BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234, 690 – 694 (1997) ARTICLE NO. RC976514 Ca 2/ Oscillations in Plant Cells: Initiation by Rapid Elevation in Cytosolic Free Ca 2/ Levels Grant M. Calder,* V. E. Franklin-Tong,† Peter J. Shaw,* and Bjørn K. Drøbak* *Department of Cell Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom; and †Wolfson Laboratory for Plant Molecular Biology, School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom Received March 5, 1997 understood about how specificity may be encoded into Temporal increases in intracellular [Ca 2/ ] are now such signals. In the present study we have investigated recognized to be key triggers for a wide range of im- the occurrence of pulsatile Ca 2/ signals in pollen tubes portant physiological events in eukaryotic cells. In from poppy (Papaver rhoeas) using time-resolved Laser mammalian cells, signal-induced Ca 2/ -elevations have Scanning Confocal Microscopy. We demonstrate the been found to be of a pulsatile nature and Ca 2/ spikes presence of a spatially distinct [Ca 2/ ] gradient in the display a high degree of spatiotemporal complexity. In apical region of growing pollen tubes and the occur- plant cells a similar picture is beginning to emerge. To rence of highly regular oscillations of this gradient. In investigate the occurrence of pulsatile Ca 2/ signals in living pollen tubes where tip growth had ceased, we plant cells we studied alterations of [Ca 2/ ] in the tip neither detected any Ca 2/ gradients nor found evidence region of pollen tubes from poppy (Papaver rhoeas). for oscillatory behaviour. However, a brief elevation of Time-Resolved Laser Scanning Confocal Microscopy cytosolic Ca 2/ , induced by photolysis of caged-Ca 2/ of pollen tubes microinjected with the Dextran-linked (Nitr-5), was found not only to re-establish the direc- Ca 2/ -indicator dyes Calcium Green or Indo-1 revealed tional Ca 2/ gradient but also to trigger the onset of that highly regular Ca 2/ oscillations occur in these regular oscillations. Changes in tip morphology and the cells. We further demonstrate that artificial elevation of cytosolic Ca 2/ by photolysis of caged-Ca 2/ (Nitr-5) resumption of directional growth was observed after can trigger the onset of oscillations.  1997 Academic Press the onset of oscillations. Although the question remains open as to whether growth is a direct consequence of the Ca 2/ oscillations or vice versa, our data confirm the presence of distinct Ca 2/ gradients and Ca 2/ oscilla- tions in growing plant cells and point to an important The role of Ca 2/ as an ubiquitous second messenger role for rapid increases in intracellular [Ca 2/ ] as trig- in eukaryotic cells is now firmly established but it is gers for the onset of oscillatory behaviour. only recently that a more detailed understanding of how Ca 2/ signals are created and propagated has begun to emerge (see e.g. 1,2). It has become clear that tonic MATERIALS AND METHODS increases in cytoplasmic Ca 2/ levels are rare occur- rences in mammalian cells and that in most cases the Plant material and germination. Pollen from Papaver rhoeas were collected and stored at 020°C, over silica gel, until required. signal-induced cellular Ca 2/ elevations are pulsatile in Pollen grains were germinated on 2% agarose in a germination me- nature and display highly complex spatio-temporal dium (0.01 % KNO 3 , 0.01 % H 3 BO 3 , 0.01% Mg(NO 3 ) 2 .6H 2 O, 0.036% patterns. Several studies have shown that Ca 2/ oscilla- CaCl 2 .2H 2 O, 7.5 % sucrose, 5 % PEG-6000; all % are given as w/v). tions also occur in plant cells (3,4), and recently we After germination at 25°C for 30-40 minutes the cells were coated with Voltalef PCTFE Oil, type 10S (Altochem, U.K.) prior to microin- demonstrated that intracellular Ca 2/ -waves can be ini- jection. tiated in pollen tubes by photolysis of caged- Ins(1,4,5)P 3 (5). There is little doubt that oscillations Micro-injection. Pollen tubes which had grown to a length of ú 200 mm were pressure micro-injected using a needle concentration and wave phenomena are very closely related since a of 2.5 mM Calcium Green-1-Dextran 10,000 MW or 5 mM Indo-1- Ca 2/ -wave can be seen merely as a spatial correlate Dextran 10,000 MW (Molecular Probes Inc., U.S.A.). Dye was deliv- of an oscillation. Little is currently known about the ered into the pollen tube by briefly increasing the pressure using an molecular mechanism(s) responsible for the initiation oil-filled hydraulic pressure injector. Micropipettes were pulled from non-filamented capillary tubes (Clark Electromedical Instruments) of pulsatile Ca 2/ signals in plant cells and even less is 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 690