Phototropin is a blue light photoreceptor for tropic responses, relocation of chloroplasts, and stomata opening in plants. Phototropin has two chromophoric domains named light-oxygenvoltage-sensing (LOV) 1 and 2 in the N-terminal half, and a serine͞threonine (Ser͞Thr) protein kinase motif in the C-terminal half. Concerning the kinase activity of phototropin, only autophosphorylation has been detected so far. However, we found that phototropin can phosphorylate a protein other than phototropin itself. Bacterially expressed Arabidopsis phototropin 2 kinase domain (KD) with GST-tag showed a constitutive kinase activity on casein, a common in vitro substrate of Ser͞Thr protein kinase. By using this in vitro assay system, the roles of each LOV domain were studied. Addition of LOV2 to KD (GST-L2-KD) inhibits the kinase activity that is canceled by light. This light activation of kinase disappeared on introduction of a mutation blocking photochemical reaction in the LOV2 domain. Accordingly, LOV2 domain acts as a major light-regulated molecular switch of casein phosphorylation. Interestingly, isolated LOV2 from the KD still binds to the KD in a light-dependent manner and functions in similar ways, indicating the role of LOV2 domain as an inhibitor of the kinase activity in the substrate phosphorylation. LOV1, in contrast, contributes little to the photoactivation in GST-L1-L2-KD; however, it acts as an attenuator of the light activation of the kinase by LOV2.ight in the wavelength region from UV to far-red is an important stimulus for plants that precisely regulates developmental and cell motility processes. To sense the light conditions including intensity, quality, and direction, plants have acquired three major photoreceptors (1): phytochrome, cryptochrome, and phototropin.Phototropin (2, 3), originally identified as a photoreceptor for tropic responses (4), has also been found to mediate chloroplast relocations (5-7), stomatal opening (8), leaf expansion (9), and rapid inhibition of hypocotyl elongation (10). Most plants have two isoforms of phototropin, named phot1 and phot2 (2). In Arabidopsis thaliana (At), phot1 and phot2 share tropic responses and also one of the chloroplast relocation responses, photoaccumulation, depending on fluence rate of light (6). Photoavoidance response, on the other hand, is mediated by only phot2 (5). In contrast, stomatal opening is regulated redundantly by both phot1 and phot2 (8).The phototropins are composed of 900-1,000 amino acid residues and two prosthetic flavin mononucleotide (FMN) molecules (2, 3) (Fig. 1A Top). The N-terminal half has a pair of FMN-binding domains with Ϸ100 residues designated lightoxygen-voltage-sensing (LOV) 1 and 2 (11), and the C-terminal half forms a serine͞threonine (Ser͞Thr) kinase domain connected to the LOV2 domain with a linker region (Fig. 1 A).Upon absorption of blue light, FMN in the two LOV domains undergoes a unique cyclic photochemical reaction through formation and breakage of a covalent bond (12) with a highly conserved cysteine found in ...

Conformational dynamics of LOV2 domain of phototropin, a plant blue light photoreceptor, is studied by the pulsed laser induced transient grating (TG) technique. The TG signal of LOV2 without the linker part to the kinase domain exhibits the thermal grating signal due to the heat releasing from the excited state and a weak population grating by the adduct formation. The diffusion coefficients of the adduct product after forming the chemical bond between the chromophore and Cys residue are found to be slightly smaller than that of the reactant, which implies that the core shrinks slightly on the adduct formation. After that change, no significant conformational change was observed. On the other hand, the signal of LOV2 with the linker part to the kinase domain clearly shows very different diffusion coefficients between the original and the adduct species. The large difference indicates significant global conformational change of the protein moiety upon the adduct formation. More interestingly, the diffusion coefficient is found to be time-dependent in the observation time range. The dynamics representing the global conformational change is a clear indication of a spectral silent intermediate between the excited triplet state and the signaling product. From the temporal profile analysis of the signal, the rate of the conformational change is determined to be 2 ms.

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Photochemical reaction of a plant blue-light photoreceptor, Arabidopsis phototropin 1-LOV (light-oxygen-voltage sensing) domain 2, was studied with a view to the diffusion coefficients (D) using the pulsed-laser-induced transient grating method. Although the reaction dynamics completes at a rate of several microseconds as long as it is monitored by the absorption change, the diffusion coefficient was found to be time-dependent in a time range of submilliseconds to seconds. The observed signal can be analyzed by the two-state model, which includes the D-value decrease from D of the reactant (9.8 +/- 0.4) x 10(-11) m2/s to D of the product (8.0 +/- 0.4) x 10(-11) m2/s. The D-value of the reactant implies that the dominant form in the ground state of phototropin 1 LOV2 is the monomeric form in a concentration range of 50-200 microM. According to the Stokes-Einstein relationship, the D-change can be explained by a volume increase of 1.8 times. Furthermore, the rate of the D-change was roughly proportional to the concentration of the sample. These two observations indicate that the LOV2 domain transiently forms a dimer upon photoexcitation. When the sample concentration is increased (>180 microM), a new signal component appears within a few milliseconds. This signal represents a D increase from 8.0 x 10(-11) m2/s to 9.8 x 10(-11) m2/s with a time constant of 300 micros. The completely opposite D-change from that observed in a lower concentration, as well as the concentration dependence, implies that a dimer is formed in the ground state in a higher concentration range, even though the fraction of the dimer is still minor in this range. This dimer is photodissociated, with a time constant of 300 micros. This research clearly shows that the time-resolved diffusion measurement is a very powerful tool for detecting spectrally silent association/dissociation processes during chemical reactions. The photoreaction of the LOV2 domain is discussed.

Phototropin is a blue-light receptor of plants and comprises two light-receptive domains, LOV1 and LOV2, Ser/Thr kinase domain and one linker region connecting the LOV2 and the kinase domains. The LOV2 domain is thought to regulate predominantly the light-dependent autophosphorylation of the kinase domain, leading to cellular signaling cascades. In this study, we constructed recombinant LOV1, LOV2, and LOV2-linker polypeptides from phototropin 1 and phototropin 2 of Arabidopsis thaliana and studied their quaternary structures and light-dependent conformational changes by small-angle X-ray scattering. The molecular weights of the polypeptides determined from scattering intensities demonstrated the dimeric associations of LOV1 polypeptides of both isoforms. In contrast, while LOV2 and LOV2-linker polypeptides of phototropin 1 were homodimers, corresponding polypeptides of phototropin 2 existed as monomeric forms. Under blue-light irradiation, the LOV2-linker polypeptide of phototropin 1 displayed small but definite changes of the scattering profile. Through simulation of low-resolution molecular structures, the changes were likely explained as structural changes of the linker region and/or a movement of the region relative to the LOV2 domain. Light-induced profile changes were not observed in the Cys(512)Ala mutated LOV2-linker polypeptide of phototropin 1 losing the phototransformation capability. Thus, it was indicated that the photoreaction in the LOV2 domain probably caused the structural changes in the LOV2-linker polypeptide of phototropin 1. On the basis of the results, the interdomain interactions in phototropin are discussed.

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Abscisic acid (ABA) is a phytohormone that regulates many physiological functions, such as plant growth, development and stress responses. The MAPK cascade plays an important role in ABA signal transduction. Several MAPK and MAPKK molecules are reported to function in ABA signaling; however, there have been few studies related to the identification of MAPKKK upstream of MAPKK in ABA signaling. In this study, we show that an Arabidopsis MAPKKK, MAPKKK18 functions in ABA signaling. The expression of MAPKKK18 was induced by ABA treatment. Yeast two-hybrid analysis revealed that MAPKKKK18 interacted with MKK3, which interacted with C-group MAPK, MPK1/2/7. Immunoprecipitated kinase assay showed that the 3xFlag-tagged MAPKKK18, expressed in Arabidopsis plants, was activated when treated with ABA. These results indicate the possibility that the MAPK cascade is composed of MAPKKK18, MKK3 and MPK1/2/7 in ABA signaling. The transgenic plants overexpressing MAPKKK18 (35S:MAPKKK18) and its kinase negative mutant (35S:MAPKKK18 KN) were generated, and their growth was monitored. Compared with the WT plant, 35S:MAPKKK18 and 35S:MAPKKK18 KN showed smaller and bigger phenotypes, respectively. Senescence of the rosette leaves was promoted in 35S:MAPKKK18, but suppressed in 35S:MAPKKK18 KN. Furthermore, ABA-induced leaf senescence was accelerated in 35S:MAPKKK18. These results suggest that MAPKKK18 controls the plant growth by adjusting the timing of senescence via its protein kinase activity in ABA dependent manners.

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