Catalina I. Pislariu

A Putative Transporter is Essential for Integrating Nutrient and Hormone Signaling with Lateral Root Growth and Nodule Development in Medicago truncatula

Legume root architecture involves not only elaboration of the root system by the formation of lateral roots but also the formation of symbiotic root nodules in association with nitrogen-fixing soil rhizobia. The Medicago truncatula LATD/NIP gene plays an essential role in the development of both primary and lateral roots as well as nodule development. We have cloned the LATD/NIP gene and show that it encodes a member of the NRT1(PTR) transporter family. LATD/NIP is expressed throughout the plant. pLATD/NIP-GFP promoter-reporter fusions in transgenic roots establish the spatial expression of LATD/NIP in primary root, lateral root and nodule meristems and the surrounding cells. Expression of LATD/NIP is regulated by hormones, in particular by abscisic acid which has been previously shown to rescue the primary and lateral root meristem arrest of latd mutants. latd mutants respond normally to ammonium but have defects in responses of the root architecture to nitrate. Taken together, these results suggest that LATD/NIP may encode a nitrate transporter or transporter of another compound.

Medicago truncatula DNF2 is a PI‐PLC‐XD‐containing protein required for bacteroid persistence and prevention of nodule early senescence and defense‐like reactions

Medicago truncatula and Sinorhizobium meliloti form a symbiotic association resulting in the formation of nitrogen-fixing nodules. Nodule cells contain large numbers of bacteroids which are differentiated, nitrogen-fixing forms of the symbiotic bacteria. In the nodules, symbiotic plant cells home and maintain hundreds of viable bacteria. In order to better understand the molecular mechanism sustaining the phenomenon, we searched for new plant genes required for effective symbiosis. We used a combination of forward and reverse genetics approaches to identify a gene required for nitrogen fixation, and we used cell and molecular biology to characterize the mutant phenotype and to gain an insight into gene function. The symbiotic gene DNF2 encodes a putative phosphatidylinositol phospholipase C-like protein. Nodules formed by the mutant contain a zone of infected cells reduced to a few cell layers. In this zone, bacteria do not differentiate properly into bacteroids. Furthermore, mutant nodules senesce rapidly and exhibit defense-like reactions. This atypical phenotype amongst Fix(-) mutants unravels dnf2 as a new actor of bacteroid persistence inside symbiotic plant cells.

From Model to Crop: Functional Analysis of a STAY-GREEN Gene in the Model Legume Medicago truncatula and Effective Use of the Gene for Alfalfa Improvement

Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.

A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod−), 51 mutants with totally ineffective nodules (Nod+ Fix−), 17 mutants with partially ineffective nodules (Nod+ Fix+/−), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/− Fix−), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/− Fix+), and 11 supernodulating mutants (Nod++Fix+/−). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN’T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod− lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.

The C2H2 Transcription Factor REGULATOR OF SYMBIOSOME DIFFERENTIATION Represses Transcription of the Secretory Pathway Gene VAMP721a and Promotes Symbiosome Development in Medicago truncatula

This work reports a plant transcription factor, RSD, that is required for symbiosome development and symbiotic nitrogen fixation (SNF) in the model legume Medicago truncatula. RSD represses the expression of the secretory pathway gene VAMP721a, which suggests that alteration in this pathway is important for SNF. Transcription factors (TFs) are thought to regulate many aspects of nodule and symbiosis development in legumes, although few TFs have been characterized functionally. Here, we describe REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD) of Medicago truncatula, a member of the Cysteine-2/Histidine-2 (C2H2) family of plant TFs that is required for normal symbiosome differentiation during nodule development. RSD is expressed in a nodule-specific manner, with maximal transcript levels in the bacterial invasion zone. A tobacco (Nicotiana tabacum) retrotransposon (Tnt1) insertion rsd mutant produced nodules that were unable to fix nitrogen and that contained incompletely differentiated symbiosomes and bacteroids. RSD protein was localized to the nucleus, consistent with a role of the protein in transcriptional regulation. RSD acted as a transcriptional repressor in a heterologous yeast assay. Transcriptome analysis of an rsd mutant identified 11 genes as potential targets of RSD repression. RSD interacted physically with the promoter of one of these genes, VAMP721a, which encodes vesicle-associated membrane protein 721a. Thus, RSD may influence symbiosome development in part by repressing transcription of VAMP721a and modifying vesicle trafficking in nodule cells. This establishes RSD as a TF implicated directly in symbiosome and bacteroid differentiation and a transcriptional regulator of secretory pathway genes in plants.

An IRE-Like AGC Kinase Gene, MtIRE, Has Unique Expression in the Invasion Zone of Developing Root Nodules in Medicago truncatula

The AGC protein kinase family (cAMP-dependent protein kinases A, cGMP-dependent protein kinases G, and phospholipid-dependent protein kinases C) have important roles regulating growth and development in animals and fungi. They are activated via lipid second messengers by 3-phosphoinositide-dependent protein kinase coupling lipid signals to phosphorylation of the AGC kinases. These phosphorylate downstream signal transduction protein targets. AGC kinases are becoming better studied in plants, especially in Arabidopsis (Arabidopsis thaliana), where specific AGC kinases have been shown to have key roles in regulating growth signal pathways. We report here the isolation and characterization of the first AGC kinase gene identified in Medicago truncatula, MtIRE. It was cloned by homology with the Arabidopsis INCOMPLETE ROOT HAIR ELONGATION (IRE) gene. Semiquantitative reverse transcription-polymerase chain reaction analysis shows that, unlike its Arabidopsis counterpart, MtIRE is not expressed in uninoculated roots, but is expressed in root systems that have been inoculated with Sinorhizobium meliloti and are developing root nodules. MtIRE expression is also found in flowers. Expression analysis of a time course of nodule development and of nodulating root systems of many Medicago nodulation mutants shows MtIRE expression correlates with infected cell maturation during nodule development. During the course of these experiments, nine Medicago nodulation mutants, including sli and dnf1 to 7 mutants, were evaluated for the first time for their microscopic nodule phenotype using S. meliloti constitutively expressing lacZ. Spatial localization of a pMtIRE-gusA transgene in transformed roots of composite plants showed that MtIRE expression is confined to the proximal part of the invasion zone, zone II, found in indeterminate nodules. This suggests MtIRE is useful as an expression marker for this region of the invasion zone.

Extreme specificity of NCR gene expression in Medicago truncatula

BackgroundLegumes 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.ResultsHere 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.ConclusionsTogether, 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.

MtSWEET11, a Nodule-Specific Sucrose Transporter of Medicago truncatula

SWEET11 is a nodule-specific sucrose transporter of Medicago truncatula. Optimization of nitrogen fixation by rhizobia in legumes is a key area of research for sustainable agriculture. Symbiotic nitrogen fixation (SNF) occurs in specialized organs called nodules and depends on a steady supply of carbon to both plant and bacterial cells. Here we report the functional characterization of a nodule-specific Suc transporter, MtSWEET11 from Medicago truncatula. MtSWEET11 belongs to a clade of plant SWEET proteins that are capable of transporting Suc and play critical roles in pathogen susceptibility. When expressed in mammalian cells, MtSWEET11 transported sucrose (Suc) but not glucose (Glc). The MtSWEET11 gene was found to be expressed in infected root hair cells, and in the meristem, invasion zone, and vasculature of nodules. Expression of an MtSWEET11-GFP fusion protein in nodules resulted in green fluorescence associated with the plasma membrane of uninfected cells and infection thread and symbiosome membranes of infected cells. Two independent Tnt1-insertion sweet11 mutants were uncompromised in SNF. Therefore, although MtSWEET11 appears to be involved in Suc distribution within nodules, it is not crucial for SNF, probably because other Suc transporters can fulfill its role(s).

Lignin Modification Leads to Increased Nodule Numbers in Alfalfa

Reducing lignin content in stems and roots of alfalfa results in an increased nodule number phenotype. Reduction of lignin levels in the forage legume alfalfa (Medicago sativa) by down-regulation of the monolignol biosynthetic enzyme hydroxycinnamoyl coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) results in strongly increased digestibility and processing ability of lignocellulose. However, these modifications are often also associated with dwarfing and other changes in plant growth. Given the importance of nitrogen fixation for legume growth, we evaluated the impact of constitutively targeted lignin modification on the belowground organs (roots and nodules) of alfalfa plants. HCT down-regulated alfalfa plants exhibit a striking reduction in root growth accompanied by an unexpected increase in nodule numbers when grown in the greenhouse or in the field. This phenotype is associated with increased levels of gibberellins and certain flavonoid compounds in roots. Although HCT down-regulation reduced biomass yields in both the greenhouse and field experiments, the impact on the allocation of nitrogen to shoots or roots was minimal. It is unlikely, therefore, that the altered growth phenotype of reduced-lignin alfalfa is a direct result of changes in nodulation or nitrogen fixation efficiency. Furthermore, HCT down-regulation has no measurable effect on carbon allocation to roots in either greenhouse or 3-year field trials.

Transcription of ENOD8 in Medicago truncatula Nodules Directs ENOD8 Esterase to Developing and Mature Symbiosomes

In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter-gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein was localized in the symbiosome membrane or symbiosome space around the bacteroids in the infected nodule cells. Immunoblot analysis of fractionated symbiosomes strongly suggested that ENOD8 protein was found in the symbiosome membrane and symbiosome space, but not in the bacteroid. Determining the localization of ENOD8 protein in the symbiosome is a first step in understanding its role in symbiosome membrane and space during nodule formation and function.