RESUMO
The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development.
Assuntos
Antocianinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Petunia/genética , Proteínas de Plantas/metabolismo , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Flores/citologia , Flores/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Homeostase , Concentração de Íons de Hidrogênio , Petunia/citologia , Petunia/fisiologia , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Vacúolos/metabolismoRESUMO
Silene vulgaris is a metallophyte of calamine, cupriferous and serpentine soils all over Europe. Its metallicolous populations are hypertolerant to zinc (Zn), cadmium (Cd), copper (Cu) or nickel (Ni), compared with conspecific nonmetallicolous populations. These hypertolerances are metal-specific, but the underlying mechanisms are poorly understood. We investigated the role of HMA5 copper transporters in Cu-hypertolerance of a S. vulgaris copper mine population. Cu-hypertolerance in Silene is correlated and genetically linked with enhanced expression of two HMA5 paralogs, SvHMA5I and SvHMA5II, each of which increases Cu tolerance when expressed in Arabidopsis thaliana. Most Spermatophytes, except Brassicaceae, possess homologs of SvHMA5I and SvHMA5II, which originate from an ancient duplication predating the appearance of spermatophytes. SvHMA5II and the A. thaliana homolog AtHMA5 localize in the endoplasmic reticulum and upon Cu exposure move to the plasma membrane, from where they are internalized and degraded in the vacuole. This resembles trafficking of mammalian homologs and is apparently an extremely ancient mechanism. SvHMA5I, instead, neofunctionalized and always resides on the tonoplast, likely sequestering Cu in the vacuole. Adaption of Silene to a Cu-polluted soil is at least in part due to upregulation of two distinct HMA5 transporters, which contribute to Cu hypertolerance by distinct mechanisms.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Arabidopsis/genética , Cobre/metabolismo , Cobre/toxicidade , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Silene/metabolismo , Sequência de Aminoácidos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana Transportadoras/química , Filogenia , Proteínas de Plantas/química , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Silene/efeitos dos fármacos , Silene/genética , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Vacúolos/metabolismoRESUMO
Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H(+) P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms. We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events. The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes.
Assuntos
Ácidos/metabolismo , Evolução Molecular , Proteínas de Membrana Transportadoras/metabolismo , Petunia/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Vacúolos/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Cátions , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/química , Rosa/genética , Homologia de Sequência de Aminoácidos , Vacúolos/metabolismo , Vitis/genéticaRESUMO
Anthocyanins are a chemically diverse class of secondary metabolites that color most flowers and fruits. They consist of three aromatic rings that can be substituted with hydroxyl, sugar, acyl, and methyl groups in a variety of patterns depending on the plant species. To understand how such chemical diversity evolved, we isolated and characterized METHYLATION AT THREE2 (MT2) and the two METHYLATION AT FIVE (MF) loci from Petunia spp., which direct anthocyanin methylation in petals. The proteins encoded by MT2 and the duplicated MF1 and MF2 genes and a putative grape (Vitis vinifera) homolog Anthocyanin O-Methyltransferase1 (VvAOMT1) are highly similar to and apparently evolved from caffeoyl-Coenzyme A O-methyltransferases by relatively small alterations in the active site. Transgenic experiments showed that the Petunia spp. and grape enzymes have remarkably different substrate specificities, which explains part of the structural anthocyanin diversity in both species. Most strikingly, VvAOMT1 expression resulted in the accumulation of novel anthocyanins that are normally not found in Petunia spp., revealing how alterations in the last reaction can reshuffle the pathway and affect (normally) preceding decoration steps in an unanticipated way. Our data show how variations in gene expression patterns, loss-of-function mutations, and alterations in substrate specificities all contributed to the anthocyanins' structural diversity.
RESUMO
Gene expression manipulation of specific metabolic pathways can be used to obtain bioaccumulation of valuable molecules and desired quality traits in plants. A single-gene approach to impact different traits would be greatly desirable in agrospace applications, where several aspects of plant physiology can be affected, influencing growth. In this work, MicroTom hairy root cultures expressing a MYB-like transcription factor that regulates the biosynthesis of anthocyanins in Petunia hybrida (PhAN4), were considered as a testbed for bio-fortified tomato whole plants aimed at agrospace applications. Ectopic expression of PhAN4 promoted biosynthesis of anthocyanins, allowing to profile 5 major derivatives of delphinidin and petunidin together with pelargonidin and malvidin-based anthocyanins, unusual in tomato. Consistent with PhAN4 features, transcriptomic profiling indicated upregulation of genes correlated to anthocyanin biosynthesis. Interestingly, a transcriptome reprogramming oriented to positive regulation of cell response to biotic, abiotic, and redox stimuli was evidenced. PhAN4 hairy root cultures showed the significant capability to counteract reactive oxygen species (ROS) accumulation and protein misfolding upon high-dose gamma irradiation, which is among the most potent pro-oxidant stress that can be encountered in space. These results may have significance in the engineering of whole tomato plants that can benefit space agriculture.
RESUMO
Homologous ("canonical") RAB5 proteins regulate endosomal trafficking to lysosomes in animals and to the central vacuole in plants. Epidermal petal cells contain small vacuoles (vacuolinos) that serve as intermediate stations for proteins on their way to the central vacuole. Here, we show that transcription factors required for vacuolino formation in petunia induce expression of RAB5a. RAB5a defines a previously unrecognized clade of canonical RAB5s that is evolutionarily and functionally distinct from ARA7-type RAB5s, which act in trafficking to the vacuole. Loss of RAB5a reduces cell height and abolishes vacuolino formation, which cannot be rescued by the ARA7 homologs, whereas constitutive RAB5a (over)expression alters the conical cell shape and promotes homotypic vacuolino fusion, resulting in oversized vacuolinos. These findings provide a rare example of how gene duplication and neofunctionalization increased the complexity of membrane trafficking during evolution and suggest a mechanism by which cells may form multiple vacuoles with distinct content and function.
Assuntos
Forma Celular/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Petunia , Transporte Proteico/genética , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The sour taste of Citrus fruits is due to the extreme acidification of vacuoles in juice vesicle cells via a mechanism that remained elusive. Genetic analysis in petunia identified two vacuolar P-ATPases, PH1 and PH5, which determine flower color by hyperacidifying petal cell vacuoles. Here we show that Citrus homologs, CitPH1 and CitPH5, are expressed in sour lemon, orange, pummelo and rangpur lime fruits, while their expression is strongly reduced in sweet-tasting "acidless" varieties. Down-regulation of CitPH1 and CitPH5 is associated with mutations that disrupt expression of MYB, HLH and/or WRKY transcription factors homologous to those activating PH1 and PH5 in petunia. These findings address a long-standing enigma in cell biology and provide targets to engineer or select for taste in Citrus and other fruits.
Assuntos
Citrus/genética , Frutas/genética , Proteínas de Plantas/genética , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/enzimologia , Citrus/metabolismo , Frutas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Concentração de Íons de Hidrogênio , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopia Confocal , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , ATPases Vacuolares Próton-Translocadoras/classificação , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/químicaRESUMO
It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV) of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.
Assuntos
Petunia/enzimologia , Proteínas de Plantas/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologia , Vacúolos/enzimologia , Flores/citologia , Flores/enzimologia , Fusão de Membrana , Petunia/citologia , Epiderme Vegetal/citologia , Transporte ProteicoRESUMO
Anthocyanins are water-soluble polyphenolic compounds with a high nutraceutical value. Despite the fact that cultivated tomato varieties do not accumulate anthocyanins in the fruit, the biosynthetic pathway can be activated in the vegetative organs by several environmental stimuli. Little is known about the molecular mechanisms regulating anthocyanin synthesis in tomato. Here, we carried out a molecular and functional characterization of two genes, SlAN2 and SlANT1, encoding two R2R3-MYB transcription factors. We show that both can induce ectopic anthocyanin synthesis in transgenic tomato lines, including the fruit. However, only SlAN2 acts as a positive regulator of anthocyanin synthesis in vegetative tissues under high light or low temperature conditions.
Assuntos
Antocianinas/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Solanum lycopersicum/metabolismo , Fatores de Transcrição/metabolismo , Temperatura Baixa , Genótipo , Luz , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , Fenótipo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
The acidification of endomembrane compartments is essential for enzyme activities, sorting, trafficking, and trans-membrane transport of various compounds. Vacuoles are mildly acidic in most plant cells because of the action of V-ATPase and/or pyrophosphatase proton pumps but are hyperacidified in specific cells by mechanisms that remained unclear. Here, we show that the blue petal color of petunia ph mutants is due to a failure to hyperacidify vacuoles. We report that PH1 encodes a P3B-ATPase, hitherto known as Mg2(+) transporters in bacteria only, that resides in the vacuolar membrane (tonoplast). In vivo nuclear magnetic resonance and genetic data show that PH1 is required and, together with the tonoplast H(+) P3A-ATPase PH5, sufficient to hyperacidify vacuoles. PH1 has no H(+) transport activity on its own but can physically interact with PH5 and boost PH5 H(+) transport activity. Hence, the hyperacidification of vacuoles in petals, and possibly other tissues, relies on a heteromeric P-ATPase pump.
Assuntos
Flores/metabolismo , Petunia/metabolismo , Pigmentação , ATPases Translocadoras de Prótons/metabolismo , Prótons , Vacúolos/metabolismo , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Petunia/enzimologia , Filogenia , ATPases Translocadoras de Prótons/genética , Vacúolos/enzimologiaRESUMO
Fluorescent protein (FP) fusions are frequently used to localize and follow the movement of proteins in living cells. However, a consensus is missing about the experimental design and controls that guarantee the reliability of the results. Here, we discuss possible artifacts and try to navigate through the many methods, preferences, and assumptions that surround protein localization in plants that make it difficult to design a universal approach to achieve reliable results.
Assuntos
Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transgenes , Expressão Gênica , Marcadores Genéticos , Vetores Genéticos , Proteínas de Fluorescência Verde , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Plantas/genética , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos Testes , Projetos de PesquisaRESUMO
The regulation of pH in cellular compartments is crucial for intracellular trafficking of vesicles and proteins and the transport of small molecules, including hormones. In endomembrane compartments, pH is regulated by vacuolar H(+)-ATPase (V-ATPase), which, in plants, act together with H(+)-pyrophosphatases (PPase), whereas distinct P-type H(+)-ATPases in the cell membrane control the pH in the cytoplasm and energize the plasma membrane. Flower colour mutants have proved useful in identifying genes controlling the pH of vacuoles where anthocyanin pigments accumulate. Here we show that PH5 of petunia encodes a P(3A)-ATPase proton pump that, unlike other P-type H(+)-ATPases, resides in the vacuolar membrane. Mutation of PH5 reduces vacuolar acidification in petals, resulting in a blue flower colour and abolishes the accumulation of proanthocyanidins (condensed tannins) in seeds. Expression of PH5 is directly activated by transcription regulators of the anthocyanin pathway, in conjunction with PH3 and PH4. Thus, flower coloration, a key-factor in plant reproduction, involves the coordinated activation of pigment synthesis and a specific pathway for vacuolar acidification.
Assuntos
Flores/enzimologia , Petunia/enzimologia , Pigmentação/fisiologia , Proteínas de Plantas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/enzimologia , Flores/citologia , Flores/ultraestrutura , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Espaço Intracelular/enzimologia , Petunia/genética , Petunia/ultraestrutura , Filogenia , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/genéticaRESUMO
The Petunia hybrida genes ANTHOCYANIN1 (AN1) and AN2 encode transcription factors with a basic-helix-loop-helix (BHLH) and a MYB domain, respectively, that are required for anthocyanin synthesis and acidification of the vacuole in petal cells. Mutation of PH4 results in a bluer flower color, increased pH of petal extracts, and, in certain genetic backgrounds, the disappearance of anthocyanins and fading of the flower color. PH4 encodes a MYB domain protein that is expressed in the petal epidermis and that can interact, like AN2, with AN1 and the related BHLH protein JAF13 in yeast two-hybrid assays. Mutation of PH4 has little or no effect on the expression of structural anthocyanin genes but strongly downregulates the expression of CAC16.5, encoding a protease-like protein of unknown biological function. Constitutive expression of PH4 and AN1 in transgenic plants is sufficient to activate CAC16.5 ectopically. Together with the previous finding that AN1 domains required for anthocyanin synthesis and vacuolar acidification can be partially separated, this suggests that AN1 activates different pathways through interactions with distinct MYB proteins.
Assuntos
Antocianinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Petunia/metabolismo , Proteínas de Plantas/fisiologia , Proteínas Proto-Oncogênicas c-myb/fisiologia , Vacúolos/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Flores/anatomia & histologia , Flores/citologia , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Sequências Hélice-Alça-Hélice , Homeostase , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Petunia/citologia , Petunia/genética , Fenótipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/metabolismo , RNA Mensageiro/análise , Alinhamento de Sequência , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
ANTHOCYANIN1 (AN1) of petunia is a transcription factor of the basic helix-loop-helix (bHLH) family that is required for the synthesis of anthocyanin pigments. Here, we show that AN1 controls additional aspects of cell differentiation: the acidification of vacuoles in petal cells, and the size and morphology of cells in the seed coat epidermis. We identified an1 alleles, formerly known as ph6, that sustain anthocyanin synthesis but not vacuolar acidification and seed coat morphogenesis. These alleles express truncated proteins lacking the C-terminal half of AN1, including the bHLH domain, at an approximately 30-fold higher level than wild-type AN1. An allelic series in which one, two, or three amino acids were inserted into the bHLH domain indicated that this domain is required for both anthocyanin synthesis and vacuolar acidification. These findings show that AN1 controls more aspects of epidermal cell differentiation than previously thought through partially separable domains.