Atpase and proton pumping activities in cotyledons and other phloem-containing tissues of Ricinus communis and the relationship to phloem loading

Williams, Lorraine Elizabeth (1987) Atpase and proton pumping activities in cotyledons and other phloem-containing tissues of Ricinus communis and the relationship to phloem loading. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The effects of various reagents including ATPase inhibitors, fusicoccin and sulphydryl reagents on ATPase activity, proton pumping and sucrose uptake in Ricinus communis var. Gibsonii have been examined to gain insight into the involvement of a plasma membrane proton pumping ATPase in the phloem loading process. A brief characterization of the microsomal fractions from phloem-containing tissues (cotyledon, bark tissue and main veins) revealed the presence of Mg²⁺-dependent ATPase activity which showed low stimulation by KCl, particularly at pH 6.5. The optimum was pH 8.5 to 9.0 although the effect of azide indicated the presence of mitochondrial ATPase. Sucrose gradient centrifugation was used to separate the cotyledon microsomal ATPase activity and purify the plasma membrane ATPase. The pellet from a three-step (30, 34, 38%) sucrose gradient was enriched in plasma membrane as determined by a combination of enzyme assays. The partially purified plasma membrane ATPase showed high sensitivity to vanadate, erythrosin B and SW26 but low sensitivity to azide and nitrate. The pH optimum was 6.5 and specificity for nucleotide triphosphates was high, greatest activity occurring with APT. Mg²⁺, K⁺- stimulated pyrophophatase activity was also found associated with the plasma membrane-enriched fraction. KI treatment of the microsomal fraction resulted in an increase in sensitivity to vanadate but reduced azide and nitrate sensitivity. This fraction was solubilized using octylglucoside and reconstituted into soybean phospholipid liposomes. The existence of ATP-driven H⁺-transport across the proteoliposomes was demonstrated by quinacrine fluorescence quenching in the presence of Mg²⁺. The quenching was gramicidin reversible and strongly inhibited by vanadate, ER B and PCMBS. Less inhibition was observed in the presence of NEM; SW26 had fairly low and variable effects. FC and sucrose had only slightly stimulatory effects. Proton pumping by intact cotyledons (detected by acidicATPase and proton pumping activities in cotyledons and other phloem-containing tissues of Ricinus communis and the relationship to phloem loading.ion of the bathing medium) was markedly stimulated by fusicoccin, inhibited by NEM and ER B, and unaffected by vanadate and SW26. The effect of PCMBS varied depending on the point of its addition. Net sucrose uptake by cotyledon discs showed strong inhibition by PCMBS, NEM and CCCP; inhibition also occurred in the presence of ER B and fusicoccin. Vanadate, SW26 and DCCD were ineffective. Sucrose uptake and microsomal ATPase activity measured at pH 6.5 varied with the developmental stage of the cotyledon but there was not a strong correlation. The results are discussed in relation to the proposed role of ATPase activity and proton pumping in phloem loading.

This record has no associated files available for download.