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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 most significant parts of forage crops such as alfalfa. Senescence is the terminal stage of leaf development and is controlled by an integrated myriad of endogenous signals and environmental stimuli. WRKY transcription factors (TFs) play essential roles in regulating leaf senescence; however, only a few studies on the analysis and identification of the WRKY TF family in Medicago Sativa have been reported. In this study, we identified 198 WRKY family members from the alfalfa (M. sativa L.) cultivar ’XinjiangDaye’ using phylogenetic analysis and categorized them into three subfamilies, Groups I, II, and III, based on their structural characteristics. Group II members were further divided into five subclasses. In addition, several hormone- and stress-related cis-acting elements were identified in the promoter regions of MsWRKYs. Furthermore, 14 aging-related MsWRKYs genes from a previous transcriptome in our laboratory were selected for RT-qPCR validation of their expression patterns, and subsequently cloned for overexpression examination. Finally, MsWRKY5, MsWRKY66, MsWRKY92, and MsWRKY141 were confirmed to cause leaf yellowing in Nicotiana benthaminana using a transient expression system. Our findings lay a groundwork for further studies on the mechanism of M. sativa leaf aging and for the creation of new germplasm resources. 

Seed physical dormancy (hard-seededness) is an interesting ecological phenomenon and important agronomic trait. The loss of seed coat impermeability/hard-seededness is a key target trait during the domestication of leguminous crops which allows seeds to germinate rapidly and uniformly. In this study, we examined the mutation of quantitative trait locus (QTL) genes, GmHs1-1 and GmqHS1, in 18 wild soybean (G. soja) and 23 cultivated soybean (G. max) accessions. The sequencing results indicate that a G-to-T substitution in GmqHS1 and a C-to-T substitution in GmHs1-1 occurred in all 23 cultivated soybean accessions but not in any of the 18 wild soybean accessions. The mutations in the two genes led to increased seed coat permeability in cultivated soybean. Therefore, we provide evidence that two genes, GmHs1-1 and GmqHS1, simultaneously contribute to the domestication of hard-seededness in soybeans. This finding is of great significance for genetic analysis and improved utilization of the soybean hard-seededness trait. 

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. 

Physical dormancy of seeds is a form of dormancy due to the presence of an impermeable seed coat layer, and it represents a feature for plants to adapt to environmental changes over an extended period of phylogenetic evolution. However, in agricultural practice, physical dormancy is problematic. because it prevents timely and uniform seed germination. Therefore, physical dormancy is an important agronomical trait to target in breeding and domestication, especially for many leguminous crops. Compared to the well-characterized physiological dormancy, research progress on physical dormancy at the molecular level has been limited until recent years, due to the lack of suitable research materials. This review focuses on the structure of seed coat, factors affecting physical dormancy, genes controlling physical dormancy, and plants suitable for studying physical dormancy at the molecular level. Our goal is to provide a plethora of information for further molecular research on physical dormancy. 

Leaves are a key forage part for livestock, and the aging of leaves affects forage biomass and quality. Preventing or delaying premature leaf senescence leads to an increase in pasture biomass accumulation and an improvement in alfalfa quality. NAC transcription factors have been reported to affect plant growth and abiotic stress responses. In this study, 48 NAC genes potentially associated with leaf senescence were identified in alfalfa under dark or salt stress conditions. A phylogenetic analysis divided MsNACs into six subgroups based on similar gene structure and conserved motif. These MsNACs were unevenly distributed in 26 alfalfa chromosomes. The results of the collinearity analysis show that all of the MsNACs were involved in gene duplication. Some cis-acting elements related to hormones and stress were screened in the 2-kb promoter regions of MsNACs. Nine of the MsNAC genes were subjected to qRT-PCR to quantify their expression and Agrobacterium-mediated transient expression to verify their functions. The results indicate that Ms.gene031485, Ms.gene032313, Ms.gene08494, and Ms.gene77666 might be key NAC genes involved in alfalfa leaf senescence. Our findings extend the understanding of the regulatory function of MsNACs in leaf senescence. 

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. 

Lipoxygenase (LOX) is associated with responses to plant hormones, environmental stresses, and signaling substances. Methyl jasmonate (MeJA) treatment triggers the production of LOX, polyphenol oxidase, and protease inhibitors in various plants, producing resistance to herbivory. To examine the response of MtLOX24 to MeJA, the phenotypic and physiological changes in Medicago truncatula MtLOX24 overexpression and lox mutant plants were investigated. Additionally, wild-type R108, the MtLOX24-overexpressing line L4, and the mutant lox-1 were utilized as experimental materials to characterize the differentially expressed genes (DEGs) and metabolic pathways in response to MeJA. The results indicate that after treatment with 200 µM of MeJA, the damage in the mutants lox-1 and lox-2 was more serious than in the overexpressing lines L4 and L6, with more significant leaf wilting, yellowing, and oxidative damage in lox-1 and lox-2. Exogenous application of MeJA induced H2O2 production and POD activity but reduced CAT activity in the lox mutants. Transcriptome analysis revealed 10,238 DEGs in six libraries of normal-growing groups (cR108, cL4, and clox1) and MeJA-treated groups (R108, L4, and lox1). GO and KEGG functional enrichment analysis demonstrated that under normal growth conditions, the DEGs between the cL4 vs. cR108 and the clox-1 vs. cR108 groups were primarily enriched in signaling pathways such as plant–pathogen interactions, flavonoid biosynthesis, plant hormone signal transduction, the MAPK signaling pathway, and glutathione metabolism. The DEGs of the R108 vs. cR108 and L4 vs. cL4 groups after MeJA treatment were mainly enriched in glutathione metabolism, phenylpropanoid biosynthesis, the MAPK signaling pathway, circadian rhythm, and α-linolenic acid metabolism. Among them, under normal growth conditions, genes like PTI5, PR1, HSPs, PALs, CAD, CCoAOMT, and CYPs showed significant differences between L4 and the wild type, suggesting that the expression of these genes is impacted by MtLOX24 overexpression. CDPKs, CaMCMLs, IFS, JAZ, and other genes were also significantly different between L4 and the wild type upon MeJA treatment, suggesting that they might be important genes involved in JA signaling. This study provides a reference for the study of the response mechanism of MtLOX24 under MeJA signaling.