Mutual regulation of plant phospholipase D and the actin cytoskeleton

Membrane lipids and cytoskeleton dynamics are intimately inter‐connected in the eukaryotic cell; however, only recently have the molecular mechanisms operating at this interface in plant cells been addressed experimentally. Phospholipase D (PLD) and its product phosphatidic acid (PA) were discovered to be important regulators in the membrane–cytoskeleton interface in eukaryotes. Here we report the mechanistic details of plant PLD–actin interactions. Inhibition of PLD by n‐butanol compromises pollen tube actin, and PA rescues the detrimental effect of n‐butanol on F‐actin, showing clearly the importance of the PLD–PA interaction for pollen tube F‐actin dynamics. From various candidate tobacco PLDs isoforms, we identified NtPLDβ1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP₂‐dependent PLD (PLDβ) is specifically enhanced by F‐actin and inhibited by G‐actin. We then identified the NtPLDβ1 domain responsible for actin interactions. Using sequence‐ and structure‐based analysis, together with site‐directed mutagenesis, we identified Asn323 and Thr382 of NtPLDβ1 as the crucial amino acids in the actin‐interacting fold. The effect of antisense‐mediated suppression of NtPLDβ1 or NtPLDδ on pollen tube F‐actin dynamics shows that NtPLDβ1 is the active partner in PLD–actin interplay. The positive feedback loop created by activation of PLDβ by F‐actin and of F‐actin by PA provides an important mechanism to locally increase membrane–F‐actin dynamics in the cortex of plant cells.     

Recent development and new insight of diversification and symbiosis specificity of legume rhizobia: mechanism and application

Currently, symbiotic rhizobia (sl., rhizobium) refer to the soil bacteria in α- and β-Proteobacteria that can induce root and/or stem nodules on some legumes and a few of nonlegumes. In the nodules, rhizobia convert the inert dinitrogen gas (N₂) into ammonia (NH₃) and supply them as nitrogen nutrient to the host plant. In general, this symbiotic association presents specificity between rhizobial and leguminous species, and most of the rhizobia use lipochitooligosaccharides, so called Nod factor (NF), for cooperating with their host plant to initiate the formation of nodule primordium and to inhibit the plant immunity. Besides NF, effectors secreted by type III secretion system (T3SS), exopolysaccharides and many microbe-associated molecular patterns in the rhizobia also play important roles in nodulation and immunity response between rhizobia and legumes. However, the promiscuous hosts like Glycine max and Sophora flavescens can nodulate with various rhizobial species harbouring diverse symbiosis genes in different soils, meaning that the nodulation specificity/efficiency might be mainly determined by the host plants and regulated by the soil conditions in a certain cases. Based on previous studies on rhizobial application, we propose a ‘1+n−N’ model to promote the function of symbiotic nitrogen fixation (SNF) in agricultural practice, where ‘1’ refers to appreciate rhizobium; ‘+n’ means the addition of multiple trace elements and PGPR bacteria; and ‘−N’ implies the reduction of chemical nitrogen fertilizer. Finally, open questions in the SNF field are raised to future think deeply and researches.     

Effects of Co-Inoculation with Arbuscular Mycorrhizal Fungi and Rhizobia on Fungal Occupancy in Chickpea Root and Nodule Determined by Real-Time PCR

Legume roots in nature are usually colonized with rhizobia and different arbuscular mycorrhizal fungi (AMF) species. Light microscopy that visualizes the presence of AMF in roots is not able to differentiate the ratio of each AMF species in the root and nodule tissues in mixed fungal inoculation. The purpose of this study was to characterize the dominant species of mycorrhiza in roots and nodules of plants co-inoculated with mycorrhizal fungi and rhizobial strains. Glomus intraradices (GI), Glomus mosseae (GM), their mix (GI + GM), and six Mesorhizobium ciceri strains were used to inoculate chickpea. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess occupancy of these fungal species in roots and nodules. Results showed that GI molecular ratio and relative density were higher than GM in both roots and nodules. These differences in molecular ratio and density between GI and GM in nodules were three folds higher than roots. The results suggested that M. ciceri strains have different effects on nodulation and mycorrhizal colonization pattern. Plants with bacterial S3 and S1 strains produced the highest root nodulation and higher fungal density in both the roots and nodules.     

A Cotyledonary Inhibitor of Root Nodulation in Pisum sativum

Root nodule formation was studied in 11-day-old seedlings of Pisum sativum L. cv. Alaska, which were inoculated with Rhizobium leguminosarum at the time of planting. On intact plants an identical nodulation pattern was observed in the lateral roots attached to the two xylem strands connected to the cotyledons. When one cotyledon was removed before germination, however, a highly significant increase in nodultion was observed on the lateral roots attached to the xylem strand which no longer was connected to a cotyledon. Excision of one cotyledon also caused an alteration in the radial location of root nodules on the primary root. Under these conditions there was a distinct promotion of nodular proliferations in the root cortex opposite the primary phloem poles. The fact that removal of one cotyledon increased nodulation on the lateral roots but had no effect on the rhizobial infection of lateral root hairs suggested that a cotyledonary inhibitor acts at a step between the infection process and the appearance of a macroscopic nodule. The data were interpreted in terms of an inhibitor of cortical cell division which is translocated from the cotyledons to the developing root via the phloem.     

A nodule endophytic Bacillus megaterium strain isolated from Medicago polymorpha enhances growth,promotes nodulation by Ensifer medicae and alleviates salt stress in alfalfa plants

A Gram‐positive, fast‐growing, endophytic bacterium was isolated from root nodules of Medicago polymorpha and identified as Bacillus megaterium. The isolate, named NMp082, co‐inhabited nodules with the symbiotic rhizobium Ensifer medicae. B. megaterium NMp082 contained nifH and nodD genes that were 100% identical to those of Ensifer meliloti, an unusual event that suggested previous lateral gene transfer from a different rhizobial species. Despite the presence of nodulation and nitrogen fixation genes, the endophyte was not able to form effective nodules; however, it induced nodule‐like unorganised structures in alfalfa roots. Axenic inoculation promoted plant growth in M. polymorpha, Medicago lupulina, Medicago truncatula and Medicago sativa, and co‐inoculation with E. medicae enhanced growth and nodulation of Medicago spp. plants compared with inoculation with either bacterium alone. B. megaterium NMp082 also induced tolerance to salt stress in alfalfa and Arabidopsis plants. The ability to produce indole acetic acid (IAA) and the 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase activity displayed by the endophyte in vitro might explain the observed plant growth promotion and salt stress alleviation. The isolate was also highly tolerant to salt stress, water deficit and to the presence of different heavy metals. The newly characterised endophytic bacterium possessed specific characteristics that point at potential applications to sustain plant growth and nodulation under abiotic stress.     

Involvement of auxin distribution in root nodule development of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

The symbiosis between legume plants and rhizobia causes the development of new organs, nodules which function as an apparatus for nitrogen fixation. In this study, the roles of auxin in nodule development in Lotus japonicus have been demonstrated using molecular genetic tools and auxin inhibitors. The expression of an auxin-reporter GH3 fused to β-glucuronidase (GUS) was analyzed in L. japonicus roots, and showed a strong signal in the central cylinder of the root, whereas upon rhizobium infection, generation of GUS signal was observed at the dividing outer cortical cells during the first nodule cell divisions. When nodules were developed to maturity, strong GUS staining was detected in vascular tissues of nodules, suggesting distinct auxin involvement in the determinate nodule development. Numbers and the development of nodules were affected by auxin transport inhibitors (1-naphthylphthalamic acid, NPA and triindobenzoic acid, TIBA), and by a newly synthesized auxin antagonist, α-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA). The common phenotypical alteration by these auxin inhibitors was the inhibition in forming lenticel which is normally developed on the nodule surface from the root outer cortex. The inhibition of lenticel formation was correlated with the inhibition of nodule vascular bundle development. These results indicate that auxin is required for the normal development of determinate nodules in a multidirectional manner.     

Stem Nodulation in Legumes: Diversity,Mechanisms, and Unusual Characteristics

Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A few aquatic legumes, however, can form nodules on their stem at dormant root primordia. The stem-nodulating legumes described so far are all members of the genera Aeschynomene, Sesbania, Neptunia, and Discolobium. Their rhizobial symbionts belong to four genera already described: Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. This review summarizes our current knowledge on most aspects of stem nodulation in legumes, the infection process and nodule development, the characterization and unusual features of the associated bacteria, and the molecular genetics of nodulation. Potential use as green manure in lowland rice of these stem-nodulating legumes, giving them agronomical importance, is also discussed.     

Effects of boron and calcium nutrition on the establishment of the Rhizobium leguminosarum–pea (Pisum sativum) symbiosis and nodule development under salt stress

The effects of modifying boron (B) and calcium (Ca²⁺) concentrations on the establishment and development of rhizobial symbiosis in Pisum sativum plants grown under salt stress were investigated. Salinity almost completely inhibited the nodulation of pea plants by Rhizobium leguminosarum bv. viciae 3841. This effect was prevented by addition of Ca²⁺ during plant growth. The capacity of root exudates derived from salt‐treated plants to induce Rhizobium nod genes was not significantly decreased. However, bacterial adsorption to roots was highly inhibited in plants grown with 75 mM NaCl. Moreover, R. leguminosarum 3841 did not grow in minimal media containing such salt concentration. High Ca²⁺ levels enhanced both rhizobial growth and adsorption to roots, and increased nodule number in the presence of high salt. Nevertheless, the nodules developed were not functional unless the B concentration was also increased. Because B has a strong effect on infection and cell invasion, these processes were investigated by fluorescence microscopy in pea nodules harbouring a R. leguminosarum strain that expresses green fluorescent protein. Salt‐stressed plants had empty nodules and only those treated with high B and high Ca²⁺ developed infection threads and exhibited enhanced cell and tissue invasion by Rhizobium. Overall, the results indicate that Ca²⁺ promotes nodulation and B nodule development leading to an increase of salt tolerance of nodulated legumes.     

Functional analysis of chimeric lysin motif domain receptors mediating Nod factor‐induced defense signaling in Arabidopsis thaliana and chitin‐induced nodulation signaling in Lotus japonicus

The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1–AtCERK1 and LjNFR5–AtCERK1, were expressed in cerk1‐2, an A. thaliana CERK1 mutant lacking chitin‐induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1‐2 expressing both chimeras accumulated reactive oxygen species, expressed chitin‐responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor‐binding protein), respectively. The chimeras, named OsCERK1–LjNFR1 and OsCEBiP–LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1‐1; nfr5‐2) carrying a GUS (β‐glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF‐induced defense signaling in non‐legumes and chitin‐induced nodulation signaling in legumes.     

Legume-Rhizobium Interactions: Cowpea Root Exudate Elicits Faster Nodulation Response by Rhizobium Species

Preinfection events in legume-Rhizobium symbiosis were analyzed by studying the different nodulation behaviors of two rhizobial strains in cowpeas (Vigna sinensis). Log-phase cultures of Rhizobium sp. strain 1001, an isolate from the plant nodule, initiated host responses leading to infection within 2 h after inoculation, whereas log-phase cultures of Rhizobium sp. strain 32H1 took at least 7 h to trigger a discernible response. The delay observed with strain 32H1 could be eliminated by incubating the rhizobial suspension, before inoculation, for 4.5 h either in the cowpea rhizosphere/rhizoplane condition or in the root exudate of cowpea plants, grown without NH₄⁺ in the rooting medium. The delay could not be eliminated by incubating the rhizobial suspension in the rooting medium of plants grown in the presence of 5 mM NH₄⁺, indicating that there is a regulatory role of combined nitrogen in triggering preinfection events by the legume. The substance(s) in the root exudate which elicited the faster nodulation response by Rhizobium sp. strain 32H1 could be separated into a high-molecular-weight fraction by Sephadex G-100 gel filtration. The data support the notion that legume roots release substances that favor the development of rhizobial features essential for infection and nodulation.     

The GmFWL1 (FW2‐2‐like) nodulation gene encodes a plasma membrane microdomain‐associated protein

The soybean gene GmFWL1 (FW2‐2‐like1) belongs to a plant‐specific family that includes the tomato FW2‐2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2‐2‐like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain‐associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process.     

Mutualism variation in the nodulation response to nitrate

The evolution of mutualisms under novel selective pressures will play a key role in ecosystem responses to environmental change. Because fixed nitrogen is traded in plant–rhizobium mutualisms, increasing N availability in the soil is predicted to alter coevolution of these interactions. Legumes typically decrease the number of associations (nodules) with rhizobia in response to nitrate, but the evolutionary dynamics of this response remain unknown. We grew plant and rhizobium genotype combinations in three N environments to assess the coevolutionary potential of the nodule nitrate response in natural communities of plants and rhizobia. We found evidence for coevolutionary genetic variation for nodulation in response to nitrate (G × G × E interaction), suggesting that the mutualism response to N deposition will depend on the combination of partner genotypes. Thus, the nitrate response is not a fixed mechanism in plant–rhizobium symbioses, but instead is potentially subject to natural selection and dynamic coevolution.     

Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation

Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [(32)P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.     

Strigolactones promote nodulation in pea

Strigolactones are recently defined plant hormones with roles in mycorrhizal symbiosis and shoot and root architecture. Their potential role in controlling nodulation, the related symbiosis between legumes and Rhizobium bacteria, was explored using the strigolactone-deficient rms1 mutant in pea (Pisum sativum L.). This work indicates that endogenous strigolactones are positive regulators of nodulation in pea, required for optimal nodule number but not for nodule formation per se. rms1 mutant root exudates and root tissue are almost completely deficient in strigolactones, and rms1 mutant plants have approximately 40% fewer nodules than wild-type plants. Treatment with the synthetic strigolactone GR24 elevated nodule number in wild-type pea plants and also elevated nodule number in rms1 mutant plants to a level similar to that seen in untreated wild-type plants. Grafting studies revealed that nodule number and strigolactone levels in root tissue of rms1 roots were unaffected by grafting to wild-type scions indicating that strigolactones in the root, but not shoot-derived factors, regulate nodule number and provide the first direct evidence that the shoot does not make a major contribution to root strigolactone levels.