RESUMO
Pyrimidine nucleotide monophosphate biosynthesis ends in the cytosol with uridine monophosphate (UMP). UMP phosphorylation to uridine diphosphate (UDP) by UMP KINASEs (UMKs) is required for the generation of all pyrimidine (deoxy)nucleoside triphosphates as building blocks for nucleic acids and central metabolites like UDP-glucose. The Arabidopsis (Arabidopsis thaliana) genome encodes five UMKs and three belong to the AMP KINASE (AMK)-like UMKs, which were characterized to elucidate their contribution to pyrimidine metabolism. Mitochondrial UMK2 and cytosolic UMK3 are evolutionarily conserved, whereas cytosolic UMK1 is specific to the Brassicaceae. In vitro, all UMKs can phosphorylate UMP, cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP), but with different efficiencies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced null mutants were generated for UMK1 and UMK2, but not for UMK3, since frameshift alleles were lethal for germline cells. However, a mutant with diminished UMK3 activity showing reduced growth was obtained. Metabolome analyses of germinating seeds and adult plants of single and higher-order mutants revealed that UMK3 plays an indispensable role in the biosynthesis of all pyrimidine (deoxy)nucleotides and UDP-sugars, while UMK2 is important for dCMP recycling that contributes to mitochondrial DNA stability. UMK1 is primarily involved in CMP recycling. We discuss the specific roles of these UMKs referring also to the regulation of pyrimidine nucleoside triphosphate synthesis.
RESUMO
Several ureides are intermediates of purine base catabolism, releasing nitrogen from the purine nucleotides for reassimilation into amino acids. In some legumes like soybean (Glycine max), ureides are used for nodule-to-shoot translocation of fixed nitrogen. Four enzymes of Arabidopsis (Arabidopsis thaliana), (1) allantoinase, (2) allantoate amidohydrolase (AAH), (3) ureidoglycine aminohydrolase, and (4) ureidoglycolate amidohydrolase (UAH), catalyze the complete hydrolysis of the ureide allantoin in vitro. However, the metabolic route in vivo remains controversial. Here, in growth and metabolite analyses of Arabidopsis mutants, we demonstrate that these enzymes are required for allantoin degradation in vivo. Orthologous enzymes are present in soybean, encoded by one to four gene copies. All isoenzymes are active in vitro, while some may be inefficiently translated in vivo. Surprisingly, transcript and protein amounts are not significantly regulated by nitrogen fixation or leaf ureide content. A requirement for soybean AAH and UAH for ureide catabolism in leaves has been demonstrated by the use of virus-induced gene silencing. Functional AAH, ureidoglycine aminohydrolase, and UAH are also present in rice (Oryza sativa), and orthologous genes occur in all other plant genomes sequenced to date, indicating that the amidohydrolase route of ureide degradation is universal in plants, including mosses (e.g. Physcomitrella patens) and algae (e.g. Chlamydomomas reinhardtii).
Assuntos
Amidoidrolases/metabolismo , Aminoidrolases/metabolismo , Arabidopsis/enzimologia , Glycine max/enzimologia , Oryza/enzimologia , Purinas/metabolismo , Ureia/metabolismo , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Teste de Complementação Genética , Cinética , Metabolômica , Modelos Biológicos , Mutação/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glycine max/genética , Frações Subcelulares/enzimologia , Ureia/análogos & derivadosRESUMO
Eugenol is a volatile that serves as an attractant for pollinators of flowers, acts as a defense compound in various plant tissues, and contributes to the aroma of fruits. Its production in a cultivated species such as strawberry (Fragaria × ananassa), therefore, is important for the viability and quality of the fruit. We have identified and functionally characterized three strawberry complementary DNAs (cDNAs) that encode proteins with high identity to eugenol synthases from several plant species. Based on a sequence comparison with the wild relative Fragaria vesca, two of these cDNAs, FaEGS1a and FaEGS1b, most likely correspond to transcripts derived from allelic gene variants, whereas the third cDNA, FaEGS2, corresponds to a different gene. Using coniferyl acetate as a substrate, FaEGS1a and FaEGS1b catalyze the in vitro formation of eugenol, while FaEGS2 catalyzes the formation of eugenol and also of isoeugenol with a lower catalytic efficiency. The expression of these genes is markedly higher in the fruit than in other tissues of the plant, with FaEGS1a and FaEGS1b mostly expressed in the green achenes, whereas FaEGS2 expression is almost restricted to the red receptacles. These expression patterns correlate with the eugenol content, which is highest in the achene at the green stage and in the receptacle at the red stage. The transient expression of the corresponding cDNAs in strawberry fruit and the subsequent volatile analyses confirm FaEGSs as genuine eugenol synthases in planta. These results provide new insights into the diversity of phenylpropene synthases in plants.
Assuntos
Biocatálise , Eugenol/metabolismo , Fragaria/anatomia & histologia , Fragaria/enzimologia , Frutas/anatomia & histologia , Frutas/enzimologia , Ligases/metabolismo , Cromatografia Líquida de Alta Pressão , Eugenol/química , Fragaria/genética , Fragaria/crescimento & desenvolvimento , Frutas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Cinética , Dados de Sequência Molecular , Filogenia , Homologia de Sequência do Ácido Nucleico , Transformação GenéticaRESUMO
The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.
Assuntos
Etilenos/metabolismo , Fragaria/crescimento & desenvolvimento , Fragaria/metabolismo , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Especificidade de Órgãos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Etilenos/biossíntese , Fragaria/enzimologia , Fragaria/genética , Frutas/enzimologia , Frutas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Metaboloma/genética , Especificidade de Órgãos/genética , Fenótipo , Plantas Geneticamente Modificadas , Receptores de Superfície Celular/metabolismoRESUMO
⢠We have reported previously that the gibberellin (GA) content in strawberry receptacle is high, peaking at specific stages, pointing to a role of this hormone in fruit development. In Arabidopsis, miR159 levels are dependent on GA concentration. This prompted us to investigate the role of two members of the miR159 family and their putative strawberry target gene, GAMYB, in relation to changes in GA content during the course of fruit development. ⢠The highest expression level of the two Fa-MIR159 genes was in the fruit's receptacle tissue, with dramatic changes observed throughout development. The lowest levels of total mature miR159 (a and b) were observed during the white stage of receptacle development, which was concurrent with the highest expression of Fa-GAMYB. A functional interaction between miR159 and Fa-GAMYB has been demonstrated in receptacle tissue. ⢠The application of bioactive GA (i.e. GA(3) ) to strawberry plants caused the down-regulated expression of Fa-MIR159a, but the expression of Fa-MIR159b was not affected significantly. Clear discrepancies between Fa-MIR159b and mature Fa-miR159b levels were indicative of post-transcriptional regulation of Fa-MIR159b gene expression. ⢠We propose that Fa-miR159a and Fa-miR159b interact with Fa-GAMYB during the course of strawberry receptacle development, and that they act in a cooperative fashion to respond, in part, to changes in GA endogenous levels.
Assuntos
Fragaria/crescimento & desenvolvimento , Fragaria/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , MicroRNAs , Sequência de Aminoácidos , Arabidopsis/genética , Sequência de Bases , Fragaria/metabolismo , Genes de Plantas , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Tropical legumes transport fixed nitrogen in form of ureides (allantoin and allantoate) over long distances from the nodules to the shoot. Ureides are formed in nodules from purine mononucleotides by a partially unknown reaction network that involves bacteroid-infected and uninfected cells. Here, we demonstrate by metabolic analysis of CRISPR mutant nodules of Phaseolus vulgaris defective in either xanthosine monophosphate phosphatase (XMPP), guanosine deaminase (GSDA), the nucleoside hydrolases 1 and 2 (NSH1, NSH2) or xanthine dehydrogenase (XDH) that nodule ureide biosynthesis involves these enzymes and requires xanthosine and guanosine but not inosine monophosphate catabolism. Interestingly, promoter reporter analyses revealed that XMPP, GSDA and XDH are expressed in infected cells, whereas NSH1, NSH2 and the promoters of the downstream enzymes urate oxidase (UOX) and allantoinase (ALN) are active in uninfected cells. The data suggest a complex cellular organization of ureide biosynthesis with three transitions between infected and uninfected cells.
Assuntos
Nitrogênio , Phaseolus , Alantoína/metabolismo , N-Glicosil Hidrolases/metabolismo , Nitrogênio/metabolismo , Phaseolus/genética , Xantina Desidrogenase/metabolismoRESUMO
⢠Enhanced Disease Susceptibility1 (EDS1) is an important regulator of plant basal and receptor-triggered immunity. Arabidopsis EDS1 interacts with two related proteins, Phytoalexin Deficient4 (PAD4) and Senescence Associated Gene101 (SAG101), whose combined activities are essential for defense signaling. The different sizes and intracellular distributions of EDS1-PAD4 and EDS1-SAG101 complexes in Arabidopsis leaf tissues suggest that they perform nonredundant functions. ⢠The nature and biological relevance of EDS1 interactions with PAD4 and SAG101 were explored using yeast three-hybrid assays, in vitro analysis of recombinant proteins purified from Escherichia coli, and characterization of Arabidopsis transgenic plants expressing an eds1 mutant (eds1(L262P) ) protein which no longer binds PAD4 but retains interaction with SAG101. ⢠EDS1 forms molecularly distinct complexes with PAD4 or SAG101 without additional plant factors. Loss of interaction with EDS1 reduces PAD4 post-transcriptional accumulation, consistent with the EDS1 physical association stabilizing PAD4. The dissociated forms of EDS1 and PAD4 are fully competent in signaling receptor-triggered localized cell death at infection foci. By contrast, an EDS1-PAD4 complex is necessary for basal resistance involving transcriptional up-regulation of PAD4 itself and mobilization of salicylic acid defenses. ⢠Different EDS1 and PAD4 molecular configurations have distinct and separable functions in the plant innate immune response.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/imunologia , Hidrolases de Éster Carboxílico/fisiologia , Proteínas de Ligação a DNA/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Imunidade Inata/genética , Mutação , Plantas Geneticamente Modificadas/imunologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
The enlargement of receptacle cells during strawberry (Fragaria × ananassa) fruit development is a critical factor determining fruit size, with the increase in cell expansion being one of the most important physiological processes regulated by the phytohormone gibberellin (GA). Here, we studied the role of GA during strawberry fruit development by analyzing the endogenous content of bioactive GAs and the expression of key components of GA signalling and metabolism. Bioactive GA(1) , GA(3) and GA(4) were monitored during fruit development, with the content of GA(4) being extremely high in the receptacle, peaking at the white stage of development. â¢Genes with high homology to genes encoding GA pathway components, including receptors (FaGID1(GIBBERELLIN-INSENSITIVE DWARF1)b and FaGID1c), DELLA (FaRGA(REPRESSOR OF GA) and FaGAI(GA-INSENSITIVE)), and enzymes involved in GA biosynthesis (FaGA3ox) and catabolism (FaGA2ox), were identified, and their expression in different tissues and developmental stages of strawberry fruit was studied in detail. The expression of all of these genes showed a stage-specific pattern during fruit development and was highest in the receptacle. FaGID1c bound GA in vitro, interacted with FaRGA in vitro and in vivo, and increased GA responses when ectopically expressed in Arabidopsis. This study thus reveals key elements of GA responses in strawberry and points to a critical role for GA in the development of the receptacle.
Assuntos
Fragaria/metabolismo , Frutas/crescimento & desenvolvimento , Giberelinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Crescimento Celular , Fragaria/genética , Fragaria/crescimento & desenvolvimento , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Giberelinas/análise , Giberelinas/biossíntese , Filogenia , Reguladores de Crescimento de Plantas/análise , Reguladores de Crescimento de Plantas/biossíntese , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Ectopic expression of the strawberry (Fragaria×ananassa) gene FaPE1 encoding pectin methyl esterase produced in the wild species Fragaria vesca partially demethylated oligogalacturonides (OGAs), which conferred partial resistance of ripe fruits to the fungus Botrytis cinerea. Analyses of metabolic and transcriptional changes in the receptacle of the transgenic fruits revealed channelling of metabolites to aspartate and aromatic amino acids as well as phenolics, flavanones, and sesquiterpenoids, which was in parallel with the increased expression of some genes related to plant defence. The results illustrate the changes associated with resistance to B. cinerea in the transgenic F. vesca. These changes were accompanied by a significant decrease in the auxin content of the receptacle of the ripe fruits of transgenic F. vesca, and enhanced expression of some auxin-repressed genes. The role of these OGAs in fruit development was revealed by the larger size of the ripe fruits in transgenic F. vesca. When taken together these results show that in cultivated F. ananassa FaPE1 participates in the de-esterification of pectins and the generation of partially demethylated OGAs, which might reinforce the plant defence system and play an active role in fruit development.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Fragaria/enzimologia , Fragaria/genética , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Oligossacarídeos/metabolismo , Transcrição Gênica , Sequência de Bases , Parede Celular/genética , Regulação para Baixo/genética , Esterificação , Fragaria/crescimento & desenvolvimento , Fragaria/metabolismo , Frutas/genética , Frutas/imunologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Ácidos Indolacéticos/metabolismo , Metilação , Modelos Biológicos , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
In plants, guanosine monophosphate (GMP) is synthesized from adenosine monophosphate via inosine monophosphate and xanthosine monophosphate (XMP) in the cytosol. It has been shown recently that the catabolic route for adenylate-derived nucleotides bifurcates at XMP from this biosynthetic route. Dephosphorylation of XMP and GMP by as yet unknown phosphatases can initiate cytosolic purine nucleotide catabolism. Here we show that Arabidopsis thaliana possesses a highly XMP-specific phosphatase (XMPP) which is conserved in vascular plants. We demonstrate that XMPP catalyzes the irreversible entry reaction of adenylate-derived nucleotides into purine nucleotide catabolism in vivo, whereas the guanylates enter catabolism via an unidentified GMP phosphatase and guanosine deaminase which are important to maintain purine nucleotide homeostasis. We also present a crystal structure and mutational analysis of XMPP providing a rationale for its exceptionally high substrate specificity, which is likely required for the efficient catalysis of the very small XMP pool in vivo.
Assuntos
Citosol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Nucleotídeos de Purina/metabolismo , Ribonucleotídeos/metabolismo , Xantina/metabolismo , Arabidopsis/classificação , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Modelos Moleculares , Mutação , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Filogenia , Plantas/classificação , Plantas/enzimologia , Plantas/genética , Ribonucleotídeos/química , Especificidade por Substrato , Xantina/químicaRESUMO
BACKGROUND: Cultivated strawberry is a hybrid octoploid species (Fragaria xananassa Duchesne ex. Rozier) whose fruit is highly appreciated due to its organoleptic properties and health benefits. Despite recent studies on the control of its growth and ripening processes, information about the role played by different hormones on these processes remains elusive. Further advancement of this knowledge is hampered by the limited sequence information on genes from this species, despite the abundant information available on genes from the wild diploid relative Fragaria vesca. However, the diploid species, or one ancestor, only partially contributes to the genome of the cultivated octoploid. We have produced a collection of expressed sequence tags (ESTs) from different cDNA libraries prepared from different fruit parts and developmental stages. The collection has been analysed and the sequence information used to explore the involvement of different hormones in fruit developmental processes, and for the comparison of transcripts in the receptacle of ripe fruits of diploid and octoploid species. The study is particularly important since the commercial fruit is indeed an enlarged flower receptacle with the true fruits, the achenes, on the surface and connected through a network of vascular vessels to the central pith. RESULTS: We have sequenced over 4,500 ESTs from Fragaria xananassa, thus doubling the number of ESTs available in the GenBank of this species. We then assembled this information together with that available from F. xananassa resulting a total of 7,096 unigenes. The identification of SSRs and SNPs in many of the ESTs allowed their conversion into functional molecular markers. The availability of libraries prepared from green growing fruits has allowed the cloning of cDNAs encoding for genes of auxin, ethylene and brassinosteroid signalling processes, followed by expression studies in selected fruit parts and developmental stages. In addition, the sequence information generated in the project, jointly with previous information on sequences from both F. xananassa and F. vesca, has allowed designing an oligo-based microarray that has been used to compare the transcriptome of the ripe receptacle of the diploid and octoploid species. Comparison of the transcriptomes, grouping the genes by biological processes, points to differences being quantitative rather than qualitative. CONCLUSIONS: The present study generates essential knowledge and molecular tools that will be useful in improving investigations at the molecular level in cultivated strawberry (F. xananassa). This knowledge is likely to provide useful resources in the ongoing breeding programs. The sequence information has already allowed the development of molecular markers that have been applied to germplasm characterization and could be eventually used in QTL analysis. Massive transcription analysis can be of utility to target specific genes to be further studied, by their involvement in the different plant developmental processes.
Assuntos
Etiquetas de Sequências Expressas/metabolismo , Fragaria/genética , Frutas/genética , Parede Celular/metabolismo , Análise por Conglomerados , Mapeamento de Sequências Contíguas , Regulação para Baixo/genética , Etilenos/metabolismo , Fragaria/crescimento & desenvolvimento , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genes de Plantas/genética , Redes e Vias Metabólicas/genética , Repetições Minissatélites/genética , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo Genético , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria x ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca. Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea. Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Fragaria/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Ácidos Urônicos/metabolismo , Botrytis/fisiologia , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Pectinas/química , Pectinas/metabolismo , Plantas Geneticamente ModificadasRESUMO
The role of auxin in ripening strawberry (Fragaria ×ananassa) fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and ripening receptacle.
RESUMO
[This corrects the article on p. 889 in vol. 8, PMID: 28611805.].
RESUMO
Although the commonly named strawberry fruit (Fragaria×ananassa) is the sum of achenes and receptacles, the true fruit in the botanical sense is the achene. Here we report the protein changes occurring in the achene when developing from immature to mature stage. We have used 2-DE followed by image analysis, and protein identification by PMF combined with MS/MS, to investigate the protein variations associated to this transition. From a total of 331 spots analyzed, the corresponding 315 proteins have been identified. Differentially accumulated proteins between immature and mature achenes mostly reflect the physiological events associated to seed development and maturation, with only a few changes related to the development of the dry pericarp. We have focused our attention on vitamin C biosynthesis. Interestingly, GDP-mannose 3',5'-epimerase, a key enzyme in the l-ascorbate biosynthesis pathway, and ascorbate peroxidase, involved in l-ascorbic acid oxidation, accumulate in immature achenes. The higher amount of these enzymes found in the green achene is coincident with a higher content of l-ascorbate, and higher expression levels of these and other gene encoding enzymes of the l-ascorbic acid biosynthesis pathway. Altogether our results suggest an important role of l-ascorbic acid at the early developmental stage of the achene. BIOLOGICAL SIGNIFICANCE: In this manuscript we report the identification of the most abundant proteins in strawberry (F.×ananassa) achenes at early and late stages of development, thus providing a proteomic view of the events that occur during the development of this organ. Despite the importance of strawberry as a commercial fruit, the molecular changes governing its growth and ripening processes are largely unknown. The lack of information is even greater in the case of the achenes, which are the true fruit and play a critical role in the developmental process of the receptacle. Our original proteomic study reported here, restricted to the achenes, completes the previous transcriptomic (very limited) and metabolomic maps of this organ, adding clarity to the role of the achene in the global ripening process. The results obtained not only complement the previous "omics" studies significantly, but also open new key questions that deserve further research (role of hormones). We finally focus on the biosynthesis of l-ascorbic acid, which appears to be tightly regulated by some specific pathways, and whose content is important in the achene. The information provided here will be of interest not only for the groups studying strawberry, but also for many other groups interested in the fruit ripening process, as well as for groups studying the regulation of l-ascorbic acid content in different plant tissues.
Assuntos
Ácido Ascórbico/biossíntese , Fragaria/metabolismo , Frutas/metabolismo , Proteínas de Plantas/biossíntese , Proteoma/biossíntese , Proteômica/métodosRESUMO
Plant innate immunity against invasive biotrophic pathogens depends on the intracellular defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). We show here that Arabidopsis thaliana EDS1 interacts in vivo with another protein, SENESCENCE-ASSOCIATED GENE101 (SAG101), discovered through a proteomic approach to identify new EDS1 pathway components. Together with PHYTOALEXIN-DEFICIENT4 (PAD4), a known EDS1 interactor, SAG101 contributes intrinsic and indispensable signaling activity to EDS1-dependent resistance. The combined activities of SAG101 and PAD4 are necessary for programmed cell death triggered by the Toll-Interleukin-1 Receptor type of nucleotide binding/leucine-rich repeat immune receptor in response to avirulent pathogen isolates and in restricting the growth of normally virulent pathogens. We further demonstrate by a combination of cell fractionation, coimmunoprecipitation, and fluorescence resonance energy transfer experiments the existence of an EDS1-SAG101 complex inside the nucleus that is molecularly and spatially distinct from EDS1-PAD4 associations in the nucleus and cytoplasm. By contrast, EDS1 homomeric interactions were detected in the cytoplasm but not inside the nucleus. These data, combined with evidence for coregulation between individual EDS1 complexes, suggest that dynamic interactions of EDS1 and its signaling partners in multiple cell compartments are important for plant defense signal relay.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Imunidade Inata/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Complexos Multiproteicos , Fenótipo , Folhas de Planta/química , Plantas Geneticamente Modificadas , Plântula/citologia , Plântula/metabolismo , Plântula/microbiologia , Alinhamento de SequênciaRESUMO
cDNA and genomic clones encoding a strawberry (Fragariaxananassa cv. Chandler) non-specific lipid transfer protein (Fxaltp gene) were isolated and characterized. The spatio-temporal expression pattern and structural features of this gene were studied for the first time in strawberry, a non-climacteric fruit of agricultural importance. The architecture and the encoded amino acid sequence of this non-climacteric fruit ltp gene were similar to those of other plant LTPs previously reported, and presents the eight cysteine residues and other features characteristic of plant LTPs. In addition, the deduced protein posseses an N-terminal signal peptide and lacks the K/HDEL retention signal, indicating that the strawberry LTP protein would enter the secretory pathway. In situ studies have shown that the Fxaltp gene is expressed in the epidermal cell layer of the strawberry fruit receptacle and achenes, flowers, and within the cell layer surrounding the endosperm. These results suggest that this Fxaltp gene promoter could be used as an endogenous promoter for biotechnological purposes in strawberry. Computer analysis using the PLACE database predicted the presence of several putative cis-regulatory sequences in response to abscisic acid and cold or wounding stresses within the Fxaltp 5'-flanking region. Accordingly, the strawberry gene responds to ABA and SA, but not to salt and heat stresses. It is also reported that ltp gene expression in strawberry is stimulated by wounding and repressed by cold stresses.