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Biochemical Studies of the Ubiquitin-26S Proteasome System in Seed Development

YU, PEIFENG
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1709894649931255

Abstract Details

2024, Doctor of Philosophy (PhD), Ohio University, Molecular and Cellular Biology (Arts and Sciences).
Functional studies of the ubiquitin (Ub)-26S proteasome system (UPS) have demonstrated that virtually all aspects of the plant’s life involve UPS-mediated turnover of abnormal or short-lived proteins. However, developmental characterization of the UPS, including in seeds and fruits, remains scarce. Unfortunately, early termination of embryogenesis limits the scope for characterizing the UPS activities in reproductive organs. In this dissertation, I utilized a biochemical approach to tackle the developmental role of UPS in plants, using Arabidopsis thaliana (Arabidopsis hereafter) as a model. The overarching goal of my research is to unravel the molecular mechanisms of UPS underpinning seed development so that new molecular breeding technologies could be developed to promote seed production. First, I systematically compared expression changes of multiple 26S proteasome subunits along with the dynamics of proteasome activity and total protein ubiquitylation in seedlings and developing siliques of Arabidopsis. Because autophagy plays the second largest role in maintaining proteome stability, I parallelly studied three late-limiting enzymes that are involved in autophagy influx. My experiments unexpectedly discovered that, in opposite to the activities in seedlings, both protein and transcript levels of six selected 26S proteasome subunits gradually decline in immature siliques toward maturation while the autophagy influx rises, albeit in a nutrient-rich condition. I also discovered a reciprocal turnover pathway between the proteasome and autophagy. While the autophagy influx is suppressed in seedlings by UPS-mediated degradation of its three key enzymes, transcriptional reprogramming dampens this process in siliques that in turn stimulates a bulk autophagy degradation of proteasomes. Collectively, my discovery about the developmental changes of the UPS and autophagy activities suggests that they relay the proteome homeostasis regulation in early seed development, which highlights their developmental interactions. In the second objective, I studied the impact of protein ubiquitylation on seed development by overexpressing a polyUb (UBQ) gene. To demonstrate the dosage-dependent impact of protein ubiquitylation, two different UBQ transgenic plants transformed with either hexa(6His-UBQ) driven by Cauliflower mosaic virus (CaMV) 35S promoter or hexa(6His-Tev-UBQ) driven by the endogenous promoter of UBQ10 were studied. Given the nature of different transgenes, I termed the two transgenic plants as HU (for 6His-UBQ) and HTU (for 6His-Tev-UBQ), respectively. I discovered that HU and HTU had contrasting effects on seed yield, as the former exhibited reduced seed size and yield, while the latter showed an opposite phenotype attributed to enhanced silique number per plant. Differences in the integrity and composition of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with the wild type (WT) plant suggestive of a contributory role of UBQ expression in proteasome assembly. Since large protein complexes or aggregates and damaged cellular organelles are targeted by protein ubiquitylation for autophagy-mediated degradation for nutrient recycle, a compromised carbon starvation response was found in both HU and HTU seedlings, further suggesting the importance of UPS in regulating plant development, including seed development. In the third objective, I aimed to apply an affinity purification-based mass spectrometry (MS) analysis to identify active Skp1-cullin (CUL)1-F-box (SCF) Ub E3 ligase complexes and substrates that may differentially regulate plant vegetative and reproductive growth. I have identified 24 F-box proteins and 590 potentially SCF substrates, providing an exciting direction to further explore the role of SCF-meditated protein ubiquitylation in plant development and growth.
Zhihua Hua (Advisor)
234 p.
Biology; Plant Biology; Plant Sciences
Ubiquitin; autophagy; protein degradation; seed development; Arabidopsis

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Citations

  • YU, P. (2024). Biochemical Studies of the Ubiquitin-26S Proteasome System in Seed Development [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1709894649931255

    APA Style (7th edition)

  • YU, PEIFENG. Biochemical Studies of the Ubiquitin-26S Proteasome System in Seed Development. 2024. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1709894649931255.

    MLA Style (8th edition)

  • YU, PEIFENG. "Biochemical Studies of the Ubiquitin-26S Proteasome System in Seed Development." Doctoral dissertation, Ohio University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1709894649931255

    Chicago Manual of Style (17th edition)

ohiou1709894649931255
© 2024, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.
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