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Medicago truncatula is a model legume for fundamental research on legume biology and symbiotic nitrogen fixation. Tnt1, a retrotransposon from tobacco, was used to generate insertion mutants in M. truncatula R108. Approximately 21 000 insertion lines have been generated and publicly available. Tnt1 retro-transposition event occurs during somatic embryogenesis (SE), a pivotal process that triggers massive methylation changes. We studied the SE of M. truncatula R108 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 of Tnt1 insertions in M. truncatula R108 and stronger hypermethylation of genes correlated with higher number of Tnt1 insertions 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 the Tnt1 retrotransposition correlates with the hyperactive methylation regions., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Though Medicago truncatula Tnt1 mutants are widely used by researchers in the legume community, they are mainly used for reverse genetics because of the availability of the BLAST-searchable large-scale flanking sequence tags database. However, these mutants should have also been used extensively for forward genetic screens, an effort that has been hindered due to the lack of a compatible genetic crossing partner for the M. truncatula genotype R108, from which Tnt1 mutants were generated. In this study, we selected three Medicago HapMap lines (HM017, HM018 and HM022) and performed reciprocal genetic crosses with R108. After phenotypic analyses in F1 and F2 progenies, HM017 was identified as a compatible crossing partner with R108. By comparing the assembled genomic sequences of HM017 and R108, we developed and confirmed 318 Indel markers evenly distributed across the eight chromosomes of the M. truncatula genome. To validate the effectiveness of these markers, by employing the map-based cloning approach, we cloned the causative gene in the dwarf mutant crs isolated from the Tnt1 mutant population, identifying it as gibberellin 3-β-dioxygenase 1, using some of the confirmed Indel markers. The primer sequences and the size difference of each marker were made available for users in the web-based database. The identification of the crossing partner for R108 and the generation of Indel markers will enhance the forward genetics and the overall usage of the Tnt1 mutants., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Legumes have unique features, such as compound inflorescences and a complex floral ontogeny. Thus, the study of regulatory genes in these species during inflorescence and floral development is essential to understand their role in the evolutionary origin of developmental novelties. The SUPERMAN (SUP) gene encodes a C2H2 zinc-finger transcriptional repressor that regulates the floral organ number in the third and fourth floral whorls of Arabidopsis thaliana. In this work, we present the functional characterization of the Medicago truncatula SUPERMAN (MtSUP) gene based on gene expression analysis, complementation and overexpression assays, and reverse genetic approaches. Our findings provide evidence that MtSUP is the orthologous gene of SUP in M. truncatula. We have unveiled novel functions for a SUP-like gene in eudicots. MtSUP controls not only the number of floral organs in the inner two whorls, but also in the second whorl of the flower. Furthermore, MtSUP regulates the activity of the secondary inflorescence meristem, thus controlling the number of flowers produced. Our work provides insight into the regulatory network behind the compound inflorescence and flower development in this angiosperm family., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed-derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1-5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1-based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plants being sessile integrate information from a variety of endogenous and external cues simultaneously to optimize growth and development. This necessitates the signaling networks in plants to be highly dynamic and flexible. One such network involves heterotrimeric G-proteins comprised of Gα, Gβ, and Gγ subunits, which influence many aspects of growth, development, and stress response pathways. In plants such as Arabidopsis, a relatively simple repertoire of G-proteins comprised of one canonical and three extra-large Gα, one Gβ and three Gγ subunits exists. Because the Gβ and Gγ proteins form obligate dimers, the phenotypes of plants lacking the sole Gβ or all Gγ genes are similar, as expected. However, Gα proteins can exist either as monomers or in a complex with Gβγ, and the details of combinatorial genetic and physiological interactions of different Gα proteins with the sole Gβ remain unexplored. To evaluate such flexible, signal-dependent interactions and their contribution toward eliciting a specific response, we have generated Arabidopsis mutants lacking specific combinations of Gα and Gβ genes, performed extensive phenotypic analysis, and evaluated the results in the context of subunit usage and interaction specificity. Our data show that multiple mechanistic modes, and in some cases complex epistatic relationships, exist depending on the signal-dependent interactions between the Gα and Gβ proteins. This suggests that, despite their limited numbers, the inherent flexibility of plant G-protein networks provides for the adaptability needed to survive under continuously changing environments., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Leaves are derived from the shoot apical meristem with three distinct axes: dorsoventral, proximodistal and mediolateral. Different regulators are involved in the establishment of leaf polarity. Members of the class III homeodomain-leucine zipper (HD-ZIPIII) gene family are critical players in the determination of leaf adaxial identity mediated by microRNA165/166. However, their roles in compound leaf development are still unclear. By screening of a retrotransposon-tagged mutant population of the model legume plant Medicago truncatula, a mutant line with altered leaflet numbers was isolated and characterized. Mutant leaves partially lost their adaxial identity. Leaflet numbers in the mutant were increased along the proximodistal axis, showing pinnate pentafoliate leaves in most cases, in contrast to the trifoliate leaves of the wild type. Detailed characterization revealed that a lesion in a HD-ZIPIII gene, REVOLUTA (MtREV1), resulted in the defects of the mutant. Overexpression of MtMIR166-insensitive MtREV1 led to adaxialized leaves and ectopic leaflets along the dorsoventral axis. Accompanying the abnormal leaf patterning, the free auxin content was affected. Our results demonstrate that MtREV1 plays a key role in determination of leaf adaxial-abaxial polarity and compound leaf patterning, which is associated with proper auxin homeostasis., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago truncatula. Using thermal asymmetric interlaced-polymerase chain reaction followed by sequencing, we recovered 388 888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1-inserted genes are significantly enriched in processes associated with 'stress', 'transport', 'signaling' and 'stimulus response'. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583 Tnt1-inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide-binding site-leucine-rich repeat genes harbored at least one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Recent studies on E3 of endoplasmic reticulum (ER)-associated degradation (ERAD) in plants have revealed homologs in yeast and animals. However, it remains unknown whether the plant ERAD system contains a plant-specific E3 ligase. Here, we report that MfSTMIR, which encodes an ER-membrane-localized RING E3 ligase that is highly conserved in leguminous plants, plays essential roles in the response of ER and salt stress in Medicago. MfSTMIR expression was induced by salt and tunicamycin (Tm). mtstmir loss-of-function mutants displayed impaired induction of the ER stress-responsive genes BiP1/2 and BiP3 under Tm treatment and sensitivity to salt stress. MfSTMIR promoted the degradation of a known ERAD substrate, CPY*. MfSTMIR interacted with the ERAD-associated ubiquitin-conjugating enzyme MtUBC32 and Sec61-translocon subunit MtSec61γ. MfSTMIR did not affect MtSec61γ protein stability. Our results suggest that the plant-specific E3 ligase MfSTMIR participates in the ERAD pathway by interacting with MtUBC32 and MtSec61γ to relieve ER stress during salt stress., (© 2019 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24 h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)