Cortical Cell Division Research Articles

Flavone-enhanced accumulation and symbiosis-related biological activity of a diglycosyl diacylglycerol membrane glycolipid from Rhizobium leguminosarum biovar trifolii

Rhizobium leguminosarum bv. trifolii is the bacterial symbiont which induces nitrogen-fixing root nodules on the leguminous host, white clover (Trifolium repens L.). In this plant-microbe interaction, the host plant excretes a flavone, 4',7-dihydroxyflavone (DHF), which activates expression of modulation genes, enabling the bacterial symbiont to elicit various symbiosis-related morphological changes in its roots. We have investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild-type R. leguminosarum bv. trifolii ANU843 when grown with DHF and the biological activities of this glycolipid bacterial factor on host and nonhost legumes. In vivo labeling studies indicated that wild-type ANU843 cells accumulate BF-7 in response to DHF, and this flavone-enhanced alteration in membrane glycolipid composition was suppressed in isogenic nodA::Tn5 and nodD::Tn5 mutant derivatives. Seedling bioassays performed under microbiologically controlled conditions indicated that subnanomolar concentrations of purified BF-7 elicit various symbiosis-related morphological responses on white clover roots, including thick short roots, root hair deformation, and foci of cortical cell divisions. Roots of the nonhost legumes alfalfa and vetch were much less responsive to BF-7 at these low concentrations. A structurally distinct diglycosyl diacylglycerol did not induce these responses on white clover, indicating structural constraints in the biological activity of BF-7 on this legume host. In bioassays using aminoethoxyvinylglycine to suppress plant production of ethylene, BF-7 elicited a meristematic rather than collaroid type of mitogenic response in the root cortex of white clover. These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 and a potent ability of this diglycosyl diacylglycerol glycolipid to perform as a bacterial factor enabling R. leguminosarum bv. trifolii to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.

Kinetics of Nodule Development in Glycine soja.

Nodule development in the interaction of Glycine soja Sieb. & Zucc. PI468.397 with Bradyrhizobium japonicum USDA110 was studied by hypochlorite clearing and methylene blue staining. Even the earliest stages of nodule development could be observed. The entire length of the primary root was examined up to 15 d postinoculation. Markedly curled root hairs and the first cell divisions in the hypodermal layer (stage I) were observed 2 d postinoculation, and by 3 d cell division activity had spread to the outer layers of the cortex (stage II). Cortical cell division centers not associated with curled root hairs, frequently observed in soybean (Glycine max [L.] Merr.), were very rare in G. soja. The cortical cell division centers that had developed a well-defined nodule meristem (at or beyond stage IV) by 6 d postinoculation continued to develop, but the less-advanced stages became arrested. Almost all nodules developed near the position of the root tip at the time of inoculation. In the parts of the root that developed after inoculation, regions with a high density of markedly curled root hairs per root length were observed. The percentage of the curled root hairs associated with cortical cell division centers, however, declined with each successive peak. Regulation of nodule development in G. soja was similar to that previously reported in soybean, although the rate of nodule development was slower.

Influence ofRhizobium leguminosarum biovartrifolii host specific nodulation genes on the ontogeny of clover nodulation

The infection of white clover seedlings byRhizobium strains with different host range properties was assessed using various microscopic techniques. Several wild-type andRhizobium leguminosarum biovarvicias hybrid strains containing definedR. l. bv.trifolii host range genes were used. The morphological changes in the root tissue of uninoculated and rhizobia inoculated white clovers were identified and compared. In particular, changes were observed in the induction of inner cortical cell division, alterations to nodule development and lateral root formation. The responses of the infected roots and the types of structures formed support the hypothesis that lateral roots and nodules may be physiologically homologous structures. To establish a normal pattern of nodulation on white clover roots, both sets of known host specific nodulation genes (operonsnod FERL andnod MNX) ofR. l. bv.trifolii were required. However, some nodule development occurred when only thenod FERL genes were present in the hybrid strain.

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.

Molecular aspects of legumes/rhizobia symbiosis: Perspectives for the '90s

Abstract The exchange of molecular signals between legume plant and rhizobia has a key role in mutual recognition and in determining bacterium-host specificity. Rhizobial nod genes are induced by plant compounds such as flavonoids and, in the case of alfalfa/R. meliloti, also by betaines like trigonelline and stachydrine, abundant in root exudates. Different legumes secrete different flavonoids inducers and a single plant may synthesise different inducers at different stages of development. Some flavonoids can also cause growth enhancement of Rhizobium, thus becoming biocontrol substances with regulation potential for the dynamic of soil microbiota. Nod factors can elicit various responses in the host legumes by inducing root hair formation, deformation and branching, infection, thread formation and cortical cell division for development of nodule primordia. The role that receptors can play in the plant, along with corresponding receptor genes in the legume family with constant and variable domains, remai...

Signaling and host range variation in nodulation.

Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts, in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes, which determine host specificity, infection, and nodulation, are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins, which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in turn in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule-meristem formation.

The role of iron in the (brady)Rhizobiumlegume symbiosis

Abstract Iron deficiency generally decreases nodule formation, leghaemoglobin production and nitrogenase activity, leading to low nitrogen concentrations in the shoots in some legumes. More iron is required for plants reliant on N2 fixation than for plants supplied with mineral nitrogen. The sensitive stage of nodulation to iron deficiency appears to be nodule initiation. Under iron deficiency, further division of root cortical cells is limited and the proliferation of (brady)rhizobia in roots is inhibited.

Bacterial-Induced Changes in Plant Form and Function

Numerous steps occur between the mutual recognition of host and Rhizobium and the establishment of a nitrogen-fixing indeterminate nodule: root hair curling and infection thread formation, cortical cell divisions and initiation of a nodule primordium, nodule meristem formation, differentiation of specific cell types and tissues, and induction of nitrogen fixation. Rhizobia with defined mutations or Agrobacterium tumefaciens transconjugants carrying R. meliloti symbiotic genes have been used to interrupt these steps. Using this approach, we have detected transcripts of the early nodulin MsENOD2 in alfalfa nodules induced by A. tumefaciens transconjugants carrying one or both symbiotic plasmids of R. meliloti. We have also specifically localized MsENOD2 transcripts by in situ hybridization to nodule parenchyma cells in nodules that form spontaneously on alfalfa roots. These bacteria-free nodules differentiate a discrete nodule meristem and other tissues that are typical of normal nodules. Transcripts for MsENOD12, a proline-rich protein normally found in the invasion zone of wildtype R. meliloti-induced nodules, were not detected in nodules induced by the Agrobacterium transconjugants or in the spontaneously formed nodules. However, we have detected MsENOD12 mRNAs in nodules formed in response to R. meliloti exoH mutants. The exoH mutants, which induce nodules without a persistent nodule meristem, infect host cells at a reduced frequency compared with wildtype or with fix or nif mutant R. meliloti. These results indicate that infection rather than the formation of a persistent nodule meristem is correlated with MsENOD12 gene expression.

Microscopic evidence on how iron deficiency limits nodule initiation in Lupinus angustifolius L.

Lupins (Lupinus angustifolius L. ev. Yandee), grown in solution culture, have been used to study the sites and process of infection by Bradyrhizobium sp. (Lupinus) and the impairment of nodulation by iron deficiency. Infection leading to nodulation occurred in an area of epidermal cells either lacking root hairs or with very young root hairs at the time of inoculation. Cells aged 13 h or over appeared not to be infected. Infection was initiated in the outer cortex. Rare, short infection threads were evident on day 4 after inoculation, 2 d after the initial division of cortical cells resulting from the bradyrhizobial inoculation. Bacteria had been released into the cytoplasm of cortical cells within 5 d after inoculation. Bacteroids multiplied in the cytoplasm, segregated passively and spread in the infection zones by repeated division of the invaded cells. Under iron deficiency, initial cell division occurred in the outer cortex of host roots, as in iron-sufficient plants after inoculation. Iron deficiency then limited further division of cortical cells. Only a few surviving infection sites developed nodules with normal structure but development was much slower than in iron-sufficient plants.

Structure and growth of infection threads in the legume symbiosis with Rhizobium leguminosarum

SummarySpecific antibodies and enzyme–gold probes were used to study the structure and development of infection threads in nodules induced by Rhizobium leguminosarum on the roots of Vicia, Pisum and Phaseolus. In Pisum nodules, the tubular infection thread wall contains polysaccharides antigenically similar to those of the cell wall, including cellulose, xyloglucan, methyl‐esterified pectin and non‐esterified pectin, but none of these wall components is present around the infection droplet structures from which bacteria are internalized by plant plasma membrane. As reported previously for pea nodules, the luminal matrix of infection threads and infection droplets contains a plant glycoprotein; this glycoprotein is also secreted by infected and uninfected cortical cells of a Vicia root at the earliest stages of nodule initiation. Synthesis of a transcellular infection thread apparently involves reorganized deposition of components normally targeted to the cell wall, and infection thread growth is orientated anticlinally through the outer cortex in the same plane observed for the deposition of new cell walls following mitosis. Both the development of infection threads in the outer cortex and the initiation of cell division in the inner cortex are preceded by a similar process of cell reactivation involving centralization of nuclei and the development of anticlinal transvacuolar strands. It is therefore suggested that the two Rhizobium‐induced processes of infection thread growth and cortical cell division may both be consequences of a similar plant cell response in the inner and outer root cortex, respectively. Phaseolus nodules contained only short intracellular infection structures which terminated within individual cells and contained no luminal matrix material. The differences in infection thread structure between Pisum and Phaseolus nodules may reflect differences in ontogeny between “indeterminate” and “determinate” nodule meristems.

The timing of exogenous 2,4-dichlorophenoxyacetate (2,4-D) application affects nodulation of soybeans,glycine max(L.) merr.

Abstract The effects of exogenous addition of 2.26 μM 2,4-dichlorophenoxyacetate (2,4-D) at the time of sowing and 3 d after on nodulation and nodule function were investigated in soybean [Glycine max (L.) Merr.] cvs. “Enrei,” “Lee,” and “Nanbushirome.” Both treatments especially that at the time of sowing considerably depressed shoot and root development. The total number of cortical cell division foci/cm lateral root was similarly increased 2-8 fold by the treatments but the increase in the number of nodules/cm lateral root in plants treated 3 d after sowing was generally twice that of the treatment at the time of sowing. Majority of the cortical cell divisions, notably in treated plants, was arrested at the nodule developmental stage before nodule emergence. The total number of nodules at harvest (24 d after sowing) relative to the control was increased in plants treated 3 d after sowing but was decreased by the earlier treatment. Nevertheless, treated plants showed a similar nitrogenase (acetylene red...

N-Acetylglutamic acid: an extracellular nod signal of Rhizobium trifolii ANU843 that induces root hair branching and nodule-like primordia in white clover roots.

An extracellular metabolite purified from Rhizobium trifolii ANU843 was established as N-acetylglutamic acid (GluNAc) by 1H NMR and Fourier transform IR spectroscopy, gas chromatography/mass spectrometry of its methylated product, and organic synthesis. TLC analyses indicated that extracellular accumulation of GluNAc by R. trifolii ANU843 grown in defined BIII culture medium was dependent on induction of its bacterial nodulation (nod) genes and the positive regulatory gene nodD on its symbiotic plasmid. 1H NMR analyses showed less GluNAc in fractionated culture supernatants of nodL and nodM mutant derivatives of R. trifolii ANU843. GluNAc induced three morphological responses on axenic roots of white clover seedlings: (i) root hair branching; (ii) tip swelling followed by resumed elongation of root hairs; and (iii) a slight increase in foci of cortical cell divisions, which developed into nodule-like primordia. These biological activities of extracellular GluNAc from R. trifolii ANU843 were confirmed with authentic standards of GluNAc. These results indicate that extracellular accumulation of N-acetylglutamic acid is linked to flavone-dependent metabolism involving nodD, nodL, and nodM in R. trifolii ANU843. This constitutes the first report on the structure of a nod-dependent extracellular signal from R. trifolii that can affect root hair and nodule development in white clover and whose biological activity on this host has been confirmed with authentic standards.

Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa

RHIZOBIUM meliloti is a symbiotic bacterium that elicits the morphogenesis of nitrogen-fixing nodules, specific organs on the roots of alfalfa (Medicago sativa)1. In R. meliloti a series of nodulation (nod) genes have been identified which are involved in root-hair curling and infection and in nodule formation1. The nodABC genes, common to all Rhizobium sp., and the host-range nodH and nodPQ genes are involved in the production of an excreted root-hair deforming factor, NodRm-1, which is a sul-phated and acylated glucosamine oligosaccharide2–7. Here we report that purified NodRm-1 and a related compound, Ac-Nod Rm-1, at concentrations in the micromolar–nanomolar range, elicit cortical cell divisions and the formation of genuine nodules on aseptically grown seedlings of alfalfa. Chemical modifications of NodRm-1, such as the removal of the sulphate group, reduction of the terminal sugar or hydrogenation of the acyl chain result in a strong decrease in the morphogenic activity. A highly specific prokaryotic lipo-oligosaccharide signal is thus able to trigger a genuine organogenesis in a higher plant.

Alfalfa Controls Nodulation during the Onset of Rhizobium-induced Cortical Cell Division

The formation of first nodules inhibits subsequent nodulation in younger regions of alfalfa (Medicago sativa L.) roots by a feedback regulatory mechanism that controls nodule number systemically (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Following inoculation with wild-type Rhizobium meliloti, almost all infections associated with cortical cell division developed into mature nodules. While the distribution of Rhizobium- induced cell divisions closely paralleled the distribution of first emergent nodules, only 9 to 15% of total cell division foci failed to become functional nodules. Nodule formation was restricted to the primary root when plants were inoculated before lateral root emergence. Excision of these primary root nodules allowed nodules to reappear in lateral roots clustered around the location of the root tip at the time of nodule removal. Apparently, this region regained susceptibility to infection within the first hours after excision of primary nodules and suppression of nodulation was restored a day later probably due to the development of new infection foci. Our results suggest that alfalfa controls nodulation during the onset of cell division in the root cortex and not during infection development as in soybean.

Ontogeny and ultrastructure of spontaneous nodules in alfalfa (Medicago sativa)

The development of spontaneous nodules, formed in the absence ofRhizobium and combined nitrogen, on alfalfa (Medicago sativa L. cv. Vernal) was investigated at the light and electron microscopic level and compared to that ofRhizobium-induced normal nodules. Spontaneous nodules were initiated from cortical cell divisions in the inner cortex next to the endodermis, i.e., the site of normal nodule development. These nodules, on uninoculated roots, were white multilobed structures, histologically composed of nodule meristems, cortex, endodermis, central zone and vascular strands. Nodules were devoid of intercellular or intracellular bacteria confirming microbiological tests. Early development of spontaneous nodules was initiated by series of anticlinal followed by periclinal divisions of dedifferentiated cells in the inner cortex of the root. These cells formed the nodular meristem from which the nodule developed. The cells in the nodule meristems divided unequally and differentiated into two distinct cell types, one larger type being filled with numerous membrane-bound starch grains, and the other smaller type with very few starch grains. There were no infection threads or bacteria in the spontaneous nodules at any stage of development. This size differentiation is suggestive of the different cell sizes seen inRhizobium-induced nodules, where the larger cell type harbours the invading bacteria and the smaller type is essential in supportive metabolic roles. The ontogenic studies further support the claim that these structures are nodules rather than aberrant lateral roots, and that plant possess all the genetic information needed to develop a nodule with distinct cell types. Our results suggest that bacteria and therefore theirnod genes are not necessarily involved in the ontogeny and morphogenesis of spontaneous and normal nodules in alfalfa.

Effect of Inoculation and Nitrogen on Isoflavonoid Concentration in Wild-Type and Nodulation-Mutant Soybean Roots

The isoflavones, daidzein and genistein, have been isolated and identified as the major inducers of nod genes of Bradyrhizobium japonicum. The common nod genes of rhizobia are in turn responsible for stimulating root hair curling and cortical root cell division, the earliest steps in the host response. This study evaluated whether there was a relationship between root isoflavonoid production and the hypernodulation phenotype of selected soybean (Glycine max [L.] Merr.) mutants. Three independently selected hypernodulating soybean mutants (NOD1-3, NOD2-4, and NOD3-7) and a nonnodulating mutant (NN5) were compared with the Williams parent for isoflavonoid concentrations. High performance liquid chromatographic analyses of soybean root extracts showed that all lines increased in daidzein, genistein, and coumestrol concentrations throughout the 12-day growth period after transplanting of both inoculated and noninoculated plants; transplanting and inoculation were done 6 days after planting. No significant differences were detected in the concentration of these compounds among the three noninoculated hypernodulating mutants and the Williams parent. In response to inoculation, the three hypernodulating mutants had higher isoflavonoid concentrations than did the Williams control at 9 to 12 days after inoculation when grown at 0 millimolar N level. However, the inoculated nonnodulating mutant also had higher isoflavonoid concentrations than did Williams. N application [urea, (NH(4))(2)SO(4) and NO(3) (-)] decreased the concentration of all three isoflavonoid compounds in all soybean lines. Application of NO(3) (-) was most inhibitory to isoflavonoid concentrations, and inhibition by NO(3) (-) was concentration dependent. These results are consistent with a conclusion that differential NO(3) (-) inhibition of nodulation may be partially due to changes in isoflavonoid levels, although the similar response of the nonnodulating mutant brings this conclusion into question. Alternatively, the nodulation control in the NN5 mutant may be due to factors totally unrelated to isoflavonoids, leaving open the possibility that isoflavonoids play a role in differential nodulation of lines genetically competent to nodulate.

Factors Determining Host Recognition in the Clover-Rhizobium Symbiosis

Rhizobia are microbes that exploit host plants as a nutritional source but cause little or no host damage. They may provide, through biological nitrogen fixation, a valuable source of nitrogen for plant growth. Different rhizobia nodulate a limited range of plants. In this review we will show that host range specificity is determined by the success or otherwise of communication events between the interacting partners. To infect different plant species, a distinct cocktail of phenolic compounds (flavonoids) is recognised. Flavonoids of the correct structure induce the expression of several bacterial nodulation (nod) and other genes required for plant infection. Flavonoids of the incorrect, but related, structure can antagonise nod gene induction. Some nod genes are responsible for the synthesis of a small family of lipo-oligosaccharides necessary for the triggering of a defined but complex series of morphological responses in the host plant including root hair curling and cortical cell division. Lipo-oligosaccharides are active at concentrations of between 10-8 and 10-12 M. The appropriate lipo-oligosaccharide required for infection of one plant host can have antagonistic effects on other non-host plants and this effect appears to be determined by minor chemical changes to the basic lipo-oligosaccharide structure. Apart from host specificity operating at the genus level, other interdependent nod gene functions determine host specificity at the cultivar level. A complex interplay between positively and negatively acting nod genes and a single host gene affects cultivar specificity in a manner analogous to, but more complex than, the gene-for-gene interactions common amongst plant-pathogen interactions.

A Lipopolysaccharide Mutant ofBradyrhizobium japonicumthat Uncouples Plant from Bacterial Differentiation

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.

Anatomical comparison of wild-type and non-nodulating mutant chickpea (Cicer arietinum)

Non-nodulating chickpea (Cicer arietinum L.) mutant PM233B was characterized anatomically via comparison with its normally nodulating parent line ICC 640. Root hair and cortical cell infection threads, cortical cell division centers, and nodule formation were observed by light microscopy in serial root sections of ICC 640, but were absent in PM233B. Scanning electron microscope observations of inoculated root sections showed that ICC 640 and PM233B were indistinguishable in adsorption of chickpea Rhizobium strain CC1192. Thus, the rhizobial infection process was blocked in PM233B at a stage subsequent to root hair adsorption of bacteria, but prior to initiation of infection threads and root cortical cell division. Reciprocal shoot grafts between ICC 640 and PM233B demonstrated that the non-nodulation phenotype of PM233B was controlled by the root, and not the shoot, genotype. Key words: chickpea, Cicer arietinum, root nodule, symbiosis, non-nodulating mutant.

Interaction of Rhizobium fredii USDA257 and nodulation mutants derived from it with the agronomically improved soybean cultivar McCall

Rhizobium fredii USDA257 does not nodulate McCall soybean (Glycine max (L.) Merr.), but two transposon-mutants derived from it, 257DH4 and 257DH5, do. All three organisms cause curling of McCall root hairs and induce the formation of underlying cortical cell divisions. The mutants produce infection threads, and many of the meristematic foci develop into nodules. In contrast, root hairs that deform in response to USDA257 lack infection threads, and meristematic activity ceases prior to the appearance of nodule meristems. Root systems nodulated by mutant 257DH4 reduce acetylene at rates similar to those of roots nodulated by reference R. fredii strain USDA191. The presence of living cells of USDA257 in inocula leads to strong inhibition of nodulation by 257DH4 but not by 257DH5. This blocking effect depends on the ratio of USDA257 cells to 257DH4 cells in the inoculum; nodules that form contain cells of 257DH4, but not those of parental strain USDA257.