RESUMO
Directional movements impact the ability of plants to respond and adjust their growth accordingly to the prevailing light environment. The plasma-membrane associated protein, ROOT PHOTOTROPISM 2 (RPT2) is a key signalling component involved in chloroplast accumulation movement, leaf positioning, and phototropism, all of which are regulated redundantly by the ultraviolet/blue light-activated AGC kinases phototropin 1 and 2 (phot1 and phot2). We recently demonstrated that members of the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)/RPT2-like (NRL) family in Arabidopsis thaliana, including RPT2, are directly phosphorylated by phot1. However, whether RPT2 is a substrate for phot2, and the biological significance of phot phosphorylation of RPT2 remains to be determined. Here, we show that RPT2 is phosphorylated by both phot1 and phot2 at a conserved serine residue (S591) within the C-terminal region of the protein. Blue light triggered the association of 14-3-3 proteins with RPT2 consistent with S591 acting as a 14-3-3 binding site. Mutation of S591 had no effect on the plasma membrane localization of RPT2 but reduced its functionality for leaf positioning and phototropism. Moreover, our findings indicate that S591 phosphorylation within the C-terminus of RPT2 is required for chloroplast accumulation movement to low level blue light. Taken together, these findings further highlight the importance of the C-terminal region of NRL proteins and how its phosphorylation contributes to phot receptor signalling in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Fototropismo/genética , Fosforilação , Fototropinas/genética , Fototropinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Plantas Geneticamente Modificadas/genética , Luz , Folhas de Planta/metabolismo , Cloroplastos/metabolismo , Fosfoproteínas/metabolismoRESUMO
The ability to enhance photosynthetic capacity remains a recognized bottleneck to improving plant productivity. Phototropin blue light receptors (phot1 and phot2) optimize photosynthetic efficiency in Arabidopsis thaliana by coordinating multiple light-capturing processes. In this study, we explore the potential of using protein engineering to improve photoreceptor performance and thereby plant growth. We demonstrate that targeted mutagenesis can decrease or increase the photocycle lifetime of Arabidopsis phototropins in vitro and show that these variants can be used to reduce or extend the duration of photoreceptor activation in planta Our findings show that slowing the phototropin photocycle enhanced several light-capturing responses, while accelerating it reduced phototropin's sensitivity for chloroplast accumulation movement. Moreover, plants engineered to have a slow-photocycling variant of phot1 or phot2 displayed increased biomass production under low-light conditions as a consequence of their improved sensitivity. Together, these findings demonstrate the feasibility of engineering photoreceptors to manipulate plant growth and offer additional opportunities to enhance photosynthetic competence, particularly under suboptimal light regimes.
Assuntos
Arabidopsis/metabolismo , Biomassa , Fotorreceptores de Plantas/metabolismo , Fototropinas/metabolismo , Engenharia de Proteínas , Cloroplastos/metabolismo , Luz , Mutagênese , Fotorreceptores de Plantas/genética , Fotossíntese , Fototropinas/genéticaRESUMO
Phototropin (phot) receptor kinases play important roles in promoting plant growth by controlling light-capturing processes, such as phototropism. Phototropism is mediated through the action of NON-PHOTOTROPIC HYPOCOTYL3 (NPH3), which is dephosphorylated following phot activation. However, the functional significance of this early signaling event remains unclear. Here, we show that the onset of phototropism in dark-grown (etiolated) seedlings of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) is enhanced by greening (deetiolation). Red and blue light were equally effective in promoting phototropism in Arabidopsis, consistent with our observations that deetiolation by phytochrome or cryptochrome was sufficient to enhance phototropism. Increased responsiveness did not result from an enhanced sensitivity to the phytohormone auxin, nor does it involve the phot-interacting protein, ROOT PHOTOTROPISM2. Instead, deetiolated seedlings showed attenuated levels of NPH3 dephosphorylation and diminished relocalization of NPH3 from the plasma membrane during phototropism. Likewise, etiolated seedlings that lack the PHYTOCHROME-INTERACTING FACTORS (PIFs) PIF1, PIF3, PIF4, and PIF5 displayed reduced NPH3 dephosphorylation and enhanced phototropism, consistent with their constitutive photomorphogenic phenotype in darkness. Phototropic enhancement could also be achieved in etiolated seedlings by lowering the light intensity to diminish NPH3 dephosphorylation. Thus, phototropism is enhanced following deetiolation through the modulation of a phosphorylation rheostat, which in turn sustains the activity of NPH3. We propose that this dynamic mode of regulation enables young seedlings to maximize their establishment under changing light conditions, depending on their photoautotrophic capacity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Estiolamento/fisiologia , Fototropismo/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Criptocromos/metabolismo , Estiolamento/efeitos dos fármacos , Estiolamento/efeitos da radiação , Proteínas de Fluorescência Verde/metabolismo , Hipocótilo/efeitos dos fármacos , Hipocótilo/fisiologia , Hipocótilo/efeitos da radiação , Ácidos Indolacéticos/farmacologia , Luz , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Fototropismo/efeitos dos fármacos , Fototropismo/efeitos da radiação , Fitocromo/metabolismo , Agregados Proteicos , Plântula/efeitos dos fármacos , Plântula/fisiologia , Plântula/efeitos da radiaçãoRESUMO
Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dicroísmo Circular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática/efeitos da radiação , Estabilidade Enzimática/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Luz , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação/efeitos da radiação , Processos Fotoquímicos , Fototropismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/efeitos da radiação , Mutação Puntual , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteínas Serina-Treonina Quinases , Estabilidade Proteica/efeitos da radiação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
How plants perceive and respond to temperature remains an important question in the plant sciences. Temperature perception and signal transduction may occur through temperature-sensitive intramolecular folding of primary mRNA transcripts. Recent studies suggested a role for retention of the first intron in the 5'UTR of the clock component LATE ELONGATED HYPOCOTYL (LHY) in response to changes in temperature. Here, we identified a set of haplotypes in the LHY 5'UTR, examined their global spatial distribution, and obtained evidence that haplotype can affect temperature-dependent splicing of LHY transcripts. Correlations of haplotype spatial distributions with global bioclimatic variables and altitude point to associations with annual mean temperature and temperature fluctuation. Relatively rare relict type accessions correlate with lower mean temperature and greater temperature fluctuation and the spatial distribution of other haplotypes may be informative of evolutionary processes driving colonization of ecosystems. We propose that haplotypes may possess distinct 5'UTR pre-mRNA folding thermodynamics and/or specific biological stabilities based around the binding of trans-acting RNA splicing factors, a consequence of which is scalable splicing sensitivity of a central clock component that is likely tuned to specific temperature environments.
Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Regiões 5' não Traduzidas/genética , Proteínas de Arabidopsis/fisiologia , Clima , Proteínas de Ligação a DNA/fisiologia , Demografia , Ensaio de Desvio de Mobilidade Eletroforética , Haplótipos , Polimorfismo de Nucleotídeo Único/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise Espacial , Temperatura , Fatores de Transcrição/fisiologiaRESUMO
Phototropin (phot1) is a blue light-activated plasma membrane-associated kinase that acts as the principal photoreceptor for shoot phototropism in Arabidopsis in conjunction with the signalling component Non-Phototropic Hypocotyl 3 (NPH3). PHOT1 is uniformly expressed throughout the Arabidopsis hypocotyl, yet decapitation experiments have localized the site of light perception to the upper hypocotyl. This prompted us to investigate in more detail the functional role of the hypocotyl apex, and the regions surrounding it, in establishing phototropism. We used a non-invasive approach where PHOT1-GFP (P1-GFP) expression was targeted to the hypocotyl apex of the phot-deficient mutant using the promoters of CUP-SHAPED COTYLEDON 3 (CUC3) and AINTEGUMENTA (ANT). Expression of CUC3::P1-GFP was clearly visible at the hypocotyl apex, with weaker expression in the cotyledons, whereas ANT::P1-GFP was specifically targeted to the developing leaves. Both lines showed impaired curvature to 0.005 µmol m-2 sec-1 unilateral blue light, indicating that regions below the apical meristem are necessary for phototropism. Curvature was however apparent at higher fluence rates. Moreover, CUC3::P1-GFP partially or fully complemented petiole positioning, leaf flattening and chloroplast accumulation, but not stomatal opening. Yet, tissue analysis of NPH3 de-phosphorylation showed that CUC3::P1-GFP and ANT::P1-GFP mis-express very low levels of phot1 that likely account for this responsiveness. Our spatial targeting approach therefore excludes the hypocotyl apex as the site for light perception for phototropism and shows that phot1-mediated NPH3 de-phosphorylation is tissue autonomous and occurs more prominently in the basal hypocotyl.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hipocótilo/metabolismo , Fosfoproteínas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Hipocótilo/genética , Fosfoproteínas/genética , Fosforilação/genética , Fosforilação/fisiologia , Fototropismo/genética , Fototropismo/fisiologia , Proteínas Serina-Treonina Quinases , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Phototropins (phots) regulate a range of adaptive processes in plants that serve to optimize photosynthetic efficiency and promote growth. Light sensing by Arabidopsis thaliana phots is predominantly mediated by the Light, Oxygen and Voltage sensing 2 (LOV2) flavin-binding motif located within the N-terminus of the photoreceptor. Here we characterize the photochemical and biochemical properties of phot from the marine picoalga Ostreococcus tauri phototropin (Otphot) and examine its ability to replace phot-mediated function in Arabidopsis. Photochemical properties of Otphot rely on both LOV1 and LOV2. Yet, biochemical analysis indicates that light-dependent receptor autophosphorylation is primarily dependent on LOV2. As found for Arabidopsis phots, Otphot associates with the plasma membrane and partially internalizes, albeit to a limited extent, in response to blue-light irradiation. Otphot is able to elicit a number of phot-regulated processes in Arabidopsis, including petiole positioning, leaf expansion, stomatal opening and chloroplast accumulation movement. However, Otphot is unable to restore phototropism and chloroplast avoidance movement. Consistent with its lack of phototropic function in Arabidopsis, Otphot does not associate with or trigger dephosphorylation of the phototropic signalling component Non-Phototropic Hypocotyl 3 (NPH3). Taken together, these findings indicate that the mechanism of action of plant and evolutionarily distant algal phots is less well conserved than previously thought.
Assuntos
Clorófitas/genética , Fototropinas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Luz , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Fototropinas/genética , Fototropismo , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , Proteínas Serina-Treonina QuinasesRESUMO
Circadian clocks allow the temporal compartmentalization of biological processes. In Arabidopsis, circadian rhythms display organ specificity but the underlying molecular causes have not been identified. We investigated the mechanisms responsible for the similarities and differences between the clocks of mature shoots and roots in constant conditions and in light : dark cycles. We developed an imaging system to monitor clock gene expression in shoots and light- or dark-grown roots, modified a recent mathematical model of the Arabidopsis clock and used this to simulate our new data. We showed that the shoot and root circadian clocks have different rhythmic properties (period and amplitude) and respond differently to light quality. The root clock was entrained by direct exposure to low-intensity light, even in antiphase to the illumination of shoots. Differences between the clocks were more pronounced in conditions where light was present than in constant darkness, and persisted in the presence of sucrose. We simulated the data successfully by modifying those parameters of a clock model that are related to light inputs. We conclude that differences and similarities between the shoot and root clocks can largely be explained by organ-specific light inputs. This provides mechanistic insight into the developing field of organ-specific clocks.
Assuntos
Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Relógios Circadianos/efeitos da radiação , Ritmo Circadiano/efeitos da radiação , Luz , Raízes de Plantas/fisiologia , Brotos de Planta/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Escuridão , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Modelos Biológicos , Especificidade de Órgãos/genética , Especificidade de Órgãos/efeitos da radiação , Fotoperíodo , Raízes de Plantas/genética , Raízes de Plantas/efeitos da radiação , Brotos de Planta/genética , Brotos de Planta/efeitos da radiaçãoRESUMO
Plants depend on the surrounding light environment to direct their growth. Blue light (300-500 nm) in particular acts to promote a wide variety of photomorphogenic responses including seedling establishment, phototropism and circadian clock regulation. Several different classes of flavin-based photoreceptors have been identified that mediate the effects of blue light in the dicotyledonous genetic model Arabidopsis thaliana. These include the cryptochromes, the phototropins and members of the Zeitlupe family. In this review, we discuss recent advances, which contribute to our understanding of how these photosensory systems are activated by blue light and how they initiate signaling to regulate diverse aspects of plant development.
Assuntos
Flavoproteínas/metabolismo , Fotorreceptores de Plantas/metabolismo , Criptocromos/metabolismo , Modelos Biológicos , Fototropinas/metabolismo , Transdução de SinaisAssuntos
Proteínas de Arabidopsis/metabolismo , Luz , Fosfoproteínas/metabolismo , Transdução de Sinais/efeitos da radiação , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Fosfoproteínas/genética , Fototropismo/genética , Filogenia , Fitocromo/genética , Fitocromo/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/genéticaRESUMO
It is well accepted that lateral redistribution of the phytohormone auxin underlies the bending of plant organs towards light. In monocots, photoreception occurs at the shoot tip above the region of differential growth. Despite more than a century of research, it is still unresolved how light regulates auxin distribution and where this occurs in dicots. Here, we establish a system in Arabidopsis thaliana to study hypocotyl phototropism in the absence of developmental events associated with seedling photomorphogenesis. We show that auxin redistribution to the epidermal sites of action occurs at and above the hypocotyl apex, not at the elongation zone. Within this region, we identify the auxin efflux transporter ATP-BINDING CASSETTE B19 (ABCB19) as a substrate target for the photoreceptor kinase PHOTOTROPIN 1 (phot1). Heterologous expression and physiological analyses indicate that phosphorylation of ABCB19 by phot1 inhibits its efflux activity, thereby increasing auxin levels in and above the hypocotyl apex to halt vertical growth and prime lateral fluxes that are subsequently channeled to the elongation zone by PIN-FORMED 3 (PIN3). Together, these results provide new insights into the roles of ABCB19 and PIN3 in establishing phototropic curvatures and demonstrate that the proximity of light perception and differential phototropic growth is conserved in angiosperms.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Fosfoproteínas/metabolismo , Fototropismo , Brotos de Planta/metabolismo , Aclimatação , Arabidopsis/crescimento & desenvolvimento , Transporte Biológico , Escuridão , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Hipocótilo/metabolismo , Mutação/genética , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Imbibed Arabidopsis (Arabidopsis thaliana) seeds are encapsulated by mucilage that is formed of hydrated polysaccharides released from seed coat epidermal cells. The mucilage is structured with water-soluble and adherent layers, with cellulose present uniquely in an inner domain of the latter. Using a reverse-genetic approach to identify the cellulose synthases (CESAs) that produce mucilage cellulose, cesa5 mutants were shown to be required for the correct formation of these layers. Expression of CESA5 in the seed coat was specific to epidermal cells and coincided with the accumulation of mucilage polysaccharides in their apoplast. Analysis of sugar composition showed that although total sugar composition or amounts were unchanged, their partition between layers was different in the mutant, with redistribution from adherent to water-soluble mucilage. The macromolecular characteristics of the water-soluble mucilage were also modified. In accordance with a role for CESA5 in mucilage cellulose synthesis, crystalline cellulose contents were reduced in mutant seeds and birefringent microfibrils were absent from adherent mucilage. Although the mucilage-modified5 mutant showed similar defects to cesa5 in the distribution of sugar components between water-soluble and adherent mucilage, labeling of residual adherent mucilage indicated that cesa5 contained less cellulose and less pectin methyl esterification. Together, the results demonstrate that CESA5 plays a major and essential role in cellulose production in seed mucilage, which is critical for the establishment of mucilage structured in layers and domains.
Assuntos
Adesivos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Celulose/biossíntese , Glucosiltransferases/metabolismo , Sementes/enzimologia , Adesividade , Alelos , Arabidopsis/citologia , Arabidopsis/ultraestrutura , Metabolismo dos Carboidratos , Diferenciação Celular , Cristalização , Substâncias Macromoleculares/metabolismo , Monossacarídeos/metabolismo , Mutação/genética , Fenótipo , Epiderme Vegetal/citologia , Epiderme Vegetal/enzimologia , Epiderme Vegetal/ultraestrutura , Sementes/citologia , Sementes/ultraestrutura , Solubilidade , Coloração e Rotulagem , ÁguaRESUMO
Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Endocitose/efeitos da radiação , Luz , Fototropinas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Cromatografia Líquida , Clatrina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endocitose/fisiologia , Imunoprecipitação , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese , Fosforilação/efeitos da radiação , Fototropinas/química , Fototropinas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/efeitos da radiação , Ligação Proteica/efeitos da radiação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em TandemRESUMO
Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hipocótilo/metabolismo , Proteínas 14-3-3/genética , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência Consenso , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Hipocótilo/genética , Luz , Fosforilação , Fototropismo/efeitos da radiação , Ligação Proteica/efeitos da radiação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Endoribonuclease E (RNase E) is a regulator of global gene expression in Escherichia coli and is the best studied member of the RNase E/G ribonuclease family. Homologues are present in other bacteria but the roles of plant RNase E/G-like proteins are not known. Arabidopsis thaliana contains a single nuclear gene (At2g04270) encoding a product with the conserved catalytic domain of RNase E/G-like proteins. At2g04270 and the adjacent At2g04280 gene form converging transcription units with a approximately 40 base overlap at their 3' ends. Several translation products were predicted from the analyses of At2g04270 cDNAs. An antibody raised against a recombinant A. thaliana RNase E/G-like protein recognized a 125 kDa protein band in purified chloroplast preparations fractionated by SDS-PAGE. The 125 kDa RNase E/G-like protein was detected in cotyledons, rosette and cauline leaves. T-DNA insertions in exon 6 or intron 11 of At2g04270 result in loss of the 125 kDa band or truncation to a 110 kDa band. Loss of At2g04270 function resulted in the arrest of chloroplast development, loss of autotrophic growth, and reduced plastid ribosomal, psbA and rbcL RNA levels. Homozygous mutant plants were pale-green, contained smaller plastids with fewer thylakoids and shorter granal stacks than wild-type chloroplasts, and required sucrose at all growth stages following germination right up to flowering and setting seeds. Recombinant A. thaliana RNase E/G-like proteins rescued an E. coli RNase E mutant and cleaved an rbcL RNA substrate. Expression of At2g04270 was highly correlated with genes encoding plastid polyribonucleotide phosphorylase, S1 RNA-binding, and CRS1/YhbY domain proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Cloroplastos/enzimologia , Processos Fototróficos , Plastídeos/enzimologia , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/química , Cloroplastos/genética , Cloroplastos/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Peso Molecular , Plastídeos/química , Plastídeos/genética , Plastídeos/ultraestrutura , Estrutura Terciária de Proteína , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Hypocotyl phototropism of etiolated Arabidopsis seedlings is primarily mediated by the blue-light receptor kinase phototropin 1 (phot1). Phot1-mediated curvature to continuous unilateral blue light irradiation (0.5 µmol m(-2) s(-1)) is enhanced by overhead pre-treatment with red light (20 µmol m(-2) s(-1) for 15 min) through the action of phytochrome (phyA). Here, we show that pre-treatment with blue light is equally as effective in eliciting phototropic enhancement and is dependent on phyA. Although blue light pre-treatment was sufficient to activate early phot1 signaling events, phot1 autophosphorylation in vivo was not found to be saturated, as assessed by subsequently measuring phot1 kinase activity in vitro. However, enhancement effects by red and blue light pre-treatment were not observed at higher intensities of phototropic stimulation (10 µmol m(-2) s(-1)). Phototropic enhancement by red and blue light pre-treatments to 0.5 µmol m(-2) s(-1) unilateral blue light irradiation was also lacking in transgenic Arabidopsis where PHOT1 expression was restricted to the epidermis. Together, these findings indicate that phyA-mediated effects on phot1 signaling are restricted to low intensities of phototropic stimulation and originate from tissues other than the epidermis.
RESUMO
Phototropin receptor kinases play an important role in optimising plant growth in response to blue light. Much is known regarding their photochemical reactivity, yet little progress has been made to identify downstream signalling components. Here, we isolated several interacting proteins for Arabidopsis phototropin 1 (phot1) by yeast two-hybrid screening. These include members of the NPH3/RPT2 (NRL) protein family, proteins associated with vesicle trafficking, and the 14-3-3 lambda (lambda) isoform from Arabidopsis. 14-3-3lambda and phot1 were found to colocalise and interact in vivo. Moreover, 14-3-3 binding to phot1 was limited to non-epsilon 14-3-3 isoforms and was dependent on key sites of receptor autophosphorylation. No 14-3-3 binding was detected for Arabidopsis phot2, suggesting that 14-3-3 proteins are specific to phot1 signalling.
Assuntos
Proteínas 14-3-3/química , Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Fosfoproteínas/metabolismo , Proteínas 14-3-3/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Fosfoproteínas/química , Fosforilação , Filogenia , Proteínas Serina-Treonina Quinases , Alinhamento de Sequência , Transdução de Sinais , Especificidade por Substrato , Técnicas do Sistema de Duplo-HíbridoRESUMO
Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser(58), Ser(85), Ser(350), and Ser(410)), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.