We previously described BT2, a BTB/POZ domain containing protein, as an activator of telomerase in Arabidopsis thaliana. In the current study, I present evidence of its interesting roles in mediating multiple hormone, stress and metabolic responses in plants. Steady-state expression of BT2 mRNA was regulated diurnally and was under the control of circadian clock, with a maximum expression in the dark. BT2 mRNA was responsive to nutrient status and to multiple biotic and abiotic stress signals. Using bt2 loss-of-function and BT2 over-expressing lines, I show that BT2 suppresses sugar and ABA-mediated responses during germination. BT2 is also essential for transcriptional gene activation mediated by CaMV 35S enhancers in Arabidopsis. Loss of BT2 in several well-characterized 35S enhancer activation-tagged lines such as yucca1d, pap1d, jaw1d etc., resulted in suppression of the activation phenotypes. The suppression of the phenotypes was due to decreased transcription of the activation-tagged genes. I further demonstrate that BT2 genetically interacts with CULLIN3. I propose that BT2 and CULLIN3 are components of a ubiquitin ligase complex. Together with associated proteins BET9 and BET10, the BT2 complex is required for CaMV 35S enhancer-mediated activation of gene expression and may regulate expression of target genes involved in multiple responses to fluctuating biotic and abiotic conditions. I also found that BT2 protein levels are tightly regulated in plants. BT2 protein was primarily localized in the nucleus and was developmentally regulated. BT2 turn-over was regulated in part by the 26S-proteosome, and rare codons present in its open reading frame affected BT2 protein accumulation. In addition to BT2, its orthologs, BT1, BT3, BT4 and BT5, also responded to light, clock and nutrients, with some differences. Moreover, BT1, BT3 and BT4 were also required for 35S enhancer-mediated activation of gene expression. I propose that BT family proteins assemble into multi-protein complexes to mediate multiple responses to changing environmental and nutritional conditions.

Considerando (1) a importância da bananicultura no Vale do Ribeira, (2) o destaque da Mata Atlântica no contexto da conservação da fauna e flora mundial, (3) a necessidade da adoção de práticas agrícolas alternativas mais eficientes do ponto de vista ambiental e econômico, (4) o papel dos hormônios etileno, acido indol-3-acético (AIA) e ácido abscísico (ABA) no contexto das respostas dos vegetais a diferentes condições ambientais e nos atributos de qualidade da banana, (5) a falta de metodologias otimizadas para a extração de compostos fenólicos solúveis totais (CFST) em bananeiras e (6) a importância do estudo da relação entre os CFST e fatores de estresse, o presente trabalho teve como objetivo avaliar a influência da proximidade do fragmento florestal de Mata Atlântica com a área de cultivo da banana (Musa acuminata AAA cv. Nanicão) sobre o amadurecimento da fruta e os CFST em folhas de bananeiras, além de otimizar uma técnica para a extração destes últimos. Foi observado que bananas colhidas próxima ao fragmento florestal apresentam vida-verde (período compreendido entre a colheita do fruto e o início do seu amadurecimento) maior quando comparados a frutos com a mesma idade fisiológica, porém colhidos em áreas sem a influência da floresta nativa. Este fato pode ser explicado, pelo menos em parte, pela diferença nos perfis de etileno, ABA e AIA ao longo do amadurecimento das bananas provenientes das diferentes áreas, que também influenciam no metabolismo amido-sacarose. Quanto aos CFST nas folhas, foi observado que a utilização de acetona 80% em água (v/v) e posterior emprego de hexano para a remoção do excesso de clorofilas é capaz de obter um bom rendimento de extração de CFST, sem extrair compostos que interferem significativamente no método de Folin-Ciocalteu. Além disso, a utilização da metodologia otimizada mostrou que bananeiras podem apresentar diferenças na sua composição de fenólicos quando influenciadas ou não pela presença de biodiversidade. Assim, a avaliação dos CFST em folhas pode fornecer informações importantes sobre as condições ambientais da planta. / Considering (1) the importance of banana production in Ribeira Valley, (2) the Atlantic Rainforest in the context of fauna and flora conservation, (3) the need for the adoption of more sustainable agricultural practices, (4) the ethylene, indole 3-acetic acid and abscisic acid responses in acclimation mechanisms of plants and in the quality attributes of the banana, (5) the lack of methodologies optimized for the extraction of total soluble phenolics compounds (TSPC) in banana leaves and (6) the importance of the relationship between the TSPC content and stress factors, the present work aimed to evaluate the influence of the Atlantic Forest fragments proximity in the banana (Musa acuminata AAA cv. Nanicão) crop area on fruit ripening and leaves TSPC levels, using a optimized methodology. It was observed that bananas harvested near to the forest fragment presented a longest greenlife (period between the harvest and the climacteric) when compared with the fruits with the same phisiologycal age, but without the influence of the native forest. This fact can be explained, at least partly, by the difference on ethylene, ABA and IAA profiles in the ripening of bananas from the different areas, which also influence the starch-sucrose metabolism. Moreover, it was observed that the extraction with acetone (80% v/v in water) and posterior hexane cycle to remove chlorophylls excess was able to obtain a good TSPC extraction yield in leaves, without extracting compounds that interfere significantly with Folin-Ciocalteu method. In additional, the use of optimized methodology showed that bananas leaves can present different TSPC amount when influenced by the presence of native forest. Thus, the evaluation of leaves TSPC profile can provide important information about the environmental conditions of the plant.

Considerando (1) a importância da bananicultura no Vale do Ribeira, (2) o destaque da Mata Atlântica no contexto da conservação da fauna e flora mundial, (3) a necessidade da adoção de práticas agrícolas alternativas mais eficientes do ponto de vista ambiental e econômico, (4) o papel dos hormônios etileno, acido indol-3-acético (AIA) e ácido abscísico (ABA) no contexto das respostas dos vegetais a diferentes condições ambientais e nos atributos de qualidade da banana, (5) a falta de metodologias otimizadas para a extração de compostos fenólicos solúveis totais (CFST) em bananeiras e (6) a importância do estudo da relação entre os CFST e fatores de estresse, o presente trabalho teve como objetivo avaliar a influência da proximidade do fragmento florestal de Mata Atlântica com a área de cultivo da banana (Musa acuminata AAA cv. Nanicão) sobre o amadurecimento da fruta e os CFST em folhas de bananeiras, além de otimizar uma técnica para a extração destes últimos. Foi observado que bananas colhidas próxima ao fragmento florestal apresentam vida-verde (período compreendido entre a colheita do fruto e o início do seu amadurecimento) maior quando comparados a frutos com a mesma idade fisiológica, porém colhidos em áreas sem a influência da floresta nativa. Este fato pode ser explicado, pelo menos em parte, pela diferença nos perfis de etileno, ABA e AIA ao longo do amadurecimento das bananas provenientes das diferentes áreas, que também influenciam no metabolismo amido-sacarose. Quanto aos CFST nas folhas, foi observado que a utilização de acetona 80% em água (v/v) e posterior emprego de hexano para a remoção do excesso de clorofilas é capaz de obter um bom rendimento de extração de CFST, sem extrair compostos que interferem significativamente no método de Folin-Ciocalteu. Além disso, a utilização da metodologia otimizada mostrou que bananeiras podem apresentar diferenças na sua composição de fenólicos quando influenciadas ou não pela presença de biodiversidade. Assim, a avaliação dos CFST em folhas pode fornecer informações importantes sobre as condições ambientais da planta. / Considering (1) the importance of banana production in Ribeira Valley, (2) the Atlantic Rainforest in the context of fauna and flora conservation, (3) the need for the adoption of more sustainable agricultural practices, (4) the ethylene, indole 3-acetic acid and abscisic acid responses in acclimation mechanisms of plants and in the quality attributes of the banana, (5) the lack of methodologies optimized for the extraction of total soluble phenolics compounds (TSPC) in banana leaves and (6) the importance of the relationship between the TSPC content and stress factors, the present work aimed to evaluate the influence of the Atlantic Forest fragments proximity in the banana (Musa acuminata AAA cv. Nanicão) crop area on fruit ripening and leaves TSPC levels, using a optimized methodology. It was observed that bananas harvested near to the forest fragment presented a longest greenlife (period between the harvest and the climacteric) when compared with the fruits with the same phisiologycal age, but without the influence of the native forest. This fact can be explained, at least partly, by the difference on ethylene, ABA and IAA profiles in the ripening of bananas from the different areas, which also influence the starch-sucrose metabolism. Moreover, it was observed that the extraction with acetone (80% v/v in water) and posterior hexane cycle to remove chlorophylls excess was able to obtain a good TSPC extraction yield in leaves, without extracting compounds that interfere significantly with Folin-Ciocalteu method. In additional, the use of optimized methodology showed that bananas leaves can present different TSPC amount when influenced by the presence of native forest. Thus, the evaluation of leaves TSPC profile can provide important information about the environmental conditions of the plant.

Orientador: Douglas Silva Domingues / Resumo: O café é uma das commodities agrícolas tropicais mais comercializadas no mundo. Coffea arabica é a principal espécie utilizada para a produção comercial de café. A espécie é originária da Etiópia. Ela é única espécie alotetraploide do gênero (2n = 4x = 44) e se reproduz predominantemente por autofecundação. As cultivares comerciais de C. arabica possuem baixa diversidade genética, o que indica a necessidade de introgressão de alelos de germoplasma para o melhoramento dessas cultivares. Acessos do centro de origem da espécie possuem maior diversidade que as cultivares comerciais e podem ser utilizados para a identificação de novos alelos. O conteúdo de macronutrientes em grãos do cafeeiro tem impacto direto na qualidade do produto. No entanto, a base molecular da composição mineral de grãos de cafeeiro ainda é pouco conhecida. Com isso, o objetivo desse trabalho foi identificar marcadores SNP possivelmente associados com a composição de macronutrientes em grãos de C. arabica. Para alcance deste objetivo, foram comparados três métodos de imputação de genótipos, bem como foi realizado o mapeamento associativo em estudo de associação genômica ampla (GWAS). Foi utilizado um painel de 110 genótipos de C. arabica, composto por genótipos elite do programa de melhoramento do Instituto Agronômico do Paraná (3), cultivares comerciais (11) e acessos selvagens (96). Foram realizadas análises da composição de cinco macronutrientes (N, P, K, Ca e Mg) em grãos de cafeeiro coletados de 70 e 1... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Coffee is one of the most traded tropical commodities in the world. Coffea arabica is the main species used for commercial production. The species is originally from Ethiopia. In the Coffea genus, C. arabica is the only allotetraploid species (2n = 4x = 44) and it reproduces predominantly by self-fertilization. The commercial cultivars of C. arabica have a narrow genetic base that indicates the need for the introgression of new alleles from germplasm into coffee breeding programs. Wild accessions of C. arabica, from Ethiopia, have higher genetic diversity and can be used to identify new alleles. The macronutrient composition of the coffee grains has a direct impact on grain quality. However, the molecular basis for the mineral composition in coffee grains still poorly understood. Thus, the aim of this work was to perform mapping association analyses using the genome-wide association study (GWAS) technique to identify single nucleotide polymorphisms (SNPs) associated with macronutrient content in coffee grains from C. arabica. We also tested three imputation methods (haplotype missing allele imputation - Beagle, K-nearest neighbors, and Random Forest) in the genotypic data, and mapped it to two C. arabica reference genomes from the cultivar Caturra red and the spontaneous dihaploid Et39. We used a panel of 110 C. arabica genotypes, including elite landraces from the IAPAR coffee breeding program (3), commercial cultivars (11) and wild accessions (96). Analysis of the compositi... (Complete abstract click electronic access below) / Doutor

<p dir="ltr">Plant immunity to pathogens involves a network of genetic, molecular, and cellular processes that culminate in activation of responses that restrict pathogen ingress and reduce diseases symptoms. Mechanisms of pathogen recognition, signaling, and activation of immune responses are well understood especially for (hemi) biotrophic pathogens. By contrast, there is paucity of knowledge on immune signaling for responses to broad host necrotrophic fungi such as <i>Botrytis cinerea (Botrytis</i><i>)</i>. Plant resistance to such pathogens is genetically complex with no complete resistance observed in any host species. However, genetic variation for quantitative resistance has been documented although the molecular mechanisms are poorly understood. In the current study, we focused on functional dissection of components of tomato immune signaling underlying quantitative resistance to <i>Botrytis</i>. Tomato BIK1-family receptor-like cytoplasmic kinases (RLCKs) were studied using gene edited mutants, and subsequent molecular, biochemical, and genomic characterizations of the mutants and the corresponding proteins. In addition, <i>Botrytis</i> induced transcriptome of Arabidopsis and tomato were compared to explain observed differences in their resistance to the fungus. The results from these studies are described in four chapters. Chapter 1 provides a review of RLCKs and their function in plant responses to biotic and abiotic stresses. Chapter 2 presents the genetic, molecular, and biochemical characterization of tomato RLCK, TPK1B RELATED PROTEIN KINASE (TPK09) in fungal resistance and responses to light stress. Chapter 3 highlights observations on the functions of TPK1B RELATED PROTEIN KINASE7 (TPK07) in fungal and bacterial resistance. Lastly, Chapter 4 covers comparative transcriptome analysis of Arabidopsis and tomato responses to <i>Botrytis</i>.</p><p dir="ltr">Specialized cell-surface receptors mediate the perception of environmental changes. A subset of plant cell surface receptors recognizes pathogen-associated molecular patterns (PAMPs), which are immunogenic pathogen or host-derived molecules, peptides (phytocytokines), lipids, and carbohydrates. PAMPs are perceived by surface receptors designated as pattern recognition receptors (PRRs) that are categorized as receptor like kinases (RLKs) or receptor like proteins (RLPs). Recognition of PAMPs through PRR is an evolutionarily conserved pathway that aids plants in specific recognition of pathogens. The signaling events initiated by PRRs are connected to PRR-associated RLCKs, which amplify the signal and activate other regulatory proteins. PRR-RLCK activation is linked to immune messengers ROS, Ca⁺, and MAPKs based on extensive research in Arabidopsis which also established the paradigm for RLCKs functions in integrating signals from various PRRs. However, insufficient attention was given to RLCK functions in crop plant responses to biotic and abiotic stressors and, thus, limited data exists on RLCKs from crops of agronomic and horticultural significance. To address this gap, we conducted genetic, genomic, and molecular studies on the biological functions of tomato TPK1B RELATED KINASES TPK09 and TPK07.</p><p dir="ltr">To determine the function of TPK09 and TPK07, mutant alleles of these two RLCKs were generated through CRISPR-Cas9 gene editing. Loss of function mutants of <i>tpk09</i> exhibited increased susceptibility to <i>Botrytis</i> but showed no altered responses to the bacterial pathogen <i>P. syringae</i>. Plants carrying mutant alleles displayed reduced immune gene expression and impaired accumulation of reactive oxygen species in response to chitin and flg22. In addition to <i>Botrytis</i> and several plant hormones, the expression of <i>TPK09</i> gene is induced by light but suppressed by darkness. The exposure of wildtype tomato plants to light-emitting diodes (LEDs) reduced hypocotyl length but <i>tpk09</i> mutants were insensitive. Furthermore, <i>tpk09</i> mutants also exhibited increased accumulation of H₂O₂ and extensive necrosis, suggesting a disturbance in cellular homeostasis in response to changes in light spectra, ultimately leading to enhanced susceptibility to <i>Botrytis</i>. Although the global impact of TPK09 on <i>Botrytis</i> induced transcriptome was limited, the expression of the tomato negative regulator of cell death SlBI-like1 gene was significantly reduced in the mutant, particularly in response to the combined effect of <i>Botrytis</i> and LED light. The data suggest TPK09 regulates SlBI-like1 gene expression, but the mechanism is unclear. Further, the impairment of the light stress response in <i>tpk09</i> mutants was substantiated by a reduction in chlorophyll content and damage to the photosynthetic machinery, along with a clear reduction in the expression of genes related to light harvesting and photosynthesis. Regulatory network analysis using RNA-seq data identified TPK09 regulated genes related to stress and oxidative damage. This was further supported from proteomic studies of tomato TPK09-HA transgenic plants. Immunoprecipitation coupled with mass spectrometry revealed that proteins associated with photosynthesis and photosystem subunits were enriched in TPK09 overexpressing plant. Our data establishes that TPK09 is required for fungal resistance and light stress by maintaining functional photosynthetic machinery and ROS homeostasis.</p><p dir="ltr">We also studied the function of tomato TPK07 which is another member of the RLCK-VII family. Through analysis of <i>tpk07</i> mutants, we show that TPK07 it required for resistance to both <i>Botrytis</i> and <i>P. syringae</i> expressing the cysteine protease type III effector AvrPphB which cleaved TPK07 protein. However, further studies are needed to understand the biological function of this cleavage. The expression of TPK07 was induced by <i>Botrytis</i> and the bacterial PAMPs flg22, flg28, and csp22. TPK07 belongs to the RLCK VII-5 subfamily and clusters in the same clade with Arabidopsis PBL34 (AT5G15080), PBL35 (AT3G01300), and PBL36 (AT3G28690) that functions in immunity to <i>P. syringae</i>. In addition, <i>tpk07</i> mutants showed reduced accumulation of ROS in response to chitin and lipopolysaccharide. Moreover, TPK07 is a plasma membrane-localized kinase with a unique N-terminal sequence, offering a research avenue for future structural analysis to understand its contribution in pathogen responses.</p><p dir="ltr">In the last chapter, we conducted comparative analyses of <i>Botrytis</i> induced transcriptome of Arabidopsis and tomato to explain the observed differences between the two plant species in their resistance to <i>Botrytis</i>. Over the years, we observed tomato is more susceptible to <i>Botrytis</i> than Arabidopsis under the same disease assay conditions, but the mechanism is not known. We sought to gain insight into the immune responses of the two species and identify targets for future functional analyses that can explain the differential pathogen responses. Many separate studies<i> </i>have demonstrated<i> </i>that <i>Botrytis</i> causes extensive transcriptional reprogramming in Arabidopsis and tomato. Our results from the comparative studies of in-house generated data identified differentially expressed genes (DEGs), Gene Ontology terms, and metabolic pathways that are specific or shared between the two species. Interestingly, some genes show distinct expression pattern in tomato and Arabidopsis consistent with previous observation that some genes in the two species show contrasting disease resistance functions. Genes with distinct expression patterns warrant further studies to better understand differences in host immune responses between the two species. In addition, transcription factors (TFs) and regulatory hub genes that could be important for further studies were identified.</p><p dir="ltr">In sum, our data establishes the functions of two tomato RLCKs in fungal resistance, interactions between fungal resistance and plant responses to light, and the conserved and contrasting gene expression profiles of tomato and Arabidopsis genes, laying the foundation for future studies.</p>