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Germins and germin-like proteins: Plant do-all proteins. But what do they do exactly?
Plant Physiol. Biochem. 39 (2001) 545−554 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942801012852/REV
Review Germins and germin-like proteins: Plant do-all proteins. But what do they do exactly? François Bernier*, Anne Berna Institut de biologie moléculaire des plantes du CNRS, Institut de botanique, 28, rue Goethe, 67083 Strasbourg cedex, France
Received 6 February 2001; accepted 23 March 2001 Abstract – Germins and germin-like proteins (GLPs) constitute a large and highly diverse family of ubiquitous plant proteins. The name germin was given because the first described member of the family, wheat germin, was discovered in germinating wheat grains. However, it is now known that proteins from this family exist in all organs and developmental stages and that several are also involved in the response to various stress conditions. It is thus increasingly obvious that germins and GLPs participate in many processes that are important for plant development and defence. However, their exact participation in these processes generally remains obscure so the main challenge now is to determine the precise activities and functions of all germins and GLPs. Molecular and biochemical studies are contributing to a better understanding of this protein family and much data has accumulated in the last 2 years. All species possess a wide range of GLPs and each GLP gene is subjected to a tight regulation. All germins and GLPs are glycoproteins somehow associated with the extracellular matrix. More specifically, three classes of functions are starting to be recognized for these proteins: some possess an enzymatic activity (oxalate oxidase or superoxide dismutase), others seem to be structural proteins while some others act as receptors. To explain the diversity and ubiquitousness of germins and GLPs, we propose that most, if not all, of them carry out a combination of different functions. © 2001 Éditions scientifiques et médicales Elsevier SAS cell wall / extracellular matrix / germin / germin-like protein / oxalate oxidase / stress response / superoxide dismutase ECM, extracellular matrix / GLP, germin-like protein / OxO, oxalate oxidase / SOD, superoxide dismutase
1. INTRODUCTION Wheat germin was first described in 1980 in a search for germination-specific proteins [57]. Hydration of dry, mature wheat embryos is completed within about 1 h post-imbibition, and most changes in the cell-free translational capacity of the mRNA population of germinating embryos are completed by 5 h post-imbibition, but the synthesis of mRNA for germin begins only between 5 and 10 h post-imbibition, after most of the change in the mRNA population of germinating embryos has been completed. Then, the quantity of germin mRNA singularly increases, together with the amount of its translation product over the next 15 h, in concert with renewed growth of the embryo. The amino acid sequence deduced from germin cDNA failed to reveal homology to any protein known *Correspondence and reprints: fax +33 3 90 24 18 84. E-mail address: [email protected] (F. Bernier).
at that time and no precise function could be attributed to this protein so the name germin was adopted [34]. It was only several years later that two proteins sharing sequence homology with germin were identified. The first one is a protein, named spherulin, synthesized by plasmodia from the slime mold Physarum polycephalum when they encyst, dehydrate and go into a dormant stage when submitted to a stress [7, 34, 37]. The second one was found in the roots of the common ice plant, Mesembryanthemum crystallinum [2, 45]. Neither of these proteins had a known activity. At that time, it could only be speculated that germin, spherulin and the M. crystallinum ‘germin-like protein’ (see below) were involved in water homeostasis: germin is synthesized during imbibition, spherulin is made during dehydration and M. crystallinum is a halophyte. It was recognized only in 1993 that wheat germin and a very closely related protein from barley were oxalate oxidases (OxO) [17, 39]. It is also at this time that the number of sequences related to germin started to increase strikingly with several fortuitous discover
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ies and also with systematic sequencing efforts. The number of different sequences related to germin is now close to a hundred. More recently, germins and related polypeptides were included in the superfamily of proteins named cupins together with spherulins, vicilin-like and legumin-like seed storage globulins, some sucrose binding proteins and also many proteins from prokaryotes and animals [18]. Cupins can be recognized by an overall low homology with a conservation of key amino acid residues that would be responsible for a conserved global three-dimensional structure containing a core β-barrel [4, 8, 53]. Cupin superfamily is also defined by the conservation of two histidine-containing motifs that could serve as a heavy metal ion binding site [18, 19]. Modelling of germin based on the three-dimensional structure of seed storage globulins has indicated that three histidines and one glutamate are indeed clustered and could thus co-ordinate a metal ion [21, 50]. Crystallographic data has confirmed the existence of an octahedral co-ordination site [64] and recent analyses have confirmed the presence of a manganese ion in some germins and GLPs (see below). A wide range of activities is found among prokaryotic and eukaryotic proteins of the cupin superfamily. However, it is worth mentioning that many of them are enzymes associated with cell wall synthesis and with stress response [20]. Since the family of cupins and the evolution of their conserved domains have been reviewed recently [20, 52], this review will concentrate on those that are found in plants: germins and GLPs.
2.1. Germins This is a very homogeneous group comprising proteins with more than 90 % identity (usually more than 95 % identity in any pairwise comparison) with most of the amino acid replacements being conservative. Germins have been extensively studied only in wheat and barley but DNA sequences for maize and ryegrass germins have been deposited in GenBank. Also, proteins immunologically related to wheat germin and sharing its biochemical properties have been reported in oat and rye whereas they could not be detected in any of the dicots that were assayed [23]. It is thus possible that germins are specific to Gramineae or even to a subset of this family. Assays for OxO activity have been carried out for several germins. From these it can be concluded that most, if not all, germins are OxO. 2.2. Germin-like proteins (GLPs) Contrary to germins, GLPs are very heterogeneous and they are present in all angiosperm families, including Gramineae, as well as in gymnosperms and mosses. In this group, amino acid identities range from about 25 to almost 100 %. Comparisons with germins reveal identities ranging from 30 % to a maximal value of 70 %. The absence of proteins showing identities between 70 and 90 % with germins further defines the germins and GLPs as very distinct groups. Phylogenetic analyses now identify four GLP subgroups [11, 12]. In this emerging view of considering relationships among these various proteins, germins, after which they were all named, can now be considered as a fifth subgroup of the germin-like protein family.
2. A FEW WORDS ON NOMENCLATURE No precise function or activity has been defined yet for most of the germin-related proteins and only a few of them have been characterized in detail at the biochemical level. In addition, oxalate oxidase activity is no longer specifically associated with members of this protein family belonging to the Gramineae (see below). In consequence, confusion still exists regarding nomenclature. Names such as ‘germins’, ‘true germins’, ‘germin-like proteins’, ‘oxalate oxidase-like proteins’, or ‘germin-like oxalate oxidase’ have been used. In the absence of known functions for most of these proteins, nomenclature has to rely on sequence comparisons and phylogenetic analysis. In this way, two very distinct groups of proteins related to wheat germin can be defined and the different names mentioned above have often been used to designate either one or the other.
3. GENE FAMILIES Because of the huge sequence diversity among GLPs, strategies relying on the use of DNA probes or antibodies will inevitably fail to evidence the actual complexity of the gene family in a given species. On the other hand, systematic sequencing programmes will yield such an information. In all species for which such a programme exists, complex GLP gene families have been discovered which encode members of all GLP subgroups. In Arabidopsis, 27 GLP genes have been evidenced by EST or genomic sequencing so the total number of genes will probably be around forty [11, 13]. EST analysis indicated that, in rice, at least eight different GLP genes are expressed ([42] and our unpubl. observ.) and the number is probably a little bit higher for soybean (Bernier et al., in prep.).
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Figure 1. Schematic representation of the typical organization of germins and GLPs. Light blue indicates moderate sequence conservation, dark blue (boxes A, B and C) indicates very high sequence conservation and red represents a highly variable region. The conserved amino acids that define boxes A, B and C are shown. The two cysteines that form an internal disulphide bridge and the four amino acids involved in the binding of a metal ion are shown in red. pep, Signal peptide; X, any hydrophobic amino acid.
In barley, fourteen different GLP sequences have recently been described [66]. The corresponding genes are expressed either in leaves or in the seed coat. All these have been isolated by screening with a single probe so it is no surprise that they all belong to a single GLP subfamily (subfamily 1 – see [11]). It is very likely that the same situation exists for the other barley GLP subfamilies, meaning that the total number of barley GLPs is probably very high. Some GLPs have been mapped in the Arabidopsis genome [43] and the genomic organization of others has been described [11]. Most of the GLP genes contain a single intron in a conserved position but this intron is lacking in the wheat germin gene [37]. Since the gene organization of GLPs has been reviewed recently [11], it will not be discussed further here.
4. CONSERVED FEATURES AND BIOCHEMICAL PROPERTIES Figure 1 shows the typical organization of germins and GLPs. All germin and GLP polypeptides are composed of around 220 amino acids, including a putative signal peptide. When N-terminal sequencing of a mature protein has been performed, the predicted signal sequence processing site has been confirmed ([11, 15, 40, 47, 48, 59, 63, 67]; and our unpubl. results for Arabidopsis AtGER3). The mature proteins are composed of three highly conserved oligopeptides (boxes A, B and C), one hypervariable region bordered by two cysteines and many conserved amino acid residues that are scattered in a backbone of moderately conserved sequence. Boxes B and C contain the three histidine and the
glutamate residues involved in metal binding as discussed above (also see table I). The simple examination of the conserved features does not tell much about the proteins, except for the presence of one or two putative N-glycosylation sites at a constant position, a few amino acids to the C-terminus of the hypervariable region (figure 1). Indeed the presence of N-glycans has been confirmed for several germins and GLPs (table I) and the structure of N-glycans occurring in wheat germin has been elucidated (see below). Another putatively important feature that has been observed in more than half of the GLPs, but not in germins, is the presence of a RGD tripeptide or, more often, of the closely related KGD (or sometimes KGE) peptides. Animal RGD-containing proteins such as fibronectin and vitronectin are adhesion proteins from the extracellular matrix (ECM) that interact with integrins and thus participate in the exchange of information between the outside and the inside of the cells. Interestingly, interactions through RGD peptides is thought to mediate the recognition between rhicadhesin and the pea root GLP [55]. More generally, RGD-dependent ECM-plasma membrane interactions have been described in plants but the proteins responsible for those have yet to be identified (see for example [3, 33]). Most of the biochemical characterization has been performed on germins, especially wheat and barley germins, since they were the first known members of the family. Germins are oligomeric glycoproteins [34]. At first, they were thought to be pentamers but recent crystallographic data has shown them to be hexamers [64, 65]. This is in fine agreement with the proposed structural similarity with seed storage globulins, that are trimers of two-domain subunits, each subunit being
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Table I. Selected biochemical properties of germins and GLPs. nd, Not determined. 1 Presence of a glycan as determined directly or by indirect methods (staining, affinity for concanavalinA, difference between the predicted MW and the MW determined by MALDI-TOF, mobility shift following glycosidase treatment). 2 In these cases, the presence of a glycan was inferred by the existence of two isoforms with slightly different mobilities on SDS-PAGE from the expression of a single transgene in tobacco. 3 Affinity for Cu2+ was used to purify this GLP but the presence of Cu2+ ion bound to the protein is unlikely. Protein Wheat germin (gf-2.8) Barley germin Barley HvGLP1/HvOxOLP Tobacco nectarin I Arabidopsis AtGER1 Arabidopsis AtGER2 Arabidopsis AtGER3 Peach ABP19/20 Pea rhicadhesin receptor Silene vulgaris GLP Moss GLP Pine GLP
Oligomers
Glycan1
Enzyme activity
Metal
+ + + + – + + + – nd + nd
+ + + + +2 +2 +2 nd + nd + +
OxO OxO
Mn2+
SOD
Mn2+
OxO SOD
(Cu2+)3 Mn2+
composed of two covalently bound cupins folds. Germins can be regarded as trimers of cupin dimers in which each pair of domains is not covalently linked [65]. Structural resemblance with seed storage proteins extends to the monomers: each one is composed of a jellyroll β-barrel domain followed by a C-terminal domain comprising three α-helices. The difference lies in the specific N-terminal extension of germin, that includes the two covalently bound cysteines and the hypervariable region (figure 1), that protrudes in the centre of the hexamer. The remarkable resistance of the oligomers to proteases, heat and other denaturing agents is explained by an exceptionally high monomer surface burial [65]. Germin exists as two isoforms, named G and G’, that have slightly different mobilities in SDS gels. The difference between the isoforms has been explained by the nature of germin’s glycan moieties. Isoform G possesses a typical complex-type glycan containing xylose and fucose while isoform G’ is lacking terminal N-acetyl glucosamine residues so its glycan would now be classified as a paucimannosidic-type N-glycan [29, 35, 41]. Germin distributes about equally between the extracellular and intracellular spaces and it is interesting to note that complex-type glycans are typically found on extracellular proteins while paucimannosidic-type N-glycans characterize vacuolar proteins [41]. Association of germin with the ECM is well documented [35, 38]. However the exact localization of the intracellular germin has never been determined. Expression of the wheat germin gene named gf-2.8 in transgenic tobacco has confirmed that a single gene
References [29, 35, 36] [50] [59, 61] [12] [44] [44] [44] [47] [55] [9] [67] [15]
is responsible for the synthesis of both isoforms. All other biochemical properties of wheat germin were also maintained in tobacco, demonstrating the usefulness of transgenic systems in the study of this protein [5]. By contrast, most GLPs are known from sequencing projects so biochemical data regarding these proteins is rather sparse. Table I summarizes the biochemical properties of GLPs, as well as germins, that have been studied at the protein level. The most important feature is that all the proteins are at least partly associated with the ECM. However, the nature of the association with the ECM can vary from one GLP to another. In the only comparative study of three GLPs from the same species (Arabidopsis), the proteins were found to be held in a cell wall fraction mostly by ionic bounds but the cation requirement for extracting them from this fraction differed from one to another [44]. Most of the GLPs that have been studied at the biochemical level behave like wheat germin, that is they migrate as doublets in SDS gels [44, 47, 61]. Although no GLP glycan moiety has been analysed in detail so far, it is tempting to speculate that the existence of isoforms differing only in the oligosaccharide side chain is a common feature in this protein family.
5. REGULATED EXPRESSION Germin was first identified in a search for germination-specific proteins [57]. Recently, a very comprehensive study of wheat gene expression during germination has been realized [10]. At the very begin
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ning of germination, germin was mostly expressed in coleorhiza and scutellum. Transcripts were then found in older root tissues and a little bit later in the vascular bundles of leaves. In young barley seedlings, OxO activity was localized in the epidermis of the root mature region and in the coleorhiza. Activity was never associated with elongating regions [16]. The interpretation is that, in addition to the originally proposed function of germin in initiating cell expansion, oxalate oxidase would also serve to provide hydrogen peroxide that would terminate cell growth by participating in the covalent cross-linking of cell wall components [10]. Data from another recent study showed that germin expression in wheat embryos was always associated with enveloping tissues and terminally differentiating vascular tissues, suggesting a role in programmed cell death [36]. Germin is also synthesized in adult leaves undergoing a stress. Indeed, germin transcription and OxO activity are stimulated by fungal infection [16, 28, 68], some heavy metal ions and by viral infection in tobacco plants transformed with the GUS reporter gene expressed under the control of the promoter of a wheat germin gene [6]. Germin transcription is also upregulated by auxins and by putrescine [6]. Finally, high OxO activity was measured in senescent ryegrass leaf sheaths, which would be at least partly responsible for the high levels of hydrogen peroxide present in the latter [49]. Most of the GLP clones have been obtained by accident or through systematic sequencing programmes so data regarding their expression is generally lacking. For the cDNA clones, at least one developmental stage and/or organ expressing the corresponding gene can usually be inferred from the origin of the mRNA. This simple analysis shows that GLPs are expressed in all parts of the plants and at all developmental stages, from root to shoot apices, in floral organs and from germination to embryo and fruit development. ESTs deposited in databanks also demonstrate the expression of GLPs in differentiating xylem cells of pine and poplar. When multiple cDNA clones corresponding to a single gene have been obtained, they have almost inevitably come from the same library. This was the case for rice GLPs [42] and for soybean GLPs (Bernier et al., unpubl.). It is thus likely that, even though all cell types contain GLPs, each GLP gene is expressed in a very specific manner and in only a small subset of plant cells. The only systematic expression study was conducted on three Arabidopsis GLP genes, AtGER1, AtGER2 and AtGER3. Northern blot analysis with
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RNAs from different organs showed that AtGER1, AtGER2 and AtGER3 transcripts predominated respectively in leaves, siliques and floral buds [43]. No transcript was detected in roots or dry seeds for any of these three genes. A more detailed analysis of AtGER3 expression was realized by fusing its promoter to the GUS reporter gene and looking for expression in transgenic Arabidopsis plants [54]. The transgene was expressed in the subepidermal cortical cells of cotyledons, young leaves, inflorescence axis and almost all floral organs, except petals and anthers. The counterpart of AtGER3 in mustard is SaGLP and its regulation was also studied. In situ hybridizations revealed the presence of SaGLP transcripts in leaf epidermis and spongy parenchyma as well as in epidermis and subepidermal cortical cells of stems and petioles [26]. Transcription of both AtGER3 and SaGLP was also regulated in a circadian manner (see below). As for germin genes, transcription of some GLP genes is influenced by stress conditions. Biotic stress will be discussed in the next section. A wheat GLP was also identified in a search for transcripts whose abundance increased in root and root tip following exposure to aluminium [24].
6. GERMINS AND GLPS IN DEFENCE AGAINST PATHOGENS Over the years, evidence has accumulated regarding the involvement of germins and GLPs in defence against pathogens. First, oxalate oxidase activity and germin transcription were shown to be induced in barley and wheat following fungal infection (see previous section). The only germin gene that has been studied in detail is both expressed during normal germination and in adult plant submitted to an infection [6]. In barley, at least some of the OxO genes are not pathogen-responsive [68]. The work was mostly pursued with barley where it was found that, more specifically, transcripts for oxalate oxidase accumulated in the mesophyll whereas a gene for a GLP was induced in the inoculated epidermis and also at a detectable level in non-inoculated epidermis of infected plants [22, 61, 69]. In addition, production of hydrogen peroxide was demonstrated in papillae and cells infected by a fungal pathogen as well as in cells responding to the infection by hypersensitive cell death whereas susceptible mutants did not display this phenomenon [27, 58]. However, the direct correlation between H2O2 production and germin remains to be established.
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In those species that do possess an OxO, this enzyme thus represents an excellent candidate for the hydrogen peroxide provider during the oxidative burst in response to an attack by a pathogen [62]. Paradoxically, these species usually contain very low levels of the OxO substrate, oxalic acid or oxalate. The solution may well lie in the fact that oxalate might be formed as needed by another enzymatic reaction, from L-ascorbic acid for example. Ascorbic acid, which is present in all plants and especially in their apoplast, is now considered as an important immediate precursor of oxalic acid [31]. The importance of germins and GLPs in plant defence was more directly demonstrated by transient expression assays in wheat epidermis [51]. Penetration of Blumeria graminis was reduced in cells expressing an intact wheat germin gene. Expression of a wheat GLP gene or of two engineered wheat germin gene encoding proteins without OxO activity produced a similar, although weaker, effect indicating that OxO activity by itself is not the sole determinant of resistance. On the other hand, the presence of a GLP whose gene is constitutively expressed in barley did not affect penetration efficiency of the pathogen [51]. Several pathogenic fungi secrete high amounts of oxalate. This is the case for the important pathogen Sclerotinia for which oxalate secretion constitutes an essential determinant of pathogenicity. Several hypotheses have been proposed to explain the effect of oxalate and it has recently been shown to act by suppressing the oxidative burst, rendering the plant more susceptible to infection [14]. OxO producing plants like wheat and barley generally resist Sclerotinia sp. so biotechnological approaches are now attempted to increase resistance of other plants by the expression of a foreign OxO gene. The advantage would in fact be double since OxO will eliminate oxalate and produce hydrogen peroxide that could contribute to defence, either directly by its antimicrobial activity or indirectly by acting as a messenger [1, 62]. This strategy has been validated in oilseed rape [56] and has also reached the stage of field trial for other species (for example, see data from the European Commission at: food.jrc.it/gmo/fr.asp).
7. PROTEINS IN SEARCH OF AN ACTIVITY 7.1. GLPs as enzymes When wheat germin and the almost identical barley protein were identified as oxalate oxidases, no GLP from Gramineae was known yet so it was hypoth-
esized that GLPs were dicot OxO. However, all the GLPs that have been assayed for this activity have turned out not to be OxO [12, 42, 47, 61, 67]. However, a Silene vulgaris protein with OxO activity was recently described [9]. It is only known from its N-terminal sequence but this nevertheless unambiguously identified this protein as a GLP. OxO activity has been described in other species (beet, banana, maize, sorghum) but knowledge about the nature of the protein (germin, GLP or unrelated protein) is lacking in all these cases. On the other hand, two GLPs have recently been identified as manganese-containing superoxide dismutases (SODs). The first one was isolated from in vitro cultured cells of the moss Barbula unguiculata [67]. The second one is the major tobacco nectar protein, named nectarin I [12]. Both SODs were shown to be extracellular, oligomeric glycoproteins (table I). In addition, proteins related to nectarin I were detected in the nectar of most plants that were assayed: they would serve to prevent microbial contamination of the nutrient-rich nectar by generating high levels of hydrogen peroxide [12]. Neither of these enzymes displays OxO activity. Following these discoveries, SOD activity has been assayed for some other GLPs as well as in germins but no activity was detected (our unpubl. observ.; S. Bornemann and B. Lane, pers. commun.; [12]).
7.2. GLPs as structural proteins Germins and GLPs are usually extracted very easily from the ECM in which they are held mostly by ionic bounds. However, some of these proteins become insolubilized following a stress. In barley, heat treatment results in increased resistance to a pathogen by a novel mechanism that does not involve papilla formation or hypersensitive cell death. Instead, cell wall modifications, including insolubilization of a few proteins, seemed to be responsible for resistance. Following heat treatment or an exposure to hydrogen peroxide or an infection, two GLPs could not be recovered in the intercellular washing fluid any more although they were not covalently linked to ECM components [59, 60]. In addition, in the wheat epidermal cell transient expression experiments described above (see section 6), the germin transgene product became cross-linked in or close to the papillae, that is at the site of hydrogen peroxide production [51]. Insolubilization following stress might not be a general property of germins and GLPs since this phenomenon was not observed with wheat germin nor Arabidopsis AtGER3 expressed in transgenic tobacco
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plants [44]. In suspension-cultured white lupin cells, an opposite phenomenon was noted. Treatment of these cells with CuCl2 caused a shift in the distribution of a GLP: it was very abundant in the culture filtrate whereas, in control cells, it was mostly entirely associated with the cell wall fraction [63].
7.3. GLPs as receptors Rhicadhesins are cell surface proteins from bacteria of the genus Agrobacterium and Rhizobium that are thought to mediate the very first steps of attachment to the root hair. The search for a pea root receptor for rhicadhesin led to the purification of a protein whose N-terminal sequence identifies it as a GLP, although the homology was not noted at the time of the discovery [55]. The search for auxin receptors also led to the purification and cloning of two closely related GLPs that were named ABP 19/20 (‘auxin-binding protein 19/20’) [47]. The physiological significance of these remains to be determined. Based on their expression patterns, some other GLPs could possibly turn out to be cell surface receptors although, in these cases, ligands have yet to be identified. In the short-day plant Pharbitis nil, the level of a GLP mRNA increases in cotyledons and leaves during a dark inductive period but this accumulation is reduced if a short exposure to light interrupts the dark treatment [48]. Although additional data will be needed, it is tempting to speculate that this GLP is involved in signalling during photoperiodic induction of flowering. Interestingly, several other GLP genes are expressed with a circadian rhythm. This was demonstrated with two very similar GLPs, one from mustard and one from Arabidopsis [26, 54] and for a barley GLP [59]. In contrast to the situation in P. nil, expression of these three GLP genes peaks at the end of the light period. Interestingly, a systematic search of Arabidopsis genes expressed at the end of the light period has mostly resulted in the identification of genes implicated in cell expansion [25]. Expression in the circadian cycle has been studied for very few GLP genes: possibly, many other genes from this family will turn out to be regulated this way. In pine, the search for markers of somatic embryogenesis led to the identification of GLPs [15]. The cDNA for one of these was cloned and expression studies confirmed that transcripts for this pine GLP are found in both zygotic and somatic embryos and in embryonic cell lines but that they are absent from non-embryonic lines [46]. Apart from a role as a receptor, the function of this GLP might be to regulate cell expansion during embryogenesis.
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8. CONCLUSION Recent years have brought much data about germins and GLPs so that some unifying themes are starting to emerge. They are ECM glycoproteins usually held in place by weak ionic bounds. Most of them exist as very stable oligomers and it is also likely that most, if not all, of them bind a metal ion, manganese being the best candidate in all cases. Regarding expression, members of the complex GLP gene families are tightly regulated but all organs and developmental stages are probably characterized by the presence of at least one GLP. Some of the GLPs are also involved in stress response. More precisely, it seems that GLP expression is generally localized in surface tissues, epidermis and cortical subepidermal cells, but very few GLPs have been studied at this level. A wide range of functions has been uncovered for germins and GLPs: some are enzymes (OxO, SOD) while others seem to be receptors or structural proteins. It is quite possible that other activities remain to be discovered. For example, some could be oxidases for substrates other than oxalate while others could be peroxidases. Affinity for very different molecules (auxins and a protein) has been described, implying that the variety of GLP ligands might itself be quite broad. How can such a wide range of activities exist inside a single gene family? Maybe the answer lies in the existence of a dual function for germins and GLPs. For example, it could be hypothesized that these proteins evolved first as cell surface receptors and that many of them, because of their subcellular location and biochemical properties, later acquired an enzymatic property. Alternatively, their first function might have been enzymatic but, because they were ECM proteins, they constituted good candidates when new receptor molecules were recruited. If indeed germins and GLPs play the role of ECM receptors, they would somehow represent plant counterpart of animal adhesion proteins. Interestingly, adhesion proteins mediate exchange of information by interacting with membrane proteins, the integrins, through the tripeptide RGD. As mentioned in the beginning of this review, the existence of ‘RGD-like’ tripeptides characterizes the majority of the GLPs. The search for plant proteins resembling animal adhesion molecules has generally yielded disappointing results and it could be that germins and GLPs represent at least part of the plant adhesion molecules that have eluded discovery so far. Interestingly, a cell-surface peroxidase from crayfish was identified in a search for molecules involved in cell adhesion and immunity.
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This protein was named peroxinectin. Peroxinectin appears to interact with integrins through a KGD peptide. It is also of interest to note that, in this invertebrate system, an extracellular SOD interacts with peroxinectin and thus participates in cell adhesion and immunity [30]. This proposed role as adhesion molecules fits well with the diversity of sequence and expression pattern observed with germins and GLPs: each cell type is surrounded by different neighbours and exposed to specific environmental conditions. This would explain why each organ needs a specific subset of GLPs, a situation reminiscent of the arabinogalactan protein family [32]. Also, animal adhesion proteins participate in signalling during development and stress response. Obviously, much work will be needed to test this hypothesis. A membrane ligand for a germin or a GLP has yet to be evidenced. Some recent studies have demonstrated interactions between an Arabidopsis GLP, AtGER1, and another protein [44]. The interacting protein was not identified though. More biochemical work with selected germins and GLPs will be the key in identifying interacting molecules and understanding the common features of this family of proteins. The importance of molecular interactions for the function of these proteins was recently illustrated by a detailed enzymologic analysis of wheat germin. Results from this work demonstrated the existence of a powerful proteinaceous OxO inhibitor in wheat soluble extracts. In contrast, highly substituted glucuronogalactoarabinoxylans, major cereal cell wall polysaccharides, had a remarkable stimulatory activity [36]. Regulation of OxO activity at the protein level following infection by a fungal pathogen has also been recently suggested [27]. Whatever germins and GLPs do, they probably participate in important aspects of cell wall remodelling that accompanies development and stress response. Elucidation of their precise activities is likely to provide valuable information about these processes.
Acknowledgments. The work in our laboratory was funded by the CNRS. We thank Prof. Byron Lane (University of Toronto, Canada) and Dr Stephen Bornemann (John Innes Centre, Norwich, UK) for a very careful reading of the manuscript before submission.
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Review Germins and germin-like proteins: Plant do-all proteins. But what do they do exactly? François Bernier*, Anne Berna Institut de biologie moléculaire des plantes du CNRS, Institut de botanique, 28, rue Goethe, 67083 Strasbourg cedex, France
Received 6 February 2001; accepted 23 March 2001 Abstract – Germins and germin-like proteins (GLPs) constitute a large and highly diverse family of ubiquitous plant proteins. The name germin was given because the first described member of the family, wheat germin, was discovered in germinating wheat grains. However, it is now known that proteins from this family exist in all organs and developmental stages and that several are also involved in the response to various stress conditions. It is thus increasingly obvious that germins and GLPs participate in many processes that are important for plant development and defence. However, their exact participation in these processes generally remains obscure so the main challenge now is to determine the precise activities and functions of all germins and GLPs. Molecular and biochemical studies are contributing to a better understanding of this protein family and much data has accumulated in the last 2 years. All species possess a wide range of GLPs and each GLP gene is subjected to a tight regulation. All germins and GLPs are glycoproteins somehow associated with the extracellular matrix. More specifically, three classes of functions are starting to be recognized for these proteins: some possess an enzymatic activity (oxalate oxidase or superoxide dismutase), others seem to be structural proteins while some others act as receptors. To explain the diversity and ubiquitousness of germins and GLPs, we propose that most, if not all, of them carry out a combination of different functions. © 2001 Éditions scientifiques et médicales Elsevier SAS cell wall / extracellular matrix / germin / germin-like protein / oxalate oxidase / stress response / superoxide dismutase ECM, extracellular matrix / GLP, germin-like protein / OxO, oxalate oxidase / SOD, superoxide dismutase
1. INTRODUCTION Wheat germin was first described in 1980 in a search for germination-specific proteins [57]. Hydration of dry, mature wheat embryos is completed within about 1 h post-imbibition, and most changes in the cell-free translational capacity of the mRNA population of germinating embryos are completed by 5 h post-imbibition, but the synthesis of mRNA for germin begins only between 5 and 10 h post-imbibition, after most of the change in the mRNA population of germinating embryos has been completed. Then, the quantity of germin mRNA singularly increases, together with the amount of its translation product over the next 15 h, in concert with renewed growth of the embryo. The amino acid sequence deduced from germin cDNA failed to reveal homology to any protein known *Correspondence and reprints: fax +33 3 90 24 18 84. E-mail address: [email protected] (F. Bernier).
at that time and no precise function could be attributed to this protein so the name germin was adopted [34]. It was only several years later that two proteins sharing sequence homology with germin were identified. The first one is a protein, named spherulin, synthesized by plasmodia from the slime mold Physarum polycephalum when they encyst, dehydrate and go into a dormant stage when submitted to a stress [7, 34, 37]. The second one was found in the roots of the common ice plant, Mesembryanthemum crystallinum [2, 45]. Neither of these proteins had a known activity. At that time, it could only be speculated that germin, spherulin and the M. crystallinum ‘germin-like protein’ (see below) were involved in water homeostasis: germin is synthesized during imbibition, spherulin is made during dehydration and M. crystallinum is a halophyte. It was recognized only in 1993 that wheat germin and a very closely related protein from barley were oxalate oxidases (OxO) [17, 39]. It is also at this time that the number of sequences related to germin started to increase strikingly with several fortuitous discover
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ies and also with systematic sequencing efforts. The number of different sequences related to germin is now close to a hundred. More recently, germins and related polypeptides were included in the superfamily of proteins named cupins together with spherulins, vicilin-like and legumin-like seed storage globulins, some sucrose binding proteins and also many proteins from prokaryotes and animals [18]. Cupins can be recognized by an overall low homology with a conservation of key amino acid residues that would be responsible for a conserved global three-dimensional structure containing a core β-barrel [4, 8, 53]. Cupin superfamily is also defined by the conservation of two histidine-containing motifs that could serve as a heavy metal ion binding site [18, 19]. Modelling of germin based on the three-dimensional structure of seed storage globulins has indicated that three histidines and one glutamate are indeed clustered and could thus co-ordinate a metal ion [21, 50]. Crystallographic data has confirmed the existence of an octahedral co-ordination site [64] and recent analyses have confirmed the presence of a manganese ion in some germins and GLPs (see below). A wide range of activities is found among prokaryotic and eukaryotic proteins of the cupin superfamily. However, it is worth mentioning that many of them are enzymes associated with cell wall synthesis and with stress response [20]. Since the family of cupins and the evolution of their conserved domains have been reviewed recently [20, 52], this review will concentrate on those that are found in plants: germins and GLPs.
2.1. Germins This is a very homogeneous group comprising proteins with more than 90 % identity (usually more than 95 % identity in any pairwise comparison) with most of the amino acid replacements being conservative. Germins have been extensively studied only in wheat and barley but DNA sequences for maize and ryegrass germins have been deposited in GenBank. Also, proteins immunologically related to wheat germin and sharing its biochemical properties have been reported in oat and rye whereas they could not be detected in any of the dicots that were assayed [23]. It is thus possible that germins are specific to Gramineae or even to a subset of this family. Assays for OxO activity have been carried out for several germins. From these it can be concluded that most, if not all, germins are OxO. 2.2. Germin-like proteins (GLPs) Contrary to germins, GLPs are very heterogeneous and they are present in all angiosperm families, including Gramineae, as well as in gymnosperms and mosses. In this group, amino acid identities range from about 25 to almost 100 %. Comparisons with germins reveal identities ranging from 30 % to a maximal value of 70 %. The absence of proteins showing identities between 70 and 90 % with germins further defines the germins and GLPs as very distinct groups. Phylogenetic analyses now identify four GLP subgroups [11, 12]. In this emerging view of considering relationships among these various proteins, germins, after which they were all named, can now be considered as a fifth subgroup of the germin-like protein family.
2. A FEW WORDS ON NOMENCLATURE No precise function or activity has been defined yet for most of the germin-related proteins and only a few of them have been characterized in detail at the biochemical level. In addition, oxalate oxidase activity is no longer specifically associated with members of this protein family belonging to the Gramineae (see below). In consequence, confusion still exists regarding nomenclature. Names such as ‘germins’, ‘true germins’, ‘germin-like proteins’, ‘oxalate oxidase-like proteins’, or ‘germin-like oxalate oxidase’ have been used. In the absence of known functions for most of these proteins, nomenclature has to rely on sequence comparisons and phylogenetic analysis. In this way, two very distinct groups of proteins related to wheat germin can be defined and the different names mentioned above have often been used to designate either one or the other.
3. GENE FAMILIES Because of the huge sequence diversity among GLPs, strategies relying on the use of DNA probes or antibodies will inevitably fail to evidence the actual complexity of the gene family in a given species. On the other hand, systematic sequencing programmes will yield such an information. In all species for which such a programme exists, complex GLP gene families have been discovered which encode members of all GLP subgroups. In Arabidopsis, 27 GLP genes have been evidenced by EST or genomic sequencing so the total number of genes will probably be around forty [11, 13]. EST analysis indicated that, in rice, at least eight different GLP genes are expressed ([42] and our unpubl. observ.) and the number is probably a little bit higher for soybean (Bernier et al., in prep.).
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Figure 1. Schematic representation of the typical organization of germins and GLPs. Light blue indicates moderate sequence conservation, dark blue (boxes A, B and C) indicates very high sequence conservation and red represents a highly variable region. The conserved amino acids that define boxes A, B and C are shown. The two cysteines that form an internal disulphide bridge and the four amino acids involved in the binding of a metal ion are shown in red. pep, Signal peptide; X, any hydrophobic amino acid.
In barley, fourteen different GLP sequences have recently been described [66]. The corresponding genes are expressed either in leaves or in the seed coat. All these have been isolated by screening with a single probe so it is no surprise that they all belong to a single GLP subfamily (subfamily 1 – see [11]). It is very likely that the same situation exists for the other barley GLP subfamilies, meaning that the total number of barley GLPs is probably very high. Some GLPs have been mapped in the Arabidopsis genome [43] and the genomic organization of others has been described [11]. Most of the GLP genes contain a single intron in a conserved position but this intron is lacking in the wheat germin gene [37]. Since the gene organization of GLPs has been reviewed recently [11], it will not be discussed further here.
4. CONSERVED FEATURES AND BIOCHEMICAL PROPERTIES Figure 1 shows the typical organization of germins and GLPs. All germin and GLP polypeptides are composed of around 220 amino acids, including a putative signal peptide. When N-terminal sequencing of a mature protein has been performed, the predicted signal sequence processing site has been confirmed ([11, 15, 40, 47, 48, 59, 63, 67]; and our unpubl. results for Arabidopsis AtGER3). The mature proteins are composed of three highly conserved oligopeptides (boxes A, B and C), one hypervariable region bordered by two cysteines and many conserved amino acid residues that are scattered in a backbone of moderately conserved sequence. Boxes B and C contain the three histidine and the
glutamate residues involved in metal binding as discussed above (also see table I). The simple examination of the conserved features does not tell much about the proteins, except for the presence of one or two putative N-glycosylation sites at a constant position, a few amino acids to the C-terminus of the hypervariable region (figure 1). Indeed the presence of N-glycans has been confirmed for several germins and GLPs (table I) and the structure of N-glycans occurring in wheat germin has been elucidated (see below). Another putatively important feature that has been observed in more than half of the GLPs, but not in germins, is the presence of a RGD tripeptide or, more often, of the closely related KGD (or sometimes KGE) peptides. Animal RGD-containing proteins such as fibronectin and vitronectin are adhesion proteins from the extracellular matrix (ECM) that interact with integrins and thus participate in the exchange of information between the outside and the inside of the cells. Interestingly, interactions through RGD peptides is thought to mediate the recognition between rhicadhesin and the pea root GLP [55]. More generally, RGD-dependent ECM-plasma membrane interactions have been described in plants but the proteins responsible for those have yet to be identified (see for example [3, 33]). Most of the biochemical characterization has been performed on germins, especially wheat and barley germins, since they were the first known members of the family. Germins are oligomeric glycoproteins [34]. At first, they were thought to be pentamers but recent crystallographic data has shown them to be hexamers [64, 65]. This is in fine agreement with the proposed structural similarity with seed storage globulins, that are trimers of two-domain subunits, each subunit being
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Table I. Selected biochemical properties of germins and GLPs. nd, Not determined. 1 Presence of a glycan as determined directly or by indirect methods (staining, affinity for concanavalinA, difference between the predicted MW and the MW determined by MALDI-TOF, mobility shift following glycosidase treatment). 2 In these cases, the presence of a glycan was inferred by the existence of two isoforms with slightly different mobilities on SDS-PAGE from the expression of a single transgene in tobacco. 3 Affinity for Cu2+ was used to purify this GLP but the presence of Cu2+ ion bound to the protein is unlikely. Protein Wheat germin (gf-2.8) Barley germin Barley HvGLP1/HvOxOLP Tobacco nectarin I Arabidopsis AtGER1 Arabidopsis AtGER2 Arabidopsis AtGER3 Peach ABP19/20 Pea rhicadhesin receptor Silene vulgaris GLP Moss GLP Pine GLP
Oligomers
Glycan1
Enzyme activity
Metal
+ + + + – + + + – nd + nd
+ + + + +2 +2 +2 nd + nd + +
OxO OxO
Mn2+
SOD
Mn2+
OxO SOD
(Cu2+)3 Mn2+
composed of two covalently bound cupins folds. Germins can be regarded as trimers of cupin dimers in which each pair of domains is not covalently linked [65]. Structural resemblance with seed storage proteins extends to the monomers: each one is composed of a jellyroll β-barrel domain followed by a C-terminal domain comprising three α-helices. The difference lies in the specific N-terminal extension of germin, that includes the two covalently bound cysteines and the hypervariable region (figure 1), that protrudes in the centre of the hexamer. The remarkable resistance of the oligomers to proteases, heat and other denaturing agents is explained by an exceptionally high monomer surface burial [65]. Germin exists as two isoforms, named G and G’, that have slightly different mobilities in SDS gels. The difference between the isoforms has been explained by the nature of germin’s glycan moieties. Isoform G possesses a typical complex-type glycan containing xylose and fucose while isoform G’ is lacking terminal N-acetyl glucosamine residues so its glycan would now be classified as a paucimannosidic-type N-glycan [29, 35, 41]. Germin distributes about equally between the extracellular and intracellular spaces and it is interesting to note that complex-type glycans are typically found on extracellular proteins while paucimannosidic-type N-glycans characterize vacuolar proteins [41]. Association of germin with the ECM is well documented [35, 38]. However the exact localization of the intracellular germin has never been determined. Expression of the wheat germin gene named gf-2.8 in transgenic tobacco has confirmed that a single gene
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is responsible for the synthesis of both isoforms. All other biochemical properties of wheat germin were also maintained in tobacco, demonstrating the usefulness of transgenic systems in the study of this protein [5]. By contrast, most GLPs are known from sequencing projects so biochemical data regarding these proteins is rather sparse. Table I summarizes the biochemical properties of GLPs, as well as germins, that have been studied at the protein level. The most important feature is that all the proteins are at least partly associated with the ECM. However, the nature of the association with the ECM can vary from one GLP to another. In the only comparative study of three GLPs from the same species (Arabidopsis), the proteins were found to be held in a cell wall fraction mostly by ionic bounds but the cation requirement for extracting them from this fraction differed from one to another [44]. Most of the GLPs that have been studied at the biochemical level behave like wheat germin, that is they migrate as doublets in SDS gels [44, 47, 61]. Although no GLP glycan moiety has been analysed in detail so far, it is tempting to speculate that the existence of isoforms differing only in the oligosaccharide side chain is a common feature in this protein family.
5. REGULATED EXPRESSION Germin was first identified in a search for germination-specific proteins [57]. Recently, a very comprehensive study of wheat gene expression during germination has been realized [10]. At the very begin
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ning of germination, germin was mostly expressed in coleorhiza and scutellum. Transcripts were then found in older root tissues and a little bit later in the vascular bundles of leaves. In young barley seedlings, OxO activity was localized in the epidermis of the root mature region and in the coleorhiza. Activity was never associated with elongating regions [16]. The interpretation is that, in addition to the originally proposed function of germin in initiating cell expansion, oxalate oxidase would also serve to provide hydrogen peroxide that would terminate cell growth by participating in the covalent cross-linking of cell wall components [10]. Data from another recent study showed that germin expression in wheat embryos was always associated with enveloping tissues and terminally differentiating vascular tissues, suggesting a role in programmed cell death [36]. Germin is also synthesized in adult leaves undergoing a stress. Indeed, germin transcription and OxO activity are stimulated by fungal infection [16, 28, 68], some heavy metal ions and by viral infection in tobacco plants transformed with the GUS reporter gene expressed under the control of the promoter of a wheat germin gene [6]. Germin transcription is also upregulated by auxins and by putrescine [6]. Finally, high OxO activity was measured in senescent ryegrass leaf sheaths, which would be at least partly responsible for the high levels of hydrogen peroxide present in the latter [49]. Most of the GLP clones have been obtained by accident or through systematic sequencing programmes so data regarding their expression is generally lacking. For the cDNA clones, at least one developmental stage and/or organ expressing the corresponding gene can usually be inferred from the origin of the mRNA. This simple analysis shows that GLPs are expressed in all parts of the plants and at all developmental stages, from root to shoot apices, in floral organs and from germination to embryo and fruit development. ESTs deposited in databanks also demonstrate the expression of GLPs in differentiating xylem cells of pine and poplar. When multiple cDNA clones corresponding to a single gene have been obtained, they have almost inevitably come from the same library. This was the case for rice GLPs [42] and for soybean GLPs (Bernier et al., unpubl.). It is thus likely that, even though all cell types contain GLPs, each GLP gene is expressed in a very specific manner and in only a small subset of plant cells. The only systematic expression study was conducted on three Arabidopsis GLP genes, AtGER1, AtGER2 and AtGER3. Northern blot analysis with
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RNAs from different organs showed that AtGER1, AtGER2 and AtGER3 transcripts predominated respectively in leaves, siliques and floral buds [43]. No transcript was detected in roots or dry seeds for any of these three genes. A more detailed analysis of AtGER3 expression was realized by fusing its promoter to the GUS reporter gene and looking for expression in transgenic Arabidopsis plants [54]. The transgene was expressed in the subepidermal cortical cells of cotyledons, young leaves, inflorescence axis and almost all floral organs, except petals and anthers. The counterpart of AtGER3 in mustard is SaGLP and its regulation was also studied. In situ hybridizations revealed the presence of SaGLP transcripts in leaf epidermis and spongy parenchyma as well as in epidermis and subepidermal cortical cells of stems and petioles [26]. Transcription of both AtGER3 and SaGLP was also regulated in a circadian manner (see below). As for germin genes, transcription of some GLP genes is influenced by stress conditions. Biotic stress will be discussed in the next section. A wheat GLP was also identified in a search for transcripts whose abundance increased in root and root tip following exposure to aluminium [24].
6. GERMINS AND GLPS IN DEFENCE AGAINST PATHOGENS Over the years, evidence has accumulated regarding the involvement of germins and GLPs in defence against pathogens. First, oxalate oxidase activity and germin transcription were shown to be induced in barley and wheat following fungal infection (see previous section). The only germin gene that has been studied in detail is both expressed during normal germination and in adult plant submitted to an infection [6]. In barley, at least some of the OxO genes are not pathogen-responsive [68]. The work was mostly pursued with barley where it was found that, more specifically, transcripts for oxalate oxidase accumulated in the mesophyll whereas a gene for a GLP was induced in the inoculated epidermis and also at a detectable level in non-inoculated epidermis of infected plants [22, 61, 69]. In addition, production of hydrogen peroxide was demonstrated in papillae and cells infected by a fungal pathogen as well as in cells responding to the infection by hypersensitive cell death whereas susceptible mutants did not display this phenomenon [27, 58]. However, the direct correlation between H2O2 production and germin remains to be established.
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In those species that do possess an OxO, this enzyme thus represents an excellent candidate for the hydrogen peroxide provider during the oxidative burst in response to an attack by a pathogen [62]. Paradoxically, these species usually contain very low levels of the OxO substrate, oxalic acid or oxalate. The solution may well lie in the fact that oxalate might be formed as needed by another enzymatic reaction, from L-ascorbic acid for example. Ascorbic acid, which is present in all plants and especially in their apoplast, is now considered as an important immediate precursor of oxalic acid [31]. The importance of germins and GLPs in plant defence was more directly demonstrated by transient expression assays in wheat epidermis [51]. Penetration of Blumeria graminis was reduced in cells expressing an intact wheat germin gene. Expression of a wheat GLP gene or of two engineered wheat germin gene encoding proteins without OxO activity produced a similar, although weaker, effect indicating that OxO activity by itself is not the sole determinant of resistance. On the other hand, the presence of a GLP whose gene is constitutively expressed in barley did not affect penetration efficiency of the pathogen [51]. Several pathogenic fungi secrete high amounts of oxalate. This is the case for the important pathogen Sclerotinia for which oxalate secretion constitutes an essential determinant of pathogenicity. Several hypotheses have been proposed to explain the effect of oxalate and it has recently been shown to act by suppressing the oxidative burst, rendering the plant more susceptible to infection [14]. OxO producing plants like wheat and barley generally resist Sclerotinia sp. so biotechnological approaches are now attempted to increase resistance of other plants by the expression of a foreign OxO gene. The advantage would in fact be double since OxO will eliminate oxalate and produce hydrogen peroxide that could contribute to defence, either directly by its antimicrobial activity or indirectly by acting as a messenger [1, 62]. This strategy has been validated in oilseed rape [56] and has also reached the stage of field trial for other species (for example, see data from the European Commission at: food.jrc.it/gmo/fr.asp).
7. PROTEINS IN SEARCH OF AN ACTIVITY 7.1. GLPs as enzymes When wheat germin and the almost identical barley protein were identified as oxalate oxidases, no GLP from Gramineae was known yet so it was hypoth-
esized that GLPs were dicot OxO. However, all the GLPs that have been assayed for this activity have turned out not to be OxO [12, 42, 47, 61, 67]. However, a Silene vulgaris protein with OxO activity was recently described [9]. It is only known from its N-terminal sequence but this nevertheless unambiguously identified this protein as a GLP. OxO activity has been described in other species (beet, banana, maize, sorghum) but knowledge about the nature of the protein (germin, GLP or unrelated protein) is lacking in all these cases. On the other hand, two GLPs have recently been identified as manganese-containing superoxide dismutases (SODs). The first one was isolated from in vitro cultured cells of the moss Barbula unguiculata [67]. The second one is the major tobacco nectar protein, named nectarin I [12]. Both SODs were shown to be extracellular, oligomeric glycoproteins (table I). In addition, proteins related to nectarin I were detected in the nectar of most plants that were assayed: they would serve to prevent microbial contamination of the nutrient-rich nectar by generating high levels of hydrogen peroxide [12]. Neither of these enzymes displays OxO activity. Following these discoveries, SOD activity has been assayed for some other GLPs as well as in germins but no activity was detected (our unpubl. observ.; S. Bornemann and B. Lane, pers. commun.; [12]).
7.2. GLPs as structural proteins Germins and GLPs are usually extracted very easily from the ECM in which they are held mostly by ionic bounds. However, some of these proteins become insolubilized following a stress. In barley, heat treatment results in increased resistance to a pathogen by a novel mechanism that does not involve papilla formation or hypersensitive cell death. Instead, cell wall modifications, including insolubilization of a few proteins, seemed to be responsible for resistance. Following heat treatment or an exposure to hydrogen peroxide or an infection, two GLPs could not be recovered in the intercellular washing fluid any more although they were not covalently linked to ECM components [59, 60]. In addition, in the wheat epidermal cell transient expression experiments described above (see section 6), the germin transgene product became cross-linked in or close to the papillae, that is at the site of hydrogen peroxide production [51]. Insolubilization following stress might not be a general property of germins and GLPs since this phenomenon was not observed with wheat germin nor Arabidopsis AtGER3 expressed in transgenic tobacco
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plants [44]. In suspension-cultured white lupin cells, an opposite phenomenon was noted. Treatment of these cells with CuCl2 caused a shift in the distribution of a GLP: it was very abundant in the culture filtrate whereas, in control cells, it was mostly entirely associated with the cell wall fraction [63].
7.3. GLPs as receptors Rhicadhesins are cell surface proteins from bacteria of the genus Agrobacterium and Rhizobium that are thought to mediate the very first steps of attachment to the root hair. The search for a pea root receptor for rhicadhesin led to the purification of a protein whose N-terminal sequence identifies it as a GLP, although the homology was not noted at the time of the discovery [55]. The search for auxin receptors also led to the purification and cloning of two closely related GLPs that were named ABP 19/20 (‘auxin-binding protein 19/20’) [47]. The physiological significance of these remains to be determined. Based on their expression patterns, some other GLPs could possibly turn out to be cell surface receptors although, in these cases, ligands have yet to be identified. In the short-day plant Pharbitis nil, the level of a GLP mRNA increases in cotyledons and leaves during a dark inductive period but this accumulation is reduced if a short exposure to light interrupts the dark treatment [48]. Although additional data will be needed, it is tempting to speculate that this GLP is involved in signalling during photoperiodic induction of flowering. Interestingly, several other GLP genes are expressed with a circadian rhythm. This was demonstrated with two very similar GLPs, one from mustard and one from Arabidopsis [26, 54] and for a barley GLP [59]. In contrast to the situation in P. nil, expression of these three GLP genes peaks at the end of the light period. Interestingly, a systematic search of Arabidopsis genes expressed at the end of the light period has mostly resulted in the identification of genes implicated in cell expansion [25]. Expression in the circadian cycle has been studied for very few GLP genes: possibly, many other genes from this family will turn out to be regulated this way. In pine, the search for markers of somatic embryogenesis led to the identification of GLPs [15]. The cDNA for one of these was cloned and expression studies confirmed that transcripts for this pine GLP are found in both zygotic and somatic embryos and in embryonic cell lines but that they are absent from non-embryonic lines [46]. Apart from a role as a receptor, the function of this GLP might be to regulate cell expansion during embryogenesis.
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8. CONCLUSION Recent years have brought much data about germins and GLPs so that some unifying themes are starting to emerge. They are ECM glycoproteins usually held in place by weak ionic bounds. Most of them exist as very stable oligomers and it is also likely that most, if not all, of them bind a metal ion, manganese being the best candidate in all cases. Regarding expression, members of the complex GLP gene families are tightly regulated but all organs and developmental stages are probably characterized by the presence of at least one GLP. Some of the GLPs are also involved in stress response. More precisely, it seems that GLP expression is generally localized in surface tissues, epidermis and cortical subepidermal cells, but very few GLPs have been studied at this level. A wide range of functions has been uncovered for germins and GLPs: some are enzymes (OxO, SOD) while others seem to be receptors or structural proteins. It is quite possible that other activities remain to be discovered. For example, some could be oxidases for substrates other than oxalate while others could be peroxidases. Affinity for very different molecules (auxins and a protein) has been described, implying that the variety of GLP ligands might itself be quite broad. How can such a wide range of activities exist inside a single gene family? Maybe the answer lies in the existence of a dual function for germins and GLPs. For example, it could be hypothesized that these proteins evolved first as cell surface receptors and that many of them, because of their subcellular location and biochemical properties, later acquired an enzymatic property. Alternatively, their first function might have been enzymatic but, because they were ECM proteins, they constituted good candidates when new receptor molecules were recruited. If indeed germins and GLPs play the role of ECM receptors, they would somehow represent plant counterpart of animal adhesion proteins. Interestingly, adhesion proteins mediate exchange of information by interacting with membrane proteins, the integrins, through the tripeptide RGD. As mentioned in the beginning of this review, the existence of ‘RGD-like’ tripeptides characterizes the majority of the GLPs. The search for plant proteins resembling animal adhesion molecules has generally yielded disappointing results and it could be that germins and GLPs represent at least part of the plant adhesion molecules that have eluded discovery so far. Interestingly, a cell-surface peroxidase from crayfish was identified in a search for molecules involved in cell adhesion and immunity.
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This protein was named peroxinectin. Peroxinectin appears to interact with integrins through a KGD peptide. It is also of interest to note that, in this invertebrate system, an extracellular SOD interacts with peroxinectin and thus participates in cell adhesion and immunity [30]. This proposed role as adhesion molecules fits well with the diversity of sequence and expression pattern observed with germins and GLPs: each cell type is surrounded by different neighbours and exposed to specific environmental conditions. This would explain why each organ needs a specific subset of GLPs, a situation reminiscent of the arabinogalactan protein family [32]. Also, animal adhesion proteins participate in signalling during development and stress response. Obviously, much work will be needed to test this hypothesis. A membrane ligand for a germin or a GLP has yet to be evidenced. Some recent studies have demonstrated interactions between an Arabidopsis GLP, AtGER1, and another protein [44]. The interacting protein was not identified though. More biochemical work with selected germins and GLPs will be the key in identifying interacting molecules and understanding the common features of this family of proteins. The importance of molecular interactions for the function of these proteins was recently illustrated by a detailed enzymologic analysis of wheat germin. Results from this work demonstrated the existence of a powerful proteinaceous OxO inhibitor in wheat soluble extracts. In contrast, highly substituted glucuronogalactoarabinoxylans, major cereal cell wall polysaccharides, had a remarkable stimulatory activity [36]. Regulation of OxO activity at the protein level following infection by a fungal pathogen has also been recently suggested [27]. Whatever germins and GLPs do, they probably participate in important aspects of cell wall remodelling that accompanies development and stress response. Elucidation of their precise activities is likely to provide valuable information about these processes.
Acknowledgments. The work in our laboratory was funded by the CNRS. We thank Prof. Byron Lane (University of Toronto, Canada) and Dr Stephen Bornemann (John Innes Centre, Norwich, UK) for a very careful reading of the manuscript before submission.
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