Guilhem Desbrosses

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.

Lotus japonicus Metabolic Profiling. Development of Gas Chromatography-Mass Spectrometry Resources for the Study of Plant-Microbe Interactions

Symbiotic nitrogen fixation (SNF) in legume root nodules requires differentiation and integration of both plant and bacterial metabolism. Classical approaches of biochemistry, molecular biology, and genetics have revealed many aspects of primary metabolism in legume nodules that underpin SNF. Functional genomics approaches, especially transcriptomics and proteomics, are beginning to provide a more holistic picture of the metabolic potential of nodules in model legumes like Medicago truncatula and Lotus japonicus. To extend these approaches, we have established protocols for nonbiased measurement and analysis of hundreds of metabolites from L. japonicus, using gas chromatography coupled with mass spectrometry. Following creation of mass spectral tag libraries, which represent both known and unknown metabolites, we measured and compared relative metabolite levels in nodules, roots, leaves, and flowers of symbiotic plants. Principal component analysis of the data revealed distinct metabolic phenotypes for the different organs and led to the identification of marker metabolites for each. Metabolites that were enriched in nodules included: octadecanoic acid, asparagine, glutamate, homoserine, cysteine, putrescine, mannitol, threonic acid, gluconic acid, glyceric acid-3-P, and glycerol-3-P. Hierarchical cluster analysis enabled discrimination of 10 groups of metabolites, based on distribution patterns in diverse Lotus organs. The resources and tools described here, together with ongoing efforts in the areas of genome sequencing, and transcriptome and proteome analysis of L. japonicus and Mesorhizobium loti, should lead to a better understanding of nodule metabolism that underpins SNF.

The Sulfate Transporter SST1 Is Crucial for Symbiotic Nitrogen Fixation in Lotus japonicus Root Nodules

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.

Simultaneous Interaction of Arabidopsis thaliana with Bradyrhizobium Sp. Strain ORS278 and Pseudomonas syringae pv. tomato DC3000 Leads to Complex Transcriptome Changes

Induced systemic resistance (ISR) is a process elicited by telluric microbes, referred to as plant growth-promoting rhizobacteria (PGPR), that protect the host plant against pathogen attacks. ISR has been defined from studies using Pseudomonas strains as the biocontrol agent. Here, we show for the first time that a photosynthetic Bradyrhizobium sp. strain, ORS278, also exhibits the ability to promote ISR in Arabidopsis thaliana, indicating that the ISR effect may be a widespread ability. To investigate the molecular bases of this response, we performed a transcriptome analysis designed to reveal the changes in gene expression induced by the PGPR, the pathogen alone, or by both. The results confirm the priming pattern of ISR described previously, meaning that a set of genes, of which the majority was predicted to be influenced by jasmonic acid or ethylene, was induced upon pathogen attack when plants were previously colonized by PGPR. The analysis and interpretation of transcriptome data revealed that 12-oxo-phytodienoic acid, an intermediate of the jasmonic acid biosynthesis pathway, is likely to be an actor in the signaling cascade involved in ISR. In addition, we show that the PGPR counterbalanced the pathogen-induced changes in expression of a series of genes.

PGPR-Arabidopsis interactions is a useful system to study signaling pathways involved in plant developmental control

Using their 1 amino cyclopropane-1-carboxylic acid (ACC) deaminase activity, many rhizobacteria can divert ACC from the ethylene biosynthesis pathway in plant roots. To investigate the role of this microbial activity in plant responses to plant growth-promoting rhizobacteria (PGPR), we analyzed the effects of acdS knock-out and wild-type PGPR strains on two phenotypic responses to inoculation –root hair elongation and root system architecture– in Arabidopsis thaliana. Our work shows that rhizobacterial AcdS activity has a negative effect on root hair elongation, as expected from the reduction of ethylene production rate in root cells, while it has no impact on root system architecture. This suggests that PGPR triggered root hair elongation is independent of ethylene biosynthesis or signaling pathway. In addition, it does indicate that AcdS activity alters local regulatory processes, but not systemic regulations such as those that control root architecture. Our work also indicates that root hair elongation induced by PGPR inoculation is probably an auxin-independent mechanism. These findings were unexpected since genetic screens for abnormal root hair development mutants led to the isolation of ethylene and auxin mutants. Our work hence shows that studying the interaction between a PGPR and the model plant Arabidopsis is a useful system to uncover new pathways involved in plant plasticity.

Lotus japonicus LjKUP Is Induced Late During Nodule Development and Encodes a Potassium Transporter of the Plasma Membrane

The KUP family of potassium transporters in plants is large but poorly characterized. We isolated and characterized the first KUP transporter from a legume, LjKUP of Lotus japonicus. Although expressed throughout plants, LjKUP transcript levels were highest in nodules. Induction of LjKUP expression occurred late during nodule development, at a time of rapid organ expansion. A high level of LjKUP expression was maintained in mature, full-sized nodules. However, induction of LjKUP expression was independent of symbiotic nitrogen fixation (SNF), and occurred in ineffective nodules resulting from mutations in either the plant or its microsymbiont, Mesorhizobium loti. Heterologous expression of LjKUP in Escherichia coli showed that the protein is able to transport potassium. Transient expression of a GFP-LjKUP fusion protein in Arabidopsis cells indicated a plasma membrane location for the transporter. Taken together, the results indicate that LjKUP is a potassium transporter of the plasma membrane, which may play roles in cell expansion during nodule development and in ion homeostasis during SNF.

Induction and spatial organization of polyamine biosynthesis during nodule development in Lotus japonicus

Putrescine and other polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase (ADC; EC 4.1.1.19), the other with ornithine decarboxylase (ODC; EC 4.1.1.17). Metabolite profiling of nitrogen-fixing Lotus japonicus nodules, using gas chromatography coupled to mass spectrometry, revealed a two- to sixfold increase in putrescine levels in mature nodules compared with other organs. Genes involved in polyamine biosynthesis in L japonicus nodules were identified by isolating cDNA clones encoding ADC (LjADC1) and ODC (LjODC) from a nodule library. Searches of the public expressed sequence tag databases revealed the presence of a second gene encoding ADC (LjADC2). Real-time reverse-transcription-polymerase chain reaction analysis showed that LjADC1 and LjADC2 were expressed throughout the plant, while LjODC transcripts were detected only in nodules and roots. Induction of LjODC and LjADC gene expression during nodule development preceded symbiotic nitrogen fixation. Transcripts accumulation was maximal at 10 days postinfection, when a 6.5-fold increase in the transcript levels of LjODC was observed in comparison with the uninfected roots, while a twofold increase in the transcript levels of LjADC1 and LjADC2 was detected. At later stages of nodule development, transcripts for ADC drastically declined, while in the case of ODC, transcript accumulation was higher than that in roots until after 21 days postinfection. The expression profile of genes involved in putrescine biosynthesis correlated well with the expression patterns of genes involved in cell division and expansion, including a L. japonicus Cyclin D3 and an alpha-expansin gene. Spatial localization of LjODC and LjADC1 gene transcripts in developing nodules revealed that both transcripts were expressed in nodule inner cortical cells and in the central tissue. High levels of LjADC1 transcripts were also observed in both nodule and connecting root vascular tissue, suggesting that putrescine and other polyamines may be subject to long-distance transport. Our results indicate that polyamines are primarily involved in physiological and cellular processes involved in nodule development, rather than in processes that support directly symbiotic nitrogen fixation and assimilation.

Differentiation of Plant Cells During Symbiotic Nitrogen Fixation

Nitrogen-fixing symbioses between legumes and bacteria of the family Rhizobiaceae involve differentiation of both plant and bacterial cells. Differentiation of plant root cells is required to build an organ, the nodule, which can feed and accommodate a large population of bacteria under conditions conducive to nitrogen fixation. An efficient vascular system is built to connect the nodule to the root, which delivers sugars and other nutrients to the nodule and removes the products of nitrogen fixation for use in the rest of the plant. Cells in the outer cortex differentiate to form a barrier to oxygen diffusion into nodules, which helps to produce the micro-aerobic environment necessary for bacterial nitrogenase activity. Cells of the central, infected zone of nodules undergo multiple rounds of endoreduplication, which may be necessary for colonisation by rhizobia and may enable enlargement and greater metabolic activity of these cells. Infected cells of the nodule contain rhizobia within a unique plant membrane called the peribacteroid or symbiosome membrane, which separates the bacteria from the host cell cytoplasm and mediates nutrient and signal exchanges between the partners. Rhizobia also undergo differentiation during nodule development. Not surprisingly, perhaps, differentiation of each partner is dependent upon interactions with the other. High-throughput methods to assay gene transcripts, proteins, and metabolites are now being used to explore further the different aspects of plant and bacterial differentiation. In this review, we highlight recent advances in our understanding of plant cell differentiation during nodulation that have been made, at least in part, using high-throughput methods.

METABOLOME ANALYSIS USING GC-MS

Global changes in gene transcription (the transcriptome), and protein amount/activity (the proteome) ultimately effect metabolism and the metabolite content (the metabolome) of cells, tissues, and organs of plants. In the past, studies of plant metabolism focussed on one or a few genes, proteins, and/or metabolites. New tools have been developed to measure levels of thousands of gene transcripts and proteins in parallel (see Chapters 4.2-4.4), facilitating nonbiased, systems-wide investigations. To complement these ‘OMICS’ technologies, we use gas chromatography coupled to mass-spectroscopy together with bioinformatics tools to monitor changes in the levels of hundreds of metabolites in different organs. The methods described in this chapter provide qualitative and quantitative information about the Lotus metabolome, which together with transcriptome and proteome analyses, enable new insights into the links between genotype and phenotype at the whole-system level.

Differential Contribution of Plant-Beneficial Functions from Pseudomonas kilonensis F113 to Root System Architecture Alterations in Arabidopsis thaliana and Zea mays

Fluorescent pseudomonads are playing key roles in plant-bacteria symbiotic interactions due to the multiple plant-beneficial functions (PBFs) they are harboring. The relative contributions of PBFs to plant-stimulatory effects of the well-known plant growth-promoting rhizobacteria Pseudomonas kilonensis F113 (formerly P. fluorescens F113) were investigated using a genetic approach. To this end, several deletion mutants were constructed, simple mutants ΔphlD (impaired in the biosynthesis of 2,4-diacetylphloroglucinol [DAPG]), ΔacdS (deficient in 1-aminocyclopropane-1-carboxylate deaminase activity), Δgcd (glucose dehydrogenase deficient, impaired in phosphate solubilization), and ΔnirS (nitrite reductase deficient), and a quadruple mutant (deficient in the four PBFs mentioned above). Every PBF activity was quantified in the wild-type strain and the five deletion mutants. This approach revealed few functional interactions between PBFs in vitro. In particular, biosynthesis of glucose dehydrogenase severely reduced the production of DAPG. Contrariwise, the DAPG production impacted positively, but to a lesser extent, phosphate solubilization. Inoculation of the F113 wild-type strain on Arabidopsis thaliana Col-0 and maize seedlings modified the root architecture of both plants. Mutant strain inoculations revealed that the relative contribution of each PBF differed according to the measured plant traits and that F113 plant-stimulatory effects did not correspond to the sum of each PBF relative contribution. Indeed, two PBF genes (ΔacdS and ΔnirS) had a significant impact on root-system architecture from both model plants, in in vitro and in vivo conditions. The current work underscored that few F113 PBFs seem to interact between each other in the free-living bacterial cells, whereas they control in concert Arabidopsis thaliana and maize growth and development.