Functional characterisation 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 localised 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 s-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

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

葉緑体が光に集まる反応を制御する新たな因子の発見. 京都大学プレスリリース. 2016-08-30.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

Root PRR7 improves the accuracy of the shoot circadian clock through nutrient transport

The circadian clock allows plants to anticipate and adapt to periodic environmental changes. Organ- and tissue-specific properties of the circadian clock and shoot-to-root circadian signaling have been reported. While this long-distance signaling is thought to coordinate physiological functions across tissues, little is known about the feedback regulation of the root clock on the shoot clock in the hierarchical circadian network. Here, we show that the plant circadian clock conveys circadian information between shoots and roots through sucrose and K⁺. We also demonstrate that K+ transport from roots suppresses the variance of period length in shoots and then improves the accuracy of the shoot circadian clock. Sucrose measurements and qPCR showed that root sucrose accumulation was regulated by the circadian clock. Furthermore, root circadian clock genes, including PSEUDO-RESPONSE REGULATOR7 (PRR7), were regulated by sucrose, suggesting the involvement of sucrose from the shoot in the regulation of root clock gene expression. Therefore, we performed time-series measurements of xylem sap and micrografting experiments using prr7 mutants and showed that root PRR7 regulates K⁺ transport and suppresses variance of period length in the shoot. Our modeling analysis supports the idea that root-to-shoot signaling contributes to the precision of the shoot circadian clock. We performed micrografting experiments that illustrated how root PRR7 plays key roles in maintaining the accuracy of shoot circadian rhythms. We thus present a novel directional signaling pathway for circadian information from roots to shoots and propose that plants modulate physiological events in a timely manner through various timekeeping mechanisms

Guard Cell Starch Degradation Yields Glucose for Rapid Stomatal Opening in Arabidopsis.

Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and β-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H+-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H+, K+, and Cl- transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation

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

Identification and Functional Characterization of Inhibitor-3, a Regulatory Subunit of Protein Phosphatase 1 in Plants1[W][OA]

Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase, and mediates diverse cellular processes in animal systems via the association of a catalytic subunit (PP1c) with multiple regulatory subunits that determine the catalytic activity, the subcellular localization, and the substrate specificity. However, no regulatory subunit of PP1 has been identified in plants so far. In this study, we identified inhibitor-3 (Inh3) as a regulatory subunit of PP1 and characterized a functional role of Inh3 in Vicia faba and Arabidopsis (Arabidopsis thaliana). We found Inh3 as one of the proteins interacting with PP1c using a yeast two-hybrid system. Biochemical analyses demonstrated that Arabidopsis Inh3 (AtInh3) bound to PP1c via the RVxF motif of AtInh3, a consensus PP1c-binding sequence both in vitro and in vivo. AtInh3 inhibited the PP1c phosphatase activity in the nanomolar range in vitro. AtInh3 was localized in both the nucleus and cytoplasm, and it colocalized with Arabidopsis PP1c in these compartments. Disruption mutants of AtINH3 delayed the progression of early embryogenesis, arrested embryo development at the globular stage, and eventually caused embryo lethality. Furthermore, reduction of AtINH3 expression by RNA interference led to a decrease in fertility. Transformation of the lethal mutant of inh3 with wild-type AtINH3 restored the phenotype, whereas that with the AtINH3 gene having a mutation in the RVxF motif did not. These results define Inh3 as a regulatory subunit of PP1 in plants and suggest that Inh3 plays a crucial role in early embryogenesis in Arabidopsis

Phototropins Promote Plant Growth in Response to Blue Light in Low Light Environments

Phototropins (phot1 and phot2) are plant-specific blue light receptors for phototropism, chloroplast movement, leaf expansion, and stomatal opening. All these responses are thought to optimize photosynthesis by helping to capture light energy efficiently, reduce photodamage, and acquire CO(2). However, experimental evidence for the promotion of plant growth through phototropins is lacking. Here, we report dramatic phototropin-dependent effects on plant growth. When plants of Arabidopsis thaliana wild type, the phot1 and phot2 mutants, and the phot1 phot2 double mutant were grown under red light, no significant growth differences were observed. However, if a very low intensity of blue light (0.1 μmol m(−2) s(−1)) was superimposed on red light, large increases in fresh weight up to threefold were found in those plants that carried functional PHOT1 genes. When the intensity of blue light was increased to 1 μmol m(−2) s(−1), the growth enhancement was also found in the phot1 single mutant, but not in the double mutant, indicating that phot2 mediated similar responses as phot1 with a lower sensitivity. The effects occurred under low photosynthetically active radiation in particular. The well-known physiological phototropin-mediated responses, including chloroplast movement, stomatal opening, and leaf expansion, in the different lines tested indicated an involvement of these responses in the blue light–induced growth enhancement. We conclude that phototropins promote plant growth by controlling and integrating a variety of responses that optimize photosynthetic performance under low photosynthetically active radiation in the natural environment