PLANT PHYSIOLOGY , Vol 101, Issue 3 1039-1045, Copyright © 1993 by American Society of Plant Biologists
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CELL BIOLOGY AND SIGNAL TRANSDUCTION |
Detection in Vivo of Very Rapid Red Light-Induced Calcium-Sensitive Protein Phosphorylation in Etiolated Wheat (Triticum aestivum) Leaf Protoplasts
K. M. Fallon, P. S. Shacklock and A. J. Trewavas
Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh, Scotland EH9 3JH
Etiolated wheat (Triticum aestivum cv Mercia) leaf protoplasts respond to
brief red-light irradiation by increasing in volume over a 10-min
incubation period (M.E. Bossen, H.A. Dassen, R.E. Kendrick, W.J. Vredenberg
[1988] Planta 174: 94-100). When the calcium-sensitive dye Fluo-3 was
incorporated into these protoplasts, red-light irradiation initiated
calcium transients lasting about 2 min (P.S. Shacklock, N.D. Read, A.J.
Trewavas [1992] Nature 358: 153-155). Release of calcium in the protoplasts
by photolysis of incorporated 1-{2-amino-5-[1-hydroxy-1-(2-nitro-4,
5-methylenedioxyphenyl)-methyl]-phenoxy}-2-(2[prime]-amino-5[prime]-methylp
henoxy)-ethane-N,N, N[prime],N[prime] -tetraccetic acid, tetrasodium salt
(caged calcium) or caged inositol trisphosphate frequently induced
transient increases in intracellular calcium levels, although the kinetics
of these changes showed variation between experiments. Upon exposure to red
light, a pronounced increase in the phosphorylation of a 70-kD and to a
lesser extent a 60-kD peptide was observed, commencing within 15 s and
continuing for up to 2 min. Simultaneous far-red and red irradiation
attenuated the response. Upon release of incorporated caged calcium by cage
photolysis, the labeling of these two peptides was greatly increased. When
incorporated caged inositol trisphosphate was photolyzed, only the labeling
of the 70-kD peptide was enhanced. Phosphorylation of the 70-kD peptide was
also increased when extracellular calcium was elevated, but it decreased
with increasing extracellular EGTA. These data thus provide direct evidence
for the operation of an in vivo transduction sequence involving red
light-dependent, calcium-sensitive protein phosphorylation.
