Characterization of the roles of the DELLA protein, PROCERA, and the O-GlcNAc transferase, SECRET AGENT, in gibberellin signaling in Solanum lycopersicum

Abstract

Gibberellins (GA) are phytohormones involved in regulating plant growth and development. DELLAs negatively regulate GA signaling and are degraded in the presence of bioactive GA. Loss of DELLA function result in increased GA response and mutants are insensitive to GA application. Gain of DELLA function result in decreased GA response. DELLAs are considered master regulators of GA signaling but evidence in tomato suggests a DELLA-independent GA signaling pathway could exists. Tomato has one DELLA protein called PROCERA (PRO) and the putative loss-of-function mutant is responsive to GA treatment. In this thesis, I used the of the genome editing tool called TALEN to induce site-specific insertion/deletion events in PRO. TALENs were successfully employed and seven heritable alleles (proTALEN) with premature stop codons were generated. All homozygous mutants exhibited phenotypes consistent with increased GA response. A new pro loss-of-function mutant (proΔGRAS) was also identified and characterized in addition to proTALEN. Complementation assays demonstrated proTALEN and proΔGRAS are on the same gene. Both mutants are tall and insensitive GA treatment. However, gene expression analysis identified GA2oxidase4 to be up-regulated along with five other genes. These results demonstrate that DELLAs are master regulators of GA signaling with respect to plant growth but some genes may have DELLA-independent regulation. I also describe how DELLA activity may be regulated by the protein SECRET AGENT (SEC). SEC is an O-linked N-acetylglucosamine transferase proposed to positively regulate GA signaling. Early studies of sec-1 and sec-2 found only subtle growth defects. A new sec-3 dwarf mutant was identified but the dwarf plant height was only partially rescued with GA treatment. This suggests SEC may be involved in regulating GA signaling and not necessarily GA levels. Thus, I investigated SEC function in tomato and identified two SEC genes (SlSEC1 and SlSEC2). Preliminary studies suggest SlSEC1 and SlSEC2 have redundant function since homozygous CRISPR/Cas9-induced mutant (slsec1_CRISPR1) exhibit wild-type phenotypes. SlSEC1 and SlSEC2 expression were also repressed using microRNAs and the suppression lines were dwarf, consistent with reduced GA signaling. Taken together, these investigations demonstrate that DELLAs are master regulators of GA signaling and DELLA repressive activity may be fine-tuned by SEC.