Unusual closed membrane vesicles containing one or more mitochondria were isolated from homogenates of aging wheat coleoptiles. Very similar (or the same) bodies were shown to exist in situ in vacuoles of undividing cells in the apical part of intact senescent coleoptiles. Vesicles isolated from coleoptile homogenate free of nuclei by 10 min centrifugation at 1700U Ug and traditional mitochondria (sedimented at between 4300U Ug and 17 400U Ug) are similar in respiration rate, composition and content of cytochromes and sensitivity to respiration inhibitors. However, vesicles contain about 2-fold more Ca 2+ ions than free mitochondria do. The specific feature of vesicles containing mitochondria in aging coleoptiles is an intensive synthesis of heavy (b b = 1.718 g/cm 3 ) mitochondrial DNA (H-mtDNA). Thus, aging in plants is accompanied by an increased selective H-mtDNA production and change in subcellular organization of mitochondria.z 1999 Federation of European Biochemical Societies.

By computer analysis of the known data bases, we have established that the open reading frames (ORF) coding for proteins that possess high degree of homology with procaryotic DNA-(amino)methyltransferases are present in the genomes of Leishmania major, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens. Conservative motifs typical for bacterial DNA-(amino)methyltransferases are detected in the amino acid sequences of these putative proteins. The ORF of all putative eucaryotic DNA-(amino)methyltransferases found are encoded in nuclear DNA. In mitochondrial genomes including a few fully sequenced higher plant mtDNA, nucleotide sequences significantly homologous to genes of procaryotic DNA-(amino)methyltransferases are not found. Thus, ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclei of protozoa, yeasts, insects, nematodes, vertebrates, higher plants, and other eucaryotes. A special search for corresponding proteins and, in particular, adenine DNA-methyltransferases in these organisms and a study of their functions are quite promising.

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Ionol (BHT), a compound having antioxidant activity, at concentrations in the range 1-50 mg/liter (0.45 x 10(-5)-2.27 x 10(-4) M), inhibits growth of etiolated wheat seedlings, changes the morphology of their organs, prolongs the coleoptile life span, and prevents the appearance of specific features of aging and apoptosis in plants. In particular, BHT prevents the age-dependent decrease in total DNA content, apoptotic internucleosomal fragmentation of nuclear DNA, appearance in the cell vacuole of specific vesicles with active mitochondria intensively producing mtDNA, and formation of heavy mitochondrial DNA rho = 1.718 g/cm3) in coleoptiles of etiolated wheat seedlings. BHT induces large structural changes in the organization of all cellular organelles (nucleus, mitochondria, plastids, Golgi apparatus, endocytoplasmic reticulum) and the formation of new unusual membrane structures in the cytoplasm. BHT distorts the division of nuclei and cells, and this results in the appearance of multi-bladed polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces intensive synthesis of pigments, presumably carotenoids, and the differentiation of plastids with formation of chloro- or chromoplasts. The observed multiple effects of BHT are due to its antioxidative properties (the structural BHT analog 3,5-di-tert-butyltoluene is physiologically inert; it has no effect similar to that of BHT). Therefore, the reactive oxygen species (ROS) controlled by BHT seem to trigger apoptosis and the structural reorganization of the cytoplasm in the apoptotic cell with formation of specific vacuolar vesicles that contain active mitochondria intensively producing mtDNA. Thus, the inactivation of ROS by BHT may be responsible for the observed changes in the structure of all the mentioned cellular organelles. This corresponds to the idea that ROS control apoptosis and mitosis including formation of cell wall, and they are powerful secondary messengers that regulate differentiation of plastids and the Golgi apparatus in plants.

It was found that production of superoxide (O(2)(*-)) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O(2)(*-) production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O(2)(*-) production was clearly observed in the period from two to four days of seedling development, then the rate of O(2)(*-) production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O(2)(*-) production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O(2)(*-) production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O(2)(*-) amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O(2)(*-) controls plant growth by cell elongation at the early stages of seedling development but later O(2)(*-) controls (induces) apoptotic DNA fragmentation and protein disintegration.