The C-terminal kinase fragment of Arabidopsis phototropin 2 triggers constitutive phototropin responses

Phototropins mediate various blue-light responses such as phototropism, chloroplast relocation, stomatal opening and leaf flattening in plants. Phototropins are hydrophilic chromoproteins that are mainly bound to the plasma membrane. One of two phototropins in Arabidopsis thaliana, phot2, associates with the Golgi apparatus in a light-dependent manner. In this study, we analyzed the biological activities of the N-terminal photosensory and C-terminal kinase domains of phot2. For this purpose, these domains were fused to green fluorescent protein (GFP) and ectopically expressed in the wild-type and a phot1 phot2 double mutant of Arabidopsis. The kinase domain fused to GFP (P2CG) was localized to the plasma membrane and the Golgi apparatus, whereas the photosensory domain fused to GFP (P2NG) was uniformly localized in the cytosol. Hence, the kinase domain rather than the photosensory domain is responsible for the membrane association. Interestingly, the P2CG plants exhibited constitutive blue-light responses even in dark conditions, i.e. stomata were open and chloroplasts were in the avoidance position. By contrast, P2CG with a mutation that abolishes the kinase activity (P2C[D720/N]G) failed to exhibit these responses. phot2 kinase is therefore suggested to be correctly localized to functional sites in the cell and to trigger light signal transduction through its kinase activity. In contrast to P2CG, P2NG did not affect the phot2 responses, except for partial inhibition of the phototropic response caused by the endogenous phototropins.     

Where and how does phototropin transduce light signals in the cell?

Light plays pivotal roles as an important environmental signal in plant growth and development. In Arabidopsis, phototropin 1 (phot1) and 2 (phot2) are the photoreceptors that mediate phototropism, chloroplast relocation, stomatal opening and leaf flattening, in response to blue light. However, little is known about how phototropins transduce the signals after the light is perceived. Changes induced by blue light in terms of intracellular localization patterns of phot2 in Arabidopsis were examined. Phot2 distributed uniformly in the plasma membrane under dark conditions. Upon irradiation with blue light, some of the phot2 associated with the Golgi apparatus. It was also shown that the kinase domain, but not the photosensory domain, is required for a plasma membrane and Golgi localization. Furthermore a kinase fragment, lacking the photosensory domain, constitutively triggered physiological responses in planta. Thus, the plasma membrane and the Golgi apparatus appear to be the most likely sites for the initial step of phot2 signal transduction. The Golgi apparatus facilitates vesicle trafficking and delivery of membrane proteins to the required locations in the cell. Therefore, this study implicates the regulation of vesicle trafficking by the Golgi apparatus as a mechanism by which phot2 elicits its cellular responses.Key words: Golgi apparatus, kinase, light signal transduction, photoreceptor, phototropin, vesicle traffickingA range of physiological responses in plants is brought about by blue (390–500 nm) and ultraviolet-A (320–390 nm) light. Phototropin, one of major classes of blue light photoreceptors in plants, mediates responses such as phototropism, chloroplast relocation, light-induced stomatal opening and leaf flattening.^1–6 The dicotyledon Arabidopsis, possesses two phototropins, termed phot1 and phot2, which have both overlapping and distinct functions.^5,7 Phototropins consist of two functional domains, a N-terminal photosensory domain, containing two LOV (Light, Oxygen, Voltage) domains (LOV1 and LOV2) and a flavin-mononucleotide (FMN) chromophore and a regulatory serine/threonine kinase domain at the C-terminus.⁸To understand the mechanism of phototropin signal transduction, we expressed phot2 derivatives with translationally-fused green fluorescent protein (GFP) in a phot1phot2 double mutant in a wild type background in Arabidopsis.^9,10 Phototropin is a membrane- associated protein lacking a membrane spanning domain.⁸ Phot1 fused to GFP (P1G) is mainly localized to the plasma membrane, regardless of the light conditions.⁶ This property was retained when phot2 was fused to GFP (P2G).⁹ A part of P2G associates with punctate structures in the cytoplasm in response to blue light. The punctate P2G colocalized with KAM1ΔC:mRFP, a Golgi marker, we therefore conclude that phot2 associated with the Golgi apparatus in a blue light-dependent manner.⁹ This association was observed even in the presence of brefeldin A (BFA), an inhibitor of the vesicle trafficking.⁹To determine which domain of phot2 is responsible for the Golgi association, fragments of phot2 were fused to GFP and expressed in protoplasts.⁹ The N-terminal fragment fused to GFP (P2NG) was distributed uniformly in the cytoplasm. By contrast, the C-terminal fragment fused to GFP (P2CG) localized to both plasma membrane and punctate structures. The latter was shown to be the Golgi apparatus with the aid of the Golgi marker, KAM1ΔC:mRFP.⁹ These observations were corroborated from data using transgenic plants.¹⁰ Hence the C-terminal kinase domain, but not the N-terminal photo-sensory domain, is essential for the association of phot2 with the plasma membrane and the Golgi apparatus.The Golgi network is a key player in vesicle trafficking, to and from ER, vacuoles, trans-Golgi network, endosome and the plasma membrane.¹¹ Membrane spanning proteins are delivered and recycled through the Golgi apparatus. Among the membrane spanning proteins that are especially interesting, with respect to phototropin function, are auxin carriers such as PIN proteins. Phototropic curvature, which is under the control of phototropin, is believed to be caused by an uneven distribution of auxin.¹² The intracellular distribution of PIN proteins is maintained and regulated by vesicle trafficking.¹³ Indeed, factors such as ADP-ribosylation factor1 (ARF1) and guanine-nucleotide exchange factors (GEFs), which are involved in vesicle trafficking, are indispensable for the proper distribution of PIN proteins.^14–17 It is intriguing that a light stimulus alters the distribution pattern of PIN proteins.¹⁸ Hence, a fascinating possibility arises that phot2 alters the intracellular distribution of PIN proteins by regulating vesicle trafficking at the level of the Golgi apparatus.Phototropins are members of the subfamily VIII of AGC kinases.¹⁹ Interestingly, PINOID, another member of the subfamily, is localized at the cell periphery and regulates the apical-basal polar distribution of PIN proteins.^20–22 Accordingly, overexpression of PINOID disturbs the auxin distribution in transgenic plants.^23,24 The kinase fragment of phototropin exhibits constitutive kinase activity in vitro.²⁵ Interestingly, the auxin distribution is disturbed in plants expressing P2CG, as is the case with PINOID.¹⁰ Hence, both PINOID and phot2 might alter the PIN protein distribution in the cell through a common mechanism, in response to distinct stimuli.To date, no authentic substrate has been described for any of the AGC VIII kinases.¹⁹ Considering the localization pattern of phototropins, the substrates are most likely to reside in the plasma membrane and/or the Golgi apparatus. NPH3, RPT2 and PKS1 are downstream factors for phototropic responses,^26–28 all associating with the plasma membrane. Although they interact preferentially with the N-terminal rather than the C-terminal domain of phot1,^26,29 it is also possible that the C-terminal kinase domain interacts transiently with these factors leading to their phosphorylation. However, at present the molecular functions of NPH3, RPT2 and PKS1 remain unclear and await future investigation.Although both phot1 and phot2 are localized to the plasma membrane, punctate structures are yet to be described for P1G. Instead, a part of phot1-GFP is released from the plasma membrane to the cytosol in response to a light stimulus.⁶ We recently reexamined the intracellular localization of P1G. A specific network-like structure in the cytoplasm in addition to intense plasma membrane staining was observed (Fig. 1). A similar pattern was observed for P2G although it is less clear.⁹ Hence, both phot1 and phot2 might be associating with a structure in the cytoplasm that has yet to be described, and which might be another site of phototropin signaling in the cell.Open in a separate windowFigure 1A light-induced network-like distribution pattern of P1G in the cytoplasm. The P1G seedlings grown under dark conditions⁶ were incubated in MS solution (diluted 50%) without (upper panels) or with (lower panels) 100 µM BFA. The cells were inspected with a confocal laser scanning microscope. Images taken before (left) or after (right) blue light illumination at 48 µmol m⁻² sec⁻¹ are shown. Bar = 10 µm.P2CG elicits some phototropin responses without a light stimulus.¹⁰ That is, chloroplasts were in the avoidance position and stomata opened without a blue light stimulus in the P2CG overexpressing plants. It is a fascinating possibility that phototropin elicits those responses through the regulation of vesicle trafficking, although other possibilities exist. Stomata open as the result of phosphorylation of the plasma membrane H⁺-ATPase³⁰ and it is unlikely that the vesicle trafficking is directly involved in this regulatory process. It is possible to conjecture that vesicle trafficking affects chloroplast positioning but how this would work remains to be determined. Overall how a single photoreceptor such as phototoropin might regulate diverse physiological responses awaits future study.     

Molecular basis of the functional specificities of phototropin 1 and 2

A blue-light photoreceptor in plants, phototropin, mediates phototropism, chloroplast relocation, stomatal opening, and leaf-flattening responses. Phototropin is divided into two functional moieties, the N-terminal photosensory and the C-terminal signaling moieties. Phototropin perceives light stimuli by the light, oxygen or voltage (LOV) domain in the N-terminus; the signal is then transduced intramolecularly to the C-terminal kinase domain. Two phototropins, phot1 and phot2, which have overlapping and distinct functions, exist in Arabidopsis thaliana. Phot1 mediates responses with higher sensitivity than phot2. Phot2 mediates specific responses, such as the chloroplast avoidance response and chloroplast dark positioning. To elucidate the molecular basis for the functional specificities of phot1 and phot2, we exchanged the N- and C-terminal moieties of phot1 and phot2, fused them to GFP and expressed them under the PHOT2 promoter in the phot1 phot2 mutant background. With respect to phototropism and other responses, the chimeric phototropin consisting of phot1 N-terminal and phot2 C-terminal moieties (P1n/2cG) was almost as sensitive as phot1; whereas the reverse combination (P2n/1cG) functioned with lower sensitivity. Hence, the N-terminal moiety mainly determined the sensitivity of the phototropins. Unexpectedly, both P1n/2cG and P2n/1cG mediated the chloroplast avoidance response, which is specific to phot2. Hence, chloroplast avoidance activity appeared to be suppressed specifically in the combination of N- and C-terminal moieties of phot1. Unlike the chloroplast avoidance response, chloroplast dark positioning was observed for P2G and P2n/1cG but not for P1G or P1n/2cG, suggesting that a specific structure in the N-terminal moiety of phot2 is required for this activity.     

A transgene encoding a blue-light receptor, phot1, restores blue-light responses in the Arabidopsis phot1 phot2 double mutant

Phototropins (phot1 and phot2) are suggested to be multifunctional blue-light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses.     

The phototropin family as photoreceptors for blue light-induced chloroplast relocation

Blue light-induced chloroplast accumulation and avoidance relocation movements are controlled by the blue light photoreceptor phototropin. The Arabidopsis thaliana genome has two phototropin genes encoding phot1 and phot2. Each of these photoreceptors contains two LOV (light oxygen and voltage) domains and a kinase domain. The LOV domains absorb blue light though an associated flavin mononucleotide chromophore, while the kinase domain is thought to be associated with signal transduction. The phototropins control not only chloroplast relocation movement, but also blue light-induced phototropic responses, leaf expansion and stomatal opening. Here I review the role of phototropin as a photoreceptor for chloroplast photorelocation movement. Electronic Publication     

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.     

Interactions between a blue-green reversible photoreceptor and a separate UV-B receptor in stomatal guard cells

Stomatal opening exhibits two main peaks of activity in the visible range-a red peak, mediated by photosynthesis, and a blue peak, mediated by one or more blue light (BL) photoreceptors. In addition, a pronounced peak in the UV-B region has been characterized, as has a smaller UV-A peak. The BL-induced stomatal opening can be reversed by green light (GL). Here we report that UV-B-induced opening is also antagonized by GL. To determine whether UV-B is being absorbed by the BL photoreceptor or by a separate UV-B receptor, the UV-B responses of two different Arabidopsis mutants, npq1 and phot1/phot2, were tested. Both putative BL-photoreceptor mutants exhibited normal stomatal opening in response to UV-B, consistent with the existence of a separate UV-B photoreceptor. Moreover, GL failed to antagonize UV-B-induced stomatal opening in the phot1/phot2 double mutant and only partially antagonized UV-B opening in npq1. Thus, both phot1 and phot 2, as well as zeaxanthin, are required for the normal GL inhibition of UV-B. A model for a photoreceptor network that regulates stomatal opening is presented. Unlike the situation in guard cells, the UV-B bending response of Arabidopsis hypocotyls during phototropism appears to be mediated by phototropins.     

The role of a 14-3-3 protein in stomatal opening mediated by PHOT2 in Arabidopsis

The 14-3-3 λ isoform is required for normal stomatal opening mediated by PHOT2 in Arabidopsis thaliana. Arabidopsis phototropin2 (PHOT2) interacts with the λ-isoform 14-3-3 protein both in yeast two-hybrid screening and in an in vitro pull-down assay. Further yeast two-hybrid analysis also showed that the PHOT2 C-terminal kinase domain was required for the interaction. Site-directed mutagenesis indicated that PHOT2 Ser-747 is essential for the yeast interaction. Phenotypic characterization of a loss-of-function 14-3-3 λ mutant in a phot1 mutant background showed that the 14-3-3 λ protein was necessary for normal PHOT2-mediated blue light-induced stomatal opening. PHOT2 Ser-747 was necessary for complementation of the blue light-activated stomatal response in a phot1 phot2 double mutant. The 14-3-3 λ mutant in the phot1 mutant background allowed normal phototropism and normal chloroplast accumulation and avoidance responses. It also showed normal stomatal opening mediated by PHOT1 in a phot2 mutant background. The 14-3-3 κ mutant had no effect on stomatal opening in response to blue light. Although the 14-3-3 λ mutant had no chloroplast movement phenotype, the 14-3-3 κ mutation caused a weaker avoidance response at an intermediate blue light intensity by altering the balance between the avoidance and accumulation responses. The results highlight the strict specificity of phototropin-mediated signal transduction pathways.     

The Arabidopsis rab5 homologs rha1 and ara7 localize to the prevacuolar compartment

Rha1, an Arabidopsis Rab5 homolog, plays a critical role in vacuolar trafficking in plant cells. In this study, we investigated the localization of Rha1 and Ara7, two Arabidopsis proteins that have highly similar amino acid sequence homology to Rab5 in animal cells. Both Ara7 and Rha1 gave a punctate staining pattern and colocalized when transiently expressed as GFP- (green fluorescent protein) or small epitope-tagged forms in Arabidopsis protoplasts. In protoplasts, transiently expressed Rha1 and Ara7 colocalized with AtPEP12p and VSR(At-1), two proteins that are known to be present at the prevacuolar compartment (PVC). Furthermore, endogenous Rha1 also gave a punctate staining pattern and colocalized with AtPEP12p to the PVC. Mutations in the first and second GTP-binding motifs alter the localizations of GFP: Rha1[S24N] in the cytosol and Rha1[Q69L] in the tonoplast of the central vacuole. Also, mutations in the effector domain and the prenylation site inhibit membrane association of Rha1. Based on these results, we propose that Rha1 and Ara7 localize to the PVC and that GTP-binding motifs as well as the effector domain are important for localization of Rha1 to the PVC.     

Blue light-induced orientation movements of chloroplasts in higher plants: Recent progress in the study of their mechanisms

The discovery of phototropins, photoreceptors for chloroplast responses in Arabidopsis thaliana, brought about renewed interest in these blue light-controlled movements. Recent progress in research on their mechanisms in higher plants is briefly summarized. Phototropins mediate phototropism, chloroplast relocations and stomatal movements. Their functions are partially overlapping, with phot1 active predominantly in weak light and phot2 active in strong light. The accumulation response of chloroplasts appears to be mediated by phot1 and phot2 whereas the avoidance response is controlled by phot2. The role of Ca²⁺ as a potential intracellular messenger has been discussed in view of the recently demonstrated blue light-induced transient increases in the cytosolic Ca²⁺ mediated differently by phot1 and phot2. Differential inhibition of accumulation and avoidance responses by wortmannin, the inhibitor of phosphoinositide-3 kinases, in Lemna trisulca points to an important role of these enzymes in the signal transduction. A new, multi-domain component controlling chloroplast positioning and movement, CHUP1, encodes an actin-binding protein in Arabidopsis.     

植物蓝光受体研究进展

植物蓝光调节的反应主要有向光性、抑制幼茎伸长、叶绿体迁移、刺激气孔张开和调节基因表达等。蓝光反应的有效波长是蓝光和近紫外光(320—400am),故蓝光受体也叫蓝光／近紫外光受体。植物蓝光受体研究近年来取得较大进展。以拟南芥为例,已得到确认的受体至少有隐花色素(CRY1、2)和向光蛋白(phototropin)两大类。转基因拟南芥对蓝光、紫外光和绿光敏感,并发现CRY1是一个可溶性蛋白。CRY2编码一个核蛋白,蓝光在转录水平对该蛋白进行调节,它的作用是增加拟南芥对蓝光的敏感性。CRY1和CRY2共同介导了拟南芥植物的向光性。隐花色素的蛋白与辅基之间以非共价键连接,可以吸收蓝光和近紫外光。CRY1和CRY2蛋白之间,尤其是N端相似性很高。向光蛋白目前只发现PHOT1和PHO12两种,向光蛋白作为丝／苏氨酸激酶蓝光受体含有两个光氧化结构域(LOV)并参与了植物向光性叶绿体运动、气孔开放等。     

Functional analyses of the activation loop of phototropin2 in Arabidopsis

Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2-mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses.     

Phototropins 1 and 2: versatile plant blue-light receptors

Blue and ultraviolet-A light regulate a wide range of responses in plants, including phototropism, chloroplast migration and stomatal opening. However, the photoreceptors for these light responses have been identified only recently. The phototropins (phot1 and phot2) represent a new class of receptor kinases that appear to be exclusive to plants. Recent genetic analysis has shown that phot1 and phot2 exhibit partially overlapping functions in mediating phototropism, chloroplast migration, and stomatal opening in Arabidopsis. Although significant progress has been made in understanding the early photochemical and biochemical events that follow phototropin excitation, the details of how this excitation activates such different responses remain to be elucidated.     

Light-induced Conformational Changes of LOV1 (Light Oxygen Voltage-sensing Domain 1) and LOV2 Relative to the Kinase Domain and Regulation of Kinase Activity in Chlamydomonas Phototropin

Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.     

Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function

Phototropins (phot1 and phot2) are autophosphorylating serine/threonine kinases that function as photoreceptors for phototropism, light-induced chloroplast movement, and stomatal opening in Arabidopsis. The N-terminal region of phot1 and phot2 contains two specialized PAS domains, designated LOV1 and LOV2, which function as binding sites for the chromophore flavin mononucleotide (FMN). Both LOV1 and LOV2 undergo a self-contained photocycle, which involves the formation of a covalent adduct between the FMN chromophore and a conserved active-site cysteine residue (Cys39). Replacement of Cys39 with alanine abolishes the light-induced photochemical reaction of LOV1 and LOV2. Here we have used the Cys39Ala mutation to investigate the role of LOV1 and LOV2 in regulating phototropin function. Photochemical analysis of a bacterially expressed LOV1 + LOV2 fusion protein indicates that LOV2 functions as the predominant light-sensing domain for phot1. LOV2 also plays a major role in mediating light-dependent autophosphorylation of full-length phot1 expressed in insect cells and transgenic Arabidopsis. Moreover, photochemically active LOV2 alone in full-length phot1 is sufficient to elicit hypocotyl phototropism in transgenic Arabidopsis, whereas photochemically active LOV1 alone is not. Further photochemical and biochemical analyses also indicate that the LOV1 and LOV2 domains of phot2 exhibit distinct roles. The significance for the different roles of the phototropin LOV domains is discussed.     

Phototropins and chloroplast activity in plant blue light signaling

In plants, phototropins 1 (phot1) and 2 (phot2) mediate chloroplast movement to blue light (BL). A recent report showed that phototropins (phot) are required for the expression of chloroplast genes in rice. The light-induced responses of phot1a rice mutants result in H₂O₂-mediated damage to chloroplast photosystems, indicating that phot-regulated responses might be associated with the other photoreceptor, such as cryptochrome (cry) BL receptor. This suggests diversification and specialization of photoreceptor signaling in plants.Key words: blue light, blue light receptor, chloroplast, cryptochrome, H₂O₂, phototropin, signalingIn order to counteract the adverse effects of environmental ight, plants have evolved sensory mechanisms that monitor their surroundings and adapt their growth and development through the use of a complex signaling network.¹ Plants sense their environmental light conditions by using three principal families of signal-transducing photoreceptors; the red/far-red (R/FR) light-absorbing phytochromes (phy) and the UV-A/blue light (BL)-absorbing cryptochromes (cry) and phototropins (phot).² The phys are reversibly photochromic biliproteins that absorb maximally in the R and FR light regions of the spectrum. Cry and phot possess a pair of flavin derivates. Two cry and two phot family members have been identified and well characterized in Arabidopsis. Photoreceptors regulate development throughout the plant lifecycle, from seed germination through to plant maturation and the onset of reproduction. BL regulates a wide variety of photoresponses in higher plants, including chloroplast movement, inhibition of hypocotyls elongation, circadian timing, regulation of gene expression and stomatal opening.^3–5 The roles of individual photoreceptors in mediating plant development have, however, often been confounded by redundant, synergistic and in some cases mutually antagonistic mechanisms of action. The mechanisms of photoreceptor signal transduction are far from being completely elucidated, but are believed to involve both cytosolic and nuclear components. The presence of putative kinase domains within photoreceptor proteins has suggested a role for phosphorylation in light signaling. The action of cry1 and cry2 has been demonstrated to involve BL-mediated autophosphorylation.^6,7 Phot1 was originally identified as a 120 kDa-membrane associated protein displaying BL-mediated autophosphorylation.⁸ It is now well accepted that phot mediates chloroplast movement, phototropism and stomatal opening in plants in response to BL.     

The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery

A yeast GTP-binding protein, the YPT1 gene product, has been found to function early in the secretion pathway. The ypt1-1 mutation causes a phenotype reminiscent of early secretion-defective mutants, including accumulation of membranes and vesicles as well as a partial defect in secretion and incomplete glycosylation of invertase. Immunofluorescence localization studies using affinity-purified antibody directed against the YPT1 protein showed punctate staining of the cytoplasm of growing yeast cells and very intense staining of small buds, where membrane growth and secretion are most active. The punctate cytoplasmic staining is changed in a mutant (sec7) under conditions that cause aberrant Golgi structures to accumulate. The pattern of immunofluorescence obtained when mouse cells were stained with the antibody coincided closely with the pattern observed with wheat germ agglutinin, suggesting that a mammalian counterpart of the yeast YPT1 protein is located in the Golgi apparatus. These results are interpreted as suggesting that GTP-binding proteins may act to direct intracellular vesicle traffic.     

Phototropins but not cryptochromes mediate the blue light-specific promotion of stomatal conductance, while both enhance photosynthesis and transpiration under full sunlight

Leaf epidermal peels of Arabidopsis (Arabidopsis thaliana) mutants lacking either phototropins 1 and 2 (phot1 and phot2) or cryptochromes 1 and 2 (cry1 and cry2) exposed to a background of red light show severely impaired stomatal opening responses to blue light. Since phot and cry are UV-A/blue light photoreceptors, they may be involved in the perception of the blue light-specific signal that induces the aperture of the stomatal pores. In leaf epidermal peels, the blue light-specific effect saturates at low irradiances; therefore, it is considered to operate mainly under the low irradiance of dawn, dusk, or deep canopies. Conversely, we show that both phot1 phot2 and cry1 cry2 have reduced stomatal conductance, transpiration, and photosynthesis, particularly under the high irradiance of full sunlight at midday. These mutants show compromised responses of stomatal conductance to irradiance. However, the effects of phot and cry on photosynthesis were largely nonstomatic. While the stomatal conductance phenotype of phot1 phot2 was blue light specific, cry1 cry2 showed reduced stomatal conductance not only in response to blue light, but also in response to red light. The levels of abscisic acid were elevated in cry1 cry2. We conclude that considering their effects at high irradiances cry and phot are critical for the control of transpiration and photosynthesis rates in the field. The effects of cry on stomatal conductance are largely indirect and involve the control of abscisic acid levels.     

Using Caveolin-1 epithelial immunostaining patterns to stratify human breast cancer patients and to predict the Caveolin-1 (P132L) mutation

Caveolin-1 (Cav-1) mutations, such as P132L, are associated with ER-positive human breast cancers. However, no immuno-histochemical methods have yet been described to predict the presence of Cav-1 mutations in human breast cancer. Since the P132L mutation acts in a dominant-negative fashion and causes the mis-localization Cav-1 in cultured cells in vitro, we hypothesized that patients carrying this mutation would show a similar Cav-1 staining pattern in vivo. Indeed, while performing histological analysis of Cav-1 immunostaining on human breast cancer samples, we noted the emergence of two distinct epithelial staining patterns: 1) punctate peri-nuclear “Golgi-like” localization; or 2) diffuse cytoplasmic staining. The punctate peri-nuclear staining pattern was associated with ER-alpha positivity and was present mainly in well-differentiated samples. In striking contrast, the diffuse staining pattern was present in poorly differentiated samples, and was not associated with ER-status. DNA sequence analysis revealed that only well-differentiated samples with a punctate staining pattern harbored the Cav-1 P132L mutation. Thus, immunostaining of Cav-1 can be used as a first step to stratify human breast patients and to predict the presence of Cav-1 mutations. As such, the punctate Cav-1 immunostaining pattern can now be used as a screening tool to select patients for Cav-1 mutational analysis.