The LysM Receptor-Like Kinase SlLYK10 Controls Lipochitooligosaccharide Signaling in Inner Cell Layers of Tomato Roots.

Establishment of arbuscular mycorrhiza relies on a plant signaling pathway that can be activated by fungal chitinic signals such as short-chain chitooligosaccharides and lipo-chitooligosaccharides (LCOs). The tomato LysM receptor-like kinase SlLYK10 has high affinity for LCOs and is involved in root colonization by arbuscular mycorrhizal fungi (AMF); however, its role in LCO responses has not yet been studied. Here, we show that SlLYK10 proteins produced by the Sllyk10-1 and Sllyk10-2 mutant alleles, which both cause decreases in AMF colonization and carry mutations in LysM1 and 2, respectively, have similar LCO-binding affinities compared to the WT SlLYK10. However, the mutant forms were no longer able to induce cell death in Nicotiana benthamiana when co-expressed with MtLYK3, a Medicago truncatula LCO co-receptor, while they physically interacted with MtLYK3 in co-purification experiments. This suggests that the LysM mutations affect the ability of SlLYK10 to trigger signaling through a potential co-receptor rather than its ability to bind LCOs. Interestingly, tomato lines that contain a calcium (Ca2+) concentration reporter [genetically encoded Ca2+ indicators (GECO)], showed Ca2+ spiking in response to LCO applications, but this occurred only in inner cell layers of the roots, while short-chain chitooligosaccharides also induced Ca2+ spiking in the epidermis. Moreover, LCO-induced Ca2+ spiking was decreased in Sllyk10-1*GECO plants, suggesting that the decrease in AMF colonization in Sllyk10-1 is due to abnormal LCO signaling.

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.

Medicago truncatula ß-glucosidase 17 contributes to drought and salt tolerance through antioxidant flavonoid accumulation.

Flavonoids are usually present in forms of glucosides in plants, which could be catabolized by ß-glucosidase (BGLU) to form their corresponding flavonoid aglycones. In this study, we isolated three abiotic-responsive BGLU genes (MtBGLU17, MtBGLU21 and MtBGLU22) from Medicago truncatula, and found only the recombinant MtBGLU17 protein could catalyse the hydrolysis of flavonoid glycosides. The recombinant MtBGLU17 protein is active towards a variety of flavonoid glucosides, including glucosides of flavones (apigenin and luteolin), flavonols (kaempferol and quercetin), isoflavones (genistein and daidzein) and flavanone (naringenin). In particular, the recombinant MtBGLU17 protein preferentially hydrolyses flavonoid-7-O-glucosides over their corresponding 3-O-glucosides. The content of luteoin-7-O-glucoside was reduced in the MtBGLU17 overexpression plants but increased in the Tnt-1 insertional mutant lines, whereas luteoin content was increased in the MtBGLU17 overexpression plants but reduced in the Tnt-1 insertional mutant lines. Under drought and salt (NaCl) treatment, the MtBGLU17 overexpression lines showed relatively higher DPPH content, and higher CAT and SOD activity than the wild type control. These results indicated that overexpression lines of MtBGLU17 possess higher antioxidant activity and thus confer drought and salt tolerance, implying MtBGLU17 could be potentially used as a candidate gene to improve plant abiotic stress tolerance.

NODULATION TRIO in Medicago truncatula: Unveiling the redundant roles of MtLYK2, MtLYK3, and MtLYK2bis.

Three Medicago truncatula LysM domain receptor kinases have redundant functions in nodulation, with multiple specificities mediating both entry and signaling responses and with distinct contributions to nodulation likely resulting from differing transcription patterns.

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.

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.

Role of the Nod Factor Hydrolase MtNFH1 in Regulating Nod Factor Levels during Rhizobial Infection and in Mature Nodules of Medicago truncatula.

Establishment of symbiosis between legumes and nitrogen-fixing rhizobia depends on bacterial Nod factors (NFs) that trigger symbiosis-related NF signaling in host plants. NFs are modified oligosaccharides of chitin with a fatty acid moiety. NFs can be cleaved and inactivated by host enzymes, such as MtNFH1 (MEDICAGO TRUNCATULA NOD FACTOR HYDROLASE1). In contrast to related chitinases, MtNFH1 hydrolyzes neither chitin nor chitin fragments, indicating a high cleavage preference for NFs. Here, we provide evidence for a role of MtNFH1 in the symbiosis with Sinorhizobium meliloti Upon rhizobial inoculation, MtNFH1 accumulated at the curled tip of root hairs, in the so-called infection chamber. Mutant analysis revealed that lack of MtNFH1 delayed rhizobial root hair infection, suggesting that excess amounts of NFs negatively affect the initiation of infection threads. MtNFH1 deficiency resulted in nodule hypertrophy and abnormal nodule branching of young nodules. Nodule branching was also stimulated in plants expressing MtNFH1 driven by a tandem CaMV 35S promoter and plants inoculated by a NF-overproducing S. meliloti strain. We suggest that fine-tuning of NF levels by MtNFH1 is necessary for optimal root hair infection as well as for NF-regulated growth of mature nodules.

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.

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.

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.

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.

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.

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.

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.

Digalactosyldiacylglycerol Synthase Gene MtDGD1 Plays an Essential Role in Nodule Development and Nitrogen Fixation.

Little is known about the genes participating in digalactosyldiacylglycerol (DGDG) synthesis during nodule symbiosis. Here, we identified full-length MtDGD1, a synthase of DGDG, and characterized its effect on symbiotic nitrogen fixation in Medicago truncatula. Immunofluorescence and immunoelectron microscopy showed that MtDGD1 was located on the symbiosome membranes in the infected cells. ß-Glucuronidase histochemical staining revealed that MtDGD1 was highly expressed in the infection zone of young nodules as well as in the whole mature nodules. Compared with the control, MtDGD1-RNA interference transgenic plants exhibited significant decreases in nodule number, symbiotic nitrogen fixation activity, and DGDG abundance in the nodules, as well as abnormal nodule and symbiosome development. Overexpression of MtDGD1 resulted in enhancement of nodule number and nitrogen fixation activity. In response to phosphorus starvation, the MtDGD1 expression level was substantially upregulated and the abundance of nonphospholipid DGDG was significantly increased in the roots and nodules, accompanied by corresponding decreases in the abundance of phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Overall, our results indicate that DGD1 contributes to effective nodule organogenesis and nitrogen fixation by affecting the synthesis and content of DGDG during symbiosis.

Engineering the unicellular alga Phaeodactylum tricornutum for high-value plant triterpenoid production.

Plant triterpenoids constitute a diverse class of organic compounds that play a major role in development, plant defence and environmental interaction. Several triterpenes have demonstrated potential as pharmaceuticals. One example is betulin, which has shown promise as a pharmaceutical precursor for the treatment of certain cancers and HIV. Major challenges for triterpenoid commercialization include their low production levels and their cost-effective purification from the complex mixtures present in their natural hosts. Therefore, attempts to produce these compounds in industrially relevant microbial systems such as bacteria and yeasts have attracted great interest. Here, we report the production of the triterpenes betulin and its precursor lupeol in the photosynthetic diatom Phaeodactylum tricornutum, a unicellular eukaryotic alga. This was achieved by introducing three plant enzymes in the microalga: a Lotus japonicus oxidosqualene cyclase and a Medicago truncatula cytochrome P450 along with its native reductase. The introduction of the L. japonicus oxidosqualene cyclase perturbed the mRNA expression levels of the native mevalonate and sterol biosynthesis pathway. The best performing strains were selected and grown in a 550-L pilot-scale photobioreactor facility. To our knowledge, this is the most extensive pathway engineering undertaken in a diatom and the first time that a sapogenin has been artificially produced in a microalga, demonstrating the feasibility of the photo-bio-production of more complex high-value, metabolites in microalgae.

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.

The protein quality control system manages plant defence compound synthesis.

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Guarding the gateway to histidine biosynthesis in plants: Medicago truncatula ATP-phosphoribosyltransferase in relaxed and tense states.

In the first committed step of histidine biosynthesis, adenosine 5'-triphosphate (ATP) and 5-phosphoribosyl-α1-pyrophosphate (PRPP), in the presence of ATP phosphoribosyltransferase (ATP-PRT, EC 2.4.2.17), yield phosphoribosyl-ATP. ATP-PRTs are subject to feedback inhibition by histidine that allosterically binds between the regulatory domains. Histidine biosynthetic pathways of bacteria, lower eukaryotes, and plants are considered promising targets for the design of antibiotics, antifungal agents, and herbicides because higher organisms are histidine heterotrophs. Plant ATP-PRTs are similar to one of the two types of their bacterial counterparts, the long-type ATP-PRTs. A biochemical and structural study of ATP-PRT from the model legume plant, Medicago truncatula (MedtrATP-PRT1) is reported herein. Two crystal structures, presenting homohexameric MedtrATP-PRT1 in its relaxed (R-) and histidine-bound, tense (T-) states allowed to observe key features of the enzyme and provided the first structural insights into an ATP-PRT from a eukaryotic organism. In particular, they show pronounced conformational reorganizations during R-state to T-state transition that involves substantial movements of domains. This rearrangement requires a trans- to cis- switch of a peptide backbone within the hinge region of MedtrATP-PRT1. A C-terminal α-helix, absent in bacteria, reinforces the hinge that is constituted by two peptide strands. As a result, conformations of the R- and T-states are significantly different from the corresponding states of prokaryotic enzymes with known 3-D structures. Finally, adenosine 5'-monophosphate (AMP) bound at the active site is consistent with a competitive (and synergistic with histidine) nature of AMP inhibition.