王 果, 馮 康, 李 倩, 薛惠云, 李麗杰, 張志勇

棉花根系和葉片質(zhì)外體汁液分離方法的改進(jìn)*

王 果, 馮 康, 李 倩, 薛惠云, 李麗杰, 張志勇**

(河南科技學(xué)院/河南省現(xiàn)代生物育種協(xié)同創(chuàng)新中心/河南省棉麥分子生態(tài)和種質(zhì)創(chuàng)新重點(diǎn)實(shí)驗(yàn)室 新鄉(xiāng) 453003)

質(zhì)外體汁液(apoplastic washing fluid, AWF)在植物生長發(fā)育和抵抗生物及非生物逆境方面發(fā)揮著重要作用。目前普遍采用真空滲透離心法提取質(zhì)外體汁液, 但具體提取流程和條件卻因植物培養(yǎng)條件、種類和器官等不同而不同。本試驗(yàn)以營養(yǎng)液培養(yǎng)的棉花幼苗為材料, 改進(jìn)了棉花根系和葉片AWF分離的取樣方法、滲透條件和離心參數(shù)等。結(jié)果表明, 相對(duì)于通常的非整體取樣, 改良后整體取樣簡單易操作且顯著降低了質(zhì)外體與共質(zhì)體的蘋果酸脫氫酶(malate dehydrogenase, MDH)活性比值, 更適宜開展質(zhì)外體汁液組分研究。根據(jù)真空滲透鮮重增加和AWF稀釋因子, 進(jìn)一步證明根系不用真空滲透; 而葉片需真空滲透, 適宜真空強(qiáng)度/時(shí)間為–60 kPa/1 min, 真空后恢復(fù)到正常壓強(qiáng)約110 s。證明葉片顏色變深面積可作為判定真空滲透強(qiáng)度或時(shí)間是否適宜的一種簡易可行方法。最后, AWF體積、質(zhì)外體與共質(zhì)體的可溶性蛋白含量比值和MDH活性比值綜合分析表明, 棉花根系適宜離心力大小/時(shí)間長度為800 ×g/10~20 min, 葉片為400 ×g/5 min。本改進(jìn)方法為棉花AWF組分, 如蛋白質(zhì)組學(xué)和代謝組學(xué)等研究結(jié)果的準(zhǔn)確性和可靠性奠定了基礎(chǔ), 為其他作物AWF分離方法的優(yōu)化提供了參考。

棉花; 蘋果酸脫氫酶; 真空滲透; 離心; 共質(zhì)體; 質(zhì)外體

高等植物的質(zhì)外體是指細(xì)胞膜以外的空間, 包括細(xì)胞壁、細(xì)胞間隙和分化成熟的木質(zhì)部, 這些空間中流動(dòng)的液體稱為質(zhì)外體汁液(apoplastic washing fluid, AWF)。質(zhì)外體汁液中含有蛋白質(zhì)、代謝物和礦質(zhì)元素等[1-3]。AWF成分對(duì)細(xì)胞分裂、伸長和分化以及信號(hào)轉(zhuǎn)導(dǎo)有重要影響, 參與了對(duì)生物和非生物脅迫的響應(yīng)[4-8]。研究AWF無機(jī)離子、代謝物和蛋白質(zhì)等物質(zhì)組成和功能的首要步驟是獲得不被或很少被共質(zhì)體(symplast)離子、代謝物和蛋白質(zhì)污染的AWF。獲取AWF的方法有壓力法、洗脫法、微透析法、濾紙條法和真空滲透離心法等。壓力法是利用壓力室將根系或葉片AWF經(jīng)由莖或葉柄壓出來, 獲得的是木質(zhì)部汁液[9-10]; 洗脫法需先將葉片表皮層揭掉, 再洗脫獲得AWF[11]; 微透析法需將微透析工具植入根系或葉片中獲得AWF, 但該方法獲得的AWF中不含蛋白質(zhì)[12]; 濾紙條法是用濾紙條吸附根系分泌的AWF[13]; 真空滲透離心法或離心法是先真空滲透進(jìn)去一部分液體再離心或不經(jīng)過真空滲透直接離心獲得AWF[14]。真空滲透離心法具有簡單、高效和重現(xiàn)性好等特點(diǎn), 研究AWF蛋白質(zhì)組時(shí), 真空滲透離心法被廣泛采用[1,15-20]。

因植物種類和器官等不同, 可采用真空滲透離心法或直接離心法獲得AWF, 并且真空滲透或離心時(shí)間、強(qiáng)度等參數(shù)也不盡相同。研究人員針對(duì)特定植物特定器官分別建立了不同的AWF分離流程, 如擬南芥()葉片[21]、菜豆()葉片[2]、水稻()葉片[17]、西紅柿()葉片[22]及水稻根系[23]等, 用于研究AWF中的蛋白質(zhì)組和代謝物等。將真空滲透離心法或直接離心法用于棉花()根系和葉片AWF分離的研究尚少見報(bào)道。本研究以營養(yǎng)液培養(yǎng)的棉花幼苗為材料, 分別研究棉花根系和葉片分離AWF時(shí)的適宜取樣方法、真空滲透的必要性和相關(guān)參數(shù)及離心力的大小和時(shí)間等, 為研究棉花根系和葉片AWF蛋白質(zhì)組和代謝物組在生長發(fā)育和響應(yīng)生物或非生物逆境方面的作用奠定基礎(chǔ)。

1 材料與方法

1.1 培養(yǎng)方法

選用美國孟山都公司的棉花品種‘DP99B’為材料。當(dāng)種子萌發(fā)3 d時(shí), 選取整齊一致的幼苗移栽至營養(yǎng)液中培養(yǎng)。營養(yǎng)液組成為: 2.5 mmol?L–1Ca(NO3)2, 2.5 mmol?L–1KCl, 1 mmol?L–1MgSO4, 0.5 mmol?L–1NH4H2PO4, 2 mmol?L–1NaCl, 2×10–4mmol?L–1CuSO4, 1×10–3mmol?L–1ZnSO4, 0.1 mmol?L–1EDTA-FeNa, 2×10–2mmol?L–1的H3BO3, 5×10–6mmol?L–1(NH4)6Mo7O24和1×10–3mmol?L–1MnSO4。幼苗培養(yǎng)時(shí)的光照強(qiáng)度為350 μmol?m–2?s–1, 光照/黑暗時(shí)間為14 h/10 h, 晝/夜溫度為30 ℃/25 ℃,相對(duì)濕度為45%。每隔3 d更換1次營養(yǎng)液, 培養(yǎng)9 d后, 分別取棉花的根系和子葉進(jìn)行AWF分離過程中取樣方法、真空滲透和真空滲透適宜度判定方法等方面的改進(jìn)。

1.2 取樣方法

1.2.1 根系取樣方法

使用解剖刀在根莖交界處切斷; 根系放入蒸餾水中輕輕沖洗2次(洗去根系表面的營養(yǎng)液和切口破碎細(xì)胞); 用吸水紙蘸干表面水分后, 單株根系稱重備用, 定義為根系整體取樣; 將蘸干表面水的根系用解剖刀將根系切成1 cm根段后, 放入蒸餾水中清洗3次(洗去切口破碎細(xì)胞), 并用吸水紙蘸干表面水分后, 立刻稱重備用, 定義為根系非整體取樣。

1.2.2 子葉取樣方法

用解剖刀在兩片子葉與葉柄交界處切斷; 葉片放入蒸餾水中輕輕沖洗2次(洗去切口破碎細(xì)胞); 用吸水紙蘸干表面水分后, 完整子葉直接稱重備用, 作為整體備用定義為葉片整體取樣; 蘸干水的葉片用內(nèi)直徑為1 cm的打孔器避開主葉脈進(jìn)行打孔, 將獲得的圓形葉片, 放入蒸餾水中清洗3次(洗去切口破碎細(xì)胞)后, 立刻稱重備用, 定義為葉片非整體取樣。

1.3 真空滲透和離心收集AWF

真空滲透: 樣品分別立即浸入盛有滲透液(50 mmol?L–1、pH 6.9的磷酸緩沖液)的小燒杯中, 燒杯放入底部盛滿碎冰的真空壓力室中, 抽真空, 用濾網(wǎng)撈出樣品, 輕輕蘸干表面水分, 立刻稱重。

離心: 將非整體樣品分別放入10 mL底部有3個(gè)1 mm孔徑的離心管中, 然后將10 mL的離心管放入30 mL的大離心管; 根系和葉片整體樣品分別從根尖開始在5 mL槍頭的槍尖上向上纏繞和用Parafilm封口膜將葉片固定在5 mL槍頭上, 槍頭尖朝下(樣品切口朝上)放入被扎有兩個(gè)1 mm孔徑和注射器頭呈等腰三角形孔的20 mL注射器中, 然后將注射器放在50 mL離心管中。樣品準(zhǔn)備好后, 在4 ℃條件下離心, 分別收集位于外部30 mL和50 mL離心管底部的液體即AWF。

1.4 共質(zhì)體汁液(symplast washing fluid, SWF)提取

將分離過AWF的根系去除主根、子葉去除主葉脈后進(jìn)行研磨, 用100 mmol?L–1含1 mmol?L–1EDTA- Na2和1% PVPP的磷酸緩沖液(pH7.5)浸提, 4℃條件下, 15 000 ×g離心20 min, 取上清液即SWF。

1.5 相對(duì)電導(dǎo)率、可溶性蛋白質(zhì)含量和蘋果酸脫氫酶(Malate dehydrogenase, MDH)活性測定和比值計(jì)算

相對(duì)電導(dǎo)率的測定參考Lutts等[24]的方法; 可溶性蛋白測定采用考馬斯亮藍(lán)法[25]; MDH活性測定采用草酰乙酸法[16], 活性定義為OD340nm?min–1?g–1(FW)。分別測定AWF和SWF的可溶性蛋白質(zhì)含量和MDH活性后, AWF中可溶性蛋白質(zhì)含量與SWF中可溶性蛋白質(zhì)含量的比值簡稱為可溶性蛋白質(zhì)含量比值, AWF中MDH活性與SWF中MDH活性的比值簡稱MDH活性比值。

1.6 AWF稀釋因子測定

AWF稀釋因子反映了根系或葉片質(zhì)外體空間被氣體所占據(jù)的空間大小。如果氣體占據(jù)空間大, 抽真空時(shí)空氣會(huì)從根系中出來, 滲透液中的水和標(biāo)記物, 如靛藍(lán)會(huì)進(jìn)入質(zhì)外體空間。真空滲透進(jìn)去的水可以更充分地溶解質(zhì)外體空間中的物質(zhì), 利于研究質(zhì)外體空間代謝物和蛋白質(zhì)組成和種類。反映了樣品在分離質(zhì)外體汁液前是否需要真空滲透。AWF稀釋因子測定參照O’Leary等的方法[2]。將樣品放入含50 μmol?L–1靛藍(lán)二磺酸鈉的水溶液或磷酸緩沖液(50 mmol?L–1,pH 6.9)中真空滲透。葉片真空滲透時(shí)呈現(xiàn)50%左右不均勻的葉色變深, 葉片比較堅(jiān)挺, 定義為半綠; 呈現(xiàn)90%~100%葉色變深, 葉片比較

堅(jiān)挺, 定義為全綠; 全部葉色變深, 且葉片變的柔軟時(shí), 定義為過綠。真空滲透后的樣品離心(4 ℃, 800×g, 20 min)分離AWF。測定真空滲透前的靛藍(lán)二磺酸鈉溶液的OD610nm值(OD610滲透液)和AWF的OD610nm值(OD610AWF)。AWF稀釋因子= OD610滲透液/(OD610滲透液–OD610AWF)。

1.7 數(shù)據(jù)分析

采用SAS軟件的ANOVA t-test或LSD對(duì)測定指標(biāo)進(jìn)行比較。利用Excel軟件進(jìn)行數(shù)據(jù)分析。

2 結(jié)果與分析

2.1 整體與非整體取樣方式之間AWF相關(guān)指標(biāo)的比較

根系非整體取樣方式的AWF體積、可溶性蛋白含量比值、MDH活性比值和相對(duì)電導(dǎo)率均高于整體取樣的方式, 且在可溶性蛋白含量比值和MDH活性比值方面達(dá)顯著水平, 分別是整體取樣方式的3.1倍和4.6倍(表1)。葉片非整體取樣方式的AWF體積、可溶性蛋白含量比值、MDH活性比值和相對(duì)電導(dǎo)率均顯著高于整體取樣方式, 分別是整體取樣方式的1.6倍、6.0倍、9.9倍和6.0倍(表1)。結(jié)果表明, 非整體取樣獲得的AWF更容易受到SWF成分的污染。

2.2 棉花根系和葉片真空滲透后的鮮重和AWF稀釋因子

棉花根系在真空滲透液A和B真空滲透后, 鮮重均有很小幅度的下降, 稀釋因子略微大于1, 且兩者之間的增重和稀釋因子均無顯著差異(表2)。棉花葉片在真空滲透后, 增重幅度為31.50%~41.41%, 稀釋因子為2.12~3.34, 且隨著滲透時(shí)間的增加, 增重量和稀釋因子顯著增加, 并且在滲透2 min時(shí), 真空滲透液A的樣品增重和稀釋因子顯著高于真空滲透液B。稀釋因子大于1則說明AWF被稀釋了, 即有抽真空滲透的必要; 如果約等于1, 則說明AWF沒被稀釋, 則沒抽真空的必要。和增重與稀釋因子趨勢一致的是, 隨著真空滲透時(shí)間延長, 葉片顏色變深的面積增大(圖1)。結(jié)果表明, 在該試驗(yàn)條件下, 用根系分離AWF時(shí)沒必要進(jìn)行真空滲透, 而葉片有必要進(jìn)行真空滲透。

2.3 棉花葉片真空滲透時(shí)的真空強(qiáng)度和時(shí)間及真空后恢復(fù)正常壓強(qiáng)所需時(shí)間

–60 kPa條件下, 棉花葉片AWF體積和可溶性蛋白含量比值均顯著高于–30 kPa和–90 kPa條件下的相應(yīng)值, 并且MDH活性比值與–90 kPa條件下相比沒差異(表3)。棉花葉片在–60 kPa下隨著真空時(shí)間延長, 葉片顏色變深面積增大, 顏色變深面積達(dá)最大時(shí), 繼續(xù)真空導(dǎo)致葉片從硬變軟(圖1)。對(duì)子葉進(jìn)行抽真空后, 在400 ×g離心力下離心5 min分離AWF蛋白時(shí), 抽真空1 min處理所得AWF體積顯著低于抽真空2 min和4 min; 抽真空1 min和2 min的可溶性蛋白比例無顯著差異, 且均顯著小于抽真空4 min; 3個(gè)抽真空時(shí)間的MDH活性比值存在顯著差異, 其中抽真空1 min處理分別是抽真空2 min和4 min的38.0%和28.4%(表4)。由此可見, 抽真空后葉片顏色和軟硬狀態(tài)可作為一個(gè)抽真空強(qiáng)度和時(shí)間組合是否適宜的簡易判斷指標(biāo)。未抽真空子葉的相對(duì)電導(dǎo)率和緩慢(110 s)恢復(fù)到壓強(qiáng)處理相比差異不顯著, 但均顯著低于快速(50 s)恢復(fù)到正常壓強(qiáng)處理, 分別為快速恢復(fù)壓強(qiáng)處理的46.6%和42.2%(表5)。

表1 棉花幼苗根系和葉片不同取樣方式的AWF體積、可溶性蛋白含量比值、MDH活性比值及相對(duì)電導(dǎo)率

可溶性蛋白含量比值: AWF中可溶性蛋白含量與SWF中可溶性蛋白含量的比值; MDH活性比值: AWF中MDH活性與SWF中MDH活性的比值。不同小寫字母表示同一器官同列指標(biāo)在不同取樣方式間差異顯著(<0.05)。Soluble protein content ratio: the ratio of soluble protein content in AWF to SWF (symplast washing fluid); MDH enzyme activity ratio: the ratio of MDH enzyme activity in AWF to SWF. Different lowercase letters in the same column indicate significant differences for the same organ between two sampling methods (< 0.05).

表2 棉花幼苗根系和子葉-60 kPa真空滲透前后的顏色變化、增重情況及AWF稀釋因子

A: 50 μmol?L–1靛藍(lán)二磺酸鈉的水溶液; B: 50 μmol?L–1靛藍(lán)二磺酸鈉的磷酸緩沖液(50 mmol?L–1, pH 6.9)。不同小寫字母表示同一器官同列指標(biāo)在不同真空滲透液間差異顯著(<0.05)。A: water solution containing 50 μmol?L–1indigotindisulfonate sodium; B: phosphate solution (pH 6.9) containing 50 μmol?L–1indigotindisulfonate sodium. Different lowercase letters in the same column indicate significant differences for the same organ between two vacuum permeates (< 0.05).

圖1 真空滲透后棉花幼苗葉片顏色的狀態(tài)

三角形箭頭指示真空滲透后顏色變深, 圓形箭頭指示真空滲透后顏色沒變化。左側(cè)圖片顯示約1/2的葉片顏色變深, 定義為半綠; 右側(cè)圖片顯示幾乎整個(gè)葉片顏色變深, 且葉片堅(jiān)挺, 定義為全綠; 如葉片變軟, 定義為過綠。The triangle arrow shows the color darkening after vacuum infiltration, and the round arrow shows no color darkening. Left leaf photo shows about half leaf darkening, defined as half green; right leaf photo shows whole leaf darkening and firmness, defined as whole green; if leaf is floppy, defined as over green.

表3 不同真空強(qiáng)度處理1 min后的棉花幼苗葉片AWF體積、可溶性蛋白含量比值和MDH活性比值

可溶性蛋白含量比值: AWF中可溶性蛋白含量與SWF中可溶性蛋白含量的比值; MDH活性比值: AWF中MDH活性與SWF中MDH活性的比值。同列不同小寫字母表示不同真空強(qiáng)度間差異顯著(<0.05)。離心力/時(shí)間為400 ×g/5 min。Soluble protein content ratio: the ratio of soluble protein content in AWF to SWF (symplast washing fluid); MDH enzyme activity ratio: the ratio of MDH enzyme activity in AWF to SWF. Different lowercase letters in the same column indicate significant differences among different vacuum intensities (< 0.05). Centrifugation strength/time is 400 ×g/5 min.

表4 –60 kPa強(qiáng)度真空處理不同時(shí)間后的棉花幼苗葉片AWF體積、可溶性蛋白含量比值和MDH活性比值

可溶性蛋白含量比值: AWF中可溶性蛋白含量與SWF中可溶性蛋白含量的比值; MDH活性比值: AWF中MDH活性與SWF中MDH活性的比值。同列不同小寫字母表示不同真空時(shí)間間差異顯著(<0.05)。離心力/時(shí)間長度為400 ×g/5 min。Soluble protein content ratio: the ratio of soluble protein content in AWF to SWF (symplast washing fluid); MDH enzyme activity ratio: the ratio of MDH enzyme activity in AWF to SWF. Different lowercase letters in the same column indicate significant differences among different vacuum times (< 0.05). Centrifugation force/time is 400 ×g/5 min.

2.4 棉花根系和葉片離心力和離心時(shí)間

離心1 200 ×g/10 min獲得的根系A(chǔ)WF體積和蛋白含量比值顯著大于其他離心力和時(shí)間組合, 獲得的MDH活性比值高于其他離心力和時(shí)間組合(1 200 ×g/5 min除外)。隨著離心力和離心時(shí)間的下降, 根系A(chǔ)WF體積、可溶性蛋白含量比值和MDH活性比值呈下降趨勢(表6)。400 ×g/5 min離心獲得的葉片AWF體積、可溶性蛋白含量比值和MDH活性比值小于400 ×g/10 min 和800 ×g/5 min組合, 僅MDH活性比值達(dá)到顯著水平, 分別是400 ×g/10 min 和800 ×g/5 min的32.1%和40.9%(表6)。

3 討論和結(jié)論

質(zhì)外體是一個(gè)具有廣泛生理功能的動(dòng)態(tài)空間。依據(jù)試驗(yàn)?zāi)康摹⒃囼?yàn)材料和培養(yǎng)條件等不同, 獲得AWF時(shí)可以將根系切成段或利用整體根系, 可以將葉片用打孔器打成小圓片、切成片段或利用葉片整體。如營養(yǎng)液培養(yǎng)水稻幼苗根系切成5 cm長根段[23]、玉米()幼苗12 mm初生根根尖及伸長區(qū)不同部位(從根尖向后3~7 mm及7~12 mm根部位)[20]、羽扇豆()和豌豆()的2 cm長根尖[14]等用于獲得AWF。玉米第5片和6片真葉被切成5.5 cm長片段[19]、青蔥()葉被切成1 cm長片段[26]、接種稻瘟菌()后的水稻葉片整體[18]、擬南芥葉片整體[21]、6周苗齡的蠶豆()完整葉片及不含中脈的11 cm2大麥(v)、菠菜()和玉米等葉片片段[16]等用于AWF分離。采用根系或葉片整體法, 可以獲取組織器官本身各部位的AWF, 有利于研究其AWF蛋白質(zhì)組或代謝組等的整體情況[1]。而且養(yǎng)分脅迫時(shí), 同一葉片的不同部位對(duì)養(yǎng)分脅迫的響應(yīng)不同。如鉀缺乏時(shí), 葉尖和邊緣先出現(xiàn)響應(yīng)癥狀[13]。因此, 研究養(yǎng)分脅迫時(shí), 采用整體葉片或根系更有利于分析養(yǎng)分對(duì)葉片或根系整個(gè)質(zhì)外體蛋白組和代謝組的影響。而且相對(duì)于獲取局部組織而言, 其操作方便簡單。同時(shí), 本試驗(yàn)結(jié)果顯示, 整體取樣的質(zhì)外體與質(zhì)內(nèi)體MDH活性比值顯著小于非整體取樣, 表明整體取樣時(shí), 質(zhì)外體蛋白質(zhì)不容易受共質(zhì)體蛋白質(zhì)污染。

質(zhì)外體空間的蛋白質(zhì)和代謝物有的以共價(jià)鍵形式結(jié)合在細(xì)胞壁上, 有的以離子鍵緊密結(jié)合在細(xì)胞壁上或以離子鍵松散結(jié)合在細(xì)胞壁上, 還有大部分溶解在質(zhì)外體空間流動(dòng)的汁液中, 獲得AWF時(shí)通常獲得溶解在AWF和以離子鍵松散結(jié)合在細(xì)胞壁上的蛋白質(zhì)和代謝物[20]。分離AWF是直接離心還是先真空滲透再離心, 取決于AWF的可獲得性。目前報(bào)道的根系有直接離心[14]或真空滲透離心[20,23], 而葉片絕大多數(shù)采用真空滲透離心[16,18-19,21], 也有直接離心的報(bào)道[3]。是否在離心前需先真空滲透, 可依據(jù)真空滲透后器官鮮重是否增加及質(zhì)外體稀釋因子的大小進(jìn)行判定。本試驗(yàn)分別對(duì)根系和葉片進(jìn)行真空滲透發(fā)現(xiàn), 根系鮮重幾乎沒變化且根系質(zhì)外體稀釋因子趨近于1, 說明滲透液未進(jìn)入根系質(zhì)外體空間; 葉片重量顯著增加且質(zhì)外體稀釋因子大于2, 說明滲透緩沖液進(jìn)入了葉片質(zhì)外體空間。由此可見, 營養(yǎng)液培養(yǎng)條件下, 棉花根系A(chǔ)WF分離可以直接離心, 和羽扇豆和豌豆根系分離AWF報(bào)道的一致[14]。但是, 水稻根系和玉米根系用于分離AWF時(shí)分別先進(jìn)行–70 kPa和–50 kPa真空滲透15 min后再離心[20,23]。棉花葉片汁液分離需先真空滲透再離心, 這和其他汁液分離葉片AWF時(shí)報(bào)道的一致[16-17,19,27]。

同列不同小寫字母表示差異顯著(<0.05)。Different lowercase letter s in the same column indicate significant differences (< 0.05).

表6 不同離心力/離心時(shí)間組合條件下棉花AWF體積、可溶性蛋白含量比值和MDH活性比值

可溶性蛋白含量比值: AWF中可溶性蛋白含量與SWF中可溶性蛋白含量的比值; MDH活性比值: AWF中MDH活性與SWF中MDH活性的比值。同列不同小寫字母表示同一器官不同組合條件間差異顯著(<0.05)。Soluble protein content ratio: the ratio of soluble protein content in AWF to SWF (symplast washing fluid); MDH enzyme activity ratio: the ratio of MDH enzyme activity in AWF to SWF. Different lowercase letters in the same column indicate significant differences among regimes for the same organ (< 0.05).

真空滲透時(shí)采取的滲透液種類和濃度有不同的報(bào)道, 如青蔥葉片用20 mmol?L–1抗壞血酸和20 mmol?L–1CaCl2[26], 大麥葉片用50 mmol?L–1MES/ KOH 緩沖液(pH 6.0, 40 mmol?L–1KCl, 2 mmol?L–1CaCl2)或50 mmol?L–1醋酸緩沖液(pH 4.5, 100 mmol?L–1KCl, 2 mmol?L–1CaCl2)[27]。對(duì)比研究發(fā)現(xiàn), 不同植物葉片分別用去離子水、10 mmol?L–1KCl、180 mmol?L–1MOPS真空滲透后離心獲得的AWF代謝物濃度差異很小[16]; 7種不同滲透液真空滲透, 發(fā)現(xiàn)用磷酸緩沖液提取的玉米葉片AWF蛋白質(zhì)含量最高, 且受SWF蛋白質(zhì)污染最少[19]。

不同植物葉片因結(jié)構(gòu)和組成的差異, 采取滲透的真空強(qiáng)度和時(shí)間以及離心力和離心時(shí)間均不同[17]。例如, 青蔥葉片滲透為真空–70 kPa/15 min[26]; 玉米葉片滲透為真空–20 kPa[19]; 擬南芥葉片滲透為真空–8 kPa/2 min, 重復(fù)5次[21]。依據(jù)AWF體積、可溶性蛋白含量比值和MDH活性比值, 本研究認(rèn)為棉花葉片滲透采用–60 kPa/1 min最為適宜, 且從真空恢復(fù)到正常壓強(qiáng)時(shí)需相對(duì)緩慢。而且本研究提供了一種根據(jù)葉片真空滲透后顏色變化直觀判定真空滲透效果的方法。真空滲透后, 蠶豆、玉米、菠菜和大麥葉片離心時(shí)的離心力分別為75 ×g、90 ×g、220 ×g和620 ×g, 離心時(shí)間均為4 min[16]; 玉米葉片400 ×g離心5 min[19]; 青蔥葉片1 500 ×g離心20 min[26]; 水稻根系1 000 ×g離心10 min[23]。

通過真空滲透和離心后, AWF中MDH活性[28-31]和6-磷酸葡萄糖脫氫酶活性[20,23]通常被作為判定AWF是否受SWF蛋白污染的指標(biāo)[32], 進(jìn)而可判定真空滲透所采用液體、真空強(qiáng)度和真空時(shí)間等及離心強(qiáng)度和時(shí)間等參數(shù)是否適宜。AWF中MDH活性和共質(zhì)體MDH活性比值通常小于5%, 比值越低說明AWF蛋白質(zhì)受SWF蛋白質(zhì)污染越低, 如小麥()莖基部小于0.5%[31], 三葉草()葉片小于3%[28], 煙草()葉片小于0.5%[29]。本試驗(yàn)中, 棉花整體根系小于0.2%, 遠(yuǎn)小于棉花根系上報(bào)道的4.2% (在該研究中棉花也為營養(yǎng)液培養(yǎng), 但根系為非整體取樣, 且在真空滲透后離心獲得質(zhì)外體汁液)[30]; 棉花整體葉片小于1%, 表明分離的AWF用于其組分如蛋白質(zhì)組和代謝組研究結(jié)果準(zhǔn)確可靠。而且, 通過抽真空后葉片顏色和軟硬狀態(tài)判斷真空強(qiáng)度和時(shí)間組合是否適宜和質(zhì)內(nèi)體受共質(zhì)體物質(zhì)污染程度的研究尚鮮見報(bào)道。該試驗(yàn)用在營養(yǎng)液培養(yǎng)條件下的根系和葉片, 進(jìn)行了AWF汁液分離方法從取樣到離心參數(shù)的綜合性改進(jìn); 如培養(yǎng)條件和器官狀態(tài)明顯變化, 建議仍然采用整體取樣法但需調(diào)整真空滲透和離心參數(shù), 范圍不超過該報(bào)道的參數(shù)上限。

綜上所述, 本試驗(yàn)改進(jìn)了取樣方式, 減免了根系的真空滲透步驟, 提出了通過葉片顏色變深面積判定真空滲透程度的簡易方法, 改進(jìn)了葉片真空滲透時(shí)的壓強(qiáng)和壓強(qiáng)恢復(fù)正常所需時(shí)間及離心力與時(shí)間, 分別針對(duì)棉花根系和葉片優(yōu)化了AWF分離的流程和方法, 不僅為棉花根系和葉片AWF組分研究的準(zhǔn)確性和可靠性奠定了基礎(chǔ), 也為其他作物AWF分離方法的優(yōu)化提供了參考和思路。

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Improved methods for separating apoplastic washing fluid from roots and leaves in cotton seedlings*

WANG Guo, FENG Kang, LI Qian, XUE Huiyun, LI Lijie, ZHANG Zhiyong**

(Henan Institute of Science and Technology / Henan Collaborative Innovation Center of Modern Biological Breeding / Henan Key Laboratory for Molecular Ecology and Germplasm Innovation of Cotton and Wheat, Xinxiang 453003, China)

Apoplast washing fluid (AWF) contains minerals, metabolites, and proteins that plays an important role in plant growth and development, as well as provides biotic and abiotic stress resistance. AWF extraction is the basis of exploring the function of AWF constituents. It is generally performed via vacuum infiltration-centrifugation technique; which varies in processes and detailed parameters depending on the plan species, organs, and culture conditions. Hydro-cultured cotton seedlings were used to investigate AWF separation processes and parameters suitable for cotton root and leaf development, and further improve methods for cotton root or leaf AWF separation. Compared with traditionally split sampling (i.e., splitting samples into segments or pieces), sampling a complete unit was simple and significantly decreased the ratio of malate dehydrogenase (MDH) activity in AWF to symplast washing fluid (SWF), which usually is used to affirm the degree of AWF substances polluted by SWF; indicating that AWF components would better to examine. Furthermore, the fresh weight increments and the AWF diluting factor after vacuum infiltration of the roots had no significant change, but significantly increased in the leaves. This indicates that vacuum infiltration is only essential for leaves, with a vacuum strength/time at-60 kPa/1 min, and about 110 s recovery from vacuum to normal atmospheric pressure. Leaf areas with dark color increased with vacuum intensity or time, which could be used as a simple indicator for determining the suitability for AWF separation. Finally, comprehensive analyses of the AWF volume, soluble protein content ratio and MDH activity ratio of AWF to SWF indicated that the suitable centrifuge forge/time was 800 ×g /10-20 min for the root, and 400 ×g /5 min for the leaf. This refined and optimized method will lay down the foundation for efficient study of AWF components such as the accuracy and reliability of proteomics and metabolomics. The approach towards establishing this method should allow it to be generally applicable to other plants.

Cotton; Malate dehydrogenase; Vacuum infiltration; Centrifuge; Symplast; Apoplast

S311

10.13930/j.cnki.cjea.200092

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* 國家自然科學(xué)基金項(xiàng)目(31571600)資助

張志勇, 主要研究方向?yàn)樽魑锬婢成?。E-mail: z_zy123@126.com

王果, 主要研究方向?yàn)橘|(zhì)外體代謝組學(xué)和蛋白質(zhì)組學(xué)。E-mail: 18336063077@163.com

2020-02-17

2020-04-09

* This study was supported by the National Natural Science Foundation of China (31571600).

, E-mail: z_zy123@126.com

Feb. 17, 2020;

Apr. 9, 2020