Protein phosphatases play significant roles in signal transduction pathways pertaining to cell proliferation, gene expression, and neurotransmission. Serine/threonine phosphatases PP1 and PP2A, which are closely related in primary structure (approximately 50%), are inhibited by a structurally diverse group of natural toxins. As part of our study toward understanding the mechanism of inhibition displayed by these toxins, we have developed research in two directions: (1) The standardization of an assay to be used in acquisition of the structure--activity relationship of inhibition data is reported. This nonradioactive assay affords detection levels of molecular phosphate released from a phosphorylated hexapeptide in subnanomolar quantities. The comparison of our IC50 values of these inhibitors against corresponding literature data provided validation for our method. (2) Computational analysis provided a global model for binding of these inhibitors to PP1. The natural toxins were shown to possess remarkably similar three-dimensional motifs upon superimposition and van der Waals minimization within the PP1 active site.

Wheat seedling grown with their shoot bottom exposed to red light (400 micromol m(-2) s(-1)) either with constant illumination or light-dark cycles did not accumulate chlorophyll. This near-etiolation response was manifested by a critical threshold intensity of red light and did not need continuous illumination. The inhibition of the greening process resulted from reduced synthesis of glutamate-1-semialdehyde and consequent reduction in tetrapyrrole precursor 5-aminolevulinic acid. Red light perceived by the shoot bottom down regulated the protein and/or gene expression of enzymes involved in the biosynthesis of tetrapyrroles. The contents of endogenous cytokinins, i.e., isopentenyl-adenosine and dihydrozeatinriboside, were reduced in seedlings grown in red light having their shoot bottom exposed. Application of exogenous cytokinin and its analogue to roots of seedlings grown in red light reversed the down regulation of the greening process. The reversal of red-light-induced near-etiolation morphogenesis by far-red (200 micromol m(-2) s(-1)) or blue (25 mumol m(-2) s(-1)) light suggests that it could be a very high red-irradiance response of phytochrome, in the meristematic layers of the shoot bottom, that works in concert with blue light receptor(s).

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Perception of red light (400 μmol photon m²/s) by the shoot bottom turned off the greening process in wheat. To understand the signaling cascade leading to this photomorphogenic response, certain signaling components were probed in seedlings grown in different light regimes. Upon analysis the gene expression of heterotrimeric Gα and Gβ were severely down-regulated in seedlings grown without vermiculite and having their shoot bottom exposed to red light (R/V-) and was similar to that of dark-grown seedlings. Supplementing the red-light-grown V- seedlings with blue light resulted in up-regulation of both Gα and Gβ expression, suggesting that blue light is able to modulate G protein expression. Treatment of cytokinin analog benzyladenine to cytokinin-deficient red-light-grown R/V- seedlings resulted in up-regulation of gene expression of both Gα and Gβ. To probe further, modulators of signal transduction pathway--AlF₃ (G protein activator), LaCl₃ (Ca(2+) channel blocker), NaF (nonspecific phosphatase inhibitor), or calmodulin (CaM) antagonists trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-nafthalene-sulfonamide (W-7)--were added along with Hoagland solution to the roots of 4-day-old etiolated seedlings, grown on germination paper and transferred to red light. AlF₃, LaCl₃, NaF failed to elicit any photomorphogenic response. However, CaM antagonists TFP and W-7 significantly reversed the red-light-induced suppression of photomorphogenesis. Phosphorylation of proteins assayed in the absence or presence of CaM antagonist TFP revealed respective up-regulation or down-regulation of phosphorylation of several plastidic proteins in R/V- seedlings. These suggest that signal transduction of red light perceived by the shoot bottom to suppress photomorphogenesis is mediated by CaM-dependent protein kinases.

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