An immunological approach using a polyclonal phosphothreonine antibody is introduced for the analysis of thylakoid protein phosphorylation in vivo. Virtually the same photosystem II (PSII) core phosphoproteins (D1, D2, CP43, and the psbH gene product) and the lightharvesting chlorophyll a/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2), as earlier identified by radiolabeling experiments, were recognized in both pumpkin and spinach leaves. Notably, the PSII core proteins and LHCII polypeptides were found to have a different phosphorylation pattern in vivo with respect to increasing irradiance. Phosphorylation of the PSII core proteins in leaf discs attained the saturation level at the growth light intensity, and this level was also maintained at high irradiances. Maximal phosphorylation of LHCII polypeptides only occurred at low light intensities, far below the growth irradiance, and then drastically decreased at higher irradiances. These observations are at variance with traditional studies in vitro, where LHCII shows a light-dependent increase in phosphorylation, which is maintained even at high irradiances. Only a slow restoration of the phosphorylation capacity for LHCII polypeptides at the low light conditions occurred in vivo after the high light-induced inactivation. Furthermore, if thylakoid membranes were isolated from the high light-inactivated leaves, no restoration of LHCII phosphorylation took place in vitro. However, both the high light-induced inactivation and low light-induced restoration of LHCII phosphorylation seen in vivo could be mimicked in isolated thylakoid membranes by incubating with reduced and oxidized dithiothreitol, respectively. We propose that stromal components are involved in the regulation of LHCII phosphorylation in vivo, and inhibition of LHCII phosphorylation under increasing irradiance results from reduction of the thiol groups in the LHCII kinase. Photosystem (PS)1 II is a multiprotein complex of the thyla- (14) protein phosphatases, active in dephosphorylation of thylakoid phosphoproteins, are reported to be present in plant chloroplasts. Distinct physiological roles have been established for the phosphorylation of LHCII polypeptides, being implicated in the regulation of excitation energy distribution between PSII and PSI (1, 2), and in the protection of PSII against photoinhibition (15,16). Under conditions where PSII becomes more excited than PSI, the redox-controlled kinase is activated and phosphorylates LHCII polypeptides. The outer LHCII subcomplex, consisting of LHCB1 and LHCB2 proteins (see, e.g., Ref. 17), then detaches from the PSII complex and migrates out from the appressed region of the thylakoid membrane, thereby reducing the excitation of PSII (1). Dephosphorylation of this mobile LHCII subcomplex allows its subsequent migration back to the stacked grana region and reassociation with PSII (16).The only function so far established for reversible phosphorylation of PSII core proteins is the regulation of the degradation of photodamaged D1 protein ...

Folds or wrinkles appear frequently in thin sections cut from botanical material embedded in epoxy resin such as Epon 812 (Polysciences) or EM-bed-812 (Electron Microscopy Sciences). Small wrinkles may occur more or less perpendicular to the cutting direction and mostly across the cell wall, causing problems in the examination of the ultrastructure. We studied the occurrence of wrinkles and folds by methodological sectioning, with regard to block hardness, use of Formvar-support film, and chloroform vapor. Methodological sectioning or changing the block hardness did not reduce the occurrence of wrinkles in sections. Chloroform vapor improved the result as did also the use of uncoated grids. Wrinkles and folds could be avoided completely by embedding the specimens in LX-112 (Ladd) or ERL-4206 (Polysciences) lowviscosity epoxy resins, in which case Formvar-coated grids could be used, and chloroform vapor was not necessary.

We present here a new, simple agar encapsulating technique, which is helpful when preparing a small quantity of isolated fragile cells, e.g. protoplasts, for electron microscopy.

Position: Post-Doctoral Fellowship (2-3 year period) Location: University of Minnesota Activity: Studies of cellular filaments and microtubules. Structural chemistry of eukaryotic microtubules (intermediate-filament-like components of ciliary and flagellar A-microtubules). Approaches will include protein chemistry, immunocytochemistry, and computer image analysis.Reply with Curriculum Vitae to: Dr. Electron Microscopy Technique 2:7-10 (1985) we have considered low viscosity epoxy resins in a broad sense, so as to include epoxy resins such as LX-112 with viscosity lower than Epon 812, which has 210 cps. Since ERL-4206 (Spurr's medium) is generally understood as a low viscosity epoxy resin we want to clarify that LX-112 has, however, higher viscosity (185 cps) as apposed to ERL-4206 (7.8 cps) has, and is so closer to Epon i n viscosity.KAARINA PIHAKASKI AND ULLA-MAIJA SUORANTA

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The unicellular green alga Micrasterias has a very differentiated form and is an excellent object for morphogenetic studies. The complicated cytoplasmic streaming system during development is an essential part of the morphogenetic process. The streamings are supported by the acto-myosin system. The presence of actin has been verified by fluorescence microscopy and both the act in-inhibitor cytochalasin B and the myosin inhibitor N-ethylmaleimide clearly inhibit the streamings.This work deals with the localization of myosin in the cell. The methods used were immunoelectronmicroscopy, polyclonal antibody/gold probe, and cryosections. In old semicells, the gold-label localized only in vesicle structures. In growing semicells, the label was abundant in growing lobes, in all levels. In control samples, without antibody, there was no label.According to the results, myosin seems to be part of the dictyosome vesicle membranes. This is clear in the case of old semicells. In growing lobes, the inner structure of the lobes was not preserved very well in cryosections, in any preparation, but there is good evidence that in new, growing parts of the cell there is generally very little material other than cell wall material vesicles. This idea is important for later studies on plant cell growth and cytoplasmic transport.

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