A gene encoding a protein with a proline-rich domain (MtPPRD1), revealed by suppressive subtractive hybridization (SSH), is specifically expressed in the Medicago truncatula embryo axis during germination (original) (raw)

Abstract

A gene MtPPRD1, encoding a protein of 132 amino acids containing a proline-rich domain (PRD), has been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of embryo axes harvested immediately before and after radicle emergence. Although at the protein level MtPPRD1 showed low homology with plant lipid transfer proteins (LTPs), it did exhibit the eight cysteine residues conserved in all plant LTPs, a characteristic signature that allows the formation of a hydrophobic cavity adapted for loading hydrophobic molecules. Expression studies of MtPPRD1 have been carried out by quantitative real time RT-PCR throughout germination and post-germination processes in control seeds and seeds in which germination was delayed by abscisic acid (ABA) or the glutamine synthetase inhibitor methionine sulphoximine (MSX) treatments. The results showed that MtPPRD1 expression is developmentally regulated, induced in the embryo axis immediately before radicle emergence, reaches its maximum expression and declines during the early post-germination phase. Organ specificity studies showed that, except for a low and probably constitutive expression in roots, MtPPRD1 is specifically expressed in the embryo axis. Based on both experimental and in silico studies several putative roles are proposed for MtPPRD1 in Medicago truncatula, this protein can intervene (i) as an LTP in membrane biogenesis and regulation of the intracellular fatty acid pool by binding and transferring fatty acids and phospholipids between membranes, (ii) in the control of a developmental process specific to late germination and to early phases of post-germination, and (iii) and/or pathogen defence.

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Introduction

Germination begins with the uptake of water by the dry seed (imbibition) and terminates with radicle emergence and elongation of the embryo axis (Bewley, 1997). Germination is characterized by an increase in both respiration and metabolic activity allowing the mobilization of storage compounds. This period is followed by post-germinative growth that leads to seedling establishment and the acquisition of autotrophy (Bewley, 1997). Several studies aimed at determining the physiological and developmental processes that allow seed germination have been focused on gene expression in relation to reserve mobilization and the breakage of dormancy. The control of carbon reserve mobilization during germination has been extensively studied, using as an example the regulation of α-amylase expression in barley aleurone cells (Fath et al., 2001; Gomez-Cadenas et al., 2001). Expression of several major genes involved in amino acid synthesis and degradation pathways has been studied in various species, improving knowledge of the role of N reserves as a source of energy and metabolites for germination (Garciarrubio et al., 1997; Limami et al., 2002).

At the genetic level germination has been shown to be controlled by a variety of positive and negative regulatory signals: ABA, GA, chilling, light, and metabolizable sugars (Finkelstein and Lynch, 2000). Interactions between sugar and hormonal signalling have been demonstrated. It has been hypothesized that ABA inhibition of germination occurs by preventing reserve mobilization, indicating the dependence of germination upon reserve mobilization (Garciarrubio et al., 1997; Finkelstein and Lynch, 2000). Indeed, the inhibitory effects of ABA were strongly repressed in the presence of glucose, sucrose, or fructose, but this repression was limited to the process of radicle emergence (Finkelstein and Lynch, 2000). However, this hypothesis has been disproved by the experiments of Pritchard et al. (2002) who showed that ABA does not inhibit the germination of Arabidopsis seeds by preventing storage reserve mobilization. Indeed, there is active lipid (triacylglycerol) mobilization in the presence of ABA and the product of this process, namely sucrose, accumulates in the ABA-treated seeds (Pritchard et al., 2002). The authors suggested the existence of two distinct programmes operating after seed imbibition, one driving germination and the other driving reserve mobilization during early post-germinative growth. Each of these programmes can progress independently (Pritchard et al., 2002).

Proteomics of Arabidopsis seeds revealed the differential accumulation during germination of the _S_-adenosylmethionine synthetase (SAMS) that catalyses the synthesis of _S_-adenosylmethionine from methionine and ATP (Gallardo et al., 2002). The SAMS protein, absent in the dry mature seed, specifically accumulated in the embryo axis concomitantly with radicle emergence. _S_-adenosylmethionine synthetase could be a fundamental component controlling metabolism in the transition from a quiescent to a highly active state during seed germination (Gallardo et al., 2002, 2003).

Several genes have been also identified in Arabidopsis thaliana that regulate the transition from embryogenesis to seedling growth; they essentially maintain embryo dormancy in order to prevent premature germination. These genes encode for phosphatase or kinase enzymes involved in signal transduction and transcription factors (Girke et al., 2000). By contrast, less is known about genes that regulate the early developmental processes responsible for germination completion in non-dormant seeds.

For this purpose, a cDNA library of genes specifically expressed in embryo axes during germination was generated using a PCR-based suppressive subtractive hybridization (SSH) technique between mRNAs of two populations of embryo axes with two correspondent imbibition times, 16 h and 23 h, that bracket radicle protrusion. The model legume Medicago truncatula was used for two major reasons: (i) although legumes comprise one of the most important agricultural taxons worldwide, providing a major source of protein for humans and animals, and nitrogen for soil improvement, few studies have been dedicated to their germination compared with the model Arabidopsis thaliana or the monocots barley and maize, and (ii) the increasing availability of genomic tools with the Medicago genome project for genome sequencing and EST libraries generation (http://medicago.toulouse.inra.fr/Mt/EST/) render Medicago truncatula very well adapted for genomic studies and gene discovery. Among the cDNAs obtained it was decided to characterize MtPPRD1, a gene encoding a protein with a proline-rich domain (PRD). In plants, proteins with a proline rich-domain have received little attention, while in mammals, particularly in humans, their functions have been extensively studied; they intervene in protein–protein interaction and signal transduction cascades by binding to the SH3 domains of protein adaptors (Mayer, 2001).

Materials and methods

Seeds and germination conditions

Medicago truncatula seeds (cv. Paraggio) were germinated in Petri dishes (diameter 9 cm) on Whatman paper soaked with 3.5 ml de-ionized water and maintained in a growth chamber in darkness at 20 °C. Three replicates of 50 seeds per Petri dish were used for the germination test.

For further expression analysis, germinated seeds were sampled at various times throughout the germination process from 0–94 h. For each sample, seed coat and albumen were removed and embryo axes were collected and frozen in liquid nitrogen before being stored at −80 °C.

For inhibition of germination tests, seeds were germinated in Petri dishes as described above on Whatman paper soaked with water (control), 5 mM methionine sulphoximine (MSX, Sigma, St Louis, Missouri, USA) or 10 μM abscissic acid (Sigma). Embryo axes were collected after 5, 21, and 48 h of imbibition.

Seeds were also sown on a mix of perlite and soil (Traysubstrat, Klasmann-Deilmann, Germany) in a growth chamber under a day/night cycle of 15 h light at 24 °C (50 μmol m−2 s−1) and 9 h dark at 24 °C, and with regular watering with deionized water. Nodules were harvested on plants aged 7 weeks, leaves, stems, and roots were harvested on plants aged 2 months, and flowers were harvested on plants aged 4 months.

RNA extraction

Total RNA were extracted from embryo axes of imbibed seeds, leaves, stems, roots, and flowers using TRIzol® Reagent (Invitrogen, Breda, The Netherlands) according to the manufacturer's protocol.

Suppressive subtractive hybridization (SSH)

Two μg of polyA RNA were purified from total RNA of embryo axes of seeds imbibed for 16 h and 23 h with PolyATract® System 1000 (Promega, Madison, Wisconsin, USA). cDNAs were generated and used for suppressive subtractive hybridization with a Clontech PCR-Select™ cDNA Subtraction Kit (Clontech, Palo Alto, California, USA).

Quantitative real-time RT-PCR

Two μg of total RNA were reverse-transcribed for 1 h at 37 °C, using 200 units of M-MLV Reverse Transcriptase (Promega, Madison, Wisconsin, USA) and 2 μg of pd(N)6 Random Hexamer (Amersham Biosciences, Freiburg, Germany) in the presence of 40 units of Recombinant Rnasin® Ribonuclease Inhibitor (Promega, Madison, Wisconsin, USA) in 50 μl final volume. Genomic DNA was removed by purifying the first strands on QIAquick® PCR Purification Kit (Qiagen, German Town, Maryland, USA).

Quantitative real-time PCR were performed on the light cycler ABI Prism 7000 SDS (Applied Biosystems, Foster City, California, USA) with the SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol. Each reaction was performed on 5 μl of 1:2 (v/v) dilution of the first cDNA strands, synthesized as described above, with 0.3 μM of each primer in a total reaction of 20 μl. The reaction were incubated for 2 min at 50 °C and 10 min at 95 °C followed by 60 cycles of 15 s at 95 °C and 1 min at 60 °C. The specificity of the PCR amplification procedures was checked with a heat dissociation protocol (from 65 °C to 95 °C) after the final cycle of the PCR. Each reaction was done in triplicate and the corresponding Ct values were determined.

Amplified fragments of each gene were cloned into the pGEM-T Easy vector (Promega, Madison, Wisconsin, USA). The plasmids were diluted several times to generate templates ranging from 105 to 102 copies used for standard curves for the estimation of copy numbers in total cDNA. The results are expressed as the ratio of the copy number of the cDNA corresponding to each gene studied and that of the constitutive control gene MSC27 (Pay et al., 1992) in 5 μl of first strands.

MtPPRD1 cDNA cloning

The 5′ and the 3′ ends of the MtPPRD1 cDNA were obtained, respectively, with a reverse primer (5′-CGAAACCCTTTGGAACTCCCTTTCC-3′) and a forward primer (5′-CTCACAAAGATCACGGCCACTCACA-3′) with the SMART™ RACE cDNA Amplification Kit (Clontech). Specific primers for MtPPRD1 were designed in the 5′ and 3′ untranslated regions (respectively 5′-GAGCACACACCGAATCAAACTA-3′ and 5′-TTTCAACACACCATAGAATTTTCG-3′) to amplify full length cDNA or genomic DNA products, which were then cloned into pGEM-T Easy vector (Promega).

In silico analysis

Database searching for ESTs encoding proteins homologous with MtPPRD1 was performed with the BLAST algorithm (Altschul et al., 1990). Identification of significant sites, patterns or profiles was performed with Scansite (http://scansite.mit.edu) and the PROSITE algorithm (http://www.expasy.org/prosite/) (Sigrist et al., 2002). Localization predictions were performed with the PSORT algorithm (http://psort.nibb.ac.jp/form.html) (Nakai and Horton, 1999).

Results

Identification and molecular cloning of MtPPRD1 cDNA

In order to identify genes involved in the control of Medicago truncatula germination, a cDNA library was constructed by suppressive subtractive hybridization (SSH) with two mRNA populations of embryo axes corresponding to seeds imbibed for 16 h and for 23 h. Only a few clones (Table 1) were obtained by this subtraction. The reliability of this cDNA library has been tested by checking for the subtraction result of two control genes. As expected the subtraction resulted in the complete extinction of the constitutively expressed MSC27 and in the appearance of a clone corresponding to _S_-adenosyl methionine synthetase (SAMS), whose expression increases during Medicago truncatula germination (Gallardo et al., 2003).

Table 1.

ESTs and genes revealed by suppressive subtractive hybridization (SSH) between two populations of mRNAs of embryo axes of seeds imbibed for 16 h and for 23 h at 20 °C in darkness

a

GenBank.

b

TIGR Medicago truncatula Gene Index.

Table 1.

ESTs and genes revealed by suppressive subtractive hybridization (SSH) between two populations of mRNAs of embryo axes of seeds imbibed for 16 h and for 23 h at 20 °C in darkness

a

GenBank.

b

TIGR Medicago truncatula Gene Index.

Among the clones obtained by the subtraction (Table 1) one corresponding to an open reading frame (ORF) coding for a protein exhibiting a proline-rich domain (PRD) with several Src homologous 3 (SH3) binding motives has been studied further.

Full-length cDNA (785 bp) coding for this clone was obtained by 5′−3′ RACE PCR. The amino acid sequence deduced from the ORF revealed that it encodes a protein of 132 amino acids which contains 11.4% of proline that was named MtPPRD1 as _Medicago truncatula_Protein with Proline-Rich Domain. Ten proline residues were localized in a N terminal 28-amino acid domain in which SH3 binding sites (PXXP) have been detected by the Scansite algorithm (Fig. 1). The PROSITE algorithm predicted one N-glycosylation site (Asn, 113–116 position) and two protein kinase C phosphorylation sites (44–46 and 54–56 positions) (Fig. 1). The PSORT algorithm allowed for the prediction of a cleavable N terminus signal sequence at position 21 and located the protein in the vacuole (probability=0.9) or outside (probability=0.82).

Fig. 1.

Comparison of the amino acid sequence of MtPPRD1 with amino acid sequences deduced from cDNAs and ESTs expressed in various species: Brassica napus LTP (U22105), tobacco LTP (D13952), Arabidopsis LTP4 (NM_125322), Phaseolus vulgaris PVR5 (U34333), Medicago LTP (AL386296), Medicago non-specific LTP (nsLTP) (AL387755), and three MtPPRD1 homologues released from the whole Medicago truncatula genome search are represented by their accession number in http://medicago.toulouse.inra.fr/Mt/EST/. The corresponding TC numbers in the TIGR Medicago truncatula Gene Index (MtGI) are TC68659 for MtC60278 and TC59336 for MtC0005. No correspondence for MtD13687 was found in the TIGR MtGI. The eight conserved cysteine residues in all plant LTPs are highlighted. MtPPRD1 proline rich domain is in bold characters. Glycosylation and phosphorylation sites are underlined differently as follows : Glycosylation, Protein kinase C phosphorylation and Casein kinase II phosphorylation.

MtPPRD1 is highly homologous to several Medicago truncatula ESTs (http://medicago.toulouse.inra.fr/Mt/EST/) coding for proteins with proline-rich domains (55% at the protein level) suggesting that MtPPRD1 belongs to a multigene family (Fig. 1). Furthermore MtPPRD1 is identical to a known Medicago truncatula EST (MtC00014) annotated in the Medicago library as a cDNA coding for a plant lipid transfer protein (TC 68049 in the TIGR Medicago truncatula gene index). At the protein level MtPPRD1 displays a relatively low homology (21–23%) with plant lipid transfer proteins (LTPs), and in particular with Medicago truncatula LTP and non-specific LTP (Fig. 1). MtPPRD1 exhibited, however, the eight cysteine residues conserved in all plant LTPs as a characteristic signature (Fig. 1). Indeed the annotation of MtC00014 as an LTP is probably due to the presence of the eight cysteines at the right spacing.

Proteins homologous to MtPPRD1 are present in several species such as Cicer arietinum, Arabidopsis thaliana, and in the legume Phaseolus vulgaris (Choi et al., 1996) but no function have been assigned to these proteins.

The genomic sequence of MtPPRD1 which has been cloned and sequenced, revealed that it is an intron-less gene (data not shown).

Developmental regulation and organ specificity of MtPPRD1

In order to check for developmental regulation and organ specificity of MtPPRD1, its expression was studied by real-time RT-PCR in embryo axes throughout the germination and post-germination processes from 5 h up to 94 h of imbibition (Fig. 2A) and in various organs (Fig. 3). Expression of SAMS has been determined in parallel as a control specifically expressed during germination sensu stricto (Fig. 2B).

Fig. 2.

(A) Bars of the histogram represent MtPPRD1 expression determined by quantitative real time RT-PCR in the Medicago truncatula embryo axis at various times throughout germination and post germination. The percentage of germinated seeds (solid symbols) correspond to the mean of three replicates of 50 seeds. (B) SAMS expression determined by quantitative real time RT-PCR in the Medicago truncatula embryo axis throughout germination and post-germination. Expression of genes in arbitrary units (A.U.) corresponds to the ratio of the copy number of cDNA of the studied gene (MtPPRD1 or SAMS) on the copy number of the constitutive control MSC27 gene in 5 μl of first strands. Bars indicate standards errors (_n_=3).

Fig. 3.

MtPPRD1 expression determined by quantitative real time RT-PCR in various organs of Medicago truncatula. Fixing nodules were harvested on plants aged 7 weeks, leaves, stems, and roots were harvested on plants aged 2 months and flowers were harvested on plants aged 4 months.

MtPPRD1 expression was undetectable at 5 h of imbibition and started to increase at 10 h, peaked at 37 h, and declined dramatically from 48 h onwards to reach a very low level at 94 h of imbibition. The expression of MtPPRD1 increased 37-fold between 15 h and 37 h. Unlike SAMS, which peaked in expression immediately before radicle emergence (100% of germination being reached at 25 h) and declined immediately afterwards, the maximum level of expression of MtPPRD1 was reached at 37 h and was still significantly high at 48 h, meaning that the expression of MtPPRD1 has been maintained during the beginning of post-germination phase.

The organ specificity of MtPPRD1 expression was checked by measuring its expression in various organs of Medicago truncatula during vegetative and reproductive development and comparing this with the embryo axis at 37 h of imbibition (Fig. 3). A very weak expression, 300–1000 times lower than that in the embryo axis was detected in cotyledons, leaves, stems, flowers, and fixing nodules. In roots, however, MtPPRD1 expression was significant, being one-sixth that in the embryo axis at 37 h.

Commitment of MtPPRD1 expression to radicle emergence during germination

In order to analyse the relationship between MtPPRD1 and germination (radicle emergence), MtPPRD1 expression was studied in the embryo axis in two situations where germination has been modified. For this purpose, seeds were germinated on media containing either abscisic acid (ABA, 10 μM and 100 μM) or the metabolic inhibitor, methionine sulphoximine (MSX, 5 mM). MSX, a specific inhibitor of glutamine synthetase (GS) was previously reported (Glevarec et al., 2004) to delay germination by altering amino acid mobilization and ammonium reassimilation.

After 21 h of imbibition, 90% of control seeds germinated, but only 80% of seeds treated with 10 μM ABA and 31% of seeds treated with MSX germinated (Fig. 4). Seeds treated with 100 μM ABA had their germination completely inhibited (data not shown). After 48 h of imbibition at least 90% of treated seeds had germinated, except those treated with 100 μM ABA that were still inhibited (Fig. 4). The result indicates that ABA at 10 μM and MSX at 5 μM did not severely alter the germination process as was the case for 100 μM ABA.

Fig. 4.

MtPPRD1 expression determined by quantitative real time RT-PCR in embryo axes at various times throughout germination and post-germination. Seeds were germinated on deionized water (control) or 5 mM MSX or 10 μM ABA. Expression of MtPPRD1 in arbitrary units (A.U.) corresponds to the ratio of the copy number of MtPPRD1 cDNA divided by the copy number of the constitutive control MSC27 gene in 5 μl of first strands. Bars indicate standards errors (_n_=3). Figures above the histogram bars indicate the percentage of germination and correspond to the mean of three replicates of 50 seeds.

Expression of MtPPRD1 has been measured in embryo axes of control and treated seeds at 5, 21, and 48 h of imbibition (Fig. 4). Before germination MtPPRD1 was not expressed. At 21 h of imbibition the level of expression of MtPPRD1 was lower in embryo axes of seeds treated with 10 μM ABA and 5 μM MSX than that in the control sample. At 48 h of imbibition the level of expression of MtPPRD1 was similar in the control and embryo axes of treated seeds. Expression of MtPPRD1 was completely inhibited in embryo axes of seeds treated with 100 μM ABA (data not shown). Expression of MtPPRD1 has also been compared in embryo axes of seeds germinating in darkness (the standard condition of germination), and in continuous light. In line with the above-mentioned results, MtPPRD1 showed the same profile of expression under both conditions, with, however, a delay under continuous light corresponding to the delay of germination (data not shown).

Discussion

The goal of the ongoing Medicago truncatula functional genomic programme of research is the identification of genes involved in the control of early developmental processes responsible for germination completion in non-dormant seeds. For this purpose, two cDNA libraries of genes specifically expressed in the embryo axis have been generated by a PCR-based suppressive subtractive hybridization (SSH) technique between mRNAs of two populations of embryo axes with two correspondent imbibition times. One SSH-cDNA library targeted genes specifically expressed in the embryo axis between 6 h and 23 h and yielded more than 300 hundred clones, and this is currently being used for the generation of a macroarray. In order to target only a few genes with specific roles in the control of germination, in particular, radicle protrusion, a SSH-cDNA library has been realized between two populations of mRNA corresponding to 16 h and 23 h, two close imbibition times that bracket radicle protrusion. As a result, in the present study only a few clones were isolated, which fell into three classes (Table 1): (i) a clone corresponding to Medicago truncatula EST of unknown function (MtPPRD1), (ii) clones corresponding to Medicago truncatula ESTs with known functions in other species but not in Medicago truncatula, and (iii) clones corresponding to genes with known functions in Medicago truncatula encoding an aquaporin, an amine oxidase, and _S_-adenosylmethionine synthetase (SAMS). Amine oxidase is the most abundant soluble protein detected in the extracellular fluids from Fabaceae seedlings (pea, lentil, and chickpea). H2O2 produced from amine degradation has been correlated with oxidative burst, cell death, as well as peroxidase-mediated lignification, suberization, and cell-wall polymer cross-linking occurring during ontogenesis and pathogen attack (Rea et al., 2002). It has been proposed that SAMS plays a decisive role in Arabidopsis thaliana and Medicago truncatula germination (Gallardo et al., 2002, 2003).

MtPPRD1 encodes an LTP-like protein with a N- terminus proline-rich domain

A clone corresponding to an ORF encoding a protein with a proline-rich domain has been selected for further studies. The full-length cDNA, obtained by 5′-3′ RACE PCR (785 bp) named MtPPRD1, corresponded to an EST, MtC00014, annotated as encoding a lipid transfer protein (LTP) in the Toulouse Medicago truncatula databank (http://medicago.toulouse.inra.fr/Mt/EST/). Lipid transfer proteins as well as proline-rich proteins have been extensively studied and their properties and potential physiological roles are relatively well known (Kader, 1996, 1997; Mayer, 2001), however, as far as is known, this is the first time a cDNA encoding a protein combining the two protein motifs has been isolated. In the opinion of the authors, this specificity justifies the analysis of MtPPRD1.

Sequence analysis of the full length MtPPRD1 cDNA, showed that it encodes a protein of 132 amino acids with a proline-rich domain of 28 amino acids containing 10 proline residues located at the N terminus. Comparison of this sequence with the available databases revealed low homologies with known LTPs. Only 21–23% identity with LTPs of tobacco, Arabidopsis thaliana, Brassica napus, and Medicago truncatula were found. The amino acid residues, that is, valine 7, proline 25 and 71, and aspartic acid 44, conserved among all plant LTPs, are not present in MtPPRD1. Moreover, the 28 amino acids N-terminus proline-rich domain of MtPPRD1 is absent from all previously reported plant lipid transfer proteins. MtPPRD1 is therefore longer than plant LTPs that are generally 91–95 amino acids long. However, MtPPRD1 shares with LTPs a major specific structural/functional feature, namely the presence of eight cysteine residues of defined spacing in the protein molecule (Soufleri et al., 1996). By forming disulphide bonds between the cysteine residues a hydrophobic cavity results that enables the protein to load and transfer molecules such as fatty acids, acyl CoA and phospholipids, phosphatidylinositol or phosphatidylcholine or hydrophobic molecules in general (Kader, 1996). MtPPRD1 exhibits a potential signal peptide of 21 amino acids capable of locating proteins to the cell wall or the vacuole suggesting that MtPPRD1 would be secreted out of the cytosol, as observed for several LTPs and non-specific LTPs that are located in the cell wall (Garcia-Garrido et al., 1998).

MtPPRD1 shares with proline-rich proteins the presence of several conserved core motifs PXXP (where P is a proline and X is any amino acid) required for binding to SH3 domains (Feng et al., 1994; Lam et al., 2001). In mammals, and particularly in human, proteins with proline-rich domain have been shown to intervene in intermolecular protein–protein interactions and signal transduction cascades by binding to SH3 domains of the Src family of tyrosine kinase, protein adaptors, and phopholipase C-γ (Mayer, 2001). The role of SH3 domains in plants has not received much attention, but Lam et al. (2001) reported the identification of several Arabidopsis ESTs that encode novel SH3-containing proteins known as AtSH3Ps. They are involved in trafficking of clathrin-coated vesicles. Possible interacting partners of AtSH3P1 have been searched for by using the predicted SH3 domain in a two-hybrid screening of an Arabidopsis cDNA library. Sequencing of the five cDNA clones isolated revealed at least one proline-rich domain with the PXXP motif per clone. A clone encoding an auxilin-like protein involved in the uncoating stage of clathrin-mediated endocytosis appeared as the best candidate to interact with AtSH3P1 (Lam et al., 2001). However, it is worth mentioning that the sequencing revealed a cyclin G-dependent protein kinase, an extensin-like protein, and a pollen-specific protein as proteins with proline–rich domains capable of binding SH3 domains (Lam et al., 2001).

MtPPRD1 is specifically expressed in the embryo axis of Medicago truncatula during radicle emergence and early post-germination

Expression studies of MtPPRD1 by real-time quantitative RT-PCR carried out on RNA from embryo axes at different times throughout germination and post-germination and in different organs produced three major results. First, the absence of expression in several organs, cotyledons, leaves, stems, and fixing nodules favours a specificity of expression of MtPPRD1 in the embryo axis. However, the low but not negligible level of expression found in the roots, equivalent to that in the embryo axis at 94 h of imbibition, suggests that MtPPRD1 would maintain a low and probably constitutive expression in roots in relation to a role played during plant development. Second, the time-course experiment of MtPPRD1 expression in the embryo axis showed that this gene is developmentally regulated. MtPPRD1 expression strongly increased in the embryo axis a few hours before radicle emergence, and this increase continued, reaching its maximum during the early phases of post-germination before declining dramatically. This profile of expression indicates that MtPPRD1 might not only be involved in developmental process linked to radicle emergence, but would also play a role in the radicle and root as suggested by the residual expression maintained in the roots. Choi et al. (1996) characterized a 14 kDa proline-rich protein (PVR5), highly homologous to MtPPRD1 preferentially expressed in the roots of Phaseolus vulgaris seedlings, but did not check for its expression during the early stages of development of germination and post-germination. Although a biological role has not been assigned to PVR5, its localization in cortical ground meristem in which maximal cell division occurs suggests an involvement in the control of a developmental processes. Furthermore, expression studies in the embryo axis of seeds, where germination has been delayed by either ABA or MSX treatments or continuous light conditions strengthened the hypothesis of the developmental regulation of MtPPRD1 and the commitment of its expression to the process of radicle emergence. Third, unlike several genes where the product of expression is stored in the dry seed, i.e. LA-ACSI an isoform of 1-aminocyclopropane-1-carboxylate synthase (Bekman et al., 2000), and PvGLP1 a germin-like protein (Aubry et al., 2003), MtPPRD1 was not expressed in the dry seed, indicating that it might not be involved in early stages of germination.

Conclusion

On the basis of sequence homologies, several putative roles are proposed for MtPPRD1 in Medicago truncatula, this protein can intervene (i) as an LTP in membrane biogenesis and regulation of the intracellular fatty acid pool by binding and transferring fatty acids and phospholipids between membranes, (ii) in the control of a developmental process specific to late germination and early phases of post-germination, and (iii) and/or in pathogen defence. MtPPRD1 would be recruited for its LTP-like characteristic, namely the presence of a hydrophobic cavity capable of interacting with hydrophobic molecules and its proline-rich domain with several PXXP motives allowing for protein–protein interactions and signal transduction cascades. Non-specific LTPs have also been shown to play different roles from LTPs, with their hydrophobic cavity they would intervene in developmental functions during embryogenesis and developmental stages (Kader, 1997) such as pollen adherence to the stigma during pollen elongation (Park et al., 2000) or the adaptation of plants to environmental changes (Kader, 1997) and pathogen–defence reactions (Garcia-Olmedo et al., 1995).

Finally, this study has allowed an interesting candidate MtPPRD1 to be isolated that could intervene in a function probably controlled by a multicomponent complex of proteins (Mayer, 2001). To study MtPPRD1 further it will be interesting to determine its possible interacting partners. Towards this aim, the protein is being produced in a heterologous system, Pischia pastoris and will be used for this purpose. In parallel, blasting the predicted SH3 domain of the Arabidopsis AtSH3P1 against the Medicago truncatula EST database allowed for the release of two interesting ESTs, MtC60438 and MtD27717, both bearing domains, respectively, 92% and 80% homologous to the Arabidopsis SH3 domain. These ESTs are annotated as proteins with an SH3 domain, but with unknown function, so they can be considered as potentially putative partners of MtPPRD1.

The authors are indebted to Dr Andreas Niebel for kindly initiating and supervising Laure Viau for analysing and screening SSH-cDNA libraries. The authors also wish to thank Professor D Herouart and his team for growing the plants in biological N2-fixing conditions and providing them with RNAs of fixing nodules. The authors wish to thank B Jettner (Seed-Co Australia Co-Operative Ltd., Hilton, Australia) for the generous gift of M. truncatula cv. Paraggio seeds.

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