高溫干旱下油菜的木質(zhì)化應(yīng)答及其在莖與根中的差異

尹能文 李加納 劉 雪 練劍平 付 春 李 威 蔣佳怡 薛雨飛 王 君 柴友榮

重慶市油菜工程技術(shù)研究中心 / 重慶市作物品質(zhì)改良重點實驗室 / 南方山地農(nóng)業(yè)教育部工程研究中心 / 西南大學(xué)農(nóng)學(xué)與生物科技學(xué)院, 重慶北碚 400715

高溫干旱下油菜的木質(zhì)化應(yīng)答及其在莖與根中的差異

尹能文**李加納**劉 雪 練劍平 付 春 李 威 蔣佳怡 薛雨飛 王 君 柴友榮*

重慶市油菜工程技術(shù)研究中心 / 重慶市作物品質(zhì)改良重點實驗室 / 南方山地農(nóng)業(yè)教育部工程研究中心 / 西南大學(xué)農(nóng)學(xué)與生物科技學(xué)院, 重慶北碚 400715

以正常生長和高溫干旱復(fù)合脅迫下甘藍型油菜中雙10號的莖和根為材料, 采用組織化學(xué)、生物化學(xué)、氣相色譜-質(zhì)譜聯(lián)用(GC-MS)分析技術(shù), 研究了木質(zhì)部結(jié)構(gòu)和木質(zhì)素成分的脅迫應(yīng)答規(guī)律及其在莖和根中的差別。冰凍切片組織化學(xué)染色顯示, 與正常生長的網(wǎng)室植株(正常植株)相比, 高溫干旱下生長的溫室植株(脅迫植株)的莖和根中木質(zhì)部均顯著加厚, 染色更深; 與此對應(yīng), 溴乙酰法測定的莖木質(zhì)素總量比對照增加31.64%。此外, 脅迫莖中的導(dǎo)管孔徑明顯變小, 但根中的導(dǎo)管孔徑和導(dǎo)管數(shù)量均明顯增加。硫代酸解法測定木質(zhì)素單體表明, 脅迫莖中被解離出的木質(zhì)素單體總量比對照降低40.08%, 說明有更高的縮合鍵比例; S/G值(1.82)比對照(1.29)大大增高, 說明S型木質(zhì)素比例增加而G型木質(zhì)素比例下降。油菜莖與根木質(zhì)化性狀比較顯示, 根木質(zhì)素比莖木質(zhì)素含有更高比例的縮合鍵, 莖中S型木質(zhì)素占主體(S/G=1.29), 而根中G型木質(zhì)素占主體(S/G=0.49)且H型木質(zhì)素含量(7.43%)比莖中(0.83%)高近10倍。H型和G型木質(zhì)素單體的苯環(huán)甲基化程度比S型低, 單體間更容易形成縮合鍵, 根中高比例H型和G型木質(zhì)素單體可能是導(dǎo)致其具有高比例縮合鍵的主要原因。

油菜; 莖; 根; 木質(zhì)素; 高溫干旱脅迫

Abstract:Using histochemical, biochemical, and gas chromatography-mass spectrometry (GC-MS) technologies, the responsive trends of xylem structure and lignin components in the stem and the root of rapeseed (Brassica napus) cultivar Zhongshuang 10 under heat and drought stresses were investigated as compared with normal-growth plants. Histochemical staining of the frozen sections showed that, compared with the cage-grown plants (normal plants), the plants grown in greenhouse (stressed plants) had a thicker xylem part in both stem and root, with deeper staining color. Correspondingly, the total lignin content in the stem of stressed plants tested by acetyl bromide method increased by 31.64% compared with that of the normal plants. Besides, heat and drought compound stress reduced vessel inner diameter in the stem, while increased both vessel number and vessel inner diameter in the root. Stem total lignin monomer yield prepared by thioacidolysis of stressed plants was 40.08% lower than that of the normal plants, indicating higher condensed bonds in stressed plants. Meanwhile, the S/G ratio (1.82) was significantly higher thanthat of the normal plants (1.29), indicating increased proportion of S units and decreased proportion of G units. Furthermore, this study also revealed the difference of lignification traits between the stem and the root. The stem had lower condensed bonds and major proportion of S units (S/G=1.29), while the root had more condensed bonds and major proportion of G units (S/G=0.49).Moreover, the H unit percentage in the root (7.43%) was almost 10 folds as that in the stem (0.83%). Since H and G units have lower methylation levels and are easier to form condensed bonds, the high proportions of H and G units might be the main reason for the high proportion of condensed bonds in root lignin structure.

Keywords:Rapeseed (Brassica napus); Stem; Root; Lignin; Heat and drought stresses

木質(zhì)素是地球上僅次于纖維素的第二豐富的生物高聚物, 是一類復(fù)雜的苯丙烷類高分子聚合物,是由木質(zhì)素單體之間以碳鍵(少數(shù))或醚鍵的形式連接聚合而成, 分子量變異很大, 從幾百至幾百萬不等[1]。木質(zhì)素對植物生長發(fā)育和適應(yīng)逆境都起著非常重要的作用, 它是一種多酚類聚合物(芳香族聚合物), 主要沉積在植物的次生壁中, 其含量可占細胞壁成分的 30%[2]。特別是在管胞、石細胞、維管束間纖維和木質(zhì)部中, 木質(zhì)素在次生細胞壁中與纖維素、半纖維素、果膠等成分交聯(lián)在一起發(fā)揮作用[3],為細胞壁提供機械支撐強度和疏水性, 以防止在植物蒸騰作用時產(chǎn)生的負壓使細胞壁塌陷[4-5]。因此,木質(zhì)部結(jié)構(gòu)和木質(zhì)素成分對植物莖稈的硬度起著至關(guān)重要的作用, 使植物在重力的環(huán)境下可以生長得高大挺拔[6-8], 保障水分、礦質(zhì)營養(yǎng)、光合產(chǎn)物的運輸[9], 抵抗病原微生物和食草動物的侵入和損傷[10-11]。所以, 木質(zhì)素的形成和木質(zhì)部的發(fā)育常被認為是陸生維管植物從水生棲息地轉(zhuǎn)向陸地生態(tài)系統(tǒng)進化過程中的一個關(guān)鍵事件[7,12]。

木質(zhì)素這種異質(zhì)多酚聚合物通常情況下在被子植物中可分為 3種主要類型, 即對羥基苯基木質(zhì)素(p-hydroxyphenyl lignin, H型木質(zhì)素), 愈創(chuàng)木基木質(zhì)素(guaiacyl lignin, G型木質(zhì)素)和紫丁香基木質(zhì)素(syringyl lignin, S型木質(zhì)素), 它們分別是由各自對應(yīng)的木質(zhì)素單體構(gòu)成, 即對香豆醇(p-coumaryl alcohol)構(gòu)成H型木質(zhì)素, 松柏醇(coniferyl alcohol)構(gòu)成G型木質(zhì)素, 芥子醇(sinapyl alcohol)構(gòu)成S型木質(zhì)素[1,13-15], 而這3種木質(zhì)素單體的主要區(qū)別就在于它們苯環(huán)的羥基化和甲基化水平不同。在被子植物中, S型木質(zhì)素主要沉積在纖維細胞, 而G型木質(zhì)素主要沉積在管狀細胞壁中[16-17]。Li等[18]指出S型木質(zhì)素在植物中擁有較高的機械支撐屬性, 然而在單子葉植株中含有高比例S型木質(zhì)素不利于動物對其進行消化吸收[19-20]。

植物中木質(zhì)素的含量及其結(jié)構(gòu)成分會受生物或是非生物脅迫的影響而改變[21], 木質(zhì)素在增強植株機械強度和抵御高溫干旱和病蟲害方面起著積極的作用。在桉樹中較高的S/G值有利于植株的抗旱[22]。Eynck等[23]報道了在對核盤菌抗性較高的亞麻薺(Camelina sativa)中, 其組成型木質(zhì)素中的S型木質(zhì)素含量比抗性較弱的植株要明顯高很多, 指出 S型木質(zhì)素促進了亞麻薺植株組成型抗病性機制。當(dāng)小麥?zhǔn)艿降蜏孛{迫的時候, 葉片中可溶性酚類物質(zhì)有所增加并且木質(zhì)素含量會有所降低, 但在根中卻是相反的[24]。杜鵑花在低溫脅迫下, 木質(zhì)素合成相關(guān)基因 C3H的表達量有明顯上調(diào)[25]。在干旱脅迫下,植株的木質(zhì)素生物合成途徑中一些關(guān)鍵基因的表達將會受到誘導(dǎo)顯著上調(diào), 植株木質(zhì)化程度也會明顯增強[26-30]。徐宇強等[31]發(fā)現(xiàn), 耐旱性強的自交系比耐旱性弱的自交系的木質(zhì)素含量升高幅度更大。

木質(zhì)部結(jié)構(gòu)的發(fā)育以及木質(zhì)素的單體比例與植物發(fā)育生物學(xué)和環(huán)境生物學(xué)息息相關(guān), 而且地上部與地下部由于所處環(huán)境及擔(dān)負功能不同, 莖與根之間也可能在木質(zhì)部結(jié)構(gòu)和木質(zhì)素單體比例上存在差異, 但這方面目前還缺乏比較研究。此外, 正因為木質(zhì)部結(jié)構(gòu)和木質(zhì)素含量的改變是植物適應(yīng)環(huán)境變化的重要基礎(chǔ), 因此揭示植物根和莖的木質(zhì)部結(jié)構(gòu)和木質(zhì)素單體含量響應(yīng)逆境脅迫的變化規(guī)律并開展比較研究, 對于植物學(xué)和作物學(xué)研究具有重要的參考和指導(dǎo)意義。

甘藍型油菜(Brassica napus)是我國和世界的重要優(yōu)質(zhì)植物食用油的來源, 還是動物飼料和生物柴油的重要原料, 但生產(chǎn)上廣泛因高溫干旱脅迫而影響產(chǎn)量和品質(zhì)[32]。本研究以甘藍型油菜常規(guī)品種中雙 10號為材料, 分別種植于網(wǎng)室(正常植株)和溫室(高溫干旱脅迫植株), 分別對莖和根的木質(zhì)部進行解剖學(xué)觀察與比較及組織化學(xué)和生物化學(xué)比較研究,探索了高溫干旱脅迫下油菜的木質(zhì)化應(yīng)答, 并揭示了油菜莖與根在木質(zhì)化上的差異。

1 材料與方法

1.1 試驗材料及處理

甘藍型油菜常規(guī)品種中雙10號(ZS10)原始種子由中國農(nóng)業(yè)科學(xué)院油料作物研究所張學(xué)昆研究員惠贈。為盡量提高試驗結(jié)果的準(zhǔn)確性, 將非脅迫植株種植在溫室臨近的田間網(wǎng)室, 接近自然條件, 整個生長期間沒有顯著的高溫和干旱脅迫。將脅迫植株種植在玻璃溫室, 在油菜植株進入抽薹期至收獲期期間, 也就是在植株木質(zhì)素合成與積累的旺盛期,玻璃溫室內(nèi)溫度在陰雨天比田間網(wǎng)室平均高 2～5℃,在強日照時平均比田間網(wǎng)室高 5～10℃ (最高達 38℃), 可以很好模擬高溫干旱脅迫環(huán)境。當(dāng)脅迫植株葉片出現(xiàn)萎蔫跡象時適當(dāng)澆水(土壤含水量長時間保持在15%以下)。

1.2 植株組織器官冰凍切片及染色觀察

取成熟期植株的中部莖稈和根, 用于冰凍切片組織化學(xué)染色觀察和木質(zhì)素總體含量極其單體成分的分析。本研究創(chuàng)立了針對油菜各組織器官的切片方法。

1.2.1 樣品包埋 首先打開 Leica CM1850冰凍切片機, 將機箱溫度降到–20℃, 切取適合大小的新鮮莖稈和根的樣品包埋, 在包埋樣品時避免樣品周圍形成氣泡。

1.2.2 回溫處理 將機箱溫度回升到–8 ～ –12℃保持30 min以上, 切片時的機箱溫度可根據(jù)切片效果再次具體優(yōu)化。

1.2.3 切片 按照切片機使用說明書切片。將切好的樣片用預(yù)冷的毛筆或毛刷刷到盛水的培養(yǎng)皿里(最好事先在冰面預(yù)冷), 然后將培養(yǎng)皿放在冰面暫時保存。

1.2.4 切片組織化學(xué)染色 采用 Phloroglucinol-HCl和 M?ule染色法[16,33]。Phloroglucinol HCl染色順序為, 滴加適量 1%間苯三酚溶液在切片上處理3～5 min→滴加適量濃HCl顯色→蓋上蓋玻片吸掉多余的染液。M?ule染色: 在切片上滴加適量的1%高錳酸鉀溶液, 浸染5 min→用蒸餾水清洗3次→滴加3% HCl浸泡 2 min→用蒸餾水清洗 3～5次→滴加29%氨水浸 2 min→蓋上蓋玻片吸掉多余的染液。M?ule染色可使G型木質(zhì)素染為棕色, 使S型木質(zhì)素染為紅色[7,16,19]。

1.2.5 顯微觀察 將染色的樣片或者沒有經(jīng)染色處理的樣片放在 Nikon SMZ1500和 Olympus MUX10體視顯微鏡以及Nikon Eclipse E600顯微鏡下, 根據(jù)儀器使用說明書觀察。

1.3 總木質(zhì)素含量和木質(zhì)素單體成分的測定

所有提取試驗過程都在通風(fēng)櫥內(nèi)完成, 依據(jù)前人研究報道的方法[34-41], 并適當(dāng)調(diào)整和優(yōu)化處理。

1.3.1 細胞壁分離 將待測材料烘干打粉, 過 80目篩; 稱取60～70 mg樣品裝入帶螺旋蓋的2 mL離心管中進行細胞壁分離[34], 完成后加入無水乙醇脫水。

1.3.2 總木質(zhì)素含量測定 采用溴乙酰法[35-37]并有改進, 測定總木質(zhì)素含量。稱取上一步所得的細胞壁成分1.5～2 mg, 加入200 μL新鮮配制的25%溴乙酰溶液(用冰醋酸配制), 50℃條件下反應(yīng) 3 h, 期間每15 min振蕩混勻一次。反應(yīng)結(jié)束后冰浴至室溫,加2 mol L–1氫氧化鈉溶液800 μL和新鮮配制的0.5 mol L–1鹽酸羥胺溶液140 μL, 振蕩混勻。將混合液轉(zhuǎn)至 15 mL具塞刻度試管中, 加冰醋酸稀釋至 15 mL, 混勻, 測定280 nm波長下的吸光度值。根據(jù)公式計算出木質(zhì)素的百分含量 ABSL, 其中 Abs為吸光度值,Coeff為消光系數(shù)且 Coeff = 23.35 g L–1cm–1[36]。

1.3.3 木質(zhì)素單體測定 采用硫代酸解法[34,38-41]并有改進, 測定木質(zhì)素單體成分。稱取第(1)步中得到的細胞壁成分 5 mg, 加入帶螺旋蓋(帶有聚四氟乙烯材質(zhì)內(nèi)墊)的5 mL刻度試管中。加新鮮配制好的裂解液1 mL (2.5%BF3和10%EtSH)。向試管中填充氮氣, 100℃加熱反應(yīng)4 h, 每1 h振蕩一次。待反應(yīng)完成后冰浴冷卻5 min。加0.4 mol L–1NaHCO3溶液300 μL, 調(diào)pH值到3～4。加蒸餾水2 mL、1 mgμL–1的用乙酸乙酯配制的二十四烷內(nèi)標(biāo)溶液100 μL和乙酸乙酯 900 μL, 充分渦旋振蕩 30 s, 靜置 10 min使溶液分層。吸取300 μL上層有機相到2 mL的離心管中。氮氣吹干, 加200 μL丙酮溶解, 繼續(xù)用氮氣吹干, 重復(fù)操作一次。最后加入500 μL乙酸乙酯溶解, 用孔徑為0.22 μm的尼龍膜過濾。

上機前樣品衍生化處理: 取100 μL上述得到的提取液到內(nèi)插管內(nèi), 加入20 μL吡啶, 100 μL N,O-雙(三甲基硅基)乙酰胺進行 TMS衍生化, 25℃條件下反應(yīng) 2 h后采用 Agilent 7000C三重四極桿GC-MS。上機條件: 氣象色譜柱采用 Agilent HP-5 MS column (30.00 mm ×0.25 mm × 0.25 μm); 進樣量1 μL, 分流模式; 進樣口和檢測器溫度設(shè)為 250℃;載氣為高純氦氣, 載氣流速1.1 mL min–1; 柱初始溫度130℃下保留3 min, 然后以3℃ min–1升溫到250℃并且保留5 min, 再回到初始溫度 130℃, 其中溶劑延遲時長為30 min。

最后根據(jù)內(nèi)標(biāo)二十四烷進行各木質(zhì)素單體的定量分析。H、G和S型分別對應(yīng)的特征質(zhì)荷比為239、269和299 m z–1, 二十四烷對應(yīng)的特征質(zhì)荷比為 57 m z–1。

2 結(jié)果與分析

2.1 高溫干旱脅迫影響維管系統(tǒng)的發(fā)育及木質(zhì)素的合成

通過對比分析發(fā)現(xiàn), 脅迫植株的莖和根中木質(zhì)化組織被Phloroglucinol-HCl染色法[42]和M?ule染色法染出的顏色都比正常植株更深, 表明高溫干旱脅迫導(dǎo)致全株木質(zhì)素積累量明顯增高(圖1)。

圖1 成熟期植株中部莖(A～D)和根切片(E～H)Fig. 1 Sections of middle stem (A–D) and root (E–H) at mature stage

對比經(jīng) M?ule染色法染色的莖稈切片(圖 1-C,圖 1-D), 很容易看出脅迫植株的維管束間纖維被染出的紅色更深, 表明其 S型木質(zhì)素含量更高。從形態(tài)學(xué)看, 脅迫植株的木質(zhì)部比正常植株更厚(圖 1),莖桿中導(dǎo)管的孔徑明顯變小(圖 1-A, 圖 1-D), 但是脅迫植株的根中導(dǎo)管的孔徑卻比正常植株明顯大很多(圖1-E和圖1-F), 尤其是在纖維細胞所占比例較小的類型中(圖 1-G, 圖 1-H), 我們將油菜植株的根分為 2個主要類型, 一種是纖維細胞所占比例較大的, 另一種是纖維細胞所占比例較小的, 另外其導(dǎo)管數(shù)量也明顯增加。以上表明, 長期高溫干旱脅迫下莖和根的木質(zhì)化程度會大大提高。

2.2 莖和根中木質(zhì)素含量及其單體成分的變化

2.2.1 木質(zhì)素總體含量分析 根中木質(zhì)素的含量比莖中顯著高很多(圖 2), 根中木質(zhì)素的百分含量(15.56%)比莖(12.29%)平均高 3.27個百分點, 這主要是因為根部木質(zhì)化組織所占體積比例比莖中高,莖中存在大量髓部。脅迫莖的木質(zhì)素含量(16.18%)比正常植株莖中(12.29%)顯著高, 平均高 3.89個百分點(圖2)。這與前面的組織化學(xué)染色觀察分析結(jié)果一致。進一步證明長期高溫干旱脅迫下的油菜植株更加傾向于合成較多木質(zhì)素, 以增加抗逆性。

2.2.2 木質(zhì)素單體成分分析 不同環(huán)境下生長的植株莖稈中木質(zhì)素單體構(gòu)成之間存在顯著差異(表1)。就被解離出的木質(zhì)素單體總量(H + G + S)而言,雖然脅迫莖的木質(zhì)素含量比正常莖高 31.64%, 但是脅迫莖被解離出的木質(zhì)素單體總量卻比正常莖降低了 40.08%, 這也說明脅迫莖木質(zhì)素的結(jié)構(gòu)中含有更高比例的縮合鍵。另外脅迫莖中S型木質(zhì)素所占比例比正常植株莖有顯著增高, 脅迫莖的S/G值(1.82)比正常植株莖(1.29)高出 41.09%。表明長期高溫干旱脅迫下植株傾向積累更高比例的S型木質(zhì)素。

油菜植株的莖稈和根的木質(zhì)素單體成分構(gòu)成之間也存在巨大差異(表1和圖3)。雖然正常植株的根中測得的木質(zhì)素含量比莖中高 26.60%, 但對于木質(zhì)素單體總量而言根卻比莖低 62.71% (表 1), 這一結(jié)果表明根中木質(zhì)素的結(jié)構(gòu)含有更高比例的縮合鍵。H型木質(zhì)素在雙子葉被子植物中普遍很低, 一般所占單體總量的比例都低于1%或1%左右。經(jīng)過測定顯示, 油菜植株根中的 H型單體所占比例基本都高于5%, 接近于莖的 10倍(表 1和圖 3)。另外, 根中 G型木質(zhì)素所含比例(62.28%)比莖中(43.35%)明顯高很多, 根的S/G值(0.49)比莖中(1.29)也就低很多, 表明油菜莖中是S型木質(zhì)素占主體, 而根中卻是G型木質(zhì)素占主體(表1和圖3)。

表1 木質(zhì)素單體含量Table 1 Lignin monomer content (%)

圖2 成熟期植株莖和根的總木質(zhì)素含量Fig. 2 Total lignin content of mature plant stem and root

圖3 GC-MS測定正常植株莖和根以及脅迫莖中木質(zhì)素單體的色譜圖Fig. 3 GC-MS detection of lignin monomers in stem and root of CK plants and in stem of stressed plants

3 討論

3.1 高溫干旱脅迫顯著改變了油菜植株莖和根中木質(zhì)素的積累和結(jié)構(gòu)組成

對比分析發(fā)現(xiàn), 脅迫植株的莖和根的切片被染出的顏色比正常生長下更深, 顯示莖和根的木質(zhì)化程度要高很多, 木質(zhì)部也更厚, 木質(zhì)素含量更高,說明高溫干旱下會促進木質(zhì)素合成和細胞壁增厚,減少水分散失, 并抵抗其他各種逆境[23,43]。

從形態(tài)學(xué)對比分析表明, 脅迫莖中木質(zhì)部面積占比更大, 導(dǎo)管孔徑更小, 但其根中的導(dǎo)管孔徑更大, 且導(dǎo)管數(shù)量更多, 推測其原因是高溫干旱下根部適應(yīng)于吸取更多水分而其維管系統(tǒng)變得更發(fā)達,地上部則適應(yīng)減少水分散失其導(dǎo)管孔徑就變小, 胞壁木質(zhì)化程度就提高?？偠灾? 油菜植株在抵御長期高溫干旱逆境時, 其維管系統(tǒng)生長發(fā)育從不同器官組織的多個方面發(fā)生針對性的適應(yīng)性改變。

硫代酸解法測定木質(zhì)素單體, 主要是使木質(zhì)素單體之間穩(wěn)定性較差的 β-O-4化學(xué)鍵斷裂, 從而解離出對應(yīng)的木質(zhì)素單體, 它并不能解離內(nèi)部含有抗性鍵(resistant inter-unit bonds)或被稱為縮合鍵(condensed bonds)類型的木質(zhì)素[38,44]。雖然脅迫莖所含木質(zhì)素含量比正常莖高出很多, 但是脅迫莖被硫代酸解出來的木質(zhì)素單體總量卻下降了不少, 脅迫莖被解離出的 H、G和 S的總量比正常莖降低了40.08%, 這也說明在高溫干旱下植株所合成的木質(zhì)素結(jié)構(gòu)中含有更高比例的縮合鍵, 造成被解離出的木質(zhì)素單體含量較少。另外脅迫莖的S/G的比值比正常莖也高 41.09%, 可能是因為長期高溫干旱脅迫下植株在增強木質(zhì)素合成以及提高木質(zhì)素單體間縮合鍵的同時, 也合成了較高比例的 S型木質(zhì)素, 從而增強植株莖稈的機械強度和致密度, 進而增強抗旱和耐高溫的能力。目前關(guān)于高溫干旱對植株木質(zhì)素結(jié)構(gòu)的影響還很少有報道。Moura-Sobczak等[22]對桉樹干旱脅迫處理發(fā)現(xiàn), 桉樹中S/G值明顯升高,并指出不管是S型木質(zhì)素含量的升高還是G型木質(zhì)素含量的下降都在桉樹適應(yīng)干旱環(huán)境中起到重要的作用。

3.2 油菜莖與根的木質(zhì)素結(jié)構(gòu)顯著不同

對比正常植株的莖和根之間解離出的木質(zhì)素單體總量, 發(fā)現(xiàn)雖然根中的木質(zhì)素含量比莖要高, 但是其被解離出的木質(zhì)素單體總量卻低很多, 說明根比莖含有更高比例的縮合鍵。其原因很可能是由于根木質(zhì)素組成中含有更高比例的H和G型木質(zhì)素單體, 因為H型和G型木質(zhì)素單體的苯環(huán)甲基化程度比 S型更低, 所以它們之間的連接就更容易形成縮合鍵[1]。Ruel等[45]指出縮合型木質(zhì)素主要含G型木質(zhì)素, 而非縮合型木質(zhì)素主要含S型木質(zhì)素。

在根中, S型木質(zhì)素單體都比G型含量明顯低很多, 并且 H型木質(zhì)素含量所占比例也比莖高, 莖中H含量所占比例為0.83%, 而根中則達到7.43%。根木質(zhì)素結(jié)構(gòu)中這種高比例H型和G型木質(zhì)素有可能是因為其硬度或剛性不必像莖稈那么強, 而韌性或可彎曲性則應(yīng)該更強, 便于其在地下彎曲生長, 高比例的H型和G型木質(zhì)素就很有可能是有助于根這種本身特性的形成。

3.3 油菜高溫干旱脅迫下根中木質(zhì)素的結(jié)構(gòu)變化有待于測定

從系統(tǒng)性講, 本研究理應(yīng)對高溫干旱脅迫處理后根木質(zhì)素的變化也進行GC-MS測定, 但卻沒有測定, 這是由于從組織切片已看出根中發(fā)生了與莖中類似的變化趨勢。現(xiàn)在看來這是本研究的一個不足,有待于以后的研究來完善, 不排除根中木質(zhì)素的成分比例和結(jié)構(gòu)變化與莖中存在不同的特點。

4 結(jié)論

高溫干旱脅迫可以顯著增強油菜植株木質(zhì)素總量的合成, 增加木質(zhì)部厚度, 且不同木質(zhì)素單體的比例和木質(zhì)素單體間的縮合方式也會發(fā)生變化, 高溫干旱促進了油菜莖中S型木質(zhì)素的積累。油菜莖與根的導(dǎo)管形態(tài)響應(yīng)長期高溫干旱的變化顯著不同,莖中導(dǎo)管孔徑變小, 而根的導(dǎo)管孔徑變大且導(dǎo)管數(shù)量變多。油菜植株的莖與根在木質(zhì)素結(jié)構(gòu)的差異非常顯著, 與莖相比, 根木質(zhì)素中含有更高比例的縮合鍵, H和G型木質(zhì)素比例也高得多。

[1]Boerjan W, Ralph J, Baucher M. Lignin biosynthesis. Annu Rev Plant Biol, 2003, 54: 519–546

[2]Scheller H V, Ulvskov P. Hemicelluloses. Annu Rev Plant Biol,2010, 61: 263–289

[3]Escamilla-Trevi?o L L, Shen H, Uppalapati S R, Ray T, Tang Y H,Hernandez T, Yin Y B, Xu Y, Dixon R A. Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties. New Phytol, 2010, 185:143–155

[4]Humphreys J M, Hemm M R, Chapple C. New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase. Proc Natl Acad Sci USA, 1999,96: 10045–10050

[5]Weng J K, Mo H P, Chapple C. Over-expression of F5H in COMT-deficient Arabidopsis leads to enrichment of an unusual lignin and disruption of pollen wall formation. Plant J, 2010, 64:898–911

[6]Rogers L A, Campbell M M. The genetic control of lignin deposition during plant growth and development. New Phytol, 2004,164: 17–30

[7]Weng J K, Chapple C. The origin and evolution of lignin biosynthesis. New Phytol, 2010, 187: 273–285

[8]Vanholme R, Van Acker R, Boerjan W. Potential of Arabidopsis systems biology to advance the biofuel field. Trends Biotechnol,2010, 28: 543–547

[9]Huang J L, Gu M, Lai Z B, Fan B F, Shi K, Zhou Y H, Yu J Q,Chen Z X. Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress. Plant Physiol, 2010, 153: 1526–1538

[10]Graven P, de Koster C G, Boon J J, Bouman F. Structure and macromolecular composition of the seed coat of the Musaceae.Ann Bot-London, 1996, 77: 105–122

[11]Zhong R Q, Taylor J J, Ye Z H. Disruption of interfascicular fiber differentiation in an Arabidopsis mutant. Plant Cell, 1997, 9:2159–2170

[12]Kenrick P, Crane P R. The origin and early evolution of plants on land. Nature, 1997, 389: 33–39

[13]Weng J K, Akiyama T, Ralph J, Chapple C. Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii. Plant Cell, 2011, 23: 2708–2724

[14]Vanholme R, Morreel K, Darrah C, Oyarce P, Grabber J H, Ralph J, Boerjan W. Metabolic engineering of novel lignin in biomass crops. New Phytol, 2012, 196: 978–1000

[15]Macmillan C P, Birke H, Bedon F, Pettolino F A. Lignin deposition in cotton cells: where is the lignin? J Plant Biochem Physiol,2013, 1: e106. doi: 10.4172/jpbp.1000e106

[16]Chapple C C S, Vogt T, Ellis B E, Somerville C R. An Arabidopsis mutant defective in the general phenylpropanoid pathway.Plant Cell, 1992, 4: 1413–1424

[17]Nakashima J, Chen F, Jackson L, Shadle G, Dixon R A. Multi-site genetic modification of monolignol biosynthesis in alfalfa(Medicago sativa): effects on lignin composition in specific cell types. New Phytol, 2008, 179: 738–750

[18]Li L, Cheng X F, Leshkevich J, Umezawa T, Harding S A,Chiang V L. The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase. Plant Cell, 2001, 13: 1567–1586

[19]Tu Y, Rochfort S, Liu Z Q, Ran Y D, Griffith M, Badenhorst P,Louie G V, Bowman M E, Smith K F, Noel J P, Mouradov A,Spangenberg G. Functional analyses of caffeic acidO-methyltransferase and cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne). Plant Cell, 2010, 22:3357–3373

[20]Giordano A, Liu Z Q, Panter S N, Dimech A M, Shang Y J, Wijesinghe H, Fulgueras K, Ran Y D, Mouradov A, Rochfort S, Patron N J, Spangenberg G C. Reduced lignin content and altered lignin composition in the warm season forage grass Paspalum dilatatum by down-regulation of a cinnamoyl CoA reductase gene.Transgenic Res, 2014, 23: 503–517

[21]Moura J C M S, Bonine C A V, De Oliveira Fernandes Viana J,Dornelas M C, Mazzafera P. Abiotic and biotic stresses and changes in the lignin content and composition in plants. J Integr Plant Biol, 2010, 52: 360–376

[22]Moura-Sobczak J, Souza U, Mazzafera P. Drought stress and changes in the lignin content and composition in Eucalyptus.BMC Proc, 2011, 5: P103

[23]Eynck C, Seguin-Swartz G, Clarke W E, Parkin I A P. Monolignol biosynthesis is associated with resistance to Sclerotinia sclerotiorum in Camelina sativa. Mol Plant Pathol, 2012, 13:887–899

[24]Olenichenko N, Zagoskina N. Response of winter wheat to cold:production of phenolic compounds and L-phenylalanine ammonia lyase activity. Appl Biochem Microbiol, 2005, 41: 600–603

[25]Wei H U I, Dhanaraj A L, Arora R, Rowland L J, Fu Y, Sun L.Identification of cold acclimation responsive Rhododendron genes for lipid metabolism, membrane transport and lignin biosynthesis: importance of moderately abundant ESTs in genomic studies. Plant Cell Environ, 2006, 29: 558–570

[26]Cruz R T, Jordan W R, Drew M C. Structural changes and associated reduction of hydraulic conductance in roots of Sorghum bicolor L. following exposure to water deficit. Plant Physiol, 1992,99: 203–212

[27]Riccardi F, Gazeau P, de Vienne D, Zivy M. Protein changes in response to progressive water deficit in maize: quantitative variation and polypeptide identification. Plant Physiol, 1998, 117:1253–1263

[28]Fan L, Linker R, Gepstein S, Tanimoto E, Yamamoto R, Neumann P M. Progressive inhibition by water deficit of cell wall extensibility and growth along the elongation zone of maize roots is related to increased lignin metabolism and progressive stelar accumulation of wall phenolics. Plant Physiol, 2006, 140: 603–612

[29]Yang L, Wang C C, Guo W D, Li X B, Lu M, Yu C L. Differential expression of cell wall related genes in the elongation zone of rice roots under water deficit. Rus J Plant Physiol, 2006, 53,390–395

[30]黃杰恒. 干旱脅迫下油菜抗倒伏相關(guān)性狀動態(tài)變化及木質(zhì)素關(guān)鍵基因表達特性分析. 西南大學(xué)博士學(xué)位論文, 重慶, 2013 Huang J H. Lodging Resistant Traits and Lignin Related Gene Analysis in B. napus under Drought Stress. PhD Dissertation of Southwest University, Chongqing, China, 2013 (in Chinese with English abstract)

[31]徐宇強, 胡軼, 付鳳玲, 李晚忱. 干旱脅迫下玉米自交系葉片木質(zhì)素含量變化及其與耐旱性的關(guān)系. 玉米科學(xué), 2007, 15(5):72–75 Xu Y Q, Hu Y, Fu F L, Li W C. Changes of lignin content in leaf of maize inbred lines under drought stress and its relationship with drought tolerance. J Maize Sci, 2007, 15(5): 72–75 (in Chinese with English abstract)

[32]Champolivier L, Merrien A. Effects of water stress applied at different growth stages to Brassica napus L. var. oleifera on yield, yield components and seed quality. Eur J Agron, 1996, 5: 153–160

[33]Chen L, Auh C, Chen F, Cheng X F, Aljoe H, Dixon R A, Wang Z Y. Lignin deposition and associated changes in anatomy, enzyme activity, gene expression, and ruminal degradability in stems of tall fescue at different developmental stages. J Agric Food Chem,2002, 50: 5558–5565

[34]Foster C E, Martin T M, Pauly M. Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part I: lignin.J Visual Exp, 2010, (37): 1745. doi: 10.3791/1745

[35]Hatfield R D, Grabber J, Ralph J, Brei K. Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes. J Agric Food Chem, 1999, 47:628–632

[36]Chang X F, Chandra R, Berleth T, Beatson R P. Rapid, microscale,acetyl bromide-based method for high-throughput determination of lignin content in Arabidopsis thaliana. J Agric Food Chem,2008, 56: 6825–6834

[37]Fukushima R S, Hatfield R D. Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method. J Agric Food Chem, 2001, 49: 3133–3139

[38]Lapierre C, Pollet B, Rolando C. New insights into the molecular architecture of hardwood lignins by chemical degradative methods. Res Chem Intermediat, 1995, 21: 397–412

[39]Yosef E, Ben-Ghedalia D. Changes in thioacidolysis products of lignin in wheat straw as affected by SO2treatment and passage through the gastro-intestine of sheep. Anim Feed Sci Tech, 1999,80: 55–65

[40]Robinson A R, Mansfield S D. Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling. Plant J,2009, 58: 706–714

[41]Yue F X, Lu F C, Sun R C, Ralph J. Syntheses of lignin-derived thioacidolysis monomers and their uses as quantitation standards.J Agric Food Chem, 2012, 60: 922–928

[42]Nakano J, Meshitsuka G. The detection of lignin. In: Lin S ed,Methods in Lignin Chemistry. Berlin, Germany: Springer-Verlag,pp 23–32

[43]Bart R S, Chern M, Vega-Sanchez M E, Canlas P, Ronald P C.Rice Snl6, a cinnamoyl-CoA reductase-like gene family member,is required for NH1-mediated immunity to Xanthomonas oryzae pv. oryzae. PLoS Genet, 2010, 6: 110–117

[44]Leplé J C, Dauwe R, Morreel K, Storme V, Lapierre C, Pollet B,Naumann A, Kang K Y, Kim H, Ruel K, Lefebvre A, Joseleau J P,Grima-Pettenati J, De Rycke R, Andersson-Gunneras S, Erban A,Fehrle I, Petit-Conil M, Kopka J, Polle A, Messens E, Sundberg B, Mansfield S D, Ralph J, Pilate G, Boerjan W. Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant Cell, 2007, 19: 3669–3691

[45]Ruel K, Berrio-Sierra J, Derikvand M M, Pollet B, Thevenin J,Lapierre C, Jouanin L, Joseleau J P. Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana.New Phytol, 2009, 184: 99–113

Lignification Response and the Difference between Stem and Root of Brassica napus under Heat and Drought Compound Stress

YIN Neng-Wen**, LI Jia-Na**, LIU Xue, LIAN Jian-Ping, FU Chun, LI Wei, JIANG Jia-Yi, XUE Yu-Fei,WANG Jun, and CHAI You-Rong*
Chongqing Rapeseed Engineering Research Center / Chongqing Key Laboratory of Crop Quality Improvement / Engineering Research Center of South Upland Agriculture of Ministry of Education / College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China

10.3724/SP.J.1006.2017.01689

本研究由教育部高等學(xué)校博士學(xué)科點專項科研基金項目(20130182110006), 國家重點基礎(chǔ)研究發(fā)展計劃(973計劃)項目(2015CB 150201), 國家自然科學(xué)基金項目(31171177, 31371238), 國家重點研發(fā)計劃試點專項(2016YFD0100506)和重慶市基礎(chǔ)科學(xué)與前沿技術(shù)研究專項重點項目(cstc2015jcyjBX0143)資助。

This study was supported by the Specialized Research Fund for the Doctoral Program of Higher Education (20130182110006), the National Basic Research Program of China (973 Program, 2015CB150201), the National Natural Science Foundation of China (31171177, 31371238),Ministry of Science and Technology of China (2016YFD0100506), and Chongqing Research Program of Basic Research and Frontier Technology (cstc2015jcyjBX0143).

*通訊作者(Corresponding author): 柴友榮, E-mail: chaiyourong@163.com**同等貢獻(Contributed equally to this work)

聯(lián)系方式: 尹能文, E-mail: nwyin80@126.com

): 2017-01-10; Accepted(接受日期): 2017-05-10; Published online(網(wǎng)絡(luò)出版日期): 2017-05-19.

URL: http://kns.cnki.net/kcms/detail/11.1809.S.20170519.1200.004.html