Frédérique C. Guinel

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants

ABSTRACT Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.

Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp.

This is the first report documenting the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium. This enzyme, previously found in free-living bacteria, yeast and fungi, degrades ACC, the immediate precursor of ethylene in higher plants. Thirteen different rhizobial strains were examined by Southern hybridization, Western blots and ACC deaminase enzyme assay. Five of them tested positive for ACC deaminase. Induction of the expression of ACC deaminase was examined in one of the positively tested strains, Rhizobium leguminosarum bv. viciae 128C53K. This rhizobial ACC deaminase had a trace basal level of expression without ACC, but could be induced by a concentration of ACC as low as 1 µM. The more ACC added to this Rhizobium the higher the expression level of the ACC deaminase.

A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3

Summary. Adenine phosphoribosyltransferase (APT) catalyzes the conversion of adenine and cytokinin bases to the corresponding nucleotides. An Arabidopsis thaliana mutant lacking the major APT isoform, APT1, is male sterile due to defects soon after meiosis. We have now used electron microscopy to define the effects of APT1 deficiency on pollen development to determine whether the changes might be attributed to adenine or cytokinin metabolism. Changes were observed in mutant anthers in both tapetal and pollen mother cells prior to meiosis with additional defects found at later stages, in both compartments. Principal changes include altered lipid accumulation in the tapetal cells, changes in pollen cell wall development, and a loss of synchrony in the development of the tapetum and microspores. Taken together our results suggest that APT1 deficiency causes a general metabolic decrease in energy metabolism, due to the lack of adenine recycling into adenylate nucleotides, which ultimately leads to pollen abortion. The early onset of meiosis in the mutant may be associated with altered cytokinin metabolism.

The Use of Plant Mutants to Study Regulation of Colonization by AM Fungi

Colonization of roots by arbuscular mycorrhizal (AM) fungi and the formation of structures that characterize the AM symbiotic association, i.e. appressoria, extraradical and intraradical hyphae, arbuscules and vesicles, involve a complex series of events. Plant mutants that show a block at some stage in the colonization process are useful in determining the factors involved in the interaction between hyphae and root cells at each step in that process. Most of the mutants identified to date are legumes that also show some impairment in nodulation. There is a tight correlation between the degree of impairment in nodulation and AM formation. For example, failure to initiate nodules (nod-) in several legume species is associated with the failure of roots to be colonized by AM fungi (myc-). Mycorrhiza formation in these mutants is blocked at the stage of appressorium formation and involves chemical and structural changes in the root epidermis. Colonization of other mutants by mycorrhizal fungi is blocked at other stages of mycorrhizal development. Although the molecular basis for the various steps in nodulation has been worked out in detail, there is less information for the AM symbiosis. It has been suggested, based on some experimental evidence, that these symbioses may share some common genes. A mutant of a non-legume species, tomato, has been isolated recently that shows various degrees of reduction in AM formation depending on the fungus species used as inoculum. Mutants of additional non-legume species are needed for the study of the developmental regulation of this symbiosis since the majority of AM fungus host plants are non-legumes.

Ethylene, a Hormone at the Center-Stage of Nodulation.

Nodulation is the result of a beneficial interaction between legumes and rhizobia. It is a sophisticated process leading to nutrient exchange between the two types of symbionts. In this association, within a nodule, the rhizobia, using energy provided as photosynthates, fix atmospheric nitrogen and convert it to ammonium which is available to the plant. Nodulation is recognized as an essential process in nitrogen cycling and legume crops are known to enrich agricultural soils in nitrogenous compounds. Furthermore, as they are rich in nitrogen, legumes are considered important as staple foods for humans and fodder for animals. To tightly control this association and keep it mutualistic, the plant uses several means, including hormones. The hormone ethylene has been known as a negative regulator of nodulation for almost four decades. Since then, much progress has been made in the understanding of both the ethylene signaling pathway and the nodulation process. Here I have taken a large view, using recently obtained knowledge, to describe in some detail the major stages of the process. I have not only reviewed the steps most commonly covered (the common signaling transduction pathway, and the epidermal and cortical programs), but I have also looked into steps less understood (the pre-infection step with the plant defense response, the bacterial release and the formation of the symbiosome, and nodule functioning and senescence). After a succinct review of the ethylene signaling pathway, I have used the knowledge obtained from nodulation- and ethylene-related mutants to paint a more complete picture of the role played by the hormone in nodule organogenesis, functioning, and senescence. It transpires that ethylene is at the center of this effective symbiosis. It has not only been involved in most of the steps leading to a mature nodule, but it has also been implicated in host immunity and nodule senescence. It is likely responsible for the activation of other hormonal signaling pathways. I have completed the review by citing three studies which makes one wonder whether knowledge gained on nodulation in the last decades is ready to be transferred to agricultural fields.

Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea

Pea has lagged behind other model legumes in the molecular study of nodulation and mycorrhizae-formation because of the difficulty to transform its roots and its poor growth on agar plates. Here we describe for pea 1) a transformation technique which permits the complementation of two known non-nodulating pea mutants, 2) a rhizobial inoculation method which allows the study of early cellular events giving rise to nodule primordia, and 3) a targeted fungal inoculation method which allows us to study short segments of mycorrhizal roots assured to be infected. These tools are certain to advance our knowledge of pea root symbioses.

The Pea Nodulation Mutant R50 ( sym16 ) Displays Altered Activity and Expression Profiles for Cytokinin Dehydrogenase

R50 (sym16) is a pleiotropic mutant of pea (Pisum sativum L.) which develops few, pale nodules and has pale young leaves. This phenotype coincides with elevated cytokinin content in vegetative organs, especially mature shoots. Because cytokinin content is known to be tightly regulated by the catabolic action of cytokinin dehydrogenase (CKX), this study focuses on whether CKX-mediated regulation of cytokinin content is involved in the R50 phenotype. Thus, we analyzed the biochemical activity of this enzyme in vitro and found that R50 displayed an aberrant activity profile. During development, PsCKX activity was significantly reduced when compared to wild-type (WT); this was observed in many tissues, specifically in mature shoots and nodules where decrease in activity correlated with elevated cytokinin content. To further address this issue, a full-length cDNA corresponding to CKX1 from pea (PsCKX1) was obtained via RACE-PCR. Although sequencing the entire PsCKX1 cDNA from R50 did not reveal any significant mutations that could have linked PsCKX1 to the sym16 mutation, relative transcript levels of PsCKX1 and of another PsCKX homolog (PsCKX2) were compared between R50 and WT using semiquantitative reverse transcriptase PCR. Interestingly, transcription of these homologs was upregulated in the tissues of R50 displaying the most aberrant phenotype, namely, the mature shoots and nodules. We propose that the R50 phenotype is linked to elevated cytokinin content as a result of deficient PsCKX activity and that transcription of two PsCKX homologs is upregulated as a means to compensate for the biochemical deficiency of this enzyme in R50 mutants.

E151 (sym15), a pleiotropic mutant of pea (Pisum sativum L.), displays low nodule number, enhanced mycorrhizae, delayed lateral root emergence, and high root cytokinin levels

Highlight The phenotype of the E151 pea mutant, unique among known legume mutants, provides the first evidence for a promoting role for cytokinins in the development of the mycorrhizal symbiosis.

Standardized mapping of nodulation patterns in legume roots

Optimizing nodulation in legumes is a target for crop improvement, and the spatial control of nodulation is just beginning to be unravelled. However, there is currently no method for standard phenotyping of nodulation patterns. Here we present a method and software for the quantitative analysis of nodulation phenotypes. Roots of nodulated peas (Pisum sativum), wild-type and two mutants, were photographed. Data from the photographs were extracted using custom image and data analysis software. The software makes it possible to extract each nodules position along primary and lateral roots, and to represent the nodulated root system in a standardized way independent of the way roots are arranged in the soil. A wide variety of nodulation and root variables are calculated, and average spatial nodulation patterns can be computed from multiple samples. Standardized spatial analysis of nodulation patterns opens the way for comparative analyses among genotypes of a single legume species, as here in pea. This approach could also be used to compare nodulation patterns among crops, among plants grown under different environmental conditions, or among plants exposed to different pharmacological treatments. The proposed method should therefore prove useful for studies on nodule organogenesis and nodule physiology and for optimizing nodulation in crops.

Getting around the legume nodule: II. Molecular biology of its peripheral zone and approaches to study its vasculature.

All legume nodules exhibit a complex cortex composed of many types of cells and tissues. This zone, which surrounds the central infected zone, plays a critical role in regulating the exchange of oxygen between the rhizosphere and the bacteroids. Not often mentioned, but of importance, are the vascular traces that develop in the nodule inner cortex. Information on their ontogeny is scarce, although their existence is critical to the symbiosis because both the nitrogenous compounds formed as a result of nitrogenase activity and the energy-rich molecules obtained from plant photosynthesis use these conduits. Here, I focus on these tissues, reviewing what we know of their appearance in pseudonodules, boron-treated legumes, and in the vasculature mutants described to date. I also examine the genes known to be expressed in the peripheral tissues and attempt to place them in functional clusters. Finally, I propose that specific vasculature markers known from the Arabidopsis literature be applied to the study of ...