Genome-wide identification, phylogeny, and gene duplication of the epigenetic regulators in Fagaceae

Epigenetic regulators are proteins involved in controlling gene expression. Informationabout the epigenetic regulators within the Fagaceae, a relevant family of trees andshrubs of the northern hemisphere ecosystems, is scarce. With the intent to character-ize these proteins in Fagaceae, we searched for orthologs of DNA methyltransferases(DNMTs) and demethylases (DDMEs) and Histone modifiers involved in acetylation(HATs), deacetylation (HDACs), methylation (HMTs), and demethylation (HDMTs) inFagus,Quercus,andCastaneagenera. Blast searches were performed in the availablegenomes, and freely available RNA-seq data were used to de novo assemble transcrip-tomes. We identified homologs of seven DNMTs, three DDMEs, six HATs, 11 HDACs,32 HMTs, and 21 HDMTs proteins. Protein analysis showed that most of them havethe putative characteristic domains found in these protein families, which suggeststheir conserved function. Additionally, to elucidate the evolutionary history of thesegenes within Fagaceae, paralogs were identified, and phylogenetic analyses were per-formed with DNA and histone modifiers. We detected duplication events in all speciesanalyzed with higher frequency inQuercusandCastaneaand discuss the evidence oftransposable elements adjacent to paralogs and their involvement in gene duplication.The knowledge gathered from this work is a steppingstone to upcoming studies con-cerning epigenetic regulation in this economically important family of Fagaceaeinfo:eu-repo/semantics/publishedVersio

The dynamics of flower development in Castanea sativa Mill

The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean tree species, being an important natural resource for the wood and fruit industries. It is a monoecious species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study of how the different flowers of C. sativa develop is fundamental to understand the reproductive success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis also suggested that changes in the canonical ABCDE protein–protein interactions may underlie the mechanisms necessary to the development of separate male and female flowers, as reported for the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the understanding of the molecular mechanisms involved in unisexual flower development in C. sativa, also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in the predominantly monoecious Fagaceae family.This work was funded by FCT/COMPETE/FEDER with the project grant POCI-01-0145- FEDER-027980/PTDC/ASP-SIL/27980/2017—“FlowerCAST—Characterisation of genetic and environmental determinants involved in reproductive development of Castanea sativa”. A.T.A. and S.A. were supported by FCT with PhD grants (ref. SFRH/BD/136834/2018 and SFRH/BD/146660/2019, respectively)

Study of flower induction and fructification mechanisms in Castanea sativa Mill

Tese de doutoramento em BiologiaThe European chestnut (Castanea sativa Mill.) is a species from the Fagaceae family that constitutes the basis of an important agroforestry system in several Mediterranean countries, being a pivotal economic resource due to its seed production. However, information regarding the molecular mechanisms underlying the life cycle of this plant is limited. Crucial events for a successful fructification, such as dormancy induction and break, flowering induction and development of unisexual male catkins and bisexual catkins (presenting male and female flowers), have not been yet molecularly characterised. Thus, the main goal of this thesis is to gather information and trace a molecular profile of the mechanisms that regulate the different stages of the life cycle of the chestnut trees. This work has provided, as far as we know, the first de novo transcriptome for C. sativa, which might be used as a platform for future studies regarding this species. As floral development is pivotal for fruit production, the molecular mechanisms that lead to development of inflorescences of separate sexes in this species were studied. Differential gene expression analysis of the homologs of homeotic genes of the ABCDE model, as well as identification of novel protein-protein interactions, suggests that the development of separate flowers in chestnut might be due to alterations in the dynamics of the ABCDE model. The mechanisms that lead to flowering induction were studied by determining the expression of putative flowering inducers during several consecutive years, in two orchards with contrasting altitudes. It was possible to determine that flowering induction occurs in summer, in the year prior to flowering, promoted by expression of CsaFLOWERING LOCUS T (CsaFT). An extensive transcriptomic analysis from buds from one growth cycle allowed the identification of genetic hubs that may be involved in the regulation of flowering, as well as the different dormancy stages and their transitions. The data obtained in this thesis constitute an advance in the comprehension of the molecular mechanisms involved in the development of unisexual flowers and the dormancy cycle of C. sativa, and may complement future studies in this species and in other Fagaceae with similar reproductive characteristics.O castanheiro europeu (Castanea sativa Mill.) é uma espécie da família Fagaceae que constitui a base de um importante sistema agroflorestal em vários países do Mediterrâneo, sendo um importante recurso económico graças à produção das suas sementes. No entanto, a informação relativa aos mecanismos genéticos envolvidos no ciclo de vida desta planta é escassa. Eventos fundamentais para uma frutificação bem-sucedida, como indução e quebra de dormência, indução de floração e desenvolvimento de amentilhos masculinos unissexuais e amentilhos bissexuais (que apresentam flores masculinas e femininas), ainda não foram caracterizados molecularmente. Como tal, o principal objetivo desta tese é obter informação e traçar um perfil molecular dos mecanismos que regulam as várias etapas do ciclo de vida do castanheiro. Este trabalho proporcionou, tanto quanto sabemos, o primeiro transcriptoma de novo de C. sativa, que poderá ser utilizado como plataforma para futuros estudos que envolvam esta espécie. Sendo o desenvolvimento floral fundamental para produção de frutos, foram estudados os mecanismos moleculares que levam ao desenvolvimento de inflorescências de diferentes sexos nesta espécie. A análise da expressão diferencial nos diferentes tipos de flores de homólogos de genes homeóticos do modelo ABCDE, aliada à identificação de novas interações proteína-proteína, sugere que o desenvolvimento de flores unissexuais no castanheiro se poderá dever a alterações nas dinâmicas do modelo ABCDE. Os mecanismos que levam à indução de floração em castanheiro foram estudados com recurso à determinação da expressão de putativos indutores florais durante vários anos consecutivos, em dois soutos localizados a diferentes altitudes. Foi possível determinar que o período de indução floral ocorre no verão do ano anterior à floração, promovido pela expressão CsaFLOWERING LOCUS T (CsaFT). Uma extensa análise transcriptómica de gomos de um ciclo de crescimento permitiu a determinação de redes genéticas que poderão estar envolvidas na regulação da floração e das diferentes etapas da dormência e suas transições. Os dados obtidos nesta tese constituem um avanço na compreensão dos mecanismos moleculares envolvidos no desenvolvimento de flores unissexuais e no ciclo de dormência de C. sativa, e poderão complementar estudos futuros nesta espécie e em outras Fagaceae com características reprodutivas semelhantes

The role of NEP-TC in the somatic embryogenesis of tamarillo (Solanum betaceum)

ALHINHO, Ana Teresa Talhinhas Barata - The role of NEP-TC in the somatic embryogenesis of tamarillo (Solanum betaceum). Coimbra : [s.n.], 2016. Dissertação de Mestrado em Biotecnologia Vegetal e Biodiversidade.O tamarilho (Solanum betaceum Cav.) é uma árvore de pequeno porte da família das solanáceas, conhecido pelos seus frutos comestíveis e nutritivos. O estudo desta espécie é bastante útil na compreensão do processo de indução de embriogénese somática (ES), uma importante ferramenta para clonagem de plantas e sua propagação in vitro. Durante este processo, tanto calos embriogénicos (CE) como não-embriogénicos (CNE) surgem no mesmo explante, num meio de cultura com reguladores de crescimento vegetal (picloram ou ácido 2,4-diclorofenoxiacetico). A NEP-TC é uma proteína expressa de forma consistente em calos não-embriogénicos de tamarilho, tendo sido associada à família SpoU de metiltransferases de RNA dependentes de SAM por comparação bioinformática com sequencias homólogas presentes na planta modelo Arabidopsis thaliana. O presente estudo tem como objetivo a elucidação do papel da NEP-TC na embriogénese somática de tamarilho, mas também fazer uma caracterização extensiva desta proteína, através da realização de ensaios de atividade específica para avaliação da ação como metiltransferase da NEP-TC recombinante expressa em Escherichia coli, assim como estudos de imunohistoquímica para determinação dos locais de expressão da proteína em tecidos embriogénicos e não-embriogénicos. Foram também realizados ensaios de bioinformática para determinação da filogenia e estrutura desta proteína. Após expressão da proteína num sistema heterólogo, os resultados de atividade mostram que a NEP-TC apresenta de facto afinidade para com RNA, não apresentando taxas de atividade em relação a DNA, confirmando desta forma estudos de bioinformática realizados previamente. Analisando a afinidade da proteína relativamente a RNA ribossomal (rRNA), esta apresenta afinidade para com este substrato. Quanto àimunolocalização, a NEP-TC mostra expressão diferencial, com uma presença consistente em tecidos não embriogénicos, e uma manifestação menos distinta em tecidos embriogénicos e tecidos em diferenciação. A análise bioinformática mostra que esta proteína pertence a uma família de genes com apenas um gene que sofreu alterações ao longo do tempo, originando os 38 genes presentes na árvore.Tamarillo (Solanum betaceum Cav.) is a small solanaceous tree known for its edible and nutritious fruits. This species is extremely useful for understanding the process of induction of somatic embryogenesis (SE), an important tool for plant cloning and in vitro propagation. During this process, both embryogenic (EC) and non-embryogenic (NEC) calli arise from the same explant, in a medium with plant growth regulators (picloram or 2,4-dichlorophenoxyacetic acid). NEP-TC (26.5 kDa) is a protein identified as being consistently present in tamarillo’s non-embryogenic calli, having been associated with the SpoU SAM-dependent RNA methyltransferase family, by bioinformatic comparison with homologous sequences found in the model plant Arabidopsis thaliana. The present study aims to elucidate the role of NEP-TC on tamarillo somatic embryogenesis, as well as perform an extensive characterization of the protein, by activity tests to evaluate the methyltransferase activity of recombinant NEP-TC expressed in Escherichia coli and immunohistochemical studies for the localization of NEP-TC on both embryogenic and non-embryogenic calli. Bioinformatic studies were also performed to determine the structure and phylogeny of this protein. After successful protein expression on a heterologous system, specific activity results show that NEP-TC has affinity towards RNA, while showing no activity rates towards DNA, thus confirming previous bioinformatic studies performed. Analyzing the protein’s affinity towards ribosomal RNA (rRNA), it is clear that the protein has affinity towards this substrate. When it comes to immunolocalization, NEP-TC shows a differential expression, with a consistent presence in peripheral areas of nonembryogenic tissues and a less distinct manifestation in embryogenic tissues and cells inthe process of differentiation. Bioinformatic analysis shows that this protein belongs to a gene family with only one gene that has been suffering alterations over time originating the 38 genes present in the genome of this tree

Vineyard calcium sprays induce changes in grape berry skin, firmness, cell wall composition and expression of cell wall-related genes

Supplementary data to this article can be found online at https://doi.org/10.1016/j.plaphy.2020.02.033.Having a central role in cell wall pectin cross-linking, calcium has been increasingly used as supplement to promote fruit firmness and extended shelf-life. However, the molecular rearrangements associated to increased fruit robustness are still a matter of debate. In this study, mechanical, histochemical and molecular assays were conducted to understand the mechanisms underlying the effects of Ca in fruit physical properties. In a two-year field trial, grapevines were sprayed with exogenous CaCl2 throughout the fruiting season. Results showed an increase in berry Ca concentration at harvest, associated to increased fruit consistency and skin resistance. Scanning electron microscopy showed that fruits from Ca-treated plants had smoother skin surfaces than control fruits, and that microcracks encircling the lenticels were less prominent. Histochemistry assays suggested higher deposition of pectin-like material in skin cell walls in grapes from Ca-treated vines, but no evident modifications in cellulose content were observed. Accordingly, the expression of cellulose synthase family gene CesA3 was not affected by exogenous Ca, while polygalacturonase-encoding genes PG1 and PG2 were downregulated, together with EXP6 belonging to expansin family, and CER9 and CYP15 involved in cuticle biosynthesis. These results suggested that Ca acts by inhibiting pectin degradation and cell wall loosening, while remodeling cuticle structure.This work was supported by the Portuguese Foundation for Science and Technology (FCT/MCTES/PIDDAC) in the framework of the strategic funding [UID/BIA/04050/2019, UID/AGR/04033/2019, UID/FIS/04650/2013 - 2019, PEst-OE/BIA/UI4046/2014; UID/MULTI/04046/2013] and the projects [PTDC/AGR-PRO/7028/2014, PTDCBIA-FBT/28165/2017, PTDC/BIA-FBT/30341/2017, SFRH/BPD/107905/2015 to V.M., SFRH/BPD/110914/2015 to P.C., and SFRH/BD/136834/2018 to A.T.A]. The authors thank Dr António Teixeira (CBMA, University of Minho) for assistance with performing Principal Component Analysis in R software.info:eu-repo/semantics/publishedVersio

Unisexual flower initiation in the monoecious Quercus suber L.: a molecular approach

Several plant species display a temporal separation of the male and female flower organ development to enhance outbreeding; however, little is known regarding the genetic mechanisms controlling this temporal separation. Quercus suber is a monoecious oak tree with accentuated protandry: in late winter, unisexual male flowers emerge adjacent to the swollen buds, whereas unisexual female flowers emerge in the axils of newly formed leaves formed during spring (4-8 weeks after male flowering). Here, a phylogenetic profiling has led to the identification of cork oak homologs of key floral regulatory genes. The role of these cork oak homologs during flower development was identified with functional studies in Arabidopsis thaliana. The expression profile throughout the year of flower regulators (inducers and repressors), in leaves and buds, suggests that the development of male and female flowers may be preceded by separated induction events. Female flowers are most likely induced during the vegetative flush occurring in spring, whereas male flowers may be induced in early summer. Male flowers stay enclosed within the pre-dormant buds, but complete their development before the vegetative flush of the following year, displaying a long period of anthesis that spans the dormant period. Our results portray a genetic mechanism that may explain similar reproductive habits in other monoecious tree species.- This work was funded by FCT/COMPETE/FEDER with the project grants FCOMP-01-0124-FEDER-019461/PTDC/AGR-GPL/118508/2010 `Characterization of Reproductive Development of Quercus suber'. R.S. was supported by funding from FCT with a PhD grant (ref. SFRH/BD/84365/2012). H.G.S. was supported by funding from FCT with a PhD grant (ref. SFRH/BD/111529/2015). M.M.R.C. was supported by funding from FCT with a grant (ref. SFRH/BSAB/113781/2015). Work supported by UIDP/04046/2020 and UIDB/04046/2020 Centre grants from FCT, Portugal to BioISI