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ABSTRACT Posttranslational tyrosine sulfation of peptides and proteins is catalysed by tyrosylprotein sulfotransferases (TPSTs). InArabidopsis, tyrosine sulfation is essential for the activities of peptide hormones, such as phytosulfokine (PSK) and root meristem growth factor (RGF). Here, we identified a TPST‐encoding gene,MtTPST, from model legumeMedicago truncatula.MtTPSTexpression was detected in all organs, with the highest level in root nodules. Apromoter:GUSassay revealed thatMtTPSTwas highly expressed in the root apical meristem, nodule primordium and nodule apical meristem. The loss‐of‐function mutantmttpstexhibited a stunted phenotype with short roots and reduced nodule number and size. Application of both of the sulfated peptides PSK and RGF3 partially restored the defective root length ofmttpst. The reduction in symbiotic nodulation inmttpstwas partially recovered by treatment with sulfated PSK peptide. MtTPST‐PSK module functions downstream of the Nod factor signalling to promote nodule initiation via regulating accumulation and/or signalling of cytokinin and auxin. Additionally, the small‐nodule phenotype ofmttpst, which resulted from decreased apical meristematic activity, was partially complemented by sulfated RGF3 treatment. Together, these results demonstrate that MtTPST, through its substrates PSK, RGF3 and other sulfated peptide(s), positively regulates nodule development and root growth. 

Abstract Long non-coding RNAs (lncRNAs) are abundant in plants, however, their regulatory roles remain unclear in most biological processes, such as response in salinity stress which is harm to plant production. Here we show a lncRNA inMedicago truncatulaidentified from salt-treated Medicagotruncatulais important for salinity tolerance. We name the lncRNALAL,LncRNAANTISENSEtoM. truncatulaLIGHT-HARVESTING CHLOROPHYLL A/B BINDING(MtLHCB)genes. LALis an antisense to four consecutiveMtLHCBgenes on chromosome 6. In salt-treatedM. truncatula,LALis suppressed in an early stage but induced later; this pattern is opposite to that of the fourMtLHCBs. Thelalmutants show enhanced salinity tolerance, while overexpressingLALdisrupts this superior tolerance in thelalbackground, which indicates its regulatory role in salinity response. The regulatory role ofLALonMtLHCB1.4is further verified by transient co-expression ofLALandMtLHCB1.4-GFPin tobacco leaves, in which the cleavage ofMtLHCB1.4and production of secondary interfering RNA is identified. This work demonstrates a lncRNA,LAL, functioning as a regulator that fine-tunes salinity tolerance via regulatingMtLHCB1s’ expression inM. truncatula. 

Abstract Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here, we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the endoplasmic reticulum (ER) exit sites and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12, and Sec24, and participates in coat protein complex II vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway. 

Abstract During nutrient scarcity, plants can adapt their developmental strategy to maximize their chance of survival. Such plasticity in development is underpinned by hormonal regulation, which mediates the relationship between environmental cues and developmental outputs. In legumes, endosymbiosis with nitrogen-fixing bacteria (rhizobia) is a key adaptation for supplying the plant with nitrogen in the form of ammonium. Rhizobia are housed in lateral root-derived organs termed nodules that maintain an environment conducive to Nitrogenase in these bacteria. Several phytohormones are important for regulating the formation of nodules, with both positive and negative roles proposed for gibberellin (GA). In this study, we determine the cellular location and function of bioactive GA during nodule organogenesis using a genetically encoded second-generation GA biosensor, GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula. We find endogenous bioactive GA accumulates locally at the site of nodule primordia, increasing dramatically in the cortical cell layers, persisting through cell divisions, and maintaining accumulation in the mature nodule meristem. We show, through misexpression of GA-catabolic enzymes that suppress GA accumulation, that GA acts as a positive regulator of nodule growth and development. Furthermore, increasing or decreasing GA through perturbation of biosynthesis gene expression can increase or decrease the size of nodules, respectively. This is unique from lateral root formation, a developmental program that shares common organogenesis regulators. We link GA to a wider gene regulatory program by showing that nodule-identity genes induce and sustain GA accumulation necessary for proper nodule formation. 

Summary Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation ofMedicago truncatula.We biochemically characterized five flavonoid‐O‐methyltransferases (OMTs) and a lux‐basednodgene reporter was used to investigate the response ofSinorhizobium medicaeNodD1 to various flavonoids.We found that chalcone‐OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4′‐dihydroxy‐2′‐methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore,chomt1andomt2showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2.We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important fornodgene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation inM. truncatulacompared to soybean, supporting a role for flavonoids in rhizobial host range. 

Abstract The adjustment of cellular redox homeostasis is essential in when responding to environmental perturbations, and the mechanism by which cells distinguish between normal and oxidized states through sensors is also important. In this study, we found thatacyl-protein thioesterase 1(APT1) is a redox sensor. Under normal physiological conditions, APT1 exists as a monomer throughS-glutathionylation at C20, C22 and C37, which inhibits its enzymatic activity. Under oxidative conditions, APT1 senses the oxidative signal and is tetramerized, which makes it functional. Tetrameric APT1 depalmitoylates S-acetylated NAC (NACsa), and NACsa relocates to the nucleus, increases the cellular glutathione/oxidized glutathione (GSH/GSSG) ratio through the upregulation ofglyoxalase Iexpression, and resists oxidative stress. When oxidative stress is alleviated, APT1 is found in monomeric form. Here, we describe a mechanism through which APT1 mediates a fine-tuned and balanced intracellular redox system in plant defence responses to biotic and abiotic stresses and provide insights into the design of stress-resistant crops. 

Abstract Vanadium (V) pollution potentially threatens human health. Here, it is found thatnsp1andnsp2,Rhizobiumsymbiosis defective mutants ofMedicago truncatula, are sensitive to V. Concentrations of phosphorus (P), iron (Fe), and sulfur (S) with V are negatively correlated in the shoots of wild‐type R108, but not in mutantnsp1andnsp2shoots. Mutations in the P transporterPHT1,PHO1, andVPTfamilies, Fe transporterIRT1, and S transporterSULTR1/3/4family confer varying degrees of V tolerance on plants. Among these gene families,MtPT1,MtZIP6,MtZIP9, andMtSULTR1; 1in R108 roots are significantly inhibited by V stress, whileMtPHO1; 2,MtVPT2, andMtVPT3are significantly induced. Overexpression ofArabidopsis thaliana VPT1orM. truncatula MtVPT3increases plant V tolerance. However, the response of these genes to V is weakened innsp1ornsp2and influenced by soil microorganisms. Mutations inNSPsreduce rhizobacterial diversity under V stress and simplify the V‐responsive operational taxonomic unit modules in co‐occurrence networks. Furthermore, R108 recruits more beneficial rhizobacteria related to V, P, Fe, and S than doesnsp1ornsp2. Thus, NSPs can modulate the accumulation and tolerance of legumes to V through P, Fe, and S transporters, ion homeostasis, and rhizobacterial community responses. 

Abstract Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class ⅡKNOTTED‐like homeobox (KNOXII) genes in the model leguminous plantMedicago truncatula. Phenotypic and genetic analyses suggest thatMtKNOX4,5are able to repress leaflet formation, whileMtKNOX3,9,10are not involved in this developmental process. Further investigations have shown that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXⅠ‐mediated CK biosynthesis. Additionally, two boundary genes,FUSED COMPOUND LEAF1(orthologue ofArabidopsisClass MKNOX) andNO APICAL MERISTEM(orthologue ofArabidopsis CUP‐SHAPED COTYLEDON), are necessary for MtKNOX4‐mediated compound leaf formation. These findings suggest, that among the members of MtKNOXⅡ, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves inM. truncatula. 

SUMMARY Although vacuolar phosphate transporters (VPTs) are essential for plant phosphorus adaptation, their role inRhizobium–legume symbiosis is unclear. In this study, homologous genes ofVPT1(MtVPTs)were identified inMedicago truncatulato assess their roles inRhizobium–legume symbiosis and phosphorus adaptation.MtVPT2andMtVPT3mainly positively responded to low and high phosphate, respectively. However, bothmtvpt2andmtvpt3mutants displayed shoot phenotypes with high phosphate sensitivity and low phosphate tolerance. The root‐to‐shoot phosphate transfer efficiency was significantly enhanced inmtvpt3but weakened inmtvpt2, accompanied by lower and higher root cytosolic inorganic phosphate (Pi) concentration, respectively. Low phosphate inducedMtVPT2andMtVPT3expressions in nodules.MtVPT2andMtVPT3mutations markedly reduced the nodule number and nitrogenase activity under different phosphate conditions. Cytosolic Pi concentration in nodules was significantly lower inmtvpt2andmtvpt3than in the wildtype, especially in tissues near the base of nodules, probably due to inhibition of long‐distance Pi transport and cytosolic Pi supply. Also,mtvpt2andmtvpt3could not maintain a stable cytosolic Pi level in the nodule fixation zone as the wildtype under low phosphate stress. These findings show thatMtVPT2and MtVPT3modulate phosphorus adaptation and rhizobia–legume symbiosis, possibly by regulating long‐distance Pi transport. 

SUMMARY Medicago truncatulais a model legume for fundamental research on legume biology and symbiotic nitrogen fixation.Tnt1, a retrotransposon from tobacco, was used to generate insertion mutants inM. truncatulaR108. Approximately 21 000 insertion lines have been generated and publicly available.Tnt1retro‐transposition event occurs during somatic embryogenesis (SE), a pivotal process that triggers massive methylation changes. We studied the SE ofM. truncatulaR108 using leaf explants and explored the dynamic shifts in the methylation landscape from leaf explants to callus formation and finally embryogenesis. Higher cytosine methylation in all three contexts of CG, CHG, and CHH patterns was observed during SE compared to the controls. Higher methylation patterns were observed in assumed promoter regions (~2‐kb upstream regions of transcription start site) of the genes, while lowest was recorded in the untranslated regions. Differentially methylated promoter region analysis showed a higher CHH methylation in embryogenesis tissue samples when compared to CG and CHG methylation. Strong correlation (89.71%) was identified between the differentially methylated regions (DMRs) and the site ofTnt1insertions inM. truncatulaR108 and stronger hypermethylation of genes correlated with higher number ofTnt1insertions in all contexts of CG, CHG, and CHH methylation. Gene ontology enrichment and KEGG pathway enrichment analysis identified genes and pathways enriched in the signal peptide processing, ATP hydrolysis, RNA polymerase activity, transport, secondary metabolites, and nitrogen metabolism pathways. Combined gene expression analysis and methylation profiling showed an inverse relationship between methylation in the DMRs (regions spanning genes) and the expression of genes. Our results show that a dynamic shift in methylation happens during the SE process in the context of CG, CHH and CHG methylation, and theTnt1retrotransposition correlates with the hyperactive methylation regions.