Study of the MADS-box genes expression during floral development in Coffea arabica L.

Orientador: Marcelo Carnier DornelasDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A família dos genes MADS-box codifica fatores de transcrição que atuam como reguladores importantes em muitas etapas no desenvolvimento de diversos organismos. Em plantas, estes genes estão envolvidos na determinação da identidade dos meristemas reprodutivos e dos órgãos florais, bem como no controle de diversos processos durante o desenvolvimento. O presente trabalho teve como objetivo estudar o padrão de expressão dos prováveis ortólogos dos genes do modelo ABC (APETALA1, APETALA3, PISTILATA e AGAMOUS de Arabidopsis thaliana, e do gene TM6 de Solanum lycopersicum) em Coffea arabica L. Estes genes pertencem à família MADS-box e estão relacionados à determinação da identidade dos órgãos florais na planta-modelo A. thaliana. A partir do banco de dados de sequências expressas de cafeeiro (CAFEST), foram identificados 23 possíveis homólogos de genes MADS-box em cafeeiro. Perfis de expressão por RT-PCR indicaram que a maioria destes genes são expressos em flor e fruto. A análise dos dados gerados pelo uso de microscopia óptica e de varredura permitiu estabelecer uma sequência de desenvolvimento para estabelecimento dos órgãos florais em cafeeiro, facilitando a identificação dos locais de expressão dos ortólogos do modelo ABC pela técnica de hibridização in situ. Sendo C. arabica uma espécie relativamente recente e com características peculiares, foi proposto um mecanismo de atuação dos genes do modelo ABC. Dessa forma, os resultados obtidos contribuem para a compreensão do estabelecimento dos órgãos florais em C. arabica. Adicionalmente, pela caracterização de um número elevado de genes da família MADS, foram identificados outros genes potencialmente envolvidos em outros processos de desenvolvimento, que futuramente poderão ser utilizados para incremento da indústria cafeeira.Abstract: The MADS-box gene family encodes transcription factors that act as key regulators in many steps in the development of various organisms. In plants, these genes are involved in determining the identity of reproductive meristems and floral organs as well as in controlling several processes during development. This work aimed to study the expression patterns of putative orthologs of the ABC model genes (APETALA1, APETALA3, AGAMOUS and PISTILATA from Arabidopsis thaliana, and TM6 from Solanum Lycopersicum) in Coffea arabica L. These genes belong to the MADS-box family and are related to the determination of floral organ identity in the model plant A. thaliana. From the CAFEST database of expressed sequence tags, 23 MADS-box gene sequences were identified in coffee. Expression profiles of these genes, determined by RT-PCR, indicated that most of these genes are expressed in flowers and fruits. The analysis of data from optical microscopy and scanning electron microscopy allowed the establishment of a developmental sequence for the establishment of floral organ, facilitating the characterization of the spatial expression patterns of orthologs of the ABC genes by in situ hybridization. A diversified role of conserved genes of the ABC model was proposed for the relatively recent and peculiar specie that is C. arabica. The obtained results aid the understanding of the establishment of floral organs in C. Arabica. Additionally, as many other coffee MADS-box genes were also characterized, other genes, potentially involved in other developmental processes that could be of interest to the industry in the future were also identified.MestradoBiologia VegetalMestre em Biologia Vegeta

Computer Vision-Aided Intelligent Monitoring of Coffee: Towards Sustainable Coffee Production

Coffee which is prepared from the grinded roasted seeds of harvested coffee cherries, is one of the most consumed beverage and traded commodity, globally. To manually monitor the coffee field regularly, and inform about plant and soil health, as well as estimate yield and harvesting time, is labor-intensive, time-consuming and error-prone. Some recent studies have developed sensors for estimating coffee yield at the time of harvest, however a more inclusive and applicable technology to remotely monitor multiple parameters of the field and estimate coffee yield and quality even at pre-harvest stage, was missing. Following precision agriculture approach, we employed machine learning algorithm YOLO, for image processing of coffee plant. In this study, the latest version of the state-of-the-art algorithm YOLOv7 was trained with 324 annotated images followed by its evaluation with 82 unannotated images as test data. Next, as an innovative approach for annotating the training data, we trained K-means models which led to machine-generated color classes of coffee fruit and could thus characterize the informed objects in the image. Finally, we attempted to develop an AI-based handy mobile application which would not only efficiently predict harvest time, estimate coffee yield and quality, but also inform about plant health. Resultantly, the developed model efficiently analyzed the test data with a mean average precision of 0.89. Strikingly, our innovative semi-supervised method with an mean average precision of 0.77 for multi-class mode surpassed the supervised method with mean average precision of only 0.60, leading to faster and more accurate annotation. The mobile application we designed based on the developed code, was named CoffeApp, which possesses multiple features of analyzing fruit from the image taken by phone camera with in field and can thus track fruit ripening in real time

A genome-wide analysis of the RNA-guided silencing pathway in coffee reveals insights into its regulatory mechanisms

microRNAs (miRNAs) are derived from self-complementary hairpin structures, while small-interfering RNAs (siRNAs) are derived from double-stranded RNA (dsRNA) or hairpin precursors. The core mechanism of sRNA production involves DICER-like (DCL) in processing the smallRNAs (sRNAs) and ARGONAUTE (AGO) as effectors of silencing, and siRNA biogenesis also involves action of RNA-Dependent RNA Polymerase (RDR), Pol IV and Pol V in biogenesis. Several other proteins interact with the core proteins to guide sRNA biogenesis, action, and turnover. We aimed to unravel the components and functions of the RNA-guided silencing pathway in a non-model plant species of worldwide economic relevance. The sRNA-guided silencing complex members have been identified in the Coffea canephora genome, and they have been characterized at the structural, functional, and evolutionary levels by computational analyses. Eleven AGO proteins, nine DCL proteins (which include a DCL1-like protein that was not previously annotated), and eight RDR proteins were identified. Another 48 proteins implicated in smallRNA (sRNA) pathways were also identified. Furthermore, we identified 235 miRNA precursors and 317 mature miRNAs from 113 MIR families, and we characterized ccp-MIR156, ccp-MIR172, and ccp-MIR390. Target prediction and gene ontology analyses of 2239 putative targets showed that significant pathways in coffee are targeted by miRNAs. We provide evidence of the expansion of the loci related to sRNA pathways, insights into the activities of these proteins by domain and catalytic site analyses, and gene expression analysis. The number of MIR loci and their targeted pathways highlight the importance of miRNAs in coffee. We identified several roles of sRNAs in C. canephora, which offers substantial insight into better understanding the transcriptional and post-transcriptional regulation of this major crop

Epigenetic regulation of the Qua-Quine Starch neogene during Arabidopsis thaliana development and its impact on starch metabolism

Orientador: Michel Georges Albert VincentzTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Metilação do quinto carbono da citosina do DNA é uma marca epigenética que pode afetar a expressão gênica. A regulação de perfis de metilação é importante para o desenvolvimento normal das plantas sendo crítica para o silenciamento de transposons, imprinting, gametogênese e o desenvolvimento inicial do embrião. Entretanto, como metilações de sequências regulatórias em cis afetam a interação entre o DNA e fatores em trans associados ao desenvolvimento para modular a expressão gênica é ainda pouco compreendida. Qua-Quine Starch (QQS) é um gene órfão de Arabidopsis thaliana que existe sob diversas formas epialélicas estavelmente herdadas e com níveis de expressão correlacionados inversamente com os níveis de metilação em seu promotor e 5¿UTR. Por meio de análises da expressão do gene marcador GUS sob o controle da sequência do promotor e da região 5¿UTR de QQS em linhagens transgênicas de Arabidopsis, inferimos o potencial de expressão de QQS nos vários órgãos e várias fases do desenvolvimento. A atividade GUS foi detectada em meristemas, folhas jovens de roseta e no pólen onde uma reprogramação epigenética deste gene foi descrita. Epialelos contrastantes de QQS apresentaram expressão diferencial ao longo do desenvolvimento. O epialelo QQS metilado (QQSme) possui diferenças marcantes de expressão entre folhas, tecidos da inflorescência e fruto, enquanto o epialelo demetilado Col*3-2 apresenta uma expressão mais homogênea entre esses vários órgãos. Estes resultados indicam que o grau de metilação das sequências regulatórias de QQS interage com fatores associados ao desenvolvimento para estabelecer o perfil de expressão deste gene. A expressão de QQSme aumenta durante o processo de envelhecimento das folhas e foi correlacionada a uma demetilação no lócus. Levantamos a hipótese de que o perfil de metilação de QQSme poderia sofrer uma reprogramação durante o desenvolvimento das folhas a partir do meristema vegetativo. Tal reprogramação poderia ocorrer de maneira ativa, mediada pela DNA glicosilase ROS1, ou passiva, como consequência da ausência ou falha do mecanismo de manutenção do padrão de metilação durante ciclos de replicação celular. De acordo com isso, mostramos que expressão de QQSme é baixa em amostras enriquecidas com células meristemáticas e aumenta gradativamente com a idade das folhas e uma redução dos níveis de metilação de QQSme foi observada em folhas mais velhas. Em alguns órgãos foram encontradas variações nos níveis de expressão de QQS não correlacionadas a variações nos níveis de metilação. Assim, os resultados também mostram que além da metilação outros fatores trans associados ao desenvolvimento são necessários para modular a expressão de QQS. O processo de demetilação de QQSme em folhas velhas não é totalmente dependente de ROS1. Porém, os níveis e padrão de expressão de QQS em mutantes ros1 são diferentes de plantas wild-type e uma análise da expressão de QQS numa população F2 resultante da autofecundação de um híbrido entre wild-type e o mutante ros1-4 revelaram que ROS1 aparentemente regula a expressão global de QQS. Dessa forma, as metilações do DNA parecem atuar como sinalizadores de sítios de ligação para fatores trans e ROS1 pode ser importante para regular e/ou definir estados epialélicos de QQS. Além disso, durante o desenvolvimento reprodutivo a expressão de QQS é detectada no pólen e correlaciona com a demetilação ativa de seu lócus mediada por DEMETER reforçando o fato de que QQS é um alvo de DNA glicosilases. Durante o desenvolvimento vegetativo QQS é descrito como um regulador negativo do metabolismo de amido. De acordo com isso, verificamos que plantas carregando epialelos QQS contrastantes acumulam diferentes quantidades de amidoAbstract: DNA methylation of the fifth carbon of cytosine is an epigenetic mark that may affect gene expression. The regulation of methylation profiles is important for normal plant development being critical for transposon silencing, imprinting, gametogenesis and early embryo development. However, how methylation of cis regulatory sequences affect the interaction between DNA and developmental associated trans factors to modulate gene expression is poorly understood. Qua-Quine Starch (QQS) is an orphan gene of Arabidopsis thaliana which exhibits several epiallelic forms stably-inherited and their expression levels correlate inversely with the methylation levels at the promoter and 5¿UTR. Through the expression analyses of the GUS reporter gene under the control of a QQS promoter sequence and QQS 5¿UTR in Arabidopsis transgenic lines, we inferred the potential of QQS expression in various organs and at several developmental stages. The GUS activity was detected in meristems, young rosette leaves and pollen where an epigenetic reprogramming of this gene has been described. Contrasting QQS epialleles presented different expression patterns during development. The methylated QQS epiallele (QQSme) showed more pronounced expression differences between leaves, inflorescence tissues and fruit, whereas the demethylated Col*3-2 epiallele presented a more homogeneous expression between this various organs. These results indicate that the methylation level of QQS cis regulatory sequence interacts with developmental-related factors to establish the expression profile of this gene. QQSme expression increases during aging of leaves and was correlated with demethylation. We hypothesized that the QQSme methylation profile could be reprogrammed during leaf development starting from the shoot apical meristem. Such process could be controlled by the DNA glycosilase ROS1, or be passive, as a consequence of the absence or failure in the maintenance mechanisms of the methylation pattern during cell replication. Accordingly, we showed that the QQSme expression is low in samples enriched in meristematic cells and increases progressively with leaf aging and reduction of QQSme methylation levels was observed in older leaves. In some organs, QQS expression variations not correlated with variation in the methylation levels were found. Thus, the results indicate that in addition to DNA methylation developmental associated trans factors are also necessary to modulate QQS expression. The demethylation process of QQSme in old leaves is not totally dependent upon ROS1. However, the levels and pattern of QQS expression in ros1 mutants are different from those in wild-type plants and the analysis of a segregating F2 population coming from selfing of a hybrid between the wild-type and ros1-4 mutant revealed that ROS1 regulates the global expression level of QQS. Therefore, DNA methylation seems to act as traffic lights of binding sites to the trans factors and ROS1 may be important to regulate and/or define QQS epiallelic states. Furthermore, during the reproductive development the expression of QQS is detected in the pollen and correlates with the active demethylation of its locus mediated by DEMETER, which reinforces the fact that QQS is a target of DNA glycosilases. During vegetative development QQS has been described as a negative regulator of starch metabolism. We verified that plants carrying contrasting QQS epialleles accumulate different starch quantitiesDoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecular2011/06413-0FAPES

WUSCHEL-related genes are expressed during somatic embryogenesis of the basal angiosperm Ocotea catharinensis Mez. (Lauraceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Ocotea catharinensis is a basal angiosperm and an endangered tree species from the Brazilian Atlantic Rain Forest. Despite its economical and ecological importance, mass-propagation of this species is hampered by seldom-produced short-lived seeds, and in vitro propagation is challenged by frequently malformed somatic embryos. Therefore, O. catharinensis somatic embryos are also a good experimental material to study the physiological and molecular mechanisms underlying in vitro morphogenesis. In an ongoing effort to characterize genes expressed during somatic embryogenesis of O. catharinensis we have cloned two Ocotea WUSCHEL-related genes. According to our RT-PCR data, both genes were preferentially expressed in embryogenic cell aggregates. One of them, OcWUS, is a possible ortholog of the Arabidopsis WUSCHEL (WUS) gene, which codes for a homeodomain-containing protein involved in the specification and maintenance of the shoot apical meristem. We analyzed the expression patterns of OcWUS and OcWOX4 by RT-PCR, and OcWUS expression was also assessed by in situ hybridization. The expression patterns of OcWUS were very similar to those described for the Arabidopsis WUS. OcWUS transcripts were generally restricted to a small group of cells in the center of the putative shoot apical meristem of O. catharinensis somatic embryos. Perturbed expression of OcWUS might be related to abnormally formed somatic embryos of O. catharinensis obtained through tissue culture.262493501Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

WUSCHEL-related genes are expressed during somatic embryogenesis of the basal angiosperm Ocotea catharinensis Mez. (Lauraceae)

Ocotea catharinensis is a basal angiosperm and an endangered tree species from the Brazilian Atlantic Rain Forest. Despite its economical and ecological importance, mass-propagation of this species is hampered by seldom-produced short-lived seeds, and in vitro propagation is challenged by frequently malformed somatic embryos. Therefore, O. catharinensis somatic embryos are also a good experimental material to study the physiological and molecular mechanisms underlying in vitro morphogenesis. In an ongoing effort to characterize genes expressed during somatic embryogenesis of O. catharinensis we have cloned two Ocotea WUSCHEL-related genes. According to our RT-PCR data, both genes were preferentially expressed in embryogenic cell aggregates. One of them, OcWUS, is a possible ortholog of the Arabidopsis WUSCHEL (WUS) gene, which codes for a homeodomain-containing protein involved in the specification and maintenance of the shoot apical meristem. We analyzed the expression patterns of OcWUS and OcWOX4 by RT-PCR, and OcWUS expression was also assessed by in situ hybridization. The expression patterns of OcWUS were very similar to those described for the Arabidopsis WUS. OcWUS transcripts were generally restricted to a small group of cells in the center of the putative shoot apical meristem of O. catharinensis somatic embryos. Perturbed expression of OcWUS might be related to abnormally formed somatic embryos of O. catharinensis obtained through tissue culture.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), BrazilCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Brazil)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Sao Paulo, BrazilFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazi

Genome-Wide Analyses of MADS-Box Genes in <i>Humulus lupulus</i> L. Reveal Potential Participation in Plant Development, Floral Architecture, and Lupulin Gland Metabolism

MADS-box transcription factors (TFs) are involved in multiple plant development processes and are most known during the reproductive transition and floral organ development. Very few genes have been characterized in the genome of Humulus lupulus L. (Cannabaceae), an important crop for the pharmaceutical and beverage industries. The MADS-box family has not been studied in this species yet. We identified 65 MADS-box genes in the hop genome, of which 29 encode type-II TFs (27 of subgroup MIKCC and 2 MIKC*) and 36 type-I proteins (26 α, 9 β, and 1 γ). Type-II MADS-box genes evolved more complex architectures than type-I genes. Interestingly, we did not find FLOWERING LOCUS C (FLC) homologs, a transcription factor that acts as a floral repressor and is negatively regulated by cold. This result provides a molecular explanation for a previous work showing that vernalization is not a requirement for hop flowering, which has implications for its cultivation in the tropics. Analysis of gene ontology and expression profiling revealed genes potentially involved in the development of male and female floral structures based on the differential expression of ABC homeotic genes in each whorl of the flower. We identified a gene exclusively expressed in lupulin glands, suggesting a role in specialized metabolism in these structures. In toto, this work contributes to understanding the evolutionary history of MADS-box genes in hop, and provides perspectives on functional genetic studies, biotechnology, and crop breeding

WUSCHEL-related genes are expressed during somatic embryogenesis of the basal angiosperm Ocotea catharinensis Mez. (Lauraceae)

Ocotea catharinensis is a basal angiosperm and an endangered tree species from the Brazilian Atlantic Rain Forest. Despite its economical and ecological importance, mass-propagation of this species is hampered by seldom-produced short-lived seeds, and in vitro propagation is challenged by frequently malformed somatic embryos. Therefore, O. catharinensis somatic embryos are also a good experimental material to study the physiological and molecular mechanisms underlying in vitro morphogenesis. In an ongoing effort to characterize genes expressed during somatic embryogenesis of O. catharinensis we have cloned two Ocotea WUSCHEL-related genes. According to our RT-PCR data, both genes were preferentially expressed in embryogenic cell aggregates. One of them, OcWUS, is a possible ortholog of the Arabidopsis WUSCHEL (WUS) gene, which codes for a homeodomain-containing protein involved in the specification and maintenance of the shoot apical meristem. We analyzed the expression patterns of OcWUS and OcWOX4 by RT-PCR, and OcWUS expression was also assessed by in situ hybridization. The expression patterns of OcWUS were very similar to those described for the Arabidopsis WUS. OcWUS transcripts were generally restricted to a small group of cells in the center of the putative shoot apical meristem of O. catharinensis somatic embryos. Perturbed expression of OcWUS might be related to abnormally formed somatic embryos of O. catharinensis obtained through tissue culture.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), BrazilCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Brazil)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Sao Paulo, BrazilFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazi

Microsporogenesis and the biosynthesis of floral small interfering RNAs in coffee have a unique pattern among eudicots, suggesting a sensitivity to climate changes

Recently, the siRNAs pathways, and especially reproductive phasiRNAs, have attracted attention in eudicots since their biological roles are still unknown and their biogenesis took different evolutionary pathways compared to monocots. In this work, we used Coffea arabica L., a recent allotetraploid formed from the hybridization of Coffea canephora and C. eugenioides unreduced gametes, to explore microsporogenesis and small RNAs-related pathways in a eudicot crop. First, we identified the microsporogenesis stages during anther development revealing that pre-meiosis occurs in anthers of 1.5&nbsp;mm inside floral buds (FBs), whereas meiosis between 1.5 and 4.2&nbsp;mm FBs, and post-meiosis in FBs larger than 4.2&nbsp;mm. These stages coincide with the Brazilian winter, a period of FBs reduced growth which suggests temperature sensitivity. Next, we identified and quantified the expression of reproductive 21- and 24-nt phasiRNAs during coffee anther development together with their canonical and novel miRNA triggers, and characterized the DCL and AGO families. Our results showed that the pattern of reproductive phasiRNA abundance in C. arabica is unique among described eudicots and the canonical trigger car-miR2275 is involved in the processing of both 21- and 24-nt phasiRNAs. Fourteen DCL genes were identified, but DCL5, related to phasiRNA biosynthesis in monocots, was not, according to its specificity for monocots. Thus, our work explored the knowledge gap about microsporogenesis and related siRNAs pathways in coffee, contributing to the control of reproductive development and the improvement of fertility in eudicots