Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses

We used the cytokinin-responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a beta-glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root-knot nematode (RKN) second-stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode-plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6-(Delta2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode-induced root galls per plant, than control hairy roots.     

The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti

Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.     

Accumulation of an Aromatic Amine, β-Phenethylamine, in Root Nodules of Adzuki Bean Vigna angularis

Neuroactive aromatic amines acting on the central nervous system are widespread in the plant kingdom. We have previously found β-phenethylamine (β-PHA), one of the aromatic alkaloids, in root nodules of various annual legume crops. The present study was undertaken to determine the site of β-PHA accumulation within root nodules of the adzuki bean Vigna angularis. High concentrations of β-PHA were always detected in the alkaloid fraction of adzuki bean root nodules. Related aromatic amines such as tyramine, dopamine, and other β-PHA derivatives, which are found in various medicinal plants, were not detected in adzuki bean root nodules. The amounts of β-PHA in root nodules varied not only with the growth stage of the host plant, but also with nodule age; β-PHA levels increased with nodule development, but declined with nodule senescence. Adzuki bean nodules, after crushing with a grinding medium, were separated into bacteroids and a nodule cytosol fraction. A large amount of β-PHA was detected in the bacteroids, while a very small amount was prsent in the nodule cytosol fraction derived from plant cells. The bacteroids in the mature nodules contained considerably higher amounts of β-PHA than did those in immature or senescent nodules. The formation of β-PHA in root-nodule bacteria was then tested using eight strains of Rhizobiaceae (Rhizobium, Bradyrhizobium and Sinorhizobium), including a strain isolated from root nodules of field-grown adzuki bean plant. None of the cultured cells produced β-PHA in liquid media in the presence or absence of phenylalanine, a putative precursor of β-PHA. Nitrogen-fixing bacteroids within nodules are the cells uniquely differentiated from root-nodule bacteria. The present results suggest that β-PHA is formed associated with the differentiation of vegetative bradyrhizobia into nitrogen-fixing bacteroids with the plant host cells.     

Production of growth hormones inCasuarina cunninghamiana root nodules induced byFrankia strain HFPCgI4

Summary Measurements of auxin and cytokinin activities in extracts ofCasuarina root nodules were made. The nodules were induced either by pure culture ofFrankia strain CgI4 or by crushed nodule inoculum. Levels of cytokinin activity were significantly higher in root nodules induced by pureFrankia culture than in those induced by crushed nodule inoculum. However, the reasons for this are unknown. Seasonal variation in levels of cytokinin activity inCasuarina nodules has also been detected.Dedicated to the memory of Professor John G. Torrey     

Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition

We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.     

LjABCB1, an ATP-binding cassette protein specifically induced in uninfected cells of Lotus japonicus nodules

Legume plants develop root nodules through symbiosis with rhizobia, and fix atmospheric nitrogen in this symbiotic organ. Development of root nodules is regulated by many metabolites including phytohormones. Previously, we reported that auxin is strongly involved in the development of the nodule vascular bundle and lenticel formation on the nodules of Lotus japonicus. Here we show that an ATP-binding cassette (ABC) protein, LjABCB1, which is a homologue of Arabidopsis auxin transporter AtABCB4, is specifically expressed during nodulation of L. japonicus. A reporter gene analysis indicated that the expression of LjABCB1 was restricted to uninfected cells adjacent to infected cells in the nodule, while no expression was observed in shoot apical meristems or root tips, in which most auxin transporter genes are expressed. The auxin transport activity of LjABCB1 was confirmed using a heterologous expression system.     

Morphogenetic Rescue of Rhizobium meliloti Nodulation Mutants by trans-Zeatin Secretion

The development of nitrogen-fixing nodules is induced on the roots of legume host plants by Rhizobium bacteria. We employed a novel strategy to probe the underlying mechanism of nodule morphogenesis in alfalfa roots using pTZS, a broad host range plasmid carrying a constitutive trans-zeatin secretion (tzs) gene from Agrobacterium tumefaciens T37. This plasmid suppressed the Nod- phenotype of Rhizobium nodulation mutants such that mutants harboring pTZS stimulated the formation of nodulelike structures. Alfalfa roots formed more or fewer of these nodules according to both the nitrogen content of the environment and the position along the root at which the pTZS+ bacteria were applied, which parallels the physiological and developmental regulation of true Rhizobium nodule formation. This plasmid also conferred on Escherichia coli cells the ability to induce root cortical cell mitoses. Both the pattern of induced cell divisions and the spatially restricted expression of an alfalfa nodule-specific marker gene (MsENOD2) in pTZS-induced nodules support the conclusion that localized cytokinin production produces a phenocopy of nodule morphogenesis.     

Phytohormones,Rhizobium mutants,and nodulation in legumes. VII. Identification and quantification of cytokinins in effective and ineffective pea root nodules using radioimmunoassay

Radioimmunoassays (RIA), employing antisera raised in rabbits against bovine serum albumin conjugates of zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine, were used to estimate levels of these cytokinins and their corresponding bases in samples of effective (nitrogen-fixing, Fix⁺), ineffective (nonnitrogen-fixing, Fix^–) pea root nodules and uninoculated roots. Assays were done on extracts of nodule tissue, 1–2 g fresh weight, or approximately 10 g fresh weight of root tissue, and high specific activity [³H]zeatin riboside was added during preparation of the extract for use as a recovery marker. Two different purification procedures were employed, each involving several purification steps. High performance liquid chromatography (HPLC) was the final step in both procedures. Fractions from HPLC were analyzed by RIA using the appropriate antiserum. The cytokinins, zeatin, zeatin riboside, dihydrozeatin riboside, isopentenyl adenine, and isopentenyladenosine were detected and quantified in nodule tissue, and similarly, in root tissue (with the exception of zeatin, which we were unable to quantify in root tissue). Cytokinin levels in nodule tissue were higher than those in root tissue. The major cytokinins detected in nodule tissue were zeatin, followed by zeatin riboside and then dihydrozeatin riboside. The levels of zeatin and zeatin riboside estimated in nodules in the present study by RIA were of the same order of magnitude, though tending to be a little higher, than values obtained previously by bioassay. Dihydrozeatin riboside was identified with confidence for the first time in nodule tissue. There was a general decline with age in cytokinin levels in nodules, but no major qualitative change in nodule cytokinins with age. For theRhizobium strains examined, the data did not indicate a clear correlation between nodule cytokinin levels and the effectiveness of nodules in nitrogen fixation.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Distribution and Seasonal Changes in Levels of Cytokinins in Alnus glutinosa (L.) Gaertn

The root nodules of both dormant and non-dormant plants of Alnusglutinosa (L.) Gaertn. were found (by the soybean callus bioassay)to contain levels of cytokinin activity greatly exceeding thoseof other parts of young vegetative plants. A large, transient increase in cytokinin activity occurred inthe nodules at the time of bud break. Similar, although muchsmaller, increases were detected also in roots and buds. Theincrease in the level of nodule cytokinin activity was observedboth in mature trees and in young pot-grown plants in two successiveyears. A second peak of cytokinin activity, considered to bederived from cytokinin nucleotides, was found in the nodulesof mature trees in midsummer. Analysis of cytokinin extracts of different plant parts by meansof a Sephadex LH20 column revealed the presence of three mainpeaks of activity, with elution volumes corresponding to thoseof zeatin-9-glucoside, zeatin riboside, and zeatin. While theglucoside-like peak -was predominant in the nodules and leavesit was not detected in root pressure sap. A zeatin ribo-side-likepeak was the major cytokinin in the roots and root pressuresap. These findings are discussed in relation to current hypothesesconcerning the production, distribution, and possible physiologicalroles of the cytokinins.     

Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation

Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.     

The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root

Biological nitrogen fixation in legumes occurs in nodules that are initiated in the root cortex following Nod factor recognition at the root surface, and this requires coordination of diverse developmental programs in these different tissues. We show that while early Nod factor signaling associated with calcium oscillations is limited to the root surface, the resultant activation of Nodule Inception (NIN) in the root epidermis is sufficient to promote cytokinin signaling and nodule organogenesis in the inner root cortex. NIN or a product of its action must be associated with the transmission of a signal between the root surface and the cortical cells where nodule organogenesis is initiated. NIN appears to have distinct functions in the root epidermis and the root cortex. In the epidermis, NIN restricts the extent of Early Nodulin 11 (ENOD11) expression and does so through competitive inhibition of ERF Required for Nodulation (ERN1). In contrast, NIN is sufficient to promote the expression of the cytokinin receptor Cytokinin Response 1 (CRE1), which is restricted to the root cortex. Our work in Medicago truncatula highlights the complexity of NIN action and places NIN as a central player in the coordination of the symbiotic developmental programs occurring in differing tissues of the root that combined are necessary for a nitrogen-fixing symbiosis.