Tomomi Suzuki

Functional Analysis of a 450–Amino Acid N-Terminal Fragment of Phytochrome B in Arabidopsis

Phytochrome, a major photoreceptor in plants, consists of two domains: the N-terminal photosensory domain and the C-terminal domain. Recently, the 651–amino acid photosensory domain of phytochrome B (phyB) has been shown to act as a functional photoreceptor in the nucleus. The phytochrome (PHY) domain, which is located at the C-terminal end of the photosensory domain, is required for the spectral integrity of phytochrome; however, little is known about the signal transduction activity of this domain. Here, we have established transgenic Arabidopsis thaliana lines expressing an N-terminal 450–amino acid fragment of phyB (N450) lacking the PHY domain on a phyB-deficient background. Analysis of these plants revealed that N450 can act as an active photoreceptor when attached to a short nuclear localization signal and β-glucuronidase. In vitro spectral analysis of reconstituted chromopeptides further indicated that the stability of the N450 Pfr form, an active form of phytochrome, is markedly reduced in comparison with the Pfr form of full-length phyB. Consistent with this, plants expressing N450 failed to respond to intermittent light applied at long intervals, indicating that N450 Pfr is short-lived in vivo. Taken together, our findings show that the PHY domain is dispensable for phyB signal transduction but is required for stabilizing the Pfr form of phyB.

CRYPTOCHROME2 in Vascular Bundles Regulates Flowering in Arabidopsis

Plants make full use of light signals to determine the timing of flowering. In Arabidopsis thaliana, a blue/UV-A photoreceptor, CRYPTOCHROME 2 (cry2), and a red/far-red photoreceptor, PHYTOCHROME B (phyB), are two major photoreceptors that control flowering. The light stimuli for the regulation of flowering are perceived by leaves. We have recently shown that phyB expression in mesophyll but not in vascular bundles suppresses the expression of a key flowering regulator, FLOWERING LOCUS T (FT), in vascular bundles. In this study, we asked where in the leaf cry2 perceives light stimuli to regulate flowering. To answer this question, we established transgenic Arabidopsis lines in which the cry2–green fluorescent protein (GFP) fusion was expressed under the control of organ/tissue-specific promoters in a cry2-deficient mutant background. Analysis of these lines revealed that expression of cry2-GFP in vascular bundles, but not in epidermis or mesophyll, rescued the late flowering phenotype. We further confirmed that cry2-GFP expressed in vascular bundles increased FT expression only in vascular bundles. Hence, in striking contrast with phyB, cry2 most likely regulates FT expression in a cell-autonomous manner.

Molecular basis of the functional specificities of phototropin 1 and 2

A blue-light photoreceptor in plants, phototropin, mediates phototropism, chloroplast relocation, stomatal opening, and leaf-flattening responses. Phototropin is divided into two functional moieties, the N-terminal photosensory and the C-terminal signaling moieties. Phototropin perceives light stimuli by the light, oxygen or voltage (LOV) domain in the N-terminus; the signal is then transduced intramolecularly to the C-terminal kinase domain. Two phototropins, phot1 and phot2, which have overlapping and distinct functions, exist in Arabidopsis thaliana. Phot1 mediates responses with higher sensitivity than phot2. Phot2 mediates specific responses, such as the chloroplast avoidance response and chloroplast dark positioning. To elucidate the molecular basis for the functional specificities of phot1 and phot2, we exchanged the N- and C-terminal moieties of phot1 and phot2, fused them to GFP and expressed them under the PHOT2 promoter in the phot1 phot2 mutant background. With respect to phototropism and other responses, the chimeric phototropin consisting of phot1 N-terminal and phot2 C-terminal moieties (P1n/2cG) was almost as sensitive as phot1; whereas the reverse combination (P2n/1cG) functioned with lower sensitivity. Hence, the N-terminal moiety mainly determined the sensitivity of the phototropins. Unexpectedly, both P1n/2cG and P2n/1cG mediated the chloroplast avoidance response, which is specific to phot2. Hence, chloroplast avoidance activity appeared to be suppressed specifically in the combination of N- and C-terminal moieties of phot1. Unlike the chloroplast avoidance response, chloroplast dark positioning was observed for P2G and P2n/1cG but not for P1G or P1n/2cG, suggesting that a specific structure in the N-terminal moiety of phot2 is required for this activity.

Mutations in N-terminal Flanking Region of Blue Light-sensing Light-Oxygen and Voltage 2 (LOV2) Domain Disrupt Its Repressive Activity on Kinase Domain in the Chlamydomonas Phototropin

Background: A plant photoreceptor “phototropin” is a light-dependent kinase containing the LOV photosensory domains. Results: Mutations in the N-terminal flanking region of LOV2 elevate kinase activity in darkness. Conclusion: The N-terminal flanking region is involved in intramolecular signaling from LOV2 to the kinase domain. Significance: This work provides insights into how the LOV domain can activate the kinase domain intramolecularly. Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.

Light-induced Conformational Changes of LOV1 (Light Oxygen Voltage-sensing Domain 1) and LOV2 Relative to the Kinase Domain and Regulation of Kinase Activity in Chlamydomonas Phototropin

Background: The plant photoreceptor “phototropin” is a light-regulated kinase containing two photosensory domains named LOV. Results: Light-induced conformational change related to the kinase activation was detected in full-length phototropin of Chlamydomonas. Conclusion: LOV1 may interact with LOV2 and modify the photosensitivity of the kinase regulation by LOV2. Significance: Configuration of LOV1, LOV2, and kinase domain in a phot molecule is first demonstrated. Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.

The phototropic response is locally regulated within the topmost light-responsive region of the Arabidopsis thaliana seedling

Phototropism is caused by differential cell elongation between the irradiated and shaded sides of plant organs, such as the stem. It is widely accepted that an uneven auxin distribution between the two sides crucially participates in this response. Plant-specific blue-light photoreceptors, phototropins (phot1 and phot2), mediate this response. In grass coleoptiles, the sites of light perception and phototropic bending are spatially separated. However, these sites are less clearly distinguished in dicots. Furthermore, the exact placement of the action of each phototropic signaling factor remains unknown. Here, we investigated the spatial aspects of phototropism using spotlight irradiation with etiolated Arabidopsis seedlings. The results demonstrated that the topmost part of about 1.1 mm of the hypocotyl constituted the light-responsive region in which both light perception and actual bending occurred. In addition, cotyledons and the shoot apex were dispensable for the response. Hence, the response was more region autonomous in dicots than in monocots. We next examined the elongation rates, the levels of phot1 and the auxin-reporter gene expression along the hypocotyl during the phototropic response. The light-responsive region was more active than the non-responsive region with respect to all of those parameters.

Auxin Contributes to the Intraorgan Regulation of Gene Expression in Response to Shade

Many genes are preferentially up-regulated by shade stimulus in the vasculature of Arabidopsis thaliana cotyledons, some of which are regulated by newly synthesized auxin in the mesophyll and/or epidermis. Plants sense and respond to light via multiple photoreceptors including phytochrome. The decreased ratio of red to far-red light that occurs under a canopy triggers shade-avoidance responses, which allow plants to compete with neighboring plants. The leaf acts as a photoperceptive organ in this response. In this study, we investigated how the shade stimulus is spatially processed within the cotyledon. We performed transcriptome analysis on microtissue samples collected from vascular and nonvascular regions of Arabidopsis (Arabidopsis thaliana) cotyledons. In addition, we mechanically isolated and analyzed the vascular tissue. More genes were up-regulated by the shade stimulus in vascular tissues than in mesophyll and epidermal tissues. The genes up-regulated in the vasculature were functionally divergent and included many auxin-responsive genes, suggesting that various physiological/developmental processes might be controlled by shade stimulus in the vasculature. We then investigated the spatial regulation of these genes in the vascular tissues. A small vascular region within a cotyledon was irradiated with far-red light, and the response was compared with that when the whole seedling was irradiated with far-red light. Most of the auxin-responsive genes were not fully induced by the local irradiation, suggesting that perception of the shade stimulus requires that a wider area be exposed to far-red light or that a certain position in the mesophyll and epidermis of the cotyledon be irradiated. This result was consistent with a previous report that auxin synthesis genes are up-regulated in the periphery of the cotyledon. Hence, auxin acts as an important intraorgan signaling factor that controls the vascular shade response within the cotyledon.