Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic ("neutral") vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PPi-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PPi-hydrolytic activity and cross reactivity with antibodies including that directed against the Na+/H+-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of "neutral" vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the "neutral" vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na+ sequestration and is accordingly equipped with a high capacity of proton pumping and Na+ uptake via the Na+/H+-antiporter.

Occurrence of the lutein-epoxide cycle in mistletoes of the Loranthaceae and Viscaceae.

The lutein-epoxide cycle (Lx cycle) is an auxiliary xanthophyll cycle known to operate only in some higher-plant species. It occurs in parallel with the common violaxanthin cycle (V cycle) and involves the same epoxidation and de-epoxidation reactions as in the V cycle. In this study, the occurrence of the Lx cycle was investigated in the two major families of mistletoe, the Loranthaceae and the Viscaceae. In an attempt to find the limiting factor(s) for the occurrence of the Lx cycle, pigment profiles of mistletoes with and without the Lx cycle were compared. The availability of lutein as a substrate for the zeaxanthin epoxidase appeared not to be critical. This was supported by the absence of the Lx cycle in the transgenic Arabidopsis plant lutOE, in which synthesis of lutein was increased at the expense of V by overexpression of epsilon-cyclase, a key enzyme for lutein synthesis. Furthermore, analysis of pigment distribution within the mistletoe thylakoids excluded the possibility of different localizations for the Lx- and V-cycle pigments. From these findings, together with previous reports on the substrate specificity of the two enzymes in the V cycle, we propose that mutation to zeaxanthin epoxidase could have resulted in altered regulation and/or substrate specificity of the enzyme that gave rise to the parallel operation of two xanthophyll cycles in some plants. The distribution pattern of Lx in the mistletoe phylogeny inferred from 18S rRNA gene sequences also suggested that the occurrence of the Lx cycle is determined genetically. Possible molecular evolutionary processes that may have led to the operation of the Lx cycle in some mistletoes are discussed.

Phenotypic subunit composition of the tobacco (Nicotiana tabacum L.) vacuolar-type H(+)-translocating ATPase.

The model plant tobacco (Nicotiana tabacum L.) was chosen for a survey of the subunit composition of the V-ATPase at the protein level. V-ATPase was purified from tobacco leaf cell tonoplasts by solubilization with the nonionic detergent Triton X-100 and immunoprecipitation. In the purified fraction 12 proteins were present. By matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and amino acid sequencing 11 of these polypeptides could be identified as subunits A, B, C, D, F, G, c, d and three different isoforms of subunit E. The polypeptide which could not be identified by MALDI analysis might represent subunit H. The data presented here, for the first time, enable an unequivocal identification of V-ATPase subunits after gel electrophoresis and open the possibility to assign changes in polypeptide composition to variations in respective V-ATPase subunits occurring as a response to environmental conditions or during plant development.