Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium

Abstract

Methods for measuring plant cytoplasmic calcium using microelectrodes or microinjected fluorescent dyes are associated with extensive technical problems, so measurements have been limited to single or small groups of cells in tissue strips or protoplasts. Aequorin is a calcium-sensitive luminescent protein from the coelenterate Aequorea victoria (A. forskalea) which is formed from apoaequorin, a polypeptide of relative molecular mass approximately 22,000, and coelenterazine, a hydrophobic luminophore. Microinjected aequorin has been widely used for intracellular calcium measurement in animal cells, but its use in plants has been limited to exceptionally large cells. We show here that aequorin can be reconstituted in transformed plants and that it reports calcium changes induced by touch, cold-shock and fungal elicitors. Reconstituted aequorin is cytoplasmic and nonperturbing; measurements can be made on whole plants and a calcium indicator can be constituted in every viable cell. Now that apoaequorin can be targeted to specific organelles, cells and tissues, with the range of coelenterazines with differing calcium sensitivities and properties available, this new method could be valuable for determining the role of calcium in intracellular signalling processes in plants.

Measurement of Ca2+ Fluxes during Elicitation of the Oxidative Burst in Aequorin-transformed Tobacco Cells

“…First, earlier studies using seedlings from the transgenic tobacco plants established that cold shock is accompanied by a flux of cytoplasmic Ca 2ϩ that has a characteristic signature, i.e. a sharp rise that returns to basal levels within a minute (38). As can be seen in Fig.…”

“…Aequorin-transformed Tobacco Suspension Cultures-Generation of aequorin-transformed tobacco (Nicotiana plumbaginofolia) plants has been previously described (38,43). Transgenic seeds from an F3 generation were a generous gift from Dr. Anthony J. Trewavas (University of Edinburgh).…”

“…Ca 2ϩ fluxes in both the cell suspension cultures and whole seedlings were similarly inhibited by EGTA and La 3ϩ , and in both systems the magnitudes of the cold shocks and Ca 2ϩ transients were roughly proportional. Although physical barriers/inequities prevent similar comparisons of Ca 2ϩ transients following mechanical perturbation, elicitor stimulation, or osmotic stress, the observation that these signals also trigger Ca 2ϩ fluxes in both single cell and whole plant systems (38,39,50,51) adds further weight to the contention that cultured cells can serve as useful models when properly validated. With this motivation in mind, it should be possible to explore in greater detail the role of Ca 2ϩ pulses in signaling a variety of defenserelated responses.…”

“…Indeed, most membrane-permeant esters of Ca 2ϩ -dependent fluorophores hydrolyze to their impermeant forms in the cell wall, preventing their penetration to their intracellular destinations. Fortunately in 1991, Knight et al (38) transformed tobacco plants with a Ca 2ϩ -sensitive luminescent protein, termed aequorin, and demonstrated that resting levels of Ca 2ϩ were insufficient to induce photon emission but that stimulus-induced Ca 2ϩ increases generated readily measurable levels of bioluminescence. Since this initial discovery, aequorin-transformed plants have been exploited to quantitate intracellular Ca 2ϩ fluxes accompanying such diverse processes/ stimuli as direct mechanical perturbation (34,38), gusts of wind (39), cold shock (38,40), and circadian oscillations (30).…”

“…Fortunately in 1991, Knight et al (38) transformed tobacco plants with a Ca 2ϩ -sensitive luminescent protein, termed aequorin, and demonstrated that resting levels of Ca 2ϩ were insufficient to induce photon emission but that stimulus-induced Ca 2ϩ increases generated readily measurable levels of bioluminescence. Since this initial discovery, aequorin-transformed plants have been exploited to quantitate intracellular Ca 2ϩ fluxes accompanying such diverse processes/ stimuli as direct mechanical perturbation (34,38), gusts of wind (39), cold shock (38,40), and circadian oscillations (30). Because the aequorin protein is expressed in the plant cell cytoplasm and because the intensity of light emitted by aequorin is directly proportional to Ca 2ϩ concentration, measurement of aequorin-derived bioluminescence enables direct quantitation of Ca 2ϩ transients in the transformed cytoplasm of the cell.…”