Microdomains in the plasma membrane (PM) have been proposed to be involved in many important cellular events in plant cells. To understand the role of PM microdomains in plant cold acclimation, we isolated the microdomains as detergent-resistant plasma membrane fractions (DRMs) from Arabidopsis seedlings and compared lipid and protein compositions before and after cold acclimation. The DRM was enriched in sterols and glucocerebrosides, and the proportion of free sterols in the DRM increased after cold acclimation. The protein-to-lipid ratio in the DRM was greater than that in the total PM fraction. The protein amount recovered in DRMs decreased gradually during cold acclimation. Cold acclimation further resulted in quantitative changes in DRM protein profiles. Subsequent mass spectrometry and Western blot analyses revealed that P-type H(+)-ATPases, aquaporins and endocytosis-related proteins increased and, conversely, tubulins, actins and V-type H(+)-ATPase subunits decreased in DRMs during cold acclimation. Functional categorization of cold-responsive proteins in DRMs suggests that plant PM microdomains function as platforms of membrane transport, membrane trafficking and cytoskeleton interaction. These comprehensive changes in microdomains may be associated with cold acclimation of Arabidopsis.

Rice proteins from nine tissues and one organelle (leaf, chloroplast, stem, root, germ, dark germinated seedling, seed, bran, chaff and callus) were isolated and then separated by two-dimensional gel electrophoresis (2-DE). The protein spots were characterized according to molecular weight, isoelectric point and partial amino-terminal sequence. Electrophoresis was carried out by isoelectric focusing (IEF), nonequilibrium pH gradient electrophoresis (NEPHGE) and immobilized pH gradient (IPG) in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. With the aid of nine marker proteins, the patterns of IEF, NEPHGE and IPG 2-DE gels were graphically combined by computer into a single synthetic image for each tissue, respectively, and these images for the nine tissues and one organelle were again combined into a single 2-DE image for the integrated rice protein spots. The rice 2-DE gel image resolved 4892 proteins. About 3% of the spots are characterized by amino-terminal sequencing.

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The extrinsic 33-kDa protein of photosystem II (PSII) was modified with various reagents, and the resulting proteins were checked for the ability to rebind to PSII and to reactivate oxygen evolution. While modification of more than eight carboxyl groups of aspartyl and glutamyl residues with glycine methyl ester did not affect the rebinding and reactivating capabilities, modification of amino groups of lysyl residues with either Nsuccinimidyl propionate or 2,4,6-trinitrobenzene sulfonic acid or modification of guanidino groups of arginyl residues with 2,3-butanedione resulted in a loss of rebinding and reactivating capabilities of the 33-kDa protein. Moreover, the number of lysyl and arginyl residues susceptible to modification was significantly decreased when the protein was bound to PSII as compared with when it was free in solution, whereas the number of carboxyl groups modified was little affected. These results suggested that positive charges are important for the electrostatic interaction between the extrinsic 33-kDa protein and PSII intrinsic proteins, whereas negative charges on the protein do not contribute to such interaction. By a combination of protease digestion and mass spectroscopic analysis, the domains of lysyl residues accessible to N-succinimidyl propionate or 2,4,6-trinitrobenzene sulfonic acid modification only when the 33-kDa protein is free in solution were determined to be

A Photosystem II (PS II) complex was purified from an acidophilic as well as a thermophilic red alga, Cyanidium caldarium. The purified PS II complex was essentially devoid of phycobiliproteins and other contaminating components, and showed a high oxygen-evolving activity of 2375 mumol O2/mg Chl per h using phenyl-p-benzoquinone as the electron acceptor. The expression of this high activity did not require addition of exogenous Ca2+, although EDTA reduced the activity by 40%. This effect of EDTA can be reversed not only by Ca2+ but also by Mg2+; a similar Mg2+ effect has been observed in purified cyanobacterial PS II but not in higher plant PS II. Immunoblotting analysis indicated the presence of major intrinsic polypeptides commonly found in PS II from cyanobacteria and higher plants as well as the extrinsic 33 kDa protein. Antibodies against the extrinsic 23 and 17 kDa proteins of higher plant PS II, however, did not crossreact with any polypeptides in the purified PS II, indicating the absence of these proteins in the red alga. In contrast, two other extrinsic proteins of 17 and 12 kDa were present in the red algal PS II; they were released by 1 M Tris or Urea/NaCl treatment but not by 1 M NaCl. The 17 kDa polypeptide was identified to be cytochrome c-550 from heme-staining, immunoblot analysis and N-terminal amino acid sequencing, and the 12 kDa protein was found to be homologous to the 12 kDa extrinsic protein of cyanobacterial PS II from its N-terminal sequence. These results indicate that PS II from the red alga is closely related to PS II from cyanobacteria rather than to that from higher plants, and that the replacement of PS II extrinsic cytochrome c-550 and the 12 kDa protein by the extrinsic 23 and 17 kDa proteins occurred during evolution from red algae to green algae and higher plants.