Phot2 Mutants Research Articles

Two types of GLR channels cooperate differently in light and dark growth of Arabidopsis seedlings

BackgroundGLutamate Receptor-like (GLR) channels are multimeric, ionotropic, ligand-gated plant transmembrane receptors. They are homologous to mammalian glutamate receptors, iGLuRs, which are critical to neuronal function. GLRs have been reported several times to play a role in photomorphogenesis. However, to date, no study has looked at the mechanism of their involvement in this process. Here we focused on examining the impact of GLRs on the regulation of early seedling growth in blue light, red light, and in the dark.ResultsWild type and six photoreceptor mutant seedlings were grown on media supplemented with known iGLuR/GLR channel antagonists: MK-801, which non-competitively blocks NMDA channels in mammalian cells, and CNQX, known for competitive blocking of AMPA channels in mammalian cells. The lengths of hypocotyls and roots were measured in seedlings of phyA, phyB, phot1, phot2, cry1, and cry2 mutants after 7 days of in vitro culture. Changes in growth parameters, both in light and in darkness upon application of chemical antagonists, show that both types of GLR channels, NMDA-like and AMPA-like, are involved in the regulation of seedling growth irrespective of light conditions. Analysis of seedling growth of photoreceptor mutants indicates that the channels are influenced by signaling from phot1, phot2, and cry1. To extend our analysis, we also evaluated the elicitation of a calcium wave, which is likely to be partially driven by GLRs, in Arabidopsis seedlings. The changes in cellobiose-induced calcium waves observed after applying GLR inhibitors suggest that both types of channels likely cooperate in shaping Arabidopsis seedling growth and development.ConclusionsOur work provides the first experimental evidence that two types of GLR channels function in plants: NMDA-like and AMPA-like. We also demonstrate that the channels are involved in seedling growth and development, at least partially through modulation of calcium signaling, but they are unlikely to play a major role in photomorphogenesis.

Enhancement of the CRISPR/Cas9-Based Genome Editing System in Lettuce (Lactuca sativa L.) Using the Endogenous U6 Promoter.

The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions as well as improving agricultural traits in various crop plants. Here, we optimized a gene-editing system in lettuce (Lactuca sativa L.) using the endogenous U6 promoter and proved that the PHOT2 gene is a versatile target gene. We isolated the LsU6-10 promoter from 10 U6 snRNA genes identified from the lettuce genome database for comparison with the AtU6-26 promoter that has been used to drive sgRNAs in lettuce. Two CRISPR/Cas9 vectors were constructed using the LsU6-10 and AtU6-26 promoters to drive sgRNA361 to target the PHOT2 gene. The chloroplast avoidance response was defective in lettuces with biallelic mutations in the targeted PHOT2 gene, as in the Arabidopsis phot2 mutant. The PHOT2 gene mutations were stably heritable from the R0 to R2 generations, and the high gene-editing efficiency enabled the selection of transgene-free lines in the R1 generation and the establishment of independent phot2 mutants in the R2 generation. Our results suggest that the LsU6-10 promoter is more effective than the AtU6-26 promoter in driving sgRNA for the CRISPR/Cas9 system in lettuce and that PHOT2 is a useful target gene to verify gene editing efficiency without any detrimental effects on plant growth, which is often a consideration in conventional target genes.

Phototropin 1 Mediates High-Intensity Blue Light-Induced Chloroplast Accumulation Response in a Root Phototropism 2-Dependent Manner in Arabidopsis phot2 Mutant Plants.

Phototropins, namely, phototropin 1 (phot1) and phototropin 2 (phot2), mediate chloroplast movement to maximize photosynthetic efficiency and prevent photodamage in plants. Phot1 primarily functions in chloroplast accumulation process, whereas phot2 mediates both chloroplast avoidance and accumulation responses. The avoidance response of phot2-mediated chloroplasts under high-intensity blue light (HBL) limited the understanding of the function of phot1 in the chloroplast accumulation process at the HBL condition. In this study, we showed that the phot2 mutant exhibits a chloroplast accumulation response under HBL, which is defective when the root phototropism 2 (RPT2) gene is mutated in the phot2 background, mimicking the phenotype of the phot1 phot2 double mutant. A further analysis revealed that the expression of RPT2 was induced by HBL and the overexpression of RPT2 could partially enhance the chloroplast accumulation response under HBL. These results confirmed that RPT2 also participates in regulating the phot1-mediated chloroplast accumulation response under HBL. In contrast, RPT2 functions redundantly with neural retina leucine zipper (NRL) protein for chloroplast movement 1 (NCH1) under low-light irradiation. In addition, no chloroplast accumulation response was detected in the phot2 jac1 double mutant under HBL, which has been previously observed in phot2 rpt2 and phot1 phot2 double mutants. Taken together, our results indicated that phot1 mediates the HBL-induced chloroplast accumulation response in an RPT2-dependent manner and is also regulated by j-domain protein required for chloroplast accumulation response 1 (JAC1).

Phototropin2-mediated hypocotyl phototropism is negatively regulated by JAC1 and RPT2 in Arabidopsis

Hypocotyl phototropism is redundantly mediated by phot1 and phot2, two blue light receptor phototropins, under the intensity of blue light>1 μmol m−2 s−1. As light intensity increases, phot1 inhibits the phot2-mediated response. To date, only Arabidopsis Root Phototropism2 (RPT2) has been shown to participate in phot1-mediated inhibition of phototropism. To dissect the signaling network that underlies phot1-mediated inhibition, we carried out a yeast two-hybrid (Y2H) screening assay for RPT2 interacting proteins and identified J-domain protein required for chloroplast accumulation response 1 (JAC1). The interaction between JAC1 and RPT2 was verified by bimolecular fluorescence complementation and Co-IP assays. JAC1 is expressed mainly in cotyledons and hypocotyls. Like RPT2, JAC1 can be induced by blue light, suggesting that it may function similarly to RPT2 in the inhibition of phototropism. Genetic analysis showed that jac1 mutation significantly enhanced the hypocotyl bending of phot1 mutants towards intermediate-intensity blue light, and this effect was inhibited by the constitutive expression of JAC1 in the phot1 jac1 mutant. The phot1 rpt2 double mutant also exhibited enhanced phototropism compared with the phot1 mutant. Taken together, our data clearly demonstrate that JAC1 cooperates with RPT2 to negatively regulate hypocotyl phototropism in plants and may act either downstream of or in parallel with phot1.

Phototropin 1 and 2 Influence Photosynthesis, UV-C Induced Photooxidative Stress Responses, and Cell Death.

Phototropins are plasma membrane-associated photoreceptors of blue light and UV-A/B radiation. The Arabidopsis thaliana genome encodes two phototropins, PHOT1 and PHOT2, that mediate phototropism, chloroplast positioning, and stomatal opening. They are well characterized in terms of photomorphogenetic processes, but so far, little was known about their involvement in photosynthesis, oxidative stress responses, and cell death. By analyzing phot1, phot2 single, and phot1phot2 double mutants, we demonstrated that both phototropins influence the photochemical and non-photochemical reactions, photosynthetic pigments composition, stomata conductance, and water-use efficiency. After oxidative stress caused by UV-C treatment, phot1 and phot2 single and double mutants showed a significantly reduced accumulation of H2O2 and more efficient photosynthetic electron transport compared to the wild type. However, all phot mutants exhibited higher levels of cell death four days after UV-C treatment, as well as deregulated gene expression. Taken together, our results reveal that on the one hand, both phot1 and phot2 contribute to the inhibition of UV-C-induced foliar cell death, but on the other hand, they also contribute to the maintenance of foliar H2O2 levels and optimal intensity of photochemical reactions and non-photochemical quenching after an exposure to UV-C stress. Our data indicate a novel role for phototropins in the condition-dependent optimization of photosynthesis, growth, and water-use efficiency as well as oxidative stress and cell death response after UV-C exposure.

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.

Contributions of cryptochromes and phototropins to stomatal opening through the day.

The UV-A/blue photoreceptors phototropins and cryptochromes are both known to contribute to stomatal opening (Δgs) in blue light. However, their relative contributions to the maintenance of gs in blue light through the whole photoperiod remain unknown. To elucidate this question, Arabidopsis phot1 phot2 and cry1 cry2 mutants (MTs) and their respective wild types (WTs) were irradiated with 200 μmolm-2s-1 of blue-, green- or red-light (BL, GL or RL) throughout a 11-h photoperiod. Stomatal conductance (gs) was higher under BL than under RL or GL. Under RL, gs was not affected by either of the photoreceptor mutations, but under GL gs was slightly lower in cry1 cry2 than its WT. Under BL, the presence of phototropins was essential for rapid stomatal opening at the beginning of the photoperiod, and maximal stomatal opening beyond 3 h of irradiation required both phototropins and cryptochromes. Time courses of whole-plant net carbon assimilation rate (Anet) and the effective quantum yield of PSII photochemistry (ΦPSII) were consistent with an Anet-independent contribution of BL on gs both in phot1 phot2 and cry1 cry2 mutants. The changing roles of phototropins and cryptochromes through the day may allow more flexible coordination between gs and Anet.

The importance of chloroplast movement, nonphotochemical quenching, and electron transport rates in light acclimation and tolerance to high light in Arabidopsis thaliana.

While essential for photosynthesis, excess light can damage plants. We investigated how growth light conditions affect two photoprotective strategies, chloroplast movement and nonphotochemical quenching (NPQ), as well as electron transport rates (ETR), and the relative importance of these processes in the short-term stress tolerance of Arabidopsis thaliana. We grew wild-type (WT) and mutant plants with impaired chloroplast movement (phot1, phot2, phot1 phot2, chup1) or NPQ (npq1) at low (160 μmol photons m-2 s-1 ) or intermediate light (400 μmol photons m-2 s-1 ) before quantifying transmission changes due to chloroplast movement, NPQ, ETR, and the ability to recover from a short-term high-light treatment. Plants with impaired chloroplast avoidance movement (phot2, phot1 phot2, chup1) did not recover as well from a short-term high light treatment as the WT or npq1 and phot1 mutants. Plants grown at intermediate light recovered more completely from the same stress treatment regardless of their genotype and despite reduced degrees of transmission changes due to chloroplast movement. This result was due in part to all genotypes having up to a 2-fold increase in ETRmax and a slight increase in NPQmax . Growth light conditions affect which mechanisms are important in dealing with short-term high-light stress. The chloroplast avoidance response is important for low-light-grown plants, while increases in ETRmax and NPQmax allow plants grown at intermediate light intensities to avoid being damaged.

Chlorophyll fluorescence parameters to assess utilization of excitation energy in photosystem II independently of changes in leaf absorption

Measurement of Pulse-Amplitude-Modulated (PAM) chlorophyll a fluorescence is widely used method for obtaining information on the functional state of photosystem II (PSII). Recently, it has been shown that some of long-established fluorescence parameters must be interpreted with caution, when the light-induced chloroplast movements occur. In our work we have analyzed the effect of chloroplast movements on these parameters. We have derived new parameters that are independent of the change in PSII absorption occurring during measurement. To verify whether there is a need for new parameters or the difference between the parameters commonly used and the newly derived ones is insignificant, we conducted an experiment with Arabidopsis thaliana wild type plants and its phot1 phot2 mutant defective in chloroplast movement. Plants were exposed to light of different qualities (450, 470, 550 or 660 nm) and quantities (100, 400 or 1200 μmol m−2 s−1) for up to 40 min. Since the blue light-induced chloroplast avoidance reaction is a photoprotective mechanism, we expected that phot1 phot2 mutant will compensate the lack of this mechanism by increasing non-photochemical quenching. However, using the light at both 450 and 470 nm, the calculation of commonly used parameter, ΦNPQ (quantum yield of regulated light-induced thermal energy dissipation in PSII) based on Hendrickson et al. [L. Hendrickson, R.T. Furbank, W.S. Chow, Photosynth. Res. 82 (2004) 73–81] showed the opposite. On the other hand, the results obtained using our newly proposed formulae to determine quantum yield of PSII thermal energy dissipation were in line with our assumption. Thus, the experimental data showed that some formulae of fluorescence parameters are dependent on the change in PSII absorption and need to be interpreted carefully. On the contrary, the formulae introduced by us can remove the effect of changes in PSII absorption that occur during measurement, without additional measurements, and give the real estimate of light-induced non-photochemical quenching.

Functional characterization of GhPHOT2 in chloroplast avoidance of Gossypium hirsutum

Chloroplast movement mediated by the plant-specific phototropin blue light photoreceptors is crucial for plants to cope with fluctuating light conditions. While chloroplasts accumulate at weak light-illuminated areas, chloroplast avoidance response mediated primarily by the phototropin2 (phot2) receptor is induced by strong light illumination. Although extensive studies have been performed on phot2-mediated chloroplast avoidance in the model plant Arabidopsis, little is known on the role of the corresponding PHOT2 orthologs in chloroplast movement in cotton. In this study, we found that chloroplast avoidance movement also occurs in the tetraploid G. hirsutum and two diploid species, G. arboreum and G. raimondii, albeit with distinct features. Further bioinformatics and genetic analysis identified the cotton PHOT2 ortholog, GhPHOT2-1, which retained a conserved role in plant chloroplast avoidance movement under strong blue light. Ghphot2-1was localized in the plasma membrane and formed aggregates after high blue light irradiation. Constitutive expression of GhPHOT2-1 restored chloroplast avoidance and accumulation response, as well as phototropism, and leaf flattening characteristics of the Arabidopsis phot2 or phot1 phot2 mutants. On the contrary, silencing of GhPHOT2-1 by virus-induced gene silencing (VIGS) disrupted high blue light-induced chloroplast avoidance movement and caused photo damage in cotton leaves. Taken together, these findings demonstrated that GhPHOT2-1 is a conserved PHOT2 ortholog in regulating chloroplast avoidance and the other aforementioned phot2-mediated responses, implicating its potential role for improving high light tolerance in cotton cultivars.

Functional Analysis of MAX2 in Phototropins-Mediated Cotyledon Flattening in Arabidopsis.

Phototropins (phot1 and phot2) are blue-light receptors that control cotyledon flattening and positioning under strong light; however, their functional redundancy restricts our understanding of the specific roles of phot2. To identify the factors responsible for phot2-dependent cotyledon flattening and growth, we screened for light-insensitive mutants among mutagenized phot1 mutants in Arabidopsis thaliana. The double mutant phot1 lea1 (leaf expansion associated 1), which is defective in cotyledon flattening and positioning but not the phototropic response was selected. This mutant phenotype could be alleviated by constitutively expressing MORE AXILLARY GROWTH 2 (MAX2), indicating that LEA1 was allelic to MAX2. The max2 mutants (max2-2 and max2-3) are defective in cotyledon flattening, which is similar to that of the phot1 phot2 mutants. Moreover, the amounts of MAX2 transcripts are inhibited in leaves of phot1 mutant. However, the additional disruption of PHOT1 gene in max2-2 or max2-3 did not affect their phenotype, including MAX2-mediated inhibition of hypocotyl elongation. By contrast, phototropins-mediated hypocotyl phototropism was not regulated by MAX2. Together, these results suggest that cotyledon flattening was mediated by both phototropins and MAX2 signaling, but the relationship between two pathways need further study.

ROC75 is an Attenuator for the Circadian Clock that Controls LHCSR3 Expression.

Strong light intensity leads to harmful overexcitation of the photosystems in green algae. In Chlamydomonas reinhardtii, LHCSR3 is required for the rapid protective response known as energy-dependent quenching (qE). Because the majority of photoacclimation analysis has been conducted under controlled laboratory conditions, physiological responses to natural environmental changes such as light/dark cycles have not been examined in detail. Regarding fitness in higher plants and microalgae, light-dark cycles represent a major Zeitgeber for synchronizing the circadian clock to multiple physiological responses, yet there is little consensus with respect to the clock response to high-intensity light in photosynthetic organisms. In a previous study, 105 circadian rhythm insertional mutants were isolated as rhythm of chloroplast (roc) mutants. Here, we report our characterization of the roc75 mutant, which exhibited a significantly higher qE value and LHCSR3 protein accumulation when grown under red light. We performed transcript analysis of ROC75 in the pcry (plant-cryptochrome) and phot mutants and found that only the former accumulated lower levels of ROC75 mRNA, suggesting that the blue light photoreceptor pCRY positively regulates ROC75. However, the degradation of pCRY by high-light exposure contributes to prevent over-accumulation of ROC75, which in turn facilitates the PHOT mediated main activation pathway for LHCSR3. Furthermore, LHCSR3 mRNA exhibited a circadian rhythm, though its basal expression level in the roc75 mutant was higher than that in WT. We therefore conclude that ROC75 acts as an attenuator of the circadian clock to control LHCSR3 expression with blue and red light as stimuli for attenuation.

Phot2-regulated relocation of NPH3 mediates phototropic response to high-intensity blue light in Arabidopsis thaliana.

Two redundant blue-light receptors, known as phototropins (phot1 and phot2), influence a variety of physiological responses, including phototropism, chloroplast positioning, and stomatal opening in Arabidopsis thaliana. Whereas phot1 functions in both low- and high-intensity blue light (HBL), phot2 functions primarily in HBL. Here, we aimed to elucidate phot2-specific functions by screening for HBL-insensitive mutants among mutagenized Arabidopsis phot1 mutants. One of the resulting phot2 signaling associated (p2sa) double mutants, phot1 p2sa2, exhibited phototropic defects that could be restored by constitutively expressing NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3), indicating that P2SA2 was allelic to NPH3. It was observed that NPH3-GFP signal mainly localized to and clustered on the plasma membrane in darkness. This NPH3 clustering on the plasma membrane was not affected by mutations in genes encoding proteins that interact with NPH3, including PHOT1, PHOT2 and ROOT PHOTOTROPISM 2 (RPT2). However, the HBL irradiation-mediated release of NPH3 proteins into the cytoplasm was inhibited in phot1 mutants and enhanced in phot2 and rpt2-2 mutants. Furthermore, HBL-induced hypocotyl phototropism was enhanced in phot1 mutants and inhibited in the phot2 and rpt2-2 mutants. Our findings indicate that phot1 regulates the dissociation of NPH3 from the plasma membrane, whereas phot2 mediates the stabilization and relocation of NPH3 to the plasma membrane to acclimate to HBL.

Corrigendum: Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study.

Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells.

Blue light–excited LOV1 and LOV2 domains cooperatively regulate the kinase activity of full-length phototropin2 from Arabidopsis

Phototropin2 (phot2) is a blue-light (BL) receptor that regulates BL-dependent activities for efficient photosynthesis in plants. phot2 comprises two BL-receiving light-oxygen-voltage-sensing domains (LOV1 and LOV2) and a kinase domain. BL-excited LOV2 is thought to be primarily responsible for the BL-dependent activation of the kinase. However, the molecular mechanisms by which small BL-induced conformational changes in the LOV2 domain are transmitted to the kinase remain unclear. Here, we used full-length wild-type and mutant phot2 proteins from Arabidopsis to study their molecular properties in the dark and under BL irradiation. Phosphorylation assays and absorption measurements indicated that the LOV1 domain assists the thermal relaxation of BL-excited LOV2 and vice versa. Using small-angle X-ray scattering and electron microscopy, we observed that phot2 forms a dimer and has a rod shape with a maximum length of 188 Å and a radius of gyration of 44 Å. Under BL, phot2 displayed large conformational changes that bent the rod shape. By superimposing the crystal structures of the LOV1 dimer, LOV2, and a homology model of the kinase to the observed changes, we inferred that the BL-dependent change consisted of positional shifts of both LOV2 and the kinase relative to LOV1. Furthermore, phot2 mutants lacking the photocycle in LOV1 or LOV2 still exhibited conformational changes under BL, suggesting that LOV1 and LOV2 cooperatively contribute to the conformational changes that activate the kinase. These results suggest that BL-activated LOV1 contributes to the kinase activity of phot2. We discuss the possible intramolecular interactions and signaling mechanisms in phot2.

Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study.

Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells.

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.

Phototropin encoded by a single-copy gene mediates chloroplast photorelocation movements in the liverwort Marchantia polymorpha.

Blue-light-induced chloroplast photorelocation movement is observed in most land plants. Chloroplasts move toward weak-light-irradiated areas to efficiently absorb light (the accumulation response) and escape from strong-light-irradiated areas to avoid photodamage (the avoidance response). The plant-specific kinase phototropin (phot) is the blue-light receptor for chloroplast movements. Although the molecular mechanisms for chloroplast photorelocation movement have been analyzed, the overall aspects of signal transduction common to land plants are still unknown. Here, we show that the liverwort Marchantia polymorpha exhibits the accumulation and avoidance responses exclusively induced by blue light as well as specific chloroplast positioning in the dark. Moreover, in silico and Southern-blot analyses revealed that the M. polymorpha genome encodes a single PHOT gene, MpPHOT, and its knockout line displayed none of the chloroplast photorelocation movements, indicating that the sole MpPHOT gene mediates all types of movement. Mpphot was localized on the plasma membrane and exhibited blue-light-dependent autophosphorylation both in vitro and in vivo. Heterologous expression of MpPHOT rescued the defects in chloroplast movement of phot mutants in the fern Adiantum capillus-veneris and the seed plant Arabidopsis (Arabidopsis thaliana). These results indicate that Mpphot possesses evolutionarily conserved regulatory activities for chloroplast photorelocation movement. M. polymorpha offers a simple and versatile platform for analyzing the fundamental processes of phototropin-mediated chloroplast photorelocation movement common to land plants.

The blue light receptor Phototropin 1 suppresses lateral root growth by controlling cell elongation

We investigated the relationship between the blue light receptor phototropin 1 (phot1) and lateral root growth in Arabidopsis thaliana seedlings. Fluorescence and confocal microscopy images, as well as PHOT1 mRNA expression studies provide evidence that it is highly expressed in the elongation zone of lateral roots where auxin is accumulating. However, treatment with the auxin transport inhibitor N-1-naphthylphthalamic acid significantly reduced PHOT1 expression in this zone. In addition, PHOT1 expression was higher in darkness than in light. The total number of lateral roots was higher in the phot1 mutant than in wild-type Arabidopsis. Cells in the elongation zone of lateral roots of the phot1 mutant were longer than those of wild-type lateral roots. These findings suggest that PHOT1 plays a role(s) in elongation of lateral roots through the control of an auxin-related signalling pathway.

TWIN SISTER OF FT, GIGANTEA, and CONSTANS Have a Positive But Indirect Effect on Blue Light-Induced Stomatal Opening in Arabidopsis

FLOWERING LOCUS T (FT) is the major regulatory component controlling photoperiodic floral transition. It is expressed in guard cells and affects blue light-induced stomatal opening induced by the blue-light receptor phototropins phot1 and phot2. Roles for other flowering regulators in stomatal opening have yet to be determined. We show in Arabidopsis (Arabidopsis thaliana) that TWIN SISTER OF FT (TSF), CONSTANS (CO), and GIGANTEA (GI) provide a positive effect on stomatal opening. TSF, which is the closest homolog of FT, was transcribed in guard cells, and light-induced stomatal opening was repressed in tsf-1, a T-DNA insertion mutant of TSF. Overexpression of TSF in a phot1 phot2 mutant background gave a constitutive open-stomata phenotype. Then, we examined whether CO and GI, which are upstream regulators of FT and TSF in photoperiodic flowering, are involved in stomatal opening. Similar to TSF, light-induced stomatal opening was suppressed in the GI and CO mutants gi-1 and co-1. A constitutive open-stomata phenotype was observed in GI and CO overexpressors with accompanying changes in the transcription of both FT and TSF. In photoperiodic flowering, photoperiod is sensed by photoreceptors such as the cryptochromes cry1 and cry2. We examined stomatal phenotypes in a cry1 cry2 mutant and in CRY2 overexpressors. Light-induced stomatal opening was suppressed in cry1 cry2, and the transcription of FT and TSF was down-regulated. In contrast, the stomata in CRY2 overexpressors opened even in the dark, and FT and TSF transcription was up-regulated. We conclude that the photoperiodic flowering components TSF, GI, and CO positively affect stomatal opening.