Unusual fast sedimenting mitochondria producing heavy DNA in the cells of aging coleoptiles of wheat seedlings

A fraction of unusual fast sedimenting (10 min at 600-1700g) particles with properties of mitochondria has been detected in wheat seedlings. This fraction conventionally called "heavy" mitochondria amounts (by protein) to about 40% of the total subcellular particle fraction sedimented by 10 min centrifugation at 17,000g. The specific feature of these "heavy" mitochondria in aging tissues is an ability to synthesize and even superproduce heavy (rho = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). The share of "heavy" mitochondria sedimented in the interval between 1000 and 1700g and possessing the maximal H-mtDNA synthesis in aging coleoptiles is about 1.5-fold higher than that in young coleoptiles. Although "heavy" mitochondria are present in young plant organs, they seem to be unable to synthesize H-mtDNA; heavy mtDNA forms only in mitochondria of aging or old cells. Thus, aging in plants is accompanied by a change in population of mitochondria and appearance of the ability for selective H-mtDNA superproduction in a certain mitochondrial fraction. Mitochondria isolated from wheat coleoptiles are practically not stimulated by uncouplers. "Heavy" (600-1700g) and usual (4,300-17,400g) mitochondria are similar in respiration rates, cytochrome compositions, cytochrome c amount (per mg protein) and sensitivities to respiration inhibitors. However, "heavy" mitochondria contain (per mg protein) cytochromes b and aa3 by 10-20% and Ca2+ by 2-3-fold more than normal mitochondria. Ultrastructural analysis showed that the isolated fraction of fast sedimenting mitochondria consists of a suspension of closed membrane vesicles filled with cytoplasm and containing one or a few mitochondria. We observed similar structures in situ in vacuoles of parenchyma cells in the apical part of intact coleoptiles. The process of formation of such structures was detected by serial ultra-thin section analysis. It was shown that tonoplast protrudes into vacuoles, the separate mitochondria translocate into these protrusions, and then these structures separate. As a result, the suspended cytoplasmic bodies containing mitochondria appear in vacuoles. Appearance of these bodies containing mitochondria and, in particular, the superproduction of H-mtDNA in them correlate with processes of aging and cell transition to apoptosis.     

Superproduction of heavy minicircular mitochondrial DNA in aging wheat coleoptiles.

On incubation of 7-day-old wheat seedlings in the presence of [3H]thymidine, the radioactivity incorporated into coleoptile DNA is found to be localized mainly (greater than 95%) in the fraction of heavy mitochondrial DNA (H-mt DNA; rho = 1.716 gm/cm3). Upon long (48-72 h) incubation of cut-off seedlings in water, the amount of this DNA shows a dramatic increase and corresponds to about 10% of the total coleoptile DNA. H-mtDNA is represented by open circular molecules with a contour length varying from 0.12 to 0.6 microns. The functional role of this DNA is still unknown.     

Zinc and copper content in developing and aging coleoptiles of wheat seedlings

The content of zinc and copper in coleoptiles of etiolated wheat (Triticum aestivum L.) seedlings was measured after seed germination. The content of these metals changed differently during coleoptile development and aging: the content of zinc increased substantially from the 8th to 14th day of seedling development, whereas the content of copper slightly increased on day 8 and later slightly decreased. These changes coincided with the period of increased proteolytic activity and the signs of coleoptile cell apoptotic death. Zinc accumulation and copper amount reduction in the aging coleoptile were most pronounced in the oldest apical segment of the coleoptile, which was most enriched in apoptotic cells. The modulations in the zinc and copper amounts observed might be related to the induction and continuation of terminal stages of apoptosis.     

In vitro replication of heavy strand DNA in permeabilized human mitochondria.

We have characterized some of the experimental conditions that are essential for initiation of human mitochondrial DNA synthesis. Mitochondria were purified from HeLa cells and were permeabilized with Triton X-100. When supplied with rNTPs and dNTPs, the permeabilized mitochondria synthesized nucleic acids that ranged in size from about 600 to 2000 nucleotides. In vitro DNA synthesis occurred on endogenous DNA templates and required a continuous supply of ATP. Analyses of the synthetic products revealed that almost all of them were of heavy-strand sequence and included authentic 7S DNA. Most of the synthetic products had 5' ends that mapped to similar locations as those previously identified for nascent heavy-strand DNA. Identification of these parameters should facilitate our efforts to achieve in vitro replication of heavy-strand mitochondrial DNA.     

Effect of the antioxidant ionol on proteolytic apparatus of aging coleoptiles of wheat seedlings grown in light

The dynamics of changes in total proteolytic activity and activities of various groups of proteases in the coleoptiles of 3- to 12-day-old wheat seedlings grown in light with and without antioxidant BHT (2,6-di-tert-butyl-4-methylphenol) was studied. It was established that the specialized proteases that easily hydrolyze specific synthetic substrates and the enzymes actively hydrolyzing histone H1 dominate in young coleoptiles of 3- to 4-day-old seedlings. Proteases that degrade equally well the majority of the studied substrates are accumulated in the cells of old coleoptiles of 11- to 12-day-old seedlings. Under the effect of BHT, the plants grown in light (in comparison with etiolated seedlings) demonstrated a somewhat changed dynamics of proteolytic activity in young coleoptiles and the disappearance of proteases active toward histone H1. An inhibitory analysis revealed a relative domination of cysteine proteases in young coleoptiles at the initial development stage of seedlings, whereas the fraction of serine proteases markedly increased in old coleoptiles. We presume that the revealed quantitative and qualitative changes in the proteolytic apparatus of the coleoptile cells induced by BHT may be largely responsible for the retardant and geroprotective effect of this antioxidant in plants.     

DNA synthesis in isolated mitochondria and mitochondrial extracts from wheat embryos

DNA synthesis was studied using purified wheat embryo mitochondria as well as mitochondrial lysates deprived of endogenous DNA. The optimal conditions for DNA synthesis are very similar in both systems: ATP stimulates dramatically mitochondrial DNA synthesis and magnesium is a better co-factor than manganese, contrary to what has been reported in animal mitochondrial systems. Wheat mitochondrial DNA synthesis is resistant to aphidicolin and strongly inhibited by dideoxythymidine triphosphate and ethidium bromide. Thus, the DNA polymerase involved in this system seems to be the same as that previously purified and characterized from wheat embryo mitochondria (Christopheet al., Plant Science Letters 21: 181, 1981). Two different approaches: restriction endonuclease digestion followed by electrophoresis, and autoradiography and cesium chloride equilibrium centrifugation of mitochondrial DNA, where BrdUTP has been incorporated instead of TTP, show that long stretches of the mitochondrial genome have been synthesized.     

Nastic curvatures of wheat coleoptiles that develop in true microgravity

Dark-grown wheat coleoptiles developed strong curvatures within 5 h of being transferred in orbit from a 1 g centrifuge to microgravity during an experiment flown on the IML-1 shuttle mission. The curving tendency was strongest in seedlings that were immature, with coleoptiles shorter than 10 mm at the time of transfer. The curvature direction was non-random, and directed away from the caryopsis (the coleptile face adjacent to the caryopsis becoming convex). The curvatures were most marked in the basal third of the coleoptiles, contrasting with phototropic responses, which occur in the apical third. We interpret these curvatures as being nastic, and related to the curvatures commonly reported to occur during clinostat rotation treatments.     

Modification of cell wall architecture of wheat coleoptiles grown under hypergravity conditions.

Cell wall structure of wheat coleoptiles grown under continuous hypergravity (300 g) conditions was investigated. Length of coleoptiles exposed to hypergravity for 2-4 days from germination stage was 60-70% of that of 1 g control. The amounts of cell wall polysaccharides substantially increased during the incubation period both in 1 g control and hypergravity-treated coleoptiles. As a results, the levels of cell wall polysaccharides per unit length of coleoptile, which mean the thickness of cell walls, largely increased under hypergravity conditions. The major sugar components of the hemicellulose fraction, a polymer fraction extracted from cell walls with strong alkali, were arabinose (Ara), xylose (Xyl) and glucose (Glc). The molar ratios of Ara and Xyl to Glc in hypergravity-treated coleoptiles were higher than those in control coleoptiles. Furthermore, the fractionation of hemicellulosic polymers into the neutral and acidic polymers by the anion-exchange column showed that the levels of acidic polymers in cell walls of hypergravity-treated coleoptiles were higher than those of control coleoptiles. These results suggest that hypergravity stimuli bias the synthesis of hemicellulosic polysaccharides and increase the proportion of acidic polymers, such as arabinoxylans, in cell walls of wheat coleoptiles. These structural changes in cell walls may contribute to plant resistance to hypergravity stimuli.     

Introduction. Oxidative stress in mitochondria disorders of aging

These special issues of Biological Signals and Receptors are intended to describe mitochondrial DNA damage, oxidative stress and human diseases, including neurodegenerative and neuromuscular diseases, disorders associated with aging, and ischemia-perfusion injury. Traditionally, mitochondria have been viewed as the 'powerhouse' of the cell, i.e., the site of the oxidative phosphorylation machinery involved in adenosine triphosphate (ATP) production. Consequently, much of the research conducted on mitochondria over the past 4 decades has focused on elucidating both those molecular events involved in ATP synthesis by oxidative phosphorylation and those involved in the biogenesis of the oxidative phosphorylation machinery. While monumental achievements have been made, and continue to be made, in the study of these remarkable but extremely complex processes essential for the life of most animal cells, it has been only in recent years that a large body of biological and biomedical scientists have come to recognize that mitochondria participate in other important processes. Two of these are cell death and aging which, not surprisingly, are related processes both involving, in part, the oxidative phosphorylation machinery. This new awareness has sparked a new and growing area of mitochondrial research that has become of great interest to a wide variety of scientists ranging from those involved in elucidating the role of mitochondria in cell death and aging to those interested in either suppressing or facilitating these processes as it relates to identifying new therapies or drugs for human disease.     

Melatonin and mitochondria in aging

The worldwide prolongation of mean life expectancy has resulted in a rapid increase of the size of the elderly population, both in numbers and as a proportion of the whole. In addition, the incidence of age-related diseases is obviously increasing as the population ages. Finding means to preserve optimal health in old age has become a primary goal of biomedical research. Aging is a multifactorial process that includes progressive cellular loss, endocrine and metabolic deficits, reduced defense mechanisms and functional losses that increase the risk of death. Mitochondria fulfill a number of essential cellular functions and play a key role in the aging process. Melatonin, which is synthesized in the pineal gland and other organs, plays a role in the biologic regulation of aging. Noctural melatonin serum levels are high during childhood and diminish substantially as people age. Melatonin preserves mitochondrial homeostasis, reduces free radical generation, e.g., by enhancing mitochondrial glutathione levels; it also safeguards proton potential and ATP synthesis by stimulating complex I and IV activities. In this article, we review the role of melatonin and mitochondria in aging.     

Localization of germin genes and their products in developing wheat coleoptiles

Germination is a process which characterized with nescient synthesis of genes. Among the genes synthesized during the germination of wheat embryos, germin genes, proteins and their enzymatic activity were defined. Germin is a water soluble homopentameric glycoprotein which is remarkable resistant to degradation by a broad range of proteases including pepsin. Germin proteins found to have strong oxalate oxidase activity which produces hydrogen peroxide by degrading oxalic acid. The current study, aimed to localize the germin genes, proteins and enzymatic activities in developing coleoptiles which is a rapidly growing protective tissue of leaf primordium and shoot apex. Non-radioactively labeled germin riboprobes were employed to localize germin mRNAs in situ. FITC (Fluorescein isothiocyanate) and alkaline phosphatase linked anti-germin antibodies were used to localize germin proteins under the fluorescence and light microscopy and finally germin enzymatic activity was localized by using appropriate enzyme assay. The results revealed that in coleoptiles germin genes, proteins and their enzymatic activity were predominantly associated with the cells of epidermis and vascular bundle sheath cells.     

Localization and properties of ATPase activity in pea stems and wheat coleoptiles

Summary Microsomal fractions from wheat coleoptiles and pea stems contain a microsomal ATPase activity that requires divalent cations (Ca²⁺ is more effective than Mg²⁺) and shows further stimulation by KCl. The effects of added indoleacetic acid were inconclusive. Cytochemical studies on both species showed most pronounced staining for ATPase in the plasmalemma at pH 7.0. However, at pH 5.5, the coleoptile cells showed heaviest staining for ATPase in the endoplasmic reticulum and dictyosomes. The results are discussed with regard to the postulated role of ATPase activity in relation to proton pumping and plant cell elongation.