Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A salinidade elevada do solo pode estar associada à escassez de K+, propiciando estresses interativos sobre a nutriÃÃo das plantas, os quais prejudicam a produÃÃo agrÃcola. Os mecanismos de transporte de Na+ e K+ nas interfaces solo-simplasto radicular-xilema sÃo decisivos para o estabelecimento da toxicidade de Na+ nas plantas submetidas ao estresse salino, especialmente sob baixa disponibilidade de K+. Assim sendo, o objetivo desse trabalho foi caracterizar fisiologicamente os mecanismos de transporte de Na+ e K+ envolvidos com a absorÃÃo e o carregamento do xilema nas raÃzes de feijÃo caupi [Vigna unguiculata (L.) Walp.]. A absorÃÃo de Na+ por mecanismos de baixa afinidade, investigada por experimentos de influxo em raÃzes destacadas, foi mediada por uma via sensÃvel e uma via insensÃvel ao Ca2+. A via sensÃvel ao Ca2+ deve envolver os canais de cÃtions nÃo-seletivos (NSCCs), enquanto a via insensÃvel deve depender dos canais de K+ e dos transportadores de K+ sensÃveis ao NH4+. A absorÃÃo de K+ por mecanismos de alta afinidade, examinada pela tÃcnica de influxo em raÃzes destacadas, envolveu uma via sensÃvel e uma via insensÃvel ao NH4+. A via sensÃvel ao NH4+ deve ser mediada pelos transportadores das famÃlias KT/HAK/KUP e HKT e a via insensÃvel, pelos canais de K+. O carregamento de Na+ e K+ no xilema, estudado por experimentos de exudaÃÃo radicular, indicou que a elevada seletividade K+/Na+ apresentada por feijÃo caupi foi colapsada sob concentraÃÃes externas de Na+ superiores a 20 mM, na presenÃa de K+ 1 mM. O acesso quase irrestrito de Na+ ao xilema radicular foi compensado pela liberaÃÃo aumentada de K+ na seiva e pela manutenÃÃo do fluxo de K+ para a parte aÃrea. A compartimentalizaÃÃo de Na+ nas raÃzes deve atuar como barreira fisiolÃgica para excluir Na+ das folhas e, conseqÃentemente, deve evitar a toxicidade desse Ãon em feijÃo caupi pela manutenÃÃo da alta relaÃÃo K+/Na+ foliar. A privaÃÃo de K+ aliada ao tratamento salino intensificou o fluxo de Na+ e restringiu o fluxo de K+ no xilema radicular, alÃm de aumentar a acumulaÃÃo de Na+ nas folhas novas, propiciando condiÃÃes menos favorÃveis à homeostase iÃnica em feijÃo caupi, em comparaÃÃo com o estresse salino aplicado isoladamente. / High soil salinity can be associated with K+ starvation enabling interactive stresses on plant nutrition that impair crop production. The transport mechanisms on the soil-root symplast-xylem boundaries are crucial to the establishment of Na+ toxicity under salt stress, especially under low K+ availability. Then, the aim of this work was the physiological characterization of Na+ and K+ uptake and xylem loading in the roots of cowpea [Vigna unguiculata (L.) Walp.]. Low-affinity Na+ uptake, investigated by influx experiments using detached roots, was mediated by Ca2+-sensitive and Ca2+-insensitive pathways. The Ca2+-sensitive pathway may involve non-selective cation channels (NSCCs), while the Ca2+-insensitive pathway may depend on K+ channels and NH4+-sensitive K+ transporters. High-affinity K+ uptake, examined by the excided root technique, involved NH4+-sensitive and NH4+-insensitive pathways. The NH4+-sensitive pathway may be mediated by K+ transporters from the KT/HAK/KUP e HKT families and the NH4+-insensitive pathway may depend on K+ channels. Xylem loading, assessed by root exudation experiments, indicates that the high K+/Na+ selectivity showed by cowpea was collapsed under external Na+ concentrations above 20 mM in the presence of 1 mM K+. The Na+ access to the root xylem was almost unrestricted and it was compensated by enhanced K+ release to the sap. The Na+ compartmentation into the root cells may act as a physiological barrier to Na+ exclusion from the leaves, avoiding Na+ toxicity in cowpea due to the maintenance of the high leaf K+/Na+ ratio. K+ starvation associated with salt stress intensified the Na+ flux and restricted the K+ flux into the root xylem, as it enhanced Na+ accumulation in the young leaves, allowing unfavourable conditions to ionic homeostasis in cowpea in comparison with salt stress applied individually.

The high affinity, ATP-dependent K+ uptake system KdpFABC of Halobacterium salinarum, is highly induced under K+ limitation. In contrast to the well-characterized Kdp system in Escherichia coli, in which the kdpFABC genes are transcriptionally regulated by the sensor kinase/response regulator system KdpD/KdpE, transcriptional regulation of the kdp genes in H. salinarum was unknown due to the absence of halobacterial homologues of KdpD/KdpE. Furthermore, the physiological relevance of the KdpFABC K+ uptake system of H. salinarum was puzzling, since hypersaline habitats usually comprise K+ concentrations which do not induce kdp expression. In order to analyze the regulation of kdp gene expression, it was essential to gain information about the transcriptional unit(s) involved. Northern blotting, primer extension analysis and real-time RT-PCR revealed the presence of a polycistronic leaderless kdpFABCQ transcript with a putative kdp terminator or at least a potential mRNA processing site downstream of kdpQ. Furthermore, promoter truncation studies verified the so far only predicted basal transcription elements together with an upstream-located operator sequence. Since deletions of this putative operator sequence did not lead to a constitutive expression, a further component has to be involved in the regulation of the kdpFABCQ genes. However, truncation and scanning mutagenesis analyses of the kdp promoter as well as translational fusions of a halophilic beta-galactosidase to the kdp promoter excluded an additional regulatory element up- or downstream of the basal transcription elements and in the kdp-coding region. These results lead to speculations of multiple basal transcription factors to be involved. Furthermore, an inducible expression vector (shuttle vector) was constructed based on the promoter of the kdpFABCQ operon due to its, K+-sensitive features. Inducible expression systems are yet not available for H. salinarum. The resulting, replicating vector pKIX is functional and enables a K+-dependent expression from the kdp promoter with rather high induction ratios of 50-fold. Expression levels could further be improved by plasmid- and additional chromosomally encoded kdpQ and mutations generated in the kdp promoter. Since transcript levels from pKIX were found to be independent of differential target genes, the general application of pKIX as an inducible expression system is strongly supported and pKIX could, thus, be made accessible to the scientific community. To decipher the physiological relevance of the halobacterial Kdp system, H. salinarum was encountered to desiccation stress and salt crystal (halite) entombment. Halite crystals grown under non-inducing K+ concentrations with entombed strains of H. salinarum and H. salinarum deleted in the kdpFABCQ genes revealed a significantly reduced survival rate of the deletion strain upon recultivation. Additionally, a kdpFABCQ-inducing desiccation stress could already be determined on agar plates under non-limiting K+ concentrations. Furthermore, the cell morphology of H. salinarum entrapped in halite crystals resembled that of H. salinarum grown under K+-limiting conditions. Therefore, the Kdp system promotes survival of H. salinarum under desiccation stress. Furthermore, the Kdp system could be identified as at least one of the systems important for long-term survival of H. salinarum in halite.