CDPK expression and activity during sprouting and leaf development in potato plants

ULLOA RM; GIAMMARIA V; GARGANTINI P; TAYLOR M

Orlando, USA

Congreso; ASPB 2004; 2004

American Society of Plant Biology

The sessile form of plants emphasizes the requirement of efficient adaptation to environmental conditions. Calcium is a second messenger that plays a main role in plant physiology and Calcium-dependent calmodulin-independent protein kinases (CDPKs) are key intermediates in calcium signaling, that couple changes in Ca2+ levels to a specific response.Solanum tuberosum is triggered in response to changing environmental conditions. Previously, we reported that StCDPK1, an active CDPK isoform (59 kDa), is transiently induced in swelling stolons while StCDPK3 is expressed in early non swelling stolons. StCDPK1 transcript appears before the induction of other tuber specific genes, such as Pin 2 and patatin, suggesting that this isoform could be involved in stolon to tuber transition. Though no significant expression or CDPK activity was detected in the dormant tuber, StCDPK1 expression was strongly enhanced in sprouting tubers. Semiquantitative RT-PCR assays were used to monitor CDPK expression in sprouts, emerging and fully expanded leaves and the PCR fragments were subcloned and sequenced. Protein extracts from the different developmental stages were obtained and CDPK activity was assayed in crude and partially purified extracts (DEAE-cellulose column). CDPK activity from younger not fully expanded leaves was significantly higher than in older ones. Soluble CDPK activities, able to phosphorylate syntide-2, GS and Histone H1, were further purified from potato leaves. CDPK activity eluted at 0.2 M NaCl from a DEAE-cellulose column and the addition of 4M urea was necessary to elute the enzyme from a Phenyl-Sepharose column suggesting that the enzyme is highly hydrophobic. Western analysis of the purified extracts with an antibody against the CLD-domain of the soybean CDPK, detected two polypeptides of 57 and 54 kDa suggesting that different CDPK isoforms or differentially phosphorylated versions of the same kinase are present in potato leaves.