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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Sistema de absorÃÃo de k+ de alta afinidade em plantas de sorgo forrageiro: papel da h+-atpase de membrana plasmÃtica e dos componentes sensÃvel e nÃo-sensÃvel ao Ãon nh4+ / System of absorption of k+ of high affinity in plants of sorgo forrageiro: paper of h+-atpase of plasmÃtica membrane and the components sensible and not-sensible to ion nh4+

Juan Carlos Alvarez Pizarro 08 March 2006 (has links)
CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O influxo de K+ na faixa de alta afinidade à controlado por transportadores dos tipos canal e carreador, os quais diferem na sua sensibilidade ao Ãon NH4 +. A atividade de ambos os componentes, nÃo-sensÃvel (canal) e sensÃvel (carreador) ao Ãon NH4 + dependem da enzima de membrana plasmÃtica, H+-ATPase. Objetivou-se a caracterizaÃÃo fisiolÃgica e molecular da absorÃÃo de K+ de alta afinidade em sorgo forrageiro [Sorghum bicolor (L.) Moench] sob a influÃncia do estado nutricional da planta e de diferentes fontes de nitrogÃnio inorgÃnico na soluÃÃo de crescimento. Sementes do genÃtipo CSF20 foram germinadas e cultivadas em soluÃÃes nutritivas contendo dois nÃveis de K+ (0,2 e 1,4 mM) e trÃs diferentes regimes de nitrogÃnio inorgÃnico (NO3 - e NH4 + a 4 mM, e a mistura NO3 -/NH4 +, ambos a 2 mM). ApÃs 15 dias de cultivo (t0) em soluÃÃes nutritivas completas, as plantas foram submetidas à deficiÃncia de K+ por 1 (t1), 2 (t2) e 3 (t3) dias. No t0, os teores de K+ da parte aÃrea foram reduzidos ignificativamente pela presenÃa do Ãon NH4 +, enquanto que, nas raÃzes a reduÃÃo foi significativa apenas nos cultivos com 0,2 mM de K+. Conforme o aumento do perÃodo de deficiÃncia, os teores de K+ nas raÃzes e na parte aÃrea tenderam a diminuir em razÃo da diluiÃÃo provocada pelo crescimento da planta. Em plantas cultivadas com 0,2 mM de K+ e com NO3 - e NO3 -/NH4 +, as eficiÃncias de absorÃÃo de K+ foram similares. Entretanto, a presenÃa do Ãon NH4 + como Ãnica fonte de nitrogÃnio, afetou severamente esse parÃmetro. O efeito do Ãon NH4 + na eficiÃncia de absorÃÃo foi mais ameno quando as plantas foram cultivadas a 1,4 mM de K+. A eficiÃncia de transporte de K+ nÃo diferiu em nenhum dos tratamentos testados. As curvas de depleÃÃo de K+ mostraram que as plantas cultivadas com NO3 -/NH4 + e NH4 + apresentaram maior capacidade para esgotar o K+ (100 μM) da soluÃÃo de depleÃÃo quando comparadas Ãquelas crescidas com NO3 -. No t2, maiores taxas de Imax foram observadas nas plantas cultivadas com NH4 + como Ãnica fonte de nitrogÃnio. O Km para o K+, das plantas provindas dos cultivos com 0,2 e 1,4 mM de K+, apresentou valores menores nas plantas tratadas com NH4 + e NO3 -/NH4 +. A induÃÃo da absorÃÃo de alta afinidade de K+ foi influenciada pelo conteÃdo de K+ dos tecidos aÃreos. Ensaios com inibidores mostraram que o influxo de K+ foi significativamente inibido pelo Ãon NH4 + a 1 mM em plantas cultivadas com NO3 - como Ãnica fonte de nitrogÃnio. Entretanto, a capacidade de absorÃÃo de K+ foi reduzida pela presenÃa de TEA nas plantas cultivadas em soluÃÃes contendo o Ãon NH4 + como Ãnica fonte de nitrogÃnio. Esses resultados sugerem que em plantas de sorgo sob deficiÃncia de K+ e na presenÃa do Ãon NH4 + no meio de crescimento, canais de K+ podem contribuir significativamente para o influxo de K+. Por outro lado, na ausÃncia do Ãon NH4 + durante o crescimento, um sistema de transporte mediado por carreadores de K+ seria a via principal para o influxo desse nutriente. A atividade de transporte de H+ da H+-ATPase de membrana plasmÃtica isolada de raÃzes mostrou que a deficiÃncia de K+ (t2) estimulou a capacidade de formaÃÃo do gradiente de H+ em presenÃa do Ãon NH4 + nas plantas provindas dos cultivos com 1,4 mM de K+, enquanto que naquelas provindas dos cultivos com 0,2 mM de K+, o Ãon NH4 + teve efeito na velocidade inicial do transporte de H+ e na hidrÃlise do ATP. A deficiÃncia de K+ na presenÃa do NO3 - nÃo estimulou as atividades da bomba de 11 H+. ImunodetecÃÃo com anticorpos especÃficos contra H+-ATPases de membrana plasmÃtica de plantas mostrou a induÃÃo de duas isoformas nas membranas plasmÃticas oriundas de plantas cultivadas com 0,2 mM de K+, independentemente da fonte de nitrogÃnio e dos perÃodos de deficiÃncia. SeqÃencias gÃnicas correspondentes a genes de H+-ATPases de membrana plasmÃtica (SBA1 e SBA2), canais (SbAKT1) e carreadores de K+ (SbHAK1) foram selecionadas no genoma do sorgo e seus nÃveis de expressÃo em raÃzes analisados por PCR em tempo real. Os genes SBA1 e SBA2 pertencem, respectivamente, aos grupos II e I da famÃlia das H+-ATPases. Em raÃzes provindas dos cultivos com 0,2 mM de K+ e na presenÃa do Ãon NH4 + os nÃveis dos transcritos de SBA1 e SBA2 foram significativamente expressos a partir do tempo t2 de deficiÃncia de K+, enquanto que na presenÃa do Ãon NO3 - eles foram reduzidos conforme o aumento do tempo de deficiÃncia. Na dose mais alta de K+, os transcritos de SBA1 tiveram sua expressÃo incrementada pela deficiÃncia de K+ em presenÃa do Ãon NH4 + como Ãnica fonte de nitrogÃnio. Ambos os genes tiveram um incremento transitÃrio dos nÃveis dos transcritos no t1 de deficiÃncia de K+ na presenÃa do Ãon NO3 -. Transcritos dos genes SbAKT1 e SbHAK1 nÃo foram detectados. AnÃlises filogenÃticas mostraram que SbAKT à um canal de K+ da famÃlia Shaker, compartilhando origem evolutiva comum com vÃrios canais de K+ de gramÃneas. Os resultados sugerem que a homeostase iÃnica do K+ à alterada pelo Ãon NH4 + em plantas de sorgo. No entanto, a adaptaÃÃo das plantas à presenÃa do Ãon NH4 + envolve a induÃÃo de um sistema altamente eficiente para a aquisiÃÃo de K+, com a participaÃÃo de canais de K+ e da H+-ATPase de membrana plasmÃtica / K+ influx in the range of high affinity is mediated by K+ carriers and channels, which can be distinguished by its differential sensibility to NH4+. The activity of the NH4+-sensitive component (carrier) and NH4+-insensitive component (channel) depend upon plasma membrane H+-ATPase. This work aimed the physiological and molecular characterization of system mediating K+ uptake in the high-affinity range of concentration in sorghum [(Sorghum bicolor (L.) Moench)]. The effect of K+ starvation and nitrogen inorganic source of the growth solution on high-affinity K+ uptake was also studied. Seeds of sorghum, genotype CSF20, were germinated and placed in modified one-fourth Hoagland solutions, which were formulated to contain 0,2 and 1,4 mM K+ and three nitrogen inorganic source, NO3- and NH4+ (4 mM) and NO3-/NH4+ in combination (2mM/2mM). Plants were grown for 15 days (t0) in complete nutritive solutions and then incubated in a K+-free solutions for one (t1), two (t2) and three (t3)days. At t0, K+ content of shoot was significantly decreased in plants grown in the presence of NH4+. In roots, the presence of NH4+ did altered the K+ content only of plants cultivated in solutions with lowest K+ concentration. The K+ content of the plant tissues was progressively reduced according to increasing of starvation period and increasing of dry matter (dilution). The highest reductions were observed in K+ content of the shoot. At the lowest level of K+ (0,2 mM), plants grown in solutions containing NO3- and NO3-/NH4+ showed similar uptake efficiency of K+. Whereas, the presence of NH4 as sole nitrogen source, reduced severely the absorption rates of K+. The effect of NH4+ on uptake efficiency of K+ was alleviated by increasing the external K+ concentration to 1,4 mM K+. At both levels of K+, the translocation rate of K+ was not altered by the presence of NH4 + and by K+ starvation. Assays of depletion in the external medium of K+ (100 μM) showed that plants growing in the presence of NO3-/NH4+ and NH4+ were more efficient to deplete external K+ than plants grown with NO3- , as sole nitrogen source. Kinetic parameters were significantly different at second day (t2) of deprivation. In the presence of NH4 +, as sole nitrogen source, Imax values were higher than those of plants grown in NO3-/NH4 + and NO3 -. On the other hand, Km values were lower in plants from solutions containing NH4+ and NO3 -/NH4 + than those cultivated in NO3-, as sole nitrogen source. High-affinity uptake of K+ responded to changes in plant K+ status, mainly to K+ content of aerial parts. K+ uptake by plants growing in NO3- was significantly inhibited by inclusion of NH4 at 1 mM in depletion solution, whereas it was not inhibited in plants grown with NH4 +, as sole nitrogen source. TEA (tetraethylammonium) inhibited the K+ influx of plants cultivated in solution containing NH4 +, as sole nitrogen source. The results suggest that factors such as presence of K+ or NH4 + during plant growth determine the relative contribution of each component to high-affinity uptake system of K+. In K+-starved plants grown in the presence of NH4 +, K+ channel could contribute to K+ uptake in the range of high-affinity concentration. On the other hand, in K+-starved plants grown without NH4 +, K+ carrier could constitute the principal route for K+ uptake. Active H+-transport driven by plasma membrane H+-ATPase of sorghum roots was stimulated by K+ deficiency and NH4 + in plants from solutions with high K+ level. However, these factors affected the initial rate 13 of H+-pumping and hydrolytic activity of ATP, but not H+-gradient formation, in plants from solutions containing 0,2 mM K+. These activities were not changed when plants were cultivated in growth solutions containing NO3 - and submitted to K+-starvation. Two specific isoforms of PM H+-ATPase by immuno-detection were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO3 --grown. cDNA sequences corresponding to plasma membrane H+-ATPase (SBA1 and SBA2), K+ channels (SbAKT) and K+ carrier (SbHAK1) from sorghum genome were identified. The expression level of these genes was analyzed by real-time PCR. SBA1 and SBA2 genes were included at subfamily II and I of plasma membrane H+-ATPase, respectively. In plants cultivated in solutions containing 0,2 mM K+ and with NH4+, the accumulation of SBA1 and SBA2 transcripts was observed after 48 h (t2) of K+ deficiency. Whereas, the expression level of these genes was reduced in plants cultivated with NO3 -. In roots came from solutions containing 1,4 mM K+ level, the accumulation of SBA1 transcripts was only observed in the presence of NH4 + and after 48 h (t2) of K+ deficiency. The transcript level of both genes increased only at t1 in roots cultivated in solution containing NO3 -, as sole nitrogen source. Transcipts of SbAKT and SbHAK1 were not detectable by real-time PCR. Phylogenetic analysis revealed that SbAKT belongs to Shaker channel family of plants and is also closely related to members of other gramineous species. The results suggest that K+ homeostasis in NH4+-grown sorghum plants may be regulated by a high capacity for K+ uptake, which is dependent upon the H+ -pumping activity of PM H+-ATPase and K+ channels

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