Medicago falcata MfSTMIR, an E3 ligase of endoplasmic reticulum-associated degradation, is involved in salt stress response.

Recent studies on E3 of endoplasmic reticulum (ER)-associated degradation (ERAD) in plants have revealed homologs in yeast and animals. However, it remains unknown whether the plant ERAD system contains a plant-specific E3 ligase. Here, we report that MfSTMIR, which encodes an ER-membrane-localized RING E3 ligase that is highly conserved in leguminous plants, plays essential roles in the response of ER and salt stress in Medicago. MfSTMIR expression was induced by salt and tunicamycin (Tm). mtstmir loss-of-function mutants displayed impaired induction of the ER stress-responsive genes BiP1/2 and BiP3 under Tm treatment and sensitivity to salt stress. MfSTMIR promoted the degradation of a known ERAD substrate, CPY*. MfSTMIR interacted with the ERAD-associated ubiquitin-conjugating enzyme MtUBC32 and Sec61-translocon subunit MtSec61γ. MfSTMIR did not affect MtSec61γ protein stability. Our results suggest that the plant-specific E3 ligase MfSTMIR participates in the ERAD pathway by interacting with MtUBC32 and MtSec61γ to relieve ER stress during salt stress.

Genome-wide profiling of expression and biochemical functions of the Medicago glutathione S-transferase gene family.

Glutathione S-transferases are ubiquitous enzyme in plants, playing vital roles in several physiological and developmental processes. In this study we identified 73 GST genes from the genome of Medicago truncatula. The Medicago GSTs were divided to eight classes with tau and phi being the most numerous. Six clusters were found on four Medicago chromosomes. The local gene duplication mainly contributed to the expansion of this large gene family. Functional divergence was found in their gene structures, gene expression patterns, and enzyme properties. A genomic comparative analysis revealed lineage-specific loss/gain events between Medicago and Glycine. This study offered new insights into the evolution of gene family between closely related species.

Non-plastidic, tyrosine-insensitive prephenate dehydrogenases from legumes.

L-Tyrosine (Tyr) and its plant-derived natural products are essential in both plants and humans. In plants, Tyr is generally assumed to be synthesized in the plastids via arogenate dehydrogenase (TyrA(a), also known also ADH), which is strictly inhibited by L-Tyr. Using phylogenetic and expression analyses, together with recombinant enzyme and endogenous activity assays, we identified prephenate dehydrogenases (TyrA(p)s, also known as PDHs) from two legumes, Glycine max (soybean) and Medicago truncatula. The identified PDHs were phylogenetically distinct from canonical plant ADH enzymes, preferred prephenate to arogenate substrate, localized outside of the plastids and were not inhibited by L-Tyr. The results provide molecular evidence for the diversification of primary metabolic Tyr pathway via an alternative cytosolic PDH pathway in plants.

Involvement of peroxidase activity in developing somatic embryos of Medicago arborea L. Identification of an isozyme peroxidase as biochemical marker of somatic embryogenesis.

The legume Medicago arborea L. is very interesting as regards the regeneration of marginal arid soils. The problem is that it does not have a good germinative yield. It was therefore decided to regenerate via somatic embryogenesis and find a marker of embryogenic potential. In this study, peroxidase activity was evaluated in non-embryogenic and embryogenic calli from M. arborea L. A decrease in soluble peroxidase activity is observed in its embryonic calli at the time at which the somatic embryos begin to appear. This activity is always lower in embryonic calli than in non-embryonic ones (unlike what happens in the case of wall-bound peroxidases). These results suggest that peroxidases can be considered to be enzymes involved in somatic embryogenesis in M. arborea. In addition, isozyme analyses were carried out on protein extracts using polyacrylamide gel electrophoresis. The band called P5 was detected only in embryogenic cultures at very early stages of development. This band was digested with trypsin and analyzed using linear ion trap (LTQ) mass spectrometer. In P5 isoform a peroxidase-L-ascorbate peroxidase was identified. It can be used as a marker that allows the identification of embryological potential.

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus, Medicago, and Phaseolus.

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.

Identification of duplicated and stress-inducible Aox2b gene co-expressed with Aox1 in species of the Medicago genus reveals a regulation linked to gene rearrangement in leguminous genomes.

In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization.

Trans-specific gene silencing of acetyl-CoA carboxylase in a root-parasitic plant.

Parasitic species of the family Orobanchaceae are devastating agricultural pests in many parts of the world. The control of weedy Orobanchaceae spp. is challenging, particularly due to the highly coordinated life cycles of the parasite and host plants. Although host genetic resistance often provides the foundation of plant pathogen management, few genes that confer resistance to root parasites have been identified and incorporated into crop species. Members of the family Orobanchaceae acquire water, nutrients, macromolecules, and oligonucleotides from host plants through haustoria that connect parasite and host plant roots. We are evaluating a resistance strategy based on using interfering RNA (RNAi) that is made in the host but inhibitory in the parasite as a parasite-derived oligonucleotide toxin. Sequences from the cytosolic acetyl-CoA carboxylase (ACCase) gene from Triphysaria versicolor were cloned in hairpin conformation and introduced into Medicago truncatula roots by Agrobacterium rhizogenes transformation. Transgenic roots were recovered for four of five ACCase constructions and infected with T. versicolor against parasitic weeds. In all cases, Triphysaria root viability was reduced up to 80% when parasitizing a host root bearing the hairpin ACCase. Triphysaria root growth was recovered by exogenous application of malonate. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that ACCase transcript levels were dramatically decreased in Triphysaria spp. parasitizing transgenic Medicago roots. Northern blot analysis identified a 21-nucleotide, ACCase-specific RNA in transgenic M. truncatula and in T. versicolor attached to them. One hairpin ACCase construction was lethal to Medicago spp. unless grown in media supplemented with malonate. Quantitative RT-PCR showed that the Medicago ACCase was inhibited by the Triphysaria ACCase RNAi. This work shows that ACCase is an effective target for inactivation in parasitic plants by trans-specific gene silencing.

Expression of a Medicago falcata small GTPase gene, MfARL1 enhanced tolerance to salt stress in Arabidopsis thaliana.

To understand the role of small GTPases in response to abiotic stress, we isolated a gene encoding a small GTPase, designated MfARL1, from a subtracted cDNA library in Medicago falcata, a native legume species in semi-arid grassland in northern China. The function of MfARL1 in response to salt stress was studied by expressing MfARL1 in Arabidopsis. Wild-type (WT) and transgenic plants constitutively expressing MfARL1 showed comparable phenotype when grown under control conditions. Germination of seeds expressing MfARL1 was less suppressed by salt stress than that of WT seeds. Transgenic seedlings had higher survival rate than WT seedlings under salt stress, suggesting that expression of MfARL1 confers tolerance to salt stress. The physiological and molecular mechanisms underlying these phenomena were elucidated. Salt stress led to a significant decrease in chlorophyll contents in WT plants, but not in transgenic plants. Transgenic plants accumulated less amounts of H(2)O(2) and malondialdehyde than their WT counterparts under salt stress, which can be accounted for by the higher catalase activities, lower activities of superoxide dismutase, and peroxidase in transgenic plants than in WT plants. Transgenic plants displayed lower Na(+)/K(+) ratio due to less accumulation of Na(+) than wild-type under salt stress conditions. The lower Na(+)/K(+) ratio may result from less accumulation of Na(+) due to reduced expression of AtHKT1 that encodes Na(+) transporter in transgenic plants under salt stress. These findings demonstrate that MfARL1 encodes a novel stress-responsive small GTPase that is involved in tolerance to salt stress.

Hydrogen peroxide and nitric oxide mediated cold- and dehydration-induced myo-inositol phosphate synthase that confers multiple resistances to abiotic stresses.

myo-Inositol phosphate synthase (MIPS) is the key enzyme of myo-inositol synthesis, which is a central molecule required for cell metabolism and plant growth as a precursor to a large variety of compounds. A full-length fragment of MfMIPS1 cDNA was cloned from Medicago falcata that is more cold-tolerant than Medicago sativa. While MfMIPS1 transcript was induced in response to cold, dehydration and salt stress, MIPS transcript and myo-inositol were maintained longer and at a higher level in M. falcata than in M. sativa during cold acclimation at 5 °C. MfMIPS1 transcript was induced by hydrogen peroxide (H(2) O(2)) and nitric oxide (NO), but was not responsive to abscisic acid (ABA). Pharmacological experiments revealed that H(2) O(2) and NO are involved in the regulation of MfMIPS1 expression by cold and dehydration, but not by salt. Overexpression of MfMIPS1 in tobacco increased the MIPS activity and levels of myo-inositol, galactinol and raffinose, resulting in enhanced resistance to chilling, drought and salt stresses in transgenic tobacco plants. It is suggested that MfMIPS1 is induced by diverse environmental factors and confers resistance to various abiotic stresses.

A nematode demographics assay in transgenic roots reveals no significant impacts of the Rhg1 locus LRR-Kinase on soybean cyst nematode resistance.

BACKGROUND: Soybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S. The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. The Glyma18g02680.1 gene at the Rhg1 locus that encodes an apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) has been proposed to be the SCN resistance gene, but its function has not been confirmed. Generation of fertile transgenic soybean lines is difficult but methods have been published that test SCN resistance in transgenic roots generated with Agrobacterium rhizogenes. RESULTS: We report use of artificial microRNA (amiRNA) for gene silencing in soybean, refinements to transgenic root SCN resistance assays, and functional tests of the Rhg1 locus LRR-kinase gene. A nematode demographics assay monitored infecting nematode populations for their progress through developmental stages two weeks after inoculation, as a metric for SCN resistance. Significant differences were observed between resistant and susceptible control genotypes. Introduction of the Rhg1 locus LRR-kinase gene (genomic promoter/coding region/terminator; Peking/PI 437654-derived SCN-resistant source), into rhg1- SCN-susceptible plant lines carrying the resistant-source Rhg4+ locus, provided no significant increases in SCN resistance. Use of amiRNA to reduce expression of the LRR-kinase gene from the Rhg1 locus of Fayette (PI 88788 source of Rhg1) also did not detectably alter resistance to SCN. However, silencing of the LRR-kinase gene did have impacts on root development. CONCLUSION: The nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs and the pSM103 vector that drives interchangeable amiRNA constructs through a soybean polyubiqutin promoter (Gmubi), with an intron-GFP marker for detection of transgenic roots, may have widespread use in legume biology. Studies in which expression of the Rhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance.

Response of nitrogen fixation in relation to nodule carbohydrate metabolism in Medicago ciliaris lines subjected to salt stress.

The effect of salt stress on nitrogen fixation, in relation to sucrose transport towards nodules and other sink organs and the potential of sucrose breakdown by nodules, was investigated in two lines of Medicago ciliaris. Under salt stress conditions, the two lines showed a decrease of total biomass production, but TNC 1.8 was less affected by salt than TNC 11.9. The chlorophyll content was not changed in TNC 1.8, in contrast to TNC 11.9. Shoot, root, and nodule biomass were also affected in the two lines, but TNC 1.8 exhibited the higher potentialities of biomass production of these organs. Nitrogen fixation also decreased in the two lines, and was more sensitive to salt than growth parameters. TNC 1.8 consistently exhibited the higher values of nitrogen fixation. Unlike nodules, leaves of both lines were well supplied in nutrients with some exceptions. Specifically, the calcium content decreased in the sensitive line leaves, and the nodule magnesium content was not changed in either line. The tolerant line accumulated more sodium in its leaves. The two lines did not show any differences in the nodule sodium content. Sucrose allocation towards nodules was affected by salt in the two lines, but this constraint did not seem to affect the repartition of sucrose between sink organs. Salt stress induced perturbations in nodule sucrolytic activities in the two lines. It inhibited sucrose synthase, but the inhibition was more marked in TNC 11.9; alkaline/neutral activity was not altered in TNC 1.8, whereas it decreased more than half in TNC 11.9. Thus, the relative tolerance of TNC 1.8 to salt stress could be attributed to a better use of these photoassimilates by nodules and a better supply of bacteroids in malate. The hypothesis of a competition for sucrose between nodules and other sink organs under salt stress could not be verified.

Regioselective synthesis of plant (iso)flavone glycosides in Escherichia coli.

The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides, and their glycosylation has a remarkable influence on their pharmacokinetic properties. The glycosyltransferases UGT71G1 and UGT73C8 from Medicago truncatula are excellent reagents for the regioselective glycosylation of (iso)flavonoids in Escherichia coli grown in Terrific broth. Ten to 20 mg/L of either genistein or biochanin A 7-O-glucoside was produced after feeding genistein or biochanin A to E. coli expressing UGT71G1, and similar levels of luteolin 4'-O- and 7-O-glucosides were produced after feeding luteolin to cultures expressing UGT73C8. For the production of kaempferol 3-O-glucoside or quercetin 3-O-glucoside, the Phe148Val or Tyr202Ala mutants of UGT71G1 were employed. Ten to 16 mg/L of either kaempferol 3-O- or quercetin 3-O-glucosides were produced on feeding kaempferol or quercetin to E. coli expressing these enzymes. More than 90% of the glucoside products were released to the medium, facilitating their isolation.

Adaptive evolution of the symbiotic gene NORK is not correlated with shifts of rhizobial specificity in the genus Medicago.

BACKGROUND: The NODULATION RECEPTOR KINASE (NORK) gene encodes a Leucine-Rich Repeat (LRR)-containing receptor-like protein and controls the infection by symbiotic rhizobia and endomycorrhizal fungi in Legumes. The occurrence of numerous amino acid changes driven by directional selection has been reported in this gene, using a limited number of messenger RNA sequences, but the functional reason of these changes remains obscure. The Medicago genus, where changes in rhizobial associations have been previously examined, is a good model to test whether the evolution of NORK is influenced by rhizobial interactions. RESULTS: We sequenced a region of 3610 nucleotides (encoding a 392 amino acid-long region of the NORK protein) in 32 Medicago species. We confirm that positive selection in NORK has occurred within the Medicago genus and find that the amino acid positions targeted by selection occur in sites outside of solvent-exposed regions in LRRs, and other sites in the N-terminal region of the protein. We tested if branches of the Medicago phylogeny where changes of rhizobial symbionts occurred displayed accelerated rates of amino acid substitutions. Only one branch out of five tested, leading to M. noeana, displays such a pattern. Among other branches, the most likely for having undergone positive selection is not associated with documented shift of rhizobial specificity. CONCLUSION: Adaptive changes in the sequence of the NORK receptor have involved the LRRs, but targeted different sites than in most previous studies of LRR proteins evolution. The fact that positive selection in NORK tends not to be associated to changes in rhizobial specificity indicates that this gene was probably not involved in evolving rhizobial preferences. Other explanations (e.g. coevolutionary arms race) must be tested to explain the adaptive evolution of NORK.

Functional analysis of members of the isoflavone and isoflavanone O-methyltransferase enzyme families from the model legume Medicago truncatula.

Previous studies have identified two distinct O-methyltransferases (OMTs) implicated in isoflavonoid biosynthesis in Medicago species, a 7-OMT methylating the A-ring 7-hydroxyl of the isoflavone daidzein and a 4'-OMT methylating the B-ring 4'-hydroxyl of 2,7,4'-trihydroxyisoflavanone. Genes related to these OMTs from the model legume Medicago truncatula cluster as separate branches of the type I plant small molecule OMT family. To better understand the possible functions of these related OMTs in secondary metabolism in M. truncatula, seven of the OMTs were expressed in E. coli, purified, and their in vitro substrate preferences determined. Many of the enzymes display promiscuous activities, and some exhibit dual regio-specificity for the 4' and 7-hydroxyl moieties of the isoflavonoid nucleus. Protein structure homology modeling was used to help rationalize these catalytic activities. Transcripts encoding the different OMT genes exhibited differential tissue-specific and infection- or elicitor-induced expression, but not always in parallel with changes in expression of confirmed genes of the isoflavonoid pathway. The results are discussed in relation to the potential in vivo functions of these OMTs based on our current understanding of the phytochemistry of M. truncatula, and the difficulties associated with gene annotation in plant secondary metabolism.

The stress kinase gene MtSK1 in Medicago truncatula with particular reference to somatic embryogenesis.

Medicago truncatula, a model for legume genomics, can be regenerated by somatic embryogensis by the use of a suitable genotype and an auxin plus cytokinin. The stress response induced by explant wounding and culture is increasingly recognized as an important component of somatic embryo induction. We have cloned and investigated the stress kinase gene MtSK1 in relation to somatic embryogenesis in M. truncatula, using the highly embryogenic mutant Jemalong 2HA (2HA) and its progenitor Jemalong. The main features of the MtSK1 protein of 351 amino acids are an N-terminal kinase domain and a C-terminal glutamic acid-rich region, which is predicted to be a coiled-coil. MtSK1 is a member of the SnRK2 subgroup of the SnRK group of plant kinases. Members of the SnRK2 kinases play a role in stress responses of plants. MtSKI expression is induced by wounding in the cultured tissue independent of auxin or cytokinin. However, in both 2HA and Jemalong, as the callus develops in response to auxin plus cytokinin, MtSK1 expression continues to increase. MtSK1 responds to salt stress in vivo, consistent with its role as a stress kinase. The likely role of MtSK1 in stress-induced signaling will facilitate the relating of stress-response pathways to auxin and cytokinin-induced signaling in the understanding of the molecular mechanisms involved in the induction of somatic embryogenesis in M. truncatula.

Enhancement of isoflavone synthase activity by co-expression of P450 reductase from rice.

Plant cytochrome P450s interact with a flavoprotein, NADPH-cytochrome P450 reductase (CPR), to transfer electrons from NADPH. The gene for rice P450 reductase (RCPR) was cloned and expressed in Saccaromyces cerevisiae, where the specific activity of the expressed RPCR was 0.91 U/mg protein. When isoflavone synthase gene (IFS) from red clover, used as a model system of plant cytochrome P450, was co-expressed with RCPR in yeast, the production of genistein from naringein increased about 4.3-fold, indicating that the RCPR efficiently interacts with cytochrome P450 to transfer electrons from NADPH.

The Medicago CDKC;1-CYCLINT;1 kinase complex phosphorylates the carboxy-terminal domain of RNA polymerase II and promotes transcription.

The Ms;CDKC;1 kinase is structurally similar to those cyclin-dependent kinases (CDKs) that are not involved directly in cell cycle regulation. The presence of a PITAIRE motif in Ms;CDKC;1 suggests that it interacts with cyclins different from known PSTAIRE/PPTALRE kinase regulatory subunits. Here we demonstrate that a Medicago CYCLINT (CYCT) protein is a specific interactor of Ms;CDKC;1 and the interaction between these two proteins gives rise to an active kinase complex that localizes to the nucleus and phosphorylates the carboxy-terminal YSPTSPS heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase II in vitro. Mutation of Ser to Ala at position 5 within the heptapeptide repeat abolishes substrate phosphorylation by the Ms;CDKC;1 kinase complex. Furthermore, our data show that addition of the Medicago CDKC;1-CYCT;1 heterodimer completely restored the transcriptional activity of a HeLa nuclear extract depleted of endogeneous CDK9 kinase complexes. Together, these results indicate that the Medicago CDKC;1-CYCT;1 complex is a positive regulator of transcription in plants and has a role similar to the CDK9/cyclin T complex of human positive transcription elongation factor P-TEFb.

Genomic characterization and physical mapping of two fucosyltransferase genes in Medicago truncatula.

Fucosyltransferases catalyse fucose transfer onto oligosaccharides. Two fucosylated structures have been identified in plants: the alpha1,4-fucosylated Lewis-a epitope and the alpha1,3-fucosylated core. Here we report the cloning, genomic characterization, and physical mapping of two genes encoding proteins similar to alpha1,4-fucosyltransferase (EC 2.4.1.65, MtFUT1) and alpha1,3-fucosyltransferase (EC 2.4.1.214, MtFUT2) in Medicago truncatula. Analysis of the genomic organization of the fucosyltransferase genes in M. truncatula, revealed the presence of two genomic variants of the MtFUT1 gene coding sequence, one containing a single intron and the other intronless, whereas in MtFUT2, the gene coding region is interrupted by four introns. Using for the first time fluorescence in situ hybridization (FISH) to physically map fucosyltransferase genes in plants, this study reveals a high genomic dispersion of these genes in Medicago. The MtFUT1 genes are mapped on chromosomes 4, 7, and 8, colocalizing on three of the five MtFUT2 loci. Chromosomes 1 and 5 carry the additional MtFUT2 loci. Moreover, the intensity of the FISH signals reveals marked differences in the number of gene copies per locus for both genes. Simultaneous mapping of rRNA genes on chromosome 5 shows that several MTFUT2 gene loci are inserted within the rDNA array. Insertions of coding DNA sequences into the rDNA repeats were never reported to date.

Stress-induced changes in protease composition are determined by nitrogen supply in non-nodulating white clover.

An inbreeding line of white clover has been identified which remains non-nodulated under appropriate physiological conditions and so the nitrogen concentration of the plant can be manipulated by altering the nitrate supply to the roots. Non-nodulating plants were used to test the hypothesis that acclimation to nitrogen limitation in white clover involves changes in protease activity and composition. These results indicate that acclimation to nitrogen limitation involves the realignment of constituent proteases without necessarily incurring significant changes in total protease activity. Plants grown at 2.5, 5.0, 7.5, and 10 mM nitrate showed a positive correlation between nitrate supply and foliar protein concentration. Protein profiles, revealed by Coomassie-stained SDS-PAGE, were unchanged between treatments for a given amount of protein. Serine, aspartate/metalloprotease, and two cysteine proteases were identified in the leaves. Although total protease activity per gram fresh weight was unchanged between treatments, the relative contributions of these four proteases was determined by nitrate supply. When plants were stressed further by withholding nitrate there was an increase in cysteine protease activity, but a senescence-related aspartate/metalloprotease was not visible. Hence, while protease expression in white clover leaves responded to the current and past nitrogen status of the plant, the proteases involved in remobilization during nutrient limitation were distinct from those involved during the main senescence period. It is suggested that nitrogen limitation induced an early, reversible stage of senescence in which perturbations in protease activity facilitated the degradation of non-essential proteins in order to increase the chances of plant survival or seed set.