Gérard Duc

Shoot control of root development and nodulation is mediated by a receptor-like kinase

In legumes, root nodule organogenesis is activated in response to morphogenic lipochitin oligosaccharides that are synthesized by bacteria, commonly known as rhizobia. Successful symbiotic interaction results in the formation of highly specialized organs called root nodules, which provide a unique environment for symbiotic nitrogen fixation. In wild-type plants the number of nodules is regulated by a signalling mechanism integrating environmental and developmental cues to arrest most rhizobial infections within the susceptible zone of the root. Furthermore, a feedback mechanism controls the temporal and spatial susceptibility to infection of the root system. This mechanism is referred to as autoregulation of nodulation, as earlier nodulation events inhibit nodulation of younger root tissues. Lotus japonicus plants homozygous for a mutation in the hypernodulation aberrant root (har1) locus escape this regulation and form an excessive number of nodules. Here we report the molecular cloning and expression analysis of the HAR1 gene and the pea orthologue, Pisum sativum, SYM29. HAR1 encodes a putative serine/threonine receptor kinase, which is required for shoot-controlled regulation of root growth, nodule number, and for nitrate sensitivity of symbiotic development.

Medicago truncatula, a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.

First report of non-mycorrhizal plant mutants (Myc^-) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.)

Abstract Genetic resistance to vesicular-arbuscular (VA) mycorrhiza formation has been obtained in spontaneous or chemically induced mutants of two mycorrhiza-forming species (Pisum sativum L. and Vicia faba L.). The eight mutants, termed myc−, are characterized by aborted infections limited to one or two host cells. Expression of the myc− character is associated with that of the nod− character in both legumes, and is likewise under recessive genetic control. Preliminary analysis of the genetic behaviour of the myc− mutants in diallel crosses has shown that at least three genes are involved in VA mycorrhiza infection.

The Medicago truncatula SUNN gene encodes a CLV1-like leucine-rich repeat receptor kinase that regulates nodule number and root length.

Four Medicago truncatula sunn mutants displayed shortened roots and hypernodulation under all conditions examined. The mutants, recovered in three independent genetic screens, all contained lesions in a leucine-rich repeat (LRR) receptor kinase. Although the molecular defects among alleles varied, root length and the extent of nodulation were not significantly different between the mutants. SUNN is expressed in shoots, flowers and roots. Although previously reported grafting experiments showed that the presence of the mutated SUNN gene in roots does not confer an obvious phenotype, expression levels of SUNN mRNA were reduced in sunn-1 roots. SUNN and the previously identified genes HAR1 (Lotus japonicus) and NARK (Glycine max) are orthologs based on gene sequence and synteny between flanking sequences. Comparison of related LRR receptor kinases determined that all nodulation autoregulation genes identified to date are the closest legume relatives of AtCLV1 by sequence, yet sunn, har and nark mutants do not display the fasciated clv phenotype. The M. truncatula region is syntenic with duplicated regions of Arabidopsis chromosomes 2 and 4, none of which harbor CLV1 or any other LRR receptor kinase genes. A novel truncated copy of the SUNN gene lacking a kinase domain, RLP1, is found immediately upstream of SUNN and like SUNN is expressed at a reduced level in sunn-1 roots.

Selection of nodulation and mycorrhizal mutants in the model plant Medicago truncatula (Gaertn.) after γ-ray mutagenesis

Abstract Seeds of Medicago truncatula cv. Jemalong line J5 were treated by γ-rays to create mutations affecting Rhizobium symbiosis. Putative mutants were selected in M 2 and true mutant phenotypes were confirmed in M 3 , M 4 , M 5 and M 6 generations. Fixed nodulation mutant lines were then screened for their endomycorrhizal characters. Eighteen mutant lines are now available. They consist of 2 non-nodulating and non-mycorrhizal (Nod − , Myc − ), 4 low-nodulating and mycorrhizal (Nod ± , Myc + ), 9 nodulating, non-fixing and mycorrhizal (Nod + Fix − , Myc + ) and 3 supernodulating, nitrate tolerant nodulation and mycorrhizal (Nod ++ Nts, Myc + ) mutant lines.

Faba bean (Vicia faba L.)

Abstract This review underlines the wide genetic variability which has been collected, characterized, and sometimes induced through mutagenesis in Vicia faba L. The comprehensive knowledge collected in the scientific community during the last 30 years has permitted major advances in faba bean breeding. These include control of major biotic and abiotic stresses, and diverse seed quality aspects. Biotechnology approaches have been developed with this species and complex breeding schemes that employ male sterility to increase outcrossing have been proposed.

Mutagenesis of pea (Pisum sativum L.) and the isolation of mutants for nodulation and nitrogen fixation

Abstract Pea mutants for nodulation have been obtained by treating seeds with ethyl methane sulfonate (EMS) followed by 2 screening procedures. In one, mutants resistant to nodulation (nod − ), or with ineffective nodules (nod + , fix − ) were obtained, whilst in the other 4 hypernodulated mutants (nod ++ ) with 5–10 times more nodules than cv. Frisson and expressing a character of nitrate tolerant symbiosis (nts) were discovered. All mutations are under the control of single recessive genes. (nod − ), (nod + , fix − ) and (nod ++ , nts) mutations result from mutation events at 6, 7 and 1 different loci respectively. Grafting experiments showed the (nod − ) and (nod + , fix − ) phenotypes are associated with the root genotypes and that (nod ++ , nts) phenotype is associated with the shoot genotype.

Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches

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.

Genetic and Cellular Analysis of Resistance to Vesicular Arbuscular (VA) Mycorrhizal Fungi in Pea Mutants

Screening of nodulation mutants of Pisum sativum has yielded mutants showing resistance to VA fungi (termed myc-). Most of these have aborted infections (myc-(1) phenotype) which are characterised by host cell reactions recalling those in certain pathogen infections. Mutants affected in later steps of mycorrhiza development (myc-(2) phenotype), with blocking of arbuscule formation, were less frequent. The myc-(1) character is recessive, segregates monogenically and occurs on at least five different, independently mutated loci, indicating that VA endomycorrhiza formation is under multiple gene control. Expression of the myc-(1) character is indissociable from that of nod’ in mutants and appears to be the result of pleiotropic effects of single genes. In late mutants where arbuscule formation is blocked, nodules develop but are inefficient (nod+,fix-). Coincidences between myc and nod characters may reflect common mechanisms in plant control over some step(s) in endomycorrhiza and nodule symbioses. Since chemical mutagenesis generally causes loss of gene function, inactivation of symbiosis-specific susceptibility genes in the mutants could affect production of signal molecules and somehow lead to stronger expression of plant resistance mechanisms to the symbionts.

Developmental Genes Have Pleiotropic Effects on Plant Morphology and Source Capacity, Eventually Impacting on Seed Protein Content and Productivity in Pea

Increasing pea (Pisum sativum) seed nutritional value and particularly seed protein content, while maintaining yield, is an important challenge for further development of this crop. Seed protein content and yield are complex and unstable traits, integrating all the processes occurring during the plant life cycle. During filling, seeds are the main sink to which assimilates are preferentially allocated at the expense of vegetative organs. Nitrogen seed demand is satisfied partly by nitrogen acquired by the roots, but also by nitrogen remobilized from vegetative organs. In this study, we evaluated the respective roles of nitrogen source capacity and sink strength in the genetic variability of seed protein content and yield. We showed in eight genotypes of diverse origins that both the maximal rate of nitrogen accumulation in the seeds and nitrogen source capacity varied among genotypes. Then, to identify the genetic factors responsible for seed protein content and yield variation, we searched for quantitative trait loci (QTL) for seed traits and for indicators of sink strength and source nitrogen capacity. We detected 261 QTL across five environments for all traits measured. Most QTL for seed and plant traits mapped in clusters, raising the possibility of common underlying processes and candidate genes. In most environments, the genes Le and Afila, which control internode length and the switch between leaflets and tendrils, respectively, determined plant nitrogen status. Depending on the environment, these genes were linked to QTL of seed protein content and yield, suggesting that source-sink adjustments depend on growing conditions.