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    黃瓜葉片可產生胞質脂滴

    2019-07-03 13:03:42 中國瓜菜2019年4期

    張超越 孫悅宸 李萌 薛夢偉 劉淑美 吳成紅 王守箐 劉林

    摘? ? 要: 胞質脂滴通常在生殖器官中產生,用透射電子顯微技術研究黃瓜葉片能否產生胞質脂滴,哪種類型的細胞能產生脂滴,以為黃瓜葉片脂滴的開發研究提供參考。結果表明,幼葉、成熟葉和衰老葉都含有胞質脂滴,即黃瓜葉片發育各階段均能產生胞質脂滴。幼葉中,表皮細胞、葉肉細胞和維管組織細胞都含有少量胞質脂滴;成熟葉中,以葉肉細胞中胞質脂滴較多;老葉中,葉肉細胞、維管薄壁細胞和伴胞都產生胞質脂滴,以伴胞中最多。老葉葉肉細胞中胞質脂滴向液泡內轉移,顯示胞質脂滴通過細胞自噬途徑降解。

    關鍵詞: 黃瓜; 葉片; 胞質脂滴

    Cucumber leaf blades produce cytosolic lipids

    ZHANG Chaoyue, SUN Yuechen, LI Meng, XUE Mengwei, LIU Shumei, WU Chenghong, WANG Shouqing, LIU Lin

    (College of Pharmacy, Linyi University, Linyi 276005, Shandong, China)

    Abstract: It is well known that cytosolic lipid droplets are produced in reproductive organs or tissues. Leaf blades of cucumber were examined by means of transmission electron microscopy, with the aim to understand whether there is occurrence of cytosolic lipid droplets, and what types of cells produce cytosolic lipid droplets. There were cytosolic lipid droplets in young,mature,and aged leaves,suggesting that cucumber leaf blades could produce cytosolic lipid droplets during all developmental stages. Cytosolic lipid droplets in young leaf blades occurred in epidermal cells,mesophyll cells,and vascular parenchyma cells. Cytosolic lipid droplets in mature leaves were present mainly in mesophyll cells. Cytosolic lipid droplets in aged leaves occurred in mesophyll cells,vascular parenchyma cells,and companion cells. Cytosolic lipid droplets in aging leaves moved to the vacuole,suggesting they underwent degradation through autophagy pathway.

    Key words:Cucumber; Leaf; Cytosolic lipid droplet

    脂滴是由磷脂單分子層膜包圍而成的囊泡狀細胞器,囊泡內積累甘油酯和其他脂溶性分子,如類胡蘿卜素和維生素E,囊泡表面結合有多種蛋白質[1-3]。所有植物都能產生脂滴,是食用油和工業用油的重要來源,也是再生能源,與人類生活關系極為密切,因此受到廣泛重視。植物脂滴分兩類,細胞質中的脂滴為胞質脂滴,葉綠體內的脂滴為質體小球[4]。葉綠體內,中性脂和其他脂溶性化合物在類囊體膜兩小葉之間積累,形成小球體,小球體脫離類囊體進入基質成為質體小球[5]。葉綠體外膜局部區域兩小葉之間積累中性脂,形成小球體,小球體脫離外膜進入細胞質而成為胞質脂滴[6]。內質網局部區域兩小葉之間積累中性脂,形成小球體,小球體脫離內質網進入細胞質而成為胞質脂滴[7]。胞質脂滴是生物膜脂成分前體及細胞代謝能源的貯藏庫[4],還參與植物發育、激素代謝、脅迫響應、信號轉導等多種生物學過程[2-4]。目前已鑒定出多種脂滴相關蛋白[8],這些蛋白的鑒定促進了對脂滴發生和功能的認識[1-3],但對胞質脂滴發生的普遍性及細胞特異性還鮮有報道[8],這些方面的信息不僅是胞質脂滴生物學功能研究的基礎,也為脂滴商業開發提供參考。植物果實、種子等生殖器官可以積累大量胞質脂滴,成為食用油和工業用油的來源,而對營養器官產生胞質脂滴的研究還非常有限,例如,目前僅發現少數種類植物葉片內產生胞質脂滴[9-11]。在這些植物葉片中,胞質脂滴發生的細胞特異性很強,例如,Lersten等[9]在71種植物葉片中觀察到胞質脂滴,都出現在葉肉細胞內;Kwiatkowska等[12-13]以及Huang等[14]觀察到天門冬目植物葉片產生胞質脂滴,都出現在表皮細胞內,不過至今還沒有維管束細胞產生胞質脂滴的報道。上述胞質脂滴發生的細胞特異性與一種基于脂滴相關蛋白鑒定結果的推測相矛盾,這一推測認為植物任何細胞都有合成胞質脂滴的裝置[11]。此外,葉片發育可分為幼期、成熟期和衰老期等3個階段,關于胞質脂滴產生的時期和階段也還鮮有報道。

    黃瓜是常見瓜類作物,莖和葉尚沒有商業用途。考慮到葉片脂滴具有潛在的開發價值,針對能否產生胞質脂滴、脂滴發生有無細胞特異性以及脂滴發生與葉片發育有無相關性,對黃瓜不同發育階段的葉片進行了透射電子顯微觀察,為進一步研究脂滴發生與黃瓜生理、產量和品質的關系奠定基礎。

    1 材料與方法

    1.1 材料

    供試黃瓜為‘錦豐2號,由臨沂市種子公司提供,2017年3月育苗, 4月移栽到臨沂大學教學科研基地,常規栽培和管理,7月取樣,間隔5 d取樣1次,共取3次。每次于晴天11:00從長勢良好植株選擇剛開始擴展的幼葉2片、充分伸展的成熟葉2片、暗綠色衰老葉2片(葉綠素下降到成熟葉的70%左右,分光光度法測定),從每葉片剪下0.5 cm×1.0 cm大小2片,立即浸入2%戊二醛固定液(0.05 mol·L-1磷酸緩沖液配制,pH 6.8),同一發育階段的4小片置于同一容器。后續工作在臨沂大學藥用植物學實驗室完成。

    1.2 透射電子顯微技術

    每次取樣后盡快將樣品帶回實驗室,在實驗室內進一步切成小于1 mm×1 mm的小片。在2% 戊二醛溶液中固定4 h后,再用1%四氧化鋨溶液(0.05 mol·L-1磷酸緩沖液配制,pH 6.8)固定4 h。固定完成后,用梯度酒精脫水,包埋于Embed-812樹脂中,制成包埋塊。將各次各發育階段樣品包埋塊用鉆石刀切片,厚度60 nm。切片用醋酸鈾和檸檬酸鉛電子染色,Jeol 1220透射電子顯微鏡下觀察。

    2 結果與分析

    2.1 幼葉中的胞質脂滴

    幼葉表皮細胞含有大液泡,液泡約占細胞體積的一半,細胞核較大,葉綠體較小,類囊體較少。細胞質中有胞質脂滴,每一細胞切片上可觀察到1~2個胞質脂滴,直徑0.2~0.4 ?m(圖1)。葉肉細胞的液泡尚小,細胞質濃,細胞核較大,葉綠體較少且體積小,類囊體和基粒都較少,積累少量淀粉,但淀粉粒很小。含有胞質脂滴,每細胞切片上可見1~2個,但葉肉組織切片上只有部分細胞可見胞質脂滴,直徑0.2~0.4 ?m(圖2)。維管組織正在分化,尚沒有成熟的木質部和韌皮部。維管組織細胞中可見胞質脂滴,但數量不多,直徑約0.3 ?m(圖2)。綜合上述觀察,幼葉產生胞質脂滴,脂滴發生在表皮細胞、葉肉細胞和維管組織細胞中,每細胞切片上可見1~2個,且葉片橫切面上僅部分細胞可見胞質脂滴,因此胞質脂滴數量不多。

    2.2 成熟葉片中的胞質脂滴

    成熟葉片表皮細胞和葉肉細胞高度液泡化,液泡占據細胞絕大部分體積。表皮細胞的細胞質呈一薄層,厚0.3~1.0 ?m(圖3-A),胞質脂滴極少觀察到。但葉肉細胞所含胞質脂滴較多,形態與質體小球類似,直徑約0.5 ?m。胞質脂滴與線粒體、過氧化物酶體和葉綠體非常接近(圖3-B)。維管薄壁細胞包括細胞質很濃的伴胞、高度液泡化的普通薄壁細胞(圖3-C)以及高度特化的篩管分子(圖3-D),這些細胞都基本不含胞質脂滴。顯然,成熟葉片以葉肉細胞所含胞質脂滴較多。

    2.3 老葉中的胞質脂滴

    衰老葉表皮細胞的細胞質更少,細胞質貼著細胞壁,僅約0.2 ?m厚,為葉綠體厚度的1/5(圖4-A),極少觀察到胞質脂滴。葉肉細胞含有較多胞質脂滴,直徑0.1~1.2 ?m,向液泡內運動,進入液泡內部(圖4-B,圖5)。此外,葉綠體中的質體小球有向外轉移的趨勢(圖4-C、D),轉移到葉綠體外成為胞質脂滴。維管組織中的胞質脂滴明顯增多(圖5),特別是伴胞中,伴胞切片可見較多胞質脂滴,直徑約0.5 ?m。普通維管薄壁細胞和篩管分子中都出現了胞質脂滴。

    3 討論與結論

    3.1 黃瓜葉發育各階段均產生胞質脂滴,但不大量積累

    本研究結果表明,黃瓜葉片能夠產生胞質脂滴,且不管是幼葉、成熟葉還是老葉,各發育階段均能產生,顯然胞質脂滴發生對葉片發育階段沒有依賴性。

    植物葉片胞質脂滴的發生可能有多方面生物學意義,作為生物膜脂成分前體及細胞代謝能的貯藏場所[4],參與發育、代謝、脅迫響應、信號轉導等生物學過程[2]。研究表明,葉片遭受溫度或干旱脅迫時會產生胞質脂滴[10,15],受到病原微生物侵染時也會產生胞質脂滴[16]。另一種觀點認為,葉片發育過程中遇到脅迫時,細胞內膜系統受到損傷而產生脂肪酸,脂肪酸游離在細胞內對細胞有毒性,因此,如果游離脂肪酸在細胞內積累,勢必對蛋白質分子造成傷害,從而影響各種生理生化活動的正常進行,而當這些有毒的脂肪酸分子轉化為中性脂儲存在脂滴中,就產生了解毒效果,達到了保護細胞的目的[8]。黃瓜葉片在正常生長條件下也會產生胞質脂滴,脂滴的產生可能是黃瓜的遺傳特征。根據上述討論,黃瓜葉片胞質脂滴發生很可能參與葉片的發育、代謝和脅迫響應,但其抗病作用及其他生理功能尚需進一步研究。

    黃瓜幼葉的各種類型細胞中均容易觀察到胞質脂滴,成熟葉片的葉肉細胞中容易觀察到脂滴,老葉除表皮細胞外,其他類型細胞均有較多脂滴,特別是維管組織細胞中明顯較多,顯然,各種類型細胞中的脂滴發生量與葉片發育階段有一定的關系,葉片衰老可能誘導維管組織中的胞質脂滴增多。雖然老葉中胞質脂滴增多,但脂滴并沒有大量積累,這與存在脂滴降解途徑有關。老葉葉肉細胞中,胞質脂滴進入液泡,這是細胞自噬,脂滴在液泡中被降解。由于脂滴通過細胞自噬途徑降解,因而胞質脂滴不會大量積累。脂滴通過自噬途徑降解的現象在西瓜葉片中也有發生[17]。脂滴通過自噬途徑降解的現象可能也發生在黃瓜葉片的其他類型細胞中,致使所有細胞都不積累大量胞質脂滴。葉片內,脂滴通過自噬途徑的降解,降解產物最終轉化成可通過韌皮部運輸的小分子蔗糖[18],蔗糖轉移到生長旺盛的部位如果實和生長點,為果實生長和新器官產生提供營養,顯然胞質脂滴的產生和降解對黃瓜產量和品質會有積極影響,不過這種相關性還需要進一步研究。

    黃瓜葉片每細胞切片含有1至數個胞質脂滴,顯然黃瓜葉片中的胞質脂滴積累水平不高。植物葉片胞質脂滴的積累水平可通過基因工程手段提高,有研究表明,細胞中的胞質脂滴積累與脂肪酸濃度之間存在動態平衡,若提高脂肪酸濃度,就可以增加脂滴的積累[8]。例如,Yurchenko等[19]通過基因工程手段提高擬南芥葉中的脂肪酸合成能力,提高脂肪濃度,胞質脂滴數量明顯增多;Slocombe等[20]通過基因工程技術抑制脂肪酸降解途徑,細胞中的脂肪水平提高,結果胞質脂滴數量增加10倍之多。因此,要想提高黃瓜葉中的脂滴積累水平,從理論上有可行性。

    3.2 黃瓜葉片不同類型的細胞都能產生胞質脂滴

    黃瓜葉胞質脂滴出現在表皮細胞、葉肉細胞和維管薄壁細胞中,表明各種類型的細胞均能產生胞質脂滴,這與過去在其他植物中的觀察結果不同。Lersten 等[9]在71種植物葉中觀察到胞質脂滴,都無一例外地出現在葉肉細胞中;而Kwiatkowska等[12-13]和Huang等[14]在天門冬目植物中發現,葉片中的胞質脂滴均出現在表皮細胞中。黃瓜葉片胞質脂滴發生在表皮、葉肉和維管組織中,顯然黃瓜葉片產生胞質脂滴的細胞類型較多,即胞質脂滴發生沒有明顯的細胞特異性。根據對胞質脂滴相關蛋白的研究,Pyc等[11]提出,植物任何細胞都有合成胞質脂滴的裝置,黃瓜葉片胞質脂滴發生的普遍性與之一致,即黃瓜葉片各種類型細胞都具有產生胞質脂滴的能力。

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