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1.
Plant Cell ; 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852772

RESUMO

Diatoms are the most diverse group of algae with at least 100,000 species. Contributing approximately 20% of annual global carbon fixation, they underpin major aquatic food webs and drive global biogeochemical cycles. Over the last two decades, Thalassiosira pseudonana and Phaeodactylum tricornutum have become the most important references for diatom molecular research, ranging from cell biology to ecophysiology, because of their rapid growth rates, small genomes and the cumulative wealth of associated genetic resources. To ascertain the significance of the evolutionary divergence of diatoms, additional references are emerging such as Fragilariopsis cylindrus and Pseudo-nitzschia multistriata. Here, we describe how functional genomics and reverse genetics have contributed to our understanding of this important class of microalgae in the context of evolution, cell biology and metabolic adaptations. Our review will also highlight promising areas of investigation into the diversity of these photosynthetic organisms, including the discovery of new molecular pathways governing the life of secondary plastid-bearing organisms in aquatic environments.

2.
Proc Natl Acad Sci U S A ; 116(26): 13137-13142, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31171659

RESUMO

Periodic light-dark cycles govern the timing of basic biological processes in organisms inhabiting land as well as the sea, where life evolved. Although prominent marine phytoplanktonic organisms such as diatoms show robust diel rhythms, the mechanisms regulating these processes are still obscure. By characterizing a Phaeodactylum tricornutum bHLH-PAS nuclear protein, hereby named RITMO1, we shed light on the regulation of the daily life of diatoms. Alteration of RITMO1 expression levels and timing by ectopic overexpression results in lines with deregulated diurnal gene expression profiles compared with the wild-type cells. Reduced gene expression oscillations are also observed in these lines in continuous darkness, showing that the regulation of rhythmicity by RITMO1 is not directly dependent on light inputs. We also describe strong diurnal rhythms of cellular fluorescence in wild-type cells, which persist in continuous light conditions, indicating the existence of an endogenous circadian clock in diatoms. The altered rhythmicity observed in RITMO1 overexpression lines in continuous light supports the involvement of this protein in circadian rhythm regulation. Phylogenetic analysis reveals a wide distribution of RITMO1-like proteins in the genomes of diatoms as well as in other marine algae, which may indicate a common function in these phototrophs. This study adds elements to our understanding of diatom biology and offers perspectives to elucidate timekeeping mechanisms in marine organisms belonging to a major, but under-investigated, branch of the tree of life.

3.
Plant Physiol ; 177(3): 953-965, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29773581

RESUMO

Marine diatoms are prominent phytoplankton organisms that perform photosynthesis in extremely variable environments. Diatoms possess a strong ability to dissipate excess absorbed energy as heat via nonphotochemical quenching (NPQ). This process relies on changes in carotenoid pigment composition (xanthophyll cycle) and on specific members of the light-harvesting complex family specialized in photoprotection (LHCXs), which potentially act as NPQ effectors. However, the link between light stress, NPQ, and the existence of different LHCX isoforms is not understood in these organisms. Using picosecond fluorescence analysis, we observed two types of NPQ in the pennate diatom Phaeodactylum tricornutum that were dependent on light conditions. Short exposure of low-light-acclimated cells to high light triggers the onset of energy quenching close to the core of photosystem II, while prolonged light stress activates NPQ in the antenna. Biochemical analysis indicated a link between the changes in the NPQ site/mechanism and the induction of different LHCX isoforms, which accumulate either in the antenna complexes or in the core complex. By comparing the responses of wild-type cells and transgenic lines with a reduced expression of the major LHCX isoform, LHCX1, we conclude that core complex-associated NPQ is more effective in photoprotection than is the antenna complex. Overall, our data clarify the complex molecular scenario of light responses in diatoms and provide a rationale for the existence of a degenerate family of LHCX proteins in these algae.


Assuntos
Diatomáceas/fisiologia , Complexos de Proteínas Captadores de Luz/metabolismo , Aclimatação , Clorofila/metabolismo , Cloroplastos/metabolismo , Diatomáceas/citologia , Fluorescência , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Luz , Complexos de Proteínas Captadores de Luz/genética , Organismos Geneticamente Modificados , Processos Fotoquímicos , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
4.
Curr Opin Plant Biol ; 37: 70-77, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28456112

RESUMO

Marine eukaryotic phytoplankton are major contributors to global primary production. To adapt and thrive in the oceans, phytoplankton relies on a variety of light-regulated responses and light-acclimation capacities probably driven by sophisticated photoregulatory mechanisms. A plethora of photoreceptor-like sequences from marine microalgae have been identified in omics approaches. Initial studies have revealed that some algal photoreceptors are similar to those known in plants. In addition, new variants with different spectral tuning and algal-specific light sensors have also been found, changing current views and perspectives on how photoreceptor structure and function have diversified in phototrophs experiencing different environmental conditions.


Assuntos
Luz , Microalgas/metabolismo , Microalgas/efeitos da radiação , Fotorreceptores de Plantas/genética , Fotorreceptores de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
5.
Phys Rev E ; 94(2-1): 022418, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27627344

RESUMO

Chain formation in diatoms is relevant because of several aspects of their adaptation to the ecosystem. However, the tools to quantify the regulation of their assemblage and infer specific mechanisms in a laboratory setting are scarce. To address this problem, we define an approach based on a statistical physics model of chain growth and separation in combination with experimental evaluation of chain-length distributions. Applying this combined analysis to data from Chaetoceros decipiens and Phaeodactylum tricornutum, we find that cells of the first species control chain separation, likely through a cell-to-cell communication process, while the second species only modulates the separation rate. These results promote quantitative methods for characterizing chain formation in several chain-forming species and in diatoms in particular.


Assuntos
Diatomáceas/crescimento & desenvolvimento , Modelos Biológicos , Diatomáceas/citologia
6.
J Exp Bot ; 67(13): 3939-51, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27225826

RESUMO

Diatoms are phytoplanktonic organisms that grow successfully in the ocean where light conditions are highly variable. Studies of the molecular mechanisms of light acclimation in the marine diatom Phaeodactylum tricornutum show that carotenoid de-epoxidation enzymes and LHCX1, a member of the light-harvesting protein family, both contribute to dissipate excess light energy through non-photochemical quenching (NPQ). In this study, we investigate the role of the other members of the LHCX family in diatom stress responses. Our analysis of available genomic data shows that the presence of multiple LHCX genes is a conserved feature of diatom species living in different ecological niches. Moreover, an analysis of the levels of four P. tricornutum LHCX transcripts in relation to protein expression and photosynthetic activity indicates that LHCXs are differentially regulated under different light intensities and nutrient starvation, mostly modulating NPQ capacity. We conclude that multiple abiotic stress signals converge to regulate the LHCX content of cells, providing a way to fine-tune light harvesting and photoprotection. Moreover, our data indicate that the expansion of the LHCX gene family reflects functional diversification of its members which could benefit cells responding to highly variable ocean environments.


Assuntos
Proteínas de Algas/genética , Diatomáceas/genética , Regulação da Expressão Gênica , Complexos de Proteínas Captadores de Luz/genética , Fitoplâncton/genética , Transdução de Sinais , Proteínas de Algas/metabolismo , Diatomáceas/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese , Fitoplâncton/metabolismo
7.
Plant Cell ; 28(3): 616-28, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26941092

RESUMO

The absorption of visible light in aquatic environments has led to the common assumption that aquatic organisms sense and adapt to penetrative blue/green light wavelengths but show little or no response to the more attenuated red/far-red wavelengths. Here, we show that two marine diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana, possess a bona fide red/far-red light sensing phytochrome (DPH) that uses biliverdin as a chromophore and displays accentuated red-shifted absorbance peaks compared with other characterized plant and algal phytochromes. Exposure to both red and far-red light causes changes in gene expression in P. tricornutum, and the responses to far-red light disappear in DPH knockout cells, demonstrating that P. tricornutum DPH mediates far-red light signaling. The identification of DPH genes in diverse diatom species widely distributed along the water column further emphasizes the ecological significance of far-red light sensing, raising questions about the sources of far-red light. Our analyses indicate that, although far-red wavelengths from sunlight are only detectable at the ocean surface, chlorophyll fluorescence and Raman scattering can generate red/far-red photons in deeper layers. This study opens up novel perspectives on phytochrome-mediated far-red light signaling in the ocean and on the light sensing and adaptive capabilities of marine phototrophs.


Assuntos
Diatomáceas/fisiologia , Transdução de Sinal Luminoso/efeitos da radiação , Fitocromo/efeitos da radiação , Plantas/efeitos da radiação , Adaptação Fisiológica , Clorofila/metabolismo , Diatomáceas/efeitos da radiação , Oceanos e Mares , Análise Espectral Raman , Luz Solar
8.
Planta ; 243(4): 909-23, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26721646

RESUMO

MAIN CONCLUSION: Arabidopsis PHS1, initially known as an actor of cytoskeleton organization, is a positive regulator of flowering in the photoperiodic and autonomous pathways by modulating both CO and FLC mRNA levels. Protein phosphorylation and dephosphorylation is a major type of post-translational modification, controlling many biological processes. In Arabidopsis thaliana, five genes encoding MAPK phosphatases (MKP)-like proteins have been identified. Among them, PROPYZAMIDE HYPERSENSITIVE 1 (PHS1) encoding a dual-specificity protein tyrosine phosphatase (DsPTP) has been shown to be involved in microtubule organization, germination and ABA-regulated stomatal opening. Here, we demonstrate that PHS1 also regulates flowering under long-day and short-day conditions. Using physiological, genetic and molecular approaches, we have shown that the late flowering phenotype of the knock-out phs1-5 mutant is linked to a higher expression of FLOWERING LOCUS C (FLC). In contrast, a decline of both CONSTANS (CO) and FLOWERING LOCUS T (FT) expression is observed in the knock-out phs1-5 mutant, especially at the end of the light period under long-day conditions when the induction of flowering occurs. We show that this partial loss of sensitivity to photoperiodic induction is independent of FLC. Our results thus indicate that PHS1 plays a dual role in flowering, in the photoperiodic and autonomous pathways, by modulating both CO and FLC mRNA levels. Our work reveals a novel actor in the complex network of the flowering regulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Flores/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Mutação , Fotoperíodo , Plantas Geneticamente Modificadas , Proteínas Tirosina Fosfatases/genética , Processamento Pós-Transcricional do RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
J Plant Physiol ; 172: 42-54, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25087009

RESUMO

Light is essential for the life of photosynthetic organisms as it is a source of energy and information from the environment. Light excess or limitation can be a cause of stress however. Photosynthetic organisms exhibit sophisticated mechanisms to adjust their physiology and growth to the local environmental light conditions. The cryptochrome/photolyase family (CPF) is composed of flavoproteins with similar structures that display a variety of light-dependent functions. This family encompasses photolyases, blue-light activated enzymes that repair ultraviolet-light induced DNA damage, and cryptochromes, known for their photoreceptor functions in terrestrial plants. For this review, we searched extensively for CPFs in the available genome databases to trace the distribution and evolution of this protein family in photosynthetic organisms. By merging molecular data with current knowledge from the functional characterization of CPFs from terrestrial and aquatic organisms, we discuss their roles in (i) photoperception, (ii) biological rhythm regulation and (iii) light-induced stress responses. We also explore their possible implication in light-related physiological acclimation and their distribution in phototrophs living in different environments. The outcome of this structure-function analysis reconstructs the complex scenarios in which CPFs have evolved, as highlighted by the novel functions and biochemical properties of the most recently described family members in algae.


Assuntos
Desoxirribodipirimidina Fotoliase/genética , Evolução Molecular , Flavoproteínas/genética , Fenômenos Fisiológicos Vegetais , Criptocromos/genética , Criptocromos/metabolismo , Desoxirribodipirimidina Fotoliase/metabolismo , Flavoproteínas/metabolismo , Luz , Fotossíntese
10.
FEBS Lett ; 589(2): 189-92, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25500270

RESUMO

Even though the plant photoreceptors cryptochromes were discovered more than 20 years ago, the mechanism through which they transduce light signals to their partner molecules such as COP1 (Constitutive Photomorphogenic 1) or SPA1 (Suppressor of Phytochrome A) still remains to be established. We propose that a negative charge induced by light in the vicinity of the flavin chromophore initiates cryptochrome 1 signalling. This negative charge might expel the protein-bound ATP from the binding pocket, thereby pushing off the C-terminus that covers the ATP pocket in the dark state of the protein. This conformational change should allow for phosphorylation of previously inaccessible amino acids. A partially phosphorylated 'ESSSSGRR-VPE' fragment of the C-terminus could mimic the sequence of the transcription factor HY5 that is essential for binding to the negative regulator of photomorphogenesis COP1. HY5 release through competition for the COP1 binding site could represent the long-sought connection between light activation of cryptochrome and modulation of photomorphogenesis.


Assuntos
Criptocromos/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Transdução de Sinais , Proteínas de Plantas/química , Plantas/química , Ligação Proteica , Fatores de Transcrição/metabolismo
11.
Sci Rep ; 4: 5175, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24898692

RESUMO

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH(·) radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·(-), from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396(-). Its negative charge could trigger conformational changes necessary for signal transduction.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Criptocromos/metabolismo , Luz , Algoritmos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Criptocromos/química , Criptocromos/efeitos da radiação , Oxirredução , Conformação Proteica , Teoria Quântica , Transdução de Sinais/efeitos da radiação , Espectrofotometria Ultravioleta
13.
Plant J ; 59(2): 316-28, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19302419

RESUMO

The proteins kinases SNF1/AMPK/SnRK1 are a subfamily of serine/threonine kinases that act as metabolite sensors to constantly adapt metabolism to the supply of, and demand for, energy. In the yeast Saccharomyces cerevisiae, the SNF1 complex is a central component of the regulatory response to glucose starvation. AMP activated protein kinase (AMPK) the mammalian homologue of SNF1, plays a central role in the regulation of energy homeostasis at the cellular as well as the whole-body levels. In Arabidopsis thaliana, SnRK1.1 and SnRK1.2 have recently been described as central integrators of a transcription network for stress and energy signalling. In this study, biochemical analysis established SnRK1.1 as the major SnRK1 isoform both in isolated cells and leaves. In order to elucidate the function of SnRK1.1 in Arabidopsis thaliana, transgenic plants over-expressing SnRK1.1 were produced. Genetic, biochemical, physiological and molecular analyses of these plants revealed that SnRK1.1 is implicated in sugar and ABA signalling pathways. Modifications of the starch and soluble sugar content were observed in the 35S:SnRK1.1 transgenic lines. Our studies also revealed modifications of the activity of essential enzymes such as nitrate reductase or ADP-glucose pyrophosphorylase, and of the expression of several sugar-regulated genes, confirming the central role of the protein kinase SnRK1 in the regulation of metabolism.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Metabolismo dos Carboidratos , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais
14.
FEBS Lett ; 583(9): 1427-33, 2009 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-19327354

RESUMO

Cryptochromes are widely distributed blue light photoreceptors involved in numerous signaling functions in plants and animals. Both plant and animal-type cryptochromes are found to bind ATP and display intrinsic autokinase activity; however the functional significance of this activity remains a matter of speculation. Here we show in purified preparations of Arabidopsis cry1 that ATP binding induces conformational change independently of light and increases the amount and stability of light-induced flavin radical formation. Nucleotide binding may thereby provide a mechanism whereby light responsivity in organisms can be regulated through modulation of cryptochrome photoreceptor conformation.


Assuntos
Trifosfato de Adenosina/metabolismo , Arabidopsis/metabolismo , Flavoproteínas/metabolismo , Proteínas de Arabidopsis , Criptocromos , Flavoproteínas/química , Flavoproteínas/isolamento & purificação , Flavoproteínas/fisiologia , Hidrólise , Oxirredução , Fotoquímica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Tripsina/metabolismo
15.
PLoS Biol ; 6(7): e160, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18597555

RESUMO

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non-light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.


Assuntos
Proteínas do Olho/metabolismo , Flavinas/metabolismo , Flavoproteínas/metabolismo , Transdução de Sinal Luminoso , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Relógios Biológicos/fisiologia , Linhagem Celular , Criptocromos , Drosophila melanogaster , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas do Olho/efeitos da radiação , Flavinas/efeitos da radiação , Flavoproteínas/efeitos da radiação , Expressão Gênica , Humanos , Organismos Geneticamente Modificados , Oxirredução , Células Fotorreceptoras de Invertebrados/efeitos da radiação , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Spodoptera , Raios Ultravioleta
16.
Mol Plant ; 1(1): 68-74, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20031915

RESUMO

Arabidopsis cryptochromes cry1 and cry2 are blue-light signalling molecules with significant structural similarity to photolyases--a class of blue-light-sensing DNA repair enzymes. Like photolyases, purified plant cryptochromes have been shown to bind both flavin and pterin chromophores. The flavin functions as a light sensor and undergoes reduction in response to blue light that initiates the signalling cascade. However, the role of the pterin in plant cryptochromes has until now been unknown. Here, we show that the action spectrum for light-dependent degradation of cry2 has a significant peak of activity at 380 nm, consistent with absorption by a pterin cofactor. We further show that cry1 protein expressed in living insect cells responds with greater sensitivity to 380 nm light than to 450 nm, consistent with a light-harvesting antenna pigment that transfers excitation energy to the oxidized flavin of cry1. The pterin biosynthesis inhibitor DHAP selectively reduces cryptochrome responsivity at 380 nm but not 450 nm blue light in these cell cultures, indicating that the antenna pigment is a folate cofactor similar to that of photolyases.


Assuntos
Arabidopsis/fisiologia , Criptocromos/fisiologia , Ácido Fólico/fisiologia , Luz , Arabidopsis/efeitos dos fármacos , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/fisiologia , Proteínas de Arabidopsis/efeitos da radiação , Criptocromos/química , Criptocromos/efeitos dos fármacos , Criptocromos/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Flavinas/fisiologia , Flavinas/efeitos da radiação , Fluorescência , Gliceraldeído 3-Fosfato/análogos & derivados , Gliceraldeído 3-Fosfato/farmacologia , Compostos Organofosforados/farmacologia , Plântula/fisiologia , Plântula/efeitos da radiação , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos da radiação , Espectrofotometria , Espectrofotometria Ultravioleta
17.
J Biol Chem ; 282(13): 9383-91, 2007 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-17237227

RESUMO

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.


Assuntos
Proteínas de Arabidopsis/metabolismo , Flavinas/metabolismo , Flavoproteínas/metabolismo , Células Fotorreceptoras/metabolismo , Raios Ultravioleta , Arabidopsis/química , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/efeitos da radiação , Relógios Biológicos/fisiologia , Relógios Biológicos/efeitos da radiação , Criptocromos , Flavinas/química , Flavinas/efeitos da radiação , Flavoproteínas/química , Flavoproteínas/efeitos da radiação , Oxirredução/efeitos da radiação , Células Fotorreceptoras/química , Células Fotorreceptoras/efeitos da radiação
18.
Plant Physiol ; 142(3): 931-44, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17028154

RESUMO

The sucrose nonfermenting-1 protein kinase (SNF1)/AMP-activated protein kinase subfamily plays a central role in metabolic responses to nutritional and environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, the beta- and gamma-noncatalytic subunits are implicated in substrate specificity and subcellular localization, respectively, and regulation of the kinase activity. The atypical betagamma-subunit has been previously described in maize (Zea mays), presenting at its N-terminal end a sequence related to the KIS (kinase interacting sequence) domain specific to the beta-subunits (Lumbreras et al., 2001). The existence of two components, SNF1-related protein kinase (SnRK1) complexes containing the betagamma-subunit and one SnRK1 kinase, had been proposed. In this work, we show that, despite its unusual features, the Arabidopsis (Arabidopsis thaliana) homolog AKINbetagamma clearly interacts with AKINbeta-subunits in vitro and in vivo, suggesting its involvement in heterotrimeric complexes located in both cytoplasm and nucleus. Unexpectedly, a transcriptional analysis of AKINbetagamma gene expression highlighted the implication of alternative splicing mechanisms in the regulation of AKINbetagamma expression. A two-hybrid screen performed with AKINbetagamma as bait, together with in planta bimolecular fluorescence complementation experiments, suggests the existence of interactions in the cytosol between AKINbetagamma and two leucine-rich repeats related to pathogen resistance proteins. Interestingly, this interaction occurs through the truncated KIS domain that corresponds exactly to a GBD (glycogen-binding domain) recently described in mammals and yeast. A phylogenetic study suggests that AKINbetagamma-related proteins are restricted to the plant kingdom. Altogether, these data suggest the existence of plant-specific SnRK1 trimeric complexes putatively involved in a plant-specific function such as plant-pathogen interactions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Doenças das Plantas , Processamento Alternativo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Deleção de Genes , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Mutação , Filogenia , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína
19.
Planta ; 224(5): 995-1003, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16703358

RESUMO

Cryptochromes are blue-light receptors controlling multiple aspects of plant growth and development. They are flavoproteins with significant homology to photolyases, but instead of repairing DNA they function by transducing blue light energy into a signal that can be recognized by the cellular signaling machinery. Here we report the effect of cry1 and cry2 blue light receptors on primary root growth in Arabidopsis thaliana seedlings, through analysis of both cryptochrome-mutant and cryptochrome-overexpressing lines. Cry1 mutant seedlings show reduced root elongation in blue light while overexpressing seedlings show significantly increased elongation as compared to wild type controls. By contrast, the cry2 mutation has the opposite effect on root elongation growth as does cry1, demonstrating that cry1 and cry2 act antagonistically in this response pathway. The site of cryptochrome signal perception is within the shoot, and the inhibitor of auxin transport, 1-N-naphthylphthalamic acid, abolishes the differential effect of cryptochromes on root growth, suggesting the blue-light signal is transmitted from the shoot to the root by a mechanism that involves auxin. Primary root elongation in blue light may thereby involve interaction between cryptochrome and auxin signaling pathways.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Flavoproteínas/fisiologia , Luz , Raízes de Plantas/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Arabidopsis , Criptocromos , Técnicas de Cultura , Flavoproteínas/antagonistas & inibidores , Flavoproteínas/genética , Expressão Gênica , Ácidos Indolacéticos/metabolismo , Mutação
20.
J Biol Chem ; 280(20): 19437-40, 2005 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-15774475

RESUMO

Cryptochromes are blue light-activated photoreceptors found in multiple organisms with significant similarity to photolyases, a class of light-dependent DNA repair enzymes. Unlike photolyases, cryptochromes do not repair DNA and instead mediate blue light-dependent developmental, growth, and/or circadian responses by an as yet unknown mechanism of action. It has recently been shown that Arabidopsis cryptochrome-1 retains photolyase-like photoreduction of its flavin cofactor FAD by intraprotein electron transfer from tryptophan and tyrosine residues. Here we demonstrate that substitution of two conserved tryptophans that are constituents of the flavin-reducing electron transfer chain in Escherichia coli photolyase impairs light-induced electron transfer in the Arabidopsis cryptochrome-1 photoreceptor in vitro. Furthermore, we show that these substitutions result in marked reduction of light-activated autophosphorylation of cryptochrome-1 in vitro and of its photoreceptor function in vivo, consistent with biological relevance of the electron transfer reaction. These data support the possibility that light-induced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flavoproteínas/metabolismo , Substituição de Aminoácidos , Antocianinas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Criptocromos , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Transporte de Elétrons , Escherichia coli/enzimologia , Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/química , Flavoproteínas/genética , Luz , Mutagênese Sítio-Dirigida , Fotobiologia , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Transdução de Sinais , Triptofano/química
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