Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling.

SummaryCalcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calciumsignals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and toconvert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits asingle calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequencyreflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. Thestretch-andthetemperature-activatedprocessesdiffer bytheirsensitivitytopharmacologicaldrugsknowntoaffect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gaugephysicalparametersof their environment.This might haveaprofound influence on the functioningof calcium-dependent processes known to control a large array of molecular events in the nucleus.Keywords: calcium signalling, aequorin, plant cell nucleus, physical stimuli sensing, BY2 cells.IntroductionCell signalling is envisaged as the intricate pathways con-necting the perception of signals by cell surface or solublereceptors and the adaptive response. In plants, the calciumion is recognized as a ubiquitous intracellular second mes-senger (Sanders et al., 2002) acting either as a switch in thesignalling process (Scrase-Field and Knight, 2003) or as asignature encoding specific information (Allen et al., 2000;McAinsh and Hetherington, 1998). To date, the messengeractionsofcalciuminplantshavebeenstudiedindepthinthecytosolwhere calciumcontrols suchwide arrayof biologicalprocesses such as movements of stomata, egg fertilization,responses to biotic and abiotic signals (White and Broadley,2003). In fact, free calcium concentrations vary in differentplant cell organelles like chloroplasts in response to day/light transition (Johnson et al., 1995) or in mitochondria(Logan and Knight, 2003) or the nucleus in response to coldand to osmotic stress (van der Luit et al., 1999; Pauly et al.,2001). Different calcium-dependent enzymes as well as cal-cium-binding proteins are present in plant cell nuclei.These include calmodulin, calmodulin-binding proteins,calcium-dependent protein kinases (Li et al., 1991) andcalmodulin-dependentproteinphosphatases(AndreevaandKutuzov, 2001; Kutuzov et al., 2001) and recent data haveillustrated the association of calmodulin with the transcrip-tion machinery in plants (Bouche et al., 2002; Yang andPoovaiah,2002).Thesefindingsclearlyshowthatchangesinnuclear-free calcium (D [Ca