Dissecting the Transcriptional Regulatory Network Underlying Plant Wound Suberin Biosynthesis
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Between 20-40% of cultivated potatoes are lost during storage equating to approximately $1.2 billion in lost potential revenue each year. Wounding during mechanical harvesting and post-harvest handling results in tuber desiccation and provides an entry point for pathogens. Poor wound healing is the major cause of these losses. Wound tissue in potato tubers and all higher plants is comprised of a large proportion of suberin that is deposited in a specialized tissue called wound periderm. Suberin is an aliphatic and aromatic-based polymer ubiquitous among higher plants that serve as the first line of defense during periods of biotic and abiotic stress. Although many genes responsible for suberin biosynthesis have been identified, the regulatory components controlling its biosynthesis and deposition are only now beginning to be deciphered. The overall objective of this dissertation is to decipher the transcriptional regulators of suberin biosynthesis with a particular emphasis on transcription factors that regulate wound suberin biosynthesis and deposition.Chapter 1 presents a comprehensive review of existing knowledge on aspects of how suberin is regulated under normal development as well as conditions of abiotic stress. This chapter describes specific conditions that either induce or repress suberin deposition as well as identifies all transcription factors known to regulate suberin biosynthesis. Chapter 2 seeks to understand why some potato cultivars suberize and store better than others. To achieve this end, we have identified two transcription factors that appear to act as positive regulators of suberin biosynthesis and deposition during tuber wound periderm formation. Using gene expression and chemical analyses, we provide further evidence that differential expression of the genes encoding these transcription factors, single amino acid polymorphisms, and the wound suberin deposition regulated by these transcription factors can, in part, explain differences in the tuber storage life of well-storing and poor-storing cultivars. This research represents an important step in understanding the transcriptional regulation of suberin biosynthesis and provides a practical foundation for targeted breeding approaches aimed at improving potato tuber storage life and reducing annual crop losses.Chapter 3 describes the identification and characterization of two transcription factors as regulators of wound suberin biosynthesis. In this chapter, it is demonstrated that Arabidopsis thaliana genes AT4G21440 and AT4G05100, members of the MYB (myeloblastosis) family of R2R3 transcription factors are regulators of wound suberin deposition. First, a system for testing and measuring wound-induced suberization responses was established in Arabidopsis. Next, knockout mutants of AT4G21440 and AT4G05100 were shown to have less accumulation of suberin like monomers and lower wound-induced suberin gene transcript abundance after wounding. Transgenic Arabidopsis lines harboring transcriptional fusions demonstrated wound site localized promoter activities of AT4G05100 and AT4G21440 as well as promoter activites in tissues normally associated with suberization. Last, transient, heterologous expression of AT4G21440 and AT4G05100 in the leaves of Nicotiana benthamiana demonstrated that these two genes are sufficient for the ectopic production and deposition of an ultrastructure essentially identical to the lamellar structures found in suberized cells as well as large increases in suberin monomers released from leaf tissues via methanolysis. These two genes represent the firsttranscription factors ever identified to regulate wound suberin biosynthesis. These transcription factors provide an important tool and opportunity to more thoroughly comprehend the complete biochemical and transcriptional networks underlying the wound healing process as it relates to suberin.Future work to identify, model, and further characterize the regulatory network responsible for the biosynthesis and deposition of suberin will provide a map to understanding how plants have evolved the ability to protect themselves from wounding, ultimately aiding in identification of targets for crop improvement.