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.

Pectins esterification in the apoplast of aluminum-treated pea root nodules.

Aiming to elucidate the possible involvement of pectins in aluminum-mediated growth inhibition the distribution of pectins in the apoplast of root nodules was investigated. Experiments were performed on the pea (Pisum sativum L.) root nodules treated with aluminum (50 µM AlCl3, for 2 or 24h). For histochemical acidic pectin localization we used ruthenium red staining. Immunolabeling techniques with monoclonal antibodies specific to high methyl-esterified pectin (JIM7), low methyl-esterified pectin (JIM5) and calcium cross-linked pectin (2F4) were used to re-examine the pattern of pectin esterification and distribution. After immunolabeling the samples were observed using a fluorescent and transmission electron microscope. Ruthenium red staining showed that acid pectin content increased in the apoplast of Al-treated nodules and immunolocalization of pectin epitopes revealed that the fraction of de-esterified pectins increased significantly under Al stress. JIM5 and 2F4 epitopes were located on the inner surface of the primary cell wall with higher intensity at cell corners lining the intercellular spaces and at infection threads (ITs) walls. By contrast, JIM 7 labels all walls uniformly throughout the nodule. In the presence of Al, the increase of JIM5 and 2F4 labeling in thick plant and IT walls, together with a decrease of JIM7 labeling was observed. These results indicate a specific role for pectin de-esterification in the process of wall thickening and growth inhibition. In particular, Al-dependent increase in pectin content and their low methyl esterification degree correlate with wall thickness and higher rigidity, and in this way, may affect IT and nodules growth.

Balanced allocation of organic acids and biomass for phosphorus and nitrogen demand in the fynbos legume Podalyria calyptrata.

Podalyria calyptrata is from fynbos soils with low availability of phosphorus (P) and nitrogen (N). We investigated the physiological basis for tolerance of low P supply in nodulated P. calyptrata and examined responses to increased supply of combined-N as Ca(NO3)2 and P. It was hypothesized that increasing supply of combined-N would stimulate P-acquisition mechanisms and enhance plant growth with high P supply. Biomass, leaf [N] and [P], organic acid and phosphatase root exudates, and phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activity in nodules and roots were examined in two N×P experiments. Low P supply decreased leaf [P] and limited growth, decreasing the nodule:root ratio but increasing nodular PEPC and MDH activity for enhanced P-acquisition or P-utilization. At low P supply, a N-induced demand for P increased root exudation of citrate and PEPC and MDH activity in roots. Greater combined-N supply inhibited nodulation more at low P supply than at high P supply. With a P-induced demand for N the plants nodulated prolifically and increased combined-N supply did not enhance plant growth. The physiological basis for N2-fixing P. calyptrata tolerating growth at low P supply and responding to greater P supply is through balanced acquisition of P and N for plant demand.

Transcriptional and physiological changes in relation to Fe uptake under conditions of Fe-deficiency and Cd-toxicity in roots of Vigna radiata L.

We investigated transcriptional and physiological changes in relation to Fe transport and uptake under various conditions of iron (Fe)-deficiency and cadmium (Cd) toxicity. Responses to four such Fe/Cd conditions were evaluated, revealing that oxidative stress was generated in the presence of Cd, followed by a decrease in Fe and an increase in Cd concentrations in green gram (Vigna radiata) material, whereas supplementation with Fe had a protective effect against Cd toxicity. The involvement of enzymes in Fe-uptake for the formation of root-nodules was largely reduced in the presence of Cd toxicity, a condition recovered by Fe-supplementation. Insufficient ferric chelate reducing activity in Fe-deprived roots in the presence of Cd was also largely improved by Fe supplementation. The expression of Fe(2+) transporters (IRT1, IRT2, and IRT3), Fe(III) chelate reductase (FRO1-FRO8) and phytochelatin synthase (PCS1, PCS2 and PCS3) genes was up regulated for the first 5 days and decreased after 10 days in roots in the presence of Cd toxicity, but was sustained with Fe-supplementation. Additionally, root biomass was fully recovered in plants in the presence of Fe during Cd toxicity. Our results suggest that Fe-status plays a significant role in ameliorating the damage in Fe transport for chelation and its uptake caused by Cd toxicity. This supports the hypothesis that leguminous plants, particularly those that are sensitive to Fe such as green gram, can cope to some extent with Cd toxicity by improving the uptake and transport of Fe.

Nitrate regulates rhizobial and mycorrhizal symbiosis in common bean (Phaseolus vulgaris L.).

Nitrogen-limited conditions are considered to be a prerequisite for legume-rhizobial symbiosis, but the effects of nitrate-rich conditions on symbiotic status remain poorly understood. We addressed this issue by examining rhizobial (Rhizobim tropici) and arbusclar mycorrhizal (Glomus intraradices) symbiosis in Phaseolus vulgaris L. cv. Negro Jamapa under nitrate pre-incubation and continuous nitrate conditions. Our results indicate that nitrate pre-incubation, independent of the concentration, did not affect nodule development. However, the continuous supply of nitrate at high concentrations impaired nodule maturation and nodule numbers. Low nitrate conditions, in addition to positively regulating nodule number, biomass, and nitrogenase activity, also extended the span of nitrogen-fixing activity. By contrast, for arbuscular mycorrhizae, continuous 10 and 50 mmol/L nitrate increased the percent root length colonization, concomitantly reduced arbuscule size, and enhanced ammonia transport without affecting phosphate transport. Therefore, in this manuscript, we have proposed the importance of nitrate as a positive regulator in promoting both rhizobial and mycorrhizal symbiosis in the common bean.

24-epibrassinolide modulates growth, nodulation, antioxidant system, and osmolyte in tolerant and sensitive varieties of Vigna radiata under different levels of nickel: a shotgun approach.

The objective of this study was to explore the response of 24-epibrassinolide to improve the biological yield of Ni-tolerant and Ni-sensitive varieties of Vigna radiata and also to test the propositions that 24-epibrassinolide induced up-regulation of antioxidant system protects the efficiency of V. radiata, grown under Ni-stress. Surface sterilized seeds of var. T-44 (Ni-tolerant) and PDM-139 (Ni-sensitive) were soaked in DDW (control), 10(-10), 10(-8), or 10(-6) M of 24-epibrassinolide for 8 h (shotgun approach). These treated seeds were then inoculated with specific Rhizobium grown in sandy loam soil supplemented with different levels of Ni 0, 50, 100, or 150 mg Ni kg(-1) of soil and were allowed to grow for 45-days. At this stage of growth, plants were sampled to assess the various growths and nodule related traits as well as selected biochemical characteristics. The remaining plants were allowed to grow to maturity to study the yield characteristics. The results indicated that plant-fresh and dry mass, number of nodules, their fresh and dry mass, leghemoglobin content, nitrogen and carbohydrate content in the nodules, leaf chlorophyll content, activities of nitrate reductase and carbonic anhydrase decreased proportionately with the increasing concentrations of soil nickel. However, the application of 24-epibrassinolide as shotgun approach (pre-sowing seed soaking) to the nickel-stressed or non-stressed plants improved growth, nodulation and enhanced the activity of various antioxidant enzymes (viz. catalase, peroxidase and superoxide dismutase) and also the content of proline. The up-regulation of antioxidant enzymes as well as proline (osmolyte) triggered by 24-epibrassinolide could have conferred tolerance to the Ni-stressed plants resulting in improved growth, nodulation and yield attributes.

Influence of cadmium stress and arbuscular mycorrhizal fungi on nodule senescence in Cajanus cajan (L.) Millsp.

Cadmium (Cd) causes oxidative damage and affects nodulation and nitrogen fixation process of legumes. Arbuscular mycorrhizal (AM) fungi have been demonstrated to alleviate heavy metal stress of plants. The present study was conducted to assess role of AM in alleviating negative effects of Cd on nodule senescence in Cajanus cajan genotypes differing in their metal tolerance. Fifteen day-old plants were subjected to Cd treatments--25 mg and 50 mg Cd per kg dry soil and were grown with and without Glomus mosseae. Cd treatments led to a decline in mycorrhizal infection (MI), nodule number and dry weights which was accompanied by reductions in leghemoglobin content, nitrogenase activity, organic acid contents. Cd supply caused a marked decrease in nitrogen (N), phosphorus (P), and iron (Fe) contents. Conversely, Cd increased membrane permeability, thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2), and Cd contents in nodules. AM inoculations were beneficial in reducing the above mentioned harmful effects of Cd and significantly improved nodule functioning. Activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) increased markedly in nodules of mycorrhizal-stressed plants. The negative effects of Cd were genotype and concentration dependent.

Organic acid accumulation may inhibit N2 fixation in phosphorus-stressed lupin nodules.

Nodulated lupins (Lupinus angustifolius cv. Wonga) were hydroponically grown under conditions of low phosphate (LP) or adequate phosphate (HP) to assess the effect of phosphoenolpyruvate carboxylase (PEPC)-derived organic acids on nitrogen assimilation in LP nodules. LP conditions are linked to altered organic acid metabolism, by the engagement of PEP metabolism via PEPC. In LP nodules, the enhanced organic acid synthesis may reduce the available organic carbon for nitrogen assimilation. The diversion of carbon between the organic acid- and amino acid pools was assessed through key nodular enzymes and (14)CO(2) metabolism. Under LP conditions, increased rates of organic acid synthesis via PEPC and malate dehydrogenase (MDH), coincided with reduced nitrogen assimilation via aspartate aminotransferase (AAT), aspartate synthetase (AS) and glutamine synthetase (GS)/glutamate synthase (GOGAT) activities. There was a preferential metabolism of nodular (14)CO(2) into organic acids and particularly into malate. High malate levels were associated with reduced N(2) fixation and synthesis of amino acids. These results indicate that phosphorus deficiency can enhance malate synthesis in nodules, but that excessive malate accumulation may inhibit N(2) fixation and nitrogen assimilation.

Growth, nitrogen fixation, yield and kernel quality of peanut in response to lime, organic and inorganic fertilizer levels.

The aim of this investigation was to study the effect of different levels of chemical fertilizers alone and in combination with farmyard manure and lime on growth, nitrogen fixation, yield and kernel quality of peanut in an acid lateritic soil. Five fertilization levels viz., no chemical fertilizer (CF) (F0), CF @ 20:40:30 (F1), CF @ 40:80:60 (F2) kg ha(-1) NPK, F1 +2.5 t ha(-1) FYM (F3) and F2 +5 t ha(-1) FYM (F4) with and without liming (2 t ha(-1)) were tested. Results revealed that integrated application of FYM+CF at F3 level significantly (P0.05) improved the nitrogen content of nodules (12.4%), kernel yield (19.3%), mineral composition, oil content (4.8%), protein content (28.2%) and hydration coefficient (11.6%) of kernels over sole CF at F1 level. Maximum level of CF or FYM+CF though improved the population of symbiotic nitrogen fixing bacteria in the peanut rhizosphere, however, could not improve nitrogen fixation, yield and kernel quality.