胡振民,趙學(xué)強(qiáng),王 超,沈仁芳*
(1 土壤與農(nóng)業(yè)可持續(xù)發(fā)展國(guó)家重點(diǎn)實(shí)驗(yàn)室(中國(guó)科學(xué)院南京土壤研究所),南京 210008;2 中國(guó)科學(xué)院大學(xué),北京 100049)
細(xì)胞壁組分在紅酵母RS1高耐鋁中的作用研究①
胡振民1,2,趙學(xué)強(qiáng)1,王 超1,沈仁芳1*
(1 土壤與農(nóng)業(yè)可持續(xù)發(fā)展國(guó)家重點(diǎn)實(shí)驗(yàn)室(中國(guó)科學(xué)院南京土壤研究所),南京 210008;2 中國(guó)科學(xué)院大學(xué),北京 100049)
紅酵母RS1是從酸性油茶土壤中分離到的一株高耐鋁微生物,能夠忍耐高達(dá)200 mmol/L以上的鋁,前期研究表明 RS1高耐鋁能力與細(xì)胞壁有關(guān),但是其具體機(jī)制還不清楚。因此,本文進(jìn)一步研究了細(xì)胞壁組分在 RS1高耐鋁中的作用,以期為RS1高耐鋁的具體機(jī)制提供新信息。結(jié)果表明,高于70 mmol/L的鋁對(duì)RS1生長(zhǎng)產(chǎn)生抑制作用,0 ~ 70 mmol/L鋁處理后細(xì)胞壁主要組分甘露糖和葡聚糖含量都沒(méi)有顯著改變,而細(xì)胞壁磷含量在70 mmol/L鋁處理后顯著升高。在高鋁處理時(shí),糖蛋白抑制劑抗生素衣霉素(tunicamycin)極大加重RS1的鋁毒害。由此說(shuō)明,細(xì)胞壁多糖組分含量并不對(duì)RS1高耐鋁起到主要作用,細(xì)胞壁結(jié)構(gòu)修飾如細(xì)胞壁磷含量響應(yīng)和細(xì)胞壁N連接的糖蛋白修飾在RS1高耐鋁中可能起到一定作用。
紅酵母;鋁毒;細(xì)胞壁;甘露糖蛋白
當(dāng)土壤pH低于5時(shí),鋁(Al)主要以毒性Al3+形式存在,是酸性土壤中對(duì)植物和微生物造成毒害的主要因子之一[1–3]。對(duì)植物的耐鋁機(jī)制研究較多,其耐鋁機(jī)制主要包括外部排斥和內(nèi)部耐受[4–6]。同時(shí),在酸性土壤中存在著高耐鋁微生物,相對(duì)植物,耐鋁微生物可以耐受高達(dá)100 mmol/L的鋁[7–9]。相對(duì)于培養(yǎng)基中高濃度鋁,耐鋁微生物能保持細(xì)胞極低鋁濃度,說(shuō)明高耐鋁微生物主要通過(guò)有效地將鋁屏蔽于細(xì)胞外達(dá)到解鋁毒目的[10–11],但關(guān)于微生物屏蔽鋁的具體機(jī)制并不清楚。對(duì)于進(jìn)入到細(xì)胞質(zhì)內(nèi)的鋁,微生物主要通過(guò)有機(jī)酸或其他螯合物質(zhì)與鋁螯合減輕細(xì)胞內(nèi)鋁毒害[12–13]。同時(shí)微生物生長(zhǎng)快、周期短、易變異、單細(xì)胞等特點(diǎn)為鋁毒研究提供了便利,其高耐鋁機(jī)制的闡明也可為植物耐鋁機(jī)制提供借鑒,在未來(lái)有望應(yīng)用于改良酸性土壤和改善生態(tài)環(huán)境[14]。
本實(shí)驗(yàn)室前期在江西鷹潭酸性油茶土壤中篩選得到一株高耐鋁紅酵母RS1,可以在200 mmol/L鋁下生長(zhǎng),并發(fā)現(xiàn)高濃度鋁處理下 RS1細(xì)胞壁增厚,這種響應(yīng)機(jī)制可能對(duì)RS1耐高濃度鋁起到重要作用[15–17],但是細(xì)胞壁在 RS1高耐鋁中的確切作用還不清楚。酵母細(xì)胞壁主要由葡聚糖和甘露糖蛋白組成,葡聚糖位于細(xì)胞壁內(nèi)側(cè),主要維持細(xì)胞壁的機(jī)械強(qiáng)度[18];甘露糖蛋白位于細(xì)胞壁表面,并廣泛被 O(絲氨酸或蘇氨酸)和 N(天冬酰胺)糖基化修飾,決定了細(xì)胞壁的疏水性、電荷及對(duì)溶質(zhì)分子的透過(guò)性[19]。釀酒酵母中與囊泡運(yùn)輸和蛋白甘露糖基化有關(guān)的基因缺失后,酵母細(xì)胞壁結(jié)構(gòu)受到影響,突變體對(duì)鋁更為敏感,說(shuō)明細(xì)胞壁相關(guān)組分及其代謝在酵母耐鋁中起到作用[20]。細(xì)胞壁糖蛋白抑制劑衣霉素,作為一種抗生素,其對(duì)酵母進(jìn)行處理后能夠抑制甘露糖N連接的糖基化修飾,會(huì)影響到細(xì)胞壁甘露糖蛋白組成及結(jié)構(gòu)[21],進(jìn)而可能會(huì)影響到RS1耐鋁性。
在本實(shí)驗(yàn)室前期研究及相關(guān)文獻(xiàn)的基礎(chǔ)上[15–21],本文進(jìn)一步研究了細(xì)胞壁在 RS1高耐鋁中的作用,并用糖蛋白抑制劑衣霉素處理細(xì)胞,以期闡明細(xì)胞壁組分在RS1耐鋁中的作用。
1.1 試驗(yàn)材料
采用紅酵母Rhodotorula taiwanensis RS1 (RS1) (=China General Microbiological Culture Collection (CGMCC) 2.4753)為試驗(yàn)材料,其分離自江西酸性
紅壤[15, 22]。
1.2 培養(yǎng)條件
試驗(yàn)采用低pH低磷低鎂 (low pH, low Phosphate and low Magnesium, LPM) 培養(yǎng)基,具體組成參照文獻(xiàn)[23],其中鎂、磷含量都降低為0.1 mmol/L,pH 3.5,同時(shí)添加50 mmol/L琥珀酸作為pH緩沖液[24]。液體培養(yǎng)在30℃、200 r/min下進(jìn)行。固體培養(yǎng)基在上述液體培養(yǎng)基基礎(chǔ)上添加2% 瓊脂糖,在30℃ 倒置靜止培養(yǎng)。鋁采用AlCl3,配備1 mol/L母液,抽濾滅菌后加入高溫滅菌LPM培養(yǎng)基中,獲得不同鋁濃度的LPM培養(yǎng)基。
1.3 RS1對(duì)鋁的耐性
混凝土的防護(hù)措施主要是對(duì)溫度的控制,為了保證混凝土可以順利的進(jìn)行硬化,可以采用相關(guān)的防護(hù)措施對(duì)于混凝土澆筑過(guò)程中的溫度進(jìn)行合理的控制,控制在混凝土規(guī)定的標(biāo)準(zhǔn)范圍之內(nèi),凝固過(guò)程中需要采取一定的手段控制好溫度,防止溫度發(fā)生急劇的變化?;炷恋酿B(yǎng)護(hù)是保障混凝土質(zhì)量的有效措施,避免混凝土在陽(yáng)光下暴曬,溫差會(huì)影響混凝土的成型。混凝土養(yǎng)護(hù)主要針對(duì)溫度、濕度的控制,高溫時(shí)可以在混凝土的表面覆蓋一層塑料布,避免了陽(yáng)光直射造成水分的蒸發(fā)[4]。
RS1預(yù)培養(yǎng)至對(duì)數(shù)期,3 000 g離心5 min收集菌體,去離子水清洗兩次,加入含不同鋁濃度 LPM培養(yǎng)基,至初始OD600= 0.01,培養(yǎng)24 h后取樣,測(cè)定OD600反應(yīng)菌體生長(zhǎng)狀況。
1.4 細(xì)胞壁組分測(cè)定
按照1.3中方法,RS1預(yù)培養(yǎng)并接種至含不同濃度鋁LPM培養(yǎng)基中,處理24 h后,離心收集菌體,加入玻璃珠(450 ~ 600 μm)置于冰上,F(xiàn)astPrep-24勻漿儀間隔破碎細(xì)胞1 min,間隔置于冰上冷卻1 min,共計(jì)破碎4個(gè)循環(huán)后,2 000 g離心10 min,用預(yù)冷的去離子水清洗數(shù)次直至上清液澄清,離心管底部沉淀即為細(xì)胞壁[25]。Labconco-2.5L臺(tái)式凍干機(jī)冷凍干燥,稱取一定量細(xì)胞壁,用 2 mol/L硫酸水解將 β-葡聚糖完全轉(zhuǎn)化為葡萄糖,甘露聚糖轉(zhuǎn)化為甘露糖,然后參照文獻(xiàn)中方法測(cè)葡萄糖和甘露糖含量[26]。
1.5 細(xì)胞壁磷含量
按照1.3中方法,不同濃度鋁處理24 h后,離心收集菌體,按照1.4中方法破壁,清洗并冷凍干燥后,稱取5 mg細(xì)胞壁,加入0.5 ml濃HNO3(優(yōu)級(jí)純)消煮,定容后用電感耦合等離子體光譜儀 PerkinElmer-Optima8000測(cè)定消煮液中磷含量。
1.6 細(xì)胞壁代謝抑制劑衣霉素對(duì)RS1耐鋁性的影響
對(duì)數(shù)期RS1細(xì)胞在含不同濃度衣霉素(0、0.05、0.1 μg/ml)液體LPM培養(yǎng)基預(yù)培養(yǎng)4 h,3 000 g離心5 min收集菌體,按照1.3中方法,分別轉(zhuǎn)接至對(duì)照和含50 mmol/L鋁的LPM培養(yǎng)基中,培養(yǎng)24 h后測(cè)定OD600。
1.7 數(shù)據(jù)分析
數(shù)據(jù)采用Excel 2013和SPSS 18.0統(tǒng)計(jì)軟件進(jìn)行分析,差異水平通過(guò) Duncan法進(jìn)行檢驗(yàn)(P<0.05)。作圖采用SigmaPlot 12.5進(jìn)行。
2.1 RS1對(duì)鋁的耐性
相對(duì)我們前面試驗(yàn)中使用的半合成培養(yǎng)基GM[15],完全合成培養(yǎng)基 LPM 中磷、鎂含量更低,都為0.1 mmol/L,這有助于降低外界因子磷和鎂緩解鋁毒的作用,從而更好地闡述紅酵母本身的鋁毒害或耐鋁機(jī)制。在LPM培養(yǎng)基中,RS1仍具有很高的耐鋁性,10 mmol/L時(shí)對(duì) RS1生長(zhǎng)有促進(jìn)作用,鋁濃度達(dá)到70 mmol/L時(shí),才對(duì)RS1生長(zhǎng)產(chǎn)生顯著抑制作用,100 ~200 mmol/L鋁對(duì)RS1生長(zhǎng)抑制更為顯著(圖 1)。
圖1 RS1在不同濃度鋁下生長(zhǎng)狀況Fig. 1 Tolerances of RS1 to different concentrations of Al
2.2 鋁對(duì)RS1細(xì)胞壁組分的影響
不同濃度鋁處理24 h后,RS1細(xì)胞壁葡聚糖、甘露糖含量都沒(méi)有顯著變化(圖 2)。細(xì)胞壁磷含量在0.1、10 mmol/L鋁處理后較對(duì)照未有顯著變化,70 mmol/L鋁處理24 h后顯著升高(圖 3)。由此可見(jiàn),RS1細(xì)胞壁多糖組分占細(xì)胞壁的比例不受鋁處理的影響,而高濃度鋁處理后細(xì)胞壁磷含量升高,暗示高濃度鋁處理導(dǎo)致細(xì)胞壁吸附了更多的磷酸根或者細(xì)胞壁磷酸化修飾程度升高。
圖2 鋁處理24 h后RS1細(xì)胞壁葡聚糖和甘露糖含量Fig. 2 Glucan and mannan contents in cell walls of RS1 after treatment with Al for 24 hours
2.3 糖蛋白抑制劑衣霉素對(duì)RS1耐鋁性的影響
圖3 鋁處理24 h后細(xì)胞壁磷含量Fig. 3 Phosphorus contents of cell walls of RS1 after treatment with Al for 24 hours
RS1在含不同濃度糖蛋白抑制劑衣霉素的LPM培養(yǎng)基中預(yù)培養(yǎng)4 h,再轉(zhuǎn)入含50 mmol/L鋁培養(yǎng)基中處理24 h,不含抑制劑預(yù)培養(yǎng)對(duì)照組細(xì)胞生長(zhǎng)基本不受鋁抑制,隨抑制劑預(yù)培養(yǎng)濃度增加,細(xì)胞生長(zhǎng)受鋁抑制逐漸增強(qiáng)(圖 4)。進(jìn)一步計(jì)算了加鋁相對(duì)于不加鋁條件下細(xì)胞的相對(duì)生長(zhǎng)率,0、0.05、0.10 μg/ml衣霉素預(yù)處理的相對(duì)生長(zhǎng)率分別為 102%、58%、37%,可見(jiàn)糖蛋白抑制劑加重了RS1鋁毒害,這說(shuō)明N連接的甘露糖蛋白在RS1耐鋁中起到作用。
圖4 衣霉素對(duì)RS1耐鋁性的影響Fig. 4 Effects of tunicamycin on growths of RS1 under control and Al condition
鋁毒害受培養(yǎng)基中多種因素影響,其中磷和鎂是培養(yǎng)基中影響鋁毒害的重要因子,磷與鋁螯合,會(huì)降低培養(yǎng)基中鋁效應(yīng)[27];細(xì)胞對(duì)鎂的吸收受低濃度鋁抑制,提高培養(yǎng)基中鎂濃度或者過(guò)表達(dá)鎂離子通道蛋白,都會(huì)提高釀酒酵母耐鋁性[28–29]。這里采用的低磷低鎂LPM培養(yǎng)基降低了外界因子磷和鎂對(duì)RS1鋁毒害的緩解作用,相對(duì) GM 培養(yǎng)基[15],更適合研究RS1本身內(nèi)在的耐鋁機(jī)制。10 mmol/L鋁能夠促進(jìn)RS1細(xì)胞生長(zhǎng),釀酒酵母中低濃度鋁能夠通過(guò)鋁劑量和細(xì)胞密度依賴的方式促進(jìn)細(xì)胞分裂,進(jìn)而促進(jìn)細(xì)胞生長(zhǎng)[30]。70 mmol/L鋁處理下RS1相對(duì)對(duì)照生長(zhǎng)率為44.7%,對(duì)RS1生長(zhǎng)起到中等抑制作用,所以后續(xù)試驗(yàn)最大鋁處理濃度選為70 mmol/L;而100 ~ 200 mmol/L鋁對(duì) RS1生長(zhǎng)抑制程度過(guò)大,所以未采用 100 ~200 mmol/L鋁處理濃度。
本實(shí)驗(yàn)室前期結(jié)果表明,RS1的高耐鋁性是本身遺傳固有的,不被誘導(dǎo)產(chǎn)生[17],細(xì)胞壁增厚可能在RS1耐高濃度鋁中起到重要作用[15]。GM培養(yǎng)基中高濃度鋁處理后RS1細(xì)胞壁增厚,細(xì)胞壁占細(xì)胞比重升高,但各多糖組分占細(xì)胞壁的比例并沒(méi)有顯著變化[15]。本試驗(yàn)中鋁處理后RS1各細(xì)胞壁多糖組分占細(xì)胞壁的比例同樣沒(méi)有顯著變化,但不同細(xì)胞壁組分間交聯(lián)程度或方式可能發(fā)生了改變,以適應(yīng)高濃度鋁脅迫。另外,雖然各多糖組分在細(xì)胞壁的比例沒(méi)有變化,但是細(xì)胞壁在鋁脅迫下厚度增加[15],這會(huì)導(dǎo)致多糖組分的總量增加。葡聚糖和甘露糖,作為細(xì)胞壁的主要組分交織在一起但并非均勻分布在細(xì)胞壁中,其中甘露糖蛋白主要位于細(xì)胞壁外側(cè),葡聚糖位于內(nèi)側(cè);內(nèi)側(cè)葡聚糖賦予細(xì)胞壁剛度,外側(cè)的甘露糖蛋白及其修飾則決定了細(xì)胞壁的孔隙度及所帶電荷[31–32],細(xì)胞壁中位于外側(cè)的甘露糖蛋白與鋁毒害關(guān)系可能最為密切。
甘露糖蛋白的修飾,如磷酸化等,使得細(xì)胞壁帶有負(fù)電荷。磷酸根在細(xì)胞壁中可能以吸附[33]或者甘露糖磷酸的形式存在,其賦予了細(xì)胞壁負(fù)電荷,決定了細(xì)胞壁與環(huán)境中陽(yáng)離子電荷的吸附能力[34]。磷能緩解水稻的鋁毒害,耐鋁品種水稻根表及根自由空間中磷濃度高于敏感品種,表現(xiàn)出更強(qiáng)的質(zhì)外體解鋁毒能力,這可能與更強(qiáng)的磷吸收效率有關(guān)[35];同時(shí)磷能增強(qiáng)鋁耐性胡枝子的耐鋁性,施磷后更多的鋁被從根尖排除[36]。可見(jiàn),根質(zhì)外體中較高的磷含量有助于緩解鋁毒,紅酵母 RS1細(xì)胞壁中的磷也可能會(huì)影響到RS1鋁毒害。70 mmol/L鋁處理后,RS1細(xì)胞壁磷含量顯著增高,此時(shí)細(xì)胞壁鋁含量也升高[15],細(xì)胞壁吸附更多的鋁,導(dǎo)致細(xì)胞壁帶有更多的正電荷,這可能會(huì)吸附更多帶負(fù)電荷的磷酸根,從而導(dǎo)致細(xì)胞壁磷含量升高。細(xì)胞壁磷含量增高也可能是由于鋁處理后細(xì)胞壁甘露糖磷酸化修飾增多造成的,對(duì)脅迫蛋白的甘露糖磷酸化修飾有助于脅迫條件下蛋白的穩(wěn)定,也可能會(huì)導(dǎo)致細(xì)胞壁表面負(fù)電荷增加[34]。磷含量增高可能是 RS1適應(yīng)高濃度鋁脅迫的響應(yīng)機(jī)制,也可能僅是高濃度鋁對(duì)細(xì)胞的毒害作用。
糖蛋白抑制劑衣霉素作為一種抗生素,能抑制N-乙酰氨基葡糖胺-1-磷酸從 UDP-N-乙酰氨基葡糖胺轉(zhuǎn)移至磷酸多萜醇,阻斷N-糖基化中蛋白和糖鏈的交聯(lián),進(jìn)而影響細(xì)胞壁甘露糖蛋白的糖基化修飾[37]。釀酒酵母用衣霉素處理后,甘露糖蛋白合成受抑制,細(xì)胞壁完整性受破壞,導(dǎo)致細(xì)胞壁具有更高的孔隙度[38],這會(huì)使得培養(yǎng)基中的物質(zhì)更容易透過(guò)細(xì)胞壁與細(xì)胞膜接觸。白念珠菌中,衣霉素能抑制細(xì)胞壁甘露糖蛋白形成,進(jìn)而抑制生物膜的形成[39]。衣霉素處理細(xì)胞后,還會(huì)抑制胞外蛋白,如果糖苷酶的分泌[40]。本試驗(yàn)中細(xì)胞壁甘露糖蛋白合成受抑制后,同時(shí)加入50 mmol/L鋁處理,極大加重了RS1鋁毒害,可見(jiàn)N連接的甘露糖蛋白在RS1耐鋁中起到重要作用。N連接的甘露糖蛋白受抑制后,可能導(dǎo)致了細(xì)胞壁結(jié)構(gòu)改變,位于細(xì)胞壁最外層的甘露糖蛋白層失去對(duì)鋁的有效屏蔽作用,從而 RS1耐鋁性下降,其中涉及到的具體機(jī)制有待進(jìn)一步試驗(yàn)驗(yàn)證。本文中采用的糖蛋白抑制劑衣霉素本身即為抗生素,高濃度時(shí)能夠抑制細(xì)胞壁合成從而完全抑制細(xì)胞生長(zhǎng),考慮到抑制劑在較低濃度下并不能達(dá)到完全、即時(shí)抑制細(xì)胞壁糖蛋白合成的效果,這可能造成了本實(shí)驗(yàn)中糖蛋白合成受抑制,鋁毒害只是加重,但并未達(dá)到50 mmol/L鋁完全抑制細(xì)胞生長(zhǎng)的效果。為闡述不同細(xì)胞壁組分在 RS1耐鋁中的作用,后續(xù)試驗(yàn)需采用細(xì)胞壁相關(guān)組分突變體。
綜上所述,RS1在LPM培養(yǎng)基中仍能夠耐受高濃度鋁,細(xì)胞壁多糖組分含量不受外部供鋁的影響,細(xì)胞壁磷含量可能對(duì) RS1耐鋁性起作用,高濃度鋁處理下 N-連接的甘露糖蛋白在 RS1耐鋁中起到作用,其具體作用機(jī)制有待進(jìn)一步研究。
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Role of Cell Wall Components in High Aluminum Tolerance of Rhodotorula taiwanensis RS1
HU Zhenmin1,2, ZHAO Xueqiang1, WANG Chao1, SHEN Renfang1*
(1 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China; 2 University of Chinese Academy of Sciences, Beijing 100049, China)
RS1 was a high Al tolerant Rhodotorula taiwanensis strain which was isolated from the acidic oil tea soil, and our previous studies showed that cell wall played an important role in the Al tolerance of RS1. However, the exact role of cell wall in the high Al tolerance of RS1 is still unclear. The role of cell wall components in the Al tolerance of RS1 was further investigated in this paper in order to clarify the mechanism underlying the high Al tolerance of RS1. The results showed that, the growth of RS1 was severely inhibited by Al higher than 70 mmol/L. The glucan and mannan contents of cell wall of RS1 did not change after Al treatment, whereas the content of phosphorus of cell wall was elevated under 70 mmol/L Al. The growth of RS1 was inhibited by Al much more after additions of cell wall metabolism inhibitor tunicamycin. In conclusion, phosphorus accumulation of cell wall and N-linked mannoproteins may play important roles in high Al tolerance of RS1.
Rhodotorula; Al toxicity; Cell wall; Mannoproteins
S154.3
A
10.13758/j.cnki.tr.2017.02.007
國(guó)家自然科學(xué)基金項(xiàng)目(41271257)、中國(guó)科學(xué)院“戰(zhàn)略性先導(dǎo)科技專項(xiàng)重點(diǎn)研究計(jì)劃”項(xiàng)目(XDB15030302)和國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃項(xiàng)目(2014CB441000)資助。
* 通訊作者(rfshen@issas.ac.cn)
胡振民(1986—),男,山東臨沂人,博士研究生,主要從事植物營(yíng)養(yǎng)研究。E-mail: zmhu@issas.ac.cn