探討過表達nbe-miR169對圓葉菸草於乾旱逆境之生理影響

Abstract

近年已有多項研究表明微型核醣核酸(microRNA, miRNA)在植物對抗非生物性逆境中扮演重要角色，其中，miR169及其目標基因-核因子轉錄因子家族(Nuclear factor Y, NF-Y)，與植株生長、發育、乾旱與缺氮逆境耐受性之調控有關。本實驗室先前研究觀察到大量表達圓葉菸草(Nicotiana benthamiana) miR169 (nbe-miR169)可使轉殖圓葉菸草(OE169)於乾旱後復水具有較好的恢復能力。本研究針對野生型(WT)及OE169圓葉菸草進行一系列的生理調查，並結合噴施ABA及ABA生合成抑制劑(Fluridone, FLU)，單獨探討植株地上部及地下部之生理反應，欲釐清nbe-miR169背後之生理機制。結果顯示，地上部噴施ABA後，OE169於乾旱狀態下可維持較WT良好之外觀及地上部鮮重，同時可維持較低的氣孔相對開啟面積、過氧化氫(H2O2)及丙二醛(MDA)之氧化逆境指標；然而噴施FLU後，OE169於乾旱狀態下植株葉片明顯下垂，且OE1-1-2與OE3-5-3於乾旱狀態下MDA濃度皆顯著高於WT，並於復水後喪失原本良好的復水能力，進而推測nbe-miR169應受ABA介導，且當OE169之ABA生合成受抑制時，可能會提升對乾旱逆境的敏感程度。另外，WT無論於正常灌溉及乾旱狀態下，氣孔密度皆顯著高於OE169，且無論是否噴施ABA及FLU，OE169於乾旱狀態下與復水後之光合作用速率及Ci/Ca比值等，皆與WT無顯著差異，顯示大量表現nbe-miR169之轉殖株，可能是透過減少氣孔密度以增加乾旱耐受性，同時以不影響光合系統之狀態下來增加植株乾旱耐受性。最後，地下部相關調查結果顯示OE169之根系外觀、鮮重、根長、吸水量及根系活力指數皆與WT無顯著差異，推測nbe-miR169可能不是透過增加根系吸收水分的面積，或是增強根系的吸水能力與活力來提升乾旱耐受性。

Several recent studies have shown that microRNAs (miRNAs) play important roles in plant’s response to abiotic stresses. Among them, miR169s and its target genes- Nuclear Factor Y (NF-Y), have been implicated in plant growth, development, drought, and nitrogen stress tolerance. Previous studies in our laboratory have observed that overexpression of Nicotiana benthamiana (tobacco) miR169 (nbe-miR169) in tobacco can make the transgenic tobacco (OE169) enhance recovery ability after drought stress. In this study, we conducted a series of physiological investigations on wild-type (WT) and OE169 tobacco to elucidate the physiological mechanisms underlying miR169-mediated responses. Specifically, we investigated the effects of foliar spray of ABA and ABA biosynthesis inhibitor (Fluridone, FLU) on the shoot and root responses of the plants under drought conditions. The results showed that after spraying ABA on the shoot, OE169 not only could maintain a better appearance, higher shoot fresh weight but also could maintain a lower stomatal relative area, hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentration (both are oxidative stress indicators) than WT under drought stress. However, FLU application caused leaf wilting in OE169 under drought stress, and both lines of OE169 (OE1-1-2 and OE3-5-3) had significantly higher MDA concentrations than WT. Additionally, three OE169 lines lost their original recovery ability after rewatering, suggesting that the miR169 response is likely ABA-mediated. Furthermore, when ABA biosynthesis is inhibited in OE169, it may increase the sensitivity to drought stress. In addition, WT showed significantly higher stomatal density than OE169 under regular irrigation and drought conditions. Additionally, regardless of ABA and FLU application, the photosynthetic rate and Ci/Ca ratio of OE169 showed no significant differences compared to WT under drought stress and after rewatering. The above results imply that OE169 may enhance drought tolerance by reducing stomatal density without affecting the photosynthetic system. Finally, the investigation of the root system revealed no significant differences in root appearance, fresh weight, length, water uptake, and root activity index between OE169 and WT. It is speculated that nbe-miR169 may not enhance drought tolerance by increasing the root's water absorption surface area or by enhancing the root's water uptake capacity and activity.