Major intrinsic proteins (MIPs) are a family of channel proteins that are mainly represented by aquaporins in plants. These are divided into TIPs (tonoplast intrinsic proteins) and PIPs (plasma membrane intrinsic proteins) according to their subcellular localization. Homologues to PIPs and TIPs were isolated from the desiccation-tolerant resurrection plant Craterostigma plantagineum by two approaches: firstly, a cDNA library constructed from RNA of dehydrated C. plantagineum leaves was screened with an Arabidopsis thaliana Ath-PIP1b cDNA probe and, secondly, a cDNA library was screened differentially to isolate early drought-induced transcripts. According to sequence homologies the isolated cDNA clones were grouped as follows: Cp-PIPa, Cp-PIPb, Cp-PIPc and Cp-TIP. Cp-PIPa, Cp-PIPc and Cp-TIP transcript accumulation was regulated by dehydration and abscisic acid (ABA). Within the Cp-PIPa group transcripts were regulated either by drought only or by drought and ABA, indicating that ABA-dependent and -independent signal transduction pathways lead to Cp-PIPa expression. Comparison of Cp-PIPa expression in detached leaves and in whole plants suggested the involvement of a signal transmitted in the whole plant in response to drought. Cp-PIPb transcript levels were constitutive in all organs tested. Antibodies raised against a Cp-PIPA protein recognized a polypeptide with an apparent molecular mass of 28 kDa. Using these antibodies it was shown that both Cp-PIPA and Cp-PIPB proteins were localized to the plasma membrane. The role of different members of the MIP group in the dehydration response is discussed.

Abbreviations and SymbolsThe H'-PPase and the H'-ATPase of the vacuolar membrane were separated during purification of tonoplast proteins ofKalanchoe daigremontiana Hamet et Perrier de la Blthie. Three membrane protein fractions prepared contained firstly, the H'-PPase protein without any subunits of the H+-ATPase, secondly, the H'-PPase protein with only minute traces of the intramembraneous 16 kDa c-subunit of the H'-ATPase, and thirdly, the H' -ATPase subunits without H+-PPase peptides as verified by SDS-PAGE. These three preparations were reconstituted into soybean (Glycine max L.)-phospholipid vesicles, and compared with proteoliposomes obtained by reconstitution of total solubilized tonoplast proteins as well as with native tonoplast vesicles. Analysis of freeze-fracture replicas prepared from these five different types of vesicles showed that there are two populations of intramembraneous particles, one with a diameter of 6.7-7.2 nm corresponding to the H+-PPase, and one with an average diameter of 9.1 nm belonging to the H+-ATPase. Thus, freeze-fracture electron microscopy allows one to visualize H'-PPase particles in addition to H+-ATPase particles in the tonoplast of Kalanchoe daigremontiana.

Summary. Differences in the activity and structure of the vacuolar H+-ATPase (V-ATPase, EC 3.6.1.3) were investigated in the C3/CAM intermediate plant Kalanchob" blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, and Hordeum vulgare cv. Carina, grown with or without 150 mM NaC1. In K. blossfeldiana ATP-hydrolysis and H+-transport activity were higher with higher expression of CAM than in plants with very weak CAM. This was mainly due to a larger amount of V-ATPase. Statistical analysis of the diameter of intramembrane particles (IMPs) on freeze-fractures of tonoplast vesicles showed that IMPs were larger in tonoplast vesicle preparations of K. blossfeldiana with strong CAM expression (9.1 nm) than in preparations of K. blossfeldiana with low CAM expression (7.3 nm). As there is evidence that the majority of IMPs on freeze-fractures of tonoplast vesicles corresponds to the V0 domain of V-ATPase, the higher activity of VATPase in K. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase in K. blossfeldiana with stronger CAM is discussed in relation to the requirement for a higher proton pumping capacity for nocturnal malate accumulation in the vacuole. The ATP-dependent H+-pumping activity in H. vulgare was higher under salt stress than in control plants, while the rates of ATP-hydrolysis and the size of IMPs were not affected by the salt treatment. The data presented here indicate that different mechanisms might increase the transport capacity of V-ATPase to meet the higher requirements of secondary active transport related to CAM expression and adaptation to salt stress.

Immuno-electron microscopy of nega-Lively stained isolated tonoplast vesicles was used to quantify stress responses of the H'-transporting tonoplast 4TPase (VoV1-ATPase; EC 3.6.1.1) of the C3/CAM intermediate Mesembryanthemum crystallinum L. and the C3 plant Hordeum vulgare L. This approach has the advantage that it relates quantitative adaptations at the level of membrane enzymes directly to membrane area and thus is independent of concomitant changes of relative amounts of other membrane proteins which may perturb conclusions when data are expressed on a tonoplast protein basis.It was shown that in M. crystallinum the amount of V,V,-ATPase per unit membrane area increased slightly with ageing and pronouncedly with salinity stress. In H. vulgare under salt stress there was an increase in VoV1-ATPase amount only in the highly salt tolerant cv. California Mariout and not in the moderately tolerant cv. Carina. This corroborates conclusions from earlier work, where results were expressed on a protein basis, although this was not to be expected a priori. In all comparative ecophysiological studies using tonoplast vesicles at least some key-point tests with the immunonegative staining technique should be included for the sake of prudence. The data obtained here via immuno-

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