Response of nitrogen fixation in relation to nodule carbohydrate metabolism in Medicago ciliaris lines subjected to salt stress

The effect of salt stress on nitrogen fixation, in relation to sucrose transport towards nodules and other sink organs and the potential of sucrose breakdown by nodules, was investigated in two lines of Medicago ciliaris. Under salt stress conditions, the two lines showed a decrease of total biomass production, but TNC 1.8 was less affected by salt than TNC 11.9. The chlorophyll content was not changed in TNC 1.8, in contrast to TNC 11.9. Shoot, root, and nodule biomass were also affected in the two lines, but TNC 1.8 exhibited the higher potentialities of biomass production of these organs. Nitrogen fixation also decreased in the two lines, and was more sensitive to salt than growth parameters. TNC 1.8 consistently exhibited the higher values of nitrogen fixation. Unlike nodules, leaves of both lines were well supplied in nutrients with some exceptions. Specifically, the calcium content decreased in the sensitive line leaves, and the nodule magnesium content was not changed in either line. The tolerant line accumulated more sodium in its leaves. The two lines did not show any differences in the nodule sodium content. Sucrose allocation towards nodules was affected by salt in the two lines, but this constraint did not seem to affect the repartition of sucrose between sink organs. Salt stress induced perturbations in nodule sucrolytic activities in the two lines. It inhibited sucrose synthase, but the inhibition was more marked in TNC 11.9; alkaline/neutral activity was not altered in TNC 1.8, whereas it decreased more than half in TNC 11.9. Thus, the relative tolerance of TNC 1.8 to salt stress could be attributed to a better use of these photoassimilates by nodules and a better supply of bacteroids in malate. The hypothesis of a competition for sucrose between nodules and other sink organs under salt stress could not be verified.     

Variability in the response of six genotypes of N<Subscript>2</Subscript>-fixing <Emphasis Type="Italic">Medicago ciliaris</Emphasis> to NaCl

Genotypic variability was assessed within six Medicago ciliaris genotypes growing symbiotically with Sinorhizobium medicae in order to identify physiological criteria (growth, ion content, and plant health) associated with salt tolerance. Response to salt stress depended on the line and the level of salt. Two lines with lower dry biomass under non-saline conditions (TNC 1.8 from a semi-arid area and TNC 10.8 from a sub-humid area), were more tolerant to NaCl, whereas the most productive lines (TNC 11.5 and TNC 11.9 from a humid bioclime) were more sensitive in terms of growth and nitrogen fixation. Susceptibility of symbiotic nitrogen fixation to saline stress was not associated with a higher accumulation of Na⁺ in nodules, since the most tolerant lines TNC 1.8 and TNC 10.8 accumulated the highest Na⁺ amount in nodules. Leaf area and net photosynthate assimilation rate were conserved in line TNC 1.8 and to a lesser extent in line TNC 10.8 potentially owing to a greater ability to protect aerial organs and nodules from Na⁺ damage and to insure a better supply of leaves with nitrogen. Our results suggest that nodule growth and number and nodule Na⁺ content should not be used as selection tools for tolerance or susceptibility, since two of the tested lines maintained consistent growth in spite of reduced nodule and high Na⁺ content. Instead, the most reliable physiological indicators for tolerance appear to be consistent growth (i.e., no growth changes) and reduced leaf Na⁺ accumulation with increasing concentrations of NaCl.     

Involvement of source-sink relationship and hormonal control in the response of Medicago ciliaris - Sinorhizobium medicae symbiosis to salt stress

In order to explore the relationship between leaf hormonal status and source-sink relations in the response of symbiotic nitrogen fixation (SNF) to salt stress, three major phytohormones (cytokinins, abscisic acid and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid), sucrose phosphate synthase activity in source leaves and sucrolytic activities in sink organs were analysed in two lines of Medicago ciliaris (salt-tolerant TNC 1.8 and salt-sensitive TNC 11.9). SNF (measured as nitrogenase activity and amount of N-fixed) was more affected by salt treatment in the TNC 11.9 than in TNC 1.8, and this could be explained by a decrease in nodule sucrolytic activities. SNF capacity was reflected in leaf biomass production and in the sink activity under salinity, as suggested by the higher salt-induced decrease in the young leaf sucrolytic activities in the sensitive line TNC 11.9, while they were not affected in the tolerant line TNC 1.8. As a consequence of maintaining sink activities in the actively growing organs, the key enzymatic activity for synthesis of sucrose (sucrose phosphate synthase) was also less affected in the mature leaves of the more tolerant genotype. Ours results showed also that the major hormone factor associated with the relative tolerance of TNC 1.8 was the stimulation of abscisic acid concentration in young leaves under salt treatment. This stimulation may control photosynthetic organ growth and also may contribute to a certain degree in the maintenance of coordinated sink-source relationships. Therefore, ABA may be an important component which conserves sucrose synthesis in source leaves.     

Phenolic content and antioxidant activity in two contrasting Medicago ciliaris lines cultivated under salt stress

The objective of this study was to determine more indepth physiological and antioxidant responses in two Medicago ciliaris lines (a salt-tolerant line TNC 1.8 and a salt-sensitive line TNC 11.9) with contrasting responses to 100 mM NaCl. Under salt stress, both lines showed a decrease in total biomass and in the growth rate for roots, but TNC 1.8 was less affected by salt than TNC 11.9 in that it maintained leaf growth even in the presence of added salt. In both lines, salt stress mainly affected micronutrient status (Fe, Mn, Cu and Zn) rather than K nutrition, but the tolerant line TNC 1.8 accumulated more Na in leaves and less in roots compared with TNC 11.9. Salt stress decreased total soluble sugars (TSS) in all organs of the sensitive line TNC 11.9, whereas TSS was only reduced in roots of the tolerant line. The salt-induced drop in growth was linked to an increase in lipid peroxidation in roots of both lines and in leaves of the sensitive line. The salt-tolerant line TNC 1.8 was more efficient at managing salt-induced oxidative damage in leaves and to a lesser extent in roots than the salt-sensitive line TNC 11.9, by preserving higher phenolic compound and superoxide dismutase levels in both organs.     

Hormonal responses of nodulated Medicago ciliaris lines differing in salt tolerance

Hormonal changes in two Medicago ciliaris lines differing in salt tolerance (TNC 1.8 being more tolerant than TNC 11.9) were studied as possible regulators of growth and symbiotic nitrogen fixation (SNF). After 21 days of saline treatment (100 mM NaCl), four major phytohormones (abscisic acid, ABA; the cytokinin trans-zeatin, t-Z; the auxin indole-3-acetic acid, IAA; and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, ACC) were analysed. Salinity differently affected hormone concentrations in vegetative tissues and nodules in both lines. Principal component analysis (PCA) revealed that growth and SNF parameters under salinity were inversely correlated to the amount of reduced nitrogen (Nred) allocated to the roots and nodules, to the sucrolytic activity (TSA) in the roots, and to the t-Z and ABA concentration in the nodules. However, those parameters were positively associated along PC1 to the Nred and TSA in the leaves and all the hormones analysed in the roots. Interestingly, the ACC concentration of all organs was positively associated with vegetative growth and with SNF under salinity, as a putative regulator of the symbiotic-parasitic relation with the bacteria. The influence of hormonal changes in relation to plant growth, SNF and symbiotic relation is discussed.     

Growth, nitrogen fixation and ion distribution in Medicago truncatula subjected to salt stress

This study compared the growth, nodulation, N₂ fixation, and ion distribution in three Medicago truncatula lines, in response to salt in nutrient solution. Two local lines (TN8.20 and TN6.18) and a reference line (Jemalong 6) were inoculated with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl) and grown in a controlled glasshouse with or without 75 mM NaCl. A genotypic variation in tolerance to salt was found: TN6.18 was the most sensitive line whereas TN8.20 was the most tolerant. The relative tolerance of TN8.20 was concomitant with the lowest leaf Na⁺ concentration and the highest nodule biomass production. However, nodule efficiency (amount of nitrogen fixed per g dry weight nodule) decreased in all lines. Results suggest that the tolerance to salt seems to depend on the host plant ability to protect its leaves against an excessive Na⁺ (and Cl^?) accumulation, and its ability to maintain the development of an abundant nodular system, which in turn determines an important rate of nitrogen fixation and allows the plants to conserve their growth potentialities. The loss of the nodular efficiency under salt stress seems to be compensated by a large nodule biomass.     

Comparative study of nitrogen fixation and carbon metabolism in two chick-pea (Cicer arietinum L.) cultivars under salt stress

Two cultivars of Cicer arietinum with differential tolerance to salinity have been compared by analysing growth, photosynthesis, nodulation, nitrogenase activity, and carbon metabolism in the nodule cytosol. The aim was to help elucidate the relationships between, on the one hand, sucrose and malate metabolism in nodules and, on the other, the inhibition of nitrogen fixation under salt stress. Chick-pea cultivars Pedrosillano (sensitive) and ILC1919 (tolerant) inoculated with Mesorhizobium ciceri strain Ch-191 were grown in a controlled environmental chamber and were treated with salt (0, 50, 75, and 100 mM NaCl) from sowing to harvest time (28 d). Plant growth and photosynthesis were more affected by salt in Pedrosillano than in ILC1919. Also the effect of salt on nodulation and nitrogen fixation was much more pronounced in Pedrosillano. The increase in nodular mass in ILC1919 can partially counteract the inhibition of nitrogenase activity. The enzymes of sucrose breakdown were inhibited by NaCl, but in ILC1919 a rise in alkaline invertase was observed with salinity, which could compensate for the lack of the sucrose synthase hydrolytic activity. The activity of PEPC was stimulated by salt in ILC1919. Also, this cultivar showed higher malate concentrations in root nodules.     

Phloem‐derived γ‐aminobutyric acid (GABA) is involved in upregulating nodule N2 fixation efficiency in the model legume Medicago truncatula

Nitrogen fixation in legumes is downregulated through a whole plant N feedback mechanism, for example, when under stress. This mechanism is probably triggered by the impact of shoot‐borne, phloem‐delivered compounds. However, little is known about any whole‐plant mechanism that might upregulate nitrogen fixation, for example, under N deficiency. We induced emerging N‐deficiency through partial excision of nodules from Medicago truncatula plants. Subsequently, the activity and composition of the remaining nodules and shifts in concentration of free amides/amino acids in the phloem were monitored. Furthermore, we mimicked these shifts through artificial feeding of γ‐aminobutyric acid (GABA) into the phloem of undisturbed plants. As a result of increased specific activity of nodules, N₂ fixation per plant recovered almost completely 4–5 d after excision. The concentration of amino acids, sugars and organic acids increased strongly in the upregulated nodules. A concomitant analysis of the phloem revealed a significant increase in GABA concentration. Comparable with the effect of nodule excision, artificial GABA feeding into the phloem resulted in an increased activity and higher concentration of amino acids and organic acids in nodules. It is concluded that GABA might be involved in upregulating nodule activity, possibly because of its constituting part of a putative amino acid cycle between bacteroids and the cytosol.     

Drought stress induced changes in some organic substances in nodules and other plant parts of two potential legumes differing in salt tolerance

A greenhouse experiment was conducted to assess the effect of water stress on growth and metabolic changes in nodules and other plant parts of two leguminous species, Phaseolus vulgaris and Sesbania aculeata, with the major objective that nodules play a vital role in drought tolerance. Imposition of water deficit conditions for 45 days to 15-day-old plants of P. vulgaris and S. aculeata reduced shoot mass and nodule mass of both species, but the reduction was more pronounced in P. vulgaris than in S. aculeata. Nitrate reductase (NR) activity was reduced more in the leaves and nodules of P. vulgaris than in S. aculeata. Soluble proteins in the nodules of S. aculeata were more decreased as compared to that in P. vulgaris. Free amino acids increased in all parts of both species due to water deficit, but a higher increase was observed in leaf and nodules of P. vulgaris than in S. aculeata. Osmoprotectants such as proline and glycine betaine increased more in the nodules and other parts of S. aculeata under drought stress. In conclusion, S. aculeata (salt tolerant) showed a higher degree of drought tolerance than P. vulgaris (salt sensitive). Drought tolerance of S. aculeata was found to be associated with a smaller reduction in number and mass of root nodules, a high activity of nitrate reductase in leaves and nodules, high accumulation of free proline in roots and nodules, and high glycine betaine content in nodules.     

Elucidation of salt-tolerance metabolic pathways in contrasting rice genotypes and their segregating progenies

Key message

Differentially expressed antioxidant enzymes, amino acids and proteins in contrasting rice genotypes, and co-location of their genes in the QTLs mapped using bi-parental population, indicated their role in salt tolerance.

Abstract

Soil salinity is a major environmental constraint limiting rice productivity. Salt-tolerant ‘CSR27’, salt-sensitive ‘MI48’and their extreme tolerant and sensitive recombinant inbred line (RIL) progenies were used for the elucidation of salt stress tolerance metabolic pathways. Salt stress-mediated biochemical and molecular changes were analyzed in the two parents along with bulked-tolerant (BT) and bulked-sensitive (BS) extreme RILs. The tolerant parent and BT RILs suffered much lower reduction in the chlorophyll as compared to their sensitive counterparts. Activities of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD) and non-enzymatic antioxidant ascorbic acid were much higher in salt-stressed CSR27 and BT RILs than MI48 and BS RILs. Further, the tolerant lines showed significant enhancement in the levels of amino acids methionine and proline in response to salt stress in comparison to the sensitive lines. Similarly, the tolerant genotypes showed minimal reduction in cysteine content whereas sensitive genotypes showed a sharp reduction. Real time PCR analysis confirmed the induction of methionine biosynthetic pathway (MBP) enzymes cystathionine-β synthase (CbS), S-adenosyl methionine synthase (SAMS), S-adenosyl methionine decarboxylase (SAMDC) and serine hydroxymethyl transferase (SHMT) genes in tolerant lines, suggesting potential role of the MBP in conferring salt tolerance in rice variety CSR27. Proteome profiling also confirmed higher expression of SOD, POD and plastidic CbS and other proteins in the tolerant lines, whose genes were co-located in the QTL intervals for salt tolerance mapped in the RIL population. The study signifies integrated biochemical-molecular approach for identifying salt tolerance genes for genetic improvement for stress tolerant rice varieties.

     

Effects of Salt Stress on Amino Acid, Organic Acid, and Carbohydrate Composition of Roots, Bacteroids, and Cytosol of Alfalfa (Medicago sativa L.)

Ethanol-soluble organic acid, carbohydrate, and amino acid constituents of alfalfa (Medicago sativa) roots and nodules (cytosol and bacteroids) have been identified by gas-liquid chromatography and high performance liquid chromatography. Among organic acids, citrate was the predominant compound in roots and cytosol, with malonate present in the highest concentration in bacteroids. These two organic acids together with malate and succinate accounted for more than 85% of the organic acid pool in nodules and for 97% in roots. The major carbohydrates in roots, nodule cytosol, and bacteroids were (descending order of concentration): sucrose, pinitol, glucose, and ononitol. Maltose and trehalose appeared to be present in very low concentrations. Asparagine, glutamate, alanine, γ-aminobutyrate, and proline were the major amino acids in cytosol and bacteroids. In addition to these solutes, serine and glutamine were well represented in roots. When alfalfa plants were subjected to 0.15 m sodium chloride stress for 2 weeks, total organic acid concentration in nodules and roots were depressed by more than 40%, whereas lactate concentration increased by 11, 27, and 94% in cytosol, roots, and bacteroids, respectively. In bacteroids, lactate became the most abundant organic acid and might contribute partly to the osmotic adjustment. On the other hand, salt stress induced a large increase in the amino acid and carbohydrate pools. Within the amino acids, proline showed the largest increase, 11.3-, 12.8-, and 8.0-fold in roots, cytosol, and bacteroids, respectively. Its accumulation reflected an osmoregulatory mechanism not only in roots but also in nodule tissue. In parallel, asparagine concentration was greatly enhanced; this amide remained the major nitrogen solute and, in bacteroids, played a significant role in osmoregulation. On the contrary, the salt treatment had a very limited effect on the concentration of other amino acids. Among carbohydrates, pinitol concentration was increased significantly, especially in cytosol and bacteroids (5.4- and 3.4-fold, respectively), in which this cyclitol accounted for more than 35% of the total carbohydrate pool; pinitol might contribute to the tolerance to salt stress. However, trehalose concentration remained low in both nodules and roots; its role in osmoregulation appeared unlikely in alfalfa.     

Understanding osmotic stress tolerance in leaves and nodules of two <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> cultivars with contrasting drought tolerance

Drought and salinity are environmental constraints that affect crop yields worldwide. In nature, both stresses are multifaceted problems that are usually associated with other adverse circumstances which limit plant performance such as water shortage and nutrient deficits. In order to assess common features of both stresses, the effects of mannitol-induced osmotic stress were monitored using two Phaseolus vulgaris cultivars, Cv. ‘Flamingo’ (tolerant) and Cv. ‘Coco Blanc’ (sensitive) which differed in their drought and salinity tolerance. Growth, water relations, organic and inorganic compound accumulation and soluble protein contents were measured in leaves and nodules of these N₂-fixing plants. The aim of the present study was to check whether osmotic stress tolerance is associated with accumulation of some of these compounds either in leaves, nodules or both organs. At the whole-plant level, Cv. ‘Flamingo’ showed a better maintenance of plant biomass and shoot water status. At the cell level, this was related to a better osmotic adjustment ability both in leaves and nodules and also to a better adjustment of the cell wall elasticity. At the metabolic level, the contrasting accumulation of the different amino acids in nodules of each cultivar suggested that amino acids pathways can be regulated to different degrees under stress conditions. At the metabolic level, it seems that symbiosis in the sink organ (the nodule) plays a crucial role in conferring drought and salinity tolerance in the common bean.     

The nodule conductance to O<Subscript>2</Subscript> diffusion increases with high phosphorus content in the <Emphasis Type="Italic">Phaseolus vulgaris-</Emphasis>rhizobia symbiosis

Although recent studies have addressed the effects of phosphorus (P) deficiency on nodule O₂ permeability, little attention has been given to the relationship between nodule P status and nodule permeability. To study these traits, four recombinant inbred lines, namely RILs 34, 83, 115, 147 and one local variety (Concesa) of common bean (Phaseolus vulgaris) were inoculated with RhM11 (a native rhizobial strain from Haouz area of Marrakesh), and grown in hydroaeroponic culture under P-sufficiency (250 μmol P plant⁻¹ week⁻¹) versus P-deficiency (75 μmol P plant⁻¹ week⁻¹) conditions. At the flowering stage, the biomass of plants and nodules and their P contents was determined after measuring O₂ uptake by nodulated roots (Conr) and nodule conductance to O₂ diffusion (g_n). The results showed that P-deficiency significantly decreased plant growth and nodulation, though there were differences between bean genotypes. P-deficiency also induced a decrease in nodule P content (31%) in both sensitive (83, 147) and tolerant lines (34, 115), a 42 and 27% reduction in shoots of sensitive and tolerant lines, respectively. These decreases were associated with significant variations in nodule surface and O₂ permeability among bean genotypes and P-nutrition. Under P-deficiency, g_n increased more for the sensitive (39%) than for the tolerant lines (27%). This increase was linked with a rise both in the P levels in nodules and shoots, as well as in the efficiency of symbiotic nitrogen fixation as determined by nodule-dependent biomass production for the sensitive lines. Furthermore, positive correlations were found between O₂ permeability, g_n and P content both in nodules and shoots (r ² = 0.94** and r ² = 0.96**). We conclude that nodule variations in Conr and g_n are related to nodule P content, and may contribute to the adaptation of energy metabolism in N₂-fixing bean nodules to P-deficiency.     

Several lanthanides activate malate efflux from roots of aluminium‐tolerant wheat

Many elements of the lanthanide series exist as trivalent cations in solution below pH 6. The present study was carried out to investigate whether lanthanides could stimulate malate efflux from wheat (Triticum aestivum L.) roots, as has been found for trivalent aluminium (Al) cations. Excised root apices treated with 100 µm of each of seven different lanthanide elements (lanthanum, praseodymium, europium, gadolinium, terbium, erbium, and ytterbium) stimulated malate efflux, with five‐ to fifty‐fold more malate being released from an Al‐tolerant wheat line than from a near‐isogenic Al‐sensitive line. As erbium stimulated the greatest malate efflux of the lanthanides tested, this response was characterized further. The characteristics of the erbium‐activated efflux were similar to the Al‐activated efflux described previously suggesting that both of these ions activate the same transport mechanism. The capacity for erbium‐activated malate efflux cosegregated with Al tolerance in wheat seedlings derived from a cross between Al‐sensitive and Al‐tolerant near‐isogenic lines. This is the first study to identify cations, other than Al, which can activate malate release from wheat roots. It also provides additional evidence that malate efflux from root apices is the primary mechanism for Al tolerance in wheat.     

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.     

Asparagine as a major factor in the N‐feedback regulation of N2 fixation in Medicago truncatula

The objective of this study was to assess whether a whole plant N‐feedback regulation impact on nitrogen fixation in Medicago truncatula would manifest itself in shifts of the composition of the amino acid flow from shoots to nodules. Detected shifts in the phloem amino acid composition were supposed to be mimicked through artificial phloem feeding and concomitant measurement of nodule activity. The amino acid composition of the phloem exudates was analyzed from plants grown under the influence of treatments (limiting P supply or application of combined nitrogen) known to reduce nodule nitrogen fixation activity. Plants in nutrient solution were supplied with sufficient (9 µM) control, limiting (1 µM) phosphorus or 3 mM NH₄NO₃ (downregulated nodule activity). Low phosphorus and the application of NH₄NO₃ reduced per plant and specific nitrogenase activity (H₂ evolution). At day 64 of growth, phloem exudates were collected from cuts of the shoot base. The amount of amino acids was strongly increased in both phloem exudates and nodules of the treatments with downregulated nodule activity. The increase in the downregulated treatments was almost exclusively the result of a higher proportion of asparagine in both phloem exudates and nodules. Leaf labeling with ¹⁵N showed that nitrogen from the leaves is retranslocated to nodules. An artificial phloem feeding with asparagine resulted in an increased concentration of asparagine in nodules and a decreased nodule activity. A possible role of asparagine in an N‐feedback regulation of nitrogen fixation in M. truncatula is discussed.     

Effects of salt stress on growth, photosynthesis and nitrogen fixation in chick-pea (Cicer arietinum L.)

Plants of chick-pea (Cicer arietinum L. cv. ILC1919) inoculated with Mesorhizobium ciceri strain ch-191 were grown in a controlled environmental chamber, and were administered salt (0, 50, 75, and 100 mM NaCl) during the vegetative period. Four harvests (4, 7, 11, and 14d after treatment) were analysed. The aim was to ascertain whether the negative effect of saline stress on nitrogen fixation is due to a limitation on the photosynthate supply to the nodule or a limitation on the nodular metabolism which sustains nitrogenase activity.Plant growth was affected only by the highest NaCl concentration, whereas nitrogenase activity was affected from 50 mM. At the first harvest, Rubisco, PEPC and MDH activities in leaves rose with salt, but fell during the following harvests. The increase of PEPC and MDH in nodules at the two first samplings was clearly related to salt concentration. While 50 mM NaCl increased GS and GOGAT in nodules at some harvests, 100 mM strongly inhibited these activities at all the harvests. The accumulation of proline, amino acids and carbohydrates was clearly related to salt especially in the leaves, whereas in the nodules the protein content was boosted by salt. Although photosynthesis declined with NaCl, the response of nitrogen fixation to salt was more pronounced. This situation, together with carbohydrate accumulation, suggests that the lack of photosynthate does not cause the inhibition of nitrogenase activity under this type of stress. The similar trend observed for the PEPC-MDH pathway and the ARA support the hypothesis concerning the limitation in the supply of energy substrate, mainly malate, to the bacteroids. The accumulation of compatible solutes is more a consequence of damage produced by salt stress than of a protective strategy.     

Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Inhibition of N2 fixation in soybean is associated with elevated ureides and amino acids

Decreased N2 fixation in soybean (Glycine max) L. Merr. during water deficits has been associated with increases in ureides and free amino acids in plant tissues, indicating a potential feedback inhibition by these compounds in response to drought. We evaluated concentrations of ureides and amino acids in leaf and nodule tissue and the concurrent change in N2 fixation in response to exogenous ureides and soil-water treatments for the cultivars Jackson and KS4895. Exogenous ureides applied to the soil and water-deficit treatments inhibited N2 fixation by 85% to 90%. Mn fertilization increased the apparent catabolism of ureides in leaves and hastened the recovery of N2 fixation following exogenous ureide application for both cultivars. Ureides and total free amino acids in leaves and nodules increased during water deficits and coincided with a decline in N2 fixation for both cultivars. N2 fixation recovered to 74% to 90% of control levels 2 d after rewatering drought-stressed plants, but leaf ureides and total nodule amino acids remained elevated in KS4895. Asparagine accounted for 82% of the increase in nodule amino acids relative to well-watered plants at 2 d after rewatering. These results indicate that leaf ureides and nodule asparagine do not feedback inhibit N2 fixation. Compounds whose increase and decrease in concentration mirrored the decline and recovery of N2 fixation included nodule ureides, nodule aspartate, and several amino acids in leaves, indicating that these are potential candidate molecules for feedback inhibition of N2 fixation.     

Selection <Emphasis Type="Italic">in vitro</Emphasis> and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 M cadmium chloride (CdCl₂) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.