Dual regulation of a heat shock promoter during embryogenesis: stage-dependent role of heat shock elements.

Transgenic tobacco expression was analysed of chimeric genes with point mutations in the heat shock element (HSE) arrays of a small heat shock protein (sHSP) gene from sunflower: Ha hsp17.7 G4. The promoter was developmentally regulated during zygotic embryogenesis and responded to heat stress in vegetative tissues. Mutations in the HSE affected nucleotides crucial for human heat shock transcription factor 1 (HSF1) binding. They abolished the heat shock response of Ha hsp17.7 G4 and produced expression changes that demonstrated dual regulation of this promoter during embryogenesis. Thus, whereas activation of the chimeric genes during early maturation stages did not require intact HSE, expression at later desiccation stages was reduced by mutations in both the proximal (-57 to -89) and distal (-99 to -121) HSE. In contrast, two point mutations in the proximal HSE that did not severely affect gene expression during zygotic embryogenesis, eliminated the heat shock response of the same chimeric gene in vegetative organs. Therefore, by site-directed mutagenesis, it was possible to separate the heat shock response of Ha hsp 17.7 G4 from its developmental regulation. The results indicate the co-existence, in a single promoter, of HSF-dependent and -independent regulation mechanisms that would control sHSP gene expression at different stages during plant embryogenesis.PPD was supported by a PhD fellowship from the Spanish "Ministerio de Educación y Ciencia". This research was supported by grants BIO96-0474 from Spanish CICYT, and CVI148 from Juanta de Andalucía, awarded to CA and JJ.Peer reviewe

Ribonucleic acid synthesis by chromatin isolated from Phaseolus aureus Roxb. : The effect of endogenous ribonuclease

Chromatin was isolated from the hypocotyls of Phaseolus aureus Roxb. and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity in vitro. The molecular size of the RNA product, measured by polyacrylamide gel electrophoresis, was found to be much smaller than that known to be synthesised in vivo and was affected by the assay temperature. Although conventional enzyme assays provided no evidence for the presence of ribonuclease in chromatin, a more sensitive technique revealed sufficient ribonuclease activity to degrade high-molecular weight RNA to smaller fragments. The inclusion of unlabelled exogenous RNA in the media for chromatin preparation and RNA polymerase assay substantially increased the molecular-weight of the RNA products synthesised in vitro.Peer reviewe

Padrão eletroforético de proteínas resistentes ao calor em sementes de milho Electrophorectic pattern of the heat resistant proteins of corn seeds

Na aquisição e manutenção da tolerância à dessecação de sementes, há vários mecanismos envolvidos, entre eles a indução das proteínas resistentes ao calor. O objetivo deste trabalho foi avaliar mudanças no padrão eletroforético das proteínas resistentes ao calor de sementes de milho submetidas a alta temperatura de secagem, associando-as à sua tolerância. Foram utilizadas sementes de linhagens, híbridos simples e híbridos recíprocos colhidas com teor de água de aproximadamente 35% e secadas a 45&deg;C. Sementes das linhagens secadas à sombra foram utilizadas como controle e sua qualidade fisiológica foi avaliada por meio do teste de germinação. As proteínas resistentes ao calor foram extraídas de eixos embrionários das sementes em tampão Tris HCl 0,05 M. Não foi possível determinar uma banda específica da fração das proteínas resistentes ao calor que possa servir como marcador da tolerância à alta temperatura de secagem. Houve estabilidade nos padrões de bandas das proteínas provenientes de sementes submetidas à secagem artificial e natural, mesmo quando foram observadas variações nos valores de germinação. Os padrões eletroforéticos das proteínas resistentes ao calor foram semelhantes entre as sementes híbridas e os respectivos recíprocos.<br>Several mechanisms are involved in the acquisition and maintenance of desiccation tolerance by the seeds. One of these mechanisms is related to the induction of heat resitant proteins. The objective of this work was to evaluate changes in the electophorectic patterns of the heat resistant proteins of corn seeds submitted to high drying temperature associating their expression with drying tolerance. Seeds of breeding lines, simple hybrids and respective reciprocals hybrids were utilized. The seeds were harvested with approximately 35% of water content and dried at 45&deg;C. Seeds of lines dried under shadow were used as control and the physiological quality was evaluated by germination test. The heat resistant proteins were extracted from embryonic axis of seeds in Tris-HCl 0.05 M buffer. It was not possible to determine a specific band of heat resistant proteins which can be of use as a molecular marker of tolerance to high drying temperature. There was stability in the patterns of the bands of proteins of seeds submitted to artificial drying compared to the ones after shadow drying even for the lines of high variation in the germination values. The electrophorectic patterns of the heat resistant proteins were similar for both the hybrid seeds and respective reciprocals