Exploring apoplast reorganization in the nodules of Lotus corniculatus L. growing on old Zn-Pb calamine wastes.

Nodulation and symbiotic nitrogen fixation are important factors that determine legume growth. A pot experiment was carried out to determine the effects of Zn-Pb contamination on nodule apoplast (cell walls and intercellular spaces) of bird's foot trefoil (Lotus corniculatus L.) that spontaneously colonized old calamine wastes. The plants were grown in pots filled with sterile calamine substrate (M, metal treated) or expanded clay (NM, untreated) and inoculated with calamine-derived Lotus-nodulating Bradyrhizobium liaoningense. Apoplast reorganization in the nodules was examined using specific dyes for cellulose, pectin and lignin detection, and immuno-histochemical techniques based on monoclonal antibodies against xyloglucan (Lm25), pectins (Jim5 and Jim7), and structural proteins (arabinogalactan protein - Lm14 and extensin - Jim12). Microscopic analysis of metal-treated nodules revealed changes in the apoplast structure and composition of nodule cortex tissues and infected cells. Wall thickening was accompanied by intensified deposition of cellulose, xyloglucan, esterified pectin, arabinogalactan protein and extensin. The metal presence redirected also lignin and suberin deposition in the walls of the nodule cortex tissues. Our results showed reorganization of the apoplast of cortex tissues and infected cells of Lotus nodules under Zn-Pb presence. These changes in the apoplast structure and composition may have created actual barriers for the toxic ions. For this reason, they can be regarded as an element of legume defense strategy against metal stress that enables effective functioning of L. corniculatus-rhizobia symbiosis on Zn-Pb polluted calamine tailings.

The Rpf84 gene, encoding a ribosomal large subunit protein, RPL22, regulates symbiotic nodulation in Robinia pseudoacacia.

MAIN CONCLUSION: A homologue of the ribosomal protein L22e, Rpf84, regulates root nodule symbiosis by mediating the infection process of rhizobia and preventing bacteroids from degradation in Robinia pseudoacacia. Ribosomal proteins (RPs) are known to have extraribosomal functions, including developmental regulation and stress responses; however, the effects of RPs on symbiotic nodulation of legumes are still unclear. Ribosomal protein 22 of the large 60S subunit (RPL22), a non-typical RP that is only found in eukaryotes, has been shown to function as a tumour suppressor in animals. Here, a homologue of RPL22, Rpf84, was identified from the leguminous tree R. pseudoacacia. Subcellular localization assays showed that Rpf84 was expressed in the cytoplasm and nucleus. Knockdown of Rpf84 by RNA interference (RNAi) technology impaired the infection process and nodule development. Compared with the control, root and stem length, dry weight and nodule number per plant were drastically decreased in Rpf84-RNAi plants. The numbers of root hair curlings, infection threads and nodule primordia were also significantly reduced. Ultrastructure analyses showed that Rpf84-RNAi nodules contained fewer infected cells with fewer bacteria. In particular, remarkable deformation of bacteroids and fusion of multiple symbiosomes occurred in infected cells. By contrast, overexpression of Rpf84 promoted nodulation, and the overexpression nodules maintained a larger infection/differentiation region and had more infected cells filled with bacteroids than the control at 45 days post inoculation, suggesting a retarded ageing process in nodules. These results indicate for the first time that RP regulates the symbiotic nodulation of legumes and that RPL22 may function in initiating the invasion of rhizobia and preventing bacteroids from degradation in R. pseudoacacia.

Phytoremediation of heavy and transition metals aided by legume-rhizobia symbiosis.

Legumes are important for nitrogen cycling in the environment and agriculture due to the ability of nitrogen fixation by rhizobia. In this review, we introduce an important and potential role of legume-rhizobia symbiosis in aiding phytoremediation of some metal contaminated soils as various legumes have been found to be the dominant plant species in metal contaminated areas. Resistant rhizobia used for phytoremediation could act on metals directly by chelation, precipitation, transformation, biosorption and accumulation. Moreover, the plant growth promoting (PGP) traits of rhizobia including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore release, and production of ACC deaminase and the volatile compounds of acetoin and 2, 3-butanediol may facilitate legume growth while lessening metal toxicity. The benefits of using legumes inoculated with naturally resistant rhizobia or recombinant rhizobia with enhanced resistance, as well as co-inoculation with other plant growth promoting bacteria (PGPB) are discussed. However, the legume-rhizobia symbiosis appears to be sensitive to metals, and the effect of metal toxicity on the interaction between legumes and rhizobia is not clear. Therefore, to obtain the maximum benefits from legumes assisted by rhizobia for phytoremediation of metals, it is critical to have a good understanding of interactions between PGP traits, the symbiotic plant-rhizobia relationship and metals.

Lupinus albus plants acquire mercury tolerance when inoculated with an Hg-resistant Bradyrhizobium strain.

One strain of Bradyrhizobium canariense (L-7AH) was selected for its metal-resistance and ability to nodulate white lupin (Lupinus albus L.) plants, from a collection of rhizobial strains previously created from soils of the Almadén mining district (Spain) with varying levels of Hg contamination. Plants were inoculated with either strain L-7AH (Hg-tolerant) or L-3 (Hg-sensitive, used as control), and watered with nutrient solutions supplemented with various concentrations (0-200 µM) of HgCl2 in a growth chamber. L. albus inoculated with L-7AH were able to nodulate even at the highest concentration of Hg while those inoculated with L-3 had virtually no nodules at Hg concentrations above 25 µM. Plants inoculated with L-7AH, but not those with the control strain, were able to accumulate large amounts of Hg in their roots and nodules. Nodulation with L-7AH allowed plants to maintain constant levels of both chlorophylls and carotenoids in their leaves and a high photosynthetic efficiency, whereas in those inoculated with L-3 both pigment content and photosynthetic efficiency decreased significantly as Hg concentration increased. Nitrogenase activity of plants nodulated with L-7AH remained fairly constant at all concentrations of Hg used. Results suggest that this symbiotic pair may be used for rhizoremediation of Hg-contaminated soils.

Growth and nodulation of symbiotic Medicago truncatula at different levels of phosphorus availability.

Medicago truncatula is an important model plant for characterization of P deficiency on leguminous plants at the physiological and molecular levels. Growth optimization of this plant with regard to P supply is the first essential step for elucidation of the role of P in regulation of nodulation. Hence, a study was carried out to address the growth pattern of M. truncatula hydroponically grown at different gradual increases in P levels. The findings revealed that M. truncatula had a narrow P regime, with an optimum P level (12 µM P) which is relatively close to the concentration that induces P toxicity. The accumulated P concentration (2.7 mg g(-1) dry matter), which is normal for other crops and legumes, adversely affected the growth of M. truncatula plants. Under P deficiency, M. truncatula showed a higher symbiotic efficiency with Sinorhizobium meliloti 2011 in comparison with S. meliloti 102F51, partially as a result of higher electron allocation to N2 versus H(+). The total composition of free amino acids in the phloem was significantly affected by P deprivation. This pattern was found to be almost exclusively the result of the increase in the asparagine level, suggesting that asparagine might be the shoot-derived signal that translocates to the nodules and exerts the down-regulation of nitrogenase activity. Additionally, P deprivation was found to have a strong influence on the contents of the nodule carbon metabolites. While levels of sucrose and succinate tended to decrease, a higher accumulation of malate was observed. These findings have provided evidence that N2 fixation of M. truncatula is mediated through an N feedback mechanism which is closely related to nodule carbon metabolism.

A phosphoenol pyruvate phosphatase transcript is induced in the root nodule cortex of Phaseolus vulgaris under conditions of phosphorus deficiency.

Although previous studies on N2-fixing legumes have demonstrated the contribution of acid phosphatases to their phosphorus (P) use efficiency under P-deficient growth conditions, localization of these enzymes in bean nodules has not been demonstrated. In this study, phosphoenol pyruvate phosphatase (PEPase) gene transcripts were localized within the nodule tissues of two recombinant inbred lines, RIL115 (P-deficiency tolerant) and RIL147 (P-deficiency sensitive), of Phaseolus vulgaris. Nodules were induced by Rhizobium tropici CIAT899 under hydroaeroponic conditions with a sufficient versus a deficient P supply. The results indicated that PEPase transcripts were particularly abundant in the nodule infected zone and cortex of both RILs. Analysis of fluorescence intensity indicated that nodule PEPase was induced under conditions of P deficiency to a significantly higher extent in RIL147 than in RIL115, and more in the inner cortex (91%) than in the outer cortex (71%) or the infected zone (79%). In addition, a significant increase (39%) in PEPase enzyme activity in the P-deficient RIL147 correlated with an increase (58%) in the efficiency of use in rhizobial symbiosis. It was concluded that nodule PEPase is upregulated under conditions of P deficiency in the P-deficiency-sensitive RIL147, and that this gene may contribute to adaptation of rhizobial symbiosis to low-P environments.

Nitrogen deposition, competition and the decline of a regionally threatened legume, Desmodium cuspidatum.

Increased nitrogen (N) deposition, resulting from the combustion of fossil fuels, production of synthetic fertilizers, growth of N(2)-fixing crops and high-intensity agriculture, is one of the anthropogenic factors most likely to cause global biodiversity changes over the next century. This influence may be especially large in temperate zone forests, which are highly N limited and occur in regions with the highest levels of N deposition. Within these ecosystems, N(2)-fixing plants, including legumes, may be more sensitive to N deposition than other plant species. Though it has long been recognized that the competitive edge conferred by N(2)-fixation diminishes with increasing soil N availability, the conservation implications of increased N deposition on native N(2)-fixers have received less attention. We focus on Desmodium cuspidatum, which has experienced dramatic population losses in the last 30-40 years in the northeastern United States. We explore competition between this regionally threatened legume and a common non-N(2)-fixing neighbor, Solidago canadensis, across a gradient of N deposition. Our data show that increased N deposition may be detrimental to N(2)-fixers such as D. cuspidatum in two ways: (1) biomass accumulation in the non-N(2)-fixer, S. canadensis, responds more strongly to increasing N deposition, and (2) S. canadensis competes strongly for available mineral nitrogen and can assimilate N previously fixed by D. cuspidatum, resulting in D. cuspidatum relying more heavily on energetically expensive N(2)-fixation when grown with S. canadensis. N deposition may thus reduce or eliminate the competitive advantage of N(2)-fixing species growing in N-limited ecosystems.

Nitrogen fixation by white lupin under phosphorus deficiency.

BACKGROUND AND AIMS: White lupin is highly adapted to growth in a low-P environment. The objective of the present study was to evaluate whether white lupin grown under P-stress has adaptations in nodulation and N2 fixation that facilitate continued functioning. METHODS: Nodulated plants were grown in silica sand supplied with N-free nutrient solution containing 0 to 0.5 mm P. At 21 and 37 d after inoculation (DAI) growth, nodulation, P and N concentration, N2 fixation (15N2 uptake and H2 evolution), root/nodule net CO2 evolution and CO2 fixation (14CO2 uptake) were measured. Furthermore, at 21 DAI in-vitro activities and transcript abundance of key enzymes of the C and N metabolism in nodules were determined. Moreover, nodulation in cluster root zones was evaluated. KEY RESULTS: Treatment without P led to a lower P concentration in shoots, roots, and nodules. In both treatments, with or without P, the P concentration in nodules was greater than that in the other organs. At 21 DAI nitrogen fixation rates did not differ between treatments and the plants displayed no symptoms of P or N deficiency on their shoots. Although nodule number at 21 DAI increased in response to P-deficiency, total nodule mass remained constant. Increased nodule number in P-deficient plants was associated with cluster root formation. A higher root/nodule CO2 fixation in the treatment without P led to a lower net CO2 release per unit fixed N, although the total CO2 released per unit fixed N was higher in the treatment without P. The higher CO2 fixation was correlated with increased transcript abundance and enzyme activities of phosphoenolpyruvate carboxylase and malate dehydrogenase in nodules. Between 21 and 37 DAI, shoots of plants grown without P developed symptoms of N- and P-deficiency. By 37 DAI the P concentration had decreased in all organs of the plants treated with no P. At 37 DAI, nitrogen fixation in the treatment without P had almost ceased. CONCLUSIONS: Enhanced nodulation in cluster root zones and increased potential for organic acid production in root nodules appear to contribute to white lupin's resilience to P-deficiency.