[Show abstract][Hide abstract] ABSTRACT: A horizontal vibrational motion of biological tissue generated by a femtosecond laser-induced impulsive force was directly detected for the first time as angular shift of the cantilever of an atomic force microscope (AFM), which was directly in contact with the tissue. The motion of a small plant stem (diameter: 160 µm) on the force loading was detected by the torsional motion of the AFM cantilever. The sensitivity of the method was evaluated by a numerical simulation with the finite element method (FEM). The results conclusively demonstrated the efficacy of this method for nano-scale detection of the horizontal motion of biological micro-objects.
[Show abstract][Hide abstract] ABSTRACT: Photobody localization of Arabidopsis phytochrome B (phyB) fused to GFP (PBG) correlates closely with the photoinhibition of hypocotyl elongation. However, the N-terminal half of phyB fused to GFP (NGB) is hypersensitive to light despite its inability to localize to photobodies. Therefore, the significance of photobodies in regulating hypocotyl growth remains debatable. Accumulating evidence indicates that under diurnal conditions, photoactivated phyB persists into darkness to inhibit hypocotyl elongation. Here, we examine whether photobodies are involved in inhibiting hypocotyl growth in darkness by comparing the PBG and NGB lines after the red-light-to-dark transition. Surprisingly, after the transition from 10 μmol m-2 s-1 red light to darkness, PBG inhibits hypocotyl elongation three times longer than NGB. The disassembly of photobodies in PBG hypocotyl nuclei correlates tightly with the accumulation of the growth-promoting transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3). Destabilizing photobodies by either decreasing the light intensity or adding monochromatic far-red treatment before the light-to-dark transition leads to faster PIF3 accumulation and a dramatic reduction in the capacity for hypocotyl growth inhibition in PBG. In contrast, NGB is defective in PIF3 degradation and its hypocotyl growth in the dark is nearly unresponsive to changes in light conditions. Together, our results support the model that photobodies are required for the prolonged, light-dependent inhibition of hypocotyl elongation in the dark by repressing PIF3 accumulation and by stabilizing the Pfr form of phyB. Our study suggests that photobody localization patterns of phyB could serve as instructive cues that control light-dependent photomorphogenetic responses in the dark.
[Show abstract][Hide abstract] ABSTRACT: In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movements and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning response. Here, we report the isolation and characterization of a Non-phototropic seedling (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark grown seedlings of Nps1mutant phototropin 1 protein accumulates at highly reduced level relative to wild type and lacks BL-induced autophosphorylation. The mutant harbors a single G1484 to A transition in the Hinge1 region of phototropin 1 homologue resulting in an arginine to histidine substitution (R495H) in a highly conserved A'α helix proximal to the LOV2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of A'α helix region in phototropic signaling of tomato.
[Show abstract][Hide abstract] ABSTRACT: 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 spot-light irradiation with etiolated Arabidopsis seedlings. The results demonstrated that a 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.
[Show abstract][Hide abstract] ABSTRACT: 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 (Cr). Full-length and LOV2-KD samples of Cr 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 to that of the phot. Small angle X-ray scattering (SAXS) 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 SAXS 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.
[Show abstract][Hide abstract] ABSTRACT: In flowering plants, light is one of the major environmental stimuli that determine the timing of the transition from the vegetative to reproductive phase. In Arabidopsis, phytochrome B (phyB); phyA; cryptochrome 2; and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 are major photoreceptors that regulate flowering. Unlike phyA; cryptochrome 2; and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1, phyB delays flowering mainly by destabilizing the CONSTANS (CO) protein, whose reduction leads to decreased expression of a florigen gene, FLOWERING LOCUS T. However, it remains unclear how the phyB-mediated CO destabilization is mechanistically regulated. Here, we identify a unique PHYTOCHROME-DEPENDENT LATE-FLOWERING (PHL) gene, which is mainly involved in the phyB-dependent regulation of flowering. Plants with mutant phl exhibited a late-flowering phenotype, especially under long-day conditions. The late-flowering phenotype of the phl mutant was completely overridden by a phyB mutation, indicating that PHL normally accelerates flowering by countering the inhibitory effect of phyB on flowering. Accordingly, PHL physically interacted with phyB both in vitro and in vivo in a red light-dependent manner. Furthermore, in the presence of phyB under red light, PHL interacted with CO as well. Taken together, we propose that PHL regulates photoperiodic flowering by forming a phyB-PHL-CO tripartite complex.
Proceedings of the National Academy of Sciences 10/2013; 110(44). DOI:10.1073/pnas.1310631110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of
plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant
organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles
in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through
the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type
and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli.
In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly
submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information
included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions
of data from all plant researchers to enhance the utility and comprehensiveness of PODB3.
[Show abstract][Hide abstract] ABSTRACT: Leaf epidermal stomata play pivotal roles in gas exchange and transpiration in higher plants. The regulatory mechanisms of the opening and closing of stomata (stomatal movement) and stomatal morphogenesis have been intensively studied. Such studies require the collection of detached epidermal cell layers ("peels") for the detailed observation of stomata under the microscope. However, there are drawbacks to the conventional techniques for preparing and handling epidermal peels, i.e., a certain level of skill or an apparatus (e. g., a Waring blender) is required. In this report, we present a simple and rapid method for preparing epidermal peels called Perforated-tape Epidermal Detachment (PED). For PED, Time Tape is adhered to the adaxial epidermis of an Arabidopsis leaf, and the abaxial epidermal layer is detached using Scotch Tape perforated with a small hole. The area inside the hole is suitable for microscopic observation because the Scotch Tape does not mask the detached epidermal layer there. PED can also be used to prepare epidermal peels from tobacco and legume leaves. The crucial advantage of PED over the conventional blender method is that the epidermal cells prepared by PED are rarely damaged, and we demonstrate that PED can be used to obtain material for physiological assays of stomatal responses to blue light and externally applied ABA. We thus believe that the PED method is suitable for preparing epidermal cell layers for physiological studies.
[Show abstract][Hide abstract] ABSTRACT: Chloroplasts change their position to adapt cellular activities to fluctuating environmental light conditions. Phototropins (phot1 and phot2 in Arabidopsis) are plant-specific blue light photoreceptors that perceive changes in light intensity and direction and mediate actin-based chloroplast photorelocation movements. Both phot1 and phot2 regulate the chloroplast accumulation response, while phot2 is mostly responsible for the regulation of the avoidance response. Although it has been widely accepted that distinct intracellular localisations of phototropins are reasonably implicated in the specificity, the mechanism underlying the phot2-specific avoidance response has remained elusive. In this study, we examined the relationship of the phot2 localisation pattern to the chloroplast photorelocation movement. First, the fusion of nuclear localisation signal with phot2, which effectively reduced the amount of phot2 in cytoplasm, retained the activity for both the accumulation and avoidance responses, indicating that membrane-localised phot2 but not cytoplasmic phot2 is functional to mediate the responses. Importantly, some fraction of phot2, and phot1 at the lesser extent, were localised on the chloroplast outer membrane. Moreover, the deletion of the C-terminal region of phot2, which are previously shown to be defective in blue light-induced Golgi localisation and avoidance response, affected the localisation pattern on the chloroplast outer membrane. Taken together, these results suggest that dynamic phot2 trafficking between the plasma membrane to the Golgi apparatus and the chloroplast outer membrane might be involved in the avoidance response.
[Show abstract][Hide abstract] ABSTRACT: Light is one of the most important environmental factors regulating the growth and development of leaves. As the primary photosynthetic organs, leaves have a laminar structure in many dicotyledonous plants. The regulation of leaf flatness is a key mechanism for the efficient absorption of light under the low light conditions. In the present study, we demonstrated that phytochrome B (phyB) promoted the development of curled leaves. Wild type leaves gently curled downwards under white light, whereas the phyB-deficient mutant (phyB) constitutively exhibited flatter leaves. In wild type, leaf flattening was promoted by end-of-day far-red light treatment (EODFR), which rapidly eliminates the active Pfr phytochrome. Interestingly, the curled-leaf phenotype in a phototropin-deficient mutant was almost completely suppressed by the phyB mutation as well as EODFR. Thus, phototropin promotes leaf flattening by suppressing the leaf-curling activity of phyB. We examined the downstream components of phyB and phototropin to further assess their antagonistic regulation of leaf flatness. Consequently, we found that a phototropin-signaling transducer, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3), was required to promote leaf flattening in phyB. The present study provides new insights into a mechanism in which leaf flatness is regulated in response to different light environmental cues.
[Show abstract][Hide abstract] ABSTRACT: Phototropins (phot1 and phot2), plant specific blue-light receptor kinases, mediate a range of physiological responses in Arabidopsis, including phototropism, chloroplast photorelocation movement, stomatal opening, and leaf flattening. Phototropins consist of two photoreceptive domains at their N-terminus, LOV1 and LOV2, and a serine/threonine kinase domain at their C-terminus. Here, we determined the molecular moiety for the membrane association of phototropins using the yeast CytoTrap and Arabidopsis protoplast systems. We then examined the physiological significance of the membrane association of phototropins. This detailed study with serial deletions narrowed down the association domain to a relatively small part of the carboxy-terminal domain of phototropin. The functional analysis of phot2 deletion mutants in the phot2-deficient Adiantum and Arabidopsis mutants revealed that the ability to mediate the chloroplast avoidance response correlated well with phot2's membrane association, especially with the Golgi apparatus. Taken together, our data suggest that a small part of the C-terminal domain of phototropins is necessary not only for membrane association but also for the physiological activities that elicit phototropin-specific responses.
[Show abstract][Hide abstract] ABSTRACT: Phytochromes are red and far-red light receptors in plants that mediate critical responses to light throughout the lifecycle. They achieve this in part by targeting negatively acting bHLH transcription factors called phytochrome-interacting factors (PIFs) for degradation within the nucleus. However, it is not known whether protein degradation is the primary mechanism by which phytochromes inhibit these repressors of photomorphogenesis. Here, we use chromatin immunoprecipitation to show that phyB inhibits the regulatory activity of PIF1 and PIF3 by releasing them from their DNA targets. The N-terminal fragment of phyB (NG-GUS-NLS; NGB) also inhibits binding of PIF3 to its target promoters. However, unlike full-length phyB, NGB does not promote PIF3 degradation, establishing the activity of NGB reflects its ability to inhibit PIF binding to DNA. We further show that Pfr forms of both full-length phyB and NGB inhibit DNA binding of PIF1 and PIF3 in vitro. Taken together, our results indicate that phyB inhibition of PIF function involves two separate processes: sequestration and protein degradation.
The Plant Journal 07/2012; 72(4). DOI:10.1111/j.1365-313X.2012.05114.x · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phytochrome is a red (R)/far-red (FR) light-sensing photoreceptor that regulates various aspects of plant development. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates atypical phytochrome responses, such as the FR high irradiance response (FR-HIR), which is elicited under prolonged FR. A proteasome-based degradation pathway rapidly eliminates active Pfr (the FR-absorbing form of phyA) under R. To elucidate the structural basis for the phyA-specific properties, we systematically constructed 16 chimeric phytochromes in which each of four parts of the phytochrome molecule, namely, the N-terminal extension plus the Per/Arnt/Sim domain (N-PAS), the cGMP phosphodiesterase/adenyl cyclase/FhlA domain (GAF), the phytochrome domain (PHY), and the entire C-terminal half, was occupied by either the phyA or phytochrome B sequence. These phytochromes were expressed in transgenic Arabidopsis thaliana to examine their physiological activities. Consequently, the phyA N-PAS sequence was shown to be necessary and sufficient to promote nuclear accumulation under FR, whereas the phyA sequence in PHY was additionally required to exhibit FR-HIR. Furthermore, the phyA sequence in PHY alone substantially increased the light sensitivity to R. In addition, the GAF phyA sequence was important for rapid Pfr degradation. In summary, distinct structural modules, each of which confers different properties to phyA, are assembled on the phyA molecule.
The Plant Cell 07/2012; 24(7):2949-62. DOI:10.1105/tpc.111.094201 · 9.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Jasmonates are phytohormones derived from oxygenated fatty acids that regulate a broad range of plant defense and developmental processes. In Arabidopsis, hypocotyl elongation under various light conditions was suppressed by exogenously supplied methyl jasmonate (MeJA). Moreover, this suppression by MeJA was particularly effective under red light condition. Mutant analyses suggested that SCFCOI1-mediated proteolysis was involved in this function. However, MeJA action still remained in the coi1 mutant, and (+)-7-iso-JA-L-Ile, a well-known active form of jasmonate, had a weaker effect than MeJA under the red light condition, suggesting that unknown signaling pathway are present in MeJA-mediated inhibition of hypocotyl elongation. EMS mutant screening identified two MeJA-insensitive hypocotyl elongation mutants, jasmonate resistance long hypocotyl 1 (jal1) and jal36, which had mutations in the phytochrome B (PHYB) gene. These analyses suggested that inhibition of hypocotyl elongation by jasmonates is enhanced under red light in phyB dependent manner.
Electronic supplementary material
The online version of this article (doi:10.1007/s10265-012-0509-3) contains supplementary material, which is available to authorized users.
Journal of Plant Research 07/2012; 126(1). DOI:10.1007/s10265-012-0509-3 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Light is an important environmental information source that plants use to modify their growth and development. Palisade parenchyma cells in leaves develop cylindrical shapes in response to blue light; however, the photosensory mechanism for this response has not been elucidated. In this study, we analyzed the palisade cell response in phototropin-deficient mutants. First, we found that two different light-sensing mechanisms contributed to the response in different proportions depending on the light intensity. One response observed under lower intensities of blue light was mediated exclusively by a blue light photoreceptor, phototropin 2 (PHOT2). Another response was elicited under higher intensities of light in a phototropin-independent manner. To determine the tissue in which PHOT2 perceives the light stimulus to regulate the response, green fluorescent protein (GFP)-tagged PHOT2 (P2G) was expressed under the control of tissue-specific promoters in the phot1 phot2 mutant background. The results revealed that the expression of P2G in the mesophyll, but not in the epidermis, promoted palisade cell development. Furthermore, a constitutively active C-terminal kinase fragment of PHOT2 fused to GFP (P2CG) promoted the development of cylindrical palisade cells in the proper direction without the directional cue provided by light. Hence, in response to blue light, PHOT2 promotes the development of cylindrical palisade cells along a predetermined axis in a tissue-autonomous manner.
The Plant Cell 10/2011; 23(10):3684-95. DOI:10.1105/tpc.111.085852 · 9.34 Impact Factor