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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. 

Leaves are the primary harvest portion in forage crops such as alfalfa (Medicago sativa). Delaying leaf senescence is an effective strategy to improve forage biomass production and quality. In this study, we employed transcriptome sequencing to analyze the transcriptional changes and identify key senescence-associated genes under age-dependent leaf senescence in Medicago truncatula, a legume forage model plant. Through comparing the obtained expression data at different time points, we obtained 1057 differentially expressed genes, with 108 consistently up-regulated genes across leaf growth and senescence. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that the 108 SAGs mainly related to protein processing, nitrogen metabolism, amino acid metabolism, RNA degradation and plant hormone signal transduction. Among the 108 SAGs, seven transcription factors were identified in which a novel bZIP transcription factor MtbZIP60 was proved to inhibit leaf senescence. MtbZIP60 encodes a nuclear-localized protein and possesses transactivation activity. Further study demonstrated MtbZIP60 could associate with MtWRKY40, both of which exhibited an up-regulated expression pattern during leaf senescence, indicating their crucial roles in the regulation of leaf senescence. Our findings help elucidate the molecular mechanisms of leaf senescence in M. truncatula and provide candidates for the genetic improvement of forage crops, with a focus on regulating leaf senescence. 

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. 

Summary Establishment of symbiosis between plants and arbuscular mycorrhizal (AM) fungi depends on fungal chitooligosaccharides (COs) and lipo‐chitooligosaccharides (LCOs). The latter are also produced by nitrogen‐fixing rhizobia to induce nodules on leguminous roots. However, host enzymes regulating structure and levels of these signals remain largely unknown.Here, we analyzed the expression of a β‐N‐acetylhexosaminidase gene ofMedicago truncatula(MtHEXO2) and biochemically characterized the enzyme. Mutant analysis was performed to study the role ofMtHEXO2during symbiosis.We found that expression ofMtHEXO2is associated with AM symbiosis and nodulation.MtHEXO2expression in the rhizodermis was upregulated in response to applied chitotetraose, chitoheptaose, and LCOs.M. truncatulamutants deficient in symbiotic signaling did not show induction ofMtHEXO2. Subcellular localization analysis indicated that MtHEXO2 is an extracellular protein. Biochemical analysis showed that recombinant MtHEXO2 does not cleave LCOs but can degrade COs intoN‐acetylglucosamine (GlcNAc).Hexo2mutants exhibited reduced colonization by AM fungi; however, nodulation was not affected inhexo2mutants.In conclusion, we identified an enzyme, which inactivates COs and promotes the AM symbiosis. We hypothesize that GlcNAc produced by MtHEXO2 may function as a secondary symbiotic signal. 

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.