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| Author(s): |
Nathalia Volpi e Silva
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2019-07-31 |
| Examining board members: |
Paulo Mazzafera;
Douglas Silva Domingues;
Paula Macedo Nobile;
Michael dos Santos Brito;
Sara Adrián López de Andrade
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| Advisor: | Igor Cesarino; Paulo Mazzafera |
| 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 (AU) | |
| FAPESP's process: | 14/17831-5 - Chlorogenic acid and its relation with lignin biosynthesis |
| Grantee: | Nathalia Volpi e Silva |
| Support Opportunities: | Scholarships in Brazil - Doctorate |