Intracellular localization of the multiple forms of ATP-ase activity in maize root tips

Summary The multiple forms of ATP-ase activity in maize root tips have been examined by separation and staining using polyacrylamide gel electrophoresis. The band pattern obtained using a whole homogenate supernatant fraction was compared with the pattern obtained from various cell organelle fractions. The kinetic properties of the ATP-ase activity in various fractions were also examined. The results support the view that ATP-ase activity exists in a number of molecular forms in the same tissue and that these forms may show specific intracellular localizations.     

Intracellular pH in rice and wheat root tips under hypoxic and anoxic conditions

The influence of anoxia and hypoxia on dynamic of intracellurar pH and ATP content in rice and wheat root tips was investigated with ³¹P-NMR spectroscopy. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. Anoxic wheat root tips were deficient in ADH induction, whereas increased activity of alcoholic fermentation enzymes took place in anoxic rice root tips, as well as in both species after hypoxic treatment. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt = 10^−pHcyt) for both hypoxic and anoxic conditions. In this addendum we want to focus the reader''s attention on the importance of adequate experimental design when hypoxia is under investigation and on some further perspectives of intracellular pH regulation in plants under anaerobic conditions.Key words: anoxia, hypoxia, rice, wheat, cytoplasmic pH regulation     

Urate oxidase in peroxisomes from maize root tips

Summary Peroxisomes isolated from maize root tips contained urate oxidase, although the supplementary enzymes allantoinase, allantoicase and NADH-glyoxylate reductase were not detected. Some glutamate-oxalacetate transaminase was present in peroxisomes. Enzymes of two other pathways occuring in plant peroxisomes, namely glycolate metabolism and the glyoxylate cycle, were not present. The root peroxisome thus resembles peroxisomes of the Arum spadix and supports the concept that peroxisomes constitute a dynamic and differentiating system.     

Ultrastructural observations of maize root tips following exposure to monensin

Summary Epidermal and outer rootcap cells of maize root tips were treated with the sodium selective ionophore, monensin, and the ultrastructural changes were studied. In the presence of 10^–5 to 10^–3 M monensin, dictyosomes became distorted, cisternae separated from the stack, and secretory vesicles were released. Released secretory vesicles disappeard from the cytoplasm suggesting that their transport to, and fusion with, the plasma membrane was unaffected. Monensin did not inhibit cytoplasmic streaming of the outer rootcap cells. No new secretory vesicles were formed on the remaining dictyosomes or dictyosome fragments. In contrast to results with animal cells, swelling of plant dictyosome cisternae was observed only after fixation in glutaraldehyde-osmium tetroxide and not after fixation in potassium permanganate. Other cell components were not altered structurally by monensin. The effects of monensin on the Golgi apparatus were reversible, and dictyosomes were either repaired or new dictyosomes were formed after the root tips were removed from the monensin.Dictyosomes in epidermal cells reacted in the same manner as those in the rootcap except that numerous secretory vesicles remained in the cytoplasm, mostly in association with dictyosome fragments. Some secretory vesicles increased in size but no evidence of vesicle-vesicle fusion was noted. Cell plate formation was partially inhibited or blocked by monensin.Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the USDA.     

Blockage of mitosis in maize root tips using colchicine-alternatives

Mitosis in plants can be blocked by colchicine which has the capacity to bind microtubule subunits. In maize (Zea mays L.) breeding, it is frequently being used for doubling chromosome numbers of haploids for producing homozygous doubled haploids. However, colchicine is highly toxic for mammals and impacts negatively on the environment. Therefore, it was interesting to find substitutes like chemical compounds and/or physical methods which would be capable of doubling chromosome numbers in maize. For this purpose, a screening system was set up using root tips of maize. Herbicides like amiprophos methyl, oryzalin, and pronamide were identified to be effective in doubling chromosome sets of maize. Additionally, the toxicity of these compounds was lower than that of colchicine and treated seedlings recovered and grew. Therefore, they could be applied in reduced concentrations showing results comparable to colchicine.     

Study of glucose starvation in excised maize root tips

Excised maize (Zea mays) root tips were used to follow the effects of a prolonged glucose starvation. Respiration rate began to decrease immediately after excision, reaching 30 to 40% of its initial value after 20 hours, and then declined more slowly until death of the tissues, which occurred after 200 hours of starvation. During the whole process, respiration could be uncoupled by 2,4-dinitrophenol and the energy charge remained high. These results suggest that in excised maize root tips, respiration rate is essentially limited by the rate of biosyntheses (ATP-utilizing processes) rather than mitochondrial number. During starvation the sugar content sharply decreased for the first 20 hours and reached zero at 120 hours. Following root excision, proteins and lipids were continuously degraded and were virtually the only substrates for respiration and biosyntheses after 20 hours of starvation. Over the first 90 hours of starvation, enzymic activities related to sugar metabolic pathways and the Krebs cycle decreased to 20% or less of their initial activity. Starvation was reversible only for the first 80 to 90 hours. Between 80 and 100 hours, there was a sharp fall in intracellular osmolarity and a 25% loss in the dry weight. The irreversibility may be due, as in senescence, to a change in membrane selective permeability.     

Intracellular pH regulation in rat round spermatids

Intracellular pH has been shown to be an important physiological parameter in cell cycle control and differentiation, aspects that are central to the spermatogenic process. However, the pH regulatory mechanisms in spermatogenic cells have not been systematically explored. In this work, measuring intracellular pH (Hi) with a fluorescent probe (BCECF), membrane potential with a fluorescent lipophilic anion (bisoxonol), and net movement of acid using a pH-stat system, we have found that rat round spermatids regulate pHi by means of a V-type H⁺-ATPase, a HCO ^{3 ?} entry pathway, a Na⁺ HCO3^?dependent transport system, and a putative proton conductive pathway. Rat spermatids do not have functional base extruder transport systems. These pH regulatory characteristics seem specially designed to withstand acid challenges, and can generate sustained alkalinization upon acid exit stimulation.     

Intracellular pH regulation in the early embryo

Intracellular pH (pHi) regulation is a homeostatic function of all cells. Additionally, the plasma membrane-based transporters controlling pHi are involved in growth factor activation, cell proliferation and salt transport – all processes active in early embryos. pHi regulation in the early embryos of many species exhibits unique features: in mouse preimplantation embryos, mechanisms for correcting excess acid apparently are inactive, while excess base is removed by the mechanism common in differentiated cells. Additionally, unlike differentiated cells, mouse preimplantation embryos are highly permeable to H⁺ until the blastocyst stage, where the epithelial cells surrounding the embryo are impermeable. In several non-mammalian species, of which the best-studied is sea urchin, cytoplasmic alkalinization at fertilization is necessary for development of the embryo, and elevated pHi must be maintained during early development. Thus, pHi regulatory mechanisms appear to be important for early embryo development in many species.     

Cytochemical localization of adenyl cyclase activity in maize root tips

Adenyl cyclase activity has been investigated in the cells of maize root tips and other tissues using a cytochemical staining procedure in which adenylyl-imidodiphosphate (AMP-PNP) is utilized as substrate. Heavy deposits of the reaction product were found in the plasma membrane, endoplasmic reticulum and nuclear membrane; no clear staining was associated with other cellular membranes. These findings are discussed in relation to the reported localization of adenyl cyclase in animal cells and to the possible role of this enzyme in plants.     

Characterization of the hexose transport system in maize root tips

Sugar-depleted excised maize (Zea mays L.) root tips were used to study the kinetics and the specificity of hexose uptake. It was found that difficulties induced by bulk diffusion and penetration barriers did not exist with root tips. Several lines of evidence indicate the existence of a complex set of uptake systems for hexoses showing an overall biphasic dependence on external sugar concentrations. The results suggest that the high and the low affinity components might be located on the same carrier. One uptake system was specific for fructose, but the high affinity component was repressed by high concentrations of external glucose. A second system was specific for glucose and its analogs (2-deoxy-d-glucose and 3-O-methyl-d-glucose), and a third one, more complex, had a high affinity for glucose and its analogs but could transport fructose when glucose was not present in the external solution. A simple method is proposed to determine the inhibitor constants in competition experiments.     

Differential responses of two wetland graminoids to high ammonium at different pH values

Enhanced soil ammonium () concentrations in wetlands often lead to graminoid dominance, but species composition is highly variable. Although is readily taken up as a nutrient, several wetland species are known to be sensitive to high concentrations or even suffer toxicity, particularly at low soil pH. More knowledge about differential graminoid responses to high availability in relation to soil pH can help to better understand vegetation changes. The responses of two wetland graminoids, Juncus acutiflorus and Carex disticha, to high (2 mmol·l^?1) versus control (20 μmol·l^?1) concentrations were tested in a controlled hydroponic set up, at two pH values (4 and 6). A high concentration did not change total biomass for these species at either pH, but increased C allocation to shoots and increased P uptake, leading to K and Ca limitation, depending on pH treatment. More than 50% of N taken up by C. disticha was invested in N‐rich amino acids with decreasing C:N ratio, but only 10% for J. acutiflorus. Although both species appeared to be well adapted to high loadings in the short term, C. disticha showed higher classic detoxifying responses that are early warning indicators for decreased tolerance in the long term. In general, the efficient aboveground biomass allocation, P uptake and N detoxification explain the competitive strength of wetland graminoids at the expense of overall biodiversity at high loading. In addition, differential responses to enhanced affect interspecific competition among graminoids and lead to a shift in vegetation composition.     

Effects of external pH,fusicoccin and butyrate on the cytoplasmic pH in barley root tips measured by 31P-nuclear magnetic resonance spectroscopy

³¹P-Nuclear magnetic resonance spectroscopy was used to measure the cytoplasmic pH (pH_c) in barley (Hordeum vulgare L.) root tips. As the external pH was raised from 4–10, pH_c was found to increase from 7.44 to 7.75. The sensitivity of pH_c to changes in external pH decreased with increasing external pH. Metabolic inhibition by sodium azide caused pH_c to fall by 0.3 units. Addition of 10 mM butyrate resulted in a gradual decline in pH_c, by approx. 0.3 units over 90 min. At a concentration of 1 mM, butyrate had no effect on pH_c even after 2 h. Fusicoccin caused pH_c to rise by 0.1–0.2 units. In maize (Zea mays L.) root tips, pH_c was shown to have a similar sensitivity to fusicoccin. The results are discussed in relation to the regulation of pH_c and the possible role of pH_c in determining transmembrane electrical potential differences.Abbreviations and symbols FC Fusicoccin - NMR nuclear magnetic resonance - p.d. membrane electrical potential difference - pH_c cytoplasmic pH - P1 inorganic phosphate - chemical shift     

Intracellular binding of lanthanum in root tips of barley (Hordeum vulgare)

Summary By means of electron microscopy and X-ray energy spectroscopy (CORA in the transmission mode) it was shown that lanthanum may enter the cytoplasmic phase of undifferentiated cells in barley root tips. The nucleolus in particular proved to be a site with a high affinity for lanthanum. This finding is discussed in relation to the inhibition of root growth which has been reported to occur when root tips are exposed to polyvalent cations.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.