FERONIA is involved in phototropin 1-mediated blue light phototropic growth in Arabidopsis

Plant shoot phototropism is triggered by the formation of a light-driven auxin gradient leading to bending growth. The blue light receptor phototropin 1 (phot1) senses light direction, but how this leads to auxin gradient formation and growth regulation remains poorly understood. Previous studies have suggested phot1's role for regulated apoplastic acidification, but its relation to phototropin and hypocotyl phototropism is unclear. Herein, we show that blue light can cause phot1 to interact with and phosphorylate FERONIA (FER), a known cell growth regulator, and trigger downstream phototropic bending growth in Arabidopsis hypocotyls. fer mutants showed defects in phototropic growth, similar to phot1/2 mutant. FER also interacts with and phosphorylates phytochrome kinase substrates, the phot1 downstream substrates. The phot1-FER pathway acts upstream of apoplastic acidification and the auxin gradient formation in hypocotyl under lateral blue light, both of which are critical for phototropic bending growth in hypocotyls. Our study highlights a pivotal role of FER in the phot1-mediated phototropic cell growth regulation in plants.     

Reduced phototropism in pks mutants may be due to altered auxin‐regulated gene expression or reduced lateral auxin transport

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1‐ and phot2‐mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi‐reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.     

Blue‐light‐induced PIN3 polarization for root negative phototropic response in Arabidopsis

Root negative phototropism is an important response in plants. Although blue light is known to mediate this response, the cellular and molecular mechanisms underlying root negative phototropism remain unclear. Here, we report that the auxin efflux carrier PIN‐FORMED (PIN) 3 is involved in asymmetric auxin distribution and root negative phototropism. Unilateral blue‐light illumination polarized PIN3 to the outer lateral membrane of columella cells at the illuminated root side, and increased auxin activity at the illuminated side of roots, where auxin promotes growth and causes roots bending away from the light source. Furthermore, root negative phototropic response and blue‐light‐induced PIN3 polarization were modulated by a brefeldin A‐sensitive, GNOM‐dependent, trafficking pathway and by phot1‐regulated PINOID (PID)/PROTEIN PHOSPHATASE 2A (PP2A) activity. Our results indicate that blue‐light‐induced PIN3 polarization is needed for asymmetric auxin distribution during root negative phototropic response.     

phot1 inhibition of ABCB19 primes lateral auxin fluxes in the shoot apex required for phototropism

It is well accepted that lateral redistribution of the phytohormone auxin underlies the bending of plant organs towards light. In monocots, photoreception occurs at the shoot tip above the region of differential growth. Despite more than a century of research, it is still unresolved how light regulates auxin distribution and where this occurs in dicots. Here, we establish a system in Arabidopsis thaliana to study hypocotyl phototropism in the absence of developmental events associated with seedling photomorphogenesis. We show that auxin redistribution to the epidermal sites of action occurs at and above the hypocotyl apex, not at the elongation zone. Within this region, we identify the auxin efflux transporter ATP-BINDING CASSETTE B19 (ABCB19) as a substrate target for the photoreceptor kinase PHOTOTROPIN 1 (phot1). Heterologous expression and physiological analyses indicate that phosphorylation of ABCB19 by phot1 inhibits its efflux activity, thereby increasing auxin levels in and above the hypocotyl apex to halt vertical growth and prime lateral fluxes that are subsequently channeled to the elongation zone by PIN-FORMED 3 (PIN3). Together, these results provide new insights into the roles of ABCB19 and PIN3 in establishing phototropic curvatures and demonstrate that the proximity of light perception and differential phototropic growth is conserved in angiosperms.     

Clathrin regulates blue light‐triggered lateral auxin distribution and hypocotyl phototropism in Arabidopsis

Phototropism is the process by which plants grow towards light in order to maximize the capture of light for photosynthesis, which is particularly important for germinating seedlings. In Arabidopsis, hypocotyl phototropism is predominantly triggered by blue light (BL), which has a profound effect on the establishment of asymmetric auxin distribution, essential for hypocotyl phototropism. Two auxin efflux transporters ATP‐binding cassette B19 (ABCB19) and PIN‐formed 3 (PIN3) are known to mediate the effect of BL on auxin distribution in the hypocotyl, but the details for how BL triggers PIN3 lateralization remain poorly understood. Here, we report a critical role for clathrin in BL‐triggered, PIN3‐mediated asymmetric auxin distribution in hypocotyl phototropism. We show that unilateral BL induces relocalization of clathrin in the hypocotyl. Loss of clathrin light chain 2 (CLC2) and CLC3 affects endocytosis and lateral distribution of PIN3 thereby impairing BL‐triggered establishment of asymmetric auxin distribution and consequently, phototropic bending. Conversely, auxin efflux inhibitors N‐1‐naphthylphthalamic acid and 2,3,5‐triiodobenzoic acid affect BL‐induced relocalization of clathrin, endocytosis and lateralization of PIN3 as well as asymmetric distribution of auxin. These results together demonstrate an important interplay between auxin and clathrin function that dynamically regulates BL‐triggered hypocotyl phototropism in Arabidopsis.     

Differential roles of auxin efflux carrier PIN proteins in hypocotyl phototropism of etiolated Arabidopsis seedlings depend on the direction of light stimulus

In a recent study, we demonstrated that although the auxin efflux carrier PIN-FORMED (PIN) proteins, such as PIN3 and PIN7, are required for the pulse-induced first positive phototropism in etiolated Arabidopsis hypocotyls, they are not necessary for the continuous-light-induced second positive phototropism when the seedlings are grown on the surface of agar medium, which causes the hypocotyls to separate from the agar surface. Previous reports have shown that hypocotyl phototropism is slightly impaired in pin3 single mutants when they are grown along the surface of agar medium, where the hypocotyls always contact the agar, producing some friction. To clarify the possible involvement of PIN3 and PIN7 in continuous-light-induced phototropism, we investigated hypocotyl phototropism in the pin3 pin7 double mutant grown along the surface of agar medium. Intriguingly, the phototropic curvature was slightly impaired in the double mutant when the phototropic stimulus was presented on the adaxial side of the hook, but was not impaired when the phototropic stimulus was presented on the abaxial side of the hook. These results indicate that PIN proteins are required for continuous-light-induced second positive phototropism, depending on the direction of the light stimulus, when the seedlings are in contact with agar medium.     

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.     

RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana

Phototropin 1 (phot1) and phot2, which are blue light receptor kinases, function in blue light-induced hypocotyl phototropism, chloroplast relocation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Previous studies have shown that the proteins RPT2 (for ROOT PHOTOTROPISM2) and NPH3 (for NONPHOTOTROPIC HYPOCOTYL3) transduce signals downstream of phototropins to induce the phototropic response. However, the involvement of RPT2 and NPH3 in stomatal opening and in chloroplast relocation mediated by phot1 and phot2 was unknown. Genetic analysis of the rpt2 mutant and of a series of double mutants indicates that RPT2 is involved in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. Biochemical analyses indicate that RPT2 is purified in the crude microsomal fraction, as well as phot1 and NPH3, and that RPT2 makes a complex with phot1 in vivo. On the other hand, NPH3 is not necessary for stomatal opening or chloroplast relocation. Thus, these results suggest that phot1 and phot2 choose different signal transducers to induce three responses: phototropic response of hypocotyl, stomatal opening, and chloroplast relocation.     

Arabidopsis ROOT PHOTOTROPISM2 Contributes to the Adaptation to High-Intensity Light in Phototropic Responses

Living organisms adapt to changing light environments via mechanisms that enhance photosensitivity under darkness and attenuate photosensitivity under bright light conditions. In hypocotyl phototropism, phototropin1 (phot1) blue light photoreceptors mediate both the pulse light-induced, first positive phototropism and the continuous light-induced, second positive phototropism, suggesting the existence of a mechanism that alters their photosensitivity. Here, we show that light induction of ROOT PHOTOTROPISM2 (RPT2) underlies photosensory adaptation in hypocotyl phototropism of Arabidopsis thaliana. rpt2 loss-of-function mutants exhibited increased photosensitivity to very low fluence blue light but were insensitive to low fluence blue light. Expression of RPT2 prior to phototropic stimulation in etiolated seedlings reduced photosensitivity during first positive phototropism and accelerated second positive phototropism. Our microscopy and biochemical analyses indicated that blue light irradiation causes dephosphorylation of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) proteins and mediates their release from the plasma membrane. These phenomena correlate closely with the desensitization of phot1 signaling during the transition period from first positive phototropism to second positive phototropism. RPT2 modulated the phosphorylation of NPH3 and promoted reconstruction of the phot1-NPH3 complex on the plasma membrane. We conclude that photosensitivity is increased in the absence of RPT2 and that this results in the desensitization of phot1. Light-mediated induction of RPT2 then reduces the photosensitivity of phot1, which is required for second positive phototropism under bright light conditions.     

Phytochrome-mediated phototropism in de-etiolated seedlings : occurrence and ecological significance

Phototropic responses to broadband far red (FR) radiation were investigated in fully de-etiolated seedlings of a long-hypocotyl mutant (lh) of cucumber (Cucumis sativus L.), which is deficient in phytochrome-B, and its near isogenic wild type (WT). Continuous unilateral FR light provided against a background of white light induced negative curvatures (i.e. bending away from the FR light source) in hypocotyls of WT seedlings. This response was fluence-rate dependent and was absent in the lh mutant, even at very high fluence rates of FR. The phototropic effect of FR light on WT seedlings was triggered in the hypocotyls and occurred over a range of fluence rates in which FR was very effective in promoting hypocotyl elongation. FR light had no effect on elongation of lh-mutant hypocotyls. Seedlings grown in the field showed negative phototropic responses to the proximity of neighboring plants that absorbed blue (B) and red light and back-reflected FR radiation. The bending response was significantly larger in WT than in lh seedlings. Responses of WT and lh seedlings to lateral B light were very similar; however, elimination of the lateral B light gradients created by the proximity of plant neighbors abolished the negative curvature only in the case of lh seedlings. More than 40% of the total hypocotyl curvature induced in WT seedlings by the presence of neighboring plants was present after equilibrating the fluence rates of B light received by opposite sides of the hypocotyl. These results suggest that: (a) phytochrome functions as a phototropic sensor in de-etiolated plants, and (b) in patchy canopy environments, young seedlings actively project new leaves into light gaps via stem bending responses elicited by the B-absorbing photoreceptor(s) and phytochrome.     

Hypocotyl growth orientation in blue light is determined by phytochrome A inhibition of gravitropism and phototropin promotion of phototropism

How developing seedlings integrate gravitropic and phototropic stimuli to determine their direction of growth is poorly understood. In this study we tested whether blue light influences hypocotyl gravitropism in Arabidopsis. Phototropin1 (phot1) triggers phototropism under low fluence rates of blue light but, at least in the dark, has no effect on gravitropism. By analyzing the growth orientation of phototropism-deficient seedlings in response to gravitropic and phototropic stimulations we show that blue light not only triggers phototropism but also represses hypocotyl gravitropism. At low fluence rates of blue light phot1 mutants were agravitropic. In contrast, phyAphot1 double mutants grew exclusively according to gravity demonstrating that phytochrome A (phyA) is necessary to inhibit gravitropism. Analyses of phot1cry1cry2 triple mutants indicate that cryptochromes play a minor role in this response. Thus the optimal growth orientation of hypocotyls is determined by the action of phyA-suppressing gravitropism and the phototropin-triggering phototropism. It has long been known that phytochromes promote phototropism but the mechanism involved is still unknown. Our data show that by inhibiting gravitropism phyA acts as a positive regulator of phototropism.     

The signal transducer NPH3 integrates the phototropin1 photosensor with PIN2-based polar auxin transport in Arabidopsis root phototropism

Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.     

NPH3- and PGP-like genes are exclusively expressed in the apical tip region essential for blue-light perception and lateral auxin transport in maize coleoptiles

Phototropic curvature results from differential growth on two sides of the elongating shoot, which is explained by asymmetrical indole-3-acetic acid (IAA) distribution. Using 2 cm maize coleoptile segments, 1st positive phototropic curvature was confirmed here after 8 s irradiation with unilateral blue light (0.33 μmol m(-2) s(-1)). IAA was redistributed asymmetrically by approximately 20 min after photo-stimulation. This asymmetric distribution was initiated in the top 0-3 mm region and was then transmitted to lower regions. Application of the IAA transport inhibitor, 1-N-naphthylphthalamic acid (NPA), to the top 2 mm region completely inhibited phototropic curvature, even when auxin was simultaneously applied below the NPA-treated zone. Thus, lateral IAA movement occurred only within the top 0-3 mm region after photo-stimulation. Localized irradiation experiments indicated that the photo-stimulus was perceived in the apical 2 mm region. The results suggest that this region harbours key components responsible for photo-sensing and lateral IAA transport. In the present study, it was found that the NPH3- and PGP-like genes were exclusively expressed in the 0-2 mm region of the tip, whereas PHOT1 and ZmPIN1a, b, and c were expressed relatively evenly along the coleoptile, and ZmAUX1, ZMK1, and ZmSAURE2 were strongly expressed in the elongation zone. These results suggest that the NPH3-like and PGP-like gene products have a key role in photo-signal transduction and regulation of the direction of auxin transport after blue light perception by phot1 at the very tip region of maize coleoptiles.     

The hypocotyl chloroplast plays a role in phototropic bending of Arabidopsis seedlings: developmental and genetic evidence

Chloroplasts of guard cells and coleoptiles have been implicated in the sensory transduction of blue light. The present study was aimed at establishing whether the chloroplast of the hypocotyl from Arabidopsis, another blue light-responding organ, has similar characteristics to that of sensory-transducing guard cell and coleoptile chloroplasts. Results showed that the phototropic curvature and arch length induced by blue light in Arabidopsis seedlings matched the distribution of mature chloroplasts in the bending hypocotyl. The bending arch consistently included the region of the hypocotyl containing mature chloroplasts, and never extended beyond that region. Manipulation of the extent of greening of dark-grown hypocotyls by varying red light pretreatments elicited blue light-stimulated curvatures and arch lengths that depended on the duration of the red light pretreatment and on the distribution of mature chloroplasts in the hypocotyl. Albino psd2 mutants of Arabidopsis, which lack mature chloroplasts, are devoid of phototropic sensitivity under conditions in which wild-type seedlings show large curvatures. The star mutant of Arabidopsis has a delayed greening and a delayed phototropic response as compared with wild type. Measurements of photosynthetic oxygen evolution and carbon fixation, dark respiration, and light-dependent zeaxanthin formation in the hypocotyl showed features similar to those of guard cells and coleoptiles, and distinctly different from those of mesophyll tissue. These results indicate that the hypocotyl chloroplast has characteristics similar to those associated with guard cell and coleoptile chloroplasts, and that phototropic bending of Arabidopsis hypocotyls appears to require mature chloroplasts.     

Clathrin light chains regulate hypocotyl elongation by affecting the polarization of the auxin transporter PIN3 in Arabidopsis

PIN-FORMED (PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers. Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.     

Arabidopsis ROOT UVB SENSITIVE2/WEAK AUXIN RESPONSE1 Is Required for Polar Auxin Transport

Auxin is an essential phytohormone that regulates many aspects of plant development. To identify new genes that function in auxin signaling, we performed a genetic screen for Arabidopsis thaliana mutants with an alteration in the expression of the auxin-responsive reporter DR5rev:GFP (for green fluorescent protein). One of the mutants recovered in this screen, called weak auxin response1 (wxr1), has a defect in auxin response and exhibits a variety of auxin-related growth defects in the root. Polar auxin transport is reduced in wxr1 seedlings, resulting in auxin accumulation in the hypocotyl and cotyledons and a reduction in auxin levels in the root apex. In addition, the levels of the PIN auxin transport proteins are reduced in the wxr1 root. We also show that WXR1 is ROOT UV-B SENSITIVE2 (RUS2), a member of the broadly conserved DUF647 domain protein family found in diverse eukaryotic organisms. Our data indicate that RUS2/WXR1 is required for auxin transport and to maintain the normal levels of PIN proteins in the root.     

PINOID functions in root phototropism as a negative regulator

The PINOID (PID) family, which belongs to AGCVIII kinases, is known to be involved in the regulation of auxin efflux transporter PIN-FORMED (PIN) proteins through changes in the phosphorylation status. Recently, we demonstrated that the PID family is necessary for phytochrome-mediated phototropic enhancement in Arabidopsis hypocotyls and that the downregulation of PID expression by red-light pretreatment results in the promotion of the PIN-mediated auxin gradient during phototropic responses. However, whether PID participates in root phototropism in Arabidopsis seedlings has not been well studied. Here, we demonstrated that negative root phototropic responses are enhanced in the pid quadruple mutant and are severely impaired in transgenic plants expressing PID constitutively. The results indicate that the PID family functions in a negative root phototropism as a negative regulator. On the other hand, analysis with PID fused to a yellow fluorescent protein, VENUS, showed that unilateral blue-light irradiation causes a lower accumulation of PID proteins on the shaded side than on the irradiated side. This result suggests that the blue-light-mediated asymmetrical distribution of PID proteins may be one of the critical responses in phototropin-mediated signals during a negative root phototropism. Alternatively, such a transverse gradient of PID proteins may result from gravitropic stimulation produced by phototropic bending.