Responses to environmental stimuli are mediated by the activation and inactivation of various signalling proteins. However, the temporal dynamics of these events in living animals are not well understood. Here we show real-time imaging of the activity of the key regulator of the MAP kinase pathway, Ras, in living Caenorhabditis elegans and that Ras is transiently activated within a few seconds in olfactory neurons in response to increase in the concentration of odorants. This fast activation of Ras is dependent on the olfactory signalling pathway and Ras guanyl nucleotide-releasing protein (RasGRP). A negative feedback loop then quickly leads to Ras inactivation despite the continued presence of the odorant. Phenotypes of Ras mutants suggest this rapid activation and inactivation of Ras is important for regulation of interneuron activities and olfactory behaviours. Our results reveal novel kinetics and biological implication of transient activation of Ras in olfactory systems.

VASCULAR-RELATED NAC-DOMAIN7 (VND7) is the master transcription factor for vessel element differentiation in Arabidopsis thaliana. To identify the cis-acting sequence(s) bound by VND7, we employed fluorescence correlation spectroscopy (FCS) to find VND7-DNA interactions quantitatively. This identified an 18-bp sequence from the promoter of XYLEM CYSTEINE PEPTIDASE1 (XCP1), a direct target of VND7. A quantitative assay for binding affinity between VND7 and the 18-bp sequence revealed the core nucleotides contributing to specific binding between VND7 and the 18-bp sequence. Moreover, by combining the systematic evolution of ligands by exponential enrichment (SELEX) technique with known consensus sequences, we defined a motif termed the Ideal Core Structure for binding by VND7 (ICSV). We also used FCS to search for VND7 binding sequences in the promoter regions of other direct targets. Taking these data together, we proposed that VND7 preferentially binds to the ICSV sequence. Additionally, we found that substitutions among the core nucleotides affected transcriptional regulation by VND7 in vivo, indicating that the core nucleotides contribute to vessel-element-specific gene expression. Furthermore, our results demonstrate that FCS is a powerful tool for unveiling the DNA-binding properties of transcription factors.

High expression of a transgene is often necessary to produce useful substances in plants. The efficiency of mRNA translation is an important determinant of the level of transgene expression. In dicotyledonous plants, the 5′UTR of certain mRNAs act as translational enhancers, dramatically improving transgene expression levels. On the other hand, translation enhancers derived from dicotyledonous plants are not so much effective in monocotyledonous plants, which are important as industrial crops and as hosts for production of useful substances. In this study, we evaluated the polysome association on a large scale with high resolution for each 5′UTR variant from multiple transcription start site in normal and heat-stressed Oryza sativa suspension cultures. Translational enhancer candidates were selected from the resultant large-scale data set, and their enhancer activities were evaluated by transient expression assay. In this manner, we obtained several translational enhancers with significantly higher activities than previously reported enhancers. Their activities were confirmed in a different monocotyledonous plant, Secale cereale, and using a different reporter gene. In addition, enhancer activities of tested 5′UTRs were different between monocotyledonous and dicotyledonous plants, suggesting that the enhancer activities were not compatible between them. Overall, we demonstrate these useful 5′UTRs as enhancer sequence for transgene expression in monocotyledonous plants.

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Signal transduction pathways play essential roles to adapt environmental changes and composed of various signaling proteins. Therefore, observation of activity of a specific protein in vivo is important for understanding the signal transduction pathways. However temporal dynamics of signaling proteins in living animals are not well known. Here we tried to monitor the activities of Ras protein which is one of the key regulators for the Ras-MAPK signal transduction pathway. It was previously reported that the Ras-MAPK pathway functions in olfactory neurons in Caenorhabditis elegans. Thus, we observed Ras activity in olfactory neuron in living animals using Raichu-Ras, a FRET-based sensor for Ras activity. We succeeded in detecting quick changes of Ras activity in response to odor stimuli.