Transcriptional modulation and characterization of plant-specific trans-acting factors in abiotic stress responses

• Ano de defesa: 2020
• Autor(a) principal: Melo, Bruno Paes de
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa; Bioquímica Aplicada
• Palavras-chave em Português: Regulação de expressão gênica; Plantas- Efeito de stress; Stress (Fisiologia); Soja- Genética; Biologia molecular; Bioquímica Molecular
• Link de acesso: https://locus.ufv.br//handle/123456789/29433

Transcription factors (TFs) are key regulators of gene expression in cells. Due to their functional plasticity and their role as pivotal players in controlling gene expression according to the environment, they may be considered hot spots to decipher the mechanisms of plant responses to multiple stresses and suitable targets for biotechnological intervention and development of adapted plants. Several plant TF families have been implicated in stress responses, including NAC, MYB, WRKY, and bZIP. The present work explores the functionality of Arabidopsis AREB-1, a well- characterized bZIP TF in drought stress response and physiological adaptation, and the soybean NAC superfamily and members associated with the control of multiple stress responses and senescence. In the first chapter, we introduced a new strategy of transcription modulation of AREB-1 by CRISPR/dCas9 in Arabidopsis for tolerance to drought. The following chapters deal with new insights toward GmNAC superfamily and functional studies of GmNAC065 and GmNAC085 based on genome- and transcriptome-wide analyses in soybean and a reverse genetics approach in Arabidopsis. Using the CRISPR activation (CRISPRa) technique, an inactive nuclease dCas9 was fused with a histone acetyl-transferase 1 (AtHAT1), optimizing gene expression via chromatin remodeling. The CRISPRa-mediated AREB1 overexpression promoted an improvement in the physiological performance of the transgenic plants under 30 days of water deprivation. The enhanced drought tolerance phenotype was associated with increased chlorophyll content, antioxidant enzyme activity, and soluble sugar content, with consequent lower reactive oxygen species (ROS) accumulation. Finally, we demonstrated that the up-regulation of AREB1 positively changed the transcription of downstream ABA-inducible genes involved in adaptive response and promoted a better plant performance under drought, validating CRISPRaas a biotechnological tool to improve specific plant traits.The NAC genes encode TFs involved in the control of plant morph-physiology and stress responses. The last soybean genome assembly (Wm82.a2.v1) raised the possibility of new NAC genes onthe soybean genome. In thisinvestigation, we identified 32 putative novel NAC genes, updating the superfamily to 180 gene members, clustered in15 phylogenetic subfamilies. We showed that 40% of the GmNACsare differentially regulated by developmental senescence. GmNAC065 and GmNAC085 display contrasting gene expression profiles in multiple stress responses and induce symptoms of leaf senescence to a different extent when transiently expressed in N. benthamiana, suggesting a divergent role of these genes. Subsequently, the soybean genome was interrogated for developmental and environmental senescence- associated genes (SAGs) belonging to the NAC superfamily. Using functionally characterized Arabidopsis SAGs as prototypes, we identified the putative NAC-SAGs in soybean, including GmNAC065 and GmNAC085, whose functions in multiple stress responses and senescence were further investigated in soybean and Arabidopsis transgenic lines. The ectopic expression of GmNAC065 in Arabidopsis leads to a delayed-senescence phenotype, with enhanced oxidative performance under multiple stresses and lower stress-induced PCD. The GmNAC085-expressing lines displayed an opposite phenotype leading to the up-regulation of several downstream SAGs in Arabidopsis, further demonstrating their divergent roles in stress responses and PCD. Finally, if we are to use soybean as a model system for genetic studies and development of new cultivars, we need to develop an efficient protocol for soybean transformation and regeneration. The final chapter of this work proposes a new methodology for soybean genetic transformation combining biolistic and Agrobacterium-mediated DNA delivery. We developed a one-step protocol for transgenic soybean recovery in approximately 30 - 40 weeks more cost-effectively and straightforwardly exploring the high regenerative capacity of shoot-apex cells in the embryonic axis. The protocol allows the direct co-cultivation and plant regeneration, avoiding contamination generated by excessive tissue-manipulation, demanded in other protocols. Therefore, we are now better positioned to translate the fundamental studies developed in this investigation into biotechnological traits to get superior crops. Keywords: Trans-acting factors. Modern crop breeding. Environmental stresses. Senescence. Plant genetic engineering.