Protein Synthesis and Breakdown during Heat Shock of Cultured Pear (Pyrus communis L.) Cells.

Cultured pear (Pyrus communis L. cv Passe Crassane) cells were subjected to temperatures of 39, 42, and 45[deg]C. Heat-shock protein (hsp) synthesis was greater at 30[deg]C than at temperatures above 40[deg]C and continued for up to 8 h. Both cellular uptake of radiolabeled methionine and total protein synthesis were progressively lower as the temperature was increased. Polysome levels decreased immediately when cells were placed at 39 or 42[deg]C, although at 39[deg]C the levels began to recover after 1 h. In cells from both temperatures, reassembly occurred after transfer of cells to 25[deg]C Four heat-shock-related mRNAs[mdash]hsp17, hsp70, and those of two ubiquitin genes[mdash]all showed greatest abundance at 39[deg]C and decreased at higher temperatures. Protein degradation increased with time at 42 and 45[deg]C, but at 39[deg]C it increased for the first 2 h and then decreased. In the presence of cycloheximide, which prevented hsp synthesis, protein degradation at 39[deg]C was as great as that at 45[deg]C in the absence of cycloheximide. The data suggest that hsps may have a role in protecting proteins from degradation at the permissive temperature of 39[deg]C. At temperatures high enough to inhibit hsp synthesis, protein degradation was enhanced. Although ubiquitin may play a role in specific protein degradation, it does not appear to be involved in increased protein degradation occurring above 40[deg]C.

Ethylene production by growing and senescing pear fruit cell suspensions in response to gibberellin.

A pear (Pyrus communis L. cv Passe Crassane) cell suspension was used as a model system to study the influence of gibberellin on processes related to fruit ripening. Growth of the cell cultures was inhibited and their loss of viability was accelerated when 0.5 millimolar gibberllic acid (GA(3)) was added to suspensions at two stages of cell development, namely, growth and quiescence. Cell respiration rate was unaffected up to 2 millimolar GA(3) but ethylene production, both basal and 1-aminocyclopropane-1-carboxylic acid-induced, was inhibited at all stages of cell development. However, the degree of inhibition decreased as the cell cultures aged. The site of ethylene inhibition by GA(3) appeared to be related to the ethylene-forming enzyme. The coincident acceleration of cell senescence and inhibition of ethylene production indicate that the pear cell suspension cannot serve as an analogous model for studying the mode of action of gibberellin in delaying ripening and senescence of fruits in its entirety, although certain specific effects might be relevant.

Conjugation of ubiquitin to proteins from green plant tissues.

Conjugation of the polypeptide ubiquitin to endogenous proteins was studied in oat (Avena sativa L.) plants, and particularly in green tissues. Conjugating activity in leaf extracts was different from that in root extracts, and in both was less than in etiolated tissue. The conjugates were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and their formation was both time- and ATP-dependent and had a pH optimum of about 8.2. The assay had a high affinity for ATP with a probable K(m) of less than 50 micromolar. The ubiquitin conjugating system was also shown to be present in isolated chloroplasts, and ubiquitin could be conjugated to endogenous proteins of lyzed chloroplasts in which the ATP concentrations were reduced by preincubation or desalting. SDS-PAGE analysis led to the suggestion that the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase) may be able to be ubiquitinated, and we have shown that ubiquitin can stimulate the in vitro breakdown of (125)I-labeled RuBPCase. These results invite the speculation that ubiquitin may be involved in the regulation of protein turnover in green plants.

Stimulation of ethylene production in bean leaf discs by the pseudomonad phytotoxin coronatine.

Coronatine is a toxin produced by Pseudomonas syringae pv. glycinea which induces the same chlorotic response in bean leaves as does infection by the bacterial pathogen. Although the structure of coronatine is known, the biological mode of action is not. One possible clue to its activity is the ethyl-substituted cyclopropane side chain of the molecule. This part structure (1-amino-2-ethycyclopropane-1-carboxylic acid or AEC) is an analog of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC).When coronatine was applied to bean leaf discs in solution, or to intact leaves through prick application, a substantial stimulation of ethylene production was measured. This stimulation was concomitant with an increase in ACC content of the tissue, and occurred under the same conditions as did the chlorotic response to the toxin. The stimulation of ethylene production was inhibited by aminoethoxyvinylglycine, an inhibitor of ACC synthesis. These results, along with those of experiments using l-[U-(14)C]methionine, indicated that the stimulation involved de novo production of ethylene via the methionine pathway.The whole, unhydrolyzed coronatine molecule is probably necessary to elicit both the ethylene and chlorosis responses since neither hydrolysis product (coronafacic acid and coronamic acid AEC]) is effective alone. A naturally occurring analog of coronatine, coronafacoylvaline, also stimulated ethylene production and caused chlorosis. However, the unrelated pseudomonad phytotoxin phaseolotoxin, which also causes chlorosis, did not stimulate ethylene production. Ethylene thus may have a specific role in the coronatine toxic syndrome.

Release of Ca2+ from plant hypocotyl microsomes by inositol-1,4,5-trisphosphate.

The effect of inositol-1,4,5-trisphosphate on Ca2+ release from microsomes isolated from dark-grown zucchini (Cucurbita pepo L.) hypocotyls was studied. Up to 30% of the Ca2+ taken up by the microsomes in the presence of 2mM ATP, was released by mumolar concentrations of inositol-1,4, 5-trisphosphate. This release was very rapid (less than 0.5 min) and was followed by a slower re-uptake of Ca2+. The microsomal levels of Ca2+ previously attained were not re-established within 5 min. External concentration of free Ca2+ was maintained in the 10(-8)M region during the release. This is the first time that inositol-1,4,5-trisphosphate has been shown to have a regulatory effect on Ca2+ in plant membrane fractions. Phosphoinositides may be important in signal transduction in plant cells, by altering the cytoplasmic Ca2+ activity, a function already known in animal cells.

Simultaneous uptake and translocation of magnesium and calcium in barley (Hordeum vulgare L.) roots.

The patterns of uptake and translocation of magnesium in different regions of the root are very similar to those of calcium. Once the endodermis has become suberized translocation of either ion to the shoot is greatly reduced and it is concluded that magnesium, like calcium, appears to move across the root cortex largely in the free space.

Effect of high temperature on calcium uptake by suspension-cultured pear fruit cells.

Uptake of Ca(2+) by suspension-cultured pear (Pyrus communis L. cv Passe Crassane) cells and protoplasts was significantly enhanced by exposure to 38 degrees C compared to 25 degrees C. The increased uptake was specific for Ca(2+) and was not due to cell wall binding. Tissues pretreated at 38 degrees C showed increased uptake even upon return to 25 degrees C. Treatment with carbonylcyanide m-chlorophenylhydrazone, salicylhydroxamic acid + KCN, or arsenite also increased Ca(2+) content of cells. Results are discussed with regard to membrane permeability changes, the cellular control of Ca(2+), and heat treatments used to inhibit softening of fruit during postharvest storage.

Effects of low temperature and respiratory inhibitors on calcium flux in plant mitochondria.

The effects of low temperature on uptake and release of (45)Ca(2+) were studied with sound, well-coupled mitochondria extracted at room temperature from avocado (Persea americana Mill, cv Fuerte) fruits. Low Ca(2+) concentrations (10 micromolar) were employed to simulate physiological conditions. At 25 degrees C, the rate of Ca(2+) uptake decreased with time, whereas at 5 degrees C the initial rate, though lower, remained linear. As a consequence total uptake at 5 degrees C was substantially greater than at 25 degrees C for periods greater than 5 min. Preincubation of mitochondria at 5 degrees C enhanced subsequent Ca(2+) uptake at 25 degrees C. Ca(2+) uptake was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and by ruthenium red, but neither KCN nor salicylhydroxamic acid separately or together had any major inhibitory effect. Preloaded mitochondria held for 60 min in a Ca-free medium lost little Ca(2+) at 25 degrees C and none at 5 degrees C, except in the presence of ruthenium red or CCCP.

Estimation of hydrogen peroxide in plant extracts using titanium(IV).

Methods for the estimation of hydrogen peroxide in acetone extracts using titanium(IV) are likely to overestimate hydrogen peroxide when applied to plant leaves. Pigments appear to co-precipitate with the titanium complex and cannot be removed by washing with solvents. Fluoride, which specifically removes the color of the titanium-peroxide complex, removes only some of the color from the reactions with plant extracts. This problem has been avoided by extracting tissues with trichloroacetic acid, and measuring peroxide against catalase-treated blanks by its reaction with the complex of titanium(IV) with 4-(2-pyridylazo) resorcinol. Levels of hydrogen peroxide in leaves of a variety of species were found to range from about 0.1 to 0.6 mumol X g-1.

Inhibition by calcium of senescence of detached cucumber cotyledons: effect on ethylene and hydroperoxide production.

The effect of Ca on senescence was followed in detached cucumber (Cucumis sativus L.) cotyledons floating on various solutions in the dark. Compared with those in water, cotyledons in 10(-4) molar CaCl(2) exhibited reduced chlorophyll loss and H(2)O(2) production, reduced and delayed ethylene production, and did not undergo a burst in CO(2) production. In contrast, Mg had little effect on cotyledon senescence, whereas K stimulated chlorophyll loss but did not increase H(2)O(2) accumulation of ethylene and CO(2) production. This reduction in the rate of senescence by Ca could also be achieved by increasing the endogenous levels of Ca in the cotyledons before excision, although the reduction was less than that with Ca in the external solution. The addition of H(2)O(2) to the solutions on which cotyledons were floated stimulated chlorophyll breakdown, but effects on ethylene and CO(2) were not consistent.

Inositol-containing lipids in suspension-cultured plant cells: an isotopic study.

Polar lipids were extracted from suspension-cultured tomato (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [(32)P]orthosphosphate, [2-(3)H]glycerol, and myo-[2-(3)H]inositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [(32)P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported.

Major changes in chromatin condensation suggest the presence of an apoptotic pathway in plant cells.

A large decrease in fluorescence intensity of propidium iodide (PI)-stained nuclei is observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was confirmed by analyses of (i) DNA accessibility to DNase I, (ii) histone disassociation induced by HCl, (iii) saturation of binding sites by the PI fluorochrome (iv), and (v) visual inspection by fluorescence and confocal microscopy. The extent of changes revealed by these assays was used to map progressive changes in chromatin condensation which allowed us to identify different stages in an apoptosis-like pathway in plants. The initial step of chromatin condensation which occurred prior to endonucleolytic DNA degradation was detected by fluorescence and confocal microscopy and confirmed by a variety of assays employing flow cytometry. The initial chromatin condensation appears to be a reversible step in the early stage of apoptosis. The loss of reversibility of chromatin condensation observed subsequently may be a critical point in the cascade of apoptotic events, leading to further irreversible changes during apoptosis in plants.