Phosphoinositides in Barley Aleurone Layers and Gibberellic Acid-Induced Changes in Metabolism

Abstract

The plant hormone gibberellin is involved in the regulation ofa number ofdiverse processes, including germination, shoot elongation, and fruit formation. Gibberellin-induced a-amylase synthesis and secretion during germination has been extensively studied and the involvement of Ca2" in this process is well established (4,12,14). However, the molecular mechanism of hormone perception, message transduction, and biochemical regulation is unknown.Membrane-bound phosphoinositides mediate signal transduction across cell membranes in a wide variety of animal cells (3,6). In these systems, ligand-receptor interaction on the extracellular face of the membrane leads to activation of a membrane-bound phospholipase-C which cleaves PtdInsP22 to diacylglycerol and Ins(1,4,5)P3. Ins(1,4,5)P3 releases calcium from endoplasmic reticulum, thereby increasing intracellular free calcium levels. At these elevated levels, calcium modifies enzyme activity by binding directly or as a Ca2+_ calmodulin complex. In addition, the released diglyceride activates protein kinase-C. 'Supported by a grant from the National Science Foundation (DCB-8609038).2Abbreviations: PtdInsP2, phosphatidylinositol bisphosphate; Ins( 1, 4, 5)P3, myo-inositol-1,4,5-trisphosphate; PtdInsP, phosphatidylinositol phosphate; PtdCh, phosphatidylcholine; LPtdCH, lysophosphatidylcholine; LPtdIns, lysophosphatidylinositol; PtdIns, phosphatidylinositol; GroPIns, glycerophosphoinositol; GroPInsP, glycerophosphoinositol phosphate; GroPInsP2, glycerophosphoinositol bisphosphate. MATERIALS AND METHODS Labeling with Radioactive PrecursorsBarley seeds (Hordeum vulgare L. cv Himalaya, 1979) were deembryonated, surface-sterilized (for 30 min in 1% [w/v] NaOCl, made by 5-fold dilution of commercial bleach), washed with sterile water, and allowed to imbibe on wet sterile sand for 4 d at room temperature. To label the myo-inositolderived compounds with tritium, aleurone layers were removed from imbibed seeds as described by Chrispeels and Varner (7)

Phosphoinositides in Barley (Hordeum vulgare L.) Aleurone Tissue

“…The inclusion of authentic standards of 32P-labeled GroPIns3P, GroPIns4P, and GroPIns(4,5)P2 obtained from S. polyrhiza allowed us to confirm an earlier report (Murthy et al, 1989) of the presence of PtdIns4P in barley aleurone cells. We have also revealed the likely presence of PtdIns3P, but at the level of labeling achieved we were unable to confirm the presence of PtdIns(4,5)P2.…”

“…We are in agreement with Murthy et al (1989) that the the level of labeling in PtdIns4P is only a small fraction of that achieved in PtdIns. We were unable at the level of labeling achieved to conclusively identify any PtdInsPz species in aleurone tissue.…”

“…We were unable at the level of labeling achieved to conclusively identify any PtdInsPz species in aleurone tissue. Our failure to detect, and the failure of Murthy et al (1989) to label, PtdInsP, appreciably, would seem to preclude the study of PtdInsPz in barley aleurone tissue. We have by the use of HPLC and the inclusion of authentic standards revealed the likely presence of PtdIns3P.…”

“…That the tissue is nonmeristematic allows us to investigate whether 3-phosphorylated PtdIns are restricted to meristematic tissues. Thus, given an early report (Murthy et al, 1989) of GA3-stimulated turnover of PtdIns in barley aleurone and the reasons stated above, we consider that the biochemistry of phosphoinositide metabolism in this tissue deserves further study. In doing so we have also addressed a report (Murthy et al, 1992) describing evidence for the presence of two isomers of PtdIns in barley aleurone layers.…”

Rice gibberellin‐binding phosphoprotein structurally related to ribulose‐1,5‐bisphosphate carboxylase/oxygenase activase

“…Murthy and Render [4] showed that phosphoinositides were involved in GAa-induced synthesis and secretion of a-amylase in the aleurone cells of barley seeds. Ladedgenskaya et al [5] showed that GA3 increased activity of Ca 2+-and phospholipid-dependent protein kinase-like enzyme in potato tubers.…”