Chloroplast Photorelocation Research Articles

Multifaceted roles of Arabidopsis heat shock factor binding protein in plant growth, development, and heat shock response

Heat shock factor-binding protein (HSBP) is a 10 kDa protein in plants and animals and consists exclusively of a coiled-coil domain. During the recovery phase following a heat shock response, HSBP relocates from the cytoplasm to the nucleus in Arabidopsis (Arabidopsis thaliana), where it interacts with heat shock factors (HSFs). Here, we found that 16 out of the 19 functional known HSFs can interact with HSBP. Besides, our results indicate that HSBP negatively regulates HSF gene expression during normal growth conditions and recovery from heat shock. Expanding our understanding of HSBP's physiological functions during regular growth, co-immunoprecipitation and mass spectrometry analysis identified 16 coiled-coil domain-containing proteins co-immunoprecipitated with HSBP. These proteins encompass HSP70s, all components of the MAIGO2 complex, COP1-interactive protein1 (CIP1), CIP1-like protein, kinesin-like protein for actin-based chloroplast movement1 and 2 (KAC1/2), and HSBP itself. By examining mutant plants lacking HSBP and its interacting proteins, we elucidated their functional relationships. Our findings underscore the indispensability of the HSBP coiled-coil heptad repeat for interacting with its partners and its crucial role in growth, development, and the heat shock response. In addition to its involvement in heat shock response, Arabidopsis HSBP, a discreet small regulatory protein, exerts multiple regulatory functions in hypocotyl elongation, flowering time, chloroplast photo-relocation, and seed development.

Light-dependent chloroplast relocation in wild strawberry (Fragaria vesca)

ABSTRACT Chloroplast photorelocation is a vital organellar response that optimizes photosynthesis in plants amid fluctuating environmental conditions. Chloroplasts exhibit an accumulation response, in which they move toward weak light to enhance photoreception, and an avoidance response, in which they move away from strong light to avoid photodamage. Although chloroplast photorelocation has been extensively studied in model plants such as Arabidopsis thaliana, little is known about this process in the economically important crop strawberry. Here, we investigated chloroplast photorelocation in leaf mesophyll cells of wild strawberry (Fragaria vesca), a diploid relative of commercially cultivated octoploid strawberry (F. × ananassa). Microscopy observation revealed that the periclinal area of leaf mesophyll cells in F. vesca is considerably smaller than that of A. thaliana. Given this small cell size, we investigated chloroplast photorelocation in F. vesca by measuring light transmittance in leaves. Weak blue light induced the accumulation response, whereas strong blue light induced the avoidance response. Unexpectedly, strong red light also induced the accumulation response in F. vesca. These findings shed light on chloroplast photorelocation as an intracellular response, laying the foundation for enhancing photosynthesis and productivity in Fragaria.

A phytochrome/phototropin chimeric photoreceptor promotes growth of fern gametophytes under limited light conditions.

Many ferns thrive even in low-light niches such as under an angiosperm forest canopy. However, the shade adaptation strategy of ferns is not well understood. Phytochrome 3/neochrome (phy3/neo) is an unconventional photoreceptor, found in the fern Adiantum capillus-veneris, that controls both red and blue light-dependent phototropism and chloroplast photorelocation, which are considered to improve photosynthetic efficiency in ferns. Here we show that phy3/neo localizes not only at the plasma membrane but also in the nucleus. Since both phototropism and chloroplast photorelocation are mediated by membrane-associated phototropin photoreceptors, we speculated that nucleus-localized phy3/neo possesses a previously undescribed biological function. We reveal that phy3/neo directly interacts with Adiantum cryptochrome 3 (cry3) in the nucleus. Plant cryptochromes are blue light receptors that transcriptionally regulate photomorphogenesis; therefore, phy3/neo may function via cry3 to synchronize light-mediated development with phototropism and chloroplast photorelocation to promote fern growth under low-light conditions. Furthermore, we demonstrate that phy3/neo regulates the expression of the Cyclin-like gene AcCyc1 and promotes prothallium expansion growth. These findings provide insight into the shade adaptation strategy of ferns and suggest that phy3/neo plays a substantial role in the survival and growth of ferns during the tiny gametophytic stage under low-light conditions, such as those on the forest floor.

CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

FKF1 Interacts with CHUP1 and Regulates Chloroplast Movement in Arabidopsis.

Plants have mechanisms to relocate chloroplasts based on light intensities in order to maximize photosynthesis and reduce photodamage. Under low light, chloroplasts move to the periclinal walls to increase photosynthesis (accumulation) and move to the anticlinal walls under high light to avoid photodamage, and even cell death (avoidance). Arabidopsis blue light receptors phot1 and phot2 (phototropins) have been reported to regulate chloroplast movement. This study discovered that another blue light receptor, FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), regulates chloroplast photorelocation by physically interacting with chloroplast unusual positioning protein 1 (CHUP1), a critical component of the chloroplast motility system. Leaf cross-sectioning and red-light transmittance results showed that overexpression of FKF1 compromised the avoidance response, while the absence of FKF1 enhanced chloroplast movements under high light. Western blot analysis showed that CHUP1 protein abundance is altered in FKF1 mutants and overexpression lines, indicating a potential regulation of CHUP1 by FKF1. qPCR results showed that two photorelocation pathway genes, JAC1 and THRUMIN1, were upregulated in FKF1-OE lines, and overexpression of FKF1 in the THRUMIN1 mutant weakened its accumulation and avoidance responses, indicating that JAC1 and THRUMIN1 may play a role in the FKF1-mediated chloroplast avoidance response. However, the precise functional roles of JAC1 and THRUMIN1 in this process are not known.

Epidermal chloroplasts are defense-related motile organelles equipped with plant immune components

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.

Spectrum of Light as a Determinant of Plant Functioning: A Historical Perspective.

The significance of the spectral composition of light for growth and other physiological functions of plants moved to the focus of “plant science” soon after the discovery of photosynthesis, if not earlier. The research in this field recently intensified due to the explosive development of computer-controlled systems for artificial illumination and documenting photosynthetic activity. The progress is also substantiated by recent insights into the molecular mechanisms of photo-regulation of assorted physiological functions in plants mediated by photoreceptors and other pigment systems. The spectral balance of solar radiation can vary significantly, affecting the functioning and development of plants. Its effects are evident on the macroscale (e.g., in individual plants growing under the forest canopy) as well as on the meso- or microscale (e.g., mutual shading of leaf cell layers and chloroplasts). The diversity of the observable effects of light spectrum variation arises through (i) the triggering of different photoreceptors, (ii) the non-uniform efficiency of spectral components in driving photosynthesis, and (iii) a variable depth of penetration of spectral components into the leaf. We depict the effects of these factors using the spectral dependence of chloroplast photorelocation movements interlinked with the changes in light penetration into (light capture by) the leaf and the photosynthetic capacity. In this review, we unfold the history of the research on the photocontrol effects and put it in the broader context of photosynthesis efficiency and photoprotection under stress caused by a high intensity of light.

PDV1 and PDV2 Differentially Affect Remodeling and Assembly of the Chloroplast DRP5B Ring.

Chloroplasts divide by binary fission, which is driven by a ring-like multiprotein complex spanning the inner and outer envelope membranes (OEMs) at the division site. The cytosolic DYNAMIN-RELATED PROTEIN 5B (DRP5B/ARC5) is a mechanochemical GTPase involved in binary fission of the chloroplast membrane in Arabidopsis (Arabidopsis thaliana), but the dynamics of its interactions with the chloroplast membranes and their regulation by guanine nucleotides and protein effectors remain poorly characterized. Using an Arabidopsis phot2 mutant with defects in chloroplast photorelocation movement, we determined that the ring structures of DRP5B at the chloroplast division site underwent subunit exchange with a cytosolic DRP5B pool. Mutant DRP5B proteins with impaired GTPase activity retained the ability to self-assemble at the constriction sites of chloroplasts, but did not rescue the chloroplast division defects in the Arabidopsis drp5B mutant. Our in vivo kinetic measurements of the DRP5B mutant T82D suggested that turnover of the DRP5B ring at the chloroplast division site is coupled to GTP hydrolysis. Furthermore, we established that DRP5B targeting to the chloroplast surface and assembly into a ring structure at the division site are specifically determined by the chloroplast outer OEM protein PLASTID DIVISION2 (PDV2), and that DRP5B-OEM dissociation is mainly mediated by PDV1, a paralog of PDV2. Thus, this study suggests that the mechanochemical properties of DRP5B on the chloroplast surface are dynamically regulated by its GTPase activity and major binding partners.

Aureochromes - Blue Light Receptors.

A variety of living organisms including bacteria, fungi, animals, and plants use blue light (BL) to adapt to changing ambient light. Photosynthetic forms (plants and algae) require energy of light for photosynthesis, movements, development, and regulation of activity. Several complex light-sensitive systems evolved in eukaryotic cells to use the information of light efficiently with photoreceptors selectively absorbing various segments of the solar spectrum, being the first components in the light signal transduction chain. They are most diverse in algae. Photosynthetic stramenopiles, which received chloroplasts from red algae during secondary symbiosis, play an important role in ecosystems and aquaculture, being primary producers. These taxa acquired the ability to use BL for regulation of such processes as phototropism, chloroplast photo-relocation movement, and photomorphogenesis. A new type of BL receptor - aureochrome (AUREO) - was identified in Vaucheria frigida in 2007. AUREO consists of two domains: bZIP (basic-region leucine zipper) domain and LOV (light-oxygen-voltage-sensing) domain, and thus this photoreceptor is a BL-sensitive transcription factor. This review presents current data on the structure, mechanisms of action, and biochemical features of aureochromes.

Actin-mediated movement of chloroplasts.

Plants are sessile and require diverse strategies to adapt to fluctuations in the surrounding light conditions. Consequently, the photorelocation movement of chloroplasts is essential to prevent damages that are induced by intense light (avoidance response) and to ensure efficient photosynthetic activities under weak light conditions (accumulation response). The mechanisms that underlie chloroplast movements have been revealed through analysis of the behavior of individual chloroplasts and it has been found that these organelles can move in any direction without turning. This implies that any part of the chloroplast periphery can function as the leading or trailing edge during movement. This ability is mediated by a special structure, which consists of short actin filaments that are polymerized at the leading edge of moving chloroplasts and are specifically localized in the space between the chloroplast and the plasma membrane, and is called chloroplast-actin. In addition, several of the genes that encode proteins that are involved in chloroplast-actin polymerization or maintenance have been identified. In this Review, we discuss the mechanisms that regulate chloroplast movements through polymerization of the chloroplast-actin and propose a model for actin-driven chloroplast photorelocation movement.

Variations in chloroplast movement and chlorophyll fluorescence among chloroplast division mutants under light stress.

Chloroplasts divide to maintain consistent size, shape, and number in leaf mesophyll cells. Altered expression of chloroplast division proteins in Arabidopsis results in abnormal chloroplast morphology. To better understand the influence of chloroplast morphology on chloroplast movement and photosynthesis, we compared the chloroplast photorelocation and photosynthetic responses of a series of Arabidopsis chloroplast division mutants with a wide variety of chloroplast phenotypes. Chloroplast movement was monitored by red light reflectance imaging of whole plants under increasing intensities of white light. The accumulation and avoidance responses were differentially affected in different mutants and depended on both chloroplast number and morphological heterogeneity. Chlorophyll fluorescence measurements during 5 d light experiments demonstrated that mutants with large-chloroplast phenotypes generally exhibited greater PSII photodamage than those with intermediate phenotypes. No abnormalities in photorelocation efficiency or photosynthetic capacity were observed in plants with small-chloroplast phenotypes. Simultaneous measurement of chloroplast movement and chlorophyll fluorescence indicated that the energy-dependent (qE) and long-lived components of non-photochemical quenching that reflect photoinhibition are affected differentially in different division mutants exposed to high or fluctuating light intensities. We conclude that chloroplast division mutants with abnormal chloroplast morphologies differ markedly from the wild type in their light adaptation capabilities, which may decrease their relative fitness in nature.

Temperature-dependent signal transmission in chloroplast accumulation response.

Chloroplast photorelocation movement, well-characterized light-induced response found in various plant species from alga to higher plants, is an important phenomenon for plants to increase photosynthesis efficiency and avoid photodamage. The signal for chloroplast accumulation movement connecting the blue light receptor, phototropin, and chloroplasts remains to be identified, although the photoreceptors and the mechanism of movement via chloroplast actin filaments have now been revealed in land plants. The characteristics of the signal have been found; the speed of signal transfer is about 1µm min-1 and that the signal for the accumulation response has a longer life and is transferred a longer distance than that of the avoidance response. Here, to collect the clues of the unknown signal substances, we studied the effect of temperature on the speed of signal transmission using the fern Adiantum capillus-veneris and found the possibility that the mechanism of signal transfer was not dependent on the simple diffusion of a substance; thus, some chemical reaction must also be involved. We also found new insights of signaling substances, such that microtubules are not involved in the signal transmission, and that the signal could even be transmitted through the narrow space between chloroplasts and the plasma membrane.

The Motor Kinesin 4II Is Important for Growth and Chloroplast Light Avoidance in the Moss <i>Physcomitrella patens</i>

Understanding how plant cells adapt dynamically to changes in the environment is a fundamental problem of plant biology. Under many conditions, plant cells respond to environmental changes by modifying their intracellular organization. A critical example of intracellular reorganization is chloroplast photo-relocation, which is required for optimal energy harvesting and avoiding photodamage. A key system responsible for the spatial organization of intracellular components is the microtubule cytoskeleton and its associated motor proteins, kinesins. Here we tested the hypothesis that members of the kinesin 4II subfamily are important for chloroplast photo-relocation in the moss Physcomitrella patens. Most land plants, including P. patens, use an actin cytoskeleton-dependent mechanism to transport chloroplasts in response to light. In addition to the actin-based system, P. patens can also transport chloroplasts via a microtubule-dependent mechanism, which is absent in flowering plants. Here, we used a P. patens line that contains an inducible RNAi system to silence all three kinesin 4-II genes present in this moss and evaluated their participation in the microtubule-dependent chloroplast light avoidance response. Because we found a significant effect on cell growth when kinesin 4IIs are silenced, we took advantage of the inducible system to establish a reproducible and quantitative assay to evaluate chloroplast photo-relocation in full-grown cells. Using a laser scanning confocal-based chloroplast light avoidance response assay, we found a reduction in chloroplast motility when kinesin 4IIs were silenced. Hence, in addition to identifying a role for kinesin 4II proteins in protonemal cell growth, our results strongly support the hypothesis that these kinesins play an important role in the chloroplast light avoidance response.

RPT2/NCH1 subfamily of NPH3-like proteins is essential for the chloroplast accumulation response in land plants

In green plants, the blue light receptor kinase phototropin mediates various photomovements and developmental responses, such as phototropism, chloroplast photorelocation movements (accumulation and avoidance), stomatal opening, and leaf flattening, which facilitate photosynthesis. In Arabidopsis, two phototropins (phot1 and phot2) redundantly mediate these responses. Two phototropin-interacting proteins, NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and ROOT PHOTOTROPISM 2 (RPT2), which belong to the NPH3/RPT2-like (NRL) family of BTB (broad complex, tramtrack, and bric à brac) domain proteins, mediate phototropism and leaf flattening. However, the roles of NRL proteins in chloroplast photorelocation movement remain to be determined. Here, we show that another phototropin-interacting NRL protein, NRL PROTEIN FOR CHLOROPLAST MOVEMENT 1 (NCH1), and RPT2 redundantly mediate the chloroplast accumulation response but not the avoidance response. NPH3, RPT2, and NCH1 are not involved in the chloroplast avoidance response or stomatal opening. In the liverwort Marchantia polymorpha, the NCH1 ortholog, MpNCH1, is essential for the chloroplast accumulation response but not the avoidance response, indicating that the regulation of the phototropin-mediated chloroplast accumulation response by RPT2/NCH1 is conserved in land plants. Thus, the NRL protein combination could determine the specificity of diverse phototropin-mediated responses.

Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana.

Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements.

Evolution of the Cp-Actin-based Motility System of Chloroplasts in Green Plants.

During the course of green plant evolution, numerous light responses have arisen that optimize their growth under fluctuating light conditions. The blue light receptor phototropin mediates several photomovement responses at the tissue, cellular and organelle levels. Chloroplast photorelocation movement is one such photomovement response, and is found not only in most green plants, but also in some red algae and photosynthetic stramenopiles. In general, chloroplasts move toward weak light to maximally capture photosynthetically active radiation (the chloroplast accumulation response), and they move away from strong light to avoid photodamage (the avoidance response). In land plants, chloroplast movement is dependent on specialized actin filaments, chloroplast-actin filaments (cp-actin filaments). Through molecular genetic analysis using Arabidopsis thaliana, many molecular factors that regulate chloroplast photorelocation were identified. In this Perspective, we discuss the evolutionary history of the molecular mechanism for chloroplast photorelocation movement in green plants in view of cp-actin filaments.

Chloroplast avoidance movement is not functional in plants grown under strong sunlight.

Chloroplast movement in nine climbing plant species was investigated. It is thought that chloroplasts generally escape from strong light to avoid photodamage but accumulate towards weak light to perform photosynthesis effectively. Unexpectedly, however, the leaves of climbing plants grown under strong sunlight showed very low or no chloroplast photorelocation responses to either weak or strong blue light when detected by red light transmittance through leaves. Direct observations of Cayratia japonica leaves, for example, revealed that the average number of chloroplasts in upper periclinal walls of palisade tissue cells was only 1.2 after weak blue-light irradiation and almost all of the chloroplasts remained at the anticlinal wall, the state of chloroplast avoidance response. The leaves grown under strong light have thin and columnar palisade tissue cells comparing with the leaves grown under low light. Depending on our analyses and our schematic model, the thinner cells in a unit leaf area have a wider total plasma membrane area, such that more chloroplasts can exist on the plasma membrane in the thinner cells than in the thicker cells in a unit leaf-area basis. The same strategy might be used in other plant leaves grown under direct sunlight.

Molecular basis of chloroplast photorelocation movement.

Chloroplast photorelocation movement is an essential physiological response for sessile plant survival and the optimization of photosynthetic ability. Simple but effective experiments on the physiological, cell biological and molecular genetic aspects have been widely used to investigate the signaling components of chloroplast photorelocation movement in Arabidopsis for the past few decades. Although recent knowledge on chloroplast photorelocation movement has led us to a deeper understanding of its physiological and molecular basis, the biochemical roles of the downstream factors remain largely unknown. In this review, we briefly summarize recent advances regarding chloroplast photorelocation movement and propose that a new high-resolution approach is necessary to investigate the molecular mechanism underlying actin-based chloroplast photorelocation movement.

Blue-light-regulated transcription factor, Aureochrome, in photosynthetic stramenopiles.

During the course of evolution through various endosymbiotic processes, diverse photosynthetic eukaryotes acquired blue light (BL) responses that do not use photosynthetic pathways. Photosynthetic stramenopiles, which have red algae-derived chloroplasts through secondary symbiosis, are principal primary producers in aquatic environments, and play important roles in ecosystems and aquaculture. Through secondary symbiosis, these taxa acquired BL responses, such as phototropism, chloroplast photo-relocation movement, and photomorphogenesis similar to those which green plants acquired through primary symbiosis. Photosynthetic stramenopile BL receptors were undefined until the discovery in 2007, of a new type of BL receptor, the aureochrome (AUREO), from the photosynthetic stramenopile alga, Vaucheria. AUREO has a bZIP domain and a LOV domain, and thus BL-responsive transcription factor. AUREO orthologs are only conserved in photosynthetic stramenopiles, such as brown algae, diatoms, and red tide algae. Here, a brief review is presented of the role of AUREOs as photoreceptors for these diverse BL responses and their biochemical properties in photosynthetic stramenopiles.

Arabidopsis phot1 and phot2 phosphorylate BLUS1 kinase with different efficiencies in stomatal opening.

In Arabidopsis thaliana, phototropins (phot1 and phot2), light-activated receptor kinases, redundantly regulate various photoresponses such as phototropism, chloroplast photorelocation movement, stomatal opening, and leaf flattening. However, it is still unclear how phot1 and phot2 signals are integrated into a common target and regulate physiological responses. In the present study, we provide evidence that phot1 and phot2 phosphorylate BLUE LIGHT SIGNALING1 (BLUS1) kinase as a common substrate in stomatal opening. Biochemical analysis revealed that the recombinant phot2 protein directly phosphorylated BLUS1 in vitro in a blue light-dependent manner, as reported for phot1. BLUS1 phosphorylation was observed in both phot1 and phot2 mutants, and phot2 mutant exhibited higher phosphorylation of BLUS1 than did phot1 mutant. Transgenic plants expressing phot1-GFP (P1G) and phot2-GFP (P2G) at a similar level under the PHOT2 promoter demonstrated that P1G initiated higher phosphorylation of BLUS1 than P2G, suggesting that phot1 phosphorylates BLUS1 more efficiently. Similarly, P1G mediated a higher activation of the plasma membrane H(+)-ATPase and stomatal opening than P2G, indicating that the phosphorylation status of BLUS1 is a key determinant of physiological response. Together, these findings provide insights into the signal integration and different properties of phot1 and phot2 signaling.