SUB1A-1在水稻淹水逆境中調控表觀遺傳及基因的轉錄

SUB1A-1 anchors a regulatory cascade for epigenetic and transcriptional control in rice under submergence

李婉嘉(Wan-Jia Lee)

指導教授 ： 施明哲

國立臺灣大學/生命科學院/植物科學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202000139

摘要

大部分的水稻都無法耐受長時間的淹水逆境。然而，部分秈稻品系如FR13A因帶有SUBMERGENCE 1A-1（SUB1A-1）基因，因此能耐受淹水逆境；帶有SUB1A-2的秈稻品系卻無法耐受淹水逆境。SUB1A屬於第七群乙烯轉錄因子（group VII ethylene-responsive factor, ERFVII）之一，含有兩個等位基因, SUB1A-1及SUB1A-2。SUB1A-1及SUB1A-2之間僅在第186的氨基酸序列不同，前者帶有Serine，而後者帶有Proline，卻因而造成只有SUB1A-1可被磷酸化。另外，SUB1A-1含有兩個下游基因，ERF66及ERF67，皆同屬於ERFVII,可共同調控淹水逆境。目前雖已找出SUB1A-1的下游基因，但SUB1A-1啟動ERF66及ERF67轉錄的分子機制仍不清楚。
本研究顯示SUB1A-1能與組蛋白乙酰化複合體ADA2b-GCN5相互結合。其中，GCN5是一個組蛋白乙酰基酶。有趣的是，SUB1A-1的磷酸化可增強其與組蛋白乙酰化複合體ADA2b-GCN5結合的能力；反之，SUB1A-2因無法被磷酸化而不能結合。此外yeast two-hybrid 顯示，ADA2b的RLR區域為ADA2b與SUB1A-1相互結合的主要區域。在淹水逆境下，ADA2b及GCN5的基因大量表現，且H3K9組蛋白的乙酰化修飾能力提高。另外，EMSA的結果顯示SUB1A-1的磷酸化能增強其結合上ERF66/ERF67啟動子的能力，且同時過量表達SUB1A-1及組蛋白乙酰化複合體ADA2b-GCN5可顯著提高ERF67的基因表現量。綜合以上所述，我們推測SUB1A-1的磷酸化能促使SUB1A-1與組蛋白乙酰化複合體ADA2b-GCN5結合，並透過改變ERF66/ERF67的染色體結構來促進基因的表達，從而增強水稻對淹水的耐受性。
淹水逆境不僅能促進H3K9的乙酰化修飾，更能提高H3K4的三甲基化修飾，表示不同的組蛋白表觀修飾之間會相互作用。此外，我們鑑定出了數個會與SUB1A-1結合的蛋白質。其中，屬於GT因子的HRA1-L1可能參與在淹水逆境的負向調控機制中。故，我們認為SUB1A-1可與多個轉錄活化因子或轉錄抑制因子作用，以便能快速應對淹水逆境。

關鍵字

淹水耐受性 ； 水稻 ； ADA2b-GCN5組蛋白乙酰化複合體 ； 組蛋白乙酰化 ； 磷酸化 ； 負向調控因子

並列摘要

Most rice (Oryza sativa) cultivars cannot survive under complete submergence. Some O. sativa ssp. indica cultivars, such a FR13A, are highly tolerant owing to SUBMERGENCE 1A-1 (SUB1A-1), which encodes a group VII ethylene-responsive factor (ERFVII), while other submergence intolerant indica cultivars contain an intolerance-specific allele SUB1A-2. In fact, the two alleles differ only by a single substitution at 186th amino acid position from serine in SUB1A-1 to proline in SUB1A-2, resulting in only SUB1A-1 could be phosphorylated. Besides, two others ERFVIIs, ERF66 and ERF67, function downstream of SUB1A-1 to form a regulatory cascade in response to submergence stress. To date, the underlying mechanism of how SUB1A-1 activates ERF66 and ERF67 transcription is poorly understood.
Herein we showed that SUB1A-1 interacts with ADA2b of the ADA2b-GCN5 acetyltransferase complex, of which GCN5 functions as a histone acetyltransferase. Interestingly, phosphorylation of SUB1A-1 enhanced interaction of SUB1A-1 with ADA2b, as SUB1A-2 cannot interact with ADA2b. In addition, yeast two-hybrid revealed that RLR region of ADA2b is required for ADA2b to interact with SUB1A-1. ADA2b and GCN5 were activated in response to submergence in FR13A and that H3K9 acetylation level was induced during submergence. Furthermore, EMSA showed that phosphorylation of SUB1A-1 enhanced binding to ERF66/ERF67 promoters. The transcript level of ERF67 was highly induced when overexpressing SUB1A-1 together with ADA2b-GCN5 acetyltransferase complex. Taken together, our results suggest that phospho-SUB1A-1 recruits the ADA2-GCN5 acetyltransferase complex to modify chromatin structure on ERF66/ERF67 promoter regions and activates gene expression, which in turn enhances rice submergence tolerance.
As well as H3K9 acetylation level, H3K4 trimethylation level was induced under submergence, suggesting that crosstalk between histone modification occurs. Besides, we identified several interacting partners of SUB1A-1. Among them, HRA1-L1, which encodes a GT factor, may mediate negative regulation under submergence. We propose that SUB1A-1 may coordinate with additional activators and repressors in order to rapidly response to submergence stress.

並列關鍵字

submergence ； rice ； ADA2b-GCN5 acetyltransferase complex ； histone acetylation ； phosphorylation ； negative regulator

參考文獻

Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., & Saltiel, A. R. (1995). PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270: 27489-27494.

Allen, M. D., Yamasaki, K., Ohme‐Takagi, M., Tateno, M., & Suzuki, M. (1998). A novel mode of DNA recognition by a β‐sheet revealed by the solution structure of the GCC‐box binding domain in complex with DNA. EMBO J. 17: 5484-5496.

Alpuerto, J. B., Hussain, R. M. F., & Fukao, T. (2016). The key regulator of submergence tolerance, SUB1A, promotes photosynthetic and metabolic recovery from submergence damage in rice leaves. Plant Cell Environ. 39: 672-684.

Aquea, F., Timmermann, T., & Herrera-Vásquez, A. (2017). Chemical inhibition of the histone acetyltransferase activity in Arabidopsis thaliana. Biochem. Bioph. Res. Co. 483: 664-668.

Ayano, M., Kani, T., Kojima, M., Sakakibara, H., Kitaoka, T., Kuroha, T., . . . Ashikari, M. (2014). Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice. Plant Cell Environ. 37: 2313-2324.