Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

• MeSH
• cystein chemie   MeSH
• flavinmononukleotid chemie   MeSH
• fotochemické procesy MeSH
• fototropiny chemie   MeSH
• glutamin chemie   MeSH
• isomerie MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• normální rozdělení MeSH
• oves chemie   MeSH
• sekvence aminokyselin MeSH
• spektrofotometrie infračervená MeSH
• Sphingomonadaceae chemie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• flavinmononukleotid MeSH
• fototropiny MeSH
• glutamin MeSH

BACKGROUND: De-etiolation is the switch from skoto- to photomorphogenesis, enabling the heterotrophic etiolated seedling to develop into an autotrophic plant. Upon exposure to blue light (BL), reduction of hypocotyl growth rate occurs in two phases: a rapid inhibition mediated by phototropin 1 (PHOT1) within the first 30-40 min of illumination, followed by the cryptochrome 1 (CRY1)-controlled establishment of the steady-state growth rate. Although some information is available for CRY1-mediated de-etiolation, less attention has been given to the PHOT1 phase of de-etiolation. RESULTS: We generated a subtracted cDNA library using the suppression subtractive hybridization method to investigate the molecular mechanisms of BL-induced de-etiolation in tomato (Solanum lycopersicum L.), an economically important crop. We focused our interest on the first 30 min following the exposure to BL when PHOT1 is required to induce the process. Our library generated 152 expressed sequence tags that were found to be rapidly accumulated upon exposure to BL and consequently potentially regulated by PHOT1. Annotation revealed that biological functions such as modification of chromatin structure, cell wall modification, and transcription/translation comprise an important part of events contributing to the establishment of photomorphogenesis in young tomato seedlings. Our conclusions based on bioinformatics data were supported by qRT-PCR analyses the specific investigation of V-H(+)-ATPase during de-etiolation in tomato. CONCLUSIONS: Our study provides the first report dealing with understanding the PHOT1-mediated phase of de-etiolation. Using subtractive cDNA library, we were able to identify important regulatory mechanisms. The profound induction of transcription/translation, as well as modification of chromatin structure, is relevant in regard to the fact that the entry into photomorphogenesis is based on a deep reprograming of the cell. Also, we postulated that BL restrains the cell expansion by the rapid modification of the cell wall.

• Klíčová slova
• Blue light, De-etiolation, Suppression subtractive hybridization, Tomato (Solanum lycopersicum L.),
• MeSH
• chromatin ultrastruktura   MeSH
• etiolizace genetika   MeSH
• fototropiny fyziologie   MeSH
• genová knihovna MeSH
• genové regulační sítě MeSH
• hypokotyl růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• semenáček genetika   růst a vývoj   MeSH
• Solanum lycopersicum genetika   růst a vývoj   MeSH
• světlo * MeSH
• upregulace MeSH
• vakuolární protonové ATPasy genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• fototropiny MeSH
• vakuolární protonové ATPasy MeSH