To determine the limiting factors in nitrate assimilation in maize and barley, the effects of nitrate on 1) steady state levels of nitrate reductase activity (NRA) and nitrate reductase protein (NRP); 2) the uptake, translocation and accumulation of nitrate in the shoots of the seedling plant were examined. Seedlings were grown on Kimpack paper containing l, 5 or 20mM KN0₃ for 7 days at 20°c (barley) or 28°C (maize). At lmM KN0₃ the rate of 3 nitrate uptake and the levels of NRA and NRP were higher in maize than in barley. In contrast., at 5 and 20mlv1 KNo₃, the rate of uptake, the accumulation of nitrate and the NRA were higher in barley than in maize. The results suggest that the synthesis of NR is induced by lower levels of nitrate in maize relative to barley. In addition, nitrate-nitrogen appears to be more efficiently converted to proteins, other than NR, in maize than in barley. At very low levels of nitrate an inactive NR protein was present. in maize. To characterize the inactive NR, maize plants were grown under conditions where high levels of NRA were detected (vermiculite:sand, l:lw/w, containing 10mM KN0₃) and under conditions where NR was present primarily in the inactive form (Kimpack paper:washed sand). Nitrate reductase was purified from primary leaves using Blue Sepharose affinity chromatography. The column was washed with NADH and KN0₃ in each case. The peaks of NR were 3 detected using Dot-immunoblotting, with an antibody prepared against maize leaf NR and by assessing the NRA. Active and inactive NR forms were found both at the NADH-and the KN0₃-wash. In the NADH-wash, the inactive NR as compared to the active form, has very low NADH (complete), FMNH₂, MV and BPB (reductase) activities. Significant levels of cyt-c and FeCN (dehydrogenase) partial activities were detected. Similarly, the inactive NR in the KN0-wash, had 3no NADH (complete), FMNH2 , MV and BPB (reductase) activities. Very low levels of cyt-c and FeCN (dehydrogenase) NR partial activities were detected, compared to the respective activities of the active enzyme in the KNO₃ wash. / Thesis / Master of Science (MSc)

No description available.

<p> The experiments described in this thesis were performed to investigate the: 1. Role of nitrate in the growth of maize seedling and 2. Role of amino acids in the assimilation of nitrate by the growing embryo.</p> <p> Nitrate stimulated the germination of Zea mays L. seeds by 10 to 15 percent. Further growth of the embryo axis, up to 6 days, however, was not affected by nitrate. During the early growth of the seedling, endosperm nitrogen was able to support the requirements of the embryo for 6 to 8 days. After a lag of 2 days, the protein content of the embryo increased linearly up to 6 days at a rate of 597 μ g a day. Some increase was observed between 6 and 8 days also. After 8 days, the protein level of embryo plateaued. Addition of 10 mM nitrate caused an increase in the protein and total nitrogen of the embryo only after 6 days. The endosperm mutant of maize, opaque-2, also responded to the nitrate in the same way.</p> <p> The protein content of the primary leaves increased linearly between 5 and 7 days. After 7 days, there is no increase in the leaf protein. Nitrate increased the protein level of primary leaves by 25 percent after 7 days. It protected against the further loss of protein in mature leaf. These results suggest that the primary role of nitrate in the growth of maize seedlings is to prevent protein loss.</p> <p> Out of 8 amino acids tested individually, only lysine and to a lesser extent arginine, inhibited the induction of nitrate reductase in the maize root tips. Different ammonium salts had no effect on the induction of nitrate reductase. The initial rate of induction in opaque-2 mutant (high lysine) was lower than the wild type, W64A (low lysine). From a comparison of the rate of induction of nitrate reductase between young and mature leaf, it was suggested that the amino acid supply from the endosperm may inhibit the induction of nitrate reductase. In the young maize seedling, this effect of amino acids may be more effective in vivo and in this way the assimilation of exogenous nitrate could be restricted, when the endosperm amino acids are supporting the growth of the embryo.</p> / Thesis / Master of Science (MSc)

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A cana-de-aÃÃcar à uma planta de grande potencial econÃmico, sendo utilizada principalmente para produÃÃo de aÃÃcar e biocombustÃveis. O Brasil tem destaque internacional na produÃÃo de cana-de-aÃÃcar, sendo o maior produtor mundial dessa cultura. Apesar dessa lideranÃa, o Brasil està localizado em uma regiÃo tropical e apresenta vÃrias adversidades climÃticas. O estudo da fisiologia da cana-de-aÃÃcar em situaÃÃes de estresse, como deficiÃncia hÃdrica, torna-se fundamental para manutenÃÃo e melhoria da produÃÃo dessa cultura. A cana-de-aÃÃcar à uma espÃcie de fotossÃntese C4, com um mecanismo de concentraÃÃo de CO2. Dessa maneira, espera-se que a crescente elevaÃÃo de CO2 na atmosfera nÃo afete a produtividade da cana-de-aÃÃcar de maneira relevante, mas os estudos acerca do assunto sÃo ainda bem escassos. Diante desse quadro, objetivou-se avaliar a regulaÃÃo da fotossÃntese por deficiÃncia hÃdrica, suprimento de nitrogÃnio e CO2 elevado em plantas de cana-de-aÃÃcar. O CapÃtulo 1 comtempla o estado da arte a respeito do tema em estudo. No capÃtulo 2, foi realizado um trabalho com o objetivo de avaliar as respostas de duas cultivares de cana-de-aÃÃcar consideradas contrastantes em relaÃÃo Ãs respostas à deficiÃncia hÃdrica, IAC SP 94-2094 e IAC SP 95-5000. Estas cultivares foram expostas a dÃficit hÃdrico severo, com suspensÃo da irrigaÃÃo por cinco dias, e recuperaÃÃo, reirrigadas por dois dias apÃs o estresse. O tratamento aplicado foi capaz de afetar fortemente as trocas gasosas, com fechamento estomÃtico nas duas cultivares, levando a fortes reduÃÃes de transpiraÃÃo e assimilaÃÃo de CO2. Os parÃmetros da fluorescÃncia da clorofila a foram afetados, mas nÃo o suficiente para apresentar fotoinibiÃÃo. A cultivar tolerante apresentou maior atividade de PEPcase no estresse, mas a atividade de Rubisco nÃo foi alterada. Na cultivar sensÃvel, nÃo houve alteraÃÃo na atividade de PEPcase enquanto que a atividade de Rubisco foi reduzida em 40% no estresse mas com total recuperaÃÃo. A expressÃo de PEPcase foi aumentada no estresse mas com maior incremento na cultivar tolerante, com padrÃo semelhante na quantidade de rubisco. No capÃtulo 3, foi realizado um trabalho com o objetivo de avaliar o sistema de defesa anti-oxidativo em plantas de cana-de-aÃÃcar expostas ao estresse hÃdrico moderado. Neste capÃtulo, apenas a cultivar IAC SP 94-2094 foi utilizada e a deficiÃncia hÃdrica foi aplicada atravÃs de ciclos de irrigaÃÃo, com plantas irrigadas a cada cinco dias por um perÃodo de 15 dias e recuperaÃÃo de trÃs dias. O estresse hÃdrico aplicado causou reduÃÃes marcantes na condutÃncia estomÃtica, na transpiraÃÃo foliar e na fotossÃntese lÃquida, com recuperaÃÃo parcial, refletindo em uma menor eficiÃncia do uso da Ãgua e eficiÃncia de carboxilaÃÃo. Os parÃmetros fotoquÃmicos apresentaram modificaÃÃes transitÃrias, completamente revertidas pela recuperaÃÃo. As atividades da Rubisco e PEPcase nÃo mostraram modificaÃÃes, mas houve aumento na quantidade relativa de PEPcase e estado de ativaÃÃo da Rubisco. O estresse hÃdrico aplicado aumentou a peroxidaÃÃo lipÃdica e o conteÃdo de H2O2, induzindo aumentos de atividades de SOD e APX, principalmente APX tilacoidal e Fe-SOD. O perfil de aÃÃcares foi alterado nas plantas estressadas, com aumento de hexoses e diminuiÃÃo do teor de amido nas folhas. O objetivo do capÃtulo 4 foi avaliar as modificaÃÃes fisiolÃgicas e bioquÃmicas em relaÃÃo aos processos fotossintÃticos em cana-de-aÃÃcar submetida a um ambiente de elevado CO2 e baixo suprimento de nitrogÃnio (-N). A fotossÃntese lÃquida foi reduzida nas plantas com CO2 elevado e -N. Os parÃmetros fotoquÃmicos foram reduzidos apenas no tratamento -N, mas sem sinal de fotoinibiÃÃo. As enzimas da carboxilaÃÃo, PEPcase e Rubisco, sofreram regulaÃÃes diferenciadas, tanto pelo alto CO2 como pelo baixo suprimento de nitrogÃnio. A determinaÃÃo de nitrato mostrou acÃmulo nas raÃzes, indicando deficiÃncia no transporte para parte aÃrea. O tratamento âN diminuiu o conteÃdo de aminoÃcidos, proteÃnas solÃveis e nitrogÃnio total. O teor de sacarose foi reduzido em todos os tratamentos. Por fim, verificamos que a cultivar IAC SP 94-2094 possui maior fotoproteÃÃo indicada pelo maior NPQ e possui ainda maior atividade e expressÃo de PEPcase, permitindo melhor performance em perÃodos de dÃficit hÃdrico severo. Quando estudada com dÃficit hÃdrico moderado, as plantas desta cultivar apresentaram uma organizada defesa anti-oxidativa para proteÃÃo contra as espÃcies reativas de oxigÃnio. Quando sob elevada concentraÃÃo de CO2, as plantas de cana-de-aÃÃcar exibem modificaÃÃes estomÃticas e bioquÃmicas, reduzindo a atividade fotossintÃtica pela reduÃÃo da assimilaÃÃo de nitrato e atividade de redutase do nitrato, mostrando uma forte relaÃÃo entre a assimilaÃÃo de carbono e nitrogÃnio. / Sugarcane is plant of great economic potential, mainly utilized to sugar and biofuel production. Brazil has an international importance, leading the world production of this culture. In despite of this high production, Brazil is located in a tropical region and presents several climatic adversities. The study of sugarcane physiology in stress situations, like water deficit, becomes essential to maintenance and improvement of the production. Sugarcane is a C4 species, with a CO2 concentration mechanism. It is expected that the growing elevation of CO2 in the atmosphere does not the sugarcane productivity in a relevant way but the studies about this subject are very scarce. Thus, it was aimed to evaluate the photosynthesis regulation by water deficit, nitrogen supply and high CO2 in sugarcane plants. Chapter 1 contains the state of the art concerning the study theme. In the chapter 2, it was performed an work in order to evaluate the responses to water deficit of two sugarcane cultivars considered contrasting about water deficit, IAC SP 94-2094 and IAC SP 95-5000. These cultivars were exposed to severe water deficit, with withholding water for five days, and recovery, irrigated after for two days after stress. The applied treatment strongly affected the gas exchanges, with stomatal closure in both cultivars, leading to great reductions in transpiration and CO2 assimilation. The chlorophyll a fluorescence parameters were affected, but without photoinhibition. Tolerant cultivar showed higher PEPcase activity in stress, but Rubisco activity was not altered. In the sensitive cultivar there was not alteration in the PEPcase activity whereas Rubisco activity was reduced 40% in stress but totally recovered. PEPcase expression was increased in stress but with a higher increment in sensitive cultivar, with a similar pattern in the Rubisco amount. In the chapter 3, it was performed an work in order to evaluate the antioxidative system in sugarcane plants exposed to mild water stress. In this chapter, IAC SP 94-2094 was utilized and the water deficit was applied with irrigation cycles, with plants irrigated each five days for fifteen days and recovery for three days. Water stress applied caused remarkable decreases in the stomatal conductance, leaf transpiration and net photosynthesis, with partial recovery, causing lower water use and carboxylation efficiencies. Photochemical parameters showed transient changes completely reversible by recovery. PEPcase and Rubisco activities did not show modifications but it was increase in the relative amount of PEPcase and Rubisco activation state. Water stress increased the lipid peroxidation and H2O2 content, inducing increases in SOD and APX activities, mainly thylakoidal APX and Fe-SOD. Sugars profile was altered in stressed plants, with increase in hexoses and decrease in starch content in leaves. The aim of the chapter 4 was evaluate the physiological and biochemical changes in concern to photosynthetic processes in sugarcane exposed to high CO2 and low supply of nitrogen (-N). Net photosynthesis was reduced in plants subjected to high CO2 and -N. Photochemical parameters were decreased only in -N treatment, but with no photoinhibition. Carboxylation enzymes, PEPcase and Rubisco, did suffer differential regulations either by high CO2 or by low nitrogen. Nitrate determination showed accumulation in roots, indicating deficiency in transport to shoot. -N treatment decreased the amino acids content, soluble proteins and total nitrogen. Sucrose content was reduced in all treatments. At last, it was verified that IAC SP 94-2094 cultivar has more photoprotection indicated by higher NPQ and more PEPcase activity and expression, conferring a better performance in severe water deficit periods. When studied under mild water stress, the plants showed an organized anti-oxidative defense for protection against reactive oxygen species. When under high CO2, sugarcane plants exhibit stomatal and biochemical changes, reducing the photosynthetic activity by reduction in nitrate assimilation and nitrate reductase activity, showing a strong relation between carbon and nitrogen assimilation.