植物生長調節劑巴克素 對阿拉伯芥以及小球藻之鏡像選擇性影響

Abstract

目前約有 30 % 農藥，成分為一個或多個鏡像異構物混合組成可稱為掌性農藥。巴克素 (Paclobutrazol, PBZ) 為三唑類農藥中的掌性農藥，可作為植物生長調節劑和殺菌劑使用。在台灣巴克素使用的相當普遍，主要用於矮化，屬殘長效性農藥，因此值得深入探討此方面研究。本研究目的為比較巴克素鏡像異構物及消旋體對阿拉伯芥以及小球藻的毒性差異，以利未來更審慎評估減少掌性農藥使用。本研究建立三唑類植物生長調節劑巴克素之鏡像異構物分離方法，研究其消旋體及不同鏡像異構物對目標生物阿拉伯芥及非目標生物小球藻的影響。利用高效液相層析方法搭配 Chiralcel® OJ-H 對掌性管柱，動相為正己烷/異丙醇 (90/10,v/v)，流速1 ml min-1之條件下，可得到有效率之巴克素異構物分離方法。以高效液相層析儀、氣相層析質譜儀、圓二色光譜分析儀及旋光度計鑑定後得到收集巴克素的鏡像異構物分別為 (2R,3R,+)-巴克素及 (2S,3S,－)-巴克素 (peak1 和 peak2)，純度達 99 %。阿拉伯芥在相同濃度的巴克素消旋體及其鏡像異構物 20 天後，外觀變化、生理指標或氧化逆境中，(2S,3S,－)-巴克素抑制植物生長顯著，例如植物面積較小。葉綠素含量、脯氨酸以及丙二醛濃度明顯提升，顯示 (2S,3S,－)-巴克素會造成阿拉伯芥較顯著逆境。且活性氧相對含量、抗氧化酵素比活性也都有相同趨勢，(2R,3R,+)-巴克素與控制組相比則無顯著差異。而 (2R,3R,+)-巴克素具有促進植物生長的現象，例如：葉片面積、根部鮮重顯著增加，與 (2S,3S,－)-巴克素相比，鏡像異構物間具相反效用。水生環境中小球藻，毒性為 (2R,3R,+)-巴克素影響較大。故未來具潛力朝單一鏡像異構物 (2S,3S,－)-巴克素農藥進行抑制植物生長的調節劑，或 (2R,3R,+)-巴克素能夠作為促進植物生長的調節劑，對於提高植物生長調節及降低農藥使用量皆有明顯的效用。此外 (2S,3S,－)-巴克素對非目標生物影響較小，具降低農藥風險能力。

From an environmentally-friendly perspective, it is ideal to eliminate an enantiomer of a less dominant activity, leaving another with higher activity. This may potentially reduce pollution and the amount of pesticide used, improve target specificity and promote sustainability. Paclobutrazol is one type that is used as both plant growth regulator and fungicide. It is frequently used in Taiwan and due to its long residual action, becomes critically researched plant growth regulator. The purpose of this study is to compare the toxicity between racemate paclobutrazol and its enantiomers on Arabidopsis thaliana and Chlorella vulgaris in order to carefully assess the future use of chiral pesticides. In this study, the effects of liquid chromatographic mobile phase composition and flow rate on separation efficiency were investigated, and the enantiomers were also collected to study the enantioselective effects of paclobutrazol. Successful enantiomeric separation of paclobutrazol by HPLC equipped with a chiral selective column was performed. The separated enantiomers were characterized by HPLC, GC-MS, CD spectrum and polarimeter. The purity of these enantiomers were 99%. The selective effects of the enantiomers of paclobutrazol on wild type A. thaliana were investigated. The appearance, physiological metabolism or oxidative stress were significantly affected by (2S,3S,-)-Paclobutrazol compared to (2R,3R,+)-Paclobutrazol. The leaf area were smaller, and moderation effect were more significant. The chlorophyll, proline content and MDA concentration were increased. ROS and antioxidant enzymes had the same trend. (2R,3R,+)-Paclobutrazol had no difference compared to control. Paclobutrazol inhibited the growth of C. vulgaris, and their IC50 values following the order: (2R,3R,+)-Paclobutrazol < (2S,3S,-)-Paclobutrazol. As such, this result showed a potential of developing effective paclobutrazol using only enantiomers of higher activities, thereby reducing usage amount and risk of pollution.