Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals1

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots1

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO2 evolution or as O2 uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 107 bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO2 production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO2, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.

Synthesis of "Nod"-like chitin oligosaccharides by the Xenopus developmental protein DG42.

The Xenopus DG42 gene is expressed only between the late midblastula and neurulation stages of embryonic development. Recent database searches show that DG42 has striking sequence similarity to the Rhizobium NodC protein. NodC catalyzes the synthesis of chitin oligosaccharides which subsequently are transformed into bacterium-plant root signaling molecules. We find that the DG42 protein made in an in vitro coupled transcription-translation system catalyzes the synthesis of an array of chitin oligosaccharides. The result suggests the intriguing possibility that a bacterium-plant type of "Nod" signaling system may operate during early stages of vertebrate embryonic development and raises issues about the use of chitin synthase inhibitors as fungal-specific drugs.

Evidence that the Plant Host Synthesizes the Heme Moiety of Leghemoglobin in Root Nodules1

Although it is well established that the plant host encodes and synthesizes the apoprotein for leghemoglobin in root nodules, the source of the heme moiety has been uncertain. We recently found that the transcript for coproporphyrinogen III oxidase, one of the later enzymes of heme synthesis, is highly elevated in soybean (Glycine max L.) nodules compared with roots. In this study we measured enzyme activity and carried out western-blot analysis and in situ hybridization of mRNA to investigate the levels during nodulation of the plant-specific coproporphyrinogen oxidase and four other enzymes of the pathway in both soybean and pea (Pisum sativum L.). We compared them with the activity found in leaves and uninfected roots. Our results demonstrate that all of these enzymes are elevated in the infected cells of nodules. Because these are the same cells that express apoleghemoglobin, the data strongly support a role for the plant in the synthesis of the heme moiety of leghemoglobin.

Independent and combined analyses of sequences from all three genomic compartments converge on the root of flowering plant phylogeny

Plant phylogenetic estimates are most likely to be reliable when congruent evidence is obtained independently from the mitochondrial, plastid, and nuclear genomes with all methods of analysis. Here, results are presented from separate and combined genomic analyses of new and previously published data, including six and nine genes (8,911 bp and 12,010 bp, respectively) for different subsets of taxa that suggest Amborella + Nymphaeales (water lilies) are the first-branching angiosperm lineage. Before and after tree-independent noise reduction, most individual genomic compartments and methods of analysis estimated the Amborella + Nymphaeales basal topology with high support. Previous phylogenetic estimates placing Amborella alone as the first extant angiosperm branch may have been misled because of a series of specific problems with paralogy, suboptimal outgroups, long-branch taxa, and method dependence. Ancestral character state reconstructions differ between the two topologies and affect inferences about the features of early angiosperms.

Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula

The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium–host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod− and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.

Regulation of Soybean Nodulation Independent of Ethylene Signaling1

Leguminous plants regulate the number of Bradyrhizobium- or Rhizobium-infected sites that develop into nitrogen-fixing root nodules. Ethylene has been implicated in the regulation of nodule formation in some species, but this role has remained in question for soybean (Glycine max). The present study used soybean mutants with decreased responsiveness to ethylene, soybean mutants with defective regulation of nodule number, and Ag+ inhibition of ethylene perception to examine the role of ethylene in the regulation of nodule number. Nodule numbers on ethylene-insensitive mutants and plants treated with Ag+ were similar to those on wild-type plants and untreated plants, respectively. Hypernodulating mutants displayed wild-type ethylene sensitivity. Suppression of nodule numbers by high nitrate was also similar between ethylene-insensitive plants, wild-type plants, and plants treated with Ag+. Ethylene insensitivity of the roots of etr1-1 mutants was confirmed using assays for sensitivity to 1-aminocyclopropane-1-carboxylic acid and for ethylene-stimulated root-hair formation. Additional phenotypes of etr1-1 roots were also characterized. Ethylene-dependent pathways regulate the number of nodules that form on species such as pea and Medicago truncatula, but our data indicate that ethylene is less significant in regulating the number of nodules that form on soybean.

Expressed Sequence Tags from a Root-Hair-Enriched Medicago truncatula cDNA Library1

The root hair is a specialized cell type involved in water and nutrient uptake in plants. In legumes the root hair is also the primary site of recognition and infection by symbiotic nitrogen-fixing Rhizobium bacteria. We have studied the root hairs of Medicago truncatula, which is emerging as an increasingly important model legume for studies of symbiotic nodulation. However, only 27 genes from M. truncatula were represented in GenBank/EMBL as of October, 1997. We report here the construction of a root-hair-enriched cDNA library and single-pass sequencing of randomly selected clones. Expressed sequence tags (899 total, 603 of which have homology to known genes) were generated and made available on the Internet. We believe that the database and the associated DNA materials will provide a useful resource to the community of scientists studying the biology of roots, root tips, root hairs, and nodulation.

Ethylene-mediated phenotypic plasticity in root nodule development on Sesbania rostrata

Leguminous plants in symbiosis with rhizobia form either indeterminate nodules with a persistent meristem or determinate nodules with a transient meristematic region. Sesbania rostrata was thought to possess determinate stem and root nodules. However, the nature of nodule development is hybrid, and the early stages resemble those of indeterminate nodules. Here we show that, depending on the environmental conditions, mature root nodules can be of the indeterminate type. In situ hybridizations with molecular markers for plant cell division, as well as the patterns of bacterial nod and nif gene expression, confirmed the indeterminate nature of 30-day-old functional root nodules. Experimental data provide evidence that the switch in nodule type is mediated by the plant hormone ethylene.

Identification of lumichrome as a Sinorhizobium enhancer of alfalfa root respiration and shoot growth

Sinorhizobium meliloti bacteria produce a signal molecule that enhances root respiration in alfalfa (Medicago sativa L.) and also triggers a compensatory increase in whole-plant net carbon assimilation. Nuclear magnetic resonance, mass spectrometry, and ultraviolet–visible absorption identify the enhancer as lumichrome, a common breakdown product of riboflavin. Treating alfalfa roots with 3 nM lumichrome increased root respiration 21% (P < 0.05) within 48 h. A closely linked increase in net carbon assimilation by the shoot compensated for the enhanced root respiration. For example, applying 5 nM lumichrome to young alfalfa roots increased plant growth by 8% (P < 0.05) after 12 days. Soaking alfalfa seeds in 5 nM lumichrome before germination increased growth by 18% (P < 0.01) over the same period. In both cases, significant growth enhancement (P < 0.05) was evident only in the shoot. S. meliloti requires exogenous CO2 for growth and may benefit directly from the enhanced root respiration that is triggered by lumichrome. Thus Sinorhizobium–alfalfa associations, which ultimately form symbiotic N2-reducing root nodules, may be favored at an early developmental stage by lumichrome, a previously unrecognized mutualistic signal. The rapid degradation of riboflavin to lumichrome under many physiological conditions and the prevalence of riboflavin release by rhizosphere bacteria suggest that events demonstrated here in the S. meliloti–alfalfa association may be widely important across many plant–microbe interactions.

Genetic ablation of root cap cells in Arabidopsis

The root cap is increasingly appreciated as a complex and dynamic plant organ. Root caps sense and transmit environmental signals, synthesize and secrete small molecules and macromolecules, and in some species shed metabolically active cells. However, it is not known whether root caps are essential for normal shoot and root development. We report the identification of a root cap-specific promoter and describe its use to genetically ablate root caps by directing root cap-specific expression of a diphtheria toxin A-chain gene. Transgenic toxin-expressing plants are viable and have normal aerial parts but agravitropic roots, implying loss of root cap function. Several cell layers are missing from the transgenic root caps, and the remaining cells are abnormal. Although the radial organization of the roots is normal in toxin-expressing plants, the root tips have fewer cytoplasmically dense cells than do wild-type root tips, suggesting that root meristematic activity is lower in transgenic than in wild-type plants. The roots of transgenic plants have more lateral roots and these are, in turn, more highly branched than those of wild-type plants. Thus, root cap ablation alters root architecture both by inhibiting root meristematic activity and by stimulating lateral root initiation. These observations imply that the root caps contain essential components of the signaling system that determines root architecture.

A cdc5+ homolog of a higher plant, Arabidopsis thaliana

We cloned and characterized a cDNA corresponding to a cdc5+ homolog of the higher plant, Arabidopsis thaliana. The cDNA, named AtCDC5 cDNA, encodes a polypeptide of 844 amino acid residues. The amino acid sequence of N-terminal one-fourth region of the predicted protein bears significant similarity to that of Schizosaccharomyces pombe Cdc5 and Myb-related proteins. Overexpression of the AtCDC5 cDNA in S. pombe cells is able to complement the growth defective phenotype of a cdc5 temperature-sensitive mutant. These results indicate that the AtCDC5 gene is a plant counterpart of S. pombe cdc5+. This is the first report of a cdc5+-like gene in a multicellular organism. We also demonstrated that a recombinant AtCDC5 protein possesses a sequence specific DNA binding activity (CTCAGCG) and the AtCDC5 gene is expressed extensively in shoot and root meristems. In addition, we cloned a PCR fragment corresponding to the DNA binding domain of human Cdc5-like protein. These results strongly suggest that Cdc5-like protein exists in all eukaryotes and may function in cell cycle regulation.

A cyclin-dependent kinase-activating kinase regulates differentiation of root initial cells in Arabidopsis

Cell division and differentiation continue throughout the plant life cycle without significant loss of control. However, little is known about the mechanisms that allow the continuous development of meristems. Cell division is controlled by a family of cyclin-dependent kinases (CDKs). CDK-activating kinases (CAKs) are known to phosphorylate and activate almost all CDKs and thus may have a crucial role in controlling CDK activities in each cell of the meristems. Here, we show that overexpression of sense or antisense gene for Cak1At in Arabidopsis by using the glucocorticoid-mediated transcriptional induction system resulted in a reduction of CDK activities. After 14–24 h of glucocorticoid treatment, starch granules appeared in columellar initials in the root meristem, and cortical initials were periclinally divided into cortical and endodermal cells. Accumulation of the cyclin∷β-glucuronidase fusion protein ceased after 72 h of glucocorticoid treatment. Our results indicate that a change of Cak1At activity leads to differentiation of initial cells, followed by cessation of cell division. Therefore, we propose that differentiation of initial cells is controlled by Cak1At but is maintained independent of cell division.

Dissection of nodulation signaling using pea mutants defective for calcium spiking induced by Nod factors and chitin oligomers

Changes in intracellular calcium in pea root hairs responding to Rhizobium leguminosarum bv. viciae nodulation (Nod) factors were analyzed by using a microinjected calcium-sensitive fluorescent dye (dextran-linked Oregon Green). Within 1–2 min after Nod-factor addition, there was usually an increase in fluorescence, followed about 10 min later by spikes in fluorescence occurring at a rate of about one spike per minute. These spikes, corresponding to an increase in calcium of ≈200 nM, were localized around the nuclear region, and they were similar in terms of lag and period to those induced by Nod factors in alfalfa. Calcium responses were analyzed in nonnodulating pea mutants, representing seven loci that affect early stages of the symbiosis. Mutations affecting three loci (sym8, sym10, and sym19) abolished Nod-factor-induced calcium spiking, whereas a normal response was seen in peas carrying alleles of sym2A, sym7, sym9, and sym30. Chitin oligomers of four or five N-acetylglucosamine residues could also induce calcium spiking, although the response was qualitatively different from that induced by Nod factors; a rapid increase in intracellular calcium was not observed, the period between spikes was lower, and the response was not as sustained. The chitin-oligomer-induced calcium spiking did not occur in nodulation mutants (sym8, sym10, and sym19) that were defective for Nod-factor-induced spiking, suggesting that this response is related to nodulation signaling. From our data and previous observations on the lack of mycorrhizal infection in some of the sym mutants, we propose a model for the potential order of pea nodulation genes in nodulation and mycorrhizal signaling.

Galactosides in the rhizosphere: Utilization by Sinorhizobium meliloti and development of a biosensor

Identifying the types and distributions of organic substrates that support microbial activities around plant roots is essential for a full understanding of plant–microbe interactions and rhizosphere ecology. We have constructed a strain of the soil bacterium Sinorhizobium meliloti containing a gfp gene fused to the melA promoter which is induced on exposure to galactose and galactosides. We used the fusion strain as a biosensor to determine that galactosides are released from the seeds of several different legume species during germination and are also released from roots of alfalfa seedlings growing on artificial medium. Galactoside presence in seed wash and sterile root washes was confirmed by HPLC. Experiments examining microbial growth on α-galactosides in seed wash suggested that α-galactoside utilization could play an important role in supporting growth of S. meliloti near germinating seeds of alfalfa. When inoculated into microcosms containing legumes or grasses, the biosensor allowed us to visualize the localized presence of galactosides on and around roots in unsterilized soil, as well as the grazing of fluorescent bacteria by protozoa. Galactosides were present in patches around zones of lateral root initiation and around roots hairs, but not around root tips. Such biosensors can reveal intriguing aspects of the environment and the physiology of the free-living soil S. meliloti before and during the establishment of nodulation, and they provide a nondestructive, spatially explicit method for examining rhizosphere soil chemical composition.