Caracterização funcional do gene VviAgl11 durante a morfogênese da semente em videira e seu potencial uso biotecnológico

A ausência de semente em videira, também chamada de apirenia, é amplamente apreciada pelo mercado consumidor de uvas de mesa. No entanto, os mecanismos moleculares que controlam a morfogênese da semente não são totalmente compreendidos. Por este trabalho, buscou-se caracterizar funcionalmente o gene candidato VviAGL11, avaliando seu papel na morfogênese de sementes de Vitis vinifera. Dados prévios permitiram determinar o padrão de expressão de VviAGL11 na camada da endotesta da casca da semente, que precisa alongar e aumentar o número de células para que haja a lignificação e a determinação do tamanho final da semente. No presente estudo, a função de VviAGL11 foi avaliada por meio de sua expressão ectópica no mutante de seedstick (AGL11) de Arabidopsis thaliana, o que restaurou o fenótipo e confirmou o papel direto deste gene no desenvolvimento da semente, sugerindo que a depleção de sua expressão é responsável pelo desenvolvimento errôneo da camada de endotesta da semente, culminando no fenótipo de apirenia típico. Além disso, a função de VviAGL11 foi avaliada em videira com o uso de plasmídeos vegetais. Os resultados permitiram demonstrar que a alta expressão de VviAGL11 na cultivar apirênica Linda, após tratamento, está relacionada com a presença de pequenas sementes que não foram encontradas nas amostras-controle não tratadas Além disso, cachos de ‘Italia’ e ‘Ruby’ tratados com o plasmídeo de silenciamento VviAGL11 mostraram diminuição da expressão desse gene, número reduzido de sementes e aumento do número de traços de sementes. Em conjunto, os resultados confirmam que VviAGL11 é um importante regulador da morfogênese de sementes em videira. Em adição, populações segregantes para ausência de sementes foram testadas com cinco marcadores SSR, dos quais três marcadores microssatélites mostraram-se relacionados à ausência de sementes e poderiam ser usados com 100% de eficiência em um haplótipo para seleção assistida. Ao mesmo tempo, nove marcadores do tipo SNPs e INDELs foram desenvolvidos com base na sequência do alelo de VviAGL11 associado à ausência de sementes em V. vinifera. Para os marcadores VvAGL11_KASP_2, VvAGL11_KASP_3, VvAGL11_KASP_8 e VvAGL11_KASP_9, polimorfismos foram observados segregando em indivíduos apirênicos genotipados, confirmando sua associação com a ausência de sementes e sugerindo seu uso na estratégia de seleção assistida rápida e eficaz de videiras apirênicas.Grapevine seedlessness, also known as apyreny, is widely appreciated by the table grape’s market. Nevertheless, the molecular mechanisms that control seed morphogenesis are not fully understood. This study aimed to characterize the function of the candidate gene VviAGL11, evaluating its role in Vitis vinifera seed morphogenesis. Previous data allowed us to determine the VviAGL11 expression pattern in the endotesta layer of the seed coat, which needs to elongate and increase in cell number to accomplish seed lignification and final seed size. In the present study VviAGL11 function was evaluated by its ectopic expression in Arabidopsis thaliana seedstick (AGL11) mutant background, which restored the phenotype and confirmed the direct role of this gene in seed development, suggesting that depletion of its expression is responsible for the erroneous development of the endotesta layer of the seed, therefore culminating in the typical seedless phenotype. Furthermore, we evaluated VviAGL11 function in grapevine with the use of plant plasmids. The results showed that a high expression of VviAGL11 in the seedless cultivar Linda, after treatment, was related with the presence of small seeds that were not found in untreated control samples Additionally, seeded ‘Italia’ and ‘Ruby’ bunches treated with a VviAGL11-silencing plasmid showed decreased gene expression, reduced number of seeds and increased number of seed traces. Taken together, the results confirm that VviAGL11 is a key master regulator of seed morphogenesis in grapevine. Moreover, segregating populations for seedlessness were tested with five SSR markers of which three microsatellite markers were proven to be related with seedlessness and could be used with 100% efficiency in a haplotype for assisted selection. Additionally, nine unique SNPs and INDELs markers were developed based on VviAGL11 allele associated with the absence of seeds in V. vinifera. For the markers VvAGL11_KASP_2, VvAGL11_KASP_3, VvAGL11_KASP_8 and VvAGL11_KASP_9, polymorphisms were observed segregating in genotyped seedless individuals, confirming their seedlessness association and suggesting their use in fast and effective assisted selection strategy for seedlessness grapevines

Regulation of DNA (de)Methylation Positively Impacts Seed Germination during Seed Development under Heat Stress

Seed development needs the coordination of multiple molecular mechanisms to promote correct tissue development, seed filling, and the acquisition of germination capacity, desiccation tolerance, longevity, and dormancy. Heat stress can negatively impact these processes and upon the increase of global mean temperatures, global food security is threatened. Here, we explored the impact of heat stress on seed physiology, morphology, gene expression, and methylation on three stages of seed development. Notably, Arabidopsis Col-0 plants under heat stress presented a decrease in germination capacity as well as a decrease in longevity. We observed that upon mild stress, gene expression and DNA methylation were moderately affected. Nevertheless, upon severe heat stress during seed development, gene expression was intensively modified, promoting heat stress response mechanisms including the activation of the ABA pathway. By analyzing candidate epigenetic markers using the mutants' physiological assays, we observed that the lack of DNA demethylation by the ROS1 gene impaired seed germination by affecting germination-related gene expression. On the other hand, we also observed that upon severe stress, a large proportion of differentially methylated regions (DMRs) were located in the promoters and gene sequences of germination-related genes. To conclude, our results indicate that DNA (de)methylation could be a key regulatory process to ensure proper seed germination of seeds produced under heat stress

ANNEXIN1 mediates calcium-dependent systemic defense in Arabidopsis plants upon herbivory and wounding.

Funder: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; Id: http://dx.doi.org/10.13039/501100002322Cellular calcium (Ca) transients are endogenous signals involved in local and systemic signaling and defense activation upon environmental stress, including wounding and herbivory. Still, not all Ca2+ channels contributing to the signaling have been identified, nor are their modes of action fully known. Plant annexins are proteins capable of binding to anionic phospholipids and can exhibit Ca channel-like activity. Arabidopsis ANNEXIN1 (ANN1) is suggested to contribute to Ca transport. Here, we report that wounding and simulated-herbivory-induced cytosolic free Ca elevation was impaired in systemic leaves in ann1 loss-of-function plants. We provide evidence for a role of ANN1 in local and systemic defense of plants attacked by herbivorous Spodoptera littoralis larvae. Bioassays identified ANN1 as a positive defense regulator. Spodoptera littoralis feeding on ann1 gained significantly more weight than larvae feeding on wild-type, whereas those feeding on ANN1-overexpressing lines gained less weight. Herbivory and wounding both induced defense-related responses on treated leaves, such as jasmonate accumulation and defense gene expression. These responses remained local and were strongly reduced in systemic leaves in ann1 plants. Our results indicate that ANN1 plays an important role in activation of systemic rather than local defense in plants attacked by herbivorous insects

Caracterização funcional do gene VviAgl11 durante a morfogênese da semente em videira e seu potencial uso biotecnológico

A ausência de semente em videira, também chamada de apirenia, é amplamente apreciada pelo mercado consumidor de uvas de mesa. No entanto, os mecanismos moleculares que controlam a morfogênese da semente não são totalmente compreendidos. Por este trabalho, buscou-se caracterizar funcionalmente o gene candidato VviAGL11, avaliando seu papel na morfogênese de sementes de Vitis vinifera. Dados prévios permitiram determinar o padrão de expressão de VviAGL11 na camada da endotesta da casca da semente, que precisa alongar e aumentar o número de células para que haja a lignificação e a determinação do tamanho final da semente. No presente estudo, a função de VviAGL11 foi avaliada por meio de sua expressão ectópica no mutante de seedstick (AGL11) de Arabidopsis thaliana, o que restaurou o fenótipo e confirmou o papel direto deste gene no desenvolvimento da semente, sugerindo que a depleção de sua expressão é responsável pelo desenvolvimento errôneo da camada de endotesta da semente, culminando no fenótipo de apirenia típico. Além disso, a função de VviAGL11 foi avaliada em videira com o uso de plasmídeos vegetais. Os resultados permitiram demonstrar que a alta expressão de VviAGL11 na cultivar apirênica Linda, após tratamento, está relacionada com a presença de pequenas sementes que não foram encontradas nas amostras-controle não tratadas Além disso, cachos de ‘Italia’ e ‘Ruby’ tratados com o plasmídeo de silenciamento VviAGL11 mostraram diminuição da expressão desse gene, número reduzido de sementes e aumento do número de traços de sementes. Em conjunto, os resultados confirmam que VviAGL11 é um importante regulador da morfogênese de sementes em videira. Em adição, populações segregantes para ausência de sementes foram testadas com cinco marcadores SSR, dos quais três marcadores microssatélites mostraram-se relacionados à ausência de sementes e poderiam ser usados com 100% de eficiência em um haplótipo para seleção assistida. Ao mesmo tempo, nove marcadores do tipo SNPs e INDELs foram desenvolvidos com base na sequência do alelo de VviAGL11 associado à ausência de sementes em V. vinifera. Para os marcadores VvAGL11_KASP_2, VvAGL11_KASP_3, VvAGL11_KASP_8 e VvAGL11_KASP_9, polimorfismos foram observados segregando em indivíduos apirênicos genotipados, confirmando sua associação com a ausência de sementes e sugerindo seu uso na estratégia de seleção assistida rápida e eficaz de videiras apirênicas.Grapevine seedlessness, also known as apyreny, is widely appreciated by the table grape’s market. Nevertheless, the molecular mechanisms that control seed morphogenesis are not fully understood. This study aimed to characterize the function of the candidate gene VviAGL11, evaluating its role in Vitis vinifera seed morphogenesis. Previous data allowed us to determine the VviAGL11 expression pattern in the endotesta layer of the seed coat, which needs to elongate and increase in cell number to accomplish seed lignification and final seed size. In the present study VviAGL11 function was evaluated by its ectopic expression in Arabidopsis thaliana seedstick (AGL11) mutant background, which restored the phenotype and confirmed the direct role of this gene in seed development, suggesting that depletion of its expression is responsible for the erroneous development of the endotesta layer of the seed, therefore culminating in the typical seedless phenotype. Furthermore, we evaluated VviAGL11 function in grapevine with the use of plant plasmids. The results showed that a high expression of VviAGL11 in the seedless cultivar Linda, after treatment, was related with the presence of small seeds that were not found in untreated control samples Additionally, seeded ‘Italia’ and ‘Ruby’ bunches treated with a VviAGL11-silencing plasmid showed decreased gene expression, reduced number of seeds and increased number of seed traces. Taken together, the results confirm that VviAGL11 is a key master regulator of seed morphogenesis in grapevine. Moreover, segregating populations for seedlessness were tested with five SSR markers of which three microsatellite markers were proven to be related with seedlessness and could be used with 100% efficiency in a haplotype for assisted selection. Additionally, nine unique SNPs and INDELs markers were developed based on VviAGL11 allele associated with the absence of seeds in V. vinifera. For the markers VvAGL11_KASP_2, VvAGL11_KASP_3, VvAGL11_KASP_8 and VvAGL11_KASP_9, polymorphisms were observed segregating in genotyped seedless individuals, confirming their seedlessness association and suggesting their use in fast and effective assisted selection strategy for seedlessness grapevines

ESTUDO AUTOECOLÓGICO Aulacoseira granulata EHRENBERG (BACILLARIOPHYTA) EM AMBIENTES LÓTICOS SUBTROPICAIS COM DIFERENTES GRAUS DE PRESERVAÇÃO

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ESTUDO AUTOECOLÓGICO Aulacoseira granulata EHRENBERG (BACILLARIOPHYTA) EM AMBIENTES LÓTICOS SUBTROPICAIS COM DIFERENTES GRAUS DE PRESERVAÇÃO

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Estrutura do gene VvAGL11 e análise da expressão durante a morfogênese da semente de videira

A videira é uma frutífera de origem antiga e atualmente é a mais cultivada do mundo, sendo de extrema importância econômica para diversos países. Certos genótipos de Vitis vinifera L., como a ‘Sultanina’, produzem bagas com apenas traços de sementes. Esta característica, denominada de apirenia, é classificada como estenoespermocarpia uma vez que a fecundação ocorre para a formação do fruto, porém é seguida de aborto do embrião devido à degeneração do endosperma. A apirenia é uma das características mais apreciadas para a uva de mesa e a compreensão dos mecanismos genéticos e moleculares controladores dessa característica é justificada pelo crescimento e pela exigência do mercado de uvas in natura. Estudos anteriores de nosso grupo permitiram identificar o gene VvAGL11 como principal candidato atuante no controle do desenvolvimento de sementes em videira. Foi nosso objetivo, pelo presente trabalho, avaliar o gene VvAGL11 nas cultivares pirênica Chardonnay e apirênica Sultanina, visando uma maior compreensão de seu papel durante a morfogênese da semente. Por meio de sequenciamento alelo-específico, foi possível caracterizar as variações alélicas do gene, o qual apresenta duas isoformas transcritas na cultivar pirênica Chardonnay. Em ‘Sultanina’, uma das isoformas identificadas possui mutações únicas. Pela avaliação do perfil transcricional de VvAGL11 em diferentes estádios de desenvolvimento, da flor e do fruto, foi observado um grande acúmulo de transcritos em sementes de 2, 4 e 6 semanas em comparação com tecidos de flor e polpa. Na cultivar Sultanina, a expressão de VvAGL11 foi extremamente baixa em todos os estádios e tecidos analisados. Análises morfo-anatômicas foram empregadas para comparar a semente e o traço de semente. Foram identificadas alterações anatômicas que sugerem a perda da identidade da camada de integumento médio do traço de semente de Sultanina, uma vez que esta camada não apresentou o padrão de diferenciação normal. O padrão de expressão espaço-temporal de VvAGL11 foi determinado pela técnica de hibridização in situ. Foram utilizadas amostras das cultivares Chardonnay e Sultanina nos mesmos estádios de desenvolvimento analisados por RT-qPCR. Os níveis de transcritos de VvAGL11 aumentaram significativamente em frutos de ‘Chardonnay’ com 2 e 4 semanas de desenvolvimento, especificamente na camada dupla do integumento médio da semente. Em ‘Sultanina’, não houve detecção de transcritos do gene em quaisquer estádios de desenvolvimento.Grapevine have ancient origins and is currently the most widely cultivated fruit in the world, with high economic importance to many countries. Certain genotypes of Vitis vinifera L. like 'Sultanina' produce berries with only seed traces. This characteristic, termed apireny, is classified as stenospermocarpy since fertilization occurs aiming the formation of fruits but it is followed by the abortion of the embryo due to endosperm degeneration. Seedlessness is one of the most appreciated features for table grapes. Thereby the understanding of genetic and molecular mechanisms that control this feature is justified by the growth and the demand of in natura grapes’ market. Our previous studies allowed us to identify a candidate gene, VvAGL11, possibly involved in the control of seed development in grapevine. Our objective with the present study was to evaluate VvAGL11 in a pirenic cultivar, Chardonnay, and in an apirenic cultivar, Sultanina, in order to better understand its role during seed morphogenesis. By allele-specific sequencing, it was possible to characterize the allelic variations of VvAGL11, which exhibited two transcript isoforms in ‘Chardonnay’. In ‘Sultanina’ one of the isoforms was identified with unique mutations. Evaluation of transcriptional profiles of VvAGL11 during different developmental stages, from flowers to mature fruits, it showed a large accumulation of transcripts in seeds of 2, 4 and 6 weeks old compared to flower and pulp tissues in the Chardonnay cultivar. In ‘Sultanina’ the expression of VvAGL11 was extremely low in all stages and tissues examined. Anato-morphological analyzes were performed in order to compare the seed and the seed trace. It was identified a loss of identity of the medium integument layer in the Sultanina seed trace since this layer neither elongated nor doubled in size as it should. The spatial-temporal expression pattern of VvAGL11 was determined by an in situ hybridization technique. Samples of ‘Chardonnay’ and ‘Sultanina’ in the same stages of development analyzed by RT-qPCR were employed. VvAGL11 transcript levels were significantly increased in ‘Chardonnay’ seeds with 2 and 4 weeks of development, specifically in the dual layer medium integument of the seed. In 'Sultanina', gene transcripts were undetectable during all stages of development

Regulation of DNA (de)Methylation Positively Impacts Seed Germination during Seed Development under Heat Stress

Seed development needs the coordination of multiple molecular mechanisms to promote correct tissue development, seed filling, and the acquisition of germination capacity, desiccation tolerance, longevity, and dormancy. Heat stress can negatively impact these processes and upon the increase of global mean temperatures, global food security is threatened. Here, we explored the impact of heat stress on seed physiology, morphology, gene expression, and methylation on three stages of seed development. Notably, Arabidopsis Col-0 plants under heat stress presented a decrease in germination capacity as well as a decrease in longevity. We observed that upon mild stress, gene expression and DNA methylation were moderately affected. Nevertheless, upon severe heat stress during seed development, gene expression was intensively modified, promoting heat stress response mechanisms including the activation of the ABA pathway. By analyzing candidate epigenetic markers using the mutants’ physiological assays, we observed that the lack of DNA demethylation by the ROS1 gene impaired seed germination by affecting germination-related gene expression. On the other hand, we also observed that upon severe stress, a large proportion of differentially methylated regions (DMRs) were located in the promoters and gene sequences of germination-related genes. To conclude, our results indicate that DNA (de)methylation could be a key regulatory process to ensure proper seed germination of seeds produced under heat stress

Improvement of crispr/CASg knock-out and base editing techniques in Apple and Pear

International audienceCRISPR/Cas9 has become the golden technique for gene knock-out in plants. In addition to gene knock-out through the generation of INDELsmutations, base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Our previous results with CRISPR/Cas9 in apple indicated the frequent production of phenotypic and edition chimeras, after edition of theeasily scorable gene phytoene desaturase (PDS). Therefore our rst goal was to determine if adding an adventitious regeneration step from leaves of the T0 plants, would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89 % werehomogeneous albino. Furthermore, the target zone sequences of twelve of these regenerated lines (T1) were studied and compared to the T0 sequences. The results showed that 99% of the T1 alleles were predicted to producing a truncated target protein and that 67% of T1 plants had less heterogeneous editing proles than the T0. This indicates that a regeneration step can efficiently reduce the initial chimerism. The second objective was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: PDS (conferring albino and dwarfphenotype by impaired chlorophyll and gibberellin synthesis) and acetolactate synthase (ALS) (conferring resistance to chlorsulfuron). The two guideRNAs (PDS+ALS) under MdU3 and MdU6 promoters, respectively, were coupled in the pDenCas9_PmCDA1_UGI vector which has a cytidine deaminase fused to a nickase Cas9. Using this cytidine-base editor, we precisely induced DNA substitutions in the target genes, leading to discrete variation in theamino-acid sequence and generating a loss-of-function allele. The successful application of base editing in the apple and pear creates new possibilities for genome engineering to explore desirable agronomic traits in these species