Shining light on the function of NPH3/RPT2-like proteins in phototropin signalling

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Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1

Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen- or voltage-sensing (LOV) domains within their N-terminus, with each binding one molecule of flavin mononucleotide (FMN) as a UV/blue light absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A′α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2-core and its flanking helical segments can uncouple this mode of light regulation. Yet, the impact of these mutations on phot function in Arabidopsis has not been explored. Here, we report that histidine substitution of Arg-472 located within the A′α-helix of Arabidopsis phot1 to histidine results in constitutively activates kinas activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo, but was unable to initiate phot1 signaling in the absence of light. Instead, we found that the phot1 R472H mutant is poorly functional under low-light conditions, but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant by in part reducing its stability, whereas the activity the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation

Interaction specificity of Arabidopsis 14-3-3 proteins with phototropin receptor kinases

Phototropin receptor kinases play an important roles in optimising plant growth in response to blue light. Much is known regarding their photochemical reactivity, yet little progress has been made to identify downstream signalling components. Here, we isolated several interacting proteins for Arabidopsis phototropin 1 (phot1) by yeast two-hybrid screening. These include members of the NPH3/RPT2 (NRL) protein family, proteins associated with vesicle trafficking, and the 14-3-3 lambda (?) isoform from Arabidopsis . 14-3-3? and phot1 were found to colocalise and interact in vivo. Moreover, 14-3-3 binding to phot1 was limited to non-epsilon 14-3-3 isoforms and was dependent on key sites of receptor autophosphorylation. No 14-3-3 binding was detected for Arabidopsis phot2, suggesting that 14-3-3 proteins represent specific mode of phot1 signalling

UV/blue light signal transduction regulating gene expression in Arabidopsis

Plants have evolved a number of mechanisms to protect themselves against environmental stresses. For example, in response to potentially harmful levels of UV light plants can accumulate flavonoids which, among other functions, act as UV-protective pigments. Chalcone synthase (CHS) is the key enzyme which commits the phenylpropanoid pathway to flavonoid biosynthesis. As the expression of CHS and other flavonoid biosynthesis genes is controlled by UV/blue light in higher plants, these genes provide ideal subjects for investigation of the cellular and molecular mechanisms involved in coupling UV/blue photoreception to transcription. To investigate the signal transduction processes concerned with the induction of CHS expression by UV/blue light, the effects of specific agonists and inhibitors were examined in a photomixotrophic Arabidopsis cell suspension culture. This cell culture behaves similarly to mature Arabidopsis leaves in the light regulation of CHS. That is CHS transcript levels are induced by UV-B and UV-A/blue light but not phytochrome. Moreover, experiments with a hy4 mutant cell suspension culture demonstrate that the effects of UV-B and UV-A/blue light on CHS expression are mediated by separate detection systems, the latter involving the CRY 1 photoreceptor. Pharmacological studies with Arabidopsis cell culture indicate that the UV-B and UV-A/blue phototransduction processes require cellular calcium. The inhibitors used suggest that an intracellular pool of calcium may be involved. However, the artificial elevation of cytosolic calcium using an ionophore is insufficient on its own to stimulate CHS expression. Similarly, in preliminary experiments with transgenic Arabidopsis containing cytosolic aequorin, no significant rapid increase in calcium is observed in response to either UV-B or UV-A/blue light. Possible reasons for this discrepancy are discussed. The UV-B induction of CHS expression appears to involve calmodulin because it is strongly inhibited by the antagonist W-7. In contrast, W-7 has little, if any, effect on the UV-A/blue response, indicating that the UV-B and UV-A/blue light signal transduction pathways regulating CHS are, at least in part, distinct. Further evidence shows that both pathways involve protein phosphorylation and require cytoplasmic protein synthesis. The involvement of chromophore excitation, ion fluxes and heterotrimeric G proteins in UV/blue light signal transduction have also been investigated. From the above pharmacological experiments, it is evident that the UV-B and UV-A/blue phototransduction pathways are distinct from the phytochrome signal transduction pathway regulating CHS expression in other species. Support for this conclusion comes from the observation that activators/inhibitors of phytochrome signalling have no effect on CHS expression in the Arabidopsis cell culture. (Abstract shortened by ProQuest.)

Deetiolation enhances phototropism by modulating NON-PHOTOTROPIC HYPOCOTYL3 phosphorylation status

Phototropin (phot) receptor kinases play important roles in promoting plant growth by controlling light-capturing processes, such as phototropism. Phototropism is mediated through the action of NON-PHOTOTROPIC HYPOCOTYL3 (NPH3), which is dephosphorylated following phot activation. However, the functional significance of this early signaling event remains unclear. Here, we show that the onset of phototropism in dark-grown (etiolated) seedlings of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) is enhanced by greening (deetiolation). Red and blue light were equally effective in promoting phototropism in Arabidopsis, consistent with our observations that deetiolation by phytochrome or cryptochrome was sufficient to enhance phototropism. Increased responsiveness did not result from an enhanced sensitivity to the phytohormone auxin, nor does it involve the phot-interacting protein, ROOT PHOTOTROPISM2. Instead, deetiolated seedlings showed attenuated levels of NPH3 dephosphorylation and diminished relocalization of NPH3 from the plasma membrane during phototropism. Likewise, etiolated seedlings that lack the PHYTOCHROME-INTERACTING FACTORS (PIFs) PIF1, PIF3, PIF4, and PIF5 displayed reduced NPH3 dephosphorylation and enhanced phototropism, consistent with their constitutive photomorphogenic phenotype in darkness. Phototropic enhancement could also be achieved in etiolated seedlings by lowering the light intensity to diminish NPH3 dephosphorylation. Thus, phototropism is enhanced following deetiolation through the modulation of a phosphorylation rheostat, which in turn sustains the activity of NPH3. We propose that this dynamic mode of regulation enables young seedlings to maximize their establishment under changing light conditions, depending on their photoautotrophic capacity

An infectious recombinant foot-and-mouth disease virus expressing a fluorescent marker protein

Foot-and-mouth disease virus (FMDV) is one of the most extensively studied animal pathogens because it remains a major threat to livestock economies worldwide. However, the dynamics of FMDV infection are still poorly understood. The application of reverse genetics provides the opportunity to generate molecular tools to further dissect the FMDV life cycle. Here, we have used reverse genetics to determine the capsid packaging limitations for a selected insertion site in the FMDV genome. We show that exogenous RNA up to a defined length can be stably introduced into the FMDV genome, whereas larger insertions are excised by recombination events. This led us to construct a recombinant FMDV expressing the fluorescent marker protein, termed iLOV. Characterization of infectious iLOV-FMDV showed the virus has a plaque morphology and rate of growth similar to the parental virus. In addition, we show that cells infected with iLOV-FMDV are easily differentiated by flow cytometry using the inherent fluorescence of iLOV and that cells infected with iLOV-FMDV can be monitored in real-time with fluorescence microscopy. iLOV-FMDV therefore offers a unique tool to characterize FMDV infection in vitro, and its applications for in vivo studies are discussed

Optogenetics in plants

The last two decades have witnessed the emergence of optogenetics; a field that has given researchers the ability to use light to control biological processes at high spatio‐temporal and quantitative resolution, in a reversible manner with minimal side effects. Optogenetics has revolutionised the neurosciences, increased our understanding of cellular signalling and metabolic networks and resulted in variety of applications in biotechnology and biomedicine. However, implementing optogenetics in plants has been less straight forward given their dependency on light for their life cycle. Here, we highlight some of the widely used technologies in microorganisms and animal systems derived from plant photoreceptor proteins and discuss strategies recently implemented to overcome the challenges for using optogenetics in plants