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.     

Mutations in the NPH1 locus of Arabidopsis disrupt the perception of phototropic stimuli.

The phototropic response is an important component of seedling establishment in higher plants because it orients the young seedlings for maximal photosynthetic light capture. Despite their obvious importance, little is known about the mechanisms underlying the perception and transduction of the light signals that induce phototropic curvatures. Here, we report the isolation of eight mutants of Arabidopsis that lack or have severely impaired phototropic responses. These nph (for nonphototropic hypocotyl) mutants comprise four genetic loci: nph1, nph2, nph3, and nph4. Physiological and biochemical characterization of the nph1 allele series indicated that the NPH1 locus may encode the apoprotein for a dual-chromophoric or multichromophoric holoprotein photoreceptor capable of absorbing UV-A, blue, and green light and that this photoreceptor regulates all the phototropic responses of Arabidopsis. It appears that the NPH1 protein is most likely a 120-kD plasma membrane-associated phosphoprotein because all of the nph1 mutations negatively affected the abundance of this protein. In addition, the putative NPH1 photoreceptor protein is genetically and biochemically distinct from the HY4 protein, which most likely acts as a photoreceptor for blue light-mediated hypocotyl growth inhibition. Furthermore, the NPH1 and HY4 proteins are not functionally redundant because mutations in either gene alone affect only one physiological response but not the other, thus providing strong support for the hypothesis that more than one blue light photoreceptor is required for the normal growth and development of a seedling.     

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.     

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.     

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.     

Genetic separation of phototropism and blue light inhibition of stem elongation

Blue light-induced regulation of cell elongation is a component of the signal response pathway for both phototropic curvature and inhibition of stem elongation in higher plants. To determine if blue light regulates cell elongation in these responses through shared or discrete pathways, phototropism and hypocotyl elongation were investigated in several blue light response mutants in Arabidopsis thaliana. Specifically, the blu mutants that lack blue light-dependent inhibition of hypocotyl elongation were found to exhibit a normal phototropic response. In contrast, a phototropic null mutant (JK218) and a mutant that has a 20- to 30-fold shift in the fluence dependence for first positive phototropism (JK224) showed normal inhibition of hypocotyl elongation in blue light. F1 progeny of crosses between the blu mutants and JK218 showed normal phototropism and inhibition of hypocotyl elongation, and approximately 1 in 16 F2 progeny were double mutants lacking both responses. Thus, blue light-dependent inhibition of hypocotyl elongation and phototropism operate through at least some genetically distinct components.     

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 phosphorylation status of NONPHOTOTROPIC HYPOCOTYL3 pr     

Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway.

Four genetic loci were recently identified by mutations that affect phototropism in Arabidopsis thaliana (L.) Heyhn. seedlings. It was hypothesized that one of these loci, NPH1, encodes the apoprotein for a phototropic photoreceptor. All of the alleles at the other three mutant loci (nph2, nph3, and nph4) contained wild-type levels of the putative NPH1 protein and exhibited normal blue-light-dependent phosphorylation of the NPH1 protein. This indicated that the NPH2, NPH3, and NPH4 proteins likely function downstream of NPH1 photoactivation. We show here that, although the nph2, nph3, and nph4 mutants are all altered with respect to their phototropic responses, only the nph4 mutants are also altered in their gravitropic responsiveness. Thus, NPH2 and NPH3 appear to act as signal carriers in a phototropism-specific pathway, whereas NPH4 is required for both phototropism and gravitropism and thus may function directly in the differential growth response. Despite their altered phototropic responses in blue and green light as etiolated seedlings, the nph2 and nph4 mutants exhibited less dramatic mutant phenotypes as de-etiolated seedlings and when etiolated seedlings were irradiated with unilateral ultraviolet-A (UV-A) light. Examination of the phototropic responses of a mutant deficient in biologically active phytochromes, hy1-100, indicated that phytochrome transformation by UV-A light mediates an increase in phototropic responsiveness, accounting for the greater phototropic curvature of the nph2 and nph4 mutants to UV-A light than to blue light.     

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.     

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.     

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.     

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.     

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。     

Roles for the N- and C-terminal domains of phytochrome B in interactions between phytochrome B and cryptochrome signaling cascades

Plants fine-tune light responses through interactions betweenphotoreceptors. We have previously reported that the greeningof Arabidopsis thaliana roots is regulated synergistically byphytochromes and cryptochromes. In the present study, we investigatedthe functions of the N- and C-terminal domains of phytochromeB (phyB) in the interactions between phyB and cryptochrome signalingcascades. Transgenic Arabidopsis expressing the phyB N-terminaldomain fused to green fluorescent protein (GFP), ß-glucuronidase(GUS) and the nuclear localization signal (NLS) showed intenseroot greening under blue light, indicating that the C-terminaldomain was dispensable for the synergistic interaction in theinduction of root greening. However, root greening under redlight was substantially reduced in the absence of the C-terminaldomain. This effect was opposite to the previous observationthat removal of the C-terminal domain enhanced the signalingactivity of phyB in the inhibition of hypocotyl elongation.In addition, we found that overexpression of the isolated C-terminaldomain of phyB enhanced the blue light response not only forroot greening but also for the inhibition of hypocotyl elongation.Analysis of this activity on various photoreceptor mutant backgroundsdemonstrated that the isolated C-terminal domain enhanced cryptochromesignaling. In summary, these results demonstrate that differentdomains of phyB can play various roles which are dependent onlight conditions as well as on the specific physiological response.     

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。     

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.     

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.