Hysiological and biochemical studies on K⁺ uptake in barley (Hordeum vulgare.L) roots

Abstract

Biophysical and kinetic studies on K⁺ uptake have led to the belief that uptake of this ion by plant roots occurs via proteinacious carriers or channels depending on the availability of this ion. Without direct evidence it has frequently been postulated that the regulation of ion fluxes may be the result of changes in carrier synthesis. Such evidence should be obtained by biochemical and molecular biological studies. The work described in this thesis was undertaken with that goal in mind. There are many observations showing that plants have a greater propensity for nutrient uptake when growing under nutrient-deficient conditions. Accordingly, barley seedlings used in this study showed increased K⁺ ( ⁸⁶Rb ) influx as a response to lowering of supply of this ion. This increase was discerned within a time period as short as 15 min from the onset of deprivation and was only partially inhibited by cycloheximide, an inhibitor of protein synthesis. This indicates that at least during the first hour of withholding K⁺, a mechanism(s) independent of carrier synthesis and incorporation regulates K⁺ influx in plant roots. However, the long-term adaptation to low K⁺ supply appeared to require synthesis of new carriers. The latter part of this thesis describes the experiments undertaken to test this hypothesis. An evaluation of the existing techniques for the isolation and characterization of membrane-enriched fractions was undertaken in order to determine the most feasible technique to be used in experiments where several treatments have to be handled simultaneously. The pros and cons of the use of available techniques in terms of obtaining a relatively high yield and considerable purity in membrane-enriched fraction in such a study are discussed. Experiments were also performed to investigate the nature of the postulated high affinity K⁺ transport system, which is suggested to be in operation under low K⁺ availability. Both in vitro ATPase activity and immunological cross-reactivity revealed that plasma membrane H⁺ ATPase activities did not increase in parallel with K⁺ influx shown by K⁺ deprived plants. This evidence, although not unequivocal, indicates that a strict coupling of K⁺ influx to H⁺ ATPase activity is unlikely. The studies on quantitative and qualitative changes in membrane proteins were quite rewarding. In spite of slight reduction in plasma membrane protein content as a response to K⁺ deprivation, several polypeptides, unique to K⁺ deprivation were expressed in the microsomal fraction. Of these a 45 kDa polypeptide was the most prominent. The expression and repression of this polypeptide were parallel with the increase and decrease of K⁺ influx with K⁺ deprivation and resupply, respectively. This polypeptide which was found to be membrane associated and highly labile in dissociated form, was lost when the microsomes were washed in the presence of 1 mM EDTA. This indicates the necessity of divalent cations for this polypeptide to be membrane associated. The possible role of this polypeptide in increased K⁺ uptake rates is discussed. The most convincing observation in the present study was the synthesis of a 43 kDa polypeptide within 12 h of K⁺ deprivation. This polypeptide appeared to be located both on plasma membrane and tonoplast. Two more polypeptides of Mr 37 and 49 were synthesized as a response to long-term K⁺ deprivation. The possibility that these polypeptides are parts of a putative K⁺ carrier is discussed. Finally, a comparison of the adaptive mechanism(s) in terms of K⁺ uptake and accumulation by barley roots as a response to low-K⁺ and low-temperature was made. The acclimatory changes shown as a response to low temperature can be considered as part of the general process of regulation of nutrient uptake while those shown as a response to low K⁺ appeared to be quite specific.