Investigation of the initial steps of jasmonic acid biosynthesis and a study of differential regulation of the ribosomal association of mRNA transcripts in jasmonate-dependent wound response
Authors:   , Holtsclaw Rebekah Elisabeth 
Published: December 2023

Jasmonic acid is an oxylipin derived hormone that is responsible for regulating plant responses against various abiotic and biotic stressors and it also serves important roles in plant development. Jasmonic acid once synthesized after elicitation is converted into a number of derivates, collectively termed jasmonates (JA). Since the late '80s, large strides in the discovery and characterization of the enzymes involved in the biosynthesis and catabolism of JA as well as the signaling components necessary for the transcriptional reprogramming after a stimulus have been made. However, there are still plenty of aspects of JA biosynthesis, signaling, and catabolism that are still unknown. At the core of what's left to discover has to do with regulation of the biosynthetic pathway and of JA-regulated post-transcriptional changes. After a leaf perceives a stimulus, such as a bite from an insect, this message is quickly relayed inside the cell and into chloroplasts to initiate the biosynthesis of JA. There are several lines of evidence to show that the production of JA precedes the transcription and translation of JA-induced transcripts, therefore there are likely post-translational modifications occurring that elicit activation of the pathway. Chapter II of this dissertation provides novel insights into the initial steps of wound induced JA biosynthesis in Nicotiana benthamiana. A time course of the JA intermediate 12-oxo-phytodienomic acid (OPDA) accumulation in wounded N. benthamiana leaves showed an immediate increase without perceivable lag implying a very fast mode of regulation. Additional insights were made by utilizing a semi-in vitro system of isolated pea chloroplasts which clearly show that JA biosynthesis is substrate limited and therefore the lipase responsible for releasing fatty acids, the primary substrate for JA biosynthesis, must be tightly regulated. The glycerolipase A1 (NbGLA1) was identified as the primary lipase responsible for JA biosynthesis in wounded leaves of N. benthamiana through virus induced gene silencing (VIGS). Transient expression of NbGLA1 was shown to trigger JA biosynthesis without wounding. However, an additional effect on NbGLA1 by wounding that could amplify JA levels by several folds, far greater than the effect of gene expression alone, implied a post-translational regulatory mechanism. Experiments with a truncated version of NbGLA1 showed that the C-terminal end may contain sequence elements that can function in a regulatory role. Finally, JA accumulation time course in wounded N. benthamiana leaves also revealed an additional phase of JA increase later in the time course that was different from a single-phased accumulation of JA reported in Arabidopsis thaliana. These results and observations beg further research into both the common and unique aspects of the regulation of JA biosynthetic pathway in N. benthamiana. In chapter III the wound induced post-transcriptional regulation of mRNAs were studied by utilizing transcriptomic, proteomic, and translatomic approaches in both wildtype (WT) and a mutant defective in JA-dependent wound response in Arabidopsis. Previous research indicated some wound-responsive JA marker proteins were less abundant in the mutant, b1b3, after wounding although their respective transcripts were unchanged. Proteomics analysis using multidimensional protein identification technology (MudPIT) confirmed the downward trend of protein accumulation in b1b3. In order to learn more about this phenomenon, translating ribosome affinity purification RNA sequencing (TRAPseq) was carried out where translating pools of mRNA were purified by the immunoprecipitation of ribosomes and the associated RNAs were subsequently sequenced. Various comparisons revealed interesting post-transcriptional regulation of transcripts induced by wounding in WT. Between WT and b1b3, even though the transcriptome was very similar, there were many transcripts were less associated with the ribosomes in b1b3. Among those genes with fewer ribosome-associated transcripts in b1b3 were genes relating to stress response, specialized metabolism, protein metabolism, ribosomal subunits, and transcription factors, consistent with the biochemical phenotypes of the mutant. These results show previously unrecognized regulation at the translational level that are affected by mis-regulation of JA homeostasis during wound response in plants. In chapter IV a structure based virtual screen was performed to create a compound screening library to find an additional ligand to interact with the COI1-JAZ1 (PDB: 3OGL) co-receptor complex. This was inspired by recent research from our lab showing that although (3R,7S)-jasmonoyl-L-isoleucine (JA-Ile) is the primary endogenous bioactive JA that binds to the hormone co-receptor complex, there is an additional JA derivative, 12-hydroxy-JA-Ile (12OH-JA-Ile), that also has bioactive properties. Two candidate molecules, 7-hydroxy-4-({[5-hydroxy-4-(1-phenylethyl)-4H-1,2,4-triazol-3-yl]sulfanyl}methyl)-2H-chromen-2-one (HTS) and 1-(2-chloro-6-fluorobenzoyl)-3-[1-(2-cyanoethyl)-3-phenyl-1H-pyrazol-5-yl]urea (CD0), were identified and tested for their ability to effect JA signaling pathway. HTS was found to have growth promoting properties in Arabidopsis and in tomato (Solanum lycopersicum). HTS was able to partially reverse the JA-induced root growth phenotype. Further biochemical studies are underway to determine the causal relationship between its putative interaction with COI1-JAZ coreceptor complex and the growth phenotype.