Nuclear Ca(2+)-fluxes and phosphoinositides in plants.

Abstract

Complex signalling events are involved in the conversion of primary extracellular signals into intracellular responses. Two signal-induced second messengers have been at the focus of attention for more than a decade; these are CaZ+ and inositol( 1,4,5)trisphosphate [ 11. Many important functions for these messengers have been identified in mammalian cells, and the possibility that Ca*+ and Ins(1,4,5)P3 also have specific roles to play in nuclear signalling events has become apparent. The nucleus in mammalian cells has, unexpectedly, been found to possess a phosphoinositide system distinct from the more familiar plasma membrane phosphoinositide system [see ref 2 for review]. Although there is still some debate about the exact location of the nuclear phosphoinositide system it appears that several of the important functional components are localized inside the nucleus rather than in the nuclear membrane($ [2]. It has further been shown that the nuclear phosphoinositide system, in several cell types, is likely to be under the control of receptors localized in the plasma membrane [3]. The nucleus in mammalian cells has been found to contain an active endoplasmic reticulum (ER)-type Caz+ pump [4] and Ins(1,4,5)P3 has been shown to be able to release Caz+ from isolated nuclear preparations [5]. Many similarities exist between signalling systems in mammalian cells and plants and both Caz+fluxes and a plant phosphoinositide system have been shown to be involved in cell signalling events [6]. To investigate the possibility that Caz+-fluxes and phosphoinositide turnover may occur in plant nuclei we have developed a novel detergent-free method for the isolation of intact nuclei from carrot (Doueus carota) protoplasts. The method relies on gentle sonication to break open the plasma membrane and the released nuclei are further purified by gradient centrifugation. The apparent purity of nuclei is high, and using a number of different criteria we have found that the nuclear membranes remain largely intact. Figure 1 shows a carrot cell nucleus stained with the membrane-specific dye DiOC,.