Nonphototropic Hypocotyl Research Articles

Membrane-Localized Orientation of NONPHOTOTROPIC HYPOCOTYL3 Affects the Necessity of Its Phosphorylation for Phototropism.

NONPHOTOTROPIC HYPOCOTYL3 (NPH3) is a key regulator of hypocotyl phototropism under both low- and high-intensity blue light (LBL/HBL), mediating phototropin1 (phot1) and phot2 signaling. NPH3 undergoes dephosphorylation and is released from the plasma membrane (PM) upon blue light irradiation. However, how its phosphorylation status and PM localization mediate phot1 and phot2 signaling in Arabidopsis (Arabidopsis thaliana) remains elusive. In this study, we found that fusing NPH3 with GFP at its C terminus (N3G) impaired its release from the PM, a defect exacerbated by a phosphorylation-deficient mutation, resulting in a dephosphorylated NPH3-GFP (N3AG). Unlike N3G, transgenic lines expressing N3AG exhibited defective hypocotyl phototropism under HBL, which could be rescued by myristoylation at the N-terminus of N3AG (mN3AG), indicating that NPH3 phosphorylation is not essential for HBL-induced phototropic responses when it is artificially anchored at the PM via its N terminus. Furthermore, genetic analysis revealed that N3AG anchored to the PM by its N terminus (as in mN3AG) only rescues phot1-mediated HBL responses, which require RPT2. However, N3AG failed to regulate phot2-mediated HBL signaling, regardless of its PM orientation. Taken together, our results revealed that NPH3 phosphorylation is essential for phot2-mediated hypocotyl phototropism under HBL, but is not required for phot1-mediated HBL signaling when the NPH3 N terminus is PM-anchored.

The photosensory function of Zmphot1 differs from that of Atphot1 due to the C-terminus of Zmphot1 during phototropic response

The role of phot1 in triggering hypocotyl phototropism and optimizing growth orientation has been well-characterized in Arabidopsis, whereas the role of Zmphot1 in maize remains largely unclear. Here, we show that Zmphot1 is involved in blue light-induced phototropism. Compared with Atphot1, Zmphot1 exhibited a weaker phototropic response to very low-fluence rates of blue light (< 0.01 μmol m−2 s−1), but stronger phototropic response to high-fluence rates of blue light (> 10 μmol m−2 s−1) than Atphot1. Notably, blue light exposure induced Zmphot1-green fluorescent protein (GFP), but not Atphot1-GFP, to form the aggregates in the cytoplasm of Nicotiana benthamiana cells. Furthermore, by generating the chimeric phot1 proteins, we found that the serine-threonine kinase (STK) domain at the C-terminus is responsible for a more volatile membrane association of Zmphot1. Consistently, the chimeric phot1 protein fusing the STK domain of Zmphot1 with other domains of Atphot1 responded similarly as Zmphot1 to both low and high fluence rates of blue light. Interestingly, although both Zmphot1 and Atphot1 interact with AtNPH3, Zmphot1 induced weaker dephosphorylation of NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) than Atphot1. Together, our findings indicate that Zmphot1 and Atphot1 exhibit different photosensory function during phototropic response and that the STK domain may play a key role in determining their properties.

Phototropin phosphorylation of ROOT PHOTOTROPISM 2 and its role in mediating phototropism, leaf positioning, and chloroplast accumulation movement in Arabidopsis.

Directional 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 leafpositioning 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.

Association genetics of early season cold and late season frost tolerance in Sorghum bicolor

AbstractSorghum [Sorghum bicolor (L.) Moench] is a multipurpose crop that is highly susceptible to low temperature stress. Early season cold (ESC) greatly affects seed germination, seedling vigor, and root architecture, whereas late‐season frost (LSF) reduces fertility, yield, and nutrition quality and content in sorghum. Here, an association mapping study was conducted to delineate the genetics of ESC and LSF tolerance in sorghum. A total of 10 single nucleotide polymorphisms (SNPs) and 17 quantitative trait loci (QTL) were identified for ESC, and 40 SNPs were identified for LSF. The diversity panel exhibited extensive variation for plant traits measured for their association with ESC and LSF tolerance. Two QTL colocalized with the classical tannin genes Tan1 (S4_61580203) and Tan2 (S2_7777693) were identified for ESC tolerance. We also identified additional putative candidate genes such as Sobic.001G157100 (Nonphototropic hypocotyl 3, NPH3), Sobic.001G156600 (Lectin receptor‐like serine/threonine kinase), and Sobic.006G061100 (Sucrose nonfermenting 1 related protein kinase 1, SnRK1) for ESC tolerance and Sobic.006G139900 (UDP‐glucoronosyl and UDP‐glucosyl transferase), Sobic.002G187400 (Serine/threonine protein kinase), and Sobic.004G333700 (Ca2+/calmodulin‐dependent protein phosphatase) for LSF tolerance. Tan1 and Tan2 play a major role in seed dormancy and induce resistance against soil‐borne pathogens under cold and wet soil conditions. Seed tannin content may be used as an indirect selection marker for identifying cold tolerant germplasm for breeding. NPH3 and SnRK1 regulate seed germinability under cold stress in various crops. Candidates identified in this study could be further exploited to develop low temperature tolerant cultivars as proxies in marker‐assisted breeding, genomic selection, and genetic engineering.

The phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 affects phot2-dependent phototropism in Arabidopsis

ABSTRACT The blue light photoreceptors, phototropin 1 (phot1) and phot2, and their signal transducer, NONPHOTOTROPIC HYPOCOTYL3 (NPH3), are activators of the phototropic responses of Arabidopsis hypocotyls. In a recent study, we reported that the control of NPH3 phosphorylation at serine 7 (S7: or S5), S213, S223, S237, S467, S474 (or S476), and S722 (or S723) contributes to the photosensory adaptation of phot1 signaling during the phototropic response. Phosphomimetic NPH3SE mutant and unphosphorylatable NPH3SA mutant on those serine residues function efficiently under blue light conditions at fluence rates of 10−5 µmol m–2 s–1 and 10–3 µmol m–2 s–1 or more, respectively. We here demonstrate that phosphomimetic NPH3SE, but not unphosphorylatable NPH3SA, promotes phot2-dependent phototropism under blue light condition at 100 µmol m–2 s–1. This result suggests that phot1 negatively controls phot2 signaling through the dephosphorylation of NPH3 at those residues and that the hyperactivation of phot1- and phot2-NPH3 complexes does not occur at the same time under high intensity blue light. We hypothesize that the dephosphorylation of NPH3 on those serine residues suppresses both phot1 and phot2 signaling, which results in different impacts on phot1- and phot2-dependent hypocotyl phototropism due to the differences in the photosensitivity and activation levels of phot1 and phot2.

Light-triggered and phosphorylation-dependent 14-3-3 association with NON-PHOTOTROPIC HYPOCOTYL 3 is required for hypocotyl phototropism

NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent plant phototropic growth response. We report that NPH3 directly binds polyacidic phospholipids, required for plasma membrane association in darkness. We further demonstrate that blue light induces an immediate phosphorylation of a C-terminal 14-3-3 binding motif in NPH3. Subsequent association of 14-3-3 proteins is causal for the light-induced release of NPH3 from the membrane and accompanied by NPH3 dephosphorylation. In the cytosol, NPH3 dynamically transitions into membraneless condensate-like structures. The dephosphorylated state of the 14-3-3 binding site and NPH3 membrane recruitment are recoverable in darkness. NPH3 variants that constitutively localize either to the membrane or to condensates are non-functional, revealing a fundamental role of the 14-3-3 mediated dynamic change in NPH3 localization for auxin-dependent phototropism. This regulatory mechanism might be of general nature, given that several members of the NPH3-like family interact with 14-3-3 via a C-terminal motif.

Regulation of plant phototropic growth by NPH3/RPT2-like substrate phosphorylation and 14-3-3 binding

Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.

Phototropin-mediated perception of light direction in leaves regulates blade flattening.

One conserved feature among angiosperms is the development of flat thin leaves. This developmental pattern optimizes light capture and gas exchange. The blue light (BL) receptors phototropins are required for leaf flattening, with the null phot1phot2 mutant showing curled leaves in Arabidopsis (Arabidopsis thaliana). However, key aspects of their function in leaf development remain unknown. Here, we performed a detailed spatiotemporal characterization of phototropin function in Arabidopsis leaves. We found that phototropins perceive light direction in the blade, and, similar to their role in hypocotyls, they control the spatial pattern of auxin signaling, possibly modulating auxin transport, to ultimately regulate cell expansion. Phototropin signaling components in the leaf partially differ from hypocotyls. Moreover, the light response on the upper and lower sides of the leaf blade suggests a partially distinct requirement of phototropin signaling components on each side. In particular, NON PHOTOTROPIC HYPOCOTYL 3 showed an adaxial-specific function. In addition, we show a prominent role of PHYTOCHROME KINASE SUBSTRATE 3 in leaf flattening. Among auxin transporters, PIN-FORMED 3,4,7 and AUXIN RESISTANT 1 (AUX1)/LIKE AUXIN RESISTANT 1 (LAX1) are required for the response while ABCB19 has a regulatory role. Overall, our results show that directional BL perception by phototropins is a key aspect of leaf development, integrating endogenous and exogenous signals.

PIN3-mediated auxin transport contributes to blue light-induced adventitious root formation in Arabidopsis

Adventitious rooting is a heritable quantitative trait that is influenced by multiple endogenous and exogenous factors in plants, and one important environmental factor required for efficient adventitious root formation is light signaling. However, the physiological significance and molecular mechanism of light underlying adventitious root formation are still largely unexplored. Here, we report that blue light-induced adventitious root formation is regulated by PIN-FORMED3 (PIN3)-mediated auxin transport in Arabidopsis. Adventitious root formation is significantly impaired in the loss-of-function mutants of the blue light receptors, PHOTOROPIN1 (PHOT1) and PHOTOROPIN2 (PHOT2), as well as the phototropic transducer, NON-PHOTOTROPIC HYPOCOTYL3 (NPH3). In addition, blue light enhanced the auxin content in the adventitious root, and the pin3 loss-of-function mutant had a reduced adventitious rooting response under blue light compared to the wild type. The PIN3 protein level was higher in plants treated with blue light than in those in darkness, especially in the hypocotyl pericycle, while PIN3-GFP failed to accumulate in nph3 PIN3::PIN3-GFP. Furthermore, the results showed that PIN3 physically interacted with NPH3, a key transducer in phototropic signaling. Taken together, our study demonstrates that blue light induces adventitious root formation through the phototropic signal transducer, NPH3, which regulates adventitious root formation by affecting PIN3-mediated auxin transport.

Phosphorylation of NONPHOTOTROPIC HYPOCOTYL3 affects photosensory adaptation during the phototropic response.

Photosensory adaptation, which can be classified as sensor or effector adaptation, optimizes the light sensing of living organisms by tuning their sensitivity to changing light conditions. During the phototropic response in Arabidopsis (Arabidopsis thaliana), the light-dependent expression controls of blue-light (BL) photoreceptor phototropin 1 (phot1) and its modulator ROOT PHOTOTROPISM2 (RPT2) are known as the molecular mechanisms underlying sensor adaptation. However, little is known about effector adaption in plant phototropism. Here, we show that control of the phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) leads to effector adaptation in hypocotyl phototropism. We generated unphosphorable and phosphomimetic NPH3 proteins on seven phosphorylation sites in the etiolated seedlings of Arabidopsis. Unphosphorable NPH3 showed a shortening of its retention time in the cytosol and caused an inability to adapt to very low fluence rates of BL (∼10-5 µmol m-2 s-1) during the phototropic response. In contrast, the phosphomimetic NPH3 proteins had a lengthened retention time in the cytosol and could not enable the adaptation to BL at fluence rates of 10-3 µmol m-2 s-1 or more. Our results indicate that the activation level of phot1 and the corresponding phosphorylation level of NPH3 determine the dissociation rate and the reassociation rate of NPH3 on the plasma membrane, respectively. These mechanisms may moderately maintain the active state of phot1 signaling across a broad range of BL intensities and contribute to the photosensory adaptation of phot1 signaling during the phototropic response in hypocotyls.

The continuing arc toward phototropic enlightenment.

Phototropism represents a simple physiological mechanism—differential growth across the growing organ of a plant—to respond to gradients of light and maximize photosynthetic light capture (in aerial tissues) and water/nutrient acquisition (in roots). The phototropin blue light receptors, phot1 and phot2, have been identified as the essential sensors for phototropism. Additionally, several downstream signal/response components have been identified, including the phot-interacting proteins NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) and PHYTOCHROME SUBSTRATE 4 (PKS4). While the structural and photochemical properties of the phots are quite well understood, much less is known about how the phots signal through downstream regulators. Recent advances have, however, provided some intriguing clues. It appears that inactive receptor phot1 is found dispersed in a monomeric form at the plasma membrane in darkness. Upon light absorption dimerizes and clusters in sterol-rich microdomains where it is signal active. Additional studies showed that the phot-regulated phosphorylation status of both NPH3 and PKS4 is linked to phototropic responsiveness. While PKS4 can function as both a positive (in low light) and a negative (in high light) regulator of phototropism, NPH3 appears to function solely as a key positive regulator. Ultimately, it is the subcellular localization of NPH3 that appears crucial, an aspect regulated by its phosphorylation status. While phot1 activation promotes dephosphorylation of NPH3 and its movement from the plasma membrane to cytoplasmic foci, phot2 appears to modulate relocalization back to the plasma membrane. Together these findings are beginning to illuminate the complex biochemical and cellular events, involved in adaptively modifying phototropic responsiveness under a wide varying range of light conditions.

Cryptochrome-mediated hypocotyl phototropism was regulated antagonistically by gibberellic acid and sucrose in Arabidopsis.

Both phototropins (phot1 and phot2) and cryptochromes (cry1 and cry2) were proven as the Arabidopsis thaliana blue light receptors. Phototropins predominately function in photomovement, and cryptochromes play a role in photomorphogenesis. Although cryptochromes have been proposed to serve as positive modulators of phototropic responses, the underlying mechanism remains unknown. Here, we report that depleting sucrose from the medium or adding gibberellic acids (GAs) can partially restore the defects in phototropic curvature of the phot1 phot2 double mutants under high‐intensity blue light; this restoration does not occur in phot1 phot2 cry1 cry2 quadruple mutants and nph3 (nonphototropic hypocotyl 3) mutants which were impaired phototropic response in sucrose‐containing medium. These results indicate that GAs and sucrose antagonistically regulate hypocotyl phototropism in a cryptochromes dependent manner, but it showed a crosstalk with phototropin signaling on NPH3. Furthermore, cryptochromes activation by blue light inhibit GAs synthesis, thus stabilizing DELLAs to block hypocotyl growth, which result in the higher GAs content in the shade side than the lit side of hypocotyl to support the asymmetric growth of hypocotyl. Through modulation of the abundance of DELLAs by sucrose depletion or added GAs, it revealed that cryptochromes have a function in mediating phototropic curvature.

Deetiolation Enhances Phototropism by Modulating NON-PHOTOTROPIC HYPOCOTYL3 Phosphorylation Status

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.

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.

Genome-wide identification and expression analysis of the BTB domain-containing protein gene family in tomato.

BTB (broad-complex, tramtrack, and bric-a-brac) family proteins are characterized by the presence of a protein-protein interaction BTB domain. BTB proteins have diverse functions, including transcriptional regulation, protein degradation, chromatin remodeling, and cytoskeletal regulation. However, little is known about this gene family in tomato (Solanum lycopersicum), the most important model plant for crop species. In this study, 38 BTB genes were identified based on tomato whole-genome sequence. Phylogenetic analysis of BTB proteins in tomato revealed that SlBTB proteins could be divided into at least 4 subfamilies. The SlBTB proteins contains 1-3 BTB domains, and several other types of functional domains, including KCTD (Potassium channel tetramerization domain-containing), the MATH (meprin and TRAF homology), ANK (Ankyrin repeats), NPR1 (nonexpressor of pathogenesis-related proteins1), NPH3 (Nonphototropic Hypocotyl 3), TAZ zinc finger, C-terminal Kelch, Skp1 and Arm (Armadillo/beta-catenin-like repeat) domains are also found in some tomato BTB proteins. Moreover, their expression patterns in tissues/stages, in response to different abiotic stress treatments and hormones were also investigated. This study provides the first comprehensive analysis of BTB gene family in the tomato genome. The data will undoubtedly be useful for better understanding the potential functions of BTB genes, and their possible roles in mediating hormone cross-talk and abiotic stress in tomato as well as in some other relative species.

Ectopic Expression of WINDING 1 Leads to Asymmetrical Distribution of Auxin and a Spiral Phenotype in Rice.

Ectopic expression of the rice WINDING 1 (WIN1) gene leads to a spiral phenotype only in shoots but not in roots. Rice WIN1 belongs to a specific class of proteins in cereal plants containing a Bric-a-Brac/Tramtrack/Broad (BTB) complex, a non-phototropic hypocotyl 3 (NPH3) domain and a coiled-coil motif. The WIN1 protein is predominantly localized to the plasma membrane, but is also co-localized to plasmodesmata, where it exhibits a punctate pattern. It is observed that WIN1 is normally expressed in roots and the shoot-root junction, but not in the rest of shoots. In roots, WIN1 is largely localized to the apical and basal sides of cells. However, upon ectopic expression, WIN1 appears on the longitudinal sides of leaf sheath cells, correlated with the appearance of a spiral phenotype in shoots. Despite the spiral phenotype, WIN1-overexpressing plants exhibit a normal phototropic response. Although treatments with exogenous auxins or a polar auxin transport inhibitor do not alter the spiral phenotype, the excurvature side has a higher auxin concentration than the incurvature side. Furthermore, actin filaments are more prominent in the excurvature side than in the incurvature side, which correlates with cell size differences between these two sides. Interestingly, ectopic expression of WIN1 does not cause either unequal auxin distribution or actin filament differences in roots, so a spiral phenotype is not observed in roots. The action of WIN1 appears to be different from that of other proteins causing a spiral phenotype, and it is likely that WIN1 is involved in 1-N-naphthylphthalamic acid-insensitive plasmodesmata-mediated auxin transport.

Impact of EHB1 and AGD12 on Root and Hypocotyl Phototropism in Arabidopsis thaliana

Previous findings concerning two proteins involved in gravity and light-induced signal transduction, (AGD12-ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN 12 and EHB1 (ENHANCED BENDING 1)), in Arabidopsis provided evidence for an antagonistic regulation of a gravitropic stimulus on roots and shoots. Both proteins share high C-terminal homology containing a canonical calcium-binding C2-domain, but EHB1 lacks a functional N-terminally located GTP-binding domain. Physiological analysis using loss-of-function mutants under clinostat conditions, where any gravitropic stimulus could be ruled out, provided evidence for diminished phototropism of agd12 mutants, but enhanced phototropism of ehb1-mutants. Data could be confirmed by seedlings overexpressing either AGD12 or EHB1, showing enhanced bending in AGD12 and reduced bending in EHB1 overexpressing seedlings. We conclude that EHB1 and AGD12 are early signaling elements of the gravitropic phototropic bending response, because both of them bind to NPH3 (NON PHOTOTROPIC HYPOCOTYL), which was described as the first element of the phototropic signal transduction chain, directly interacting with phot1. Both proteins affect phototropic bending antagonistically. In contrast to the total incapability of agd12 and ehb1 mutants to respond to increased calcium concentration in response to gravity, agd12 nor ehb1 seedlings differ only gradually in their calcium induced bending response to blue light. This would indicate that EHB1 and AGD12 might be triggered by other signal elements within the phototropism signal transduction chain.

Functional characterization of Arabidopsis phototropin 1 in the hypocotyl apex.

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.

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 Ostreococcus tauri phototropin.

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.