HDA6和HDA19藉由與AS1及AS2交互作用調控阿拉伯芥葉片形態

Abstract

在阿拉伯芥中，去乙醯化酵素可分成四大類，前三類是酵母菌及哺乳類動物中 RPD3、 HDA1和SIR2的同源基因，而第四類為HD2則是植物特有的。在RPD3中根據氨基酸的相似性又可細分為三個族群，其中class I包含了HDA6、HDA7、HDA9及HDA19。早期的研究發現HDA6和HDA19參與了很多生理途徑，例如調控胚胎發育，花的生長與開花時間以及植物逆境調節等，除此之外，HDA19也參與了光的調節路徑。然而， HDA7及HDA9的功能到目前為止還不是很明確。 在我們實驗研究中發現，HDA9的突變株開花時間提早以及果莢末端呈現膨大的現象，這表示了HDA9有可能參與開花調控路徑以及影響果實的發育。除此之外，在HDA6的突變株中，發現其葉片會產生波浪狀的型態。同樣的，HDA19 突變株中葉片也呈現鋸齒形狀，表示HDA6和HDA19有可能參與調控葉片的生長發育。 在阿拉伯芥裡，ASYMMETRIC LEAVES1 (AS1)及 AS2這兩個轉錄因子會去抑制class 1 KNOX 基因的表現控制葉片生長。經由雙分子螢光互補分析(BiFC) 及Pull- down assay發現，HDA6和HDA19會和AS1及AS2產生蛋白質交互作用。進一步分析KNOX基因表現發現，其中一個KNOX基因KNATM的表現在hda6和hda19突變株中有非常顯著的提升，說明HDA6和HDA19很有可能會藉由AS1及AS2一起作用去影響KNATM的表現。 染色質免疫沉澱的實驗結果表明，KNATM基因上組蛋白H3K9K14乙醯化程度的確在HDA6突變株中有明顯的增加，然而在HDA19突變株中組蛋白H3K9K14乙醯化程度和野生型比較並沒有太大的差異，然而，在組蛋白H3K4me2程度在HDA6及HDA19突變株中都有顯著的提升，說明HDA6和HDA19會影響KNATM的組蛋白甲基化而調控葉子的發育。

There are four groups of histone deacetylases (HDACs) in Arabidopsis, there of them are homologous to RPD3, HDA1 and SIR2 found in yeast and animal cells. The fourth group of histone deacetylases, HD2, is plant- specific. Base on the amino acid sequences, the RPD3-type HDACs can be further classified into three classes. The class I of RPD3-type HDACs contains HDA6, HDA7, HDA9 and HDA19. It is known that HDA6 and HDA19 are global repressors involved in embryonic and flower development as well as stress responses. In addition, HDA19 also participates in light responses. However, the functions of HDA7 and HDA9 are still not clear. In this study, it is found that HDA9 mutant was early flowering and had blunt silique tips, indicating that HDA9 may participate in flowering pathway and affect fruit development. The hda6 mutant, axe1-5, displayed the curling leaf phenotype. Similarly, hda19-1 T-DNA insertion mutant displayed the serrated leaves. These observations indicated that HDA6 and HDA19 may be involved in leaf development. In Arabidopsis, ASYMMETRIC LEAVES1 (AS1) and AS2 are required for the repression of class 1 KNOX genes for the leaf polarity establishment. The bimolecular fluorescence complementation and in vitro pull-down assays indicate that HDA6 and HDA19 can interact with AS1 and AS2, suggesting that HDA6 and HDA19 may form complex with the AS1 and AS2 to regulate KNOX gene expression. The expression of KNATM gene, one of the KNOX family genes, was up-regulated in both axe1-5 and hda19-1 mutants. Furthermore, chromatin immunoprecipitation assay showed that the level of H3K9K14 acetylation of the KNATM was increased in axe1-5 mutants, but not in hda19-1 Nevertheless, the H3K4 trimethylation of KNATM was elevated in these two mutants. These results indicate that HDA6 and HDA19 may affect KNATM gene expression by interacting with AS1 and AS2 to control leaf development.