Research

Arabidopsis E3 ubiquitin ligase protein interacting networks

阿拉伯芥泛素連結酶的蛋白交互作用網路

The ubiquitin-proteasome system (UPS) is a highly-regulated and specific protein degradation pathway. In UPS degraded proteins are marked with ubiquitin, allowing them to be recognized and degraded by the proteasome. The E3 ubiquitin ligase (E3) binds to the protein to be degraded and labels it with ubiquitin, promoting its degradation. Conversely, deubiquitinating enzymes (DUBs) bind to ubiquitinated proteins and remove ubiquitin, stabilizing the protein. The actions of E3 ligases and DUBs edit the ubiquitin code on proteins, which not only regulates their stabilities but also may change their functions or cellular localizations. However, while there are nearly 1400 E3 ubiquitin ligases in Arabidopsis, the majority of their functions, the proteins interacting with them, and their ubiquitin codes remain unknown.

We collaborated with Dr. Joshua Gendron's laboratory at Yale University to establish a high-throughput yeast screening system for approximately 300 Arabidopsis E3 ubiquitin ligases. By using yeast two-hybrid assays to rapidly identify proteins that bind to E3 ubiquitin ligases, we aim to understand the regulatory mechanisms between these E3s and their binding proteins. These research results will be helpful in understanding the roles that E3 ubiquitin ligases and their binding proteins play in the cell.

Arabidopsis circadian clock under higher temperatures

阿拉伯芥生物時鐘在高溫下的調控與反應

生物因應地球自轉產生晝夜規律的光和溫度變化，進而演化成24小時週期的生物時鐘 (circadian clock)。生物時鐘可調節生物體的代謝及生理途徑，使其有效利用資源，以利適應環境及生存。植物的生物時鐘和其他生物有許多相似之處，但因為植物根生於地，不斷地感知可預期和不可預期的環境變化，其調控機制又更為有趣且複雜。目前的研究發現，植物的生物時鐘除了調控周而復始的光合作用、醣類合成分解、植物激素(Phytohormone)及二次代謝物(secondary metabolites)的合成等基礎生理途徑，植物也可藉由生物時鐘來計算日照的長短以分辨季節，進而調控植物季節性的生長、開花、冬眠等。除此之外，植物的生物時鐘可預知環境物理性(如:光、溫度、水分等)及生物性(如:真菌、昆蟲)週期的變化，並調整一天內對生物性(biotic)及非生物性(abiotic)的反應靈敏度，以調整植物生理及生長。生物時鐘調控植物生理的現象和機制已逐漸應用於在農業上，例如：調控開花時間、增加作物產量、增進抗逆境的反應,或是增加農藥施用的效率等。然而，仍有許多植物生理時鐘調控的生理途徑尚未被發掘，而且大部分生物時鐘調控植物生理途徑的機制仍未被完全了解。

隨著氣候變遷地球暖化，高溫對植物的生長發育有極大的影響。而溫度也會影響生物時鐘的節律溫度，使其週期變快（<24 小時），為了維持生物時鐘植物也會慢慢將週期調整回24小時，稱為溫度補償效應（Temperature compensation)。我們找到一些E3泛素連結酶和去泛素化酶(DUB)在高溫下參與泛素化調控生物時鐘，此外我們也用影像分析系統和阿拉伯芥近交種(natural accession)欲找尋新的調控因子。

Due to the rotation of the Earth resulting in rhythmic light and temperature cycles, living organisms have evolved an approximately 24-hour circadian clock. The circadian clock regulates the metabolism and physiological pathways of organisms, allowing them to efficiently use resources and adapt to their environment for survival. While the circadian clocks of plants share similarities with those of other organisms, they are particularly complex due to the immobile lifestyle of plants. They constantly perceive expected and unexpected environmental changes and then respond to the environment. Studies have found that plant circadian clocks regulate basic physiological pathways such as photosynthesis, carbohydrate biosynthesis and catabolism, as well the synthesis of phytohormones and secondary metabolites. Additionally, plant circadian clocks can calculate the day-length to distinguish between seasons to egulate plant seasonal growth, flowering, dormancy, etc. Furthermore, plant circadian clocks can predict the periodic changes in environmental factors (such as light, temperature, and water) and biotic factors (such as fungi and insects), and adjust the sensitivity of biotic and abiotic responses within a day to regulate plant physiology and growth. The phenomenon and mechanism of the circadian clock-regulated physiology have gradually been applied in agriculture, such as regulating the flowering time, increasing crop yield, enhancing stress response, or improving pesticide application efficiency, etc. However, many physiological pathways regulated by the plant circadian clock have yet to be discovered, and the mechanisms by which the circadian clock governs plant physiology are still not fully understood.

As global warming and climate change, high temperature has significant impacts on plant growth and development. Temperature also affects the rhythm of the circadian clock, causing the clock runs faster (<24 hours). In order to maintain the circadian clock, plants slowly adjust their cycle back to 24 hours, which is called "temperature compensation." We have found that some E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) are involved in the ubiquitination regulation of the circadian clock at high temperatures. Also, we apply imaging systems and Arabidopsis natural accession to search for new regulators.

Ubiquitin-mediated protein degradation in Arabidopsis heat stress responses

 泛素化調控阿拉伯芥高溫逆境反應

Plants have different levels of tolerance to stress. In high-temperature stress, some plants can produce heat shock factors (HSFs) and heat shock proteins (HSPs) under high temperatures to alleviate the misfolding of proteins caused by high temperatures and maintain protein and cell functions. In addition, degrading some misfolded proteins or degrading heat shock transcription proteins when plants return to normal temperatures is also an important part of high-temperature stress regulation. We are currently studying how E3 ubiquitin ligases and protein ubiquitination participate in regulating protein balance under heat stress. 

ubiquitin signaling-regulated RNA processing in Arabidopsis floral regulatory pathways

泛素化藉由影響RNA剪切在開花時間的調控

   植物開花受到環境因子和內生的生理途徑共同調控，以確保植物在最適當的時間開花並成功繁衍子代。在眾多的調控路徑中RNA剪切(RNA splicing)被認為重要但仍未大量探索的方向。RNA 剪切由U1、U2、U4/U6 、U5等SnRNPs及其他調控蛋白組成剪切體(spliceosome)，而剪切體會與一個具保守性的MAC 蛋白體(MAC complex)結合並調節剪切體功能，進而影響 RNA剪切的結果。
在MAC蛋白體中有一對E3泛素連結酶MAC3A和MAC3B， 它們被認為可以泛素化U4 snRNP，而在過去的研究上也被發現它們的突變體中將近50%RNA剪切受到影響。此外這個突變體在生物時鐘、開花、逆境反應等都有缺失。我們目前以MAC3A與MAC3B泛素化調控為題，以探究MAC3A和MAC3B在RNA 剪切及開花調控上扮演的角色。

 Flowering time is tightly regulated by both environmental factors and endogenous physiological pathways to ensure plants flower at the optimal time and successfully reproduce offspring. Among many regulatory pathways, RNA splicing is considered an important but still largely unexplored field. Spliceosome, which is composed of U1, U2, U4/U6, U5 snRNPs and other regulatory proteins, executes RNA splicing. The spliceosome binds to a conserved MAC complex to regulate its function, thereby affecting the outcome of RNA splicing. Within the MAC complex, there is a pair of E3 ubiquitin ligases, MAC3A and MAC3B, which are thought to ubiquitinate U4 snRNP. In previous studies,  it has been found that the splicing of approximately 50% of transcripts is affected in the MAC3A/MAC3B mutants. Furthermore, this mutant showed defects in the circadian clock, flowering time, and stress responses. Currently, we are studying the regulation of MAC3A and MAC3B ubiquitination to explore their roles in RNA splicing and flowering regulation.