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1.  Local and Systemic Regulation of Plant Root System Architecture and Symbiotic Nodulation by a Receptor-Like Kinase 
PLoS Genetics  2014;10(12):e1004891.
In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions.
Author Summary
Despite the essential functions of roots in plant access to water and nutrients, root system architecture has not been directly considered for crop breeding improvement, but it is now considered key for a “second green revolution.” In this study, we aimed to decipher integrated molecular mechanisms coordinating lateral organ development in legume roots: lateral roots and nitrogen-fixing symbiotic nodules. The compact root architecture 2 (cra2) mutant form an increased number of lateral roots and a reduced number of symbiotic nitrogen-fixing nodules. This mutant is affected in a CLAVATA1-like Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that has not previously been linked to root development. Grafting experiments showed that CRA2 negatively controls lateral root formation and positively controls nodule development through local and systemic pathways, respectively. Overall, our results can be integrated in the framework of regulatory pathways controlling the symbiotic nodule number, the so-called “Autoregulation of Nodulation” (AON), involving another LRR-RLK that also acts systemically from the shoots, SUNN (Super Numeric Nodules). A coordinated function of the CRA2 and SUNN LRR-RLKs may thereby permit the dynamic fine tuning of the nodule number depending on the environmental conditions.
PMCID: PMC4270686  PMID: 25521478
2.  Extreme specificity of NCR gene expression in Medicago truncatula 
BMC Genomics  2014;15(1):712.
Legumes form root nodules to house nitrogen fixing bacteria of the rhizobium family. The rhizobia are located intracellularly in the symbiotic nodule cells. In the legume Medicago truncatula these cells produce high amounts of Nodule-specific Cysteine-Rich (NCR) peptides which induce differentiation of the rhizobia into enlarged, polyploid and non-cultivable bacterial cells. NCRs are similar to innate immunity antimicrobial peptides. The NCR gene family is extremely large in Medicago with about 600 genes.
Here we used the Medicago truncatula Gene Expression Atlas (MtGEA) and other published microarray data to analyze the expression of 334 NCR genes in 267 different experimental conditions. We find that all but five of these genes are expressed in nodules but in no other plant organ or in response to any other biotic interaction or abiotic stress tested. During symbiosis, none of the genes are induced by Nod factors. The NCR genes are activated in successive waves during nodule organogenesis, correlated with bacterial infection of the nodule cells and with a specific spatial localization of their transcripts from the apical to the proximal nodule zones. However, NCR expression is not associated with nodule senescence. According to their Shannon entropy, a measure expressing tissue specificity of gene expression, the NCR genes are among the most specifically expressed genes in M. truncatula. Moreover, when activated in nodules, their expression level is among the highest of all genes.
Together, these data show that the NCR gene expression is subject to an extreme tight regulation and is only activated during nodule organogenesis in the polyploid symbiotic cells.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-712) contains supplementary material, which is available to authorized users.
PMCID: PMC4168050  PMID: 25156206
Symbiosis; Legume nitrogen fixation; Nodulation; Bacteroid; Medicago truncatula; Sinorhizobium meliloti; NCR; Defensin; Gene expression; Transcriptome compendium
3.  Growth Conditions Determine the DNF2 Requirement for Symbiosis 
PLoS ONE  2014;9(3):e91866.
Rhizobia and legumes are able to interact in a symbiotic way leading to the development of root nodules. Within nodules, rhizobia fix nitrogen for the benefit of the plant. These interactions are efficient because spectacularly high densities of nitrogen fixing rhizobia are maintained in the plant cells. DNF2, a Medicago truncatula gene has been described as required for nitrogen fixation, bacteroid’s persistence and to prevent defense-like reactions in the nodules. This manuscript shows that a Rhizobium mutant unable to differentiate is not sufficient to trigger defense-like reactions in this organ. Furthermore, we show that the requirement of DNF2 for effective symbiosis can be overcome by permissive growth conditions. The dnf2 knockout mutants grown in vitro on agarose or Phytagel as gelling agents are able to produce nodules fixing nitrogen with the same efficiency as the wild-type. However, when agarose medium is supplemented with the plant defense elicitor ulvan, the dnf2 mutant recovers the fix− phenotype. Together, our data show that plant growth conditions impact the gene requirement for symbiotic nitrogen fixation and suggest that they influence the symbiotic suppression of defense reactions in nodules.
PMCID: PMC3954807  PMID: 24632747
4.  To be or noot to be 
Plant Signaling & Behavior  2013;8(8):e24969.
Legume plants develop symbiosis specific organs on their roots as a result of their interaction with rhizobia. These organs, called nodules, house the nitrogen fixing bacteria. The molecular mechanisms governing the identity and maintenance of this organ are still poorly understood, but it is supposed that root and nodule development share common features. We have identified the Medicago truncatula nodule root (NOOT) and Pisum sativum cochleata (COCH) orthologous genes as necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis blade-on-petiole (BOP) orthologs and NOOT and COCH show functions in leaf and flower development in M. truncatula and P. sativum respectively that are conserved with the functions of BOP in Arabidopsis. The characterization of the noot and coch mutants highlights the root evolutionary origin of nodule vascular strands and suggests that the NOOT and COCH genes were recruited to repress root identity in the legume symbiotic organ.
PMCID: PMC4004616  PMID: 23733067
root nodule symbiosis; meristem; homeosis; organogenesis; auxin; evolution
5.  Fine Mapping Links the FTa1 Flowering Time Regulator to the Dominant Spring1 Locus in Medicago 
PLoS ONE  2013;8(1):e53467.
To extend our understanding of flowering time control in eudicots, we screened for mutants in the model legume Medicago truncatula (Medicago). We identified an early flowering mutant, spring1, in a T-DNA mutant screen, but spring1 was not tagged and was deemed a somaclonal mutant. We backcrossed the mutant to wild type R108. The F1 plants and the majority of F2 plants were early flowering like spring1, strongly indicating that spring1 conferred monogenic, dominant early flowering. We hypothesized that the spring1 phenotype resulted from over expression of an activator of flowering. Previously, a major QTL for flowering time in different Medicago accessions was located to an interval on chromosome 7 with six candidate flowering- time activators, including a CONSTANS gene, MtCO, and three FLOWERING LOCUS T (FT) genes. Hence we embarked upon linkage mapping using 29 markers from the MtCO/FT region on chromosome 7 on two populations developed by crossing spring1 with Jester. Spring1 mapped to an interval of ∼0.5 Mb on chromosome 7 that excluded MtCO, but contained 78 genes, including the three FT genes. Of these FT genes, only FTa1 was up-regulated in spring1 plants. We then investigated global gene expression in spring1 and R108 by microarray analysis. Overall, they had highly similar gene expression and apart from FTa1, no genes in the mapping interval were differentially expressed. Two MADS transcription factor genes, FRUITFULLb (FULb) and SUPPRESSOR OF OVER EXPRESSION OF CONSTANS1a (SOC1a), that were up-regulated in spring1, were also up-regulated in transgenic Medicago over-expressing FTa1. This suggested that their differential expression in spring1 resulted from the increased abundance of FTa1. A 6255 bp genomic FTa1 fragment, including the complete 5′ region, was sequenced, but no changes were observed indicating that the spring1 mutation is not a DNA sequence difference in the FTa1 promoter or introns.
PMCID: PMC3538541  PMID: 23308229
6.  Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria 
Current Genomics  2011;12(2):147-152.
Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project ‘GLIP’: Grain Legumes Integrated Project, funded by the European Union ( In the project ‘IFCOSMO’: Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.
PMCID: PMC3129049  PMID: 21966253
Insertional mutagenesis; legume genomics; Medicago truncatula; Lotus japonicus; phenotyping; Tnt1 mutants.
7.  Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches 
PLoS ONE  2010;5(3):e9519.
The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix− nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this “nodule-specific transcriptome” were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.
PMCID: PMC2832008  PMID: 20209049
8.  An Italian functional genomic resource for Medicago truncatula 
BMC Research Notes  2008;1:129.
Medicago truncatula is a model species for legumes. Its functional genomics have been considerably boosted in recent years due to initiatives based both in Europe and US. Collections of mutants are becoming increasingly available and this will help unravel the genetic control of important traits for many species of legumes.
Our report is on the production of three complementary mutant collections of the model species Medicago truncatula produced in Italy in the frame of a national genomic initiative. Well established strategies were used: Tnt1 mutagenesis, TILLING and activation tagging. Both forward and reverse genetics screenings proved the efficiency of the mutagenesis approaches adopted, enabling the isolation of interesting mutants which are in course of characterization. We anticipate that the reported collections will be complementary to the recently established functional genomics tools developed for Medicago truncatula both in Europe and in the United States.
PMCID: PMC2633015  PMID: 19077311

Results 1-8 (8)