青蔥幼苗於淹水及復氧期間之轉錄與生理變化

Abstract

青蔥(Allium fistulosum L.)在台灣容易遭受颱風與強降雨造成的淹水逆境，使生產受阻，然而青蔥的耐淹水性評估與其生理機制之研究資訊相當有限。本研究探討台灣常見的青蔥栽培品種‘三星蔥’ (‘Sanxingcong’)及‘北蔥’ (‘Beicong’)於淹水逆境下的反應，從外觀性狀、內部抗氧化生理反應，及缺氧相關基因之表現量，探討青蔥之耐淹水性與淹水逆境下之生理調控機制。‘北蔥’在淹水及退水後的復氧期間具有比‘三星蔥’佳的生長勢，推測‘北蔥’為較耐淹水品種。淹水導致的缺氧逆境及退水後回復期造成的氧化逆境會增加活性氧化物(reactive oxygen species, ROS)的產生，其中‘三星蔥’具有較高之過氧化氫(hydrogen peroxide, H2O2)含量。在抗氧化能力方面，‘北蔥’的抗壞血酸(ascorbic acid, AsA)相對含量在復氧期間高於‘三星蔥’，丙二醛(malondialdehyde, MDA)含量較‘三星蔥’低。抗氧化酵素活性方面，‘北蔥’之抗壞血酸過氧化酶(ascorbate peroxidase, APX)活性較‘三星蔥’高。為了解青蔥於淹水及復氧期間之基因表現變化，本研究藉由次世代核醣核酸定序分析進行青蔥轉錄體之從頭組裝定序，共獲得120,076個被註釋的unigenes。分析缺氧反應相關基因表現之結果顯示，乙烯反應因子(ethylene response factor, ERF) ERF5、ERF4及ERFVIIs家族的RAP2.12在兩青蔥品種淹水6小時後表現提升，其中‘三星蔥’的ERF5表現量在淹水及復氧期間均較‘北蔥’高；無氧呼吸相關基因ALCOHOL DEHYDROGENASE1(ADH1)在‘北蔥’淹水6小時後具有明顯的上調，推測北蔥具有在淹水逆境下維持能量生產的能力；‘三星蔥’於淹水後具較高的ROS生合成基因RESPIRATION BURST OXIDASE HOMOLOG D (ROBHD)表現量，可能與其累積較多之ROS相關。綜觀所述，造成兩青蔥品種耐淹水性差異之原因可能來自於抗氧化能力與無氧呼吸的誘導。本研究有助於理解青蔥抵抗淹水逆境之機制，並建立青蔥耐淹水性狀之篩選平台，其中假莖長、相對含水量、H2O2、MDA、AsA含量、APX活性具有做為耐淹水青蔥篩選指標之潛力。

Welsh onion (Allium fistulosum L.) yields are often reduced due to flooding stress caused by typhoons and intense rainfall in Taiwan. However, the research on the physiological mechanisms of Welsh onion under flooding is limited. This study aims to explore the response of two Taiwan common Welsh onion varieties, ‘Sanxingcong’ and ‘Beicong’, after submergence. The phenotypes, antioxidant physiology, and hypoxia-related gene expression were measured to evaluate the flooding tolerance and physiology regulatory mechanism of Welsh onion. 'Beicong' was verified as a submergence-tolerant cultivar with its better shoot growth after submergence. Hypoxia stress caused by submergence and oxidative stress during reoxygenation after submergence will increase reactive oxygen species (ROS) generation. To survey the content of reactive oxygen species, and superoxide anion (O2-) was observed with nitro blue tetrazolium chloride (NBT). O2- accumulation started in the leaf tips of ‘Sanxingcong’ after submergence for 24 hours. For antioxidant ability, ‘Beicong’ has higher relative content of ascorbic acid (AsA) than ‘Sanxingcong’ during reoxygenation, and the content of malondialdehyde (MDA) is lower in contrast. Ascorbate peroxidase (APX) activity in ‘Beicong’ is higher than in ‘Sanxingcong’. To identify gene expression during submergence and desubmergence, the transcriptome of Welsh onion was sequenced using next generation RNA sequencing (RNA-Seq) technology. After de novo assembly, a total of 120,076 annotated unigenes were obtained. Genes related to hypoxia response, including ETHYLENE RESPONSE FACTOR 5 (ERF5), ERF4 and the group ERFVIIs family gene RAP2.12 were up regulated after submergence for 6 hours in two Welsh onion cultivars. ‘Sanxingcong’ have higher ERF5 expression during submergence and desubmergence. Fermentation related gene ALCOHOL DEHYDROGENASE1 (ADH1) was highly enriched in ‘Beicong’ after submergence for 6 hours, which is evaluated that ‘Beicong’ can keep producing energy by fermentation when suffering from flooding stress. The expression level of ROS synthesis gene RESPIRATION BURST OXIDASE HOMOLOG D (ROBHD) was higher in ‘Sanxingcong’, which might relate to the higher accumulation of ROS. In summary, the reasons contributing to the different flooding tolerance between two Welsh onions may be the antioxidant ability and the induction of anaerobic respiration. This study contributes to understanding the regulatory mechanism of Welsh onion in response to flooding stress and to setting the selection platform of flooding tolerance in Welsh onion. The indicators such as pseudostem length, pseudostem width, relative water content (RWC), the content of H2O2, MDA and AsA, and activity of APX are potential markers for submergence tolerant selection to Welsh onion.