Light-induced stomatal opening requires phosphorylation of the C-terminal autoinhibitory domain of plasma membrane H+-ATPase.

Plasma membrane H+-ATPase provides the driving force for light-induced stomatal opening. However, the mechanisms underlying the regulation of its activity remain unclear. Here, we show that the phosphorylation of two Thr residues in the C-terminal autoinhibitory domain is crucial for H+-ATPase activation and stomatal opening in Arabidopsis thaliana. Using phosphoproteome analysis, we show that blue light induces the phosphorylation of Thr-881 within the C-terminal region I, in addition to penultimate Thr-948 in AUTOINHIBITED H+-ATPASE 1 (AHA1). Based on site-directed mutagenesis experiments, phosphorylation of both Thr residues is essential for H+ pumping and stomatal opening in response to blue light. Thr-948 phosphorylation is a prerequisite for Thr-881 phosphorylation by blue light. Additionally, red light-driven guard cell photosynthesis induces Thr-881 phosphorylation, possibly contributing to red light-dependent stomatal opening. Our findings provide mechanistic insights into H+-ATPase activation that exploits the ion transport across the plasma membrane and light signalling network in guard cells.

Thin Glass Micro Force Plate Supported by Planar Spiral Springs for Measuring Minute Forces.

Microforce plates are indispensable tools for quantitatively evaluating the behavior of small objects such as tiny insects or microdroplets. The two main measurement principles for microforce plates are: the formation of strain gauges on the beam that supports the plate and the measurement of the deformation of the plate using an external displacement meter. The latter method is characterized by its ease of fabrication and durability as strain concentration is not required. To enhance the sensitivity of the latter type of force plates with a planar structure, thinner plates are generally desired. However, brittle material force plates that are both thin and large and can be fabricated easily have not yet been developed. In this study, a force plate consisting of a thin glass plate with a planar spiral spring structure and a laser displacement meter placed under the plate center is proposed. The plate deforms downward when a force is exerted vertically on its surface, resulting in the determination of the applied force using Hooke's law. The force plate structure is easily fabricated by laser processing combined with the microelectromechanical system (MEMS) process. The fabricated force plate has a radius and thickness of 10 mm and 25 µm, respectively, with four supporting spiral beams of sub-millimeter width. A fabricated force plate featuring a sub-N/m spring constant achieves a resolution of approximately 0.01 µN.

Neuroimaging features of FOXR2-activated CNS neuroblastoma: A case series and systematic review.

BACKGROUND AND PURPOSE: CNS neuroblastoma, FOXR2-activated (CNS NB-FOXR2) is a newly recognized tumor type in the 2021 World Health Organization classification of central nervous system (CNS) tumors. We aimed to investigate the clinical and neuroimaging findings of CNS NB-FOXR2 and systematically review previous publications and three new cases. METHODS: We searched PubMed, SCOPUS, and Embase databases for patients with pathologically proven CNS NB-FOXR2 with sufficient information for preoperative CT and MRI findings. Two board-certified radiologists reviewed the studies and imaging data. RESULTS: Thirty-one patients from six previous publications and 3 patients from our hospital comprised the study population (median age, 4.2 [range: 1.4-16] years; 19 girls). Clinically, CNS NB-FOXR2 mainly affected children between 2 and 6 years (24/34, 67.6%). Nausea/vomiting and seizures were reported as the main presenting symptoms (100% in total). The tumors frequently showed hyperdensity compared to the cortex on nonenhanced CT (4/5, 80%) with calcification along the inner rim of the tumor (4/5, 80%). More than half of patients showed susceptibility artifacts indicating intratumoral hemorrhage and/or calcification (15/28, 53.6%) on T2*- and/or susceptibility-weighted imaging. Elevated relative cerebral blood volume and flow and percentile signal recovery were observed in one case with dynamic susceptibility contrast MRI. CONCLUSIONS: Characteristic imaging features including hyperdense attenuation of the solid components and calcification along the inner rim on CT and susceptibility-weighted imaging may assist with preoperative diagnosis of CNS NB-FOXR2 in pediatric patients.

A Polyimide Film-Based Simple Force Plate for Measuring the Body Mass of Tiny Insects.

Insects exhibit excellent maneuvers such as running and flying despite their small bodies; therefore, their locomotion mechanism is expected to provide a design guideline for micromachines. Numerical simulations have been performed to elucidate this mechanism, whereby it is important to develop a model that is physically identical to the target insect's parts to reproduce kinematic dynamics. In particular, in flight, the shape and mass of wings, which flap at high frequencies, are significant parameters. However, small insects such as fruit flies have small, thin, and light wings; thus, their mass cannot be easily measured. In this study, we proposed a high-resolution and simple force plate to measure the mass of each part of a tiny insect. The device consists of a circular plate supported by flat spiral springs made of polyimide film, and a laser displacement meter that detects the displacement of the center of the plate. The simple plate fabrication process requires only a couple of minutes. A fabricated force plate with a sub-N/m spring constant achieved a resolution of less than 2 µg. As a demonstration, the wing mass of the fruit flies was measured. The experimental results suggest that the wings accounted for approximately 0.4% of the body mass.

Distribution and possible function of galanin about headache and immune system in the rat dura mater.

Galanin (GAL) is a nociceptive transmitter or modulator in the trigeminal sensory system. In this study, GAL expression was investigated in the rat dura mater to demonstrate its possible function in headache using immunohistochemical techniques. The cerebral falx and cerebellar dura mater received abundant blood and nerve supply, and were significantly thicker compared to other portions in the cerebral dura mater. GAL-immunoreactivity was expressed by cell and nerve fiber profiles. Presumed macrophages and dendritic cells contained GAL-immunoreactivity, and co-expressed with CD11b-immunoreactivity. Many isolated and perivascular nerve fibers also showed GAL-immunoreactivity. In addition, GAL-immunoreactive nerve fibers were present in the vicinity of macrophages and dendritic cells with either GAL- or ED1-immunoreactivity. GAL-immunoreactive cells and nerve fibers were common in the cerebral falx and cerebellar dura mater and infrequent in other portions. And, GAL-immunoreactive nerve fibers usually co-expressed calcitonin gene-related peptide (CGRP)-immunoreactivity. In the trigeminal ganglion, a substantial proportion of sensory neurons innervating the dura mater contained GAL-immunoreactivity (mean ± SD, 3.4 ± 2.2%), and co-expressed CGRP-immunoreactivity (2.7 ± 2.1%). The present study may suggest that GAL is associated with nociceptive transduction or modulation in the dura mater. GAL also possibly plays a role in the immune mechanism of the dura mater.

Distribution of alpha-synuclein in the rat cranial sensory ganglia, and oro-cervical regions.

BACKGROUND: Alpha-synuclein (Syn), an unfolded soluble cytosolic protein, is known as a disease-associated protein in the brain. However, little is known about distribution of this protein in the peripheral nervous system. In this study, expression of Syn was investigated in the sensory ganglia of the cranial nerves V, IX and X. METHODS: To analyze distribution of Syn and its co-expression with calcitonin gene-related peptide (CGRP) or the transient receptor potential cation channel subfamily V member 1 (TRPV1), immunohistochemical techniques were used in the rat cranial sensory ganglia and their peripheral tissues. RESULTS: Syn-immunoreactive (-ir) neurons were abundant in the sensory ganglia of the petrosal (56.7%), jugular (28.3%) and nodose ganglia (82.5%). These neurons had small to medium-sized cell bodies (petrosal, mean ± S.D. = 667.4 ± 310.8 µ m2; jugular, 625.1 ± 318.4 µ m2; nodose, 708.3 ± 248.3 µ m2), and were distributed throughout the ganglia. However, the trigeminal ganglion was mostly free of Syn-ir neurons. By double and triple immunofluorescence staining, Syn-ir neurons co-expressed CGRP and TRPV1 in the petrosal and jugular ganglia. Syn-immunoreactivity was expressed by nerve fibers in the epithelium and taste bud of oral and cervical viscerae. These nerve fibers were abundant in the naso-pharynx, epiglottis and laryngeal vestibule. Some taste bud cells were also immunoreactive for Syn. In addition, Syn-ir nerve fibers were detected in the vicinity of macrophages, dendritic cells and Langerhans cells. CONCLUSIONS: Syn was abundant in the visceral sensory neurons but not in somatic sensory neurons. This protein may play a role in nociceptive and chemosensory transduction in the glossopharyngeal and vagal sensory ganglia. It is possible that Syn has a function about the immune mechanism of the upper air way.

A BLUS1 kinase signal and a decrease in intercellular CO2 concentration are necessary for stomatal opening in response to blue light.

Light-induced stomatal opening stimulates CO2 uptake and transpiration in plants. Weak blue light under strong red light effectively induces stomatal opening. Blue light-dependent stomatal opening initiates light perception by phototropins, and the signal is transmitted to a plasma membrane H+-ATPase in guard cells via BLUE LIGHT SIGNALING 1 (BLUS1) kinase. However, it is unclear how BLUS1 transmits the signal to H+-ATPase. Here, we characterized BLUS1 signaling in Arabidopsis thaliana, and showed that the BLUS1 C-terminus acts as an auto-inhibitory domain and that phototropin-mediated Ser-348 phosphorylation within the domain removes auto-inhibition. C-Terminal truncation and phospho-mimic Ser-348 mutation caused H+-ATPase activation in the dark, but did not elicit stomatal opening. Unexpectedly, the plants exhibited stomatal opening under strong red light and stomatal closure under weak blue light. A decrease in intercellular CO2 concentration via red light-driven photosynthesis together with H+-ATPase activation caused stomatal opening. Furthermore, phototropins caused H+-ATPase dephosphorylation in guard cells expressing constitutive signaling variants of BLUS1 in response to blue light, possibly for fine-tuning stomatal opening. Overall, our findings provide mechanistic insights into the blue light regulation of stomatal opening.

CIPK23 regulates blue light-dependent stomatal opening in Arabidopsis thaliana.

Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL-interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull-down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin-mediated responses. We further found that blue light activation of inward-rectifying K+ (K+ in ) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+ -ATPase was not. The blue light activation of K+ in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+ in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+ -ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.

Autophagy controls reactive oxygen species homeostasis in guard cells that is essential for stomatal opening.

Reactive oxygen species (ROS) function as key signaling molecules to inhibit stomatal opening and promote stomatal closure in response to diverse environmental stresses. However, how guard cells maintain basal intracellular ROS levels is not yet known. This study aimed to determine the role of autophagy in the maintenance of basal ROS levels in guard cells. We isolated the Arabidopsis autophagy-related 2 (atg2) mutant, which is impaired in stomatal opening in response to light and low CO2 concentrations. Disruption of other autophagy genes, including ATG5, ATG7, ATG10, and ATG12, also caused similar stomatal defects. The atg mutants constitutively accumulated high levels of ROS in guard cells, and antioxidants such as ascorbate and glutathione rescued ROS accumulation and stomatal opening. Furthermore, the atg mutations increased the number and aggregation of peroxisomes in guard cells, and these peroxisomes exhibited reduced activity of the ROS scavenger catalase and elevated hydrogen peroxide (H2O2) as visualized using the peroxisome-targeted H2O2 sensor HyPer. Moreover, such ROS accumulation decreased by the application of 2-hydroxy-3-butynoate, an inhibitor of peroxisomal H2O2-producing glycolate oxidase. Our results showed that autophagy controls guard cell ROS homeostasis by eliminating oxidized peroxisomes, thereby allowing stomatal opening.

Guard cell photosynthesis is crucial in abscisic acid-induced stomatal closure.

Reactive oxygen species (ROS) are ubiquitous signaling molecules involved in diverse physiological processes, including stomatal closure. Photosynthetic electron transport (PET) is the main source of ROS generation in plants, but whether it functions in guard cell signaling remains unclear. Here, we assessed whether PET functions in abscisic acid (ABA) signaling in guard cells. ABA-elicited ROS were localized to guard cell chloroplasts in Arabidopsis thaliana, Commelina benghalensis, and Vicia faba in the light and abolished by the PET inhibitors 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea and 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. These inhibitors reduced ABA-induced stomatal closure in all three species, as well as in the NADPH oxidase-lacking mutant atrboh D/F. However, an NADPH oxidase inhibitor did not fully eliminate ABA-induced ROS in the chloroplasts, and ABA-induced ROS were still observed in the guard cell chloroplasts of atrboh D/F. This study demonstrates that ROS generated through PET act as signaling molecules in ABA-induced stomatal closure and that this occurs in concert with ROS derived through NADPH oxidase.

Transient receptor potential melastatin-3 in the rat sensory ganglia of the trigeminal, glossopharyngeal and vagus nerves.

Transient receptor potential melastatin-3 (TRPM3) is a nonselective cation channel, has permeability of Ca2+, and probably participates in thermosensitive nociception. In this study, immunohistochemistry for TRPM3 was conducted in the rat trigeminal, glossopharyngeal and vagal sensory ganglia. TRPM3-immunoreactivity was expressed by half of sensory neurons in the trigeminal (TG), petrosal (PG) and jugular ganglia (JG), and by about 80% of sensory neurons in the nodose ganglion (NG). They mostly had small to medium-sized cell bodies. A trichrome immunofluorescence method showed co-existence of TRPM3 with TRP vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP). Approximately 70% of TRPM3-immunoreactive (-IR) neurons contained TRPV1-immunoreactivity in all the examined ganglia. More than 40% of TRPM3-IR neurons exhibited CGRP-immunoreactivity in the TG, PG and JG. Only a few sensory neurons co-expressed TRPM3- and CGRP-immunoreactivity in the NG. In addition, more than 40% of TRPM3-IR neurons bound to isolectin B4 in all the examined ganglia. By combination of retrograde tracing method and immunohistochemistry, half of TG neurons innervating the facial skin and incisive papilla expressed TRPM3-immunoreactivity whereas approximately 20% of those innervating the tooth pulp contained TRPM3-immunoreactivity. Co-expression of TRPM3-immunoreactivity with TRPV1- or CGRP-immunoreactivity was common among cutaneous and papillary TG neurons but not among pulpal TG neurons. More than 60% of PG and JG neurons innervating the external ear canal skin and circumvallate papilla contained TRPM3-immunoreactivity. Co-expression of TRPM3 with TRPV1 or CGRP was common among PG and JG neurons innervating the external ear canal skin. However, a smaller number of TRPM3-IR neurons co-expressing TRPV1- or CGRP-immunoreactivity innervate the circumvallate papilla in the PG. The present study suggests that expression of TRPM3 and its co-existence with TRPV1 and CGRP in sensory neurons depend on the variety of their peripheral targets in the trigeminal, glossopharyngeal and vagal nervous systems.

Stomatal response to blue light in crassulacean acid metabolism plants Kalanchoe pinnata and Kalanchoe daigremontiana.

Blue light (BL) is a fundamental cue for stomatal opening in both C3 and C4 plants. However, it is unknown whether crassulacean acid metabolism (CAM) plants open their stomata in response to BL. We investigated stomatal BL responses in the obligate CAM plants Kalanchoe pinnata and Kalanchoe daigremontiana that characteristically open their stomata at night and close them for part of the day, as contrasted with C3 and C4 plants. Stomata opened in response to weak BL superimposed on background red light in both intact leaves and detached epidermal peels of K. pinnata and K. daigremontiana. BL-dependent stomatal opening was completely inhibited by tautomycin and vanadate, which repress type 1 protein phosphatase and plasma membrane H+-ATPase, respectively. The plasma membrane H+-ATPase activator fusicoccin induced stomatal opening in the dark. Both BL and fusicoccin induced phosphorylation of the guard cell plasma membrane H+-ATPase in K. pinnata. These results indicate that BL-dependent stomatal opening occurs in the obligate CAM plants K. pinnata and K. daigremontiana independently of photosynthetic CO2 assimilation mode.

The transient receptor potential cation channel subfamily V members 1 and 2, P2X purinoceptor 3 and calcitonin gene-related peptide in sensory neurons of the rat trigeminal ganglion, innervating the periosteum, masseter muscle and facial skin.

OBJECTIVE: Distribution of the transient receptor potential cation channel subfamily V members 1 (TRPV1) and 2 (TRPV2), and P2X purinoceptor 3 (P2 × 3) was investigated in rat trigeminal ganglion neurons innervating the periosteum, masseter muscle and facial skin. DESIGN: Double immunofluorescence method for TRPV1 and TRPV2 ion channels or ATP receptor P2 × 3 with calcitonin gene-related peptide (CGRP) was performed on trigeminal ganglion neurons retrogradely labeled from the mandibular periosteum, masseter muscle, or facial skin in 15 male Wistar rats. RESULTS: The cell size of periosteum neurons (mean ± S.D. = 810.7 ± 36.1 µ m2) was smaller than that of masseter muscle neurons (927.0 ± 75.6 µ m2), and larger than that of facial skin neurons (661.3 ± 82.2 µ m2). Periosteum neurons contained TRPV1- (26.7%), TRPV2- (47.1%) and P2 × 3-immunoreactivity (50.1%). Expression of TRPV2-immunoreactivity was more abundant among periosteum neurons than among facial skin neurons (16.1%). Regarding to TRPV1 and P2 × 3 expression, however, there was no significant difference between periosteum neurons and, masseter muscle and facial skin neurons. TRPV1- immunoreactive trigeminal ganglion neurons which innervated the periosteum, masseter muscle and facial skin mostly had small and medium-sized cell bodies, whereas TRPV2- and P2 × 3-immunoreactive trigeminal ganglion neurons innervating those tissues were of various cell body sizes. Approximately 20% of periosteum (19.2%), masseter muscle (19.2%) and facial skin (21.5%) neurons contained both TRPV1- and CGRP-immunoreactivity. Some periosteum neurons also co-expressed CGRP-immunoreactivity with TRPV2- (10.9%) or P2 × 3- immunoreactivity (11.1%). Distributions of perivascular and free nerve fibers containing CGRP and either TRPV1, TRPV2, or P2 × 3 were occasionally very similar in the mandibular periosteum. CONCLUSIONS: The present study indicated that trigeminal ganglion nociceptors innervating the periosteum as well as those innervating the masseter muscle and facial skin have vanilloid, acidic, thermal, mechanical and ATP sensors. In some periosteum neurons, CGRP may act as inflammatory mediator through activation of TRPV1, TRPV2 and P2 × 3.

Blue light and CO2 signals converge to regulate light-induced stomatal opening.

Stomata regulate gas exchange between plants and atmosphere by integrating opening and closing signals. Stomata open in response to low CO2 concentrations to maximize photosynthesis in the light; however, the mechanisms that coordinate photosynthesis and stomatal conductance have yet to be identified. Here we identify and characterize CBC1/2 (CONVERGENCE OF BLUE LIGHT (BL) AND CO2 1/2), two kinases that link BL, a major component of photosynthetically active radiation (PAR), and the signals from low concentrations of CO2 in guard cells. CBC1/CBC2 redundantly stimulate stomatal opening by inhibition of S-type anion channels in response to both BL and low concentrations of CO2. CBC1/CBC2 function in the signaling pathways of phototropins and HT1 (HIGH LEAF TEMPERATURE 1). CBC1/CBC2 interact with and are phosphorylated by HT1. We propose that CBCs regulate stomatal aperture by integrating signals from BL and CO2 and act as the convergence site for signals from BL and low CO2.

Reconstitution of Abscisic Acid Signaling from the Receptor to DNA via bHLH Transcription Factors.

The plant hormone abscisic acid (ABA) confers drought tolerance in plants through stomatal closure and regulation of gene expression. The complex consisting of the ABA receptor PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR), type 2C protein phosphatase (PP2C), and SNF1-related protein kinase 2 (SnRK2) has a key role in ABA signaling. Basic helix-loop-helix (bHLH) transcriptional activator ABA-RESPONSIVE KINASE SUBSTRATE1 (AKS1, also known as FBH3) is released from DNA by phosphorylation-induced monomerization in response to ABA in guard cells. Here we reconstituted the release of AKS1 from DNA via the ABA signaling core complex in vitro. We first obtained evidence to confirm that AKS1 is an endogenous substrate for SnRK2s. Phosphorylation of AKS1 and activation of SnRK2 showed the same time course in response to ABA in guard cells. AKS1 was bound to SnRK2.6 in vivo. Three ABA-responsive SnRK2s (SnRK2.2/SRK2D, SnRK2.3/SRK2I, and SnRK2.6/SRK2E/OST1) phosphorylated AKS1 in vitro, and the phosphorylation was eliminated by the triple mutation of SnRK2s in plants. We reconstituted the AKS1 phosphorylation in vitro via the signaling complex containing the ABA receptor PYR1, a PP2C, HYPERSENSITIVE TO ABA1 (HAB1), and a protein kinase, SnRK2.6 in response to ABA We further reconstituted the release of AKS1 from the target gene of POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1 (KAT1) via the complex in response to ABA These results demonstrate that AKS1 provides a link between the signaling complex and ABA-responsive genes and furnish evidence for a minimal signaling mechanism from ABA perception to DNA.

Brassinosteroid Involvement in Arabidopsis thaliana Stomatal Opening.

Stomata within the plant epidermis regulate CO2 uptake for photosynthesis and water loss through transpiration. Stomatal opening in Arabidopsis thaliana is determined by various factors, including blue light as a signal and multiple phytohormones. Plasma membrane transporters, including H+-ATPase, K+ channels and anion channels in guard cells, mediate these processes, and the activities and expression levels of these components determine stomatal aperture. However, the regulatory mechanisms involved in these processes are not fully understood. In this study, we used infrared thermography to isolate a mutant defective in stomatal opening in response to light. The causative mutation was identified as an allele of the brassinosteroid (BR) biosynthetic mutant dwarf5. Guard cells from this mutant exhibited normal H+-ATPase activity in response to blue light, but showed reduced K+ accumulation and inward-rectifying K+ (K+in) channel activity as a consequence of decreased expression of major K+in channel genes. Consistent with these results, another BR biosynthetic mutant, det2-1, and a BR receptor mutant, bri1-6, exhibited reduced blue light-dependent stomatal opening. Furthermore, application of BR to the hydroponic culture medium completely restored stomatal opening in dwarf5 and det2-1 but not in bri1-6. However, application of BR to the epidermis of dwarf5 did not restore stomatal response. From these results, we conclude that endogenous BR acts in a long-term manner and is required in guard cells with the ability to open stomata in response to light, probably through regulation of K+in channel activity.

Determination of H+-ATPase Activity in Arabidopsis Guard Cell Protoplasts through H+-pumping Measurement and H+-ATPase Quantification.

The opening of stomata in plants in response to blue light is driven by the plasma membrane H+-ATPase in guard cells. To evaluate the activation of the H+-ATPase in vivo, we can use H+-pumping by guard cells in response to blue light and fusicoccin. To do this, it is required to prepare a large amount of guard cell protoplasts and measure H+-pumping in the protoplasts. It is also necessary to determine the protein amount of H+-ATPase. In this protocol, we describe the procedures required for these preparations and measurements.

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.

Functional characterization of Arabidopsis phototropin 1 in the hypocotyl apex.

Phototropin (phot1) is a blue light-activated plasma membrane-associated kinase that acts as the principal photoreceptor for shoot phototropism in Arabidopsis in conjunction with the signalling component Non-Phototropic Hypocotyl 3 (NPH3). PHOT1 is uniformly expressed throughout the Arabidopsis hypocotyl, yet decapitation experiments have localized the site of light perception to the upper hypocotyl. This prompted us to investigate in more detail the functional role of the hypocotyl apex, and the regions surrounding it, in establishing phototropism. We used a non-invasive approach where PHOT1-GFP (P1-GFP) expression was targeted to the hypocotyl apex of the phot-deficient mutant using the promoters of CUP-SHAPED COTYLEDON 3 (CUC3) and AINTEGUMENTA (ANT). Expression of CUC3::P1-GFP was clearly visible at the hypocotyl apex, with weaker expression in the cotyledons, whereas ANT::P1-GFP was specifically targeted to the developing leaves. Both lines showed impaired curvature to 0.005 µmol m-2  sec-1 unilateral blue light, indicating that regions below the apical meristem are necessary for phototropism. Curvature was however apparent at higher fluence rates. Moreover, CUC3::P1-GFP partially or fully complemented petiole positioning, leaf flattening and chloroplast accumulation, but not stomatal opening. Yet, tissue analysis of NPH3 de-phosphorylation showed that CUC3::P1-GFP and ANT::P1-GFP mis-express very low levels of phot1 that likely account for this responsiveness. Our spatial targeting approach therefore excludes the hypocotyl apex as the site for light perception for phototropism and shows that phot1-mediated NPH3 de-phosphorylation is tissue autonomous and occurs more prominently in the basal hypocotyl.

The Plasma Membrane H+-ATPase AHA1 Plays a Major Role in Stomatal Opening in Response to Blue Light.

Stomata open in response to a beam of weak blue light under strong red light illumination. A blue light signal is perceived by phototropins and transmitted to the plasma membrane H(+)-ATPase that drives stomatal opening. To identify the components in this pathway, we screened for mutants impaired in blue light-dependent stomatal opening. We analyzed one such mutant, provisionally named blus2 (blue light signaling2), and found that stomatal opening in leaves was impaired by 65%, although the magnitude of red light-induced opening was not affected. Blue light-dependent stomatal opening in the epidermis and H(+) pumping in guard cell protoplasts were inhibited by 70% in blus2 Whole-genome resequencing identified a mutation in the AHA1 gene of the mutant at Gly-602. T-DNA insertion mutants of AHA1 exhibited a similar phenotype to blus2; this phenotype was complemented by the AHA1 gene. We renamed blus2 as aha1-10 T-DNA insertion mutants of AHA2 and AHA5 did not show any impairment in stomatal response, although the transcript levels of AHA2 and AHA5 were higher than those of AHA1 in wild-type guard cells. Stomata in ost2, a constitutively active AHA1 mutant, did not respond to blue light. A decreased amount of H(+)-ATPase in aha1-10 accounted for the reduced stomatal blue light responses and the decrease was likely caused by proteolysis of misfolded AHA1. From these results, we conclude that AHA1 plays a major role in blue light-dependent stomatal opening in Arabidopsis and that the mutation made the AHA1 protein unstable in guard cells.