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Summary of recently completed project Title: Effect of temperature, salt and stress on biochemical parameters in cells from Chenopodium and crop seedlings

Key words: Actin rings, Chenopodium rubrum, Extracellular pH, Extracellular phosphatase

Collaboration: Chulalongkorn University, Bangkok, Thailand

Funding: Royal Thai Government Doctoral Scholarship Programme (Anchalee Chaidee)

Period: November 2003 – October 2007

Aims. Heat shock and heat stress are principal problems for plant growth especially in southern countries like Thailand. Heat shock transcription factors and heat shock proteins reflecting the significance of heat shock have been described already in detail. However, the role of principal cellular components like membrane transport, metabolism and the cytoskeleton is still largely unknown. Therefore, the aim of the present study was to explore a model system of higher plant cells e.g. Chenopodium rubrum for specific cellular responses and physiological markers for heat shock.

Results and Discussion. Early responses of Chenopodium rubrum cells to a brief heat shock were investigated by biochemical and fluorescence microscopic methods. It was clearly demonstrated that heat shock induces extracellular proton flux with extreme Q10. Thus, extracellular pH was shown to constitute a convenient parameter for cellular stress induced by heat shock. In addition to pH change, release of phosphatase is induced by heat shock at physiological temperature. Vitality tests together with the release of protein, osmotic compounds, and betalain provided supplementary information about membrane integrity. The phosphatase released in response to heat shock was characterized by enzymatic and protein assays. Investigations of kinetics of activity, pH dependence, inhibitor effects, and substrate specificity revealed an alkaline phosphatase activated by heat shock. Native-PAGE and SDS-PAGE with regeneration treatment and in-gel phosphatase staining demonstrated a particular heat shock-induced protein and its apparent molecular mass. Concanavalin A blotting and endoglycosidase H treatment allowed the characterization of a glycosylated 100-kD phosphatase with a small N-linked oligosaccharide residue. The heat shock-induced actin reorganization associated with changes of extracellular pH was investigated by confocal laser scanning microscopy. In addition to heat shock, inhibitors affecting ion gradients across cellular membranes and inhibitors of actin and myosin were used to characterize the heat shock-induced cellular changes. We found the formation of actin rings specifically associated with resistance to perturbations of cellular homeostasis, suggesting a signaling function. In addition, temperature and pharmacology data revealed that actin rings are not a prerequisite for extracellular alkalinization. Interestingly, it was found that extracellular free calcium of the culture medium reduces the number of heat shock-induced actin rings by 50%. This indicates a calcium-dependent protection against shock. Correlations between extracellular pH and phosphatase, and between extracellular pH and actin organization after heat shock and chemical treatments were detected. Evidence for a close correlation between extracellular pH and phosphatase was demonstrated by treatments with stimuli affecting pH, e.g. benzoic acid, fusicoccin, hydrochloric acid, and nigericin, in both heterotroph and autotroph cells. Especially, the concerted induction of extracellular alkalinization and formation of actin rings by heat shock at 37°C was investigated. The combined changes of these two master parameters may cause the specific cellular sensitivity for temperatures around 37°C. Moreover, a cell-specific response to heat shock of the actin organization was found in heterotroph cells and not detectable in autotroph cells.

Presentations and publications of the project. Chaidee A, Pfeiffer W (2004) Abstract: Effect of heat shock on different cellular parameters in Chenopodium rubrum. In: Book of abstracts Botanikertagung Braunschweig 2004, p. 17 - Chaidee A, Pfeiffer W (2005) Abstract: Early responses of Chenopodium rubrum cells to heat shock. In: Book of abstracts XVII International Botanical Congress Vienna, p. 250 - Chaidee A (2006a) Talk: Early heat shock effects in plant cells. University Salzburg Colloquium of Plant Physiology (16.01.2006) - Chaidee A, Pfeiffer W (2006b) Abstract: Characterization of a tyrosine phosphatase released by heat shock. In: Book of abstracts XV FESPB Congress Lyon 2006, p. 155 - Chaidee A, Pfeiffer W (2006) Parameters for cellular viability and membrane function in Chenopodium cells show a specific response of extracellular pH to heat shock with extreme Q10. Plant Biology 7: 42-51 – Chaidee A (2007) Heat shock and stress signaling by extracellular pH and phosphatase, and the actin organization in heterotroph cells of Chenopodium rubrum L. Doctoral Thesis University Salzburg, Austria, Europe - Chaidee A, Foissner I, Pfeiffer W (2008a) Cell-specific association of heat shock-induced proton flux with actin ring formation in Chenopodium cells: comparison of auto- and heterotroph cultures. Protoplasma 234: 33-50 - Chaidee A, Wongchai C, Pfeiffer W (2008b) Extracellular alkaline phosphatase is a sensitive marker for cellular stimulation and exocytosis in heterotroph cell cultures of Chenopodium rubrum. Journal of Plant Physiology 165: 1655-1666

Project list:

Detection of repellent activity in Chitosan and Plant-Animal-Interactions – Chatchawal Wongchai (Post Doctoral project 1. April 2014 – 30. June 2014, OEAD-Thailand, Technologiestipendium Südostasien TSA Post Doc, Grant No. OEAD ICM-2013-05667)

Effect of temperature, salt and stress on biochemical parameters in cells from Chenopodium and crop seedlings - Anchalee Chaidee (Doctoral project 2003-2007 Royal Thai Government)

Salt stress effects on proton transporters – Zhujun Zhu (Post Doctoral project 2000-2001, OEAD-PR China)

Cell death and vitality in Chenopodium cells – Eugenia Maximova (Post Doctoral project 2000, Academy of Sciences of Moldova)

Function and regulation of the vacuolar PPase of Chenopodium rubrum – Wolfgang Kranewitter (Doctoral project 1994-1998 FWF project P09788-MOB, Vienna, Austria)

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