Photoreaction Dynamics of Full-Length Phototropin from Chlamydomonas reinhardtii.

Phototropin (phot) is a blue light sensor involved in the light responses of several species from green algae to higher plants. Phot consists of two photoreceptive domains (LOV1 and LOV2) and a Ser/Thr kinase domain. These domains are connected by a hinge and a linker domain. So far, studies on the photochemical reaction dynamics of phot have been limited to short fragments, and the reactions of intact phot have not been well elucidated. Here, the photoreactions of full-length phot and of several mutants from Chlamydomonas reinhardtii (Cr) were investigated by the transient grating and circular dichroism (CD) methods. Full-length Cr phot is in monomeric form in both dark and light states and shows conformational changes upon photoexcitation. When LOV1 is excited, the hinge helix unfolds with a time constant of 77 ms. Upon excitation of LOV2, the linker helix unfolds initially followed by a tertiary structural change of the kinase domain with a time constant of 91 ms. The quantum yield of conformational change after adduct formation of LOV2 is much smaller than that of LOV1, indicating that reactive and nonreactive forms exist. The conformational changes associated with the excitations of LOV1 and LOV2 occur independently and additively, even when they are excited simultaneously. Hence, the role of LOV1 is not to enhance the kinase activity in addition to LOV2 function; we suggest LOV1 has different functions such as regulation of intermolecular interactions.

Photoreaction Dynamics of LOV1 and LOV2 of Phototropin from Chlamydomonas reinhardtii.

Phototropin is a blue light sensor protein found in higher plants and green algae. Photochemical reactions of a variety of differently truncated constructs of a phototropin from Chlamydomonas reinhardtii (Cr) (LOV1, LOV1-hinge, LOV2, LOV2-linker, and hinge-LOV2) are investigated. In the dark state, LOV1 is in dynamic equilibrium between the monomer and dimer, and the main photochemical reaction is dimerization of the monomer and dissociation of the dimer. On the other hand, LOV1-hinge exists as the monomer and the photochemical reaction is the dimerization reaction associated with the unfolding of the helix of the hinge domain. LOV2 in the dark state is monomeric. The conformation changes after the photoexcitation of LOV2 and LOV2-linker are minor, which differs notably from the reaction of LOV2-Jα and LOV2-linker from Arabidopsis thaliana (At). The linker region, including the Jα helix, is rather stable upon photoexcitation. The helix of the hinge domain of hinge-LOV2 is slightly unfolded in the dark state, and the major photoreaction is the dimerization event. The dark recovery rate of LOV2 was found to decrease significantly in the presence of the hinge domain. These photochemical properties of Cr phot are considerably different from those of At phot regarding conformational changes and their kinetics, although Cr phot has been reported to rescue the phot function in At. The differences and the diversity of phots are discussed.

Digital gene expression profiling by 5'-end sequencing of cDNAs during reprogramming in the moss Physcomitrella patens.

Stem cells self-renew and repeatedly produce differentiated cells during development and growth. The differentiated cells can be converted into stem cells in some metazoans and land plants with appropriate treatments. After leaves of the moss Physcomitrella patens are excised, leaf cells reenter the cell cycle and commence tip growth, which is characteristic of stem cells called chloronema apical cells. To understand the underlying molecular mechanisms, a digital gene expression profiling method using mRNA 5'-end tags (5'-DGE) was established. The 5'-DGE method produced reproducible data with a dynamic range of four orders that correlated well with qRT-PCR measurements. After the excision of leaves, the expression levels of 11% of the transcripts changed significantly within 6 h. Genes involved in stress responses and proteolysis were induced and those involved in metabolism, including photosynthesis, were reduced. The later processes of reprogramming involved photosynthesis recovery and higher macromolecule biosynthesis, including of RNA and proteins. Auxin and cytokinin signaling pathways, which are activated during stem cell formation via callus in flowering plants, are also activated during reprogramming in P. patens, although no exogenous phytohormone is applied in the moss system, suggesting that an intrinsic phytohormone regulatory system may be used in the moss.