UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA Nathalia Volpi e Silva CHLOROGENIC ACID AND ITS RELATIONSHIP WITH LIGNIN BIOSYNTHESIS ÁCIDO CLOROGÊNICO E SUA RELAÇÃO COM A BIOSSÍNTESE DE LIGNINA CAMPINAS - SP 2019 NATHALIA VOLPI E SILVA CHLOROGENIC ACID AND ITS RELATIONSHIP WITH LIGNIN BIOSYNTHESIS ÁCIDO CLOROGÊNICO E SUA RELAÇÃO COM A BIOSSÍNTESE DE LIGNINA Thesis presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Genetic and Molecular Biology in the area of Plant Genetics and Breeding. Tese apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para obtenção do Título de Doutor em Genética e Biologia Molecular, na área de Genética Vegetal e Melhoramento. Supervisor / Orientador: Prof. Dr. Paulo Mazzafera Co-supervisor / Co-Orientador: Prof. Dr. Igor Cesarino ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA NATHALIA VOLPI E SILVA, ORIENTADA PELO PROF. DR. PAULO MAZZAFERA. CAMPINAS - SP 2019 Agência de fomento: FAPESP Agência de fomento: Capes N° Processo: 2014/17831-5, 2016/15834-2 N° Processo: 001 Nº processo:0 Nº processo:0 Campinas, 31de julho de 2019 EXAMINATION COMMITTEE Banca examinadora Dr. Paulo Mazzafera (Supervisor/Orientador) Dr. Paula Macêdo Nobile Dra. Sara Adrian Lopez de Andrade Dr. Douglas Silva Domingues Dr. Michael dos Santos Brito Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno. ACKNOWLEDGMENT I would like to thank the São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo) Grant (Processo) nº 2014/17831-5, FAPESP and n° 2016/15834-2, FAPESP for the grant/fellowship and all financial support to develop this thesis. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. I am profoundly grateful to my supervisor Dr. Paulo Mazzafera and my co-supervisor Dr. Igor Cesarino for all knowledge shared and patience to guide me through this journey. I am also extremely grateful to Dra. Nicola J Patron for receiving me in her laboratory at Earlham Institute (Norwich – UK) and teach me about genome editing. My sincere thanks to Tatiane Gregório, Felipe Tolentino, Ewerton Ribeiro, Dr. Oleg Raitskin, Dra. Juliana Mayer and Dr. Eduardo Kiyota for helping me with my experiments when I needed. I could not forget to thank Rafaela Bulgarelli, Dra. Sarah Caroline Ribeiro and Uiara Romero Souza for all the help in taking care of my plants while I was in maternity leave. Dr. Adilson Domingues Junior, Dr. Franklin Magnum Silva, Dr. Luciano Pereira, and Dra. Flávia Shimpl also gave me invaluable help with my writing and academic talk. I also would like to thank all LAFIMP’s team for friendship and support. My research would have been impossible without the support of my family: my husband, my parents, my daughter, my sister. You have always been there for me with unfailing support and continuous encouragement, thank you. My husband, my mother and my mother- in-law help taking care of my daughter Laura were essential while I was writing my thesis. ABSTRACT Phenylpropanoids are compounds derived from phenylalanine and are involved in several aspects related to the defense of biotic and abiotic stresses. One of the phenylpropanoids present in most plants is chlorogenic acid (CGA). CGA biosynthesis is mediated by the enzyme hydroxycinnamoyl quinate transferase (HQT). Although never proved, some papers have suggested that the CGA pool could be related to lignin biosynthesis in plants. Another enzyme, hydroxycinnamoyl shikimate transferase (HCT), appears to be involved with both lignin and CGA pathway. Like HCT, HQT uses p-coumaroyl CoA for the formation of the hydroxycinnamoyl shikimate or hydroxycinnamoyl quinate esters, respectively. In addition, recently the enzyme caffeoyl shikimate esterase (CSE) has been described as involved in the conversion of caffeoyl shikimate to caffeic acid, which is subsequently converted to caffeoyl CoA in lignin route. CSE shares the substrate with the HCT enzyme, thus suggesting that a change in its expression may interfere not only with lignin metabolism, but also with CGA. Because they present common intermediates, it is possible that CGA can act as a donor of carbon skeletons for lignin biosynthesis. In Chapter 1 we brought a review discussing the interconnection among the main genes involved in CGA and lignin interdependency, HCT, HQT, and CSE. In Chapter 2 we focused on the relationship between the genes CSE and HCT, bringing important data to reinforce the importance of shikimate shunt in both pathways. In Chapter 3 we constructed and validated CRISPR/Cas9 constructions to genome edit HCT, CSE, and CCoAOMT aiming the development of tobacco stable mutants. The construction of mutant and double mutants overexpressing and silencing the HCT, HQT and CSE genes may help to clarify the nature of this interdependence between the CGA pool and lignin, as well as to validate the role of the CSE enzyme as a common component in the lignin pathway. Bioinformatics analyses identified four putative isoforms of the HCT gene and two of CSE in Nicotiana tabacum, the species chosen for this study. In order to obtain mutants for these genes we designed several transformation constructions: pCaMV35S::CSE (CSE overexpression), pCaMV35S::HCT (HCT overexpression); pCaMV35S::HQT (HQT overexpression); pCaMV35S::amiRNACSE (CSE downregulation), pCaMV35S::HCT::pCsVMV::amiRNAHQT (HCT overexpression combined with HQT downregulation); pCaMV35S::HQT::pCsVMV::amiRNAHCT (HQT overexpression combined with HCT downregulation), and pCaMV35S::HCT::pCsVMV::amiRNACSE (HCT overexpression combined with CSE downregulation). CSE silencing plants (amiCSE) showed severe dwarfed phenotype and did not produce any descendants indicating the importance of CSE in plant normal development. On the other hand, HCTamiCSE and CSE developed normally and were carried to generation T1 where it was conducted further analyses. The mutants were assayed for phenotype, gene expression, lignin, plant cell wall polysaccharides, saccharification, and phenolic profiling. The plants analyzed showed no alteration in the composition of lignin, but presented alterations in the metabolism of chlorogenic acid, especially the plants overexpressing CSE, indicating a probable role of CGA as carbon skeleton of the lignin pathway. In addition, we also successfully constructed and validated vectors using CRISPR/Cas9 tool for the CCoAOMT, CSE and HCT genes in tobacco leaves. Although several studies suggest the interconnection between the lignin and chlorogenic acid routes, most of the analyzes shown are in vitro. The fact that our mutants have the chlorogenic acid composition affected strongly suggests that these pathways are interconnected and that CSE may play a decisive role in the biosynthesis of chlorogenic acid in tobacco plants. RESUMO Fenilpropanóides são compostos derivados da fenilalanina e estão envolvidos em vários aspectos relacionados à defesa de plantas. Alguns desses fenilpropanóides são os ácidos clorogênicos (CGA). A biossíntese de CGA é mediada pela enzima hidroxicinamoil quinato transferase (HQT). Embora nunca provado, alguns trabalhos sugerem que o pool de CGA poderia estar relacionado com a biossíntese de lignina. Outra enzima, a hidroxiciamoil chiquimato esterase (HCT), parece estar envolvida nas rotas de biossíntese de lignina e ácido clorogênico. Assim como a HCT, a HQT utiliza p-coumaoil CoA para formação de ésteres de hidrocinamoil chiquimato ou hidroxicinamoil quinato, respectivamente. Além disso, a enzima cafeoil chiquimato esterase (CSE) foi descrita como envolvida na conversão de cafeoil chiquimato em ácido cafeico, o qual é convertido em cafeoil CoA. Dessa forma, CSE compartilha o substrato com a enzima HCT, sugerindo que uma mudança em sua expressão deva interferir não apenas no metabolismo de lignina, mas também no metabolismo de CGA. Por terem intermediários em comum, é possível que haja interdependência entre essas vias, e CGA possa atuar como doadora de esqueleto de carbono para a biossíntese de lignina. Desta forma, este trabalho objetiva trazer mais informações a fim de entender a relação entre estas duas vias de biossíntese. No Capítulo 1 trouxemos uma revisão com foco na relação entre os principais genes envolvidos na interdependência entre CGA e lignina, os genes HCT, HQT e CSE, com objetivo de conectar os dados disponíveis na literatura que tratam deste assunto. No Capítulo 2 focamos na relação entre estas vias e os genes HCT e CSE, trazendo dados importante que reforça a importância do braço da rota que utiliza chiquimato tanto para lignina como para CGA. No Capítulo 3 construímos e validamos vetores para edição de genoma os genes HCT, CSE e CCoAOMT com objetivo de futuramente desenvolvermos plantas mutantes para estes genes via CRISPR/Cas9. A construção de mutantes e duplos mutantes super- expressando e silenciando os genes HCT, HQT e CSE pode contribuir para esclarecer a natureza dessa interdependência entre o pool de CGA e lignina, assim como validar o papel da enzima CSE como um componente da via de lignina em Nicotiana tabacum. Análises de bioinformática identificaram quatro isoformas putativas do gene HCT e duas do gene CSE em N. tabacum, a espécie escolhida para estudo. Com objetivo de obtermos mutantes para estes genes foram desenhadas várias construções para transformação