Target enrichment sequencing coupled with GWAS identifies MdPRX10 as a candidate gene in the control of budbreak in apple

The timing of floral budbreak in apple has a significant effect on fruit production and quality. Budbreak occurs as a result of a complex molecular mechanism that relies on accurate integration of external environmental cues, principally temperature. In the pursuit of understanding this mechanism, especially with respect to aiding adaptation to climate change, a QTL at the top of linkage group (LG) 9 has been identified by many studies on budbreak, but the genes underlying it remain elusive. Here, together with a dessert apple core collection of 239 cultivars, we used a targeted capture sequencing approach to increase SNP resolution in apple orthologues of known or suspected A. thaliana flowering time-related genes, as well as approximately 200 genes within the LG9 QTL interval. This increased the 275 223 SNP Axiom® Apple 480 K array dataset by an additional 40 857 markers. Robust GWAS analyses identified MdPRX10, a peroxidase superfamily gene, as a strong candidate that demonstrated a dormancy-related expression pattern and down-regulation in response to chilling. In-silico analyses also predicted the residue change resulting from the SNP allele associated with late budbreak could alter protein conformation and likely function. Late budbreak cultivars homozygous for this SNP allele also showed significantly up-regulated expression of C-REPEAT BINDING FACTOR (CBF) genes, which are involved in cold tolerance and perception, compared to reference cultivars, such as Gala. Taken together, these results indicate a role for MdPRX10 in budbreak, potentially via redox-mediated signaling and CBF gene regulation. Moving forward, this provides a focus for developing our understanding of the effects of temperature on flowering time and how redox processes may influence integration of external cues in dormancy pathways

Perfil transcricional de genes relacionados à dormência em gemas de macieira

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado que possui grande importância econômica mundialmente, sendo sua produtividade intimamente relacionada à saída do processo de dormência hibernal. Este processo pode ser definido como a incapacidade da planta iniciar o crescimento meristemático mesmo sob condições favoráveis e os mecanismos de controle molecular da dormência em macieiras ainda são pouco compreendidos. O objetivo do presente trabalho foi investigar o perfil gênico diferencial entre cultivares de macieiras contrastantes para requerimento de frio. As cultivares selecionadas foram Gala e sua mutante espontânea Castel Gala, as quais apresentam alto e baixo requerimento de frio, respectivamente. A técnica de hibridização supressiva subtrativa (SSH) permitiu a identificação de 28 genes candidatos à regulação da dormência. Análises de RT-qPCR foram realizadas visando a validação da expressão diferencial dos genes selecionados, assim como caracterizá-los transcricionalmente em três cultivares distintas durante um ciclo de crescimento e de dormência. Dos 28 genes candidatos, 17 apresentaram o mesmo perfil diferencial identificado por SSH. Um acúmulo sazonal de transcritos durante o inverno foi identificado para alguns genes e as cultivares de maior requerimento de frio apresentaram acúmulo de transcritos por mais tempo. Este perfil permitiu-nos sugerir que estes genes podem estar atuando na regulação dos processos de dormência e de aclimatação ao frio. Dos 17 genes validados, aqueles codificadores de proteínas DAM, desidrinas, GAST1, LTI65, NAC, histonas variantes H2A.Z e RAP2.12 apresentaram os maiores contrastes transcricionais entre as cultivares analisadas durante o inverno e constituem-se como fortes candidatos a participantes do processo de progressão da dormência em macieiras. Finalmente, a família de genes codificadores de desidrinas de macieira teve seus membros identificados e caracterizados transcricionalmente. Análises in silico permitiram a identificação de oito modelos gênicos preditos de desidrinas no genoma de macieira. As cadeias peptídicas deduzidas foram classificadas conforme a presença dos segmentos conservados YnSKn. Um perfil sazonal de regulação da expressão foi identificado, com a presença de um pico de acúmulo de transcritos durante o inverno, o que sugere a presença de um mecanismo similar de regulação entre genes de desidrinas de macieira.Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related with the release from a bud dormancy process. This process is defined as the plant inability to initiate growth from meristems under favorable conditions and molecular information about dormancy control in apple trees is limited. The aim of the present work was to investigate the differential gene expression profiles between apple tree cultivars contrasting in chilling requirement for breaking dormancy. The selected apple cultivars were Gala and its derived bud sport Castel Gala, which displays high and low chilling requirement, respectively. A suppression subtractive hybridization (SSH) assay yielded 28 candidate genes putatively associated to dormancy cycling. RT-qPCR analyses were performed in order to validate the differential expression profiles and also to transcriptionally characterize the selected genes in three distinct apple tree cultivars during a growth to dormancy cycle. Among the 28 candidate genes, 17 confirmed the differential expression profile predicted by SSH. A seasonal transcript accumulation during the winter was identified to some genes, with high chilling requirement cultivars presenting higher levels of transcripts. This profile allowed us to suggest that these genes may be acting on dormancy regulation and cold acclimation. Out of the 17 candidate genes, those coding for DAM, dehydrins, GAST1, LTI65, NAC, histone variants H2A.Z and RAP2.12 displayed major differences in gene expression between cultivars through the winter and are strong candidates to play key roles on dormancy progression in apple trees. Finally, we identified and transcriptionally characterized the dehydrin gene family in apple trees. In silico analyses allowed us to identify eight predicted gene models for dehydrins in the apple genome. Deduced polypeptides were classified according to the presence of the conserved YnSKn segments. A seasonal regulation of gene expression was observed, with higher transcript accumulation during the winter. This data suggests that a similar mechanism of transcript regulation is acting through the apple dehydrin genes

Análise funcional e potencial biotecnológico de desidrinas e galactinol sintases de macieira

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado de grande importância econômica, e sua produtividade está diretamente relacionada à dormência. Além dos genes responsáveis pelo controle molecular, uma série de proteínas e metabólitos também é recrutada para proteger a integridade da gema dormente, destacando-se as desidrinas (DHN) e as enzimas galactinol sintases (GolS). As DHNs são proteínas que atuam na resposta adaptativa vegetal a estresses abióticos, enquanto que GolS são enzimas responsáveis pela síntese de galactinol, essencial à síntese de oligossacarídeos da família da rafinose (RFOs), os quais se acumulam em resposta a estresses abióticos. O objetivo do presente trabalho foi explorar a adaptação das gemas a condições de estresse a que são submetidas na dormência, visando identificar genes com potencial uso biotecnológico. Para tal, foram identificados e caracterizados os genes codificadores de DHNs e GolS no genoma da macieira por meio da utilização de ferramentas in silico para estudar a evolução, experimentos a campo e sob condições controladas, análises de expressão, localização subcelular, e geração de plantas transgênicas. As análises evolutivas sugerem que eventos de duplicação do genoma inteiro (WGD) foram responsáveis por moldar a evolução e diversificação dos genes GolS em macieira, enquanto que no caso das DHN eventos de duplicação em tandem e WGD nortearam a sua evolução. Nossos resultados sugerem que DHNs, galactinol e rafinose integram uma série de mecanismos que agem em conjunto durante a dormência a fim de proteger a integridade da gema, além dos carboidratos constituírem uma fonte de energia para a brotação. Ao longo da evolução, o aparecimento de novas estruturas e programas de desenvolvimento, tais como a gema e a dormência, necessitaram de adaptação de vias moleculares já estabelecidas, o que ajuda a explicar por que as dormências de gemas e de sementes compartilham rotas moleculares comuns. Finalmente, o gene MdDHN11 foi funcionalmente caracterizado e nossos resultados fornecem evidências de que MdDHN11 desempenha importantes papéis durante o desenvolvimento da semente de maçã, protegendo o embrião e o endosperma de alterações no status da água. Além disso, apenas a planta superexpressando MdDHN11 sobreviveu ao ensaio de simulação de seca, confirmando o potencial uso biotecnológico de DHNs de macieira no aumento da tolerância ao déficit hídrico.Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related to bud dormancy. Besides genes responsible for the molecular control of the process, a number of proteins and metabolites are also recruited to protect bud integrity, such as dehydrins (DHN) and galactinol synthases (GolS). DHNs are proteins that act on plant adaptive responses to abiotic stresses, while GolS are enzymes that catalyze for the synthesis of galactinol, an essential carbohydrate in the synthesis of raffinose family oligosaccharides (RFOs), which also accumulate in response to abiotic stresses. The objective of this work was to explore bud adaptation to stress conditions that occur during dormancy to identify genes with potential biotechnological applications. DHN and GolS genes were identified and characterized in the apple genome employing in silico tools, experiments under field and controlled conditions, expression analysis, subcellular localization assays, and the generation of transgenic plants. Evolutionary analyses suggest that whole genome duplication (WGD) events were responsible for shaping the evolution and diversification of GolS genes in apple, whereas WGD and tandem duplication events could be held accountable for DHN evolution. Our results suggest that DHNs, galactinol and raffinose integrate a series of mechanisms that act together during dormancy in order to protect bud integrity, besides the carbohydrates being an energy source for budbreak. During evolution, the appearance of new structures and developmental programs, such as buds and dormancy, required the adaptation of already established molecular pathways, partially explaining why bud and seed dormancy share common pathways. Finally, the MdDHN11 gene has been functionally characterized and our results provide evidences that MdDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from water deficit. Moreover, only the plant overexpressing MdDHN11 survived the water withholding assay, confirming the potential biotechnological use of apple DHNs in increasing tolerance to drought

Análise funcional e potencial biotecnológico de desidrinas e galactinol sintases de macieira

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado de grande importância econômica, e sua produtividade está diretamente relacionada à dormência. Além dos genes responsáveis pelo controle molecular, uma série de proteínas e metabólitos também é recrutada para proteger a integridade da gema dormente, destacando-se as desidrinas (DHN) e as enzimas galactinol sintases (GolS). As DHNs são proteínas que atuam na resposta adaptativa vegetal a estresses abióticos, enquanto que GolS são enzimas responsáveis pela síntese de galactinol, essencial à síntese de oligossacarídeos da família da rafinose (RFOs), os quais se acumulam em resposta a estresses abióticos. O objetivo do presente trabalho foi explorar a adaptação das gemas a condições de estresse a que são submetidas na dormência, visando identificar genes com potencial uso biotecnológico. Para tal, foram identificados e caracterizados os genes codificadores de DHNs e GolS no genoma da macieira por meio da utilização de ferramentas in silico para estudar a evolução, experimentos a campo e sob condições controladas, análises de expressão, localização subcelular, e geração de plantas transgênicas. As análises evolutivas sugerem que eventos de duplicação do genoma inteiro (WGD) foram responsáveis por moldar a evolução e diversificação dos genes GolS em macieira, enquanto que no caso das DHN eventos de duplicação em tandem e WGD nortearam a sua evolução. Nossos resultados sugerem que DHNs, galactinol e rafinose integram uma série de mecanismos que agem em conjunto durante a dormência a fim de proteger a integridade da gema, além dos carboidratos constituírem uma fonte de energia para a brotação. Ao longo da evolução, o aparecimento de novas estruturas e programas de desenvolvimento, tais como a gema e a dormência, necessitaram de adaptação de vias moleculares já estabelecidas, o que ajuda a explicar por que as dormências de gemas e de sementes compartilham rotas moleculares comuns. Finalmente, o gene MdDHN11 foi funcionalmente caracterizado e nossos resultados fornecem evidências de que MdDHN11 desempenha importantes papéis durante o desenvolvimento da semente de maçã, protegendo o embrião e o endosperma de alterações no status da água. Além disso, apenas a planta superexpressando MdDHN11 sobreviveu ao ensaio de simulação de seca, confirmando o potencial uso biotecnológico de DHNs de macieira no aumento da tolerância ao déficit hídrico.Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related to bud dormancy. Besides genes responsible for the molecular control of the process, a number of proteins and metabolites are also recruited to protect bud integrity, such as dehydrins (DHN) and galactinol synthases (GolS). DHNs are proteins that act on plant adaptive responses to abiotic stresses, while GolS are enzymes that catalyze for the synthesis of galactinol, an essential carbohydrate in the synthesis of raffinose family oligosaccharides (RFOs), which also accumulate in response to abiotic stresses. The objective of this work was to explore bud adaptation to stress conditions that occur during dormancy to identify genes with potential biotechnological applications. DHN and GolS genes were identified and characterized in the apple genome employing in silico tools, experiments under field and controlled conditions, expression analysis, subcellular localization assays, and the generation of transgenic plants. Evolutionary analyses suggest that whole genome duplication (WGD) events were responsible for shaping the evolution and diversification of GolS genes in apple, whereas WGD and tandem duplication events could be held accountable for DHN evolution. Our results suggest that DHNs, galactinol and raffinose integrate a series of mechanisms that act together during dormancy in order to protect bud integrity, besides the carbohydrates being an energy source for budbreak. During evolution, the appearance of new structures and developmental programs, such as buds and dormancy, required the adaptation of already established molecular pathways, partially explaining why bud and seed dormancy share common pathways. Finally, the MdDHN11 gene has been functionally characterized and our results provide evidences that MdDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from water deficit. Moreover, only the plant overexpressing MdDHN11 survived the water withholding assay, confirming the potential biotechnological use of apple DHNs in increasing tolerance to drought

Perfil transcricional de genes relacionados à dormência em gemas de macieira

A macieira (Malus x domestica Borkh.) é uma frutífera de clima temperado que possui grande importância econômica mundialmente, sendo sua produtividade intimamente relacionada à saída do processo de dormência hibernal. Este processo pode ser definido como a incapacidade da planta iniciar o crescimento meristemático mesmo sob condições favoráveis e os mecanismos de controle molecular da dormência em macieiras ainda são pouco compreendidos. O objetivo do presente trabalho foi investigar o perfil gênico diferencial entre cultivares de macieiras contrastantes para requerimento de frio. As cultivares selecionadas foram Gala e sua mutante espontânea Castel Gala, as quais apresentam alto e baixo requerimento de frio, respectivamente. A técnica de hibridização supressiva subtrativa (SSH) permitiu a identificação de 28 genes candidatos à regulação da dormência. Análises de RT-qPCR foram realizadas visando a validação da expressão diferencial dos genes selecionados, assim como caracterizá-los transcricionalmente em três cultivares distintas durante um ciclo de crescimento e de dormência. Dos 28 genes candidatos, 17 apresentaram o mesmo perfil diferencial identificado por SSH. Um acúmulo sazonal de transcritos durante o inverno foi identificado para alguns genes e as cultivares de maior requerimento de frio apresentaram acúmulo de transcritos por mais tempo. Este perfil permitiu-nos sugerir que estes genes podem estar atuando na regulação dos processos de dormência e de aclimatação ao frio. Dos 17 genes validados, aqueles codificadores de proteínas DAM, desidrinas, GAST1, LTI65, NAC, histonas variantes H2A.Z e RAP2.12 apresentaram os maiores contrastes transcricionais entre as cultivares analisadas durante o inverno e constituem-se como fortes candidatos a participantes do processo de progressão da dormência em macieiras. Finalmente, a família de genes codificadores de desidrinas de macieira teve seus membros identificados e caracterizados transcricionalmente. Análises in silico permitiram a identificação de oito modelos gênicos preditos de desidrinas no genoma de macieira. As cadeias peptídicas deduzidas foram classificadas conforme a presença dos segmentos conservados YnSKn. Um perfil sazonal de regulação da expressão foi identificado, com a presença de um pico de acúmulo de transcritos durante o inverno, o que sugere a presença de um mecanismo similar de regulação entre genes de desidrinas de macieira.Apple tree (Malus x domestica Borkh.) is a temperate fruit crop of great economic importance worldwide and its productivity is related with the release from a bud dormancy process. This process is defined as the plant inability to initiate growth from meristems under favorable conditions and molecular information about dormancy control in apple trees is limited. The aim of the present work was to investigate the differential gene expression profiles between apple tree cultivars contrasting in chilling requirement for breaking dormancy. The selected apple cultivars were Gala and its derived bud sport Castel Gala, which displays high and low chilling requirement, respectively. A suppression subtractive hybridization (SSH) assay yielded 28 candidate genes putatively associated to dormancy cycling. RT-qPCR analyses were performed in order to validate the differential expression profiles and also to transcriptionally characterize the selected genes in three distinct apple tree cultivars during a growth to dormancy cycle. Among the 28 candidate genes, 17 confirmed the differential expression profile predicted by SSH. A seasonal transcript accumulation during the winter was identified to some genes, with high chilling requirement cultivars presenting higher levels of transcripts. This profile allowed us to suggest that these genes may be acting on dormancy regulation and cold acclimation. Out of the 17 candidate genes, those coding for DAM, dehydrins, GAST1, LTI65, NAC, histone variants H2A.Z and RAP2.12 displayed major differences in gene expression between cultivars through the winter and are strong candidates to play key roles on dormancy progression in apple trees. Finally, we identified and transcriptionally characterized the dehydrin gene family in apple trees. In silico analyses allowed us to identify eight predicted gene models for dehydrins in the apple genome. Deduced polypeptides were classified according to the presence of the conserved YnSKn segments. A seasonal regulation of gene expression was observed, with higher transcript accumulation during the winter. This data suggests that a similar mechanism of transcript regulation is acting through the apple dehydrin genes

The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

The floral transition occurs at the shoot apical meristem (SAM) in response to favourable external and internal signals. Among these signals, variations in daylength (photoperiod) act as robust seasonal cues to activate flowering. In Arabidopsis, long-day photoperiods stimulate production in the leaf vasculature of a systemic florigenic signal that is translocated to the SAM. According to the current model, FLOWERING LOCUS T (FT), the main Arabidopsis florigen, causes transcriptional reprogramming at the SAM, so that lateral primordia eventually acquire floral identity. FT functions as a transcriptional coregulator with the bZIP transcription factor FD, which binds DNA at specific promoters. FD can also interact with TERMINAL FLOWER 1 (TFL1), a protein related to FT that acts as a floral repressor. Thus, the balance between FT-TFL1 at the SAM influences the expression levels of floral genes targeted by FD. Here, we show that the FD-related bZIP transcription factor AREB3, which was previously studied in the context of phytohormone abscisic acid signalling, is expressed at the SAM in a spatio-temporal pattern that strongly overlaps with FD and contributes to FT signalling. Mutant analyses demonstrate that AREB3 relays FT signals redundantly with FD, and the presence of a conserved carboxy-terminal SAP motif is required for downstream signalling. AREB3 shows unique and common patterns of expression with FD, and AREB3 expression levels are negatively regulated by FD thus forming a compensatory feedback loop. Mutations in another bZIP, FDP, further aggravate the late flowering phenotypes of fd areb3 mutants. Therefore, multiple florigen-interacting bZIP transcription factors have redundant functions in flowering at the SAM

Correction: The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

[This corrects the article DOI: 10.1371/journal.pgen.1010766.]

Unraveling the role of MADS transcription factor complexes in apple tree dormancy

International audienceA group of MADS transcription factors (TFs) are believed to control temperature-mediated bud dormancy. These TFs, called DORMANCY-ASSOCIATED MADS-BOX (DAM), are encoded by genes similar to SHORT VEGETATIVE PHASE (SVP) from Arabidopsis. MADS proteins form transcriptional complexes whose combinatory composition defines their molecular function. However, how MADS multimeric complexes control the dormancy cycle in trees is unclear. Apple MdDAM and other dormancy-related MADS proteins form complexes with MdSVPa, which is essential for the ability of transcriptional complexes to bind to DNA. Sequential DNA-affinity purification sequencing (seq-DAP-seq) was performed to identify the genome-wide binding sites of apple MADS TF complexes. Target genes associated with the binding sites were identified by combining seq-DAP-seq data with transcriptomics datasets obtained using a glucocorticoid receptor fusion system, and RNA-seq data related to apple dormancy. We describe a gene regulatory network (GRN) formed by MdSVPa-containing complexes, which regulate the dormancy cycle in response to environmental cues and hormonal signaling pathways. Additionally, novel molecular evidence regarding the evolutionary functional segregation between DAM and SVP proteins in the Rosaceae is presented. MdSVPa sequentially forms complexes with the MADS TFs that predominate at each dormancy phase, altering its DNA-binding specificity and, therefore, the transcriptional regulation of its target genes

Spring is coming: genetic analyses of the bud break date locus reveal candidate genes from the cold perception pathway to dormancy release in apple (Malus x domestica Borkh.)

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Evolutionary diversification of galactinol synthases in Rosaceae: adaptive roles of galactinol and raffinose during apple bud dormancy

International audienceGalactinol synthase (GolS) is a key enzyme in the biosynthetic pathway of raffinose family oligosaccharides (RFOs), which play roles in carbon storage, signal transduction, and osmoprotection. The present work assessed the evolutionary history of GolS genes across the Rosaceae using several bioinformatic tools. Apple (Malus x domestica) GolS genes were transcriptionally characterized during bud dormancy, in parallel with galactinol and raffinose measurements. Additionally, MdGolS2, a candidate to regulate seasonal galactinol and RFO content during apple bud dormancy, was functionally characterized in Arabidopsis. Evolutionary analyses revealed that whole genome duplications have driven GolS gene evolution and diversification in Rosaceae speciation. The strong purifying selection identified in duplicated GolS genes suggests that differential gene expression might define gene function better than protein structure. Interestingly, MdGolS2 was differentially expressed during bud dormancy, concomitantly with the highest galactinol and raffinose levels. One of the intrinsic adaptive features of bud dormancy is limited availability of free water; therefore, we generated transgenic Arabidopsis plants expressing MdGolS2. They showed higher galactinol and raffinose contents and increased tolerance to water deficit. Our results suggest that MdGolS2 is the major GolS responsible for RFO accumulation during apple dormancy, and these carbohydrates help to protect dormant buds against limited water supply