Genetic specificity of nitrogen nutrition in leguminous plants

Summary Mineral nitrogen did not increase grain yield and seed protein levels ofVicia faba L. andLupinus luteus L. in field trials and pot experiments. Fixed N₂ was substituted by mineral nitrogen in these cases because of inhibition of N₂ fixation by mineral nitrogen. Contrary to these results mineral nitrogen increased grain yields and seed protein amounts ofLupinus albus L.,Pisum sativum L., andGlycine max. (L.) Merr. The nitrogen effect was caused at an early stage by saving energy due to inhibition of N₂ fixation (measurement of gas exchange by means of IRGA). In case of the N application after flowering grain, yields and seed protein levels increased because the mineral N was an additional nitrogen source for plants. At this stage the plants had ceased fixing atmospheric nitrogen. The high sink activity of growing fruits induced a lack of assimilates in nodules (determined by means of¹⁴CO₂ application). The N effect was therefore the consequence of the lower assimilate pool for supplying root nodules in these plants in comparison withVicia faba L. andLupinus luteus L. Hence it follows that response to mineral nitrogen can be a criterion for discovering more effective Rhizobium-host combinations.     

Nitrogen regime and isoenzyme changes in Vicia faba

Plants of Vicia faba were supplied either with fertilizer inorganic nitrogen or they received only nitrogen fixed by their root nodules. The esterase and transaminase isoenzyme profiles and the total water-soluble protein complement of seed cotyledons and of pollen did not vary irrespective of the N regime employed, whereas those of the leaf did. Possible causes and the implications of these differences are discussed.     

Plant symbiotic mutants as a tool to analyse nitrogen nutrition and yield relationship in field-growth peas (Pisum sativum L.)

Pisum sativum L. is known for high seed and protein yields but also for.yield instability. Because legumes utilize two sources of nitrogen (atmospheric N₂ fixed in nodules and assimilation of soil mineral N), studies on their nitrogen nutrition is more complex than in other plants. In this work, pea symbiotic mutants (with no nodules at all ([Nod^-]), with inefficient nodules ([Nod⁺Fix^-]) or showing an hypernodulating and a ‘nitrate-tolerant symbiosis’ character ([Nod⁺⁺Nts]), their semi-leafless isogenic homologues and the parental control line cv Frisson were fertilized with three levels of mineral nitrogen (0, 25 or 50 g N m^-2) to generate a range of mineral nitrogen regimes in the same genetic background. Impact of the source and level of nitrogen nutrition was measured on reproductive development, growth, nitrogen accumulation and seed yield. It was shown that a N deficiency induced flowering termination. It also led to a large decrease in the number of seeds produced and the amount of N accumulated in forage and in seeds, when little effect was observed on the progression rates of reproductive stages along the stem. The single seed weight and the amount of dry matter accumulated in forage neither responded strongly to N deficiency. The source of nitrogen was shown to be of little importance to yield but the application of about 50 g N m^-2 was necessary to reach the yield of the control cv Frisson when exclusive assimilation was ensuring the N requirements of the plant. Despite the fact that the nitrate-tolerant and hypernodulating mutant P64 used in this study did not yield as well as the parent cv Frisson, it is proposed that [Nod⁺⁺Nts] characters could act as a yield regulating factor.     

Carbon Metabolism, Nitrogen Assimilation, and Seed Yield of Cowpea (Vigna unquiculata L. Walp.) Grown in an Adverse Temperature Regime

When grown in an environment known not to favour the productionof large seed yields (warm days-cool nights; 33–19 °C),non-nodulated plants of cowpea cv. K 2809 supplied with abundantinorganic nitrogen not only assimilated N more rapidly but alsoproduced larger total dry weights and seed yields than plantsdependent on Rhizobium CB 756. Remobilization of nitrogen fromvegetative organs started sooner in nitrate-dependent than innodulated plants and contributed 69 and 47%, respectively, tothe N content of mature fruits. Plants dependent on nodulesrelied more on current assimilation of nitrogen during the laterstages of fruit growth than those given inorganic N; they alsoutilized a larger proportion of shoot-derived photosynthatesin growth of organs below ground and in the respiratory activitiesof both nodules and supporting roots. Although nitrate-dependentplants developed larger shoot systems than those relying onnodules, the distribution of carbon and nitrogen to leaves decreasedmarkedly as branches extended during early reproductive growth.The respiration of roots on nodulated plants became more efficientduring the later stages of fruit growth whereas the populationof secondary nodules present at this stage of development respiredless efficiently (mg C consumed per mg N assimilated) than theprimary nodules present earlier during development.     

野生大豆（Glycine soja）酰脲含量与根瘤固氮活力的关系

野生大豆（Ｇｌｙｃｉｎｅｓｏｊａ）酰脲含量与根瘤固氮活力的关系朱长甫,苗以农,刘学军,许守民（东北师范大学生命科学学院,长春１３００２４）郑惠玉,徐豹（吉林省农业科学院大豆研究所,公主岭１３６１００）关键词：野生大豆,固氮活力,酰脲,蛋白质根据固氮豆...     

Developmental genes have pleiotropic effects on plant morphology and source capacity, eventually impacting on seed protein content and productivity in pea

Increasing pea (Pisum sativum) seed nutritional value and particularly seed protein content, while maintaining yield, is an important challenge for further development of this crop. Seed protein content and yield are complex and unstable traits, integrating all the processes occurring during the plant life cycle. During filling, seeds are the main sink to which assimilates are preferentially allocated at the expense of vegetative organs. Nitrogen seed demand is satisfied partly by nitrogen acquired by the roots, but also by nitrogen remobilized from vegetative organs. In this study, we evaluated the respective roles of nitrogen source capacity and sink strength in the genetic variability of seed protein content and yield. We showed in eight genotypes of diverse origins that both the maximal rate of nitrogen accumulation in the seeds and nitrogen source capacity varied among genotypes. Then, to identify the genetic factors responsible for seed protein content and yield variation, we searched for quantitative trait loci (QTL) for seed traits and for indicators of sink strength and source nitrogen capacity. We detected 261 QTL across five environments for all traits measured. Most QTL for seed and plant traits mapped in clusters, raising the possibility of common underlying processes and candidate genes. In most environments, the genes Le and Afila, which control internode length and the switch between leaflets and tendrils, respectively, determined plant nitrogen status. Depending on the environment, these genes were linked to QTL of seed protein content and yield, suggesting that source-sink adjustments depend on growing conditions.     

Genetic variability in nodulation and root growth affects nitrogen fixation and accumulation in pea

BACKGROUND AND AIMS: Legume nitrogen is derived from two different sources, symbiotically fixed atmospheric N(2) and soil N. The effect of genetic variability of root and nodule establishment on N acquisition and seed protein yield was investigated under field conditions in pea (Pisum sativum). In addition, these parameters were related to the variability in preference for rhizobial genotypes. METHODS: Five different spring pea lines (two hypernodulating mutants and three cultivars), previously identified in artificial conditions as contrasted for both root and nodule development, were characterized under field conditions. Root and nodule establishment was examined from the four-leaf stage up to the beginning of seed filling and was related to the patterns of shoot dry matter and nitrogen accumulation. The genetic structure of rhizobial populations associated with the pea lines was obtained by analysis of nodule samples. The fraction of nitrogen derived from symbiotic fixation was estimated at the beginning of seed filling and at physiological maturity, when seed protein content and yield were determined. KEY RESULTS: The hypernodulating mutants established nodules earlier and maintained them longer than was the case for the three cultivars, whereas their root development and nitrogen accumulation were lower. The seed protein yield was higher in 'Athos' and 'Austin', the two cultivars with increased root development, consistent with their higher N absorption during seed filling. CONCLUSION: The hypernodulating mutants did not accumulate more nitrogen, probably due to the C cost for nodulation being higher than for root development. Enhancing exogenous nitrogen supply at the end of the growth cycle, by increasing the potential for root N uptake from soil, seems a good option for improving pea seed filling.     

Nitrogen fixation and carbon metabolism in legume nodules

A large amount of energy is utilized by legume nodules for the fixation of nitrogen and assimilation of fixed nitrogen (ammonia) into organic compounds. The source of energy is provided in the form of photosynthates by the host plant. Phosphoenol pyruvate carboxylase (PEPC) enzyme, which is responsible for carbon dioxide fixation in C4 and crassulacean acid metabolism plants, has also been found to play an important role in carbon metabolism in legume root nodule. PEPC-mediated CO2 fixation in nodules results in the synthesis of C4 dicarboxylic acids, viz. aspartate, malate, fumarate etc. which can be transported into bacteroids with the intervention of dicarboxylate transporter (DCT) protein. PEPC has been purified from the root nodules of few legume species. Information on the relationship between nitrogen fixation and carbon metabolism through PEPC in leguminous plants is scanty and incoherent. This review summarizes the various aspects of carbon and nitrogen metabolism in legume root nodules.     

Effect of Molybdenum and Inorganic Nitrogen on Molybdenum Redistribution in Black Gram (Vigna mungo L. Hepper) with Particular Reference to Seed Fill

Seeds used to plant a crop may contain sufficient molybdenum(Mo) to prevent subsequent Mo deficiency in the crop even whenthey are sown on Mo deficient soils. However, little is knownabout either the sources of the Mo acquired by the seed, orthe timing of its redistribution during seed development. Aglasshouse experiment was set up to examine the effect of Mosupply and nitrogen source on the redistribution of Mo withinblack gram, from full flowering to seed maturity. Treatmentscomprised two sources of N (symbiotic N₂fixation, NH₄NO₃), twolevels of Mo supply [nil (-Mo), 0.64 mg Mo kg^-1soil (+Mo)] andfour harvests (full flowering, early pod setting, late pod fillingand seed maturity). The redistribution of Mo in black gram wasexamined by determining changes over time in the content ofMo in plant parts at each growth stage. Molybdenum supply and the plant growth stage strongly affectedthe redistribution of Mo to the seed. In -Mo plants relianton symbiotic N₂fixation, Mo redistributed from roots, stemsand leaves was the only source of Mo for reproductive developmentsince, from full flowering until maturity, there was no netincrease in whole plant Mo. For pod and early seed development,the roots were the major source of Mo in -Mo plants. After latepod filling, nodules replaced roots as the major source of Mofor seed fill in -Mo plants. By contrast, for +Mo plants relianton symbiotic N₂fixation, Mo taken up from the soil after fullflowering could have supplied nearly 50% of the seed Mo. Themajor sources of Mo for seed filling in +Mo plants were middlestem leaves during early podding, and middle stems and pod wallsfrom late podding. Supplying NH₄NO₃to plants from sowing had little effect on Modistribution or redistribution in +Mo black gram plants. However,in -Mo plants it accelerated the loss of Mo from middle stemsand their leaves compared to nodulated plants. Black gram; Vigna mungo L. Hepper; distribution; molybdenum; nitrogen; nodules; redistribution; seed fill     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Effect of soil moisture stress on legume-rhizobium symbiosis in soybeans

Summary In a pot culture experiment, the influence of soil moisture stress at different physiological stages of soybean, cv. Hark, on nodulation, symbiosis and nitrogen accumulation was studied. Moisture stress reduced leghemoglobin content of root nodules and nitrogen uptake by plants. It had no effect on number of bacteroids. Stress at mid bloom and rapid pod filling stages reduced yield and seed protein content. However, these parameters were not affected by stress at nodule initiation and early flowering stages, though, flower initiation and maturity of the plant were delayed. Moisture stress at any stage did not alter nitrogen status of roots.     

Overexpression of the ASN1 gene enhances nitrogen status in seeds of Arabidopsis

In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.     

Effect of Source of Nitrogen on the Growth of Fiskeby Soya Bean: the Carbon Economy of Whole Plants

Fiskeby V soya bean was grown from seed germination to seedmaturation with two contrasting patterns of nitrogen metabolism:either wholly dependent on dinitrogen fixation, or with an abundantsupply of nitrate nitrogen, but lacking root nodules. The carbonand nitrogen economies of the plants were assessed at frequentintervals by measurements of photosynthesis, shoot and rootrespiration, and organic and inorganic nitrogen contents. Plantsfixing atmospheric nitrogen assimilated only 25–30 percent as much nitrogen as equivalent plants given nitrate nitrogen:c. 40 per cent of the nitrogen of ‘nitrate’ plantswas assimilated after dinitrogen fixation had ceased in ‘nodulated’plants. The rates of photosynthesis and respiration of the shootsof soya bean were not markedly affected by source of nitrogen;in contrast, the roots of ‘nodulated’ plants respiredtwice as rapidly during intense dinitrogen fixation as thoseof ‘nitrate’ plants. The magnitude of this respiratoryburden was calculated to increase the daily whole-plant respiratory loss of assimilate by 10–15 per cent over thatof plants receiving abundant nitrate. It is concluded that ‘nodulated’plants grew more slowly than ‘nitrate’ plants inthese experiments for at least two reasons: firstly, the symbioticassociation fixed insufficient nitrogen for optimum growth and,secondly, the assimila tion of the nitrogen which was fixedin the root nodules was more energy-demanding in terms of assimilatethan that of plants which assimilated nitrogen by reducing nitratein their leaves.     

施氮水平对小麦籽粒发育过程中氨基酸含量的影响

施氮能提高小麦籽粒蛋白质氨基酸的含量,并与施氮水平呈正相关;但对普通小麦必需氨基酸与蛋白质氨基酸的比值没有影响,而硬粒小麦4286随施氮水平的提高,该比值下降。在开花后32d以前,籽粒发育过程中游离氨基酸与施氮水平呈正相关,以后,籽粒中游离氨基酸趋于相近,表明施氮增加了游离氨基酸的库源,不同基因型小麦对施氮水平的反应不同,在同等施氮水平和栽培条件下,籽粒中蛋白质氨基酸和游离氨基酸含量为硬粒小麦4286＞小偃6号＞小偃107,不同施氮水平下,籽粒中氨基酸含量为高氮＞中氮＞低氮。     

Partitioning and Utilization of Carbon and Nitrogen in Nodulated Roots and Nodules of Chickpea (Cicer arietinum) Grown at Two Moisture Levels

The partitioning and utilization of carbon (C) and nitrogen(N) in nodulated roots and nodules of chickpea (Cicer arietinumL.) was studied at two moisture levels at 10-d intervals 40–140d after sowing (DAS). More C was used in respiration and lessin growth of nodulated roots and nodules under water stresscompared to controls during all growth stages except at theearly vegetative stage. Similarly, less nitrogen was investedin dry matter of both nodules and nodulated roots under stress,except during the vegetative stage where more nitrogen was used.Calculated over the entire growth period, as much as 14 and20% of the total nitrogen and 3 and 4% of the total carbon fixedby the plant was lost to the rooting medium under controlledand stressed conditions, respectively. The efficiency of nitrogen fixation with respect to net C utilizationwas maximal during seed filling under both control and stressconditions However, the efficiency of nitrogen fixation wasalways greater under drier conditions. Carbon, chickpea (Cicer arietinum L.), nitrogen, nitrogen fixation, partitioning     

Effect of applied nitrate on growth and N2-fixation inCicer arietinum L.

Summary Application of nitrate, either weekly or at the time of nodulation and pod-filling, significantly retarded nodule development and exerted a delay effect on the rate of N₂-fixating. However, after a certain period of time, its effect on nitrogenase became less conspicuous. Nitrate enhanced nitrate reductase activity in leaves as well as in nodules. At the initial stages, nitrate treated plants accumulated dry mass at a higher rate than those growing exclusively on atmospheric nitrogen. Nitrate induced premature senescence of plants towards the final stages of growth and lowered both the seed number per plant as well as weight of individual seed.     

Genetic dissection of nitrogen nutrition in pea through a QTL approach of root, nodule, and shoot variability

Pea (Pisum sativum L.) is the third most important grain legume worldwide, and the increasing demand for protein-rich raw material has led to a great interest in this crop as a protein source. Seed yield and protein content in crops are strongly determined by nitrogen (N) nutrition, which in legumes relies on two complementary pathways: absorption by roots of soil mineral nitrogen, and fixation in nodules of atmospheric dinitrogen through the plant–Rhizobium symbiosis. This study assessed the potential of naturally occurring genetic variability of nodulated root structure and functioning traits to improve N nutrition in pea. Glasshouse and field experiments were performed on seven pea genotypes and on the ‘Cameor’ × ‘Ballet’ population of recombinant inbred lines selected on the basis of parental contrast for root and nodule traits. Significant variation was observed for most traits, which were obtained from non-destructive kinetic measurements of nodulated root and shoot in pouches, root and shoot image analysis, ¹⁵N quantification, or seed yield and protein content determination. A significant positive relationship was found between nodule establishment and root system growth, both among the seven genotypes and the RIL population. Moreover, several quantitative trait loci for root or nodule traits and seed N accumulation were mapped in similar locations, highlighting the possibility of breeding new pea cultivars with increased root system size, sustained nodule number, and improved N nutrition. The impact on both root or nodule traits and N nutrition of the genomic regions of the major developmental genes Le and Af was also underlined.     

The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in Lotus japonicus nodules

Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.     

A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max [L.] Merr.)

Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.