<p>\ / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O presente trabalho teve por objetivo estudar as respostas fisiolÃgicas e bioquÃmicas de clones de cajueiro anÃo-precoce (Anacardium occidentale L.) ao estresse salino. Os experimentos foram conduzidos em casa de vegetaÃÃo, sendo as plÃntulas cultivadas em vasos plÃsticos contendo vermiculita. No primeiro experimento, cinco clones de cajueiro anÃo-precoce foram submetidos aos tratamentos com NaCl a 0 (controle), 8 e 16 dS.m-1 de condutividade elÃtrica e objetivou selecionar clones com tolerÃncias diferenciadas ao estresse salino. Para isso, foram estudados os efeitos da salinidade no crescimento, nas trocas gasosas, no teor de Ãgua, na suculÃncia foliar, no potencial osmÃtico, nas concentraÃÃes de prolina, N-aminossolÃveis e carboidratos solÃveis e nos teores dos Ãons inorgÃnicos (Na+, Cl- e K+). A salinidade reduziu o crescimento das plÃntulas de todos os clones estudados. Os efeitos inibitÃrios do NaCl foram mais conspÃcuos na parte aÃrea do que nas raÃzes. O clone CCP 06 foi aquele que apresentou maior reduÃÃo no crescimento foliar, enquanto os clones BRS 189 e CCP 09 foram os que apresentaram as menores reduÃÃes. A salinidade inibiu a mobilizaÃÃo das reservas cotiledonÃrias, principalmente, na dose mais elevada de sal. A reduÃÃo no crescimento, pela salinidade, correlacionou-se com a reduÃÃo na taxa de fotossÃntese lÃquida. Os clones CCP 06 e BRS 189 apresentaram, respectivamente, a maior e a menor reduÃÃo na taxa fotossintÃtica a 8 dS.m-1. Embora a salinidade tenha reduzido a condutÃncia estomÃtica dos clones de cajueiro anÃo-precoce, essa reduÃÃo nÃo foi acompanhada por mudanÃas nas concentraÃÃes internas de CO2. Os clones estudados nÃo apresentaram alteraÃÃes, em funÃÃo da salinidade, no estado hÃdrico das folhas e raÃzes, porÃm, apresentaram reduÃÃes no potencial osmÃtico, favorecendo o ajustamento osmÃtico e, consequentemente, a manutenÃÃo da turgescÃncia dos tecidos. Sob condiÃÃes de estresse salino, os clones BRS 189 e CCP 09 foram os mais eficientes na regulaÃÃo do transporte do Ãon Na+ para a parte aÃrea da plÃntula, acumulando-o nas raÃzes. Em relaÃÃo ao Cl-, o clone CCP 09 mostrou-se o mais eficiente no controle do transporte desse Ãon. PorÃm, CCP 06 foi o clone que mais acumulou ambos os Ãons tÃxicos na parte aÃrea da planta. Com o aumento da salinidade, os teores de potÃssio dos clones estudados tiveram seus valores reduzidos apenas nas raÃzes. Na dose de 8 dS.m-1, o BRS 189 foi o clone que mais aumento suas concentraÃÃes de N-aminosolÃveis e prolina no suco radicular. Nesse mesmo nÃvel de sal, a salinidade aumentou a concentraÃÃo de carboidratos apenas nos clones CCP 06 e BRS 189. De posse destes resultados, o segundo experimento foi realizado com os clones CCP 06 e BRS 189 que foram os que se mostraram, respectivamente, o menos e o mais tolerante à salinidade. Esse experimento teve por objetivo estudar os efeitos da salinidade (NaCl a 8 dS.m-1) na atividade da H+-ATPase e na composiÃÃo e peroxidaÃÃo dos lipÃdios de membrana plasmÃtica isoladas de raÃzes das plÃntulas dos dois clones contrastantes. A salinidade estimulou a atividade da H+-ATPase apenas no clone tolerante, o BRS 189, sendo esse clone o que apresentou maior conteÃdo de esterÃis totais e menor relaÃÃo fosfolipÃdios totais (PLt)/ esterÃis totais (Et), tanto em condiÃÃes controle como de estresse. Esses resultados foram concordantes com o fato de ter sido o BRS 189 o clone que melhor excluiu o Na+ da parte aÃrea. Nesse clone nÃo foram observadas alteraÃÃes nos teores de malondialdeÃdo, diferentemente do que ocorreu com o CCP 06, cujos teores aumentaram com o estresse salino. A maior proteÃÃo da membrana plasmÃtica do clone BRS 189 ao dano oxidativo està de acordo com os maiores acÃmulos de prolina e N-aminossolÃveis observados nesse clone. Os principais fosfolipÃdios da membrana plasmÃtica isolada de raÃzes do clone BRS 189 foram fosfatilglicerol (PG), fosfatidiletalonamina (PE) e fosfatilserina (PS). A salinidade provocou alteraÃÃes nas proporÃÃes relativas dos fosfolipÃdios, sendo PE e fosfatidilinositol (PI) os que apresentaram maiores aumentos em relaÃÃo ao total, enquanto que fosfatidilglicerol (PG) e Ãcido fosfatÃdico (PA) foram os que apresentaram maiores reduÃÃes. A percentagem de PS, em relaÃÃo ao total, nÃo foi afetada pela salinidade. No entanto, a relaÃÃo entre essas mudanÃas na composiÃÃo lipÃdica do BRS 189 pela salinidade e o aumento na atividade da H+-ATPase necessita ser melhor investigada. / Early-dwarf cashew seedlings (Anacardium occidentale L.) were used in order to investigate the physiological and biochemical changes induced by salt stress. The seeds (nuts) were sown in plastics pots containing vermiculite moistened with either distilled water (control treatment) or NaCl solutions at 8 and 16 dS.m-1 of electrical conductivity (saline treatment), and kept in greenhouse throughout the experimental period. Uniform 28-day-old seedlings were used for the analyses. The first experiment aimed to select, among five clones (CCP 06, CCP 09, CCP 76, Embrapa 51 and BRS 189), the ones showing contrasting salt-tolerance. The effect of salinity on the growth, gas exchange, water content, leaf succulence, osmotic potential and inorganic (Na+, Cl-, K+) and organic (proline, soluble carbohydrates, quaternary ammonium compounds) solute concentration for both salt-sensitive and salt-tolerant clones was studied. Salinity inhibited the growth of all clones studied, being the inhibitory effect on shoot growth more conspicuous than in root growth. Clone CCP 06 leaf area was the most inhibited by salt stress, while clones BRS 189 and CCP 09 leaf areas were the least affected by salinity. Salt stress caused a great decrease in the cotyledon reserve mobilization especially at 16 dS.m-1. Growth reduction was correlated to the reduction in net photosynthetic rate. CCP 06 and BRS 189 showed the greatest and the lowest reduction in photosynthetic rate at 8 dS.m-1, respectively. Although, salinity reduced stomatal conductance, this reduction was not followed by changes in CO2 internal concentration. The water status, expressed as water content in relation to dry mass, was not changed by salt-stress. Salinity induced the lowering of osmotic potential both in leaves and roots of all clones studied. This osmotic adjustment might have lead to turgor maintenance of those tissues. The concentrations of Cl- and Na+ increased with increasing salt stress. Clones BRS 189 and CCP 09 accumulated more Na+ in the roots, and this could explain their efficiency in maintaining a lower ion concentration in shoots, i.e. they regulated more efficiently the transport of Na+ from roots to shoots. The regulation of Cl- transport to shoots was more efficient in clone CCP 09 than in the others. Salinity did not induce significant changes in leaves and stems K+ concentration, but it induced a reduction of K+ concentration in roots. Salinity also induced increases of quaternary ammonium compounds and proline concentration in BRS 189 root at 8 dS.m-1. In addition, this level of salinity increased soluble carbohydrates in the root sap especially in clones BRS 189 and CCP 06. During the second experiment, the effect of salt stress (NaCl at 8 dS.m-1) on the activity of H+-ATPase, lipid composition and peroxidation of root plasma membrane of both salt-tolerant (BRS 189) and salt- sensitive (CCP 06) clones were studied. The vanadate-sensitive H+-ATPase activity was studied in plasma membrane-enriched vesicles isolated by discontinuous sucrose gradient centrifugation from roots. ATP hidrolizing activity in this fraction was mostly inhibited by vanadate and scarcely, by azide and molybdate, indicating that it was essentially enriched in plasma membrane vesicles. Salinity induced a 1.3-fold increase in the H+-ATPase specific activity in roots of BRS 189 seedlings. Salinity had no appreciable effect on the hydrolytic activity of this enzyme during the growth of CCP 06 seedlings. Likewise, clone BRS 189 roots plasma membrane showed higher sterol content and lower phospholipids/total sterol ratio than clone CCP 06. Both properties could contribute to the decrease in Na+ influx or increase in Na+ efflux or âexclusionâ from roots. This could result in less Na+ being transported to the shoot, and thus explaining the higher salt-tolerance of clone BRS 189. The higher degree of root plasma membrane lipid peroxidation of clone, and the lower proline and ammonium quaternary compounds contents of CCP 06 when compared to BRS 189 could also explain the differences in salt-tolerance between the two clones. These organic solutes could protect and stabilize plasma membrane against oxidative stress. Phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were the major phospholipids in the plasma membrane from BRS 189 roots. Salinity induced increases in the relative proportions of PE and phosphatidylinositol (PI), while PG and PA were reduced. No changes were detected in PS in relation to control plant. The importance of lipid composition changes on H+-ATPase activity must be more studied.

In plants, calcium is a ubiquitous signaling molecule and changes in cytosolic calcium levels reported in response to various abiotic and biotic stresses like salt stress, drought, pathogen attack and phytohormone signaling. Any calcium- mediated signal transduction process involves the establishment of a signal-specific change in the cytosolic calcium concentration termed as ‗calcium signature‘ which is decoded by the specific group of proteins called ‗calcium sensors‘ (eg: Calmodulin (CaM) and Ca2+ - regulated kinases). Plants have a novel group of kinases designated as Ca2+- dependent protein kinases (CDPK; EC 2.7.1.37). CDPKs are biochemically distinct from other Ca2+- dependent kinases, such as Ca2+- and phospholipid- dependent protein kinases, as they are activated directly by Ca2+-and are independent of CaM. They exist as monomeric serine/threonine protein kinases and consist of four domains namely an amino-terminal variable domain, a kinase domain, an autoinhibitory domain and a calmodulin-like domain (CaM-LD). CDPKs represent a unique class of Ca2+ sensors, having protein kinase as well as CaM-LD in a single polypeptide chain, enabling them to couple the calcium sensor directly to its responder (kinase). In the absence of calcium signature, CDPKs activity is inhibited by the autoinhibitory domain, which acts as a pseudo-substrate of kinase domain and thus blocks the active site of the enzyme. In the presence of calcium signature, CDPKs undergo conformational changes leading to removal of the inhibition. Besides plants, CDPKs are also reported in few protozoans viz Plasmodium falciparam, Paramecium and Taxoplasma. However, CDPKs are not found in the eukaryotic genome of yeast, nematodes, fruitflies and humans. In the current study, we have cloned CDPK1 gene from Cicer arietinum (CaCDPK1) in pRSET-A expression vector and expressed it in Escherichia coli BL21pLysS strain. However, while expressing the recombinant CaCDPK1 in E.coli, most of the recombinant CaCDPK1 protein was expressed as insoluble form. Therefore, we focused our efforts on optimizing the culture conditions for achieving the maximum yield of soluble recombinant CaCDPK1. Expression of the soluble CaCDPK1 was achieved by optimizing the different conditions like IPTG concentrations, temperature and growth time after induction. Maximum amount of soluble expression of recombinant CaCDPK1 was achieved by inducing the bacterial culture with 0.1 mM IPTG at 0.6 OD and growing it further for 4 h at 25°C. As with several other CDPKs, CaCDPK1 was found to get autophosphorylated in a calcium-dependent manner. To find the significance of autophosphorylation, we measured the substrate phosphorylation activity of the native and autophosphorylated CaCDPK1, which revealed that the autophosphorylation enhances the kinase activity of CaCDPK1 by 2-fold. Autophosphorylation was linearly dependant on concentrations of the enzyme suggesting that the autophosphorylation in CaCDPK1 occurs via an intra-molecular mechanism. Further analysis of autophosphorylation shows that autophosphorylation happens before substrate phosphorylation and provides calcium -independent substrate phosphorylation property. It also reduces the lag phase for activation of the enzyme and utilizes both ATP and GTP as phosphor-donor, but ATP is preferred over GTP. Autophosphorylation was found to occur at serine and threonine residues. The MALDI MS/MS analysis of the cold ATP autophosphorylated CaCDPK1 showed Thr- 339, Ser- 357, and Ser- 367 residues could be the potential autophosphorylation sites in CaCDPK1. Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress conditions. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported in this study. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in Vmax of the enzyme activity by these phospholipids significantly decreased the Km indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K½ = 1.3 nM) when compared to PA (K½ = 56 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response. In the current study we also investigated CaCDPK1 interactions with calcium ions to address the Ca2+ -induced conformational changes in CaCDPK1 by using circular dichroism (CD), fluorescence spectroscopy and isothermal titration (ITC). Isothermal calorimetric analysis of calcium binding to CaCDPK1 shows a biphasic curve with two Kd of 27 nM and 1.72 µM respectively. The fluorescence measurements showed quenching in fluorescence intensity with a 5 nm red shift. The plot of changes in intensity against calcium concentrations again showed a biphasic curve, indicating that there may be more than one kind of Ca2+ binding sites. 8-anilinonaphthalene-1-sulfonic acid (ANS) binding showed that calcium bound form of CaCDPK1 exposes hydrophobic surfaces which may act as binding sites for other proteins. CD analysis of CaCDPK1 showed that it‘s an alpha helical rich protein and its helical content increases after binding to calcium. Taken all together this study describes the successful heterologous expression of Cicer arietinum CDPK isoform 1 in E.coli. and demonstrates that the autophoshorylation happens via an intra-molecular mechanism and it increases the kinase activity of CaCDPK1 at least by 2-fold. We also report here that CaCDPK1 prefers ATP as phosphodonor over GTP. The present study also shows the activation of CaCDPK1 by PC and PA, but not by PE or diacylglycerol. Both phospholipids were able to bind to CaCDPK1 and increased its Vmax and affinity towards the exogenous substrate, histone III-S. The current study also shows that calicum binding induces conformational changes in CaCDPK1 and the all four EF hand motifs of CaCDPK1 do not function in an equivalent manner.