RESUMO
Global proliferative arrest (GPA) is a phenomenon in monocarpic plants in which the activity of all aboveground meristems generally ceases in a nearly coordinated manner after the formation of a certain number of fruits. Despite the fact that GPA is a biologically and agriculturally important event, the underlying molecular mechanisms are not well understood. In this study, we attempted to elucidate the molecular mechanism of GPA regulation by identifying the gene responsible for the Arabidopsis mutant fireworks (fiw), causing an early GPA phenotype. Map-based cloning revealed that the fiw gene encodes CYSTEIN-RICH RECEPTOR-LIKE KINASE 14 (CRK14). Genetic analysis suggested that fiw is a missense, gain-of-function allele of CRK14. Since overexpression of the extracellular domain of CRK14 resulted in delayed GPA in the wild-type background, we concluded that CRK14 is involved in GPA regulation. Analysis of double mutants revealed that fiw acts downstream of or independently of the FRUITFULL-APETALA2 (AP2)/AP2-like pathway, which was previously reported as an age-dependent default pathway in GPA regulation. In addition, fiw is epistatic to clv with respect to GPA control. Furthermore, we found a negative effect on WUSCHEL expression in the fiw mutants. These results thus suggest the existence of a novel CRK14-dependent signaling pathway involved in GPA regulation.
RESUMO
Phytochromes play a central role in mediating adaptive responses to light and temperature throughout plant life cycles. Despite evidence for adaptive importance of natural variation in phytochromes, little information is known about molecular mechanisms that modulate physiological responses of phytochromes in nature. We show evolutionary divergence in physiological responses relevant to thermal stability of a physiologically active form of phytochrome (Pfr) between two sister species of Brassicaceae growing at different latitudes. The higher latitude species (Cardamine bellidifolia; Cb) responded more strongly to light-limited conditions compared with its lower latitude sister (C. nipponica; Cn). Moreover, CbPHYB conferred stronger responses to both light-limited and warm conditions in the phyB-deficient mutant of Arabidopsis thaliana than CnPHYB: that is Pfr CbphyB was more stable in nuclei than CnphyB. Our findings suggest that fine tuning Pfr stability is a fundamental mechanism for plants to optimise phytochrome-related traits in their evolution and adapt to spatially varying environments, and open a new avenue to understand molecular mechanisms that fine tune phytochrome responses in nature.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/genética , Luz , Fitocromo B/genéticaRESUMO
Hydathodes are typically found at leaf teeth in vascular plants and are involved in water release to the outside. Although morphological and physiological analysis of hydathodes has been performed in various plants, little is known about the genes involved in hydathode function. In this study, we performed fluorescent protein-based imaging and tissue-specific RNA-seq analysis in Arabidopsis hydathodes. We used the enhancer trap line E325, which has been reported to express green fluorescent protein (GFP) at its hydathodes. We found that E325-GFP was expressed in small cells found inside the hydathodes (named E cells) that were distributed between the water pores and xylem ends. No fluorescence of the phloem markers pSUC2:GFP and pSEOR1:SEOR1-YFP was observed in the hydathodes. These observations indicate that Arabidopsis hydathodes are composed of three major components: water pores, xylem ends, and E cells. In addition, we performed transcriptome analysis of the hydathode using the E325-GFP line. Microsamples were collected from GFP-positive or -negative regions of E325 leaf margins with a needle-based device (~130 µm in diameter). RNA-seq was performed with each single microsample using a high-throughput library preparation method called Lasy-Seq. We identified 72 differentially expressed genes. Among them, 68 genes showed significantly higher and four genes showed significantly lower expression in the hydathode. Our results provide new insights into the molecular basis for hydathode physiology and development.
Assuntos
Arabidopsis/fisiologia , Folhas de Planta/fisiologia , Água/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis , RNA-Seq , Xilema/fisiologiaRESUMO
The biogenesis of the photosynthetic apparatus in developing seedlings requires the assembly of proteins encoded on both nuclear and chloroplast genomes. To coordinate this process there needs to be communication between these organelles, but the retrograde signals by which the chloroplast communicates with the nucleus at this time are still essentially unknown. The Arabidopsis thaliana genomes uncoupled (gun) mutants, that show elevated nuclear gene expression after chloroplast damage, have formed the basis of our understanding of retrograde signaling. Of the 6 reported gun mutations, 5 are in tetrapyrrole biosynthesis proteins and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes. However, the molecular consequences of the strongest of the gun mutants, gun1, are poorly understood, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other porphyrins, reduces flux through the tetrapyrrole biosynthesis pathway to limit heme and protochlorophyllide synthesis, and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism, supporting a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fotossíntese/fisiologia , Tetrapirróis/biossíntese , Proteínas de Arabidopsis/genética , Vias Biossintéticas/genética , Vias Biossintéticas/fisiologia , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Proteínas de Ligação a DNA/genética , Ferroquelatase , Regulação da Expressão Gênica de Plantas , Heme/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Mutação , Transdução de Sinais/fisiologiaAssuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Plastídeos/metabolismo , Fatores de Transcrição/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Lincomicina , Biogênese de Organelas , Piridazinas , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Plants sense and respond to light via multiple photoreceptors including phytochrome. The decreased ratio of red to far-red light that occurs under a canopy triggers shade-avoidance responses, which allow plants to compete with neighboring plants. The leaf acts as a photoperceptive organ in this response. In this study, we investigated how the shade stimulus is spatially processed within the cotyledon. We performed transcriptome analysis on microtissue samples collected from vascular and nonvascular regions of Arabidopsis (Arabidopsis thaliana) cotyledons. In addition, we mechanically isolated and analyzed the vascular tissue. More genes were up-regulated by the shade stimulus in vascular tissues than in mesophyll and epidermal tissues. The genes up-regulated in the vasculature were functionally divergent and included many auxin-responsive genes, suggesting that various physiological/developmental processes might be controlled by shade stimulus in the vasculature. We then investigated the spatial regulation of these genes in the vascular tissues. A small vascular region within a cotyledon was irradiated with far-red light, and the response was compared with that when the whole seedling was irradiated with far-red light. Most of the auxin-responsive genes were not fully induced by the local irradiation, suggesting that perception of the shade stimulus requires that a wider area be exposed to far-red light or that a certain position in the mesophyll and epidermis of the cotyledon be irradiated. This result was consistent with a previous report that auxin synthesis genes are up-regulated in the periphery of the cotyledon. Hence, auxin acts as an important intraorgan signaling factor that controls the vascular shade response within the cotyledon.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Cotilédone/genética , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Luz , Células do Mesofilo/fisiologia , Oxigenases/genética , Epiderme Vegetal/fisiologia , Plantas Geneticamente ModificadasAssuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Transdução de Sinais , Tetrapirróis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Cloroplastos/genética , Cloroplastos/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , Fenótipo , Plântula/genética , Plântula/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Expression of Photosynthesis-Associated Nuclear Genes (PhANGs) is controlled by environmental stimuli and plastid-derived signals ("plastid signals") transmitting the developmental and functional status of plastids to the nucleus. Arabidopsis genomes uncoupled (gun) mutants exhibit defects in plastid signaling, leading to ectopic expression of PhANGs in the absence of chloroplast development. GUN5 encodes the plastid-localized Mg-chelatase enzyme subunit (CHLH), and recent studies suggest that CHLH is a multifunctional protein involved in tetrapyrrole biosynthesis, plastid signaling and ABA responses in guard cells. To understand the basis of CHLH multifunctionality, we investigated 15 gun5 missense mutant alleles and transgenic lines expressing a series of truncated CHLH proteins in a severe gun5 allele (cch) background (tCHLHs, 10 different versions). Here, we show that Mg-chelatase function and plastid signaling are generally correlated; in contrast, based on the analysis of the gun5 missense mutant alleles, ABA-regulated stomatal control is distinct from these two other functions. We found that none of the tCHLHs could restore plastid-signaling or Mg-chelatase functions. Additionally, we found that both the C-terminal half and N-terminal half of CHLH function in ABA-induced stomatal movement.
RESUMO
Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1) and null (fc1-2) mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1) and null (fc2-2) mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.
RESUMO
Gene expression analysis is a key technology that is used to understand living systems. Multicellular organisms, including plants, are composed of various tissues and cell types, each of which exhibits a unique gene expression pattern. However, because of their rigid cell walls, plant cells are difficult to isolate from the whole plant. Although laser dissection has been used to circumvent this problem, the plant sample needs to be fixed beforehand, which presents several problems. In the present study, we developed an alternative method to conduct highly reliable gene expression profiling. First, we assembled a dissection apparatus that used a narrow, sharpened needle to dissect out a microsample of fresh plant tissue (0.1-0.2 mm on each side) automatically from a target site within a short time frame. Then, we optimized a protocol to synthesize a high-quality cDNA library on magnetic beads using a single microsample. The cDNA library was amplified and subjected to high-throughput sequencing. In this way, a stable and reliable system was developed to conduct gene expression profiling in small regions of a plant. The system was used to analyze the gene expression patterns at successive 50 µm intervals in the shoot apex of a 4-day-old Arabidopsis seedling. Clustering analysis of the data demonstrated that two small, adjacent domains, the shoot apical meristem and the leaf primordia, were clearly distinguishable. This system should be broadly applicable in the investigation of the spatial organization of gene expression in various contexts.
Assuntos
Arabidopsis/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Microdissecção/métodos , Proteínas de Arabidopsis/genética , Análise por Conglomerados , Cotilédone/genética , Perfilação da Expressão Gênica/instrumentação , Hipocótilo/genética , Meristema/genética , Microdissecção/instrumentação , Agulhas , Epiderme Vegetal/genética , Folhas de Planta/genética , Brotos de Planta/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The shade avoidance response, which allows plants to escape from nearby competitors, is triggered by a reduction in the PFR form of phytochrome in response to shade. Classic physiological experiments have demonstrated that the shade signal perceived by the leaves is transmitted to the other parts of the plant. Recently, a simple method was developed to analyze the transcriptome in a single microgram tissue sample. In the present study, we adopted this method to conduct organ-specific transcriptomic analysis of the shade avoidance response in Arabidopsis seedlings. The shoot apical samples, which contained the meristem, basal parts of leaf primordia and short fragments of vasculature, were collected from the topmost part of the hypocotyl and subjected to RNA sequencing analysis. Unexpectedly, many more genes were up-regulated in the shoot apical region than in the cotyledons. Spotlight irradiation demonstrated that the apex-responsive genes were mainly controlled by phytochrome in the cotyledons. In accordance with the involvement of many auxin-responsive genes in this category, auxin biosynthesis was genetically shown to be essential for this response. In contrast, organ-autonomous regulation was more important for the genes that were up-regulated preferentially either in the cotyledons or in both the cotyledons and the apical region. Their responses to shade depended variously on auxin and PIFs (phytochrome-interacting factors), indicating the mechanistic diversity of the organ-autonomous response. Finally, we examined the expression of the auxin synthesis genes, the YUC genes, and found that three YUC genes, which were differently spatially regulated, co-ordinately elevated the auxin level within the shoot apical region.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Plântula/genética , Transcriptoma/efeitos da radiação , Proteínas de Arabidopsis/genética , Cotilédone/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Hipocótilo/genética , Ácidos Indolacéticos/farmacologia , Meristema/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reguladores de Crescimento de Plantas/farmacologia , Brotos de Planta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Transcriptoma/efeitos dos fármacosRESUMO
To elucidate the molecular mechanisms of stomatal opening and closure, we performed a genetic screen using infrared thermography to isolate stomatal aperture mutants. We identified a mutant designated low temperature with open-stomata 1 (lost1), which exhibited reduced leaf temperature, wider stomatal aperture, and a pale green phenotype. Map-based analysis of the LOST1 locus revealed that the lost1 mutant resulted from a missense mutation in the Mg-chelatase I subunit 1 (CHLI1) gene, which encodes a subunit of the Mg-chelatase complex involved in chlorophyll synthesis. Transformation of the wild-type CHLI1 gene into lost1 complemented all lost1 phenotypes. Stomata in lost1 exhibited a partial ABA-insensitive phenotype similar to that of rtl1, a Mg-chelatase H subunit missense mutant. The Mg-protoporphyrin IX methyltransferase (CHLM) gene encodes a subsequent enzyme in the chlorophyll synthesis pathway. We examined stomatal movement in a CHLM knockdown mutant, chlm, and found that it also exhibited an ABA-insensitive phenotype. However, lost1 and chlm seedlings all showed normal expression of ABA-induced genes, such as RAB18 and RD29B, in response to ABA. These results suggest that the chlorophyll synthesis enzymes, Mg-chelatase complex and CHLM, specifically affect ABA signaling in the control of stomatal aperture and have no effect on ABA-induced gene expression.
Assuntos
Ácido Abscísico/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Metiltransferases/metabolismo , Estômatos de Plantas/enzimologia , Adenosina Trifosfatases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clorofila/biossíntese , Metiltransferases/genética , Mutação , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , TermografiaRESUMO
The Arabidopsis thaliana L. SOUL/haem-binding proteins, AtHBPs belong to a family of five members. The Arabidopsis cytosolic AtHBP1 (At1g17100) and AtHBP2 (At2g37970) have been shown to bind porphyrins and metalloporphyrins including haem. In contrast to the cytosolic localization of these haem-binding proteins, AtHBP5 (At5g20140) encodes a protein with an N-terminal transit peptide that probably directs targeting to the chloroplast. In this report, it is shown that AtHBP5 binds haem and interacts with the haem oxygenase, HY1, in both yeast two-hybrid and BiFC assays. The expression of HY1 is repressed in the athbp5 T-DNA knockdown mutant and the accumulation of H(2)O(2) is observed in athbp5 seedlings that are treated with methyl jasmonate (MeJA), a ROS-producing stress hormone. In contrast, AtHBP5 over-expressing plants show a decreased accumulation of H(2)O(2) after MeJA treatment compared with the controls. It is proposed that the interaction between the HY1 and AtHBP5 proteins participate in an antioxidant pathway that might be mediated by reaction products of haem catabolism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Heme Oxigenase-1/metabolismo , Hemeproteínas/metabolismo , Estresse Oxidativo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Heme/metabolismo , Heme Oxigenase-1/genética , Proteínas Ligantes de Grupo Heme , Hemeproteínas/genética , Ligação ProteicaRESUMO
Phytochrome is a red (R)/far-red (FR) light-sensing photoreceptor that regulates various aspects of plant development. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates atypical phytochrome responses, such as the FR high irradiance response (FR-HIR), which is elicited under prolonged FR. A proteasome-based degradation pathway rapidly eliminates active Pfr (the FR-absorbing form of phyA) under R. To elucidate the structural basis for the phyA-specific properties, we systematically constructed 16 chimeric phytochromes in which each of four parts of the phytochrome molecule, namely, the N-terminal extension plus the Per/Arnt/Sim domain (N-PAS), the cGMP phosphodiesterase/adenyl cyclase/FhlA domain (GAF), the phytochrome domain (PHY), and the entire C-terminal half, was occupied by either the phyA or phytochrome B sequence. These phytochromes were expressed in transgenic Arabidopsis thaliana to examine their physiological activities. Consequently, the phyA N-PAS sequence was shown to be necessary and sufficient to promote nuclear accumulation under FR, whereas the phyA sequence in PHY was additionally required to exhibit FR-HIR. Furthermore, the phyA sequence in PHY alone substantially increased the light sensitivity to R. In addition, the GAF phyA sequence was important for rapid Pfr degradation. In summary, distinct structural modules, each of which confers different properties to phyA, are assembled on the phyA molecule.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Luz , Fitocromo A/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Escuridão , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/fisiologia , Hipocótilo/efeitos da radiação , Mutação , Fitocromo A/genética , Fitocromo B/genética , Fitocromo B/metabolismo , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Plântula/efeitos da radiaçãoRESUMO
Heme functions not only as a prosthetic group of hemoproteins but also as a regulatory molecule, suggesting the presence of 'free' heme. Classically, total non-covalently bound heme is extracted from plant samples with acidic acetone after removal of pigments with basic and neutral acetone. Earlier work proposed that free heme can be selectively extracted into basic acetone. Using authentic hemoproteins, we confirmed that acidic acetone can quantitatively extract heme, while no heme was extracted into neutral acetone. Meanwhile, a certain amount of heme was extracted into basic acetone from hemoglobin and myoglobin. Moreover, basic acetone extracted loosely bound heme from bovine serum albumin, implying that the nature of hemoproteins largely influences heme extraction into basic acetone. Using a highly sensitive heme assay, we found that basic and neutral acetone can extract low levels of heme from plant samples. In addition, neutral acetone quantitatively extracted free heme when it was externally added to plant homogenates. Furthermore, the level of neutral acetone-extractable heme remained unchanged by precursor (5-aminolevulinic acid) feeding, while increased by norflurazon treatment which abolishes chloroplast biogenesis. However, changes in these heme levels did not correlate to genomes uncoupled phenotypes, suggesting that the level of unbound free heme would not affect retrograde signaling from plastids to the nucleus. The present data demonstrate that the combination of single-step acetone extraction following a sensitive heme assay is the ideal method for determining total and free heme in plants.
Assuntos
Acetona/química , Fracionamento Químico/métodos , Heme/isolamento & purificação , Células Vegetais/química , Ácidos/química , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacologia , Animais , Arabidopsis/química , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/química , Núcleo Celular , Cloroplastos/química , Cloroplastos/efeitos dos fármacos , Heme/química , Hemeproteínas/análise , Hemeproteínas/química , Hemoglobinas/química , Mioglobina/química , Oxirredução , Células Vegetais/efeitos dos fármacos , Piridazinas/farmacologia , Sensibilidade e Especificidade , Soroalbumina Bovina/química , SolubilidadeRESUMO
Phytochrome regulates various physiological and developmental processes throughout the life cycle of plants. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates the far-red light high irradiance response (FR-HIR), which is elicited by continuous far-red light. In FR-HIR, nuclear accumulation of phyA, which precedes physiological responses, is proposed to be required for the response. In contrast to FR, red light induces rapid degradation of phyA to suppress undesirable long-term photomorphogenic responses of phyA. In the present study, we compared biological activities between phyA derivatives to which either a nuclear localization (NLS) or export (NES) signal sequence was attached. Those derivatives were expressed under the control of the PHYA promoter in the Arabidopsis phyA mutant. Detailed microscopic observation revealed that the phyA-green fluorescent protein (GFP) without a signal sequence is localized exclusively in the cytoplasm in darkness. Rapid nuclear entry was observed after exposure to both red and far-red light. Interestingly, both phyA-GFP-NLS and phyA-GFP-NES were rapidly degraded under continuous red light. Furthermore, a proteasome inhibitor delayed degradation equally under these two conditions. Therefore, similar mechanisms for phyA degradation may exist in the cytoplasm and nucleus. As expected from previous reports, phyA-GFP-NLS, but not phyA-GFP-NES, mediated different aspects of FR-HIR, such as inhibition of hypocotyl elongation and rapid induction of gene expression, confirming that phyA nuclear localization is required for FR-HIR. In addition, a detailed time course analysis of phyA-GFP and phyA-GFP-NLS responses revealed that they were almost indistinguishable, raising the question of the physiological relevance of phyA cytoplasmic retention in darkness.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fitocromo A/metabolismo , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Luz , Microscopia Confocal , Mutação , Sinais de Exportação Nuclear , Sinais de Localização Nuclear , Fitocromo A/genética , Fitocromo A/efeitos da radiação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras GenéticasRESUMO
Tetrapyrroles such as chlorophyll and heme are co-factors for essential proteins involved in a wide variety of crucial cellular functions. Nearly 2% of the proteins encoded by the Arabidopsis thaliana genome are thought to bind tetrapyrroles, demonstrating their central role in plant metabolism. Although the enzymes required for tetrapyrrole biosynthesis are well characterized, there are still major questions about the regulation of the pathway, and the transport of tetrapyrroles within cells. These issues are important, as misregulation of tetrapyrrole metabolism can lead to severe photo-oxidative stress, and because tetrapyrroles have been implicated in signaling pathways coordinating interactions between plant organelles. In this review, we discuss the cell biology of tetrapyrrole metabolism and its implications for tetrapyrroles as signaling molecules.
Assuntos
Plantas/metabolismo , Tetrapirróis/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/metabolismo , Humanos , Plastídeos/metabolismo , Ligação ProteicaRESUMO
The first step of chlorophyll biosynthesis is catalyzed by a Mg-chelatase composed of the subunits CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis that can be attributed to CHLI. Arabidopsis has two CHLI isoforms, CHLI1 and CHLI2, that have similar expression profiles, but it has been suggested that CHLI2 has limited function in the Mg-chelatase complex. Recently, we showed that Arabidopsis CHLI1 is an ATPase and a target of chloroplast thioredoxin. Here, we demonstrate that CHLI2 also has ATPase activity but with a lower Vmax and higher Km ATP than CHLI1. We confirmed the thioredoxin-dependent reduction of a disulfide bond in CHLI2 and thiol-modulation of its ATPase activity. We then examined the physiological contribution of CHLI2 using a chli2 T-DNA knockout line. Although visible phenotype of homozygous chli2 mutants was almost comparable to wild type, the mutant accumulated significantly less chlorophyll. Furthermore, cs/cs; chli2/chli2 double mutants were almost albino. There were three phenotypes among progenies segregated from the cs/cs; CHLI2/chli2 parent: cs-like pale green, yellow, and almost albino were obtained in the approximate ratio of 1:2:0.7. PCR analysis confirmed that the chli2 mutation is semidominant on a homozygous cs background. These results reveal that although CHLI2 plays a limited role in chlorophyll biosynthesis, this subunit certainly contributes to the assembly of the Mg-chelatase complex.
Assuntos
Arabidopsis/enzimologia , Isoenzimas/metabolismo , Liases/metabolismo , Subunidades Proteicas/metabolismo , Arabidopsis/metabolismo , Catálise , Clorofila/biossíntese , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica/genética , Isoenzimas/química , Isoenzimas/genética , Cinética , Liases/química , Liases/genética , Mutação , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The plastid plays a vital role in various cellular activities within plant cells including photosynthesis and other metabolic pathways. It is believed that the functional status of the plastid is somehow monitored by the nucleus to optimize the expression of genes encoding plastid proteins. The currently dominant model for plastid-derived signaling ("plastid signaling") proposes that Mg-protoporphyrin IX (MgProto) is a negative signal that represses the expression of a wide range of nuclear genes encoding plastid-localized proteins when plastid development is inhibited. In this study, we have re-evaluated this hypothesis by quantifying the steady-state levels of MgProto (as well as its neighboring intermediates protoporphyrin IX and Mg-Proto monomethyl ester [MgProtoMe]) in Arabidopsis plants with altered plastid signaling responses as monitored by expression of the Lhcb1, RBCS, HEMA1, BAM3 and CA1 genes. In addition, we have examined the correlation between gene expression and MgProto (MgProtoMe) in a range of mutants and conditions in which the steady-state levels of MgProto (MgProtoMe) have been modified. Overall we found that there was no correlation between the steady-state levels of MgProto (MgProtoMe) and Lhcb1 expression or with any of the other genes tested. Taking these results together, we propose that the current model on plastid signaling must be revised.
Assuntos
Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Plastídeos/metabolismo , Protoporfirinas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Herbicidas/farmacologia , Mutação , Plastídeos/efeitos dos fármacos , Plastídeos/genética , Piridazinas/farmacologia , Transdução de SinaisRESUMO
The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.