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In leaf mesophyll cells of transgenic Arabidopsis thaliana plants expressing GFP in the chloroplast, stromules (stroma-filled tubules) with a length of up to 20 mum and a diameter of about 400-600 nm are observed in cells with spaces between the chloroplasts. They appear extremely dynamic, occasionally branched or polymorphic. In order to investigate the effect of temperature on chloroplasts, we have constructed a special temperature-controlled chamber for usage with a light microscope (LM-TCC). This LM-TCC enables presetting of the temperature for investigation directly at the microscope stage with an accuracy of +/-0.1 degrees C in a temperature range of 0 degrees C to +60 degrees C. With the LM-TCC a temperature-dependent appearance of chloroplast protrusions has been found. These structures have a considerably smaller length-to-diameter ratio than typical stromules and reach a length of 3-5 mum. At 5-15 degrees C (low temperatures), almost no chloroplast protrusions are observed, but they appear with increasing temperatures. At 35-45 degrees C (high temperatures), numerous chloroplast protrusions with a beaklike appearance extend from a single chloroplast. Interaction of stromules with other organelles has also been investigated by transmission electron microscopy. At 20 degrees C, transverse sections of stromules are frequently observed with a diameter of about 450 nm. A close membrane-to-membrane contact of stromules with the nucleus and mitochondria has been visualised. Golgi stacks and microbodies are found in the spatial vicinity of stromules. At 5 degrees C, virtually no chloroplast protrusions or stromules are observed. At 35 degrees C, chloroplast protrusions are present as broader thylakoid-free stroma-filled areas, resulting in an irregular chloroplast appearance.

The various metabolic activities of plastids require continuous exchange of reactants and products with other organelles of the plant cell. Physical interactions between plastids and other organelles might therefore enhance the efficiency of plant metabolism. We have observed a close apposition of plastids and nuclei in various organs of Nicotiana tabacum and Arabidopsis thaliana. In hypocotyl epidermal cells, plastids and stromules, stroma-filled tubular extensions of the plastid envelope membrane, were observed to reside in grooves and infoldings of the nuclear envelope, indicating a high level of contact between the two organelle membranes. In a number of non-green tissues, including suspension-cultured cells, perinuclear plastids were frequently associated with long stromules that extended from the cell center to the cell membrane. In cotyledon petioles, cells lying adjacent to one another frequently contained stromules that met on either side of the shared cell wall, suggesting a means of intercellular communication. Our results therefore suggest that stromules have diverse roles within plant cells, perhaps serving as pathways between nuclei and more distant regions of the cell and possibly even other cells.

Cytoplasmic male sterility arises when mitochondrial activities are disrupted that are essential for pollen development. Rearrangements in the mitochondrial genome that create novel open reading frames are strongly correlated with CMS phenotypes in a number of systems. The morphological aberrations which indicate CMS-associated degeneration are frequently restricted to the male sporogenous tissue and a limited number of vegetative tissues. In several cases, this tissue specificity may result from interactions between the mitochondrial genome and nuclear genes that regulate mitochondrial gene expression. A molecular mechanism by which CMS might be caused has not been conclusively demonstrated for any system. Several hypotheses for general mechanisms by which mitochondrial dysfunction might disrupt pollen development are discussed, based on similarities between the novel CMS-associated genes from a number of systems.

Selection and screening methods were devised which resulted in the identification of a number of somatic hybrid callus clones following fusion of Lycopersicon esculentum protoplasts and L. pennellii suspension culture protoplasts. Visual selection for callus morphology combined with a high fusion frequency and irradiation of one parental protoplast type ((137)Cs source, 1.5 Krads) resulted in selection of a callus clone population containing a high proportion of somatic hybrids. Analysis of a dimeric isozyme for the presence of a heterodimeric form was found to be satisfactory for distinguishing parental-type calli, somatic hybrid calli, and mixed calli derived from both types of unfused parental cells. No somatic hybrid calli produced shoots, although the sexual hybrid between L. esculentum and L. pennellii regenerated well under the culture conditions employed. This result suggests that the non-regenerable growth habit of the L. pennellii suspension culture was dominant in the somatic hybrid. The culture conditions described here are suitable for obtaining regenerated plants from L. esculentum mesophyll protoplasts. L. esculentum protoplast calli from fusion cultures gave rise to shoots with L. esculentum phenotype at higher frequency than calli from control unfused L. esculentum mesophyll protoplast cultures. The use of probes for species-specific organelle DNA fragments allowed identification of organelle DNA restriction fragments in digests of total DNA from small samples of individual callus clones. The callus clones analyzed either carried predominantly one parental plastid DNA type or mixtures of both types. Use of a mitochondrial DNA (mtDNA) probe which distinguishes two parental mtDNA fragments revealed that the L. pennellii-specific fragment was present in all clones examined, but the L. esculentum fragment was absent or in low proportion.

The chloroplast (cp) and mitochondrial (mt) DNAs of Petunia somatic hybrid plants, which were derived from the fusion of wild-type P. parodii protoplasts with albino P. inflata protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization. Using (32)P-labelled probes that distinguished the two parental cpDNAs at a BamH1 site and at a HpaII site, only the P. parodii chloroplast genome was detected in the 10 somatic hybrid plants analyzed. To examine whether cytoplasmic mixing had resulted in rearrangement of the mitochondrial genome in the somatic hybrids, restriction patterns of purified somatic hybrid and parental mtDNAs were analyzed. Approximately 87% of those restriction fragments which distinguish the two parental genomes are P. inflata-specific. Restriction patterns of the somatic hybrid mtDNAs differ both from the parental patterns and from each other, suggesting that an interaction occurred between the parental mitochondrial genomes in the somatic fusion products which resulted in generation of the novel mtDNA patterns. Southern blot hybridization substantiates this conclusion. In addition, somatic hybrid lines derived from the same fusion product were observed to differ in mtDNA restriction pattern, reflecting a differential sorting-out of mitochondrial genomes at the time the plants were regenerated.

A single somatic hybrid callus clone was identified following the fusion of Lycopersicon esculentum protoplasts and Solanum rickii suspension culture protoplasts. The hybrid nature of the callus and the plants regenerating from it was determined by assaying phosphoglucomutase-2 isozyme expression. The chloroplast genome present in four somatic hybrid plants was characterized by probing digests of total DNA with nick translated L. esculentum chloroplast DNA(cpDNA). All four somatic hybrid plants had inherited S. rickii cpDNA. Two clones of plant mitochondrial DNA (mtDNA), soybean 18S and 5S rDNA and maize cytochrome oxidase subunit II were used to characterize the mtDNA present in total DNA digests of four somatic hybrid plants. In both cases, the somatic hybrid plants had inherited most but not all of the S. rickii specific fragments, but none of the L. esculentum specific fragments.

The genetic stability of plants regenerated from either mesophyll protoplasts or leaf slices of the F1 hybrid between Lycopersicon esculentum and L. pennellii was assayed by comparing the ploidy level, leaf morphology and isozyme patterns of the regenerants with their somatic parents. Regenerants from protoplasts were predominantly tetraploid, regenerants from leaf slices were predominantly diploid; both classes of regenerants had isozyme patterns identical to those of the parent plant. Callus was analyzed that grew up from cultures containing fused protoplasts from either irradiated or untreated protoplasts of L. esculentum and L. pennellii. The L. pennellii cell line used was 18 months old and could no longer regenerate. Out of 75 calli scored at 3 isozyme loci, 51 were heterozygous at only one or two of the loci. Irradiation of the two parental lines was not necessary to produce fusion products exhibiting asymmetric expression of parental genes.