Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system.

The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm. To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein alpha-1,2 mannosidase I. The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal alpha-1,2 mannosidases. Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots. Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks. In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 microm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion. In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior. We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.

Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers.

Nuclear transformation of intact (walled) cells of the green alga Chlamydomonas reinhardtii was achieved by agitating the cells in the presence of plasmid DNA and silicon carbide (SiC) whiskers. The protocol was used to introduce the wild-type nitrate reductase structural gene into the nitrate reductase-deficient mutant strain nit1-305. Using SiC whiskers, 10-100 transformants per 10(7) cells were routinely produced, which is comparable to transformation rates achieved by agitating the cells with glass beads. In contrast to the glass bead protocol, cell viability was very high following treatment with SiC, with greater than 80% cell survival after agitation for 10 min. Agitation with SiC whiskers appears to be an efficient method for introducing DNA into intact C. reinhardtii cells and may prove to be applicable to other algal species for which cell wall mutants or protoplasting procedures are unavailable.

Immunogold localization of intra- and extra-cellular proteins and polysaccharides of plant cells.

This paper illustrates post-embedding immunogold labelling of protein and polysaccharide molecules of plant cells. For EM studies, one is restricted (for most plant cells) to the post-embedding approach because the surrounding cell wall prevents access of antibodies (and secondary gold-tagged markers) to internal sites. The large size of many plant cells also does not lend itself to diffusional entry of antibodies. The molecules localized include seed storage proteins that are large and present in major quantities, a smaller less abundant, water soluble albumin, an oxygen-binding protein, components of the photosynthetic electron transport chain, and complex sugars from the cell wall. A range of preparative procedures and embedding plastics are used.

Phosphorylation of spinach chlorophyll-protein complexes. CPII, but not CP29, CP27, or CP24, is phosphorylated in vitro.

Previous studies have indicated that the reversible phosphorylation of a population of antenna complexes that can donate energy to PS II ('mobile LHC II') plays a regulatory role in the state 1-state 2 transition in thylakoid membranes. The relationship of phosphorylated LHC II to the multiple PS II-associated chlorophyll a/b-proteins resolvable on green gels is currently unclear. We have used a high resolution gel system to analyze thylakoids phosphorylated in vitro. The only PS II-associated antenna complex to become phosphorylated is CPII, indicating that this complex represents the mobile LHC II. The other putative PS II antenna complexes, CP29, CP24, and the new complex designated CP27 which comigrates with CPII, are not phosphorylated and are probably components of the bound 'LHC II' antenna.

Isolation and Characterization of a New Minor Chlorophyll a/b-Protein Complex (CP24) from Spinach.

We have identified a new minor chlorophyll a/b-protein complex in the thylakoid membranes of spinach (Spinacia oleracea L.), which migrates as a green band below CPII on mildly denaturing polyacrylamide gels. This complex, designated CP24, was isolated from octyl glucoside/sodium dodecyl sulfate solubilized spinach grana membrane fractions by preparative gel electrophoresis and has been characterized as to its spectral properties and polypeptide composition. CP24 has a room temperature absorption maximum at 668 nanometers, a chlorophyll a/b ratio between 0.8 and 1.2, and contains three or four polypeptides between 20 and 23 kilodaltons. CP24 was also identified in grana membrane preparations from peas (Pisum sativum) and barley (Hordeum vulgare). We postulate that CP24 functions as a linker component in photosystem II, acting to orient the photosystem II light harvesting components to ensure efficient energy transfer to the reaction center.

Isolation of photosystem I complexes from octyl glucoside/sodium dodecyl sulfate solubilized spinach thylakoids : characterization and reconstitution into liposomes.

We have used the nonionic detergent octyl-beta-d-glucopyranoside in combination with sodium dodecyl sulfate to isolate two novel Photosystem I (PSI) complexes from spinach (Spinacea oleracea L.) thylakoid membranes. These complexes have been characterized as to their spectral properties, content of PSI reaction center chlorophyll P(700), and protein composition. PSI-B, purified from solubilized membranes by sucrose density gradient centrifugation, is a putative native PSI complex. PSI-B contains four polypeptides between 21 and 25 kilodaltons in addition to the components of the PSI antenna complex (LHCI); three of these polypeptides have not previously been associated with PSI. A second complex, CPI(*), is purified from octyl glucoside/sodium dodecyl sulfate solubilized thylakoids by two cycles of preparative gel electrophoresis under mildly denaturing conditions. Electrophoresis under these conditions releases a discrete set of polypeptides from PSI producing a complex composed only of the PSI reaction center and the LHCI antenna.In addition, the PSI reaction center complex CPI isolated from preparative gels and PSI-B were reconstituted into lecithin liposomes for structural analysis using freeze-fracture electron microscopy. The results suggest that the native PSI complex produces 12- to 13-nanometer particles, while the PSI reaction center, depleted of LHCI and peripheral proteins, produces particles with an average diameter of 10 nanometers.