The receptor kinase RiSho1 in Rhizophagus irregularis regulates arbuscule development and drought tolerance during arbuscular mycorrhizal symbiosis.

In terrestrial ecosystems, most plant species can form beneficial associations with arbuscular mycorrhizal (AM) fungi. Arbuscular mycorrhizal fungi benefit plant nutrient acquisition and enhance plant tolerance to drought. The high osmolarity glycerol 1 mitogen-activated protein kinase (HOG1-MAPK) cascade genes have been characterized in Rhizophagus irregularis. However, the upstream receptor of the HOG1-MAPK cascade remains to be investigated. We identify the receptor kinase RiSho1 from R. irregularis, containing four transmembrane domains and one Src homology 3 (SH3) domain, corresponding to the homologue of Saccharomyces cerevisiae. Higher expression levels of RiSho1 were detected during the in planta phase in response to drought. RiSho1 protein was localized in the plasma membrane of yeast, and interacted with the HOG1-MAPK module RiPbs2 directly by protein-protein interaction. RiSho1 complemented the growth defect of the yeast mutant ∆sho1 under sorbitol conditions. Knock-down of RiSho1 led to the decreased expression of downstream HOG1-MAPK cascade (RiSte11, RiPbs2, RiHog1) and drought-resistant genes (RiAQPs, RiTPSs, RiNTH1 and Ri14-3-3), hampered arbuscule development and decreased plants antioxidation ability under drought stress. Our study reveals the role of RiSho1 in regulating arbuscule development and drought-resistant genes via the HOG1-MAPK cascade. These findings provide new perspectives on the mechanisms by which AM fungi respond to drought.

Genome-wide identification and characterization of the heavy metal ATPase (HMA) gene family in Medicago truncatula under copper stress.

In nature, the normal growth, development, and quality of plants are significantly affected by many abiotic stresses, such as drought, salinity, low temperature, and heavy metals. Among heavy metals, copper is an essential element for plant growth and development but also has a toxic effect on plants when its concentration is excessive. Therefore, plants have evolved a complex regulatory network to regulate the balance of copper ions in cells. Heavy metal ATPases (HMAs), which transport heavy metals to intracellular compartments or detoxify heavy metals present at excessive concentrations, have been extensively studied in model plant species. However, no comprehensive and systematic surveys of members of the HMA gene family have been conducted in the model legume species Medicago truncatula. Here, nine putative MtHMAs were identified in the M. truncatula genome. These MtHMAs were phylogenetically divided into two distinct groups. The members in each group had a relatively conserved gene structure and motif composition. The number of introns in the MtHMAs varied from 5 to 16, with the majority of these genes containing 8 introns. The expression patterns showed that MtHMAs exhibit preferential or distinct expression patterns among different tissues. Finally, the expression patterns of the members of this gene family were verified in the leaves and roots of plants under Cu stress. Our findings will be valuable for the functional investigation and application of members of this gene family in M. truncatula and other related legume species.

Genome-Wide Identification and Analyses of Drought/Salt-Responsive Cytochrome P450 Genes in Medicago truncatula.

Cytochrome P450 monooxygenases (P450s) catalyze a great number of biochemical reactions and play vital roles in plant growth, development and secondary metabolism. As yet, the genome-scale investigation on P450s is still lacking in the model legume Medicago truncatula. In particular, whether and how many MtP450s are involved in drought and salt stresses for Medicago growth, development and yield remain unclear. In this study, a total of 346 MtP450 genes were identified and classified into 10 clans containing 48 families. Among them, sixty-one MtP450 genes pairs are tandem duplication events and 10 MtP450 genes are segmental duplication events. MtP450 genes within one family exhibit high conservation and specificity in intron-exon structure. Meanwhile, many Mt450 genes displayed tissue-specific expression pattern in various tissues. Specifically, the expression pattern of 204 Mt450 genes under drought/NaCl treatments were analyzed by using the weighted correlation network analysis (WGCNA). Among them, eight genes (CYP72A59v1, CYP74B4, CYP71AU56, CYP81E9, CYP71A31, CYP704G6, CYP76Y14, and CYP78A126), and six genes (CYP83D3, CYP76F70, CYP72A66, CYP76E1, CYP74C12, and CYP94A52) were found to be hub genes under drought/NaCl treatments, respectively. The expression levels of these selected hub genes could be induced, respectively, by drought/NaCl treatments, as validated by qPCR analyses, and most of these genes are involved in the secondary metabolism and fatty acid pathways. The genome-wide identification and co-expression analyses of M. truncatulaP450 superfamily genes established a gene atlas for a deep and systematic investigation of P450 genes in M. truncatula, and the selected drought-/salt-responsive genes could be utilized for further functional characterization and molecular breeding for resistance in legume crops.

Phosphorylation of MtRopGEF2 by LYK3 mediates MtROP activity to regulate rhizobial infection in Medicago truncatula.

The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.

Structural and mechanistic insights into the bifunctional HISN2 enzyme catalyzing the second and third steps of histidine biosynthesis in plants.

The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme-HISN2. The enzyme consists of two distinct domains, active respectively as a phosphoribosyl-AMP cyclohydrolase (PRA-CH) and phosphoribosyl-ATP pyrophosphatase (PRA-PH). The domains are analogous to single-domain enzymes encoded by bacterial hisI and hisE genes, respectively. The calculated sequence similarity networks between HISN2 analogs from prokaryotes and eukaryotes suggest that the plant enzymes are closest relatives of those in the class of Deltaproteobacteria. In this work, we obtained crystal structures of HISN2 enzyme from Medicago truncatula (MtHISN2) and described its architecture and interactions with AMP. The AMP molecule bound to the PRA-PH domain shows positioning of the N1-phosphoribosyl relevant to catalysis. AMP bound to the PRA-CH domain mimics a part of the substrate, giving insights into the reaction mechanism. The latter interaction also arises as a possible second-tier regulatory mechanism of the HBP flux, as indicated by inhibition assays and isothermal titration calorimetry.

Identification of the trehalose-6-phosphate synthase gene family in Medicago truncatula and expression analysis under abiotic stresses.

Trehalose-6-phosphate synthase (TPS) exerts important functions related to plant desiccation tolerance and responses to environmental stimuli. However, in Medicago truncatula, the TPS family has not been reported to date. This study found 11 MtTPS genes in the genome of M. truncatula, which could be divided into two subfamilies: Class I and Class II. All TPS family members have a TPS domain (Glyco transf_20) at the N-terminus and a TPP domain (Trehalose_PPase) at the C-terminus. Interestingly, the genetic structures differ between Class I and Class II, Class I members have more introns than Class II members. Furthermore, transcriptome and real-time PCR analysis showed that five MtTPS genes could be induced by drought, salt or cold. Specifically, MtTPS2, MtTPS8, MtTPS9, MtTPS11 were up-regulated under both drought and salt treatment, particularly, MtTPS8 and MtTPS9 can also be induced by cold, while MtTPS7 only responded to salt stress. In summary, this study provides the foundation for further research on TPS genes in M. truncatula and their regulatory function in response to abiotic stresses.

Identification and Characterization of Abiotic Stress Responsive CBL-CIPK Family Genes in Medicago.

The calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) play important roles in plant signal transduction and response to abiotic stress. Plants of Medicago genus contain many important forages, and their growth is often affected by a variety of abiotic stresses. However, studies on the CBL and CIPK family member and their function are rare in Medicago. In this study, a total of 23 CBL and 58 CIPK genes were identified from the genome of Medicago sativa as an important forage crop, and Medicaog truncatula as the model plant. Phylogenetic analysis suggested that these CBL and CIPK genes could be classified into five and seven groups, respectively. Moreover, these genes/proteins showed diverse exon-intron organizations, architectures of conserved protein motifs. Many stress-related cis-acting elements were found in their promoter region. In addition, transcriptional analyses showed that these CBL and CIPK genes exhibited distinct expression patterns in various tissues, and in response to drought, salt, and abscisic acid treatments. In particular, the expression levels of MtCIPK2 (MsCIPK3), MtCIPK17 (MsCIPK11), and MtCIPK18 (MsCIPK12) were significantly increased under PEG, NaCl, and ABA treatments. Collectively, our study suggested that CBL and CIPK genes play crucial roles in response to various abiotic stresses in Medicago.

The R2R3-MYB transcription factor MtMYB134 orchestrates flavonol biosynthesis in Medicago truncatula.

KEY MESSAGE: Our results provide insights into the flavonol biosynthesis regulation of M. truncatula. The R2R3-MYB transcription factor MtMYB134 emerged as tool to improve the flavonol biosynthesis. Flavonols are plant specialized metabolites with vital roles in plant development and defense and are known as diet compound beneficial to human health. In leguminous plants, the regulatory proteins involved in flavonol biosynthesis are not well characterized. Using a homology-based approach, three R2R3-MYB transcription factor encoding genes have been identified in the Medicago truncatula reference genome sequence. The gene encoding a protein with highest similarity to known flavonol regulators, MtMYB134, was chosen for further experiments and was characterized as a functional flavonol regulator from M. truncatula. MtMYB134 expression levels are correlated with the expression of MtFLS2, encoding a key enzyme of flavonol biosynthesis, and with flavonol metabolite content. MtMYB134 was shown to activate the promoters of the A. thaliana flavonol biosynthesis genes AtCHS and AtFLS1 in Arabidopsis protoplasts in a transactivation assay and to interact with the Medicago promoters of MtCHS2 and MtFLS2 in yeast 1-hybrid assays. To ascertain the functional aspect of the identified transcription factor, we developed a sextuple mutant, which is defective in anthocyanin and flavonol biosynthesis. Ectopic expression of MtMYB134 in a multiple myb A. thaliana mutant restored flavonol biosynthesis. Furthermore, overexpression of MtMYB134 in hairy roots of M. truncatula enhanced the biosynthesis of various flavonol derivatives. Taken together, our results provide insight into the understanding of flavonol biosynthesis regulation in M. truncatula and provides MtMYB134 as tool for genetic manipulation to improve flavonol synthesis.

Genome-wide identification and expression profiling of glutathione S-transferase family under multiple abiotic and biotic stresses in Medicago truncatula L.

Glutathione transferases (GSTs) constitute an ancient, ubiquitous, multi-functional antioxidant enzyme superfamily that has great importance on cellular detoxification against abiotic and biotic stresses as well as plant development and growth. The present study aimed to a comprehensive genome-wide identification and functional characterization of GST family in one of the economically important legume plants-Medicago truncatula. Here, we have identified a total of ninety-two putative MtGST genes that code for 120 proteins. All these members were classified into twelve classes based on their phylogenetic relationship and the presence of structural conserved domain/motif. Among them, 7 MtGST gene pairs were identified to have segmental duplication. Expression profiling of MtGST transcripts revealed their high level of organ/tissue-specific expression in most of the developmental stages and anatomical tissues. The transcripts of MtGSTU5, MtGSTU8, MtGSTU17, MtGSTU46, and MtGSTU47 showed significant up-regulation in response to various abiotic and biotic stresses. Moreover, transcripts of MtGSTU8, MtGSTU14, MtGSTU28, MtGSTU30, MtGSTU34, MtGSTU46 and MtGSTF8 were found to be highly upregulated in response to drought treatment for 24h and 48h. Among the highly stress-responsive MtGST members, MtGSTU17 showed strong affinity towards its conventional substrates reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) with the lowest binding energy of-5.7 kcal/mol and -6.5 kcal/mol, respectively. Furthermore, the substrate-binding site residues of MtGSTU17 were found to be highly conserved. These findings will facilitate the further functional and evolutionary characterization of GST genes in Medicago.

Comprehensive identification and characterization of abiotic stress and hormone responsive glycosyl hydrolase family 1 genes in Medicago truncatula.

ß-glucosidases (BGLUs) hydrolyze the ß-D-glycosidic bond with retention of anomeric configuration. BGLUs were associated with many aspects of plant physiological processes, in particular biotic and abiotic stresses through the activation of phytohormones and defense compounds. However, studies on systematic analysis of the stress- or hormone-responsive BGLU genes in plant are still rare. In this study, total 51 BGLU genes of the glycoside hydrolase family 1 were identified in the genome of the model legume plant Medicago truncatula, and they were classified into five distinct clusters. Sequence alignments revealed several conserved and characteristic motifs among these MtBGLU proteins. Analyses of their putative signal peptides and N-glycosylation site suggested that the majority of MtBGLU members have dual targeting to the vacuole and/or chloroplast. Many regulatory elements possibly related with abiotic stresses and phytohormones were identified in MtBGLU genes. Moreover, Microarray and qPCR analyses showed that these MtBGLU genes exhibited distinct expression patterns in various tissues, and in response to different abiotic stress and hormonal treatments. Notably, MtBGLU21, MtBGLU22, MtBGLU28, and MtBGLU30 in cluster I were dramatically activated by NaCl, PEG, IAA, ABA, SA and GA3 treatments. Collectively, our genome-wide characterization, evolutionary analysis, and expression pattern analysis of MtBGLU genes suggested that BGLU genes play crucial roles in response to various abiotic stresses and hormonal cues in M. truncatula.

Reconstruction of the Evolutionary Histories of UGT Gene Superfamily in Legumes Clarifies the Functional Divergence of Duplicates in Specialized Metabolism.

Plant uridine 5'-diphosphate glycosyltransferases (UGTs) influence the physiochemical properties of several classes of specialized metabolites including triterpenoids via glycosylation. To uncover the evolutionary past of UGTs of soyasaponins (a group of beneficial triterpene glycosides widespread among Leguminosae), the UGT gene superfamily in Medicago truncatula, Glycine max, Phaseolus vulgaris, Lotus japonicus, and Trifolium pratense genomes were systematically mined. A total of 834 nonredundant UGTs were identified and categorized into 98 putative orthologous loci (POLs) using tree-based and graph-based methods. Major key findings in this study were of, (i) 17 POLs represent potential catalysts for triterpene glycosylation in legumes, (ii) UGTs responsible for the addition of second (UGT73P2: galactosyltransferase and UGT73P10: arabinosyltransferase) and third (UGT91H4: rhamnosyltransferase and UGT91H9: glucosyltransferase) sugars of the C-3 sugar chain of soyasaponins were resulted from duplication events occurred before and after the hologalegina-millettoid split, respectively, and followed neofunctionalization in species-/ lineage-specific manner, and (iii) UGTs responsible for the C-22-O glycosylation of group A (arabinosyltransferase) and DDMP saponins (DDMPtransferase) and the second sugar of C-22 sugar chain of group A saponins (UGT73F2: glucosyltransferase) may all share a common ancestor. Our findings showed a way to trace the evolutionary history of UGTs involved in specialized metabolism.

S-adenosylmethionine synthases in plants: Structural characterization of type I and II isoenzymes from Arabidopsis thaliana and Medicago truncatula.

S-adenosylmethionine synthases (MATs) are responsible for production of S-adenosylmethionine, the cofactor essential for various methylation reactions, production of polyamines and phytohormone ethylene, etc. Plants have two distinct MAT types (I and II). This work presents the structural analysis of MATs from Arabidopsis thaliana (AtMAT1 and AtMAT2, both type I) and Medicago truncatula (MtMAT3a, type II), which, unlike most MATs from other domains of life, are dimers where three-domain subunits are sandwiched flat with one another. Although MAT types are very similar, their subunits are differently oriented within the dimer. Structural snapshots along the enzymatic reaction reveal the exact conformation of precatalytic methionine in the active site and show a binding niche, characteristic only for plant MATs, that may serve as a lock of the gate loop. Nevertheless, plants, in contrary to mammals, lack the MAT regulatory subunit, and the regulation of plant MAT activity is still puzzling. Our structures open a possibility of an allosteric activity regulation of type I plant MATs by linear compounds, like polyamines, which would tighten the relationship between S-adenosylmethionine and polyamine biosynthesis.

Analysis of Aldo-Keto Reductase Gene Family and Their Responses to Salt, Drought, and Abscisic Acid Stresses in Medicago truncatula.

Salt and drought stresses are two primary abiotic stresses that inhibit growth and reduce the activity of photosynthetic apparatus in plants. Abscisic acid (ABA) plays a key role in abiotic stress regulation in plants. Some aldo-keto reductases (AKRs) can enhance various abiotic stresses resistance by scavenging cytotoxic aldehydes in some plants. However, there are few comprehensive reports of plant AKR genes and their expression patterns in response to abiotic stresses. In this study, we identified 30 putative AKR genes from Medicago truncatula. The gene characteristics, coding protein motifs, and expression patterns of these MtAKRs were analyzed to explore and identify candidate genes in regulation of salt, drought, and ABA stresses. The phylogenetic analysis result indicated that the 52 AKRs in Medicago truncatula and Arabidopsis thaliana can be divided into three groups and six subgroups. Fifteen AKR genes in M. truncatula were randomly selected from each group or subgroup, to investigate their response to salt (200 mM of NaCl), drought (50 g·L-1 of PEG 6000), and ABA (100 µM) stresses in both leaves and roots. The results suggest that MtAKR1, MtAKR5, MtAKR11, MtAKR14, MtAKR20, and MtAKR29 may play important roles in response to these stresses.

Sinorhizobium meliloti succinylated high-molecular-weight succinoglycan and the Medicago truncatula LysM receptor-like kinase MtLYK10 participate independently in symbiotic infection.

The formation of nitrogen-fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen-fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin-motif receptor-like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild-type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild-type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan-defective strains was achieved by in trans rescue with a Nod factor-deficient S. meliloti mutant. While the Nod factor-deficient strain was always more abundant inside nodules, the succinoglycan-deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.

Distribution and evolution of the serine/aspartate racemase family in plants.

Previous studies have shown that several d-amino acids are widely present in plants, and serine racemase (SerR), which synthesizes d-serine in vivo, has already been identified from three plant species. However, the full picture of the d-amino acid synthesis pathway in plants is not well understood. To clarify the distribution of amino acid racemases in plants, we have cloned, expressed and characterized eight SerR homologous genes from five plant species, including green alga. These SerR homologs exhibited racemase activity towards serine or aspartate and were identified on the basis of their maximum activity as SerR or aspartate racemase (AspR). The plant AspR gene is identified for the first time from Medicago truncatula, Manihot esculenta, Solanum lycopersicum, Sphagnum girgensohnii and Spirogyra pratensis. In addition to the AspR gene, three SerR genes are identified in the former three species. Phylogenetic tree analysis showed that SerR and AspR are widely distributed in plants and form a serine/aspartate racemase family cluster. The catalytic efficiency (kcat/Km) of plant AspRs was more than 100 times higher than that of plant SerRs, suggesting that d-aspartate, as well as d-serine, can be synthesized in vivo by AspR. The amino acid sequence alignment and comparison of the chromosomal gene arrangement have revealed that plant AspR genes independently evolved from SerR in each ancestral lineage of plant species by gene duplication and acquisition of two serine residues at position 150 to 152.

Structural basis of methotrexate and pemetrexed action on serine hydroxymethyltransferases revealed using plant models.

Serine hydroxymethyltransferases (SHMTs) reversibly transform serine into glycine in a reaction accompanied with conversion of tetrahydrofolate (THF) into 5,10-methylene-THF (5,10-meTHF). In vivo, 5,10-meTHF is the main carrier of one-carbon (1C) units, which are utilized for nucleotide biosynthesis and other processes crucial for every living cell, but hyperactivated in overproliferating cells (e.g. cancer tissues). SHMTs are emerging as a promising target for development of new drugs because it appears possible to inhibit growth of cancer cells by cutting off the supply of 5,10-meTHF. Methotrexate (MTX) and pemetrexed (PTX) are two examples of antifolates that have cured many patients over the years but target different enzymes from the folate cycle (mainly dihydrofolate reductase and thymidylate synthase, respectively). Here we show crystal structures of MTX and PTX bound to plant SHMT isozymes from cytosol and mitochondria-human isozymes exist in the same subcellular compartments. We verify inhibition of the studied isozymes by a thorough kinetic analysis. We propose to further exploit antifolate scaffold in development of SHMT inhibitors because it seems likely that especially polyglutamylated PTX inhibits SHMTs in vivo. Structure-based optimization is expected to yield novel antifolates that could potentially be used as chemotherapeutics.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity.

Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31-Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

Two-step d-ononitol epimerization pathway in Medicago truncatula.

Methylated inositol, d-pinitol (3-O-methyl-d-chiro-inositol), is a common constituent in legumes. It is synthesized from myo-inositol in two reactions: the first reaction, catalyzed by myo-inositol-O-methyltransferase (IMT), consists of a transfer of a methyl group from S-adenosylmethionine to myo-inositol with the formation of d-ononitol, while the second reaction, catalyzed by d-ononitol epimerase (OEP), involves epimerization of d-ononitol to d-pinitol. To identify the genes involved in d-pinitol biosynthesis in a model legume Medicago truncatula, we conducted a BLAST search on its genome using soybean IMT cDNA as a query and found putative IMT (MtIMT) gene. Subsequent co-expression analysis performed on publicly available microarray data revealed two potential OEP genes: MtOEPA, encoding an aldo-keto reductase and MtOEPB, encoding a short-chain dehydrogenase. cDNAs of all three genes were cloned and expressed as recombinant proteins in E. coli. In vitro assays confirmed that putative MtIMT enzyme catalyzes methylation of myo-inositol to d-ononitol and showed that MtOEPA enzyme has NAD+ -dependent d-ononitol dehydrogenase activity, while MtOEPB enzyme has NADP+ -dependent d-pinitol dehydrogenase activity. Both enzymes are required for epimerization of d-ononitol to d-pinitol, which occurs in the presence of NAD+ and NADPH. Introduction of MtIMT, MtOEPA, and MtOEPB genes into tobacco plants resulted in production of d-ononitol and d-pinitol in transformants. As this two-step pathway of d-ononitol epimerization is coupled with a transfer of reducing equivalents from NADPH to NAD+ , we speculate that one of the functions of this pathway might be regeneration of NADP+ during drought stress.

Characterization of plant glutamine synthetase S-nitrosation.

The identification of S-nitrosated substrates and their target cysteine residues is a crucial step to understand the signaling functions of nitric oxide (NO) inside the cells. Here, we show that the key nitrogen metabolic enzyme glutamine synthetase (GS) is a S-nitrosation target in Medicago truncatula and characterize the molecular determinants and the effects of this NO-induced modification on different GS isoenzymes. We found that all the four M. truncatula GS isoforms are S-nitrosated, but despite the high percentage of amino acid identity between the four proteins, S-nitrosation only affects the activity of the plastid-located enzymes, leading to inactivation. A biotin-switch/mass spectrometry approach revealed that cytosolic and plastid-located GSs share an S-nitrosation site at a conserved cysteine residue, but the plastidic enzymes contain additional S-nitrosation sites at non-conserved cysteines, which are accountable for enzyme inactivation. By site-directed mutagenesis, we identified Cys369 as the regulatory S-nitrosation site relevant for the catalytic function of the plastid-located GS and an analysis of the structural environment of the SNO-targeted cysteines in cytosolic and plastid-located isoenzymes explains their differential regulation by S-nitrosation and elucidates the mechanistic by which S-nitrosation of Cys369 leads to enzyme inactivation. We also provide evidence that both the cytosolic and plastid-located GSs are endogenously S-nitrosated in leaves and root nodules of M. truncatula, supporting a physiological meaning for S-nitrosation. Taken together, these results provide new insights into the molecular details of the differential regulation of individual GS isoenzymes by NO-derived molecules and open new paths to explore the biological significance of the NO-mediated regulation of this essential metabolic enzyme.

Medicago TERPENE SYNTHASE 10 Is Involved in Defense Against an Oomycete Root Pathogen.

In nature, plants interact with numerous beneficial or pathogenic soil-borne microorganisms. Plants have developed various defense strategies to expel pathogenic microbes, some of which function soon after pathogen infection. We used Medicago truncatula and its oomycete pathogen Aphanomyces euteiches to elucidate early responses of the infected root. A. euteiches causes root rot disease in legumes and is a limiting factor in legume production. Transcript profiling of seedlings and adult plant roots inoculated with A. euteiches zoospores for 2 h revealed specific upregulation of a gene encoding a putative sesquiterpene synthase (M. truncatula TERPENE SYNTHASE 10 [MtTPS10]) in both developmental stages. MtTPS10 was specifically expressed in roots upon oomycete infection. Heterologous expression of MtTPS10 in yeast led to production of a blend of sesquiterpenes and sesquiterpene alcohols, with NMR identifying a major peak corresponding to himalachol. Moreover, plants carrying a tobacco (Nicotiana tabacum) retrotransposon Tnt1 insertion in MtTPS10 lacked the emission of sesquiterpenes upon A. euteiches infection, supporting the assumption that the identified gene encodes a multiproduct sesquiterpene synthase. Mttps10 plants and plants with reduced MtTPS10 transcript levels created by expression of an MtTPS10-artificial microRNA in roots were more susceptible to A. euteiches infection than were the corresponding wild-type plants and roots transformed with the empty vector, respectively. Sesquiterpenes produced by expression of MtTPS10 in yeast also inhibited mycelial growth and A. euteiches zoospore germination. These data suggest that sesquiterpene production in roots by MtTPS10 plays a previously unrecognized role in the defense response of M. truncatula against A. euteiches.