Efficient binding of NC2.TATA-binding protein to DNA in the absence of TATA.

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2.TBP with DNA. We show that NC2.TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP.TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2.TBP displays a K(D) for DNA of approximately 2 x 10(-9) m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP.DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.

A heat-activated MAP kinase in tomato: a possible regulator of the heat stress response.

Adaptation to elevated temperatures is of major importance for the survival of plants. The role of kinases in heat stress response was studied in tomato by in gel and in solution kinase assays using myelin basic protein as substrate. The application of heat stress in a naturally occurring temperature range resulted in a fast and transient activation of a 50 kDa mitogen-activated protein (MAP) kinase both in a photoautotrophic cell suspension culture and in leaves of mature plants. The heat activation of the MAP kinase was shown to be calcium-dependent. The specific phosphorylation of tomato heat stress transcription factor HsfA3 by a partially purified preparation of the heat-activated MAP kinase supports a physiological role of the identified kinase activity in transducing the heat stress signal.

Metabolizable and non-metabolizable sugars activate different signal transduction pathways in tomato.

To gain insight into the regulatory mechanisms of sugar signaling in plants, the effect of derivatives of the transport sugar sucrose (Suc), the Suc isomers palatinose and turanose, and the Suc analog fluoro-Suc were tested. Photo-autotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used to study their effect on the regulation of marker genes of source and sink metabolism, photosynthesis, and the activation of mitogen-activated protein kinases (MAPKs). Suc and glucose (Glc) resulted in reverse regulation of source and sink metabolism. Whereas the mRNA level of extracellular invertase (Lin6) was induced, the transcript level of small subunit of ribulose bisphosphate carboxylase (RbcS) was repressed. In contrast, turanose, palatinose, and fluoro-Suc only rapidly induced Lin6 mRNA level, whereas the transcript level of RbcS was not affected. The differential effect of the metabolizable and non-metabolizable sugars on RbcS mRNA regulation was reflected by the fact that only Suc and Glc inhibited photosynthesis and chlorophyll fluorescence. The activation of different signal transduction pathways by sugars was further supported by the analysis of the activation of MAPKs. MAPK activity was found to be strongly activated by turanose, palatinose, and fluoro-Suc, but not by Suc and Glc. To analyze the role of sugars in relation to pathogen perception, an elicitor preparation of Fusarium oxysporum lycopersici was used. The strong activation of MAPKs and the fast and transient induction of Lin6 expresssion by the fungal elicitor resembles the effect of turanose, palatinose, and fluoro-Suc and indicates that non-metabolizable sugars are sensed as stress-related stimuli.

Biochemical evidence for the activation of distinct subsets of mitogen-activated protein kinases by voltage and defense-related stimuli.

Activation of mitogen-activated protein (MAP) kinases is a common reaction of plant cells in defense-related signal transduction pathways. To gain insight into the mechanisms that determine specificity in response to a particular stimulus, a biochemical approach has been employed. Photoautotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used as experimental system to characterize MAP kinase activation by different stress-related stimuli. An elicitor preparation of the tomato-specific pathogen Fusarium oxysporum lycopersici was shown to result in the simultaneous induction of four kinase activities that could be separated by ion-exchange chromatography. The simultaneous activation of multiple MAP kinases was further substantiated by distinct pharmacological and immunological properties: a differential sensitivity toward various protein kinase inhibitors and a differential cross-reaction with isoform-specific MAP kinase antibodies. In contrast to the two fungal elicitors chitosan and the F. oxysporum lycopersici preparation, the plant-derived stimuli polygalacturonic acid and salicylic acid were shown to activate distinctly different subsets of MAP kinases. Application of a voltage pulse was introduced as a transient stress-related stimulus that does not persist in the culture. Voltage application activates a distinct set of MAP kinases, resembling those activated by salicylic acid treatment, and generates a refractory state for the salicylic acid response. The inhibitory effect of nifedipine indicates that current application may directly affect voltage-gated calcium channels, thus, providing a tool to study various calcium-dependent pathways.