Search for: All records

Medicago (Medicago truncatula) establishes a symbiosis with the rhizobia Sinorhizobium sp, resulting in the formation of nodules where the bacteria fix atmospheric nitrogen. The loss of immunity repression or early senescence activation compromises symbiont survival and leads to the formation of nonfunctional nodules (fix−). Despite many studies exploring an overlap between immunity and senescence responses outside the nodule context, the relationship between these processes in the nodule remains poorly understood. To investigate this phenomenon, we selected and characterized three Medicago mutants developing fix− nodules and showing senescence responses. Analysis of specific defense (PATHOGENESIS-RELATED PROTEIN) or senescence (CYSTEINE PROTEASE) marker expression demonstrated that senescence and immunity seem to be antagonistic in fix− nodules. The growth of senescence mutants on non-sterile (sand/perlite) substrate instead of sterile in vitro conditions decreased nodule senescence and enhanced defense, indicating that environment can affect the immunity/senescence balance. The application of wounding stress on wild-type (WT) fix+ nodules led to the death of intracellular rhizobia and associated with co-stimulation of defense and senescence markers, indicating that in fix+ nodules the relationship between the two processes switches from opposite to synergistic to control symbiont survival during response to the stress. Our data show that the immune response in stressed WT nodules is linked to the repression of DEFECTIVE IN NITROGEN FIXATION 2 (DNF2), Symbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (SymCRK), and REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD), key genes involved in symbiotic immunity suppression. This study provides insight to understand the links between senescence and immunity in Medicago nodules.

 

Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. Medicago truncatula is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, M. truncatula reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. M. truncatula R108 is more attractive for genetic studies due to its high transformation efficiency and Tnt1-insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for M. truncatula cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the Tnt1 insertion landscape on this reference assembly presents an important resource for M. truncatula functional genomics by supporting efficient mutant gene identification in Tnt1 insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future. 

Cu⁺‐chaperones are a diverse group of proteins that allocate Cu⁺ions to specific copper proteins, creating different copper pools targeted to specific physiological processes.

Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required.

MtNCC1 is a nodule‐specific Cu⁺‐chaperone encoded in theMedicago truncatulagenome, with a N‐terminus Atx1‐like domain that can bind Cu⁺with picomolar affinities. MtNCC1 is able to interact with nodule‐specific Cu⁺‐importer MtCOPT1.MtNCC1is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation,ncc1mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper‐dependent cytochromecoxidase activity.

A subset of the copper proteome is also affected in thencc1mutant nodules. Many of these proteins can be pulled down when using a Cu⁺‐loaded N‐terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper‐dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

 

Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants. Floral organ identity is controlled by the well-known ABC model, which has been generalized to many flowering plants. Here, we report a previously uncharacterized MYB-like gene,AGAMOUS-LIKE FLOWER(AGLF), involved in flower development in the model legumeMedicago truncatula. Loss-of-function ofAGLFresults in flowers with stamens and carpel transformed into extra whorls of petals and sepals. Compared with the loss-of-function mutant of the class C geneAGAMOUS(MtAG) inM. truncatula, the defects in floral organ identity are similar betweenaglfandmtag, but the floral indeterminacy is enhanced in theaglfmutant. Knockout ofAGLFin the mutants of the class A geneMtAP1or the class B geneMtPIleads to an addition of a loss-of-C-function phenotype, reflecting a conventional relationship ofAGLFwith the canonical A and B genes. Furthermore, we demonstrate thatAGLFactivatesMtAGin transcriptional levels in control of floral organ identity. These data shed light on the conserved and diverged molecular mechanisms that control flower development and morphology among plant species.

 

Flowering of the reference legumeMedicago truncatulais promoted by winter cold (vernalization) followed by long‐day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacksFLCandCO, key regulators of Arabidopsis VLD flowering.Most plants have twoINHIBITOR OF GROWTH(ING) genes (ING1andING2), encoding proteins with an ING domain with two anti‐parallel alpha‐helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described.In Medicago,Mting1gene‐edited mutants developed and flowered normally, but anMting2‐1 Tnt1insertion mutant and gene‐editedMting2mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels.Mting2mutants had reduced expression of activators of flowering, including theFT‐like geneMtFTa1, and increased expression of the candidate repressorMtTFL1c, consistent with the delayed flowering of the mutant.MtING2overexpression complementedMting2‐1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weaklyin vitro, but analysis of gene‐edited mutants indicated that it was dispensable to MtING2 function in wild‐type plants. RNA sequencing experiments indicated that >7000 genes are mis‐expressed in theMting2‐1mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP‐seq analysis identified >5000 novel H3K4me3 locations in the genome ofMting2‐1mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plantING2gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism.

 

l‐Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4‐hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrA_a), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrA_p), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterizedTnt1‐transposon mutants ofMtPDH1(pdh1) inMedicago truncatulato investigate PDH function. The pdh1mutants lackedPDHtranscript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence thatMtPDH1is responsible for the PDH activity detected inM. truncatula. Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity ofpdh1 mutants were not significantly reduced compared with wild‐type (Wt) during symbiosis with nitrogen‐fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume‐specific specialized metabolism.

 

Patterned leaf coloration in plants generates remarkable diversity in nature, but the underlying mechanisms remain largely unclear.

Here, usingMedicago truncatulaleaf marking as a model, we show that the classicM. truncatulaleaf anthocyanin spot trait depends on two R2R3 MYB paralogous regulators, RED HEART1 (RH1) and RH2.

RH1 mainly functions as an anthocyanin biosynthesis activator that specifically determines leaf marking formation depending on its C‐terminal activation motif. RH1 physically interacts with theM. truncatulabHLH protein MtTT8 and the WDR family member MtWD40‐1, and this interaction facilitates RH1 function in leaf anthocyanin marking formation. RH2 has lost transcriptional activation activity, due to a divergent C‐terminal domain, but retains the ability to interact with the same partners, MtTT8 and MtWD40‐1, as RH1, thereby acting as a competitor in the regulatory complex and exerting opposite effects. Moreover, our results demonstrate that RH1 can activate its own expression and that RH2‐mediated competition can repressRH1expression.

Our findings reveal the molecular mechanism of the antagonistic gene paralogs RH1 and RH2 in determining anthocyanin leaf markings inM. truncatula, providing a multidimensional paralogous–antagonistic regulatory paradigm for fine‐tuning patterned pigmentation.

 

Yellow Stripe‐Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non‐proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 inMedicago truncatulanodules. MtYSL7, which encodes a plasma membrane‐bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides.M.truncatulatransposon insertion mutants with decreased expression ofMtYSL7had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation.