Drought causes considerable reduction of legume productivity and significantly threatens the food security, and this situation is expected to be aggravated due to climate change. In soybean and common bean, water resource capturing through plant root architectural plasticity and the role of symbiotic nitrogen fixation have not been investigated in greater detail yet. This study was therefore conducted to identify and apply useful morphological and physiological performance markers (traits) for selection of drought-tolerant common bean and soybean cultivars under both controlled phytotron and field conditions that might be applicable as markers in future legume breeding programs. In soybean, traits related to above ground performance, such as photosynthesis, biomasses, and stomatal conductance, were related to parameters for nitrogen acquisition in nodules. The ability to maintain vigorous shoot growth under drought-induced nitrogen limitation was identified as an important trait that can be used to select for improved drought tolerance. Further, experiments carried out growing different common bean inbred lines under controlled phytotron conditions revealed the importance of growth and gas exchange parameters as well as nitrogen fixing ability as performance markers to select superior performing bean lines for growth under drought. As a further result, the strong association of symbiotic nitrogen fixation with CO2 assimilation and stomatal conductance was also ascertained. In field experiments the effective use of water through enhanced lateral root development and maintaining the water status of the plant was found to be crucial for enhanced productivity under drought, with root morphology traits (root length, area and volume) as well as root architectural traits (first whorl angle, basal root number and adventitious root branching density) significantly related to seed yield. Measurement of these traits might be added to future bean varietal improvement programs. Further, a direct relationship between both water use efficiency (WUE) estimated using carbon isotope discrimination (CID) and nitrogen fixation (15N abundance) with root morphological and architectural traits (root length, area and volume, basal root number, 1st as well as 2nd whorl angles) was identified. CID (WUE) and 15N abundance (SNF ability) had a direct relationship with each other and also with productivity traits (seed yield and pod harvest index). Soybean field experiments verified the importance of root system architecture and morphology for providing drought tolerance with root architectural traits, tap and lateral roots (diameter and branching density) and morphological traits (root length, surface area and volume) contributing to better performance under drought. Moreover, the strong association of CID (WUE) with ä15N (SNF), root traits as well as seed yield in soybean exposed to drought was ascertained. Findings suggested that higher performance in CID under drought stress may be due to higher CO2 assimilation and better N2 fixation resulting in better root system architecture and morphology of the drought-tolerant cultivar through maintenance of the water status of the plant for efficient biological activity. Overall the study has generated new knowledge about the use of physiological markers (traits) that can be used widely for legume evaluation under drought suitable for both phytotron and field studies. / Thesis (PhD)--University of Pretoria, 2012. / Plant Science / unrestricted

Sinorhizobium meliloti is a model bacterium for the study of symbiotic nitrogen fixation (SNF). It infects the roots of alfalfa as well as some other legumes and differentiates into N2-fixing bacteroids within the plant cells of specialized nodule organs. To understand genes essential for SNF and, in the longer term, to facilitate the manipulation of this SNF process for agricultural purposes, it is highly desirable to construct the minimal genome for SNF in this organism. S. meliloti harbors two replicons required for SNF, a 1.7-Mb chromid (pSymB) and a 1.4-Mb megaplasmid (pSymA). A previous deletion analysis revealed that only four gene regions, accounting for <12% of the total sequences of pSymA and pSymB that, were essential for SNF. In the first part of the thesis, I report the cloning of these two pSymA SNF-essential regions on a plasmid (pTH3255) in Escherichia coli, and the integration of this plasmid into the genome of a ∆pSymA S. meliloti derivative strain (the strain was named as RmP4291 after integration). Plant root dry weight and nitrogenase-catalyzed acetylene reduction assays were carried out on RmP4291 with four host plants, including Medicago sativa, Medicago truncatula, Melilotus alba and Melilotus officinalis. Nodule kinetic assays were also performed on RmP4291 and RmP110(wt). The results showed that the SNF-essential regions from pSymA were sufficient to restore the symbiotic capabilities to the ∆pSymA derivative strain with all the host plants tested, except a significant reduction (~40%) in SNF by RmP4291 was noticed on M. officinalis compared to that by wildtype S. meliloti. A higher alfalfa nodulation efficiency of RmP4291 compared to that of wildtype RmP110 was also discovered. In the second part of the thesis, a histochemical staining method for S. meliloti nodules was developed by integrating the marker genes gusA (β-glucuronidase) and celB (β-glucosidase) into the S. meliloti genome. This staining method was found to be useful in the study of nodule competitiveness. A nodule competition assay was carried out between RmP4291 and RmP110 using the new staining method. RmP4291 was found to be significantly reduced in nodulation competitiveness compared to wildtype S. meliloti. The development of the histochemical staining method for S. meliloti nodules will accelerate the identification of genes required for nodule competitiveness in the organism, which will be of crucial importance to the construction of the minimal genome strains with high SNF efficiency. / Thesis / Master of Science (MSc) / Nitrogen is one of the critical elements for life. Biological nitrogen fixation plays a crucial role in providing fixed nitrogen for the ecosystem on Earth. Our Laboratory has endeavored to establish a minimal symbiotic genome in Sinorhizobium meliloti, a model nitrogen fixing bacterium which forms symbiosis with certain kinds of legumes. Building this minimal symbiotic genome will improve our understanding of the symbiotic nitrogen fixation process in S. meliloti at gene level. It may also help in eventually introducing a nitrogen fixation system into other organisms. In this study, the minimal symbiotic genome of the pSymA replicon in S. meliloti was constructed. In addition, a staining method to detect specific S. meliloti strains in nodules was established. This method is potentially useful in finding genes related to nodule competitiveness, and these are potentially important for augmenting the genes that constitute the minimal symbiotic genome.

Made available in DSpace on 2014-06-11T19:22:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-27Bitstream added on 2014-06-13T19:48:25Z : No. of bitstreams: 1 ferrarineto_j_me_botfca.pdf: 394206 bytes, checksum: 6b64f3f5824ba5c1c97d98a5556b8a6a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Áreas que vem sendo cultivadas com pastagens por vários anos podem apresentar deficiências nutricionais e a população de bactérias fixadoras do nitrogênio atmosférico pode estar reduzida. O molibdênio é um micronutriente que faz parte da enzima nitrogenase, a qual é responsável pela fixação simbiótica do nitrogênio nas leguminosas, e está presente nos bacteróides nodulares. Com base nisso, o presente trabalho teve por objetivo avaliar o efeito da aplicação de molibdênio e inoculante, na cultura do amendoim em semeadura direta sobre palhada de forrageiras. Foram conduzidos quatro experimentos, dois no ano agrícola 2008/09 e dois no ano agícola 2009/10, onde a cultura do amendoim foi semeada, respectivamente, sobre as palhadas da Brachiaria brizantha, Brachiaria ruziziensis, Brachiaria brizantha e Brachiaria decumbens no delineamento de blocos casualizados, em esquema de parcelas subdivididas, com quatro repetições. As parcelas dos experimentos foram constituídas por dois tratamentos, com e sem aplicação de inoculante (1- inoculação artificial com estirpe específica de bactéria Bradyrhizobium spp. para a cultura do amendoim e 2- sem inoculação artificial) e as subparcelas dos experimentos do ano agrícola 2008/09 por três doses de molibdênio (27,5, 55, 110g ha-1) aplicadas via tratamento de sementes, na forma de molibdato de amônio e a testemunha. As subparcelas dos experimentos do ano agrícola 2009/10 constituíram-se de três doses de molibdênio (50, 100, 200g ha-1) aplicadas via foliar, na forma de molibdato de amônio e a testemunha. Foram avaliados o estado nutricional das plantas, a nodulação (número e matéria seca de nódulos por planta), a atividade da enzima nitrogenase pelo método da redução do acetileno, a atividade específica da nitrogenase, os componentes da produção... / The areas that have been cultivated with pasture for several years may have nutritional deficiencies and the population of atmospheric nitrogen fixing bacteria may be reduced. Molybdenum, a nutrient that is part of the nitrogenase enzyme, is responsible for symbiotic nitrogen fixation in legumes and is present in bacteria from the nodules. This study aimed at evaluating the effect of molybdenum and inoculant application, in peanut crop sown on a straw of forages. Four experiments were carried out, two in 2008/09 crop year and two in 2009/10 crop year, where peanut crop was directly sown, respectively, on the straws of Brachiaria brizantha, Brachiaria ruziziensis, Brachiaria brizantha and Brachiaria decumbens in the design of randomized blocks in a split-plot with four replications. The plots comprised two treatments, with and without inoculant application (1 - artificial inoculation with specific strain of Bradyrhizobium spp. for peanut crop and 2 - without artificial inoculation) and the subplots of the experiments carried out in the 2008/09 crop year in three molybdenum rates (27,5, 55 e 110 g ha-1) applied in the form of ammonium molybdate by seeds treatment and a control. The subplots of the experiments carried out in the 2009/10 crop year comprised three molybdenum rates (50, 100 e 200 g ha-1) applied as ammonium molybdate by foliar spraying and a control. The nutritional status of plants, nodulation (number and mass of dry matter of the nodules per plant), the activity of nitrogenase enzyme by acetylene reduction method, the specific activity of nitrogenase, yield components and the yield of pods and grains were evaluated. The data of each experiment were subjected to analysis of variance following the split plot model. To compare the average of inoculation, the method of comparison of unprotected average... (Complete abstract click electronic access below)

Orientador: Carlos Alexandre Costa Crusciol / Banca: Denizart Bolonhez / Banca: Edson Lazarini / Resumo: Áreas que vem sendo cultivadas com pastagens por vários anos podem apresentar deficiências nutricionais e a população de bactérias fixadoras do nitrogênio atmosférico pode estar reduzida. O molibdênio é um micronutriente que faz parte da enzima nitrogenase, a qual é responsável pela fixação simbiótica do nitrogênio nas leguminosas, e está presente nos bacteróides nodulares. Com base nisso, o presente trabalho teve por objetivo avaliar o efeito da aplicação de molibdênio e inoculante, na cultura do amendoim em semeadura direta sobre palhada de forrageiras. Foram conduzidos quatro experimentos, dois no ano agrícola 2008/09 e dois no ano agícola 2009/10, onde a cultura do amendoim foi semeada, respectivamente, sobre as palhadas da Brachiaria brizantha, Brachiaria ruziziensis, Brachiaria brizantha e Brachiaria decumbens no delineamento de blocos casualizados, em esquema de parcelas subdivididas, com quatro repetições. As parcelas dos experimentos foram constituídas por dois tratamentos, com e sem aplicação de inoculante (1- inoculação artificial com estirpe específica de bactéria Bradyrhizobium spp. para a cultura do amendoim e 2- sem inoculação artificial) e as subparcelas dos experimentos do ano agrícola 2008/09 por três doses de molibdênio (27,5, 55, 110g ha-1) aplicadas via tratamento de sementes, na forma de molibdato de amônio e a testemunha. As subparcelas dos experimentos do ano agrícola 2009/10 constituíram-se de três doses de molibdênio (50, 100, 200g ha-1) aplicadas via foliar, na forma de molibdato de amônio e a testemunha. Foram avaliados o estado nutricional das plantas, a nodulação (número e matéria seca de nódulos por planta), a atividade da enzima nitrogenase pelo método da redução do acetileno, a atividade específica da nitrogenase, os componentes da produção... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The areas that have been cultivated with pasture for several years may have nutritional deficiencies and the population of atmospheric nitrogen fixing bacteria may be reduced. Molybdenum, a nutrient that is part of the nitrogenase enzyme, is responsible for symbiotic nitrogen fixation in legumes and is present in bacteria from the nodules. This study aimed at evaluating the effect of molybdenum and inoculant application, in peanut crop sown on a straw of forages. Four experiments were carried out, two in 2008/09 crop year and two in 2009/10 crop year, where peanut crop was directly sown, respectively, on the straws of Brachiaria brizantha, Brachiaria ruziziensis, Brachiaria brizantha and Brachiaria decumbens in the design of randomized blocks in a split-plot with four replications. The plots comprised two treatments, with and without inoculant application (1 - artificial inoculation with specific strain of Bradyrhizobium spp. for peanut crop and 2 - without artificial inoculation) and the subplots of the experiments carried out in the 2008/09 crop year in three molybdenum rates (27,5, 55 e 110 g ha-1) applied in the form of ammonium molybdate by seeds treatment and a control. The subplots of the experiments carried out in the 2009/10 crop year comprised three molybdenum rates (50, 100 e 200 g ha-1) applied as ammonium molybdate by foliar spraying and a control. The nutritional status of plants, nodulation (number and mass of dry matter of the nodules per plant), the activity of nitrogenase enzyme by acetylene reduction method, the specific activity of nitrogenase, yield components and the yield of pods and grains were evaluated. The data of each experiment were subjected to analysis of variance following the split plot model. To compare the average of inoculation, the method of comparison of unprotected average... (Complete abstract click electronic access below) / Mestre

Legumes play an important role in agriculture as major food sources for humans and as feed for animals. Bioavailable nitrogen is a limiting nutrient for crop growth. Legumes are important because they can form a symbiotic relationship with soil bacteria called rhizobia that results in nitrogen-fixing root nodules. In this symbiosis, rhizobia provide nitrogen to the legumes and the legumes provide carbon sources to the rhizobia. The Medicago truncatula NPF1.7/NIP/LATD gene is essential for root nodule development and also for proper development of root architecture. Work in our lab on the MtNPF1.7/MtNIP/LATD gene has established that it encodes a nitrate transporter and strongly suggests it has another function. Mtnip-1/latd mutants have pleiotropic defects, which are only partially explained by defects in nitrate transport. MtNPF1.7/NIP/LATD is a member of the large and diverse NPF/NRT1(PTR) transporter family. NPF/NRT1(PTR) members have been shown to transport other compounds in addition to nitrate: nitrite, amino acids, di- and tri-peptides, dicarboxylates, auxin, abscisic acid and glucosinolates. In Arabidopsis thaliana, the AtNPF6.3/NRT1.1( CHL1) transporter was shown to transport auxin as well as nitrate. Atchl1 mutants have defects in root architecture, which may be explained by defects in auxin transport and/or nitrate sensing. Considering the pleiotropic phenotypes observed in Mtnip-1/latd mutant plants, it is possible that MtNPF1.7/NIP/LATD could have similar activity as AtNPF6.3/NRT1.1(CHL1). Experimental evidence shows that the MtNPF1.7/NIP/LATD gene is able to restore nitrate-absent responsiveness defects of the Atchl1-5 mutant. The constitutive expression of MtNPF1.7/NIP/LATD gene was able to partially, but not fully restore the wild-type phenotype in the Atchl1-5 mutant line in response to auxin and cytokinin. The constitutive expression of MtNPF1.7/NIP/LATD gene affects the lateral root density of wild-type Col-0 plants differently in response to IAA in the presence of high (1mM) or low (0.1 mM) nitrate. MtNPF1.7/NIP/LATD gene expression is not regulated by nitrate at the concentrations tested and MtNPF1.7/NIP/LATD does not regulate the nitrate-responsive MtNRT2.1 gene. Mtnip-1 plants have an abnormal gravitropic root response implicating an auxin defect. Together with these results, MtNPF1.7/NIP/LATD is associated with nitrate and auxin; however, it does not act in a homologous fashion as AtNPF6.3/NRT1.1(CHL1) does in A. thaliana.

Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence of higher auxin in both the Mtnip-1 mutant and in M. truncatula plants constitutively expressing MtNPF1.7 was observed. Previous experiments showed MtNPF1.7 expression is hormone regulated and the MtNPF1.7 promoter is active in root and nodule meristems and in the vasculature. Two potential binding sites for an auxin response factors (ARFs) were found in the MtNPF1.7 promoter. Chromatin immunoprecipitation-qRT-PCR confirmed MtARF1 binding these sites. Mutating the MtARF1 binding sites increases MtNPF1.7 expression, suggesting a mechanism for auxin repression of MtNPF1.7. Consistent with these results, constitutive expression of an ARF in wild-type plants partially phenocopies Mtnip-1 mutants’ phenotypes.

In this dissertation I am using M. truncatula as a model legume that forms indeterminate nodules with rhizobia under limited nitrogen conditions. I take advantage of an M. truncatula Tnt1 mutant population that provides a useful resource to uncover and characterize novel genes. Here, I focused on several objectives. First, I carried out forward and reverse genetic screening of M. truncatula Tnt1 mutant populations to uncover novel genes involved in symbiotic nitrogen fixation. Second, I focused on reverse genetic screening of two genes, identified as encoding blue copper proteins, and characterization of their mutants' potential phenotypes. Third, I further characterized a nodule essential gene, M. truncatula vacuolar iron transporter like 8 (MtVTL8), which encodes a nodule specific iron transporter. I characterized the expression pattern, expression localization and function of MtVTL8. Additionally, I characterized several residues predicted to be essential to function using a model based on the known crystal structure of Eucalyptus grandis vacuolar iron transporter 1 (EgVIT1), a homologous protein to MtVTL8. I identified several potential essential residues of the MtVTL8 protein, mutagenized them, and through complementation experiments in planta and in yeast assessed functionality of the resulting protein. This helped us to better understand the potential mechanism by which MtVTL8 functions.

O Níquel (Ni) é um micronutriente para as plantas, por ser componente estrutural das enzimas urease e hidrogenase, que desempenham função no metabolismo do nitrogênio (N) nas plantas leguminosas. A aplicação de Ni via tratamento de semente em soja pode potencializar o processo de fixação biológica de nitrogênio (FBN) com a finalidade de proporcionar maior produção de biomassa da parte aérea e de grãos. Objetivou-se com este trabalho avaliar o efeito da aplicação de doses de Ni, via tratamento de semente, no processo de FBN em plantas de soja, por meio da atividade de nitrogenase (ANase) e da abundância natural de 15N (? 15N?). Foi realizado um experimento em casa de vegetação, em condições controladas. O genótipo de soja BMX POTÊNCIA RR foi cultivado em solo arenoso e submetido à aplicação de 0; 45; 90; 135; 180; 360; e 540 mg kg-1 de Ni aplicado via tratamento de semente. As plantas de soja foram conduzidas até o estádio fenológico R7 (maturação dos grãos). Foram avaliadas a concentração de macro e micronutrientes nas folhas utilizadas para diagnose, determinou-se o teor de clorofila, a atividade enzimática da urease, bem como se quantificou a massa seca de nódulos, no estádio fenológico R1 (Início da floração: até 50% das plantas com flor). Foram ainda determinados: as produções de massa seca de parte aérea e de grãos, o índice de colheita, os acúmulos de Ni e de N na semente e a contribuição da fixação biológica de nitrogênio, por meio da técnica de abundância natural de 15N e indiretamente através da técnica da redução do acetileno (atividade da nitrogenase). Observou-se que as doses de Ni exerceram efeitos em todos os parâmetros avaliados. A aplicação de Ni na dose de 45 mg kg-1 aumentou a fixação biológica de N na ordem de 12% em relação ao tratamento controle (sem adição de Ni) e incrementou o acúmulo de Ni e de N no grão, com efeito na atividade da enzima urease. A aplicação de 45 e 90 mg kg-1 de níquel via semente resultou, respectivamente, no aumento de 67% e 77% da atividade da NAase em relação ao controle. Pela análise da abundância natural de 15N no grão (?15N?) observou-se que houve diferença na FBN entre os tratamentos e que a aplicação da dose de 45 mg kg-1, de Ni via semente, revelou que 99% do N acumulado no grão foi proveniente da FBN, enquanto que no tratamento sem adição de Ni via semente, a contribuição da FBN foi de apenas 77%. Nas condições do experimento, a aplicação de Ni na semente refletiu em maior produção de massa seca de parte aérea e na produção de grãos de soja / Nickel (Ni) is an essential micronutrient for plants due to its role on structural component of the enzymes urease and hydrogenase, which perform nitrogen (N) metabolism in legumes plants. Seed treatment with Ni in soybean might improve the symbiotic or biological nitrogen fixation (BNF) process, by increasing biomass production and grain yield. The objective of this study was to evaluate the effect of soybean seed treatment with Ni rates on the biological nitrogen fixation (BNF) process by evaluating the nitrogenase activity (NAase), the natural 15N abundance (? 15N ?) and urease activity in soybean plants, as well. The experiment was carried out in greenhouse with controlled conditions. The soybean cultivar used was BMX POTÊNCIA RR, which was grown in sandy soil, and submitted to application of 0, 45, 90,135, 180, 360 and 540 mg kg-1 of Ni on the seed treatment. Soybean plants were grown up to the R7 developmental stage (grain maturity). Macro and micronutrients concentration in the leaves used for diagnosis, as well as the chlorophyll content and urease activity have been determined on these tissues, and dry weight of nodules were evaluated in developmental stage R1 (flowering stage). The following measurements also were made: dry matter yield of plant top, dry matter yield of grains, the harvest index, uptake of Ni and N in the seed and the contribution of biological nitrogen fixation by 15N natural abundance and to indirectly way by the reduction of acetylene technique (nitrogenase activity), as well. It was observed that Ni rates influenced all parameters. Application of Ni rate of 45 mg kg-1 increased biological nitrogen fixation in the order of 12% as compared to the control. Ni rates of 45 and 90 mg kg-1 via seed treatment increased the NAase activity, respectively, in 67% and 77% compared to the control. Other benefits of Ni-soybean seeds treatment were the increased accumulation of Ni and N on the grain and its positive influence on the urease activity. By analyzing the natural abundance of 15N on the grain (?15N ?) it was observed that there were differences between treatments in the symbiotic nitrogen fixation. Around 99% of total N taken up in the seeds came from biological nitrogen fixation by supplying Ni-seed application rate of 45 mg kg-1, whereas in the control, the contribution of BNF was only 77%. In these experimental conditions, seed treatment with Ni reflected in higher dry matter production of plant top and grain yield

O Níquel (Ni) é um micronutriente para as plantas, por ser componente estrutural das enzimas urease e hidrogenase, que desempenham função no metabolismo do nitrogênio (N) nas plantas leguminosas. A aplicação de Ni via tratamento de semente em soja pode potencializar o processo de fixação biológica de nitrogênio (FBN) com a finalidade de proporcionar maior produção de biomassa da parte aérea e de grãos. Objetivou-se com este trabalho avaliar o efeito da aplicação de doses de Ni, via tratamento de semente, no processo de FBN em plantas de soja, por meio da atividade de nitrogenase (ANase) e da abundância natural de 15N (? 15N?). Foi realizado um experimento em casa de vegetação, em condições controladas. O genótipo de soja BMX POTÊNCIA RR foi cultivado em solo arenoso e submetido à aplicação de 0; 45; 90; 135; 180; 360; e 540 mg kg-1 de Ni aplicado via tratamento de semente. As plantas de soja foram conduzidas até o estádio fenológico R7 (maturação dos grãos). Foram avaliadas a concentração de macro e micronutrientes nas folhas utilizadas para diagnose, determinou-se o teor de clorofila, a atividade enzimática da urease, bem como se quantificou a massa seca de nódulos, no estádio fenológico R1 (Início da floração: até 50% das plantas com flor). Foram ainda determinados: as produções de massa seca de parte aérea e de grãos, o índice de colheita, os acúmulos de Ni e de N na semente e a contribuição da fixação biológica de nitrogênio, por meio da técnica de abundância natural de 15N e indiretamente através da técnica da redução do acetileno (atividade da nitrogenase). Observou-se que as doses de Ni exerceram efeitos em todos os parâmetros avaliados. A aplicação de Ni na dose de 45 mg kg-1 aumentou a fixação biológica de N na ordem de 12% em relação ao tratamento controle (sem adição de Ni) e incrementou o acúmulo de Ni e de N no grão, com efeito na atividade da enzima urease. A aplicação de 45 e 90 mg kg-1 de níquel via semente resultou, respectivamente, no aumento de 67% e 77% da atividade da NAase em relação ao controle. Pela análise da abundância natural de 15N no grão (?15N?) observou-se que houve diferença na FBN entre os tratamentos e que a aplicação da dose de 45 mg kg-1, de Ni via semente, revelou que 99% do N acumulado no grão foi proveniente da FBN, enquanto que no tratamento sem adição de Ni via semente, a contribuição da FBN foi de apenas 77%. Nas condições do experimento, a aplicação de Ni na semente refletiu em maior produção de massa seca de parte aérea e na produção de grãos de soja / Nickel (Ni) is an essential micronutrient for plants due to its role on structural component of the enzymes urease and hydrogenase, which perform nitrogen (N) metabolism in legumes plants. Seed treatment with Ni in soybean might improve the symbiotic or biological nitrogen fixation (BNF) process, by increasing biomass production and grain yield. The objective of this study was to evaluate the effect of soybean seed treatment with Ni rates on the biological nitrogen fixation (BNF) process by evaluating the nitrogenase activity (NAase), the natural 15N abundance (? 15N ?) and urease activity in soybean plants, as well. The experiment was carried out in greenhouse with controlled conditions. The soybean cultivar used was BMX POTÊNCIA RR, which was grown in sandy soil, and submitted to application of 0, 45, 90,135, 180, 360 and 540 mg kg-1 of Ni on the seed treatment. Soybean plants were grown up to the R7 developmental stage (grain maturity). Macro and micronutrients concentration in the leaves used for diagnosis, as well as the chlorophyll content and urease activity have been determined on these tissues, and dry weight of nodules were evaluated in developmental stage R1 (flowering stage). The following measurements also were made: dry matter yield of plant top, dry matter yield of grains, the harvest index, uptake of Ni and N in the seed and the contribution of biological nitrogen fixation by 15N natural abundance and to indirectly way by the reduction of acetylene technique (nitrogenase activity), as well. It was observed that Ni rates influenced all parameters. Application of Ni rate of 45 mg kg-1 increased biological nitrogen fixation in the order of 12% as compared to the control. Ni rates of 45 and 90 mg kg-1 via seed treatment increased the NAase activity, respectively, in 67% and 77% compared to the control. Other benefits of Ni-soybean seeds treatment were the increased accumulation of Ni and N on the grain and its positive influence on the urease activity. By analyzing the natural abundance of 15N on the grain (?15N ?) it was observed that there were differences between treatments in the symbiotic nitrogen fixation. Around 99% of total N taken up in the seeds came from biological nitrogen fixation by supplying Ni-seed application rate of 45 mg kg-1, whereas in the control, the contribution of BNF was only 77%. In these experimental conditions, seed treatment with Ni reflected in higher dry matter production of plant top and grain yield

A large proportion of the world’s nitrogen needs is derived from symbiotic nitrogen fixation (SNF), which contributes substantially to agricultural sustainability. The partnership between legumes and rhizobia result in the formation of specialised structures called root nodules. Within these nodules SNF is supported by the sucrose transported from the leaves to the nodules for respiration. The end products of SNF in soybean (Glycine max (L.) Merr.) root nodules, namely ureides, are transported to the upper parts of the plant to supply nitrogen. Symbiotic nitrogen fixation provides a vital advantage for the production of soybean compared with most grain crops in that soybean fixes the nitrogen required for its growth and for the production of the high-protein content in seed and oil. The process of SNF is dramatically affected by drought, salt, cold and heavy metal stresses. Since SNF is such an important yield-determining factor, a lack in understanding these complexes inevitably delays progress towards the genetic improvement of soybean genotypes and also complicates decisions with regard to the suitability of certain genotypes for the various soybean producing areas in South Africa. The largest soybean producing areas in South Africa are situated at high altitudes, with minimum daily temperatures which can be critically low and impeding the production of soybean. Soybean is chilling sensitive, with growth, development and yield being affected negatively at temperatures below 15°C. Dark chilling (low night temperature) stress has proved to be one of the most important restraints to soybean production in South Africa. Among the symptoms documented in dark chilling sensitive soybean genotypes are reduced growth rates, loss of photosynthetic capacity and pigment content, as well as premature leaf senescence and severely inhibited SNF. Existing knowledge about stress-induced nodule senescence is based on fragmented information in the literature obtained in numerous, and often diverse, legume species. The precise nature and sequence of events participating in nodule senescence has not yet been fully explained. The main objectives of this investigation were to characterise the natural senescence process in soybean nodules under optimal growth conditions and to characterise the alteration of the key processes of SNF in a chilling sensitive soybean genotype during dark chilling. Moreover, to establish whether recovery in nodule functionality following a long term dark chilling period occured, or whether nodule senescence was triggered, and if sensitive biochemical markers of premature nodule senescence could be identified. A known chilling sensitive soybean genotype, PAN809, was grown under controlled growth conditions in a glasshouse. To determine the baseline and change over time for key parameters involved in SNF, a study was conducted under optimal growing conditions over a period of 6 weeks commencing 4 weeks after sowing. The cluster of crown nodules were monitored weekly and analysis included nitrogenase activity, ureide content, respiration rate, leghemoglobin content, sucrose synthase (SS) activity and sucrose content. Further investigations focused on induced dark chilling effects on nodule function to determine the alterations in key parameters of SNF. Plants were subjected to dark chilling (6˚C) for 12 consecutive nights and kept at normal day temperatures (26˚C). The induced dark chilling was either only shoot (SC) exposure or whole plant chilling (WPC). These treatments were selected since, in some areas in South Africa cold nights result not only in shoot chilling (SC) but also in low soil temperatures causing direct chilling of both roots and shoots. To determine if premature nodule senescence was triggered, the recovery following 12 consecutive nights of chilling treatment was monitored for another 4 weeks. It was established that the phase of optimum nitrogenase activity under optimal growing conditions occurred during 4 to 6 weeks after sowing where after a gradual decline commenced. This decline was associated with a decline in nitrogenase protein content and an increase in ureide content. The stability of SS activity and nodule respiration showed that carbon-dependent metabolic processes were stable for a longer period than previously mentioned parameters. The negative correlation that was observed between nitrogenase activity and nodule ureide content pointed towards the possible presence of a feedback inhibition trigger on nitrogenase activity. A direct effect of dark chilling on nitrogenase activity and nodule respiration rate led to a decline in nodule ureide content that occurred without any limitations on the carbon flux of the nodules (i.e. stable sucrose synthase activity and nodule sucrose content). The effect on SC plants was much less evident but did indicate that currently unknown shoot-derived factors could be involved in the minor inhibition of SNF. It was concluded that the repressed rates of respiration might have led to increased O2 concentrations in the nodule, thereby inhibiting the nitrogenase protein and so the production of ureides. It was found that long term chilling severely disrupted nitrogenase activity and ureide synthesis in nodules. Full recovery in all treatments occurred after 2 weeks of suspension of dark chilling, however, this only occurred when control nodules already commenced senescence. This points toward reversible activation of the nitrogenase protein with no evidence in support of premature nodule senescence. An increase in intercellular air space area was induced by long term dark chilling in nodules, specifically by the direct chilling of nodules (WPC treatment). The delayed diminishment of intercellular air space area back to control levels following dark chilling may be an important factor involved in the recovery of nitrogenase activity because enlarged air spaces would have favoured gaseous diffusion, and hence deactivation of nitrogenase, in an elevated O2 environment (due to supressed nodule respiration rates). These findings revealed that dark chilling did not close the diffusion barrier, as in the case of drought and other stress factors, but instead opened it due to an increase in air space areas in all regions of the nodule. In conclusion, this study established that dark chilling did not initiate premature nodule senescence and that SNF demonstrated resilience, with full recovery possible following even an extended dark chilling period involving low soil temperatures. / Thesis(PhD (Botany))--North-West University, Potchefstroom Campus, 2013