There have been numerous recent reports documenting phosphorylation of DNA-binding proteins [Montminy and Bilezikjian (1987); Sorger, Lewis, and Pelham (1987); Hoeffler, Kovelman, and Roeder (1988); Jones et al. (1988); Prywes et al. (1988); Sorger and Pelham (1988); Yamamoto et al. (1988)], and the transcriptional regulatory activity of at least one of these proteins appears to be modulated by this modification [Montminy and Bilezikjian (1987); Yamamoto et al. (1988)]. We report here on a plant nuclear protein, the DNA-binding activity of which is strongly affected by phosphorylation. This protein, AT-1, binds to specific AT-rich elements (the AT-1 box) within promoters of certain nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the polypeptide components of the light-harvesting chlorophyll a/b protein complex. A consensus sequence of AATATTTTTATT was derived for the AT-1 box. We demonstrate that the DNA-binding ability of AT-1, from nuclear extracts of pea, can be reversibly modulated by phosphorylation. AT-1 is active in the nonphosphorylated form and loses all DNA-binding ability as a result of phosphorylation. The kinase that phosphorylates AT-1 uses both Mg-ATP and Mg-GTP as a substrate and is inhibited by heparin and spermine, indicative of an NII-type casein kinase.

No abstract

A protein identifiable as calmodulin has been isolated from oat (Avena sativa, var Garry) tissues. This protein is relatively heat stable, binds to hydrophobic gels, and phenothiazines in a calcium-dependent fashion, and binds to antibody to rat testes calmodulin. Based on its migration on sodium dodecyl sulfate-polyacrylamide gels, ultraviolet absorption spectrum, and amino acid composition, oat calmodulin is essentially identical to calmodulin isolated from other higher plants. Radioimmunoassays indicate that calmodulin is associated with isolated oat protoplasts, mitochondria, etioplasts, and nuclei and also appears to be a component of oat cell wall fractions.Much evidence has accumulated recently to support the hypothesis that Ca 2 plays a major role in mediating the adaptations of plants to certain environmental changes (12,22). As in animals, at least some Ca2-imediated responses in plants are controlled by Ca2-binding regulatory proteins. Among these, the most studied and best characterized is calmodulin (1).We have published reports suggesting a possible role for calmodulin in mediating phytochrome and gravitropic responses in Avena saliva (oats) (2, 21). As part of our ongoing research on this question, we have isolated and characterized calmodulin from oats and have estimated its content, both in intact tissue and in isolated subcellular fractions, by radioimmunoassay. Here we report the results of these experiments.MATERIALS AND METHODS Plant Material. Except where indicated, the starting material for all extractions was taken from the coleoptiles and primary leaves of 3-to 4-d-old dark-grown oat (A vena sativa, var. Garry) seedlings, harvested 5 to 7.5 mm above the seed. The oats were grown on water-saturated vermiculite at 27C.Calmodulin Isolation. Two extraction methods were employed.'Supported by grants from the National Aeronautics and Space Administration (NSG 7480), The National Science Foundation (PCM 81-03429), and The Robert A. Welch Foundation (F 858) to S. J. R.

There have been numerous recent reports documenting phosphorylation of DNA-binding proteins [Montminy and Bilezikjian (1987); Sorger, Lewis, and Pelham (1987); Hoeffler, Kovelman, and Roeder (1988); Jones et al. (1988); Prywes et al. (1988); Sorger and Pelham (1988); Yamamoto et al. (1988)], and the transcriptional regulatory activity of at least one of these proteins appears to be modulated by this modification [Montminy and Bilezikjian (1987); Yamamoto et al. (1988)]. We report here on a plant nuclear protein, the DNA-binding activity of which is strongly affected by phosphorylation. This protein, AT-1, binds to specific AT-rich elements (the AT-1 box) within promoters of certain nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the polypeptide components of the light-harvesting chlorophyll a/b protein complex. A consensus sequence of AATATTTTTATT was derived for the AT-1 box. We demonstrate that the DNA-binding ability of AT-1, from nuclear extracts of pea, can be reversibly modulated by phosphorylation. AT-1 is active in the nonphosphorylated form and loses all DNA-binding ability as a result of phosphorylation. The kinase that phosphorylates AT-1 uses both Mg-ATP and Mg-GTP as a substrate and is inhibited by heparin and spermine, indicative of an NII-type casein kinase.

Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a lambda Zap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907-8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be up-regulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.