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Biochemical and functional analysis of Arabidopsis metacaspases
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Wang, Jianqiao. Biochemical and functional analysis of Arabidopsis metacaspases. Retrieved from https://doi.org/doi:10.7282/t3-sd50-8k41

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TitleBiochemical and functional analysis of Arabidopsis metacaspases
NameWang, Jianqiao (author); Lam, Eric (chair); Belanger, Faith (internal member); Tumer, Nilgun (internal member); Gallavotti, Andrea (internal member); Bidle, Kay (outside member); Rutgers University; School of Graduate Studies
Date Created2018
Other Date2018-10 (degree)
SubjectPlant Biology, Cysteine proteinases, Arabidopsis thaliana
Extent1 online resource (157 pages : illustrations)
DescriptionMetacaspases are a group of cysteine proteases found in plants, protozoa and fungi, which share structural similarities and are considered functional homologs of metazoan caspases. Although metacaspases were discovered almost two decades ago, research on their characteristics, functions and mechanisms remain rather limited. In order to get a better understanding of plant metacaspases, I conducted several studies on metacaspases from the model plant Arabidopsis thaliana.
My first project presented in Chapter Two is domain functional analysis of AtMC4 and AtMC9, two type II Arabidopsis metacaspases (AtMCs) that are distinct in many of their biochemical characteristics. Through domain swapping, I generated six chimeric type II MCs (called Domain Swap Proteins or DSPs) for all possible permutations of functional domains of AtMC4 and AtMC9. Subsequent activity assays and immunoblots of these DSPs (DSP1-DSP6) identified essential domain elements involved in each specific biochemical trait of either AtMC4 or AtMC9. Also, conserved "signature" residues in each domain of both AtMC4 and AtMC9 were identified, which relates to the origins of those metacaspases and eventually links AtMC9-like proteases with the evolution of more complex root archetypes in terrestrial plants.
My second project presented in Chapter Three is about the in planta function of AtMC3. AtMC3 is an atypical member of the type I Arabidopsis metacaspases gene family. Research record on this metacaspases gene is close to none, which could be due to lack of proper knockout mutants. Utilizing the newly developed CRISPR/Cas9 technology, I created atmc3 knockout mutants. atmc3 mutant alone or in combination with other type I atmc mutants do not produce any visible abnormal phenotypes. However, when crossed into lsd1-related mutant background, atmc3 mutant displays an alleviating effect on the runaway cell death phenotype of lsd1, indicating that AtMC3 may act as a positive regulator in the HR cell death pathway.
My third project presented in Chapter Four is about characterization of the underlying biochemical properties of type I AtMCs in vitro, for which no relevant research result are available so far. I expressed all three type I AtMC recombinant proteins in E.coli and tested their activation pH optima, calcium dependency as well as the potential effect of prodomains on those proteases’ activity levels. I also examined the significance of the conserved cysteines in those type I AtMCs through point mutations and found that most of those cysteines do not play a catalytic role in those AtMCs, although a slight exception was seen with AtMC1. My study provided the first available data set for biochemical characterization of Arabidopsis type I metacaspases.
NotePh.D.
NoteIncludes bibliographical references
NoteIncludes vita
Genretheses, ETD doctoral
Persistent URLhttps://doi.org/doi:10.7282/t3-sd50-8k41
Languageeng
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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