阿拉伯芥生長素相關基因之R-loop對其基因表現量之影響

Abstract

R-loop是由DNA-RNA雜合體 (hybrid) 及一未配對單股DNA所構成之〝三股核酸結構〞，R-loop已在許多物種中被發現參與轉錄層次的調控。然而植物中關於R-loop之研究仍剛起步。本實驗室先前研究顯示，在阿拉伯芥側根發育中扮演重要角色的生長素相關基因LBD18 (LATERAL ORGAN BOUNDARIES DOMAIN18)，會在體外實驗中 (in vitro) 形成R-loop。本研究利用吲哚-3-乙酸 (IAA) 與可促進R-loop累積之DNA拓樸異構酶抑制劑喜樹鹼 (CPT) 處理，探討在阿拉伯芥中R-loop對生長素相關基因之調控。研究結果顯示CPT處理會抑制側根的生長，且針對在生長素訊息傳遞路徑中具代表性的十二個基因分析其mRNA表現量，結果指出CPT處理會降低LAX3、PIN1、IAA14及LBD16的表現量約30-50%，暗示了此四基因較有可能形成R-loop。為探討上述結果確實是由R-loop所造成，進一步使用可辨認R-loop之DNA-RNA雜合體 (hybrid) 的S9.6單株抗體，進行DNA-RNA免疫沉澱法接續qPCR (DRIP-qPCR) 測量各基因之R-loop多寡，結果顯示在未處理CPT或IAA的情況下，PIN1、IAA14及LBD18之R-loop就比預期不受R-loop調控之ARF19高出約4-6倍，此外CPT處理後PIN1之R-loop增加約40-50%，且其mRNA表現量減少50%，PIN1為生長素的運輸者，在側根發育前期扮演建立生長素最大濃度的角色，綜合上述結果，R-loop可能藉由參與PIN1之調控，進而影響阿拉伯芥的側根發育。

R-loop is a “three-stranded nucleic acids complex” including a DNA-RNA hybrid and an unpaired single-stranded DNA. R-loop has been identified to participate in transcriptional regulation in various organisms. However, the R-loop research in plants is still limited. Previous studies in our laboratory suggested that LBD18 (LATERAL ORGAN BOUNDARIES DOMAIN18), one of auxin-related genes, could form R-loop in vitro. Indole-3-acetic acid (IAA) and camptothecin (CPT, a DNA topoisomerase 1 inhibitor) were used in this study to investigate the roles of R-loop in the regulation of auxin-related genes in Arabidopsis. It is expected that CPT treatment will lead to R-loop accumulation. Phenotypic analysis showed that the lateral root growth was inhibited in CPT-treated Arabidopsis. Twelve representative auxin-related genes were selected from the auxin signaling pathway to analyze their mRNA expression level. The results showed that the expression of LAX3, PIN1, IAA14 and LBD16 decreased about 30-50% after the CPT treatment. It implied that these four genes had higher R-loop forming potential than that in other genes. To further investigate if above results were actually caused by R-loops, S9.6 monoclonal antibody was employed to recognize DNA-RNA hybrids of R-loops to conduct DRIP-qPCR (DNA-RNA immunoprecipitation followed by qPCR) to quantify R-loop enrichment of individual gene. The results showed that R-loops at PIN1, IAA14 and LBD18 were 4-6 fold higher than ARF19 which was expected not to be regulated by R-loops under the condition of no IAA or CPT treatment. In addition, R-loops at PIN1 increased about 40-50% while its mRNA expression decreased 50% after the CPT treatment. PIN1 functions as an auxin transporter that establishes of an auxin maximum in the early stage of lateral root development. In summary, R-loops might modulate lateral root development via participating in the regulation of PIN1 in Arabidopsis.