaceae, Taxaceae, and Pinaceae) and they describe ca 36 different protein structures. A large number of these structures (18) describe a single pollen allergen of birch, the BET V allergen, and a similar number (17) a hydroxynitryle lyase from Hevea. Overall, nearly half of the forest tree structures describe poplar proteins illustrating how genome sequencing influences downstream the structural field (poplar was the first genome tree sequenced and fully annotated). Overall, 19 of the poplar structures describe a single protein plastocyanin either from NMR or crystallographic data in complex with a variety of ligands, one of the structures describing plastocyanin in complex with cytochrome f. In reality, the effort concerning plastocyanin started well before the genome sequence of poplar became available. Our own contribution to the structural field has resulted in 17 structures describing 11 different poplar proteins in various states solved either by NMR or X-ray crystallography. They are marked with a star in Table 1. It now seems clear that poplar is bound to become a model organism for structural biology especially when coupled to molecular biology allowing the production of recombinant proteins. Still it is remarkable that the first plastocyanin structure was obtained with the poplar protein well before the advent of all those molecular techniques (Guss and Freeman 1983). To conclude on those aspects of structural biology, it should be mentioned that poplar is and will most likely become the model species for trees but it is of course not the only one for plants in general and remarkable advances have been done in the past with spinach or pea high molecular weight enzymes as RubisCO and LHC (Knight et al. 1990; Standfuss et al. 2005; Barros et al. 2009).
Table 1

Known forest tree protein structures

PDB code

Family

Source

Enzyme/protein

1B6F

Betulaceae

Betula pendula (birch)

BET V 1 allergen

1BTV

Betulaceae

Betula pendula (birch)

BET V 1 allergen

1BV1

Betulaceae

Betula pendula (birch)

BET V 1 allergen

1FM4

Betulaceae

Betula pendula (birch)

BET V 1 allergen

1LLT

Betulaceae

Betula pendula (birch)

BET V 1 allergen

1QMR

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A80

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A81

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A83

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A84

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A85

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A86

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A87

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A88

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A8G

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A8U

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4A8V

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4B9R

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4BK6

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4BK7

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4BKC

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4BKD

Betulaceae

Betula pendula (birch)

BET V 1 allergen

4BTZ

Betulaceae

Betula pendula (birch)

BET V 1 allergen

1H4B

Betulaceae

Betula pendula (birch)

BET V 4 allergen

1CQA

Betulaceae

Betula pendula (birch)

Profilin

1R8N

Caesalpinaceae

Delonix regia

Kunitz trypsin inhibitor

3QQQ

Cannabaceae

Trema tomentosa

Plant hemoglobin

1PXZ

Cupressaceae

Cedar

Allergen

1QYD

Cupressaceae

Thuya

Pinoresinol reductase

1P9G

Eucommiaceae

Eucommia ulmoides

Antifungal peptide

1P9Z

Eucommiaceae

Eucommia ulmoides

Antifungal peptide

4MPI

Euphorbiaceae

Hevea

Chitin-binding module of a chitinase-like protein

4HPG

Euphorbiaceae

Hevea

Glycosylated glucanase

4IIS

Euphorbiaceae

Hevea

Glycosylated glucanase

1HVQ

Euphorbiaceae

Hevea

Hevamin A

1LLO

Euphorbiaceae

Hevea

Hevamin A

2HVM

Euphorbiaceae

Hevea

Hevamin A

1KQY

Euphorbiaceae

Hevea

Hevamin mutant

1KQZ

Euphorbiaceae

Hevea

Hevamin mutant

1KR0

Euphorbiaceae

Hevea

Hevamin mutant

1KR1

Euphorbiaceae

Hevea

Hevamin mutant

1HEV

Euphorbiaceae

Hevea

Hevein

1QB9

Euphorbiaceae

Hevea

Hevein

1T0W

Euphorbiaceae

Hevea

Hevein

1WKX

Euphorbiaceae

Hevea

Hevein

4YAS

Euphorbiaceae

Hevea

Hydroxy nitryle lyase

5YAS

Euphorbiaceae

Hevea

Hydroxy nitryle lyase

6YAS

Euphorbiaceae

Hevea

Hydroxy nitryle lyase

7YAS

Euphorbiaceae

Hevea

Hydroxy nitryle lyase

1QJ4

Euphorbiaceae

Hevea

Hydroxynitryle lyase

1SC9

Euphorbiaceae

Hevea

Hydroxynitryle lyase

1SCI

Euphorbiaceae

Hevea

Hydroxynitryle lyase

1SCK

Euphorbiaceae

Hevea

Hydroxynitryle lyase

1SCQ

Euphorbiaceae

Hevea

Hydroxynitryle lyase

1YAS

Euphorbiaceae

Hevea

Hydroxynitryle lyase

1YB6

Euphorbiaceae

Hevea

Hydroxynitryle lyase

1YB7

Euphorbiaceae

Hevea

Hydroxynitryle lyase

2G4L

Euphorbiaceae

Hevea

Hydroxynitryle lyase

2YAS

Euphorbiaceae

Hevea

Hydroxynitryle lyase

3C6X

Euphorbiaceae

Hevea

Hydroxynitryle lyase

3C6Y

Euphorbiaceae

Hevea

Hydroxynitryle lyase

3C6Z

Euphorbiaceae

Hevea

Hydroxynitryle lyase

3C70

Euphorbiaceae

Hevea

Hydroxynitryle lyase

3YAS

Euphorbiaceae

Hevea

Hydroxynitryle lyase

1G5U

Euphorbiaceae

Hevea

Latex profilin

1FNY

Fabaceae

Robinia

Lectin

1FNZ

Fabaceae

Robinia

Lectin

2VLC

Laraceae

Cinnamomum camphora (camphor)

Cinnamomin (ribosome inactivating protein)

2LVF

Lecythidoideae

Bertholletia excelsa (Brazil nut)

Allergen

1J4S

Moraceae

Artocarpus

Artocarpin

1J4T

Moraceae

Artocarpus

Artocarpin

1J4U

Moraceae

Artocarpus

Artocarpin

1VBO

Moraceae

Artocarpus

Artocarpin

1VBP

Moraceae

Artocarpus

Artocarpin

1JAC

Moraceae

Artocarpus

Jacalin

1WS4

Moraceae

Artocarpus

Jacalin

1WS5

Moraceae

Artocarpus

Jacalin

3P8S

Moraceae

Artocarpus

Jacalin

1KU8

Moraceae

Artocarpus

Jacalin (lectin)

1KUJ

Moraceae

Artocarpus

Jacalin (lectin)

1M26

Moraceae

Artocarpus

Jacalin (lectin)

1PXD

Moraceae

Artocarpus

Jacalin (lectin)

1UGW

Moraceae

Artocarpus

Jacalin (lectin)

1UGX

Moraceae

Artocarpus

Jacalin (lectin)

1UGY

Moraceae

Artocarpus

Jacalin (lectin)

1UH0

Moraceae

Artocarpus

Jacalin (lectin)

1UH1

Moraceae

Artocarpus

Jacalin (lectin)

1TOQ

Moraceae

Artocarpus

Lectin

1TP8

Moraceae

Artocarpus

Lectin

4AK4

Moraceae

Artocarpus

Lectin

4AKB

Moraceae

Artocarpus

Lectin

4AKC

Moraceae

Artocarpus

Lectin

4AKD

Moraceae

Artocarpus

Lectin

4CUO

Moraceae

Ficus bengalensis

Banyan peroxidase

1JOT

Moraceae

Maclura pomifera

Agglutinin

3LLY

Moraceae

Maclura pomifera

Agglutinin

3LLZ

Moraceae

Maclura pomifera

Agglutinin

3LM1

Moraceae

Maclura pomifera

Agglutinin

3S9V

Pinaceae

Abies

Abietadiene synthase

3SAE

Pinaceae

Abies

Sesquiterpene synthase

3SDQ

Pinaceae

Abies

Sesquiterpene synthase

3SDR

Pinaceae

Abies

Sesquiterpene synthase

3SDT

Pinaceae

Abies

Sesquiterpene synthase

3SDU

Pinaceae

Abies

Sesquiterpene synthase

3SDV

Pinaceae

Abies

Sesquiterpene synthase

3HBD

Pinaceae

Picea

Chitinase

3HBE

Pinaceae

Picea

Chitinase

3HBH

Pinaceae

Picea

Chitinase

1QYC

Pinaceae

Pinus

Phenylcoumaran benzylic ether reductase

1UOU

Pinaceae

Pinus

Stilbene synthase

1XES

Pinaceae

Pinus

Stilbene synthase

1XET

Pinaceae

Pinus

Stilbene synthase

4LEJ

Pinaceae

Pinus

Vicilin

4N6T

Salicaceae

Populus (poplar)

Adhiron (de novo prot)

4N6U

Salicaceae

Populus (poplar)

Adhiron (de novo prot)

3A9U

Salicaceae

Populus (poplar)

Coumarate CoA ligase

3A9V

Salicaceae

Populus (poplar)

Coumarate CoA ligase

3NI2

Salicaceae

Populus (poplar)

Coumarate CoA ligase

1Z7P

Salicaceae

Populus (poplar)

Glutaredoxin C1a

1Z7R

Salicaceae

Populus (poplar)

Glutaredoxin C1a

2E7P

Salicaceae

Populus (poplar)

Glutaredoxin C1a

No deposit

Salicaceae

Populus (poplar)

Glutaredoxin C4 NMR crystallographya

3FZ9

Salicaceae

Populus (poplar)

Glutaredoxin S12a

3FZA

Salicaceae

Populus (poplar)

Glutaredoxin S12a

2LKU

Salicaceae

Populus (poplar)

Glutaredoxin S14 apo NMRa

2LKU

Salicaceae

Populus (poplar)

Glutaredoxin S14 NMRa

2P5Q

Salicaceae

Populus (poplar)

Glutathione peroxidase Gpx5a

2P5R

Salicaceae

Populus (poplar)

Glutathione peroxidase Gpx5a

4PQH

Salicaceae

Populus (poplar)

Glutathione S transferase L1a

4PQI

Salicaceae

Populus (poplar)

Glutathione S transferase L3a

3N0F

Salicaceae

Populus (poplar)

Isoprene synthase

3N0G

Salicaceae

Populus (poplar)

Isoprene synthase

2 J89

Salicaceae

Populus (poplar)

Methionine sulfoxide reductase Aa

1JXG

Salicaceae

Populus (poplar)

Plastocyanin

1PLC

Salicaceae

Populus (poplar)

Plastocyanin

1PNC

Salicaceae

Populus (poplar)

Plastocyanin

1PND

Salicaceae

Populus (poplar)

Plastocyanin

2PCY

Salicaceae

Populus (poplar)

Plastocyanin

3PCY

Salicaceae

Populus (poplar)

Plastocyanin

4DP0

Salicaceae

Populus (poplar)

Plastocyanin

4DP1

Salicaceae

Populus (poplar)

Plastocyanin

4DP2

Salicaceae

Populus (poplar)

Plastocyanin

4DP4

Salicaceae

Populus (poplar)

Plastocyanin

4DP5

Salicaceae

Populus (poplar)

Plastocyanin

4DP6

Salicaceae

Populus (poplar)

Plastocyanin

4DP7

Salicaceae

Populus (poplar)

Plastocyanin

4DP8

Salicaceae

Populus (poplar)

Plastocyanin

4DP9

Salicaceae

Populus (poplar)

Plastocyanin

4DPA

Salicaceae

Populus (poplar)

Plastocyanin

4DPB

Salicaceae

Populus (poplar)

Plastocyanin

4DPC

Salicaceae

Populus (poplar)

Plastocyanin

4PCY

Salicaceae

Populus (poplar)

Plastocyanin

5PCY

Salicaceae

Populus (poplar)

Plastocyanin

6PCY

Salicaceae

Populus (poplar)

Plastocyanin

1TKW

Salicaceae

Populus (poplar)

Plastocyanin cyt f complex

1SI9

Salicaceae

Populus (poplar)

Protein SP1 heat stable

1TRO

Salicaceae

Populus (poplar)

Protein SP1 heat stable

1YQD

Salicaceae

Populus (poplar)

Sinapyl alcohol dehydrogenase

1YQX

Salicaceae

Populus (poplar)

Sinapyl alcohol dehydrogenase

1TI3

Salicaceae

Populus (poplar)

Thioredoxin h1a

3D21

Salicaceae

Populus (poplar)

Thioredoxin h4a

3D22

Salicaceae

Populus (poplar)

Thioredoxin h4a

1TP9

Salicaceae

Populus (poplar)

Type II peroxiredoxina

1UMZ

Salicaceae

Populus (poplar)

Xyloglucan endotransglycolase

1UN1

Salicaceae

Populus (poplar)

Xyloglucan endotransglycolase

2YII

Taxaceae

Taxus

Phenylalanine amino mutase

4BAA

Taxaceae

Taxus

Phenylalanine amino mutase

4BAB

Taxaceae

Taxus

Phenylalanine amino mutase

4C5R

Taxaceae

Taxus

Phenylalanine amino mutase

4C5S

Taxaceae

Taxus

Phenylalanine amino mutase

4C5U

Taxaceae

Taxus

Phenylalanine amino mutase

4C6G

Taxaceae

Taxus

Phenylalanine amino mutase

4CQ5

Taxaceae

Taxus

Phenylalanine amino mutase

3NZ4

Taxaceae

Taxus

Taxadiene synthase

3P5P

Taxaceae

Taxus

Taxadiene synthase

3P5R

Taxaceae

Taxus

Taxadiene synthase

aIndicates the structures deposited by the authors of this paper

Table 2

Known non-forest tree protein structures

pdf #

Family

Source

Enzyme/protein

3G4F

Malvaceae

Cotton, Gossypium arboreum

Cadinene synthase

3G4D

Malvaceae

Cotton, Gossypium arboreum

Cadinene synthase

3BRX

Malvaceae

Cotton, Gossypium hirsutum

Annexin

1N00

Malvaceae

Cotton, Gossypium hirsutum

Annexin

2JON

Oleaceae

Olive

Allergen

1SS3

Oleaceae

Olive

Allergen

1B8G

Rosaceae

Apple

ACC synthase

1M4N

Rosaceae

Apple

ACC synthase

1M7Y

Rosaceae

Apple

ACC synthase

1YNU

Rosaceae

Apple

ACC synthase

3PIU

Rosaceae

Apple

ACC synthase

3ZS3

Rosaceae

Apple

Thaumatin like

1IQQ

Rosaceae

Pyrus, pear

Pistil ribonuclease

1E09

Rosaceae

Prunus

Pru Av1 cherry allergen

1H2O

Rosaceae

Prunus

Pru Av1 cherry allergen

1JU2

Rosaceae

Prunus

Hydroxy nitryle lyase

2AHN

Rosaceae

Prunus

Pru Av2 allergen

2ALG

Rosaceae

Prunus

Pru P (LTP)

2B5S

Rosaceae

Prunus

Pru P (LTP)

3EHK

Rosaceae

Prunus

Allergenic protein

3FZ3

Rosaceae

Prunus

Almond Pru I allergenic

3GDN

Rosaceae

Prunus

Hydroxy nitryle lyase

3GDP

Rosaceae

Prunus

Hydroxy nitryle lyase

3RED

Rosaceae

Prunus

Hydroxy nitryle lyase

2EFJ

Rubiaceae

Coffee

3,7-Dimethylxanthine methyltransferase

2EG5

Rubiaceae

Coffee

Xanthosine methyl transferase

4G22

Rubiaceae

Coffee

Lys HCT

4G0B

Rubiaceae

Coffee

Lys HCT

4G2M

Rubiaceae

Coffee

Lys HCT

2M70

Rutaceae

Citrus

Poly(A)-binding protein 1

3WD7

Rutaceae

Citrus

Polyketide synthase

3WD8

Rutaceae

Citrus

Polyketide synthase

4JJM

Rutaceae

Citrus

Cyclophilin

9 Tree system biology: what could be contribution of structural proteomics?

Massive information from genome sequencing needs now to be organized into a systems biology database in order to understand the structure-function relationships of individual proteins (Dietz et al. 2010). Besides, the characterization of protein-protein interactions obviously requires working at the protein level. Still, even the genome organization helps in this respect. We have given above a couple examples explaining how the genes coding for separate proteins working together in poplar can be found linked together in bacteria, leading to the production of hybrid enzymes. A take home message is that one should systematically look for such possible associations in prokaryotic genomes. The results of this systematic genomic data-mining can be sometimes extremely informative. Alternatively, genetic engineering allows the production of recombinant enzymes that can be used for biochemical determination and also for protein structural work. Very elegant fishing techniques such as the yeast two-hybrid system or bimolecular fluorescence complementation experiments can be used to fish or validate potential protein ligands for a given protein. Still another avenue for research is the structural determination of tree proteins, though it requires first access to the genes of a given organism. Given the present high-throughput of tree genome sequencing, we postulate that tree protein structural genomics should soon take off. As we have detailed in this paper, the field is still very much in infancy with the structures of only ca 70 individual tree proteins that have been solved up to now. We believe that the future of tree protein structure determination is very bright indeed, given its interesting potential for systems biology improvement.

Notes

Acknowledgments

This work was supported by a grant overseen by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” program (ANR-11-LABX-0002-01, Lab of Excellence ARBRE). The visit of Desirée Gütle is supported by a grant overseen by the French National Research Agency (ANR) as part of the “Investissements d’Avenir” program (ANR-11-LABX-0002-01, Lab of Excellence ARBRE). Her PhD grant is from the French MENRT

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Copyright information

© INRA and Springer-Verlag France 2014

Authors and Affiliations

  • Jean-Pierre Jacquot
    • 1
    • 2
  • Jérémy Couturier
    • 1
    • 2
  • Claude Didierjean
    • 3
    • 4
  • Eric Gelhaye
    • 1
    • 2
  • Mélanie Morel-Rouhier
    • 1
    • 2
  • Arnaud Hecker
    • 1
    • 2
  • Christophe Plomion
    • 5
  • Desirée D. Gütle
    • 1
    • 2
    • 6
    • 7
  • Nicolas Rouhier
    • 1
    • 2
  1. 1.Université de Lorraine, Interactions Arbres - Microorganismes, UMR1136Vandoeuvre-lès-NancyFrance
  2. 2.INRA, Interactions Arbres - Microorganismes, UMR1136ChampenouxFrance
  3. 3.Université de Lorraine, CRM2, Equipe BioMod, UMR 7036, Faculté des Sciences et TechnologiesVandoeuvre-lès-NancyFrance
  4. 4.CNRS, CRM2, Equipe BioMod, UMR 7036, Faculté des Sciences et TechnologiesVandoeuvre-lès-NancyFrance
  5. 5.INRA, UMR1202, BIOGECOCestasFrance
  6. 6.Plant Biotechnology, Faculty of BiologyUniversität FreiburgFreiburgGermany
  7. 7.Spemann Graduate School of Biology and Medicine (SGBM)University of FreiburgFreiburgGermany

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