Increase In Number Of Nodules Research Articles

Bradyrhizobium Inoculants Enhance Grain Yields of Soybean and Cowpea in Northern Ghana.

This study evaluated the symbiotic effectiveness and economic evaluation of Rhizobium inoculants with the objective of recommending the most effective inoculant strain for soybean and cowpea production in Northern Ghana. Field experiments were established in three locations using randomized complete block design with five blocks. A total of four treatments (Legumefix, Biofix, 100 kg N ha-1 and uninoculated control for soybean and BR 3267, BR 3262, 100 kg N ha-1 and uninoculated control for cowpea) were applied. At Nyankpala, inoculation of soybean with Legumefix and Biofix led to significant (P < 0.05) increases in nodule number (90–118%), nodule dry weight (>two-folds), and grain yield (12–19%) relative to the control. The Biofix effect on soybean grain yield was 1.5-fold of Legumefix. Similarly, inoculation of cowpea with BR 3262 and BR 3267 significantly (P < 0.05) increased nodule number (41–68%), nodule dry weight (45–65%), and grain yield (11–38%) relative to the control. Strain BR 3267 performed consistently (>two-folds) better than BR 3262 on grain yield. At Nyagli, there was no significant effect of inoculation on cowpea. Wilks lambda values (0.067, 0.039; P = 0.00) indicated that 93.3 and 96.1% of the variations observed in soybean and cowpea, respectively, were due to the applied inoculants. Biofix and BR 3267 were economically profitable with VCR ratio of 8.7 and 4.6, respectively. Based on grain yield and economic returns observed, Biofix and BR 3267 can be recommended in Nyankpala for inoculation of soybean and cowpea, respectively.

Nodulation, Nutrient Uptake and Yield of Common Bean Inoculated with Rhizobia and Trichoderma in an Acid Soil

Common bean is an important source of protein, fat, carbohydrate, vitamins and minerals for both human beings and livestock. In Zambia, common bean is produced mostly by smallholder farmers whose current yields fall far short of the potential 2 t/ha due to various challenges. Among the biophysical constraints, poor soil fertility and acidity pose the greatest challenges. This study investigated the individual and dual inoculation of Rhizobia and Trichoderma to common bean in a phosphorus deficient, acid soil. Soil in which the common bean was grown was characterized for selected chemical properties before planting and at harvest using standard laboratory procedures. Soils were amended with nitrogen and phosphorus fertilizer at the rates of 100 kg N and 80 kg P2O5 per ha; 1 g/kg of seed of Trichoderma harzianum; 100 g/kg of seed of Rhizobium tropici; or a combination of both at the recommended rates at planting. Nodulation and nodule effectiveness were determined at 51 days after planting. Nitrogen and phosphorus accumulation in the above ground biomass, biomass and grain yields were determined at maturity. To determine differences among soil amendments, data were analysed using Analysis of Variance and Least Significant Difference at 95% confidence limit. Relationships among parameters were determined using correlation analysis. The results showed that amending soils with inorganic N at high rates can depress nodulation even in the presence of high levels of inorganic phosphorus. Inoculating common bean with Rhizobia and Trichoderma either singly or in combination increases nodule number and effectiveness per plant but may not result in higher nitrogen and phosphorus accumulation, or in an increase in subsequent biomass or grain yields. The low phosphorus accumulation in Trichoderma inoculated plants observed in this study needs to be investigated further by studying the extent of colonization and the accompanying changes in root volume.

Gene expression and localization of a β-1,3-glucanase of Lotus japonicus.

Phytohormone abscisic acid (ABA) inhibits root nodule formation of leguminous plants. LjGlu1, a β-1,3-glucanase gene of Lotus japonicus, has been identified as an ABA responsive gene. RNA interference of LjGlu1 increased nodule number. This suggests that LjGlu1 is involved in the regulation of nodule formation. Host legumes control nodule number by autoregulation of nodulation (AON), in which the presence of existing root nodules inhibits further nodulation. For further characterization of LjGlu1, we focused on the expression of LjGlu1 in relation to AON. In a split-root system, LjGlu1 expression peaked when AON was fully induced. Hairy roots transformed with LjCLE-RS1, a gene that induces AON, were generated. Expression of LjGlu1 was greater in the transgenic roots than in untransformed roots. LjGlu1 was not induced in a hypernodulating mutant inoculated with Mesorhizobium loti. These results suggest that the expression of LjGlu1 is involved in the system of AON. However, neither hypernodulation nor enlarged nodulation zone was observed on the transgenic hairy roots carrying LjGlu1-RNAi, suggesting that LjGlu1 is not a key player of AON. Recombinant LjGlu1 showed endo-β-1,3-glucanase activity. LjGlu1-mOrange fusion protein suggested that LjGlu1 associated with M. loti on the root hairs. Exogenous β-1,3-glucanase inhibited infection thread formation by both the wild type and the mutant, and nodule numbers were reduced. These results suggest that LjGlu1 is expressed in response to M. loti infection and functions outside root tissues, resulting in the inhibition of infection.

Interactions between ethylene, gibberellins, and brassinosteroids in the development of rhizobial and mycorrhizal symbioses of pea.

The regulation of arbuscular mycorrhizal development and nodulation involves complex interactions between the plant and its microbial symbionts. In this study, we use the recently identified ethylene-insensitive ein2 mutant in pea (Pisum sativum L.) to explore the role of ethylene in the development of these symbioses. We show that ethylene acts as a strong negative regulator of nodulation, confirming reports in other legumes. Minor changes in gibberellin1 and indole-3-acetic acid levels in ein2 roots appear insufficient to explain the differences in nodulation. Double mutants produced by crosses between ein2 and the severely gibberellin-deficient na and brassinosteroid-deficient lk mutants showed increased nodule numbers and reduced nodule spacing compared with the na and lk single mutants, but nodule numbers and spacing were typical of ein2 plants, suggesting that the reduced number of nodules innaandlkplants is largely due to the elevated ethylene levels previously reported in these mutants. We show that ethylene can also negatively regulate mycorrhizae development when ethylene levels are elevated above basal levels, consistent with a role for ethylene in reducing symbiotic development under stressful conditions. In contrast to the hormone interactions in nodulation, ein2 does not override the effect of lk or na on the development of arbuscular mycorrhizae, suggesting that brassinosteroids and gibberellins influence this process largely independently of ethylene.

Exploring plant growth-promotion actinomycetes from vermicompost and rhizosphere soil for yield enhancement in chickpea.

The main objective of the present study was to isolate and characterize actinomycetes for their plant growth-promotion in chickpea. A total of 89 actinomycetes were screened for their antagonism against fungal pathogens of chickpea by dual culture and metabolite production assays. Four most promising actinomycetes were evaluated for their physiological and plant growth-promotion properties under in vitro and in vivo conditions. All the isolates exhibited good growth at temperatures from 20 °C to 40 °C, pH range of 7–11 and NaCl concentrations up to 8%. These were also found highly tolerant to Bavistin, slightly tolerant to Thiram and Captan (except VAI-7 and VAI-40) but susceptible to Benlate and Ridomil at field application levels and were found to produce siderophore, cellulase, lipase, protease, chitinase (except VAI-40), hydrocyanic acid (except VAI-7 and VAI-40), indole acetic acid and β-1,3-glucanase. When the four actinomycetes were evaluated for their plant growth-promotion properties under field conditions on chickpea, all exhibited increase in nodule number, shoot weight and yield. The actinomycetes treated plots enhanced total N, available P and organic C over the un-inoculated control. The scanning electron microscope studies exhibited extensive colonization by actinomycetes on the root surface of chickpea. The expression profiles for indole acetic acid, siderophore and β-1,3-glucanase genes exhibited up-regulation for all three traits and in all four isolates. The actinomycetes were identified as Streptomyces but different species in the 16S rDNA analysis. It was concluded that the selected actinomycetes have good plant growth-promotion and biocontrol potentials on chickpea.

GmEXPB2, a Cell Wall β-Expansin, Affects Soybean Nodulation through Modifying Root Architecture and Promoting Nodule Formation and Development.

Nodulation is an essential process for biological nitrogen (N2) fixation in legumes, but its regulation remains poorly understood. Here, a β-expansin gene, GmEXPB2, was found to be critical for soybean (Glycine max) nodulation. GmEXPB2 was preferentially expressed at the early stage of nodule development. β-Glucuronidase staining further showed that GmEXPB2 was mainly localized to the nodule vascular trace and nodule vascular bundles, as well as nodule cortical and parenchyma cells, suggesting that GmEXPB2 might be involved in cell wall modification and extension during nodule formation and development. Overexpression of GmEXPB2 dramatically modified soybean root architecture, increasing the size and number of cortical cells in the root meristematic and elongation zones and expanding root hair density and size of the root hair zone. Confocal microscopy with green fluorescent protein-labeled rhizobium USDA110 cells showed that the infection events were significantly enhanced in the GmEXPB2-overexpressing lines. Moreover, nodule primordium development was earlier in overexpressing lines compared with wild-type plants. Thereby, overexpression of GmEXPB2 in either transgenic soybean hairy roots or whole plants resulted in increased nodule number, nodule mass, and nitrogenase activity and thus elevated plant N and phosphorus content as well as biomass. In contrast, suppression of GmEXPB2 in soybean transgenic composite plants led to smaller infected cells and thus reduced number of big nodules, nodule mass, and nitrogenase activity, thereby inhibiting soybean growth. Taken together, we conclude that GmEXPB2 critically affects soybean nodulation through modifying root architecture and promoting nodule formation and development and subsequently impacts biological N2 fixation and growth of soybean.

Pea nodule gradients explain N nutrition and limited symbiotic fixation in hypernodulating mutants

Legumes fix atmospheric nitrogen by symbiosis with soil bacteria in root nodules. Legume yields are limited by the low capacity of N2 fixation. Hypernodulating mutants have been selected decades ago to try to increase nodule number. However, literature data show that N fixation of hypernodulating mutants was not increased compared to parental lines. Here, we study the functional basis of limited N fixation associated to hypernodulation. We grew two wild type genotypes and nine hypernodulating mutants of pea in hydroponics in three greenhouse experiments. We measured the following traits related to N nutrition during the vegetative period: nodule number, plant N uptake, nodule-specific activity, and plant and nodule concentrations. Genetic and environmental variations induced nodule gradients. These gradients were used to set quantitative relationships between N nutrition traits and nodule number. We compared the relationships obtained for hypernodulating and for wild types. N nutrition traits were analysed together with C nutrition traits, through correlation networks. Our results show that higher nodule number of hypernodulating mutants is correlated with lower levels of nodule activity, from −25 to −60 %, by comparison to the wild type. Higher nodule number of hypernodulating mutants is also correlated with lower total N uptake by symbiotic fixation, from −0 to −60 %, by comparison to the wild type. Findings demonstrate that N nutrition is not a factor limiting growth in hypernodulating mutants, as shown by N nutrition index higher than 1, indicating N nutrition in excess. The correlations suggest that limited N2 fixation in hypernodulating mutants arises from restricted shoot growth, which limits the plant capacity to accumulate N. Furthermore, symbiotic efficiency decreased with increasing nodule number, down to a minimal value for hypernodulating mutants. Thus, to overcome the trade-off between N benefits from N2 fixation and carbon nodulation costs, the hypernodulation trait should be associated with high shoot growth capacity in breeding programs.

Tripartite mutualism: facilitation or trade-offs between rhizobial and mycorrhizal symbionts of legume hosts.

• Multiple mutualist effects (MMEs) are common in nature, yet we lack a predictive understanding of how two mutualists on the same host will influence each other and whether these effects will be positive or negative. Leguminous plants maintain root symbioses with two nutritional mutualists: rhizobia that fix atmospheric nitrogen and arbuscular mycorrhizal fungi (AMF) that increase phosphorus uptake. Both symbionts receive plant carbon, and host genetic networks that regulate colonization are partially shared by both symbioses; whether these factors generate trade-offs or facilitation between rhizobial and AMF symbionts of legumes is not well known.• We evaluated host allocation to each symbiont in three settings. First, in situ in a remnant prairie, then in a greenhouse experiment with multiple plant populations, and finally under manipulated rhizobium densities in the greenhouse.• In the remnant prairie, rhizobium nodule number and colonization of AMF were positively correlated, and plants with increased nodule number had higher fitness in the field, generating indirect selection on the colonization of AMF. In the greenhouse experiment, allocation to each symbiont was genetically variable among populations, with some suggestion that rhizobium and AMF colonization are positively genetically correlated. Finally, increasing the number of rhizobia in the soil decreased AMF colonization.• Our results suggest that trade-offs between plant colonization by rhizobia and AMF are context dependent and might not be common under field conditions, but that physiological and/or genetic drivers couple these two symbioses in nature.

Phytosulfokine Is Involved in Positive Regulation of Lotus japonicus Nodulation.

Phytosulfokine (PSK) is a tyrosine-sulfated peptide that is widely distributed in plants, participating in cell proliferation, differentiation, and innate immunity. The potential role of PSK in nodulation in legumes has not been reported. In this work, five PSK precursor genes were identified in Lotus japonicas, designated as LjPSK1 to LjPSK5. Three of them (LjPSK1, LjPSK4, and LjPSK5) were found to be expressed in nitrogen-fixing root nodules. LjPSK1 and LjPSK4 were not induced at the early stage of nodulation. Interestingly, while the expression of LjPSK4 was also found in spontaneous nodules without rhizobial colonization, LjPSK1 was not induced in these pseudo nodules. Promoter-β-glucuronidase analysis revealed that LjPSK1 was highly expressed in enlarged symbiotic cells of nodules. Exogenous addition of 1 1M synthetic PSK peptide resulted in increased nodule numbers per plant. Consistently, the number of mature nodules but not the events of rhizobial infection and nodule initiation was increased by overexpressing LjPSK1 in transgenic hairy roots, in which the expression of jasmonate-responsive genes was found to be repressed. These results suggest that PSK is a new peptide signal that regulates nodulation in legumes, probably through cross-talking with other phytohormones.

Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase activity shows enhanced growth when grown under N2-fixing conditions.

Overexpression of SPS in alfalfa is accompanied by early flowering, increased plant growth and an increase in elemental N and protein content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS; EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome of overexpression of SPS in different plants using transgenic approaches has been quite varied, but the general consensus is that increased SPS activity is associated with the production of new sinks and increased sink strength. In legumes, the root nodule is a strong C sink and in this study our objective was to see how increasing SPS activity in a legume would affect nodule number and function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed that overexpression of SPS in alfalfa, is accompanied by an increase in nodule number and mass and an overall increase in nitrogenase activity at the whole plant level. The nodules exhibited an increase in the level of key enzymes contributing to N assimilation including glutamine synthetase and asparagine synthetase. Moreover, the stems of the transformants showed higher level of the transport amino acids, Asx, indicating increased export of N from the nodules. The transformants exhibited a dramatic increase in growth both of the shoots and roots, and earlier flowering time, leading to increased yields. Moreover, the transformants showed an increase in elemental N and protein content. The overall conclusion is that increased SPS activity improves the N status and plant performance, suggesting that the availability of more C in the form of sucrose enhances N acquisition and assimilation in the nodules.

Inoculation of Phaseolus vulgaris with the nodule-endophyte Agrobacterium sp. 10C2 affects richness and structure of rhizosphere bacterial communities and enhances nodulation and growth.

Agrobacterium sp. 10C2 is a nonpathogenic and non-symbiotic nodule-endophyte strain isolated from root nodules of Phaseolus vulgaris. The effect of this strain on nodulation, plant growth and rhizosphere bacterial communities of P. vulgaris is investigated under seminatural conditions. Inoculation with strain 10C2 induced an increase in nodule number (+54%) and plant biomass (+16%). Grains also showed a significant increase in phosphorus (+53%), polyphenols (+217%), flavonoids (+62%) and total antioxidant capacity (+82%). The effect of strain 10C2 on bacterial communities was monitored using terminal restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. When the initial soil was inoculated with strain 10C2 and left 15days, the Agrobacterium strain did not affect TRF richness but changed structure. When common bean was sown in these soils and cultivated during 75days, both TRF richness and structure were affected by strain 10C2. TRF richness increased in the rhizosphere soil, while it decreased in the bulk soil (root free). The taxonomic assignation of TRFs induced by strain 10C2 in the bean rhizosphere revealed the presence of four phyla (Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria) with a relative preponderance of Firmicutes, represented mainly by Bacillus species. Some of these taxa (i.e., Bacillus licheniformis, Bacillus pumilus, Bacillus senegalensis, Bacillus subtilis, Bacillus firmus and Paenibacillus koreensis) are particularly known for their plant growth-promoting potentialities. These results suggest that the beneficial effects of strain 10C2 observed on plant growth and grain quality are explained at least in part by the indirect effect through the promotion of beneficial microorganisms.

Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula.

Small, post-translationally modified and secreted peptides regulate diverse plant developmental processes. Due to low natural abundance, it is difficult to isolate and identify these peptides. Using an improved peptide isolation protocol and Orbitrap mass spectrometry, nine 15-amino-acid CEP peptides were identified that corresponded to the two domains encoded by Medicago truncatula CEP1 (MtCEP1). Novel arabinosylated and hydroxylated peptides were identified in root cultures overexpressing MtCEP1. The five most abundant CEP peptides were hydroxylated and these species were detected also in low amounts in vector control samples. Synthetic peptides with different hydroxylation patterns differentially affected root development. Notably, the domain 1 peptide hydroxylated at Pro4 and Pro11 (D1:HyP4,11) imparted the strongest inhibition of lateral root emergence when grown with 5mM KNO3 and stimulated the highest increase in nodule number when grown with 0mM KNO3. Inhibition of lateral root emergence by D1:HyP4,11 was not alleviated by removing peptide exposure. In contrast, the domain 2 peptide hydroxylated at Pro11 (D2:HyP11) increased stage III-IV lateral root primordium numbers by 6-fold (P < 0.001) which failed to emerge. Auxin addition at levels which stimulated lateral root formation in wild-type plants had little or no ameliorating effect on CEP peptide-mediated inhibition of lateral root formation or emergence. Both peptides increased and altered the root staining pattern of the auxin-responsive reporter GH3:GUS suggesting CEPs alter auxin sensitivity or distribution. The results showed that CEP primary sequence and post-translational modifications influence peptide activities and the improved isolation procedure effectively and reproducibly identifies and characterises CEPs.

English

Cold-tolerant plant growth-promoting rhizobacteria (PGPR) are of immense agronomic importance due to the fact that the winter season crop growing cycle is subject to varying spells of low temperatures and such PGPRs are metabolically functional at cold conditions. Thirty-five (35) rhizobacterial isolates were isolated from lentil rhizospheric soil, six isolates showing maximum exponential growth at both 10 and 15&deg;C after 24 h were selected and characterized belonging to genera Bacillus and Pseudomonas. Production of indole acetic acid (IAA) ranged from 12.7-17.5 &micro;gml-1 at 10&deg;C to 18.4-20.4 &mu;g/ml at 15&deg;C. At 15&deg;C, an isolate J-17 was found to be strong HCN producer whereas at 10&deg;C, only two isolates: J-26 and J-30 were moderate producers. P-solubilizion at 15&deg;C, ranged from 62.5 - 77.2 &micro;gml-1. However, at 10&deg;C none of the isolates solubilized P. At 15&deg;C, catechol type siderophore production ranged from 523.4 - 606.1 &micro;gml-1. On the basis of the PGP traits, four isolates J-3, J-17, J-18 and J-30 were selected for evaluation under field conditions of lentil at research farm of PAU, Ludhiana, Punjab, India. Coinoculation exhibited a significant increase in nodule number, nodule dry weight, plant and root dry weight, chlorophyll and leghaemoglobin content over Rhizobium alone. Application of J-3, J-17, J-18 and J-30 along with Rhizobium further enhanced the grain yield (1.8, 4.4, 3 and 1.4%). Key words: HCN production, indole acetic acid, siderophore, P-solubilization.

Endophytic bacteria improve nodule function and plant nitrogen in soybean on co-inoculation with Bradyrhizobium japonicum MN110

The potential of bacterial endophytes to improve symbiotic efficiency through synergistic interactions with rhizobia can help to improve nodulation and nitrogen fixation in legume plants. In the present study, we compared the effect of endophytic plant growth-promoting bacteria on nodulation and effective rhizobial symbiosis in soybean. Nodule endophyte Bacillus megaterium LNL6 isolated from root nodules of Lesperdeza sp. and plant endophyte Methylobacterium oryzae CBMB20 isolated from rice leaves were selected as endophytic co-inoculants. Treatment of Bradyrhizobium japonicum MN110 along with B. megaterium LNL6 and M. oryzae CBMB20 exhibited an increase in nodule number in pots at 35 days after sowing compared to single inoculation of MN110. Additionally, both the co-inoculation treatments showed significant increase in nodule activity which was measured in terms of nodule leghemoglobin content, nodulated root ARA and total plant nitrogen content compared to solitary inoculation of B. japonicum MN110. Though ACCD activity of the co-inoculated strains can be attributed to increase in nodule number, the observed increase in root nitrogenase activity and leghemoglobin content in the nodules is understood as due to plant growth promotion traits of the specific endophytic strains. High levels of IAA produced by B. megaterium LNL6 can be considered to have aided development of mature nodules which thereby improved the nodular nitrogen fixation. Thus, endophytic lifestyle combined with plant growth-promoting traits, such as IAA production, ACC deaminase, cellulase and nitrogenase activity by B. megaterium LNL6 and M. oryzae CBMB20, contributes to the improvement of overall symbiotic nitrogen fixation by the plant.

A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal increases nodule numbers in Medicago truncatula independent of autoregulation.

N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate cell-density dependent behaviors in many gram-negative bacteria, in particular those important for plant-microbe interactions. AHLs can also be recognized by plants, and this may influence their interactions with bacteria. Here we tested whether the exposure to AHLs affects the nodule-forming symbiosis between legume hosts and rhizobia. We treated roots of the model legume, Medicago truncatula, with a range of AHLs either from its specific symbiont, Sinorhizobium meliloti, or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis. We found increased numbers of nodules formed on root systems treated with the S. meliloti-specific AHL, 3-oxo-C14-homoserine lactone, at a concentration of 1 μM, while the other AHLs did not result in significant changes to nodule numbers. We did not find any evidence for altered nodule invasion by the rhizobia. Quantification of flavonoids that could act as nod gene inducers in S. meliloti did not show any correlation with increased nodule numbers. The effects of AHLs were specific for an increase in nodule numbers, but not lateral root numbers or root length. Increased nodule numbers following 3-oxo-C14-homoserine lactone treatment were under control of autoregulation of nodulation and were still observed in the autoregulation mutant, sunn4 (super numeric nodules4). However, increases in nodule numbers by 3-oxo-C14-homoserine lactone were not found in the ethylene-insensitive sickle mutant. A comparison between M. truncatula with M. sativa (alfalfa) and Trifolium repens (white clover) showed that the observed effects of AHLs on nodule numbers were specific to M. truncatula, despite M. sativa nodulating with the same symbiont. We conclude that plant perception of the S. meliloti-specific 3-oxo-C14-homoserine lactone influences nodule numbers in M. truncatula via an ethylene-dependent, but autoregulation-independent mechanism.

Phaseolus vulgaris-rhizobia symbiosis increases the phosphorus uptake and symbiotic N2 fixation under insoluble phosphorus

Five north Tunisian native rhizobia were tested in symbiosis with ‘Coco-blanc’ variety under two phosphorus (P) supplies [P-sufficient and insoluble P (IP)] at 250 μmol P plant−1. At flowering stage, responses against IP were assessed and indicated that inoculation with P.Ps.09 produced a high nodule biomass (0.12g). Similarly, P.Tb.09 produced 0.08 g nodule biomass under both P and P.Bj.09 that significantly increased nodule number under IP. Shoot biomass exhibited high dry weight with P.Tb.09 and CIAT899 (1.6 g plant−1). Inoculation with CIAT899 produced a high root biomass under both P. Analysis of a halo diameter of clear zone rounding each colony under IP indicated that P.Tb.09 has developed high halo Ø (1.7cm). These variations were associated under IP with a decrease of pH by P.Ar.09 and with increase in shoot phosphatase activity by P.Ar.09, P.Bj.09, P.OM.09 and P.Ps.09. The use of P-efficient rhizobia may constitute an adaptive mechanism against P-deficiency tolerance.

Lignin modification leads to increased nodule numbers in alfalfa.

Reduction of lignin levels in the forage legume alfalfa (Medicago sativa) by down-regulation of the monolignol biosynthetic enzyme hydroxycinnamoyl coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) results in strongly increased digestibility and processing ability of lignocellulose. However, these modifications are often also associated with dwarfing and other changes in plant growth. Given the importance of nitrogen fixation for legume growth, we evaluated the impact of constitutively targeted lignin modification on the belowground organs (roots and nodules) of alfalfa plants. HCT down-regulated alfalfa plants exhibit a striking reduction in root growth accompanied by an unexpected increase in nodule numbers when grown in the greenhouse or in the field. This phenotype is associated with increased levels of gibberellins and certain flavonoid compounds in roots. Although HCT down-regulation reduced biomass yields in both the greenhouse and field experiments, the impact on the allocation of nitrogen to shoots or roots was minimal. It is unlikely, therefore, that the altered growth phenotype of reduced-lignin alfalfa is a direct result of changes in nodulation or nitrogen fixation efficiency. Furthermore, HCT down-regulation has no measurable effect on carbon allocation to roots in either greenhouse or 3-year field trials.

Effect of Nitrogen and Sulphur Nutrition on Nitrogen assimilating Enzymes in Soybean Roots and Nodules

Soybean is an important legume crop with high protein content but deficient in sulphur (S) containing amino acids viz methionine and cysteine. Soybean protein quality can be improved by nutrient fertilization. Present studies report the effect of nitrogen (N) and S nutrition on nodulation, activities of ammonia assimilating enzymes and biochemical parameters in soybean roots and nodules. Nitrogen as urea @ 31.25 kg ha−1 and sulphur as gypsum @ 20 kg ha−1 alone significantly increased nodule number, fresh and dry weight per nodule, root length and nodular leghemoglobin content whereas total soluble proteins, free amino acids, glucose and sucrose content did not vary significantly in nodules and roots. Treatment of N or S significantly increased glutamate dehydrogenase activity in cytosol and bacteroidal fractions from 70 to 90 days after sowing whereas aspartate aminotransferase, glutamate synthase, glutamine synthetase and alanine aminotransferase activities showed non-significant variations in roots at different stages of development under the influence of N or S alone or their combination. Results suggested that N and S could improve nodulation and vegetative growth in soybean but ammonia assimilating enzyme activities in roots/nodules did not vary significantly.

A Nodule-Specific Lipid Transfer Protein AsE246 Participates in Transport of Plant-Synthesized Lipids to Symbiosome Membrane and Is Essential for Nodule Organogenesis in Chinese Milk Vetch

Rhizobia in legume root nodules fix nitrogen in symbiosomes, organelle-like structures in which a membrane from the host plant surrounds the symbiotic bacteria. However, the components that transport plant-synthesized lipids to the symbiosome membrane remain unknown. This study identified and functionally characterized the Chinese milk vetch (Astragalus sinicus) lipid transfer protein AsE246, which is specifically expressed in nodules. It was found that AsE246 can bind lipids in vitro. More importantly, AsE246 can bind the plant-synthesized membrane lipid digalactosyldiacylglycerol in vivo. Immunofluorescence and immunoelectron microscopy showed that AsE246 and digalactosyldiacylglycerol localize in the symbiosome membrane and are present in infection threads. Overexpression of AsE246 resulted in increased nodule numbers; knockdown of AsE246 resulted in reduced nodule numbers, decreased lipids contents in nodules, diminished nitrogen fixation activity, and abnormal development of symbiosomes. AsE246 knockdown also resulted in fewer infection threads, nodule primordia, and nodules, while AsE246 overexpression resulted in more infection threads and nodule primordia, suggesting that AsE246 affects nodule organogenesis associated with infection thread formation. Taken together, these results indicate that AsE246 contributes to lipids transport to the symbiosome membrane, and this transport is required for effective legume-rhizobium symbiosis.

MiR172 Regulates Soybean Nodulation

Micro-RNAs (miRNAs) play a pivotal role in the control of gene expression and regulate plant developmental processes. miRNA 172 (miR172) is a conserved miRNA in plants reported to control the expression of genes involved in developmental phase transition, floral organ identity, and flowering time. However, the specific role of miR172 in legume nodulation is undefined. Ectopic expression of soybean miR172 resulted in an increase in nodule numbers in transgenic roots and an increase in the expression of both symbiotic leghemoglobin and nonsymbiotic hemoglobin. These nodules showed higher levels of nitrogenase activity. Further analysis revealed a complex regulatory circuit in which miR156 regulates miR172 expression and controls the level of an AP2 transcription factor. The latter, either directly or indirectly, controls the expression of nonsymbiotic hemoglobin, which is essential for regulating the levels of nodulation.