RESUMEN
In order to capture the drought impacts on seed quality acquisition in Brassica napus and its potential interaction with early biotic stress, seeds of the 'Express' genotype of oilseed rape were characterized from late embryogenesis to full maturity from plants submitted to reduced watering (WS) with or without pre-occurring inoculation by the telluric pathogen Plasmodiophora brassicae (Pb + WS or Pb, respectively), and compared to control conditions (C). Drought as a single constraint led to significantly lower accumulation of lipids, higher protein content and reduced longevity of the WS-treated seeds. In contrast, when water shortage was preceded by clubroot infection, these phenotypic differences were completely abolished despite the upregulation of the drought sensor RD20. A weighted gene co-expression network of seed development in oilseed rape was generated using 72 transcriptomes from developing seeds from the four treatments and identified 33 modules. Module 29 was highly enriched in heat shock proteins and chaperones that showed a stronger upregulation in Pb + WS compared to the WS condition, pointing to a possible priming effect by the early P. brassicae infection on seed quality acquisition. Module 13 was enriched with genes encoding 12S and 2S seed storage proteins, with the latter being strongly upregulated under WS conditions. Cis-element promotor enrichment identified PEI1/TZF6, FUS3 and bZIP68 as putative regulators significantly upregulated upon WS compared to Pb + WS. Our results provide a temporal co-expression atlas of seed development in oilseed rape and will serve as a resource to characterize the plant response towards combinations of biotic and abiotic stresses.
Asunto(s)
Brassica napus , Sequías , Regulación de la Expresión Génica de las Plantas , Semillas , Estrés Fisiológico , Brassica napus/genética , Brassica napus/fisiología , Semillas/genética , Semillas/crecimiento & desarrollo , Estrés Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmodiophorida/fisiología , Transcriptoma/genéticaRESUMEN
The spatiotemporal pattern of deposition, final amount, and relative abundance of oleic acid (cis-ω-9 C18:1) and its derivatives in the different lipid fractions of the seed of Arabidopsis (Arabidopsis thaliana) indicates that omega-9 monoenes are synthesized at high rates in this organ. Accordingly, we observed that four Δ9 stearoyl-ACP desaturase (SAD)-coding genes (FATTY ACID BIOSYNTHESIS2 [FAB2], ACYL-ACYL CARRIER PROTEIN5 [AAD5], AAD1, and AAD6) are transcriptionally induced in seeds. We established that the three most highly expressed ones are directly activated by the WRINKLED1 transcription factor. We characterized a collection of 30 simple, double, triple, and quadruple mutants affected in SAD-coding genes and thereby revealed the functions of these desaturases throughout seed development. Production of oleic acid by FAB2 and AAD5 appears to be critical at the onset of embryo morphogenesis. Double homozygous plants from crossing fab2 and aad5 could never be obtained, and further investigations revealed that the double mutation results in the arrest of embryo development before the globular stage. During later stages of seed development, these two SADs, together with AAD1, participate in the elaboration of the embryonic cuticle, a barrier essential for embryo-endosperm separation during the phase of invasive embryo growth through the endosperm. This study also demonstrates that the four desaturases redundantly contribute to storage lipid production during the maturation phase.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Oxigenasas de Función Mixta/genética , Semillas/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Oxigenasas de Función Mixta/metabolismo , Mutación , Plantas Modificadas Genéticamente , Semillas/genética , Semillas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Acyl lipids are important constituents of the plant cell. Depending on the cell type, requirements in acyl lipids vary greatly, implying a tight regulation of fatty acid and lipid metabolism. The discovery of the WRINKLED1 (WRI1) transcription factors, members of the AP2-EREBP (APETALA2-ethylene-responsive element binding protein) family, has emphasized the importance of transcriptional regulation for adapting the rate of acyl chain production to cell requirements. Here, we describe the identification of another activator of the fatty acid biosynthetic pathway, the Arabidopsis MYB92 transcription factor. This MYB and all the members of the subgroups S10 and S24 of MYB transcription factors can directly activate the promoter of BCCP2 that encodes a component of the fatty acid biosynthetic pathway. Two adjacent MYB cis-regulatory elements are essential for the binding and activation of the BCCP2 promoter by MYB92. Overexpression of MYB92 or WRI1 in Nicotiana benthamiana induces the expression of fatty acid biosynthetic genes but results in the accumulation of different types of acyl lipids. In the presence of WRI1, triacylglycerol biosynthetic enzymes coded by constitutively expressed genes efficiently channel the excess fatty acids toward reserve lipid accumulation. By contrast, MYB92 activates both fatty acid and suberin biosynthetic genes; hence, the remarkable increase in suberin monomers measured in leaves expressing MYB92. These results provide additional insight into the molecular mechanisms that control the biosynthesis of an important cell wall-associated acylglycerol polymer playing critical roles in plants.
Asunto(s)
Ácidos Grasos/biosíntesis , Nicotiana/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/metabolismo , Ácidos Grasos/metabolismo , Lípidos/biosíntesis , Plantas Modificadas GenéticamenteRESUMEN
In angiosperm seeds, the endosperm develops to varying degrees and accumulates different types of storage compounds remobilized by the seedling during early post-germinative growth. Whereas the molecular mechanisms controlling the metabolism of starch and seed-storage proteins in the endosperm of cereal grains are relatively well characterized, the regulation of oil metabolism in the endosperm of developing and germinating oilseeds has received particular attention only more recently, thanks to the emergence and continuous improvement of analytical techniques allowing the evaluation, within a spatial context, of gene activity on one side, and lipid metabolism on the other side. These studies represent a fundamental step toward the elucidation of the molecular mechanisms governing oil metabolism in this particular tissue. In particular, they highlight the importance of endosperm-specific transcriptional controls for determining original oil compositions usually observed in this tissue. In the light of this research, the biological functions of oils stored in the endosperm of seeds then appear to be more diverse than simply constituting a source of carbon made available for the germinating seedling.
Asunto(s)
Grano Comestible/crecimiento & desarrollo , Endospermo/metabolismo , Aceites de Plantas/metabolismo , Proteínas de Plantas/genética , Grano Comestible/genética , Grano Comestible/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Metabolismo de los LípidosRESUMEN
During seed germination, proteins are translated not only from mRNAs newly transcribed upon imbibition but also from long-lived mRNAs that are synthesized during seed maturation and stored in the mature dry seeds. To clarify the distinct roles of proteins translated from long-lived mRNAs and de novo transcribed mRNAs in germinating rice embryos, proteome analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining the use of a transcriptional inhibitor was performed. We observed that α-amanitin significantly represses transcription in germinating embryos; nevertheless, the embryos could germinate, albeit slowly. The proteomic analysis revealed that a total of 109 proteins were translated from long-lived mRNAs associated with germination as well as 222 proteins whose expression were dependent on de novo transcription upon imbibition. Transcriptomic datasets available in public databases demonstrated that mRNAs of the 222 proteins notably increased during germination while those of the 109 proteins highly accumulated in dry embryos and constitutively expressed upon imbibition. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that many of the 109 proteins from long-lived mRNAs are implicated in energy production such as glycolysis or annotated as nucleotide binding proteins, while the 222 proteins are involved in pathways such as pyruvate metabolism and TCA cycle following glycolysis, and momilactones biosynthesis. We propose that long-lived mRNAs support initial energy production and activation of translational machinery upon imbibition whereas de novo transcription accelerates the energy production after glycolysis, which enables rice seeds to germinate vigorously.
Asunto(s)
Oryza/metabolismo , ARN Mensajero/metabolismo , Semillas/metabolismo , Alfa-Amanitina/metabolismo , Germinación/fisiología , ProteómicaRESUMEN
In angiosperms, double fertilization of the embryo sac initiates the development of the embryo and the endosperm. In Arabidopsis thaliana, an exalbuminous species, the endosperm is reduced to one cell layer during seed maturation and reserves such as oil are massively deposited in the enlarging embryo. Here, we consider the strikingly different fatty acid (FA) compositions of the oils stored in the two zygotic tissues. Endosperm oil is enriched in ω-7 monounsaturated FAs, that represent more than 20 mol% of total FAs, whereas these molecular species are 10-fold less abundant in the embryo. Two closely related transcription factors, MYB118 and MYB115, are transcriptionally induced at the onset of the maturation phase in the endosperm and share a set of transcriptional targets. Interestingly, the endosperm oil of myb115 myb118 double mutants lacks ω-7 FAs. The identification of two Δ9 palmitoyl-ACP desaturases responsible for ω-7 FA biosynthesis, which are activated by MYB115 and MYB118 in the endosperm, allows us to propose a model for the transcriptional control of oil FA composition in this tissue. In addition, an initial characterization of the structure-function relationship for these desaturases reveals that their particular substrate specificity is conferred by amino acid residues lining their substrate pocket that distinguish them from the archetype Δ9 stearoyl-ACP desaturase.
Asunto(s)
Arabidopsis/metabolismo , Endospermo/metabolismo , Oxigenasas de Función Mixta/metabolismo , Semillas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endospermo/genética , Ácidos Grasos Monoinsaturados/metabolismo , Oxigenasas de Función Mixta/genética , Semillas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In Arabidopsis (Arabidopsis thaliana), transcriptional control of seed maturation involves three related regulators with a B3 domain, namely LEAFY COTYLEDON2 (LEC2), ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (ABI3/FUS3/LEC2 [AFLs]). Although genetic analyses have demonstrated partially overlapping functions of these regulators, the underlying molecular mechanisms remained elusive. The results presented here confirmed that the three proteins bind RY DNA elements (with a 5'-CATG-3' core sequence) but with different specificities for flanking nucleotides. In planta as in the moss Physcomitrella patens protoplasts, the presence of RY-like (RYL) elements is necessary but not sufficient for the regulation of the OLEOSIN1 (OLE1) promoter by the B3 AFLs. G box-like domains, located in the vicinity of the RYL elements, also are required for proper activation of the promoter, suggesting that several proteins are involved. Consistent with this idea, LEC2 and ABI3 showed synergistic effects on the activation of the OLE1 promoter. What is more, LEC1 (a homolog of the NF-YB subunit of the CCAAT-binding complex) further enhanced the activation of this target promoter in the presence of LEC2 and ABI3. Finally, recombinant LEC1 and LEC2 proteins produced in Arabidopsis protoplasts could form a ternary complex with NF-YC2 in vitro, providing a molecular explanation for their functional interactions. Taken together, these results allow us to propose a molecular model for the transcriptional regulation of seed genes by the L-AFL proteins, based on the formation of regulatory multiprotein complexes between NF-YBs, which carry a specific aspartate-55 residue, and B3 transcription factors.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Semillas/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Bases , Sitios de Unión/genética , Briófitas/metabolismo , ADN de Plantas/metabolismo , Inmunoprecipitación , Modelos Biológicos , Regiones Promotoras Genéticas , Unión Proteica/genética , Protoplastos/metabolismoRESUMEN
In the exalbuminous species Arabidopsis thaliana, seed maturation is accompanied by the deposition of oil and storage proteins and the reduction of the endosperm to one cell layer. Here, we consider reserve partitioning between embryo and endosperm compartments. The pattern of deposition, final amount, and composition of these reserves differ between the two compartments, with the embryo representing the principal storage tissue in mature seeds. Complex regulatory mechanisms are known to prevent activation of maturation-related programs during embryo morphogenesis and, later, during vegetative growth. Here, we describe a regulator that represses the expression of maturation-related genes during maturation within the endosperm. MYB118 is transcriptionally induced in the maturing endosperm, and seeds of myb118 mutants exhibit an endosperm-specific derepression of maturation-related genes associated with a partial relocation of storage compounds from the embryo to the endosperm. Moreover, MYB118 activates endosperm-induced genes through the recognition of TAACGG elements. These results demonstrate that the differential partitioning of reserves between the embryo and endosperm in exalbuminous Arabidopsis seeds does not only result from developmental programs that establish the embryo as the preponderant tissue within seeds. This differential partitioning is also regulated by MYB118, which regulates the biosynthesis of reserves at the spatial level during maturation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Arabidopsis/metabolismo , Endospermo/embriología , Endospermo/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Endospermo/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica/genética , Factores de Transcripción/genética , Activación Transcripcional/genéticaRESUMEN
Acyl lipids are essential constituents of all cells, but acyl chain requirements vary greatly and depend on the cell type considered. This implies a tight regulation of fatty acid production so that supply fits demand. Isolation of the Arabidopsis thaliana WRINKLED1 (WRI1) transcription factor established the importance of transcriptional regulation for modulating the rate of acyl chain production. Here, we report the isolation of two additional regulators of the fatty acid biosynthetic pathway, WRI3 and WRI4, which are closely related to WRI1 and belong to the APETALA2-ethylene-responsive element binding protein family of transcription factors. These three WRIs define a family of regulators capable of triggering sustained rates of acyl chain synthesis. However, expression patterns of the three WRIs differ markedly. Whereas only WRI1 activates fatty acid biosynthesis in seeds for triacylglycerol production, the three WRIs are required in floral tissues to provide acyl chains for cutin biosynthesis and prevent adherence of these developing organs and subsequent semisterility. The targets of these WRIs encode enzymes providing precursors (acyl chain and glycerol backbones) for various lipid biosynthetic pathways, but not the subsequent lipid-assembling enzymes. These results provide insights into the developmental regulation of fatty acid production in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Grasos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Lípidos de la Membrana/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triglicéridos/metabolismoRESUMEN
Carotenoid cleavage, catalyzed by the 9-cis-epoxycarotenoid dioxygenase (NCED) constitutes a key step in the regulation of ABA biosynthesis. In Arabidopsis, this enzyme is encoded by five genes. NCED3 has been shown to play a major role in the regulation of ABA synthesis in response to water deficit, whereas NCED6 and NCED9 have been shown to be essential for the ABA production in the embryo and endosperm that imposes dormancy. Reporter gene analysis was carried out to determine the spatiotemporal pattern of NCED5 and NCED9 gene expression. GUS activity from the NCED5 promoter was detected in both the embryo and endosperm of developing seeds with maximal staining after mid-development. NCED9 expression was found at early stages in the testa outer integument layer 1, and after mid-development in epidermal cells of the embryo, but not in the endosperm. In accordance with its temporal- and tissue-specific expression, the phenotypic analysis of nced5 nced6 nced9 triple mutant showed the involvement of the NCED5 gene, together with NCED6 and NCED9, in the induction of seed dormancy. In contrast to nced6 and nced9, however, nced5 mutation did not affect the gibberellin required for germination. In vegetative tissues, combining nced5 and nced3 mutations reduced vegetative growth, increased water loss upon dehydration, and decreased ABA levels under both normal and stressed conditions, as compared with nced3. NCED5 thus contributes, together with NCED3, to ABA production affecting plant growth and water stress tolerance.
Asunto(s)
Ácido Abscísico/metabolismo , Arabidopsis/enzimología , Dioxigenasas/genética , Regulación de la Expresión Génica de las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Semillas/crecimiento & desarrollo , Ácido Abscísico/análisis , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Deshidratación , Dioxigenasas/metabolismo , Sequías , Genes Reporteros , Germinación , Giberelinas/metabolismo , Mutación , Especificidad de Órganos , Fenotipo , Latencia en las Plantas , Reguladores del Crecimiento de las Plantas/análisis , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/fisiología , Semillas/enzimología , Semillas/genética , Semillas/fisiología , Agua/metabolismoRESUMEN
The WRINKLED1 (WRI1) protein is an important regulator of oil accumulation in maturing Arabidopsis seeds. WRI1 is a member of a plant-specific family of transcription factors (AP2/EREBP) that share either one or two copies of a DNA-binding domain called the AP2 domain. Here, it is shown that WRI1 acts as a transcriptional enhancer of genes involved in carbon metabolism in transgenic seeds overexpressing this transcription factor. PKp-beta1 and BCCP2, two genes encoding enzymes of the glycolysis and fatty acid biosynthetic pathway, respectively, have been chosen to investigate the regulatory action exerted by WRI1 over these pathways. Using the reporter gene uidA, it was possible to demonstrate in planta that WRI1 regulates the activity of both PKp-beta1 and BCCP2 promoters. Electrophoretic mobility-shift assays and yeast one-hybrid experiments showed that WRI1 was able to interact with the BCCP2 promoter. To further elucidate the regulatory mechanism controlling the transcription of these genes, functional dissections of PKp-beta1 and BCCP2 promoters were performed. Two enhancers, of 54 and 79 bp, respectively, have thus been isolated that are essential to direct the activity of these promoters in oil-accumulating tissues of the embryo. A consensus site is present in these enhancers as well as in other putative target promoters of WRI1. Loss of this consensus sequence in the BCPP2 promoter decreases both the strength of the interaction between WRI1 and this promoter in yeast and the activity of the promoter in planta.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Ácidos Grasos/biosíntesis , Glucólisis/genética , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Secuencia de Bases , Secuencia de Consenso , ADN de Plantas/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Semillas/genética , Semillas/metabolismo , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
In plants, light-dependent activation of de novo fatty acid synthesis (FAS) is partially mediated by acetyl-CoA carboxylase (ACCase), the first committed step for this pathway. However, it is not fully understood how plants control light-dependent FAS regulation to meet the cellular demand for acyl chains. We report here the identification of a gene family encoding for three small plastidial proteins of the envelope membrane that interact with the α-carboxyltransferase (α-CT) subunit of ACCase and participate in an original mechanism restraining FAS in the light. Light enhances the interaction between carboxyltransferase interactors (CTIs) and α-CT, which in turn attenuates carbon flux into FAS. Knockouts for CTI exhibit higher rates of FAS and marked increase in absolute triacylglycerol levels in leaves, more than 4-fold higher than in wild-type plants. Furthermore, WRINKLED1, a master transcriptional regulator of FAS, positively regulates CTI1 expression by direct binding to its promoter. This study reveals that in addition to light-dependent activation, "envelope docking" of ACCase permits fine-tuning of fatty acid supply during the plant life cycle.
Asunto(s)
Acetil-CoA Carboxilasa/metabolismo , Arabidopsis/metabolismo , Ácidos Grasos/biosíntesis , Membranas Intracelulares/metabolismo , Acetatos/metabolismo , Acetil-CoA Carboxilasa/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Radioisótopos de Carbono , Regulación del Desarrollo de la Expresión Génica , Luz , Simulación del Acoplamiento Molecular , Plastidios/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Protoplastos/metabolismoRESUMEN
The hydroxysteroid dehydrogenase HSD1, identified in the proteome of oil bodies from mature Arabidopsis seeds, is encoded by At5g50600 and At5g50700, two gene copies anchored on a duplicated region of chromosome 5. Using a real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) approach, the accumulation of HSD1 mRNA was shown to be specifically and highly induced in oil-accumulating tissues of maturing seeds. HSD1 mRNA disappeared during germination. The activity of HSD1 promoter and the localization of HSD1 transcripts by in situ hybridization were consistent with this pattern. A complementary set of molecular and genetic analyses showed that HSD1 is a target of LEAFY COTYLEDON2, a transcriptional regulator able to bind the promoter of HSD1. Immunoblot analyses and immunolocalization experiments using anti-AtHSD1 antibodies established that the pattern of HSD1 deposition faithfully reflected mRNA accumulation. At the subcellular level, the study of HSD1:GFP fusion proteins showed the targeting of HSD1 to the surface of oil bodies. Transgenic lines overexpressing HSD1 were then obtained to test the importance of proper transcriptional regulation of HSD1 in seeds. Whereas no impact on oil accumulation could be detected, transgenic seeds exhibited lower cold and light requirements to break dormancy, germinate and mobilize storage lipids. Interestingly, overexpressors of HSD1 over-accumulated HSD1 protein in seeds but not in vegetative organs, suggesting that post-transcriptional regulations exist that prevent HSD1 accumulation in tissues deprived of oil bodies.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Semillas/metabolismo , Triglicéridos/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Germinación/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Semillas/genética , Semillas/fisiología , Factores de Transcripción/metabolismoAsunto(s)
Enfermedades Gastrointestinales , Síndrome de Horner , Degeneraciones Espinocerebelosas , Masculino , Animales , Síndrome de Horner/diagnóstico , Síndrome de Horner/veterinaria , Dolor/veterinaria , Enfermedades Gastrointestinales/veterinaria , Diagnóstico Bucal , Degeneraciones Espinocerebelosas/veterinaria , Ataxia/veterinariaRESUMEN
Omega-7 monoenoic fatty acids (ω-7 FAs) are increasingly exploited both for their positive effects on health and for their industrial potential. Some plant species produce fruits or seeds with high amounts of ω-7 FAs. However, the low yields and poor agronomic properties of these plants preclude their commercial use. As an alternative, the metabolic engineering of oilseed crops for sustainable ω-7 FA production has been proposed. Two palmitoyl-ACP desaturases (PADs) catalyzing ω-7 FA biosynthesis were recently identified and characterized in Arabidopsis thaliana, together with MYB115 and MYB118, two transcription factors that positively control the expression of the corresponding PAD genes. In the present research, we examine the biotechnological potential of these new actors of ω-7 metabolism for the metabolic engineering of plant-based production of ω-7 FAs. We placed the PAD and MYB115 coding sequences under the control of a promoter strongly induced in seeds and evaluated these different constructs in A. thaliana. Seeds were obtained that exhibit ω-7 FA contents ranging from 10 to >50% of the total FAs, and these major compositional changes have no detrimental effect on seed germination.
Asunto(s)
Arabidopsis/genética , Ácidos Grasos/metabolismo , Genes de Plantas , Semillas/genética , Arabidopsis/embriología , Arabidopsis/metabolismo , Germinación , Regiones Promotoras GenéticasRESUMEN
The Arabidopsis abscisic acid (ABA) insensitive (ABI)5 transcription factor participates in the ABA-dependent induction of late embryogenesis abundant (LEA) genes in the final stages of seed development. We tested whether the VP16 transcriptional activation domain is sufficient to provide ABI5 with the ability to activate the AtEm LEA genes in vegetative tissues. We took advantage of a new transgenic seed selection assay based on green fluorescent protein (GFP) fluorescence and found that VP16-ABI5 triggered growth retardation and ABA-independent induction of AtEm1 in seedlings. These results indicate that ABI5 activation potential is a limiting step and might be a target for ABA signaling.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Plantas Modificadas Genéticamente , Semillas/genética , Factores de Transcripción/genética , Alelos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Etopósido/farmacología , Regulación de la Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Estructura Terciaria de Proteína , Semillas/crecimiento & desarrollo , Activación TranscripcionalRESUMEN
The WRINKLED1 (WRI1) transcription factor has been shown to play a role of the utmost importance during oil accumulation in maturing seeds of Arabidopsis thaliana. However, little is known about the regulatory processes involved. In this paper, comprehensive functional analyses of three new mutants corresponding to null alleles of wri1 confirm that the induction of WRI1 is a prerequisite for fatty acid synthesis and is important for normal embryo development. The strong expression of WRI1 specifically detected at the onset of the maturation phase in oil-accumulating tissues of A. thaliana seeds is fully consistent with this function. Complementation experiments carried out with various seed-specific promoters emphasized the importance of a tight regulation of WRI1 expression for proper oil accumulation, raising the question of the factors controlling WRI1 transcription. Interestingly, molecular and genetic analyses using an inducible system demonstrated that WRI1 is a target of LEAFY COTYLEDON2 and is necessary for the regulatory action of LEC2 towards fatty acid metabolism. In addition to this, quantitative RT-PCR experiments suggested that several genes encoding enzymes of late glycolysis, the fatty acid synthesis pathway, and the biotin and lipoic acid biosynthetic pathways are targets of WRI1. Taken together, these results indicate new relationships in the regulatory model for the control of oil synthesis in maturing A. thaliana seeds. In addition, they exemplify how metabolic and developmental processes affecting the developing embryo can be coordinated at the molecular level.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Ácidos Grasos/metabolismo , Semillas/fisiología , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ADN Bacteriano/genética , ADN de Plantas/genética , Eliminación de Gen , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Hibridación in Situ , Plantas Modificadas Genéticamente , Factores de Transcripción/genéticaRESUMEN
A novel abscisic acid (ABA)-deficient mutant, aba4, was identified in a screen for paclobutrazol-resistant germination. Compared with wild-type, the mutant showed reduced endogenous ABA levels in both dehydrated rosettes and seeds. Carotenoid composition analysis demonstrated that the defective locus affects neoxanthin synthesis. The ABA4 gene was identified by map-based cloning, and found to be a unique gene in the Arabidopsis genome. The predicted protein has four putative helical transmembrane domains and shows significant similarity to predicted proteins from tomato, rice and cyanobacteria. Constitutive expression of the ABA4 gene in Arabidopsis transgenic plants led to increased accumulation of trans-neoxanthin, indicating that the ABA4 protein has a direct role in neoxanthin synthesis. aba4 mutant phenotypes were mild compared with previously identified ABA-deficient mutants that exhibit vegetative tissue phenotypes. Indeed, ABA levels in seeds of aba4 mutants were higher than those of aba1 mutants. As aba1 mutants are also affected in a unique gene, this suggests that ABA can be produced in the aba4 mutant by an alternative pathway using violaxanthin as a substrate. It appears, therefore, that in Arabidopsis both violaxanthin and neoxanthin are in vivo substrates for 9-cis-epoxycarotenoid dioxygenases. Furthermore, significantly reduced levels of ABA were synthesized in the aba4 mutant on dehydration, demonstrating that ABA biosynthesis in response to stress must occur mainly via neoxanthin isomer precursors.
Asunto(s)
Ácido Abscísico/genética , Ácido Abscísico/metabolismo , Arabidopsis/genética , Carotenoides/genética , Mutación , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/genética , Secuencia Conservada , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Eliminación de Gen , Genotipo , Cinética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismoRESUMEN
In Arabidopsis thaliana, four major regulators (ABSCISIC ACID INSENSITIVE3 [ABI3], FUSCA3 [FUS3], LEAFY COTYLEDON1 [LEC1], and LEC2) control most aspects of seed maturation, such as accumulation of storage compounds, cotyledon identity, acquisition of desiccation tolerance, and dormancy. The molecular basis for complex genetic interactions among these regulators is poorly understood. By analyzing ABI3 and FUS3 expression in various single, double, and triple maturation mutants, we have identified multiple regulatory links among all four genes. We found that one of the major roles of LEC2 was to upregulate FUS3 and ABI3. The lec2 mutation is responsible for a dramatic decrease in ABI3 and FUS3 expression, and most lec2 phenotypes can be rescued by ABI3 or FUS3 constitutive expression. In addition, ABI3 and FUS3 positively regulate themselves and each other, thereby forming feedback loops essential for their sustained and uniform expression in the embryo. Finally, LEC1 also positively regulates ABI3 and FUS3 in the cotyledons. Most of the genetic controls discovered were found to be local and redundant, explaining why they had previously been overlooked. This works establishes a genetic framework for seed maturation, organizing the key regulators of this process into a hierarchical network. In addition, it offers a molecular explanation for the puzzling variable features of lec2 mutant embryos.