Nitrogen fixation control under drought stress. Localized or systemic?

Legume-Rhizobium nitrogen fixation is dramatically affected under drought and other environmental constraints. However, it has yet to be established as to whether such regulation of nitrogen fixation is only exerted at the whole-plant level (e.g. by a systemic nitrogen feedback mechanism) or can also occur at a local nodule level. To address this question, nodulated pea (Pisum sativum) plants were grown in a split-root system, which allowed for half of the root system to be irrigated at field capacity, while the other half was water deprived, thus provoking changes in the nodule water potential. Nitrogen fixation only declined in the water-deprived, half-root system and this result was correlated with modifications in the activities of key nodule's enzymes such as sucrose synthase and isocitrate dehydrogenase and in nodular malate content. Furthermore, the decline in nodule water potential resulted in a cell redox imbalance. The results also indicate that systemic nitrogen feedback signaling was not operating in these water-stressed plants, since nitrogen fixation activity was maintained at control values in the watered half of the split-root plants. Thus, the use of a partially droughted split-root system provides evidence that nitrogen fixation activity under drought stress is mainly controlled at the local level rather than by a systemic nitrogen signal.     

STABILIZING MECHANISMS IN A LEGUME–RHIZOBIUM MUTUALISM

Preferential rewarding of more beneficial partners may stabilize mutualisms against the invasion of less beneficial, that is cheater, genotypes. Recent evidence suggests that both partner choice and sanctioning may play roles in preventing the invasion of less-beneficial rhizobia in legume–rhizobium mutualisms. The importance of these mechanisms in natural communities, however, remains unclear. We grew 12 Medicago truncatula maternal families with a mixture of three rhizobium strains from their native range for three plant generations and estimated the symbiotic benefits (nodule number and size) conferred to each rhizobium strain. In this experiment, the majority of M. truncatula genotypes formed more nodules with more beneficial rhizobium strains, providing evidence for adaptive partner choice. We also found that three generations of symbiosis resulted in an increase in the relative frequency of rhizobium strains that were most beneficial to plants—suggesting that partner choice affects rhizobium fitness. By contrast, we found no evidence that plants differentially rewarded rhizobia postnodulation via sanctioning leading to differences in nodule size. Taken together, our data suggest that plants have evolved to recognize beneficial rhizobial signals during the early stages of symbiosis, and that signaling between plants and rhizobia may be subject to coevolutionary pressures.     

The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations

The plant hormone ethylene negatively regulates bacterial infection and nodule formation in legumes in response to symbiotic rhizobia, but the molecular mechanism(s) of ethylene action in symbiosis remain obscure. We have identified and characterized multiple mutant alleles of the MtSkl1 gene, which controls both ethylene sensitivity and nodule numbers. We show that this locus encodes the Medicago truncatula ortholog of the Arabidopsis ethylene signaling protein EIN2. In addition to the well-characterized role of MtSkl1 in rhizobial symbiosis, we show that MtSkl1 is involved in regulating early phases of the symbiotic interaction with mycorrhizal fungi, and in mediating root responses to cytokinin. MtSkl1 also functions in the defense against Rhizoctonia solani and Phytophthora medicaginis , with the latter interaction likely to involve positive feedback amplification of ethylene biosynthesis. Overexpression of the C-terminal domain of MtEIN2 is sufficient to block nodulation responses, consistent with previous reports in Arabidopsis on the activation of ethylene signaling. This same C-terminal region is uniquely conserved throughout the EIN2 homologs of angiosperms, which is consistent with its role as a higher plant-specific innovation essential to EIN2 function.     

Metabolite profiles of nodulated alfalfa plants indicate that distinct stages of nodule organogenesis are accompanied by global physiological adaptations

An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago sativa is dependent on a balanced physiological interaction enabling the microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic interaction is regulated by still poorly understood control mechanisms. A first step toward a better understanding of nodule metabolism was the determination of characteristic metabolites for alfalfa root nodules. Furthermore, nodules arrested at different developmental stages were analyzed in order to address metabolic changes induced during the progression of nodule formation. Metabolite profiles of bacteroid-free pseudonodule extracts indicated that early nodule developmental processes are accompanied by photosynthate translocation but no massive organic acid formation. To determine metabolic adaptations induced by the presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains lacking the nitrogenase protein were analyzed. The bacteroids are unable to provide ammonium to the host plant, which is metabolically reflected by reduced levels of characteristic amino acids involved in ammonium fixation. Elevated levels of starch and sugars in Fix(-) nodules provide strong evidence that plant sanctions preventing a transformation from a symbiotic to a potentially parasitic interaction are not strictly realized via photosynthate supply. Instead, metabolic and gene expression data indicate that alfalfa plants react to nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis and an early induction of senescence. Noneffective symbiotic interactions resulting from plants nodulated by mutant rhizobia also are reflected in characteristic metabolic changes in leaves. These are typical for nitrogen deficiency, but also highlight metabolites potentially involved in sensing the N status.     

Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.     

Response of purely symbiotic and NO3-fed nodulated plants of Lupinus luteus and Vicia atropurpurea to ultraviolet-B radiation

The effects of elevated UV-B radiation on growth, symbiotic function and concentration of metabolites were assessed in purely symbiotic and NO3-fed nodulated plants of Lupinus luteus and Vicia atropurpurea grown outdoors either on tables under supplemental UV-B radiation or in chambers covered with different types of plexi-glass to attenuate solar ultraviolet radiation. Moderately and highly elevated UV-B exposures simulating 15% and 25% ozone depletion as well as sub- ambient UV-B did not alter organ growth, plant total dry matter and N content per plant in both L. luteus and V. atropurpurea. In contrast, elevated UV-B increased (P <0.05) flavonoid and anthocyanin concentrations in roots and leaves of L. luteus, but not of V. atropurpurea. Feeding nodulated plants of L. luteus under elevated UV-B radiation with 2 mM NO3 increased (P <0.05) nodule, leaf and total dry matter, and whole plant N content. With V. atropurpurea, NO3 reduced (P <0.05) nodule activity, root %N and concentrations of flavonoids, anthocyanins in roots and leaves and soluble sugars in roots, in contrast to an observed increase (P <0.05) in nodule dry matter per plant. Similarly, supplying 2 mM NO3 to L. luteus plants exposed to sub-ambient UV-B radiation significantly reduced individual organ growth, plant total biomass, nodule dry matter, nodule %N, and whole plant N content, as well as root concentrations of flavonoids, anthocyanins, soluble sugars, and starch of L. luteus, but not V. atropurpurea plants. These results show no adverse effect of elevated UV-B radiation on growth and symbiotic function of L. luteus and V. atropurpurea plants. However, NO3 supply promoted growth in L. luteus plants exposed to the highly elevated UV-B radiation.     

Symbiotic dinitrogen fixation and host-plant growth during development of and recovery from phosphorus deficiency

Characterization of nodule growth and function, phosphorus and nitrogen status of plant tissues and host-plant growth of nodulated soybean ( Glycine max L. Merr.) plants developing and recovering from phosphorus deficiency was used to evaluate the role of phosphorus in symbiotic dinitrogen fixation. The sequence of physiological responses during recovery from phosphorus deficiency was; (1) rapid uptake of phosphorus, (2) rapid increases in the phosphorus concentration of leaves and nodules, (3) enhanced growth and function of nodules, (4) increased nitrogen concentrations in all plant organs and (5) enhanced plant growth. The sequence of physiological responses to onset of phosphorus deficiency was; (1) decreased phosphorus uptake, (2) decreased phosphorus concentrations in leaves and nodules, (3) decreased nodule function, (4) decreased nitrogen concentration in plant organs and (5) decreased plant growth. These results, in conjunction with previously published data (Sa and Israel, Plant Physiol. 97: 928–935, 1991), support an interpretation that the total response of symbiotic dinitrogen fixation in soybean plants to altered phosphorus supply is a function of both indirect effects on host-plant growth and more direct effects on the metabolic function of nodules.     

Nitrate-induced CLE35 signaling peptides inhibit nodulation through the SUNN receptor and miR2111 repression

Legume plants form nitrogen (N)-fixing symbiotic nodules when mineral N is limiting in soils. As N fixation is energetically costly compared to mineral N acquisition, these N sources, and in particular nitrate, inhibit nodule formation and N fixation. Here, in the model legume Medicago truncatula, we characterized a CLAVATA3-like (CLE) signaling peptide, MtCLE35, the expression of which is upregulated locally by high-N environments and relies on the Nodule Inception-Like Protein (NLP) MtNLP1. MtCLE35 inhibits nodule formation by affecting rhizobial infections, depending on the Super Numeric Nodules (MtSUNN) receptor. In addition, high N or the ectopic expression of MtCLE35 represses the expression and accumulation of the miR2111 shoot-to-root systemic effector, thus inhibiting its positive effect on nodulation. Conversely, ectopic expression of miR2111 or downregulation of MtCLE35 by RNA interference increased miR2111 accumulation independently of the N environment, and thus partially bypasses the nodulation inhibitory action of nitrate. Overall, these results demonstrate that the MtNLP1-dependent, N-induced MtCLE35 signaling peptide acts through the MtSUNN receptor and the miR2111 systemic effector to inhibit nodulation.

Expression of a Medicago truncatula signaling peptide is upregulated by nitrate and inhibits nodulation through repression of the accumulation of a microRNA.     

Symbiotic N2 fixation activity in relation to C economy of Pisum sativum L. as a function of plant phenology

The relationships between symbiotic nitrogen fixation (SNF) activity and C fluxes were investigated in pea plants (Pisum sativum L. cv. Baccara) using simultaneous 13C and 15N labelling. Analysis of the dynamics of labelled CO2 efflux from the nodulated roots allowed the different components associated with SNF activity to be calculated, together with root and nodule synthetic and maintenance processes. The carbon costs for the synthesis of roots and nodules were similar and decreased with time. Carbon lost by turnover, associated with maintenance processes, decreased with time for nodules while it increased in the roots. Nodule turnover remained higher than root turnover until flowering. The effect of the N source on SNF was investigated using plants supplied with nitrate or plants only fixing N2. SNF per unit nodule biomass (nodule specific activity) was linearly related to the amount of carbon allocated to the nodulated roots regardless of the N source, with regression slopes decreasing across the growth cycle. These regression slopes permitted potential values of SNF specific activity to be defined. SNF activity decreased as the plants aged, presumably because of the combined effects of both increasing C costs of SNF (from 4.0 to 6.7 g C g-1 N) and the limitation of C supply to the nodules. SNF activity competed for C against synthesis and maintenance processes within the nodulated roots. Synthesis was the main limiting factor of SNF, but its importance decreased as the plant aged. At seed-filling, SNF was probably more limited by nodule age than by C supply to the nodulated roots.     

Interplay of flg22-induced defence responses and nodulation in Lotus japonicus

In this study the interplay between the symbiotic and defence signalling pathways in Lotus japonicus was investigated by comparing the responses to Mesorhizobium loti, the symbiotic partner of L. japonicus, and the elicitor flg22, a conserved peptide motif present in flagellar protein of a wide range of bacteria. It was found that defence and symbiotic pathways overlap in the interaction between L. japonicus and M. loti since similar responses were induced by the mutualistic bacteria and flg22. However, purified flagellin from M. loti did not induce any response in L. japonicus, which suggests the production of other elicitors by the symbiotic bacteria. Defence responses induced by flg22 caused inhibition of rhizobial infection and delay in nodule organogenesis, as demonstrated by the negative effect of flg22 in the formation of spontaneous nodules in the snf1 L. japonicus mutant, and the inhibition of NSP1 and NSP2 genes. This indicates the antagonistic effect of the defence pathway on the nodule formation in the initial rhizobium-legume interaction. However, the fact that flg22 did not affect the formation of new nodules once the symbiosis was established indicates that after the colonization of the host plant by the symbiotic partner, the symbiotic pathway has prevalence over the defensive response. This result is also supported by the down-regulation of the expression levels of the flg22 receptor FLS2 in the nodular tissue.     

Is genotypic variation of H⁺ efflux under P deficiency linked with nodulated-root respiration of N₂ fixing common-bean (Phaseolus vulgaris L.)?

To examine genotypic variation of common bean in growth, phosphorus uptake, nodulated-root proton release, and nodule gas permeability, seven common bean recombinant inbred lines (RIL) from the cross of BAT477×DOR364 were inoculated by Rhizobium tropici CIAT 899 and grown in hydroaeroponic culture under glasshouse conditions. A positive correlation was observed between shoot and nodule biomass for most of the studied RILs. Under P deficiency, the tolerant common bean RILs acidified more of their rhizosphere than the sensitive ones. The proton release of the RILs 147, 124, 104, 75 and RIL34 was positively correlated with nodule O? permeability. We conclude that nodulated common bean plants release a substantial amount of H? into the rhizosphere that is linked to the symbiotic N? fixation. It depends upon the nodule permeability to O? diffusion, and varies with genotype.     

Nitric oxide is formed in Medicago truncatula-Sinorhizobium meliloti functional nodules

Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant development and response to environmental cues. Its role is particularly crucial for plant-pathogen interactions, during which it participates in the control of plant defense response and resistance. Indication for the presence of NO during symbiotic interactions has also been reported. Here, we defined when and where NO is produced during Medicago truncatula-Sinorhizobium meliloti symbiosis. Using the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate, NO production was detected by confocal microscopy in functional nodules. NO production was localized in the bacteroid-containing cells of the nodule fixation zone. The infection of Medicago roots with bacterial strains impaired in nitrogenase or nitrite reductase activities lead to the formation of nodules with an unaffected NO level, indicating that neither nitrogen fixation nor denitrification pathways are required for NO production. On the other hand, the NO synthase inhibitor N-methyl-L-arginine impaired NO detection, suggesting that a NO synthase may participate to NO production in nodules. These data indicate that a NO production occurs in functional nodules. The location of such a production in fully metabolically active cells raises the hypothesis of a new function for NO during this interaction unrelated to defense and cell-death activation.     

Integration of local and systemic signaling pathways for plant N responses

Nitrogen (N) is an essential macronutrient and a signal that has profound impacts on plant growth and development. In order to cope with changing N regimes in the soil, plants have developed complex regulatory mechanisms that involve short-range and long-range signaling pathways. These pathways act at the cellular and whole plant scale to coordinate plant N metabolism, growth and development according to external and internal N status. Although molecular components of local and systemic N signaling have been identified and characterized, an integrated view of how plants coordinate and organize the N response is still lacking. In this review, we discuss recent advances toward understanding the mechanisms of local and systemic N responses and provide an integrated model for how these responses are orchestrated.     

Symbiosis between the nodule bacterium Sinorhizobium meliloti and alfalfa (Medicago sativa) under salinization conditions

Two hundred forty-three isolates of alfalfa nodule bacteria (Sinorhizobium meliloti) were obtained from legume nodules and soils sampled in the northern Aral region, experiencing secondary salinization. Isolates obtained from nodules (N isolates) were significantly more salt-tolerant than those from soils (S isolates) when grown in a liquid medium with 3.5% NaCl. It was found that wild species of alfalfa, melilot, and trigonella preferably formed symbioses with salt-tolerant nodule bacteria in both salinized and nonsalinized soils. Only two alfalfa species, Medicago falcata and M. trautvetteri, formed efficient symbioses in soils contrasting in salinity. The formation of efficient symbiosis with alfalfa in the presence of 0.6% NaCl was studied in 36 isolates (N and S) differing in salt tolerance and symbiotic efficiency. Fifteen isolates formed efficient symbioses in the presence of salt. The increase in the dry weight of the plants was 25-68% higher than in the control group. The efficiency of symbiotic interaction under salinization conditions depended on the efficiency of the isolates under standard conditions but did not correlate with the source of nodule bacteria (soil or nodule) or their salt tolerance. The results indicate that nodule bacterium strains forming efficient symbioses under salinization conditions can be found.     

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.     

Partner choice in Medicago Truncatula–Sinorhizobium symbiosis

In nitrogen-fixing symbiosis, plant sanctions against ineffective bacteria have been demonstrated in previous studies performed on soybean and yellow bush lupin, both developing determinate nodules with Bradyrhizobium sp. strains. In this study, we focused on the widely studied symbiotic association Medicago truncatula–Sinorhizobium meliloti, which forms indeterminate nodules. Using two strains isolated from the same soil and displaying different nitrogen fixation phenotypes on the same fixed plant line, we analysed the existence of both partner choice and plant sanctions by performing split-root experiments. By measuring different parameters such as the nodule number, the nodule biomass per nodule and the number of viable rhizobia per nodule, we showed that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step. However, no sanction mechanism, described as a decrease in rhizobia fitness inside the nodules, was detected. Consequently, even if partner choice seems to be widespread among legumes, sanction of non-effective rhizobia might not be universal.     

一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。