淹水後復氧期間氧化逆境對甘藍淹水耐受性之角色

Abstract

甘藍(Brassica oleracea L. var. capitata)是全球重要的蔬菜作物之一。在台灣，颱風伴隨之暴雨經常導致淹水災情使得甘藍產量大量損失，使得現今產業上有發展耐淹水甘藍品種之需求。了解甘藍於淹水後反應有助於耐淹水甘藍品種之選育，故本研究欲利用淹水耐受性不同之甘藍品種，探討淹水後回復期甘藍耐淹水之機制。期望挑選耐淹水標誌以應用於品種改良。 首先為了定義不同甘藍品種之耐淹水能力，調查三甘藍商業品種‘大蕊’、‘初秋’及‘228’避光淹水後之性狀。分析甘藍品種生理特性，‘大蕊’於回復期之有最小之葉片傷害及株高萎縮量，且其鮮重及相對水含量也較穩定，由此推測‘大蕊’為三品種中較耐淹水之品種。淹水的主要傷害在退水後之回復期。退水後由於高光強度、高氧氣含量使氧化逆境產生，導致細胞傷害嚴重。為了呈現三甘藍品種淹水後氧化逆境之差異，利用3',3'-diaminobenzendine (DAB)染色及nitro blue tetrazolium chloride (NBT)做組織化學染色。過氧化氫(hydrogen peroxide, H2O2)和超氧陰離子(superoxide anion, O2∙−)於‘初秋’及‘228’葉子累積明顯，而於‘大蕊’中並無此現象。此外，‘初秋’及‘228’之H2O2及丙二醛(malondialdehyde, MDA)含量多於‘大蕊’。以此結果可推測耐淹水品種‘大蕊’有更強之能力以緩解氧化逆境。本研究接著探索回復期之淹水耐受性跟活性氧化物(reactive oxygen species, ROS)生合成基因與抗氧化能力之關聯。就甘藍品種生化特性而言，於回復期間，‘大蕊’相較於‘初秋’及‘228’ 有較高之過氧化酶(peroxidase, POX)活性及還原態抗壞血酸(ascorbic acid, AsA)含量。就基因表現量來說，‘初秋’及‘228’有較高之ROS生合成基因RESPIRATORY BURST OXIDASE HOMOLOG D-LIKE (RBOHD-L)表現量，可能進而導致ROS之累積。作用於防止粒線體過多ROS形成之ALTERNATIVE OXIDASE1a (AOX1a)基因於‘大蕊’中表現量較高，暗示‘大蕊’緩解氧化逆境傷害能力較佳。 總體而言，本研究將有助於了解甘藍耐淹水機制。其中，測量指標如葉片受傷程度、鮮重、株高及相對水含量、H2O2、MDA和AsA之含量及RBOHD-L和AOX1a之基因表現量，具作為淹水耐受性之指標之潛力，期望有助於加速耐淹水甘藍品種之育種。

Cabbage (Brassica oleracea L. var. capitata) is one of the most important vegetable crops grown worldwide. In Taiwan, strong typhoons with torrential rain flood many areas and often result in large losses in cabbage production. There is a need to develop submergence tolerant cabbage cultivars in commercial. Understanding the response of cabbage after flooding is helpful to the selection of submergence tolerant cabbage cultivars. Therefore, our study intends to use cabbage cultivars with different submergence tolerance and investigated mechanisms of their tolerance upon recovery stage after submergence. We expect to develop markers that are closely linked to submergence tolerance for breeding. To define submergence tolerance in different genotypes of cabbages, phenotypes were assessed for three commercial cabbage cultivars ‘Fuyudori’, ‘K-Y cross’ and ‘228’ after submerged in dark. With respect to physiological observation, ‘Fuyudori’ had least damaged leaves and wilted shoots in height upon recovery stage, and its fresh weight and relative water content (RWC) were relative stable, suggesting that ‘Fuyudori’ is the most flooding-tolerant cultivar among the three cultivars tested. Major damage from submergence occurs during recovery stage after flooding. Desubmergence leads to serious cellular injury due to oxidative stress caused by higher light intensity and oxygen concentration. To visualize the difference of oxidative stress among three cabbage cultivars after submergence, histochemical staining were carried out using 3',3'-diaminobenzendine (DAB) and nitro blue tetrazolium chloride (NBT) staining. The accumulation of hydrogen peroxide (H2O2) and superoxide anion (O2∙−) was pronounced in ‘K-Y cross’ and ‘228’ leaves but not in ‘Fuyudori’. Moreover, ‘K-Y cross’ and ‘228’ had higher amount of H2O2 and malondialdehyde (MDA) than ‘Fuyudori’ did. These results suggest submergence tolerant cultivar ‘Fuyudori’ has higher ability to alleviate oxidative stress. We further explored the relationship among submergence tolerance, reactive oxygen species (ROS) synthesis gene and antioxidant ability. With respect to biochemical observation, ‘Fuyudori’ had higher activity of peroxidase (POX) and content of reduced ascorbic acid (AsA) than ‘K-Y cross’ and ‘228’ did throughout the recovery. In terms of gene expression level, transcript level of ROS synthesis gene RESPIRATORY BURST OXIDASE HOMOLOG D-LIKE (RBOHD-L) was higher in ‘K-Y cross’ and ‘228’, which suggests a higher accumulation of ROS. The transcript level of ALTERNATIVE OXIDASE1a (AOX1a) which functions to prevent excess mitochondrial ROS formation was higher in ‘Fuyudori’, suggesting higher ability to alleviate oxidative stress. Taken together, this study contributes to our understanding of the regulatory mechanisms underlying the flooding tolerance in cabbage. The indicators such as leaf damage, fresh weight, height and RWC, the content of H2O2, MDA and AsA, and transcript level of RBOHD-L and AOX1a are potential markers for submergence tolerant selection to accelerate cabbage breeding for submergence tolerance.