Involvement of protein kinase C in the response of Neurospora crassa to blue light

As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa. Received: 4 December 1998 / Accepted: 21 May 1999     

Involvement of protein kinase C in the response of Neurospora crassa to blue light

As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa.     

Mapping the interaction sites of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> phytochrome FphA with the global regulator VeA and the White Collar protein LreB

Aspergillus nidulans senses red and blue-light and employs a phytochrome and a Neurospora crassa White Collar (WC) homologous system for light perception and transmits this information into developmental decisions. Under light conditions it undergoes asexual development and in the dark it develops sexually. The phytochrome FphA consists of a light sensory domain and a signal output domain, consisting of a histidine kinase and a response regulator domain. Previously it was shown that the phytochrome FphA directly interacts with the WC-2 homologue, LreB and another regulator, VeA. In this paper we mapped the interaction of FphA with LreB to the histidine kinase and the response regulator domain at the C-terminus in vivo using the bimolecular fluorescence complementation assay and in vitro by co-immunoprecipitation. In comparison, VeA interacted with FphA only at the histidine kinase domain. We present evidence that VeA occurs as a phosphorylated and a non-phosphorylated form in the cell. The phosphorylation status of the protein was independent of the light receptors FphA, LreB and the WC-1 homologue LreA.     

VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation

Blue light regulates many physiological and developmental processes in fungi. Most of the blue light responses in the ascomycete Neurospora crassa are dependent on the two blue light regulatory proteins White Collar (WC)-1 and -2. WC-1 has recently been shown to be the first fungal blue light photoreceptor. In the present study, we characterize the Neurospora protein VIVID. VIVID shows a partial sequence similarity with plant blue light photoreceptors. In addition, we found that VIVID non-covalently binds a flavin chromophore. Upon illumination with blue light, VIVID undergoes a photocycle indicative of the formation of a flavin-cysteinyl adduct. VVD is localized in the cytoplasm and is only present after light induction. A loss-of-function vvd mutant was insensitive to increases in light intensities. Furthermore, mutational analysis of the photoactive cysteine indicated that the formation of a flavin-cysteinyl adduct is essential for VIVID functions in vivo. Our results show that VIVID is a second fungal blue light photoreceptor which enables Neurospora to perceive and respond to daily changes in light intensity.     

How temperature affects the circadian clock of Neurospora crassa

Light and temperature are major environmental cues that influence circadian clocks. The molecular effects of these zeitgebers on the circadian clock of Neurospora crassa have been studied intensively during the last decade. While signal transduction of light into the circadian clock is quite well characterized, we have only recently begun to understand the molecular mechanisms that underlie temperature sensing. Here we summarize briefly the current knowledge about the effects of temperature on the circadian clock of Neurospora crassa.     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

The effects of light on sex determination in gametophytes of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

The sexuality of homosporous fern gametophytes is usually determined by antheridiogen, a pheromone that promotes maleness. In this work the effect of photomorphogenically active light on antheridiogen-induced male development was examined for gametophytes of Ceratopteris richardii. Although blue light did not affect sensitivity to Ceratopteris antheridiogen (A_Ce) in wild-type gametophytes, it was found that the gametophytes of the her1 mutant, which are insensitive to A_Ce, developed into males when grown under blue light in the presence of A_Ce. Thus, we conclude that another A_Ce-signal transduction pathway activated by blue light exists latently in the gametophytes of C. richardii. Red light, on the other hand, suppressed male development. Because simultaneous red and blue light-irradiation did not promote male development in the her1 gametophytes, the action of red light seems to dominate that of blue light. The results of experiments with a photomorphogenic mutant also suggested that phytochrome may be involved in the action of red light.     

Ca uptake and plasma membrane depolarization associated with blue light-sensitive exogenous NADH oxidation by Cuscuta protoplasts

Protoplasts isolated from the apical segments of Cuscuta reflexa exhibited blue light-sensitive PM-linked NADH oxidase activity and increased rate of Ca²⁺-uptake in presence of NADH in dark, which was also stimulated by blue light. Contrary to marginal inhibition by Con A treatment, the ATPase inhibitors significantly inhibited the Ca²⁺ uptake by the protoplasts both in dark and under blue light. The Ca²⁺-calmodulin antagonists, W-7 and calmidazolium, also inhibited Ca²⁺-uptake by protoplasts under similar conditions. The state of PM polarization was monitored by the fluorescent dye 9-amino acridine. It was observed that PM-linked NADH oxidation caused hyperpolarization of the membrane, the exposure of which to blue light resulted in membrane depolarization. The presence of Ca²⁺-calmodulin antagonists or Con A treatment completely abolished the blue light-induced membrane depolarization. It is argued that these actities at the PM, having some glycoproteic components, are functionally closely involved in blue light-induced signal transduction in Cuscuta     

模式生物粗糙脉孢菌光受体的研究进展

粗糙脉孢菌是一种重要的模式生物,在遗传调节机制、昼夜节律运行以及真菌光应答反应研究中起重要的作用.本综述主要介绍粗糙脉孢菌光受体WC-1和VVD的结构与功能,以及它们参与调节昼夜节律和光适应机制方面的研究进展.在该真菌中,所有已知的光应答反应都受蓝光调节,由光受体WC-1和VVD介导.WC-1是该真菌的转录因子,介导最初的光反应过程,产生VVD等多种光反应蛋白,而VVD通过负反馈机制抑制WC-1的转录作用.此外,vvd基因已经用于构建在哺乳动物中表达的光调节基因元件.     

Effect of 5-Azacytidine on the Light-Sensitive Formation of Sexual and Asexual Reproductive Structures in wc-1 and wc-2 Mutants of Neurospora crassa

Under nitrogen starvation conditions, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit condition (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in the light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.     

Polar auxin transport is required for the inhibition by blue light of the elongation-related LeEXT tomato gene

Hypocotyl elongation is an early developmental process regulated antagonistically by light and auxin. To highlight the interaction between both signals, we studied the photoregulation of the auxin-induced tomato (Lycopersicon esculentum Mill.) gene LeEXT involved in this process. RNA gel blot analysis indicated that this gene is down-regulated in response to blue light. We demonstrate that this response is principally mediated by the blue light photoreceptor cry1, but an interaction with the red/far-red light photoreceptors phyA, phyB1 and phyB2 has also been established. Furthermore, the polar auxin transport inhibitor NPA reverts the blue light inhibition of Lycopersicon esculentum gene encoding xyloglucan endotransglycosylase (LeEXT) expression, when it has the opposite effect in the dark or under red light. These results provide strong support for a specific interaction between auxin and blue light transduction pathways in the control of LeEXT expression, and therefore, of hypocotyl elongation in tomato.     

Genetic and molecular analysis of light-regulated plant development

Light regulates many physiological and developmental events in plants through the action of multiple sensory pigment systems. Although our understanding of the regulatory photoreceptors, including phytochromes (that principally absorb red and far-red energy) and blue light receptors, has advanced considerably in the recent past, the mechanisms of light signal transduction in higher plants are poorly understood. To unravel the molecular events associated with light-regulated plant development, a large number of photomorphogenic mutants have been isolated in several different plant species, including Arabidopsis, cucumber, tomato, pea, Brassica and Sorghum, which are either impaired in normal perception of light signal (photoreceptor mutants) or are affected in some specific or a sub-set of phenotypic traits (signal transduction mutants). Their physiological and molecular analysis is proving to be valuable in (1) assigning specific function to discrete phytochrome species, (2) elucidation of elements that constitute the transduction pathway downstream of signal perception, and (3) determining how different photosensory systems regulate many diverse responses. The progress made in the analysis of photomorphogenic mutants, as reviewed in this article, clearly indicates that multiple photoreceptors, either of the same or different class, interact through an intricate network of signal transduction pathways to finally determine the light-dependent phenotype of both monocots and dicots.     

Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axrl-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.     

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