Rapid Changes in Protein Phosphorylation Associated with Light-Induced Gravity Perception In Corn Roots

Abstract

The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,NN',N' tetrmacetic acid and A23187 prevented light-induced changes in protein phosphorylation.We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.Significant advances have been made in our understanding of gravity signal perception and transduction. Recent (3-5, 13, 14, 16). In this paper, we report lightinduced rapid changes in protein phosphorylation, and suggest that these changes participate in the development of the gravitropic response.In animal systems, it is well known that protein phosphorylation plays a key role in signal transduction (2). Signal reception can trigger changes in protein kinases such as protein kinase C (15,17,19 reported in plants (7,18,23,24), and attempts have been made to isolate plant protein kinases and determine their substrates (6,7,19,20). Previous work from this laboratory has provided evidence for calcium-and calmodulin-dependent protein phosphorylation in corn roots (18). The data presented here extends that work by showing rapid and specific calcium-dependent changes in protein phosphorylation induced by light treatment of dark-grown roots of Merit corn.MATERIALS AND METHODS Plant Material. Corn seeds (Zea mays L., cv Merit) were imbibed for 12 h, then planted on plastic trays in paper towels under dim green light. Seedlings were grown for 2 d in darkness, then 1 cm apical segments (100 per treatment) were harvested under dim green light, using a Safelight Kodak filter (Wratten Series 3) and a 7 W bulb.In Vivo Protein Phosphorylation. One hundred segments per treatment were preloaded with 32P in the dark by incubating in 1 ml of buffer (25 mm MES, pH 6.2, 5 mm MgSO4, 250 mm sucrose) with 0.5 mCi of carrier free [32P]H3PO4 for 1 h. Then excess label was removed with two washes of 2 ml each ofbuffer. Preloading was done to prevent the possibility that treatments could affect uptake of label and alter the cytoplasmic phosphate pool. After removal of label, 1 ml of buffer was added, and root segments were treated in one ofthree ways: (a) incubation for 15 min in darkness, then frozen in liquid N2; (b) incubation for 8 min in darkness, then 7 min in light, and frozen in liquid N2; (c) incubation for 8 min in darkness in the presence of 1 jM A23 187 and 5 mM EGTA,2 then 7 min in light, and frozen in liquid N2.During preliminary experiments, light-induced changes in phosphorylation were measured after 1, 7, and 15 min. Significant increases in phosphorylation were observed after 7 min. Hence, further experiments were carried out after 7 min of light exposure. Experiments were repeated four times.Separation of Phosphoproteins. After freezing, roots were homogenized, and labeled proteins were separated by two-dimensional gel electrophoresis as described previou...

Characterization and Distribution of a Maize cDNA Encoding a Peptide Similar to the Catalytic Region of Second Messenger Dependent Protein Kinases

“…In roots, light-, calcium-, and calmodulin-dependent phosphorylation of specific peptides have been reported, supporting the view that in plants, as in other eukaryotes, protein kinases transduce environmental stimuli (10,13). Because these data suggest a role for calcium as a second messenger in plants (13) it is probable that specific calcium-dependent protein kinases ubiquitous in other eukaryotes (e.g.…”

“…For both of these stimuli the root cap is believed to be the site ofperception, and also is hypothesized involved with the transduction of the stimulus leading to a particular developmental response. Recent studies have revealed that a few specific proteins are rapidly phosphorylated in response to light and calcium application, and that calmodulin is probably involved in this response (10,14). Here we present further evidence for the existence of a second messenger-dependent protein kinase in maize roots, and also describe the primary structure and the spatial distribution of gene expression within a tissue known to transduce a variety of environmental stimuli.…”