Author : Jessica D. Weaver
Publisher :
Page : 88 pages
File Size : 23,19 MB
Release : 2015
Category : Abscisic acid
ISBN :
Book Description
Since plants are sessile, they rely on rapid, well-adapted responses to environmental stressors. These responses include the activation of specialized metabolism and the production of defense compounds. The medicinal plant Catharanthus roseus serves as a useful model for studying specialized metabolism, as it produces numerous pharmaceutically valuable Terpenoid Indole Alkaloids (TIAs), including the anticancer drugs vincristine and vinblastine, in response to stressors such as wounding or the presence of a pathogen. Because of their low concentrations in the plant, however, these anticancer drugs are very expensive. Over the last decade, there has been remarkable advancement in the understanding of TIA biosynthesis, including the sequencing of biosynthetic enzymes, discovery of transcription factors (TFs) regulating biosynthesis, transcriptomic analysis under various conditions, elucidation of the pathway induced by the phytohormone Jasmonate (JA) and recently sequencing of the genome. However, attempts to overexpress individual enzymes in the pathway have been largely unsuccessful; bottlenecks simply shift downstream, and there is no significant increase in the desired products. This suggests that the pathway is tightly regulated and increasing production of TIAs will require the manipulation of multiple steps. Specialized metabolism, including TIA production, is primarily regulated at the transcriptional level through the action of TFs. Since TFs can mediate the expression of multiple genes, manipulating their expression may be a more efficient strategy for increasing production of TIAs. Although several TFs have been identified in TIA biosynthesis, including the activator Octadecanoid-derivative Responsive Catharanthus AP2-domain (ORCA) and repressor Zinc Finger Catharanthus Transcription factor (ZCT), little is known about the network of TFs and how they crosstalk and interact with one another to regulate TIA biosynthesis. JA induces expression of the transcriptional repressor gene Zct1, and ZCT1 negatively regulates TIA biosynthesis. Here, we investigate the regulation of Zct1 expression in order to gain a better understanding of its place within the regulatory network. In Chapter 1 of this dissertation, we adapted and optimized a transient expression system, the Fast Agro-mediated Seedling Transformation (FAST) method, in C. roseus. In addition to effectively delivering DNA into the plant tissue, the FAST method elicits the defense response and increases expression of Zct1. In Chapter 2, we sequenced and analyzed the Zct1 promoter in silico and determined the Transcription Start Site (TSS). Several hormone-responsive elements were identified, including two JA-responsive As-1 elements. Since the FAST method strongly induces expression of Zct1, this method is not suitable to study hormonal induction of Zct1. Therefore, we created a stable transgenic hairy root line with the Zct1 promoter upstream of the Beta-glucuronidase (Gus) reporter gene. We found the Zct1 promoter was responsive to the phytohormones JA, abscisic acid (ABA) and gibberellin (GA). A series of deletions of the Zct1 promoter and one containing mutant As-1 elements were used to drive expression of the Gus reporter gene. We determined that the As-1 elements contribute significantly to the response of Zct1 to the defense response elicited by the FAST method. We also found that the activator ORCA3 transactivates expression of the Gus reporter gene driven by the Zct1 promoter. In vitro binding studies confirmed that ORCA3 can physically bind to the Zct1 promoter. In Chapter 3, we further investigate Zct1's role in growth and GA-signaling. Transient silencing of Zct1 via virus-induced gene silencing (VIGS) resulted in disruption of meristematic growth of the seedlings. We show that Zct1's expression is modulated by GA treatment and identified additional putative GA-responsive elements on the Zct1 promoter, including the binding site for indeterminate domain (IDD) proteins. IDD proteins act as intermediate proteins between DELLAs (named for conserved amino acids in DELLA domain) and their downstream gene targets in GA signaling. Unlike other DELLA binding partners, such as Jasmonate ZIM-domain (JAZ) and Phytochrome Interacting Factors (PIF), whose DNA-binding capacity is abolished when bound to DELLA, IDDs act as intermediate DNA-binding proteins to allow DELLA to activate expression of downstream gene targets. We identified three Idd transcripts and one Della in the C. roseus transcriptome and found that CrDella expression responds to GA treatment in C. roseus seedlings. The predicted protein sequences of the three CrIDDs are highly similar to the IDDs found in Arabidopsis thaliana. Experiments are currently underway to determine if CrDELLA binds to CrJAZ, CrPIF and CrIDD proteins using yeast two-hybrid and if IDD binds to the Zct1 promoter using an electrophoretic mobility shift assay (EMSA). ZCT1 may lie at the nexus of multiple phytohormone pathways. Taken together, the results presented herein suggest ZCT1 plays a significant role in the transcriptional network that regulates plant specialized metabolism, defense, and growth. The highly conserved DELLA protein in conjunction with IDDs that are involved in GA-signaling may regulate Zct1 expression in addition to the defense related JA signaling. Further investigation of the transcriptional network that regulates TIA production and the key repressor ZCT1 may yield valuable insight into overcoming limitations in TIA biosynthesis. Furthermore, understanding phytohormone signaling in plants in general and how plants direct resources to growth and development versus specialized metabolism and defense in particular has broad implications for the future of plant-based pharmaceuticals, sustainable agriculture and alternative energy resources.