The Apparent Turnover of 1-Aminocyclopropane-1-Carboxylate Synthase in Tomato Cells Is Regulated by Protein Phosphorylation and Dephosphorylation.

In suspension-cultured cells of tomato (Lycopersicon esculentum Mill.), the activity of 1-aminocyclopropane-1-carboxylate synthase (ACC-S) rapidly increases in response to fungal elicitors. The effect of inhibitors of protein kinases and protein phosphatases on the regulation of ACC-S was studied. K-252a, an inhibitor of protein kinases, prevented induction of the enzyme by elicitors and promoted its apparent turnover in elicitor-stimulated cells, causing a 50% loss of activity within 4 to 8 min in both the presence and absence of cycloheximide. Calyculin A, an inhibitor of protein phosphatases, caused a rapid increase of ACC-S in the absence of elicitors and an immediate acceleration of the rate of ACC-S increase in elicitor-stimulated cells. In the presence of cycloheximide there was no such increase, indicating that the effect depended on protein synthesis. Cordycepin, an inhibitor of mRNA synthesis, did not prevent the elicitor-induced increase in ACC-S activity but strongly reduced the K-252a-induced decay and the calyculin A-induced increase of its activity. In vitro, ACC-S activity was not affected by K-252a and calyculin A or by treatments with protein phosphatases. These results suggest that protein phosphorylation/dephosphorylation is involved in the regulation of ACC-S, not by regulating the catalytic activity itself but by controlling the rate of turnover of the enzyme.

Rapid changes of protein phosphorylation are involved in transduction of the elicitor signal in plant cells.

Plant cells have an acute sense for pathogen-derived chemical stimuli, so-called elicitors, which induce the plant's defense response. To investigate the molecular basis of chemosensory transduction, elicitor-treated tomato cells were labeled with 1-min pulses of [32P] phosphate. This technique revealed drastic changes in protein phosphorylation in vivo within minutes of stimulation. The protein kinase inhibitors K-252a and staurosporine completely prevented these elicitor-induced changes in protein phosphorylation. They also blocked two early biochemical responses to elicitors, extracellular alkalinization and biosynthesis of ethylene. The ability of K-252a, staurosporine, and benzoylated staurosporine derivatives to inhibit elicitor responses in vivo correlated with their ability to inhibit tomato microsomal protein kinase in vitro. When K-252a was given to elicited cells 1 min after the[32] phosphate, the radioactivity in certain newly labeled phosphoprotein bands disappeared again within minutes. This correlated with an arrest of alkalinization within minutes when K-252a was applied in midcourse of elicitation. These data show that phosphorylation of protein substrates by K-252a-sensitive protein kinases is essential for transduction of elicitor signals in plant cells and that continuous phosphorylation of these proteins is required to maintain the elicited state.

Elicitor-induced ethylene biosynthesis in tomato cells: characterization and use as a bioassay for elicitor action.

The induction of ethylene biosynthesis by an elicitor partially purified from yeast extract was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells. Unstimulated cells produced little ethylene during exponential growth and even less in stationary phase. Treatment with elicitor stimulated ethylene biosynthesis 10-fold to 20-fold in the exponentially growing cells and more than 100-fold in stationary cells. Activities of both 1-aminocyclopropane-1-carboxylate (ACC) synthase, measured in vitro, and ethylene-forming enzyme (EFE), measured in vivo, increased strongly in response to elicitor treatments. During exponential growth, cells contained large pools of ACC, and the elicitor stimulated ethylene biosynthesis primarily through induction of EFE. In the stationary phase, cells contained almost no ACC, and the elicitor stimulated ethylene biosynthesis primarily through its effect on ACC synthase activity. Cordycepin did not affect the increase in activity of ACC synthase but blocked that of EFE, indicating that the former was posttranscriptionally regulated, the latter transcriptionally regulated. Removal of elicitor by washing or inactivation of a biotinylated derivative of the elicitor by complexation with avidin caused a rapid cessation of the increase in ACC synthase activity, suggesting that continuous presence of stimulus is necessary for the response. Using induction of ethylene production to measure amounts of elicitor, it was found that the elicitor disappeared from the incubation medium during the course of the treatment.

K-252a inhibits the response of tomato cells to fungal elicitors in vivo and their microsomal protein kinase in vitro.

Two characteristic responses of plant cells to fungal elicitors, induction of phenylalanine ammonia-lyase activity and of ethylene biosynthesis, were studied in suspension-cultured tomato cells. Induction of both responses was completely blocked by 500 nM K-252a, a known inhibitor of mammalian protein kinases. About 100 nM K-252a caused half-maximal inhibition. In vitro, K-252a inhibited protein kinase activity in microsomal preparations from tomato cells. Inhibition was competitive with respect to ATP and had a Ki of about 15 nM. Thus, protein kinases sensitive to K-252a occur in plants and might be important for the plant's response to fungal elicitors.

Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings.

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a (2)H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.

Amyloplast development in etiolated and ethylene-treated pea epicotyls.

The amyloplasts found in the apical hook cells of etiolated pea (Pisum sativum L.) epicotyls were randomly distributed. Sedimentation of endodermal amyloplasts in the direction of gravity became apparent in the transition from the hook to the top of the main axis of the epicotyl. Cortical amyloplasts in this region were not, however, sedimented. These patterns of sedimentation could not be related to changes in amyloplast size, and it is proposed that cytoplasmic properties determine amyloplast behaviour.The differentiation of plastids in the hook differed between the amyloplast-containing endodermal cells and the cortical cells, in which amoeboid plastids predominated over amyloplasts. Amyloplasts disappeared from the cortical cells in the main axis of the epicotyl, but in the endodermal cells sedimented amyloplasts were found throughout the upper epicotyl.Etiolated epicotyls induced to grow horizontally by treatment with ethylene had a normal content of amyloplasts, sedimented in the direction of gravity.