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.     

D6PK AGCVIII Kinases Are Required for Auxin Transport and Phototropic Hypocotyl Bending in Arabidopsis

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.     

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.     

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.     

Plasma membrane H+‐ATPase regulation is required for auxin gradient formation preceding phototropic growth

Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H⁺‐ATPases that are required to control apoplastic pH. Our results show that H⁺‐ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H⁺‐ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH.     

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.     

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.     

Mapping of the phosphorylation sites on the phototropic signal transducer, NPH3

The phototropic response in Arabidopsis thaliana is initiated by the blue-light photoreceptors, phototropin (phot)1 and phot2. A recent study has shown that one of the phototropic signal transducers, NPH3, is phosphorylated under dark conditions and dephosphorylated by blue-light irradiation. To further understand the function of phosphorylation and dephosphorylation of NPH3 during this phototropic response, we have mapped the phosphorylation sites of NPH3 in our current study. The NPH3 protein has at least three phosphorylation sites at serine residues, Ser212, Ser222, and Ser236, and these sites are dephosphorylated by blue-light irradiation. By immunoblotting analysis, these phosphorylation sites in phot1 mutants are not dephosphorylated following blue-light irradiation at both low and high fluence rates, even though such irradiations induce the phot2-dependent phototropic response in phot1. These results suggest that the dephosphorylated NPH3 is involved in the phot1-dependent phototropic response and is not essential for the phot2-dependent phototropic response. We generated two types of transgenic nph3 plants expressing a NPH3^{S212A/S222A/S232A/S236A} protein and a NPH3^Δ212–238 protein in which the phosphorylation region is deleted, and assessed the phototropic phenotype of these. Based upon our present findings, we discuss the role of dephosphorylated and phosphorylated NPH3 in mediating the 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.     

PHOT2介导拟南芥下胚轴向光弯曲调节子的筛选与鉴定

向光素(PHOT1和PHOT2)功能冗余调节单侧强蓝光诱导的拟南芥(Arabidopsis thaliana)黄化苗下胚轴向光弯曲表现功能冗余,限制了人们对PHOT2信号转导机制的深入研究。通过化学诱变剂甲基磺酸乙酯(EMS)诱变拟南芥phot1突变体,避开PHOT1基因的干扰,寻找PHOT2下游信号分子。研究结果表明,已成功筛选到1株遗传稳定的下胚轴向蓝光不弯曲突变体。遗传分析结果显示,该突变体可能是PHOT2下游信号分子突变,将其命名为p2sa1(phototropin2 signaling associated1)。用100μmol·m–2·s–1强蓝光单侧照射,phot1p2sa1下胚轴向光弯曲缺失,呈现phot1phot2双突变的表型,然而phot1p2sa1在强蓝光下叶绿体避光正常,明显不同于phot1phot2。实验证实P2SA1可能位于PHOT2的下游,参与调节PHOT2介导的拟南芥下胚轴向光弯曲反应。     

Regulation of phototropic signaling in Arabidopsis via phosphorylation state changes in the phototropin 1-interacting protein NPH3

Phototropism, or the directional growth (curvature) of various organs toward or away from incident light, represents a ubiquitous adaptive response within the plant kingdom. This response is initiated through the sensing of directional blue light (BL) by a small family of photoreceptors known as the phototropins. Of the two phototropins present in the model plant Arabidopsis thaliana, phot1 (phototropin 1) is the dominant receptor controlling phototropism. Absorption of BL by the sensory portion of phot1 leads, as in other plant phototropins, to activation of a C-terminal serine/threonine protein kinase domain, which is tightly coupled with phototropic responsiveness. Of the five phot1-interacting proteins identified to date, only one, NPH3 (non-phototropic hypocotyl 3), is essential for all phot1-dependent phototropic responses, yet little is known about how phot1 signals through NPH3. Here, we show that, in dark-grown seedlings, NPH3 exists as a phosphorylated protein and that BL stimulates its dephosphorylation. phot1 is necessary for this response and appears to regulate the activity of a type 1 protein phosphatase that catalyzes the reaction. The abrogation of both BL-dependent dephosphorylation of NPH3 and development of phototropic curvatures by protein phosphatase inhibitors further suggests that this post-translational modification represents a crucial event in phot1-dependent phototropism. Given that NPH3 may represent a core component of a CUL3-based ubiquitin-protein ligase (E3), we hypothesize that the phosphorylation state of NPH3 determines the functional status of such an E3 and that differential regulation of this E3 is required for normal phototropic responsiveness.     

Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.     

A negative effector of blue light-induced and gravitropic bending in Arabidopsis

Although sessile, plants are able to grow toward or away from an environmental stimulus. Important examples are stem or leaf orientation of higher plants in response to the direction of the incident light. The responsible photoreceptors belong to the phototropin photoreceptor family. Although the mode of phototropin action is quite well understood, much less is known of how the light signal is transformed into a bending response. Several lines of evidence indicate that a lateral auxin gradient is responsible for asymmetric cell elongation along the light gradient within the stem. However, some of the molecular key players leading to this asymmetric auxin distribution are, as yet, unidentified. Previously, it was shown that phototropin gets autophosphorylated upon illumination and binds to a scaffold protein termed NPH3 (for nonphototropic hypocotyl 3). Using a yeast three-hybrid approach with phototropin and NPH3 as a bait complex, we isolated a protein, termed EHB1 (for enhanced bending 1), with a so far unknown function, which binds to this binary complex. This novel interacting factor negatively affects hypocotyl bending under blue light conditions in Arabidopsis (Arabidopsis thaliana) and thus seems to be an important component regulating phototropism. Interestingly, it could be shown that the gravitropic response was also affected. Thus, it cannot be ruled out that this protein might also have a more general role in auxin-mediated bending toward an environmental stimulus.     

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.     

Modulation of phototropic responsiveness in Arabidopsis through ubiquitination of phototropin 1 by the CUL3-Ring E3 ubiquitin ligase CRL3(NPH3)

Plant phototropism is an adaptive response to changes in light direction, quantity, and quality that results in optimization of photosynthetic light harvesting, as well as water and nutrient acquisition. Though several components of the phototropic signal response pathway have been identified in recent years, including the blue light (BL) receptors phototropin1 (phot1) and phot2, much remains unknown. Here, we show that the phot1-interacting protein NONPHOTOTROPIC HYPOCOTYL3 (NPH3) functions as a substrate adapter in a CULLIN3-based E3 ubiquitin ligase, CRL3(NPH3). Under low-intensity BL, CRL3(NPH3) mediates the mono/multiubiquitination of phot1, likely marking it for clathrin-dependent internalization from the plasma membrane. In high-intensity BL, phot1 is both mono/multi- and polyubiquitinated by CRL3(NPH3), with the latter event targeting phot1 for 26S proteasome-mediated degradation. Polyubiquitination and subsequent degradation of phot1 under high-intensity BL likely represent means of receptor desensitization, while mono/multiubiquitination-stimulated internalization of phot1 may be coupled to BL-induced relocalization of hormone (auxin) transporters.     

Molecular genetic analysis of phototropism in Arabidopsis

Plant life is strongly dependent on the environment, and plants regulate their growth and development in response to many different environmental stimuli. One of the regulatory mechanisms involved in these responses is phototropism, which allows plants to change their growth direction in response to the location of the light source. Since the study of phototropism by Darwin, many physiological studies of this phenomenon have been published. Recently, molecular genetic analyses of Arabidopsis have begun to shed light on the molecular mechanisms underlying this response system, including phototropin blue light photoreceptors, phototropin signaling components, auxin transporters, auxin action mechanisms and others. This review highlights some of the recent progress that has been made in further elucidating the phototropic response, with particular emphasis on mutant phenotypes.     

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.     

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.     

植物下胚轴向光弯曲机制研究进展

植物向光弯曲生长主要是由于其向光和背光面生长素的不对称分布引起。近年来研究发现,在不同强度的蓝光单侧照射下,植物可能存在不同的向光弯曲调节机制。目前,关于向光素PHOT1介导弱蓝光引起的下胚轴弯曲研究较为详细,即PHOT1感受蓝光后,与其下游的信号蛋白NPH3、RPT2和PKS1相互作用,调控生长素运输蛋白的活性及定位,诱导生长素的不对称分布引起向光弯曲。PHOT1和PHOT2以功能冗余方式调节强蓝光引起的植物下胚轴向光弯曲,NPH3可能作为共享调节因子,引发不同的信号转导通路实现功能互补。此外,其他光受体、激素、蛋白激酶、蛋白磷酸酶以及Ca2＋也参与了植物向光弯曲的调节。本文就近年来有关植物下胚轴向光弯曲信号组分及可能的网络关系进行总结,并对该研究领域存在的问题及今后可能的研究方向进行展望。