Phototropins are blue-light receptors that regulate diverse light-driven processes, including phototropism, chloroplast movement, stomatal opening, leaf positioning, and leaf flattening, all of which optimise photosynthesis and promote plant growth. These autophosphorylating serine/threonine kinases initiate signalling from the plasma membrane by altering protein phosphorylation patterns. Nearly 3 decades of research have uncovered numerous phosphorylation events pivotal for distinct phototropin-mediated responses. Here, we summarise major advances in understanding phototropin autophosphorylation, the identification of substrate proteins, and how phosphorylation at specific sites, collectively termed the phototropin phosphocode, controls protein function. We also discuss the emerging role of protein phosphatases in modulating this phosphocode and the potential to engineer these regulatory mechanisms to enhance photosynthetic performance and plant biomass.

Phototropin receptor kinases (phot1 and phot2) enhance photosynthesis by coordinating light responses such as phototropism and chloroplast repositioning under low blue light conditions. These processes rely on NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)/ROOT PHOTOTROPISM 2-like (NRL) proteins. The Arabidopsis (Arabidopsis thaliana) NRL protein NPH3 is phosphorylated by phot1 at S744, a modification that alters its localization and promotes hypocotyl phototropism. Although reversible phosphorylation of S744 is necessary for reassembling an active phot1-NPH3 complex at the plasma membrane, the mechanisms driving NPH3 dephosphorylation remain unclear. Here, we show that clade L TYPE 2C PROTEIN PHOSPHATASES (PP2C19 and PP2C35) regulate S744 phosphorylation and NPH3 function. Mutants lacking PP2C19 exhibit sustained S744 phosphorylation and reduced phototropism, with greater defects observed in pp2c19 pp2c35 double mutants, indicating functional redundancy between these phosphatases. Moreover, clade L pp2c mutants display altered RPT2 phosphorylation and abundance and modulate the phosphorylation status of NRL PROTEIN FOR CHLOROPLAST MOVEMENT 1 (NCH1), suggesting broader regulatory control over phot1-NRL targets. Consistent with this, phototropin-dependent chloroplast accumulation is impaired in both Arabidopsis and Marchantia pp2c19 mutants. These findings identify clade L PP2Cs as key regulators of auxin-dependent (phototropism) and auxin-independent (chloroplast accumulation movement) light responses, which, together with their additional role in regulating hypocotyl gravitropism, are particularly important for seedling emergence and establishment from beneath the soil surface.

SUMMARYDirectional movements impact the ability of plants to respond and adjust their growth accordingly to the prevailing light environment. The plasma‐membrane associated protein, ROOT PHOTOTROPISM 2 (RPT2) is a key signalling component involved in chloroplast accumulation movement, leaf positioning, and phototropism, all of which are regulated redundantly by the ultraviolet/blue light‐activated AGC kinases phototropin 1 and 2 (phot1 and phot2). We recently demonstrated that members of the NON‐PHOTOTROPIC HYPOCOTYL 3 (NPH3)/RPT2‐like (NRL) family in Arabidopsis thaliana, including RPT2, are directly phosphorylated by phot1. However, whether RPT2 is a substrate for phot2, and the biological significance of phot phosphorylation of RPT2 remains to be determined. Here, we show that RPT2 is phosphorylated by both phot1 and phot2 at a conserved serine residue (S591) within the C‐terminal region of the protein. Blue light triggered the association of 14‐3‐3 proteins with RPT2 consistent with S591 acting as a 14‐3‐3 binding site. Mutation of S591 had no effect on the plasma membrane localization of RPT2 but reduced its functionality for leaf positioning and phototropism. Moreover, our findings indicate that S591 phosphorylation within the C‐terminus of RPT2 is required for chloroplast accumulation movement to low level blue light. Taken together, these findings further highlight the importance of the C‐terminal region of NRL proteins and how its phosphorylation contributes to phot receptor signalling in plants.

Polarity underlies all plant physiology and directional growth responses such as phototropism. Yet, our understanding of how plant tropic responses are established is far from complete. The plasma-membrane associated BTB-containing protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by AGC kinases known as the phototropins (phots). However, the mechanism by which phots initiate phototropic signalling via NPH3, and other NPH3/RPT2-like (NRL) members, has remained unresolved. Here we demonstrate that NPH3 is directly phosphorylated by phot1 both in vitro and in vivo . Light-dependent phosphorylation within a conserved consensus sequence (RxS) located at the extreme C-terminus of NPH3 is necessary to promote its functionality for phototropism and petiole positioning in Arabidopsis . Phosphorylation of this region by phot1 also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Seedlings expressing mutants of NPH3 that are unable to bind or constitutively bind 14-3-3s show compromised functionality that is consistent with a model where signalling outputs arising from a gradient in NPH3 RxS phosphorylation/localisation across the stem are a major contributor to phototropic responsiveness. Our current findings provide further evidence that 14-3-3 proteins are instrumental components regulating auxin-dependent growth and show for the first time that NRL proteins are direct phosphorylation targets for plant AGC kinases. Moreover, the C-terminal phosphorylation site/14-3-3-binding motif of NPH3 is conserved in several members of the NRL family, suggesting a common mechanism of regulation.

The ability to enhance photosynthetic capacity remains a recognized bottleneck to improving plant productivity. Phototropin blue light receptors (phot1 and phot2) optimize photosynthetic efficiency in Arabidopsis thaliana by coordinating multiple light-capturing processes. In this study, we explore the potential of using protein engineering to improve photoreceptor performance and thereby plant growth. We demonstrate that targeted mutagenesis can decrease or increase the photocycle lifetime of Arabidopsis phototropins in vitro and show that these variants can be used to reduce or extend the duration of photoreceptor activation in planta. Our findings show that slowing the phototropin photocycle enhanced several light-capturing responses, while accelerating it reduced phototropin's sensitivity for chloroplast accumulation movement. Moreover, plants engineered to have a slow-photocycling variant of phot1 or phot2 displayed increased biomass production under low-light conditions as a consequence of their improved sensitivity. Together, these findings demonstrate the feasibility of engineering photoreceptors to manipulate plant growth and offer additional opportunities to enhance photosynthetic competence, particularly under suboptimal light regimes.

Phototropin (phot) receptor kinases play important roles in promoting plant growth by controlling light-capturing processes, such as phototropism. Phototropism is mediated through the action of NON-PHOTOTROPIC HYPOCOTYL3 (NPH3), which is dephosphorylated following phot activation. However, the functional significance of this early signaling event remains unclear. Here, we show that the onset of phototropism in dark-grown (etiolated) seedlings of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) is enhanced by greening (deetiolation). Red and blue light were equally effective in promoting phototropism in Arabidopsis, consistent with our observations that deetiolation by phytochrome or cryptochrome was sufficient to enhance phototropism. Increased responsiveness did not result from an enhanced sensitivity to the phytohormone auxin, nor does it involve the phot-interacting protein, ROOT PHOTOTROPISM2. Instead, deetiolated seedlings showed attenuated levels of NPH3 dephosphorylation and diminished relocalization of NPH3 from the plasma membrane during phototropism. Likewise, etiolated seedlings that lack the PHYTOCHROME-INTERACTING FACTORS (PIFs) PIF1, PIF3, PIF4, and PIF5 displayed reduced NPH3 dephosphorylation and enhanced phototropism, consistent with their constitutive photomorphogenic phenotype in darkness. Phototropic enhancement could also be achieved in etiolated seedlings by lowering the light intensity to diminish NPH3 dephosphorylation. Thus, phototropism is enhanced following deetiolation through the modulation of a phosphorylation rheostat, which in turn sustains the activity of NPH3. We propose that this dynamic mode of regulation enables young seedlings to maximize their establishment under changing light conditions, depending on their photoautotrophic capacity.

How plants perceive and respond to temperature remains an important question in the plant sciences. Temperature perception and signal transduction may occur through temperature-sensitive intramolecular folding of primary mRNA transcripts. Recent studies suggested a role for retention of the first intron in the 5'UTR of the clock component LATE ELONGATED HYPOCOTYL (LHY) in response to changes in temperature. Here, we identified a set of haplotypes in the LHY 5'UTR, examined their global spatial distribution, and obtained evidence that haplotype can affect temperature-dependent splicing of LHY transcripts. Correlations of haplotype spatial distributions with global bioclimatic variables and altitude point to associations with annual mean temperature and temperature fluctuation. Relatively rare relict type accessions correlate with lower mean temperature and greater temperature fluctuation and the spatial distribution of other haplotypes may be informative of evolutionary processes driving colonization of ecosystems. We propose that haplotypes may possess distinct 5'UTR pre-mRNA folding thermodynamics and/or specific biological stabilities based around the binding of trans-acting RNA splicing factors, a consequence of which is scalable splicing sensitivity of a central clock component that is likely tuned to specific temperature environments.

Plasma membrane receptors play fundamental roles in shaping plant growth and development. A large proportion of these are autophosphorylating Ser/Thr kinases (De Smet et al., 2009). Some function as dualspecificity kinases, autophosphorylating additionally on Tyr residues, one of which is the extensively studied steroid receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1; Oh et al., 2009). Light regulation of plant growth also is mediated by plasma membrane-bound Ser/Thr kinases known as the phototropins (phots; Fankhauser and Christie, 2015). Seed plants contain two phots (phot1 and phot2) that have important roles in regulating leaf positioning and expansion, chloroplast photorelocation movement, stomatal opening, and phototropism, all of which serve to optimize photosynthetic efficiency (Christie et al., 2015). Phots are members of the AGCVIII kinase family (Rademacher and Offringa, 2012) but are distinct from transmembrane receptor kinases such as BRI1, as they are hydrophilic and bind to the intracellular side of the plasma membrane (Kong et al., 2013) to initiate signaling (Preuten et al., 2015). Although the ability to associate with the plasma membrane is conserved in algal phots (Sullivan et al., 2016a), the mechanism underlying this attachment is still not known, but it is thought to involve some form of lipid binding/ modification. Kinase-inactive versions of phot1 and phot2 are nonfunctional, highlighting the importance of receptor autophosphorylation in phot signaling (Inoue et al., 2008a, 2011). Autophosphorylation occurs predominantly on multiple Ser residues. At least 21 and 29 phosphorylation sites have been identified for Arabidopsis (Arabidopsis thaliana) phot1 and phot2, respectively (Christie et al., 2015). Most of these sites are found in the N terminus of the protein, which contains two light-sensing modules known as LIGHT, OXYGEN, OR VOLTAGE SENSING (LOV) domains known as LOV1 and LOV2 (Christie et al., 2012). Autophosphorylation of Ser-350, Ser-376, and Ser-410 within the LOV-linker region promotes the binding of 14-3-3 regulatory proteins to phot1 (Inoue et al., 2008a; Sullivan et al., 2009). However, the biological significance of this interaction is still not known. The occurrence of some these phosphorylation sites also is fluence rate dependent (Salomon et al., 2003) and is thought to play a role in receptor desensitization (Christie and Murphy, 2013). By contrast, the autophosphorylation of two conserved Ser residues within the activation loop of the C-terminal kinase domain (Ser-849 and Ser-851 in phot1 and Ser-761 or Ser-763 in phot2) is necessary for receptor signaling (Inoue et al., 2008a, 2011). Mutation of these sites to Ala impairs phot1 function in Arabidopsis, whereas phosphomimetic substitutions to Asp are without effect (Inoue et al., 2008a).

Plasma membrane receptors play fundamental roles in shaping plant growth and development. A large proportion of these are autophosphorylating Ser/Thr kinases ([De Smet et al., 2009][1]). Some function as dual-specificity kinases, autophosphorylating additionally on Tyr residues, one of which is the

Phototropins (phots) are plasma membrane–associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light–absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A′α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A′α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation.

Update on Blue Light Signaling 1 2 Shining Light on the Function of NPH3/RPT2-like Proteins in Phototropin Signalling 3 4 John M. Christie*, Noriyuki Suetsugu, Stuart Sullivan and Masamitsu Wada 5 6 Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life 7 Sciences, Bower Building, University of Glasgow, Glasgow G12 8QQ, UK 8 Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan 9 Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, 10 Japan 11 12 Corresponding Author: John M. Christie 13 Tel: +44 141 330 239; Email john.christie@glasgow.ac.uk 14 15 This work was supported by funding from the UK Biotechnology and Biological Sciences 16 Research Council (BB/M002128/1 to J.M.C) and the Grant-in-Aid for Scientific Research Grant 17 from the Japan Society for the Promotion of Science (15KK0254 to N.S.). 18 19 Summary: NRL proteins coordinate different aspects of phototropin signaling through signaling 20 processes that are conserved in land plants and algae 21 22 Plant Physiology Preview. Published on July 18, 2017, as DOI:10.1104/pp.17.00835

Summary Circadian clocks allow the temporal compartmentalization of biological processes. In Arabidopsis, circadian rhythms display organ specificity but the underlying molecular causes have not been identified. We investigated the mechanisms responsible for the similarities and differences between the clocks of mature shoots and roots in constant conditions and in light : dark cycles. We developed an imaging system to monitor clock gene expression in shoots and light‐ or dark‐grown roots, modified a recent mathematical model of the Arabidopsis clock and used this to simulate our new data. We showed that the shoot and root circadian clocks have different rhythmic properties (period and amplitude) and respond differently to light quality. The root clock was entrained by direct exposure to low‐intensity light, even in antiphase to the illumination of shoots. Differences between the clocks were more pronounced in conditions where light was present than in constant darkness, and persisted in the presence of sucrose. We simulated the data successfully by modifying those parameters of a clock model that are related to light inputs. We conclude that differences and similarities between the shoot and root clocks can largely be explained by organ‐specific light inputs. This provides mechanistic insight into the developing field of organ‐specific clocks.

Hypocotyl phototropism of etiolated Arabidopsis seedlings is primarily mediated by the blue-light receptor kinase phototropin 1 (phot1). Phot1-mediated curvature to continuous unilateral blue light irradiation (0.5 µmol m-2 s-1) is enhanced by overhead pre-treatment with red light (20 µmol m-2 s-1 for 15 min) through the action of phytochrome (phyA). Here, we show that pre-treatment with blue light is equally as effective in eliciting phototropic enhancement and is dependent on phyA. Although blue pre-treatment was sufficient to activate early phot1 signalling events, phot1 autophosphorylation in vivo was not found to be saturated, as assessed by subsequently measuring phot1 kinase activity in vitro. However, enhancement effects to red and blue pre-treatment were not observed at higher intensities of phototropic stimulation (10 µmol m-2 s-1). Phototropic enhancement to red and blue pre-treatments to 0.5 µmol m-2 s-1 unilateral blue light irradiation was also lacking in transgenic Arabidopsis where PHOT1 expression was restricted to the epidermis. Together, these findings indicate that phyA-mediated effects on phot1 signalling are restricted to low intensities of phototropic stimulation and originate from tissues other than the epidermis.

Phototropins (phots) regulate a range of adaptive processes in plants that serve to optimize photosynthetic efficiency and promote growth. Light sensing by Arabidopsis thaliana phots is predominantly mediated by the Light, Oxygen and Voltage sensing 2 (LOV2) flavin-binding motif located within the N-terminus of the photoreceptor. Here we characterize the photochemical and biochemical properties of phot from the marine picoalga Ostreococcus tauri phototropin (Otphot) and examine its ability to replace phot-mediated function in Arabidopsis. Photochemical properties of Otphot rely on both LOV1 and LOV2. Yet, biochemical analysis indicates that light-dependent receptor autophosphorylation is primarily dependent on LOV2. As found for Arabidopsis phots, Otphot associates with the plasma membrane and partially internalizes, albeit to a limited extent, in response to blue-light irradiation. Otphot is able to elicit a number of phot-regulated processes in Arabidopsis, including petiole positioning, leaf expansion, stomatal opening and chloroplast accumulation movement. However, Otphot is unable to restore phototropism and chloroplast avoidance movement. Consistent with its lack of phototropic function in Arabidopsis, Otphot does not associate with or trigger dephosphorylation of the phototropic signalling component Non-Phototropic Hypocotyl 3 (NPH3). Taken together, these findings indicate that the mechanism of action of plant and evolutionarily distant algal phots is less well conserved than previously thought.

SummaryPhototropin (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.

Plants depend on the surrounding light environment to direct their growth. Blue light (300-500 nm) in particular acts to promote a wide variety of photomorphogenic responses including seedling establishment, phototropism and circadian clock regulation. Several different classes of flavin-based photoreceptors have been identified that mediate the effects of blue light in the dicotyledonous genetic model Arabidopsis thaliana. These include the cryptochromes, the phototropins and members of the Zeitlupe family. In this review, we discuss recent advances, which contribute to our understanding of how these photosensory systems are activated by blue light and how they initiate signaling to regulate diverse aspects of plant development.

For centuries, phototropism has given researchers a convenient experimental system to study how light directs plant growth. Yet, despite over a century of research, deciphering how plants reorientate their growth to maximize photosynthetic light capture and promote seedling survival has proved remarkably challenging. In this article, we present a brief historical overview of our current understanding of the molecular mechanisms underlying shoot phototropism in higher plants and discuss recent progress that now provides new insights on where this adaptive growth response is initiated in dicotyledonous seedlings.

Imbibed Arabidopsis seeds are encapsulated by mucilage that is formed of hydrated polysaccharides released from seed coat epidermal cells. The mucilage is structured with water-soluble and adherent layers, with cellulose present uniquely in an inner domain of the latter. Using a reverse-genetic approach to identify the cellulose synthases (CESAs) that produce mucilage cellulose, cesa5 mutants were shown to be required for the correct formation of these layers. Expression of CESA5 in the seed coat was specific to epidermal cells and coincided with the accumulation of mucilage polysaccharides in their apoplast. Analysis of sugar composition showed that although total sugar composition or amounts were unchanged, their partition between layers was different in the mutant, with redistribution from adherent to water-soluble mucilage. The macromolecular characteristics of the water-soluble mucilage were also modified. In accordance with a role for CESA5 in mucilage cellulose synthesis, crystalline cellulose contents were reduced in mutant seeds, and birefringent microfibrils were absent from adherent mucilage. Although the mum5 mutant showed similar defects to cesa5 in the distribution of sugar components between water-soluble and adherent mucilage, labelling of residual adherent mucilage indicated that cesa5 contained less cellulose and less pectin methyl-esterification. Together the results demonstrate that CESA5 plays a major and essential role in cellulose production in seed mucilage, which is critical for the establishment of mucilage structured in layers and domains. www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved.

Imbibed Arabidopsis (Arabidopsis thaliana) seeds are encapsulated by mucilage that is formed of hydrated polysaccharides released from seed coat epidermal cells. The mucilage is structured with water-soluble and adherent layers, with cellulose present uniquely in an inner domain of the latter. Using a reverse-genetic approach to identify the cellulose synthases (CESAs) that produce mucilage cellulose, cesa5 mutants were shown to be required for the correct formation of these layers. Expression of CESA5 in the seed coat was specific to epidermal cells and coincided with the accumulation of mucilage polysaccharides in their apoplast. Analysis of sugar composition showed that although total sugar composition or amounts were unchanged, their partition between layers was different in the mutant, with redistribution from adherent to water-soluble mucilage. The macromolecular characteristics of the water-soluble mucilage were also modified. In accordance with a role for CESA5 in mucilage cellulose synthesis, crystalline cellulose contents were reduced in mutant seeds and birefringent microfibrils were absent from adherent mucilage. Although the mucilage-modified5 mutant showed similar defects to cesa5 in the distribution of sugar components between water-soluble and adherent mucilage, labeling of residual adherent mucilage indicated that cesa5 contained less cellulose and less pectin methyl esterification. Together, the results demonstrate that CESA5 plays a major and essential role in cellulose production in seed mucilage, which is critical for the establishment of mucilage structured in layers and domains.