Structural and functional changes in root cells induced by calcium ionophore A23187

The shifts of Ca²⁺, K⁺ and proton homeostasis of wheat (Triticum aestivum L. M. cv Ljuba) root cells induced by the Ca²⁺-ionophore A23187 caused different responses, depending on the time of exposure to the ionophore. Oxygen consumption and heat production by roots were increased when the Ca²⁺-specific effect of A23187 was expressed. Ultrastructural re-organization of cell organelles was found to follow the ion shifts. The endoplasmic reticulum, Golgi apparatus and mitochondria rearranged their membranes following treatment. The increased ion permeability of root cell membranes is proposed to cause an excessive energy expenditure for the restoration of ion homeostasis.     

Changes in mitochondrial shape in wheat root cells exposed to mitochondrial poisons

It is found that mitochondrial poisons not only modify the functional activity of the organelles but also change the shape of some mitochondria. The ring-shaped organelles were found to appear in root cells of 5- to 6-day-old seedlings of spring wheat (Triticum aestivum L., cvs. Moskovskaya 35 and Lyuba). Using a technique of serial sections, we have shown that the circular profiles of mitochondria within the cell correspond in most cases to bowl-shaped organelles. Spatial reorganization of mitochondria did not depend directly on the respiration rate, duration of exposure to inhibitors, and inhibitor specificity. These observations indicate the reversibility of spatial rearrangements and general nonspecific nature of the detected morphological changes.     

Wheat root cells functioning under inhibition of I and II complexes of mitochondrial respiratory chain

A joint effect of rotenone and malonate on the intensity of respiration, output of K+ and ultrastructure of wheat root cells treated for 6 h was studied. The addition of malonate to rotenone containing solution, in which wheat roots had been incubated for an hour, caused further decrease in respiration intensity and K+ output into external medium. Many mitochondria acquired torus shape in 2h after malonate addition. The increase in respiratory intensity and re-entry of K+ from the incubation medium into the cells were observed during following hours of incubation. We assume that reparation and adaptation processes took place in this case. The observed contacts of endoplasmic reticulum lumens with mitochondria are indicative of possible synthesis of an enzyme able to metabolize malonate to acetyl-CoA and CO2. We propose that torus shape of mitochondria is due to the increase in their outer surfaces, that, in turn, is a result of activation of external NAD(P)H-dehydrogenase. These findings may be evidence of possible adaptation of the root cells to the joint effect of the inhibitors.     

Synthesis of polyglycerol phosphate by Bacillus stearothermophilus.

A particulate enzyme preparation from Bacillus stearothermophilus synthesized 1,3-poly(glycerol phosphage) from CDPglycerol at an optimum pH of 8.0 and the reaction was stimulated by divalent cations. Km for CDPglycerol was 0.18 mM. The synthesis was inhibited by CMP, CDP, and CTP and by concentrations of CDP-glycerol above 0.49 mM. The reaction was irreversible, The product had an average chain length of 8 glycerol units. About two thirds of the polymers were synthesized in entirety while the ramainder were attached to some acceptor by their phosphate end. The enzome was able to synthesize only a limited amount of polymer.     

Localization of ATPase activity, respiration and ultrastructure of wheat root cells with modulated ion conductivity of plasma membrane

Changes in the localization of ATPase activity, respiration and ultrastructure of wheat root cells with modulated ion conductivity of plasma membrane were studied. A 2 h treatment of excised root with valinomycin (20 microM), N,N-dicyclohexylcarbodiimid (100 microM), gramicidin S (20 microM) and chlorpromazine (100 microM) caused an increased loss of potassium by cells, a decreased respiration and changes in the localization of ATPase activity and in cell ultrastructure. Differences in the observed changes may be conditioned by different mechanisms of action of the membrane active compounds used. It is concluded that changes in the localization of ATPase activity and ultrastructure may indicate some early specific responses of root cells, whereas the increase in the ion conductivity and decrease in respiration under disruption of ion homeostasis caused by membrane active compounds indicate unspecific responses of cells.     

Ultrastructural characteristics of mitochondria during cell adaptation to rotenone

A study was made of respiration, heat production, K+ output and ultrastructure of wheat root cells treated for 6 h with rotenone (10 microM), an inhibitor of HADH-ubiquinone oxidoreductase (Complex I). Besides, the involvement of alternative pathways for adaptation to this inhibitor was studied. After 20 min of treatment, a brightened mitochondrial matrix and mitochondria with torus shapes were observed. We propose that the outer area of mitochondria increases due to their torus shapes, and this can point to the activating of extremal NAD(P)H-dehydrogenase, which uses enternal NAD(P)H. Further on the normal ultrastructure of mitochondria was observed, which may result from activation of succinate dehydrogenase and rotenone resistant NAD(P)H-dehydrogenase. After 1 h of treatment, a decrease in respiration, heat production, K+ output and pH increase of incubation medium were observed. Starting from 2 h of incubation and up to the end of the experiment, an increase of respiration and heat production was observed, pointing to the activation of oxidative phosphorilation. Besides, re-entry of K+ and pH decrease in the incubation medium were observed. We conclude that these findings may indicate to a possible adaptation of root cells to this inhibitor. We propose that the torus shape of mitochondria may be associated with function of external NAD(P)H-dehydrogenase.     

Effect of malonate on the structural and functional changes of wheat Triticum aestivum L. root cells

A study was made of respiration, output of K+ and ultrastructure of wheat root cells treated for 6 h with malonic acid (MA) (15 mM), an inhibitor of succinate dehydrogenase. After a 1 h treatment, on the background of a decrease in respiration, and output of K+ an increased number of lumens of smooth endoplasmic reticulum was observed. These changes may be the result of lipid biosynthesis. Within first hours of treatment with MA, the mitochondrial matrix was becoming more brightened, and after 3 h all organelles became transparent. Moreover, mitochondria increased in size and almost lacked cristae. After 4 h mitochondria assumed their normal sizes due, presumably, to a competitive action of malonate. After 5 h the matrix was brightened again, mitochondria augmented in size, several organelles acquired torus shapes, and their outer area was eventually increased. We found contacts of endoplasmic reticulum lumens with mitochondria, which may suggest the synthesis of an enzyme, able to transform to malonate. After a 6 h exposure of MA, we observed the increase of respiration, re-entry of K+ and normal ultrastructure of mitochondria. Based on our experiments, we conclude that adaptation of root cells may be a result of external NADPH-dehydrogenase activity and MA detoxification.     

Dynamics of structural and functional changes in wheat root cells under the action of protonophore

Structural and functional changes in wheat root cells during long-term action of a protonophore--carbonyl cyanide 3-chlorophenylhydrazone (CCCP)--were studied. It was demonstrated that CCCP affected the electrical potential and inward resistance of cells, increased K+ ions release to the incubation medium, inhibits oxygen uptake for 1-4 h, which was followed by oxygen uptake stimulation for 6 h of treatment. These changes of physiological processes were accompanied with a variety of ultrastructural changes in cell organization, namely in the structure of mitochondria, endoplasmic reticulum canals, and the nucleus. The role of protons is discussed, in particular, in the regulation of metabolic state of mitochondria, and in general regulation of structural and functional conditions of cells.     

Peculiarities of functioning of wheat cut-off root cells at inhibition of complexes I and II of mitochondrial respiratory chain

Combined action of rotenone and malonate, inhibitors of complexes I and II of the mitochondrial electron transport chain (ETC), on wheat cut-off root seedlings was studied after 6 h of incubation. Intensity of oxygen consumption and release of potassium ions into incubation medium were determined simultaneously with the study of changes in cell ultrastructure. Malonic acid was added 1 h after the root incubation in the rotenone solution and produced inhibition of respiration, as well as a greater release of K⁺ into the incubation solution as compared with effect of rotenone alone. After 2 h of the combined action of these inhibitors, many mitochondria acquired a toroidal shape, thereby increasing the outer surface. For the ensuing hours, stimulation of oxygen consumption by the roots and a decrease of K⁺ content in the incubation medium were observed. Mitochondria once again acquired a round or oval shape and compensation-reparation processes took place. Contacts of endoplasmic reticulum channels with mitochondria were observed, which seems to be due to the synthesis of the enzyme splitting malonate to acetyl-CoA, which in turn can be included both into the Krebs cycle and into lipogenesis. It is suggested that the toroidal form of mitochondria is associated with the activation of the external NAD(P)H-dehydrogenase of the inner mitochondrial membrane, as under these conditions, at the inhibition of the ETC complexes I and II, the activity of other dehydrogenises is blocked. Thus, the use of the external NAD(P)H allows the activity of the ETC mitochondria to be restored, which facilitates the course of the reparation processes and allows cells to be adapted to this action.