Studies On The Molecular Mechanism Of Cytokinin Action: Involvement Of Ca2+, Protein Kinase And Concurrent Protein Synthesis In Signaling Of Cytokinin-Induced Expression Of Pathogenesis-Related Enzymes In Cucumber

Abstract

Phytohormones act as signals to regulate plant growth and development by modulation of gene expression in response to internal developmental cues or external environmental stimuli, such as light and pathogen infection. There are five major classes of phytohormones, viz. auxins, cytokinins, gibberellins, ethylene and abscisic acid. Of these, cytokinins, 6N substituted adenine derivatives, are of special importance owing to their possible diverse roles in plant growth and development. They induce cell division, cell expansion in cotyledon, chloroplast and etioplast development, suppression of apical dominance and senescence, and differentiation of in vitro cultured cells. However, very little is known about the mechanism of cytokinin action at the molecular level. Cytokinins have been demonstrated to modulate the expression of genes coding for several enzymes including nitrate reductase, ribulose-l,5-bisphosphate carboxylase, RNA polymerase I, and pathogenesis-related (PR) enzymes, i.e. chitinases and β-1,3- glucanases. One of the important questions regarding cytokinin regulation of enzyme activities and/or the accumulation of their corresponding proteins and mRNAs is how the cytokinin signal is transduced.

There is considerable evidence from earlier reports demonstrating that pathogens alter hormone physiology of the host plant and it has been proposed that the infection-associated enzyme changes might be mediated by phytohormones. In the present study, two PR enzymes, viz. cucumber chitinase and β-l,3-glucanase, have been chosen to examine the mode of regulation of their gene expression by cytokinins, including the identification of cytokinin signal transduction components. Plant chitinases and glucanases are important enzymes in plant defense mechanisms against fungal pathogens as they degrade the major fungal cell wall components, chitin and β-1,3-glucan, respectively. Besides their role in plant defense, they are known to be involved in diverse physiological and developmental processes, such as embryogenesis, seed germination and flower development, and are also developmentally and hormonally regulated.

Initially, in order to study the effects of various cytokinins on chitinase and β-1,3-gIucanase enzyme activities and their gene expression, cotyledons excised from seven-day-old dark-grown cucumber seedlings were treated with water, and cytokinins, viz. benzyladenine, kinetin, zeatin and zeatin riboside. It was observed that chitinase and β-l,3-ghucanase enzyme activities and their transcripts were induced to varying extents following treatments of cotyledons with the cytokinins tested. However, a maximum increase in enzyme activities and their transcript levels was noticed in zeatin-treated cotyledons. Therefore, zeatin was used for further studies.

The main objective of the present study was to investigate the cytokinin signal transduction mechanism involving the induction of expression of chitinase and β-1-3-glucanase. In order to obtain insights into the downstream components of the cytokinin-signaling pathway, effects of several agonists and antagonists of the signal transduction components on zeatin-induced chitinase and β-l,3-glucanase activities, and their protein and transcript levels were monitored by enzyme assay, by immunoblot analysis, and by northern analysis, respectively. Treatment of excised dark-grown cucumber cotyledons with staurosporine, a broad spectrum protein kinase inhibitor, reduced the zeatin-induced chitinase and β-l,3-glucanase enzyme activities and the accumulation of their proteins and transcripts. On the other hand, treatment with sodium fluoride, a general inhibitor of protein phosphatases, stimulated the basal chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation, whereas it had no effect on the zeatin-induced enzyme activities and their protein and transcript accumulation. These findings suggested that protein phosphorylation is critical in the cytokinin induction of expression of chitinase and β-l,3-glucanase.

Since Ca2+ is known to be an important second messenger in several plant signal transduction pathways, the possible involvement of Ca2+ in the cytokinin-induced expression of chitinase andβ-l,3-glucanase was examined. The results of the present investigation showed that the chitinase and β-1,3-ghicanase activities and their proteins and transcripts were appreciably increased by exogenous CaCl2 treatment in control cotyledons. Treatment of cotyledons with zeatin plus CaCl2 did not result in a further increase in either these enzyme activities or their protein and transcript accumulation as compared to zeatin or CaCl2 treatment alone. The lack of additivity of zeatin plus CaCl2 treatment indicated a common mechanism of action of zeatin and Ca2+ in the induction of these enzyme activities and their gene expression. To test the occurrence of influx of extracellular Ca2+ by cytokinin, cotyledons were treated with the plasma membrane Ca2+ channel blocker, verapamil, and Ca2+ ionophore A23187. Verapamil treatment inhibited the zeatin-induced chitinase and β-1,3-ghicanase enzyme activities and their protein and transcript accumulation. An increase in the intracellular Ca2+ levels by means of Ca2+ ionophore treatment resulted in a significant increase in basal chitinase and β-l,3-glucanase activities and their protein and transcript accumulation. These results suggested that an influx of extracellular Ca2+ leading to increased levels of cytosolic Ca2+ is required for the cytokinin induction of expression of these enzymes.

The correlation of chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation in the zeatin-treated cotyledons suggested that the cytokinin zeatin stimulates chitinase and β-l,3-glucanase accumulation at the mRNA level and that the increase in enzyme activities is due to an increase in the amount of the enzyme protein and not by the activation of the existing enzyme. Further, the effect of zeatin on both the enzyme activities and their transcript levels under conditions that inhibit protein synthesis was studied. Treatment of excised dark-grown cucumber cotyledons with cycloheximide, an inhibitor of protein synthesis, in the presence of zeatin, completely nullified the stimulatory effect of zeatin. These results indicated the requirement of cytokinin-induced enhanced concurrent protein synthesis in the observed stimulation of chitinase and β-l,3-glucanase enzyme activities as well as their transcript accumulation Ca2+

In an attempt to isolate the full length cucumber β-l,3-glucanase cDNA from a cucumber cDNA library, we isolated and sequenced one cDNA clone, which was 978 bp long and had a potential polyadenylation signal A ATA A starting 172 bases before the polyadenylation tail A deduced amino acid sequence of the cDNA, which was 242 amino acids in length, apparently encoded a partial β-amyrin synthase. Sequence comparison of the deduced partial amino acid sequence of cucumber β-amyrin synthase with other known plant β-amyrin synthase sequences available in databases revealed significant homologies to β-amyrin synthases from Panax, Pisum and Glycyrrhiza. Southern blot analysis indicated that there was only one β-amyrin synthase gene in the cucumber genome. RT-PCR analysis performed on total RNA isolated from zeatin- and salicylic acid-treated cotyledons using forward and reverse primers designed from the internal regions of the cDNA showed that the transcript levels of β-amyrin synthase were enhanced by both zeatin and salicylic acid.

In conclusion, we have demonstrated that chitinase and β-l,3-glucanase accumulation is stimulated by exogenous cytokinin treatment of excised cucumber cotyledons, and this effect is correlated with the content of chitinase and β-1,3-glucanase transcripts as judged by northern analyses. Further, the findings reported in the thesis suggested that Ca2+ influx from extracellular space, protein phosphorylation by staurosporine-sensitive protein kinase(s) and concurrent protein synthesis are required for the signaling of cytokinin-induced expression of both these pathogenesis-related enzymes.