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.

SummaryIt is a common belief that plant mesophyll cells are occupied up to 95% by a single multipurpose vacuole. The common ice plant, Mesembryanthemum crystallinum L., however, requires two contrasting functions of the vacuole under salt stress. Large amounts of NaCl have to be sequestered permanently for osmotic purpose and for protecting the cytoplasm from NaCl toxicity. A dynamic exchange with the cytoplasm is required because photosynthesis proceeds under these conditions via the metabolic cycle of crassulacean acid metabolism (CAM). Nocturnally acquired CO 2 must be kept as malate in the vacuole and re-mobilized in the daytime. Here, we show that two large independent types of vacuoles with different transport properties meet the requirements for the contrasting functions within the same cell.

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

Summary• Salinity tolerance in plants involves controlled Na + transport at the site of Na + accumulation and intracellular Na + compartmentation. McNhaD in chloroplasts and demonstrates that the chloroplast is one of the compartments involved in the response of cells to salt stress.

Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H ϩ -transport by H ϩ -pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, ⌬ Hϩ , across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, J mal rel , of apparent ATP-dependent malate-stimulated H ϩ -transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. J mal rel was high in the obligate crassulacean acid metabolism (CAM) species Kalanchoë daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO 3 Ϫ nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher J mal rel . It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO 3 Ϫ nutrition and that a putative malate transporter must be within the 32-to 33-kD polypeptide fraction of tonoplast proteins.