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马尾松(Pinus massoniana Lamb.)作为我国亚热带特有树种,具有分布广、速生、丰产、适生能力较强等优良特性,是我国南方最主要工业用材树种之一,种植面积约占2/3以上[1],同时也是典型的先锋树种和外生菌根树种[2]。由于酸沉降、马尾松自身凋落物和根系分泌物的作用使得马尾松林地土壤酸化严重,并随连栽代数的增加而酸化增强[3]。我国南方马尾松林土壤心土层pH值一般小于4.5,交换性铝含量很高[4],因而马尾松常表现出中等以上程度的受害,甚至枯死,进而导致林地生态功能失调,水土保持功能下降。20世纪80年代,由于铝毒作用导致我国南方酸雨地区发生了马尾松林衰亡现象[5]。已有大量研究表明,真菌和植物根系形成的共生体——菌根,不仅能促进植物的生长,还能通过外延菌丝扩大植物根系的吸收面积,增强植物对营养和水分的摄取能力,从而提升宿主对干旱胁迫、盐分胁迫、低磷胁迫、重金属毒害等的耐受性[6-10]。菌根化育苗造林必将成为林业生态工程综合治理的重要前沿技术途径。而且,在自然界也存在既耐铝又耐贫瘠的优良菌株,如彩色豆马勃(Pisolithus tinctorius)[11],筛选更多的优良本土菌株,研究其提高寄主植物耐铝机制对退化森林的恢复具有重要意义。
松属植物(包括马尾松)是各种外生菌根真菌的天然宿主[12-13]。马尾松可与约 36 种外生菌根真菌形成菌根,其中,以牛肝菌科(Boletaceace)和红菇科(Russulaceae)的真菌占优势[14]。接种外生菌根真菌,可以提高马尾松在酸性土壤中的生存能力和耐铝性,是因为外生菌根真菌可以将重金属元素吸附、固定并积聚在菌根菌丝等部位,限制其向植物体内运输,也可以通过菌套和哈蒂氏网等菌根结构吸收过滤有毒物质[15]。而且菌根分泌的有机酸降低了活性铝含量,菌根还可以促进土壤中难溶养分的溶解,促进植物对土壤养分的吸收[4, 9, 16]。陈展等[17]也发现,模拟酸雨处理下接种外生菌根真菌的马尾松幼苗根系中Al含量下降,抵消了酸雨胁迫对马尾松的影响。 接种褐环乳牛肝菌(Suillus luteus)的马尾松幼苗,还改变了马尾松幼苗根系结构从而提高了马尾松幼苗的耐铝能力[18]。
植物根系细胞壁是与铝接触的最初部位,铝在根尖细胞壁上的积累是铝对植物根尖产生毒害的先决条件[19]。铝与细胞结合后,会影响一系列的细胞过程,如影响细胞壁酶活性[20]、营养物质交换[21]、细胞器损伤[22]、降低细胞延展性[23]等。目前,关于铝的亚细胞定位仍然是以推测性的为主[21, 23],而铝在马尾松菌根细胞的亚细胞定位还未见报道。因此,本试验利用前期筛选出的优良外生菌根真菌褐环乳牛肝菌接种马尾松幼苗,采用砂培盆栽浇铝法,分别设置4个Al3+浓度梯度处理,通过比较研究马尾松菌根/非菌根幼苗的生理、根尖细胞超微结构的变化以及Al的亚细胞分布,分析菌根化苗木对铝的响应及其提高寄主植物耐铝性的可能机理,为外生菌根真菌在提高寄主植物抗逆性和育苗造林应用等方面提供理论依据。
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随铝浓度的升高,菌根和非菌根苗根系SOD、CAT活性均呈总体上升趋势,POD活性则呈先降低后升高趋势(图1),且未接菌NE组的抗氧化酶活性均高于接菌组,表明随着铝浓度的提升,根系抗氧化酶活性增强,并且,未接菌NE组受到铝的影响程度更大,接种SL在一定程度上缓解了铝毒害。其中,未接菌NE组在高铝(0.8 mmol·L−1)水平时SOD活性最高,达到201.14 U·g−1,且显著高于其他处理(P<0.05),其他各处理间差异不显著。CAT活性随铝浓度的提高呈上升趋势,在未接菌NE组中,高铝和中铝(0.4 mmol·L−1)水平下的CAT活性与无铝比差异显著,低铝(0.2 mmol·L−1)与无铝比差异不显著,接菌的SL组内,高铝和中铝水平下CAT活性与无铝比差异显著,低铝与无铝比差异不显著。接菌和未接菌组的POD活性均在低铝水平时最低,但与无铝比差异不显著,中铝与无铝比差异不显著,高铝水平下的POD活性显著高于无铝,且在同一铝浓度下,接菌组的POD均低于未接菌组。
图 1 不同Al3+水平对马尾松根系抗氧化酶活性的影响
Figure 1. Effects on antioxidant enzyme activity in the root of Pinus massoniana seedlings at different aluminum levels
从组织染色(图2)也可以看出,马尾松未接菌NE组根尖DAB、NBT染色深度和DCHF-DA荧光强度均随着铝浓度的升高而逐渐加深,且各铝处理也使得根尖膨大、变粗、弯曲,表现出明显的铝毒害症状。而接菌SL组的各项染色均不明显,表明接菌后根尖的H2O2、O2·−及总活性氧ROS积累的含量不高,明显低于NE组,且各铝浓度间差异也不明显。
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丙二醛(MDA)是表征植物细胞膜脂过氧化程度的重要指标。从图3a可以看出,随着外源铝浓度的升高,未接菌NE组的MDA含量呈逐渐上升趋势,在高铝(0.8 mmol·L−1)水平时含量最高,达5.10 nmol·g−1。接菌SL组的MDA含量呈现先降低后升高的趋势,在低铝(0.2 mmol·L−1)水平时MDA含量最低,为3.50 nmol·g−1。并且与无铝处理相比,从低铝到高铝,未接菌组MDA的增幅均大于接菌组,表明接菌苗缓解了根系细胞的膜脂过氧化程度,降低了铝对马尾松幼苗根尖的毒害作用。从Schiff’s reagent染色(图3b)的根尖也可以看出,随着铝浓度的升高,未接菌NE组的根尖染色均有不同程度的加深,表明铝处理加剧了马尾松幼苗根系膜脂过氧化程度。而接菌SL组的染色不明显,外源Al3+对菌根的膜脂过氧化程度影响较小。
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在马尾松未接菌NE组中(图4A~D),在无铝和低铝(0.2 mmol·L−1)水平下,根尖细胞排列紧密,细胞核、核仁、线粒体、液泡和都清晰可见,在无铝处理中还观察到淀粉粒,液泡较大且多;在低铝时,液泡则变得少且小,线粒体数量明显增加,且膨大变圆,核仁膨大。随着外源Al3+浓度的增加,在中铝(0.4 mmol·L−1)时,细胞壁明显增厚,细胞内液泡、细胞核等细胞器的膜结构遭到严重破坏,细胞空泡化现象十分严重,线粒体被挤压堆积在一侧,细胞核核质散入细胞质中。到高铝(0.8 mmol·L−1)时,细胞间隙增大,局部胞间层出现空隙,除细胞内各细胞器遭到严重破坏外,由于大量铝离子的进入而出现大面积黑色斑块。在接菌SL组(图4E~H),无铝处理的根尖边缘细胞较小,由于菌丝侵入皮层细胞间,形成哈蒂氏网,因此胞间层较厚且颜色较深。低铝时,根尖细胞内液泡增多且小,细胞核、核仁、线粒体等均清晰可见,线粒体结构完整。中铝时,细胞内线粒体膨大变圆,同时,由于铝不断向液泡转运,并在液泡中沉淀为黑色不溶颗粒,使液泡内颜色变深,直至结构遭到破坏,细胞质颜色变深。细胞核遭到破坏固缩,核物质凝聚,核膜不清晰,核仁膨大,有解体趋势。高铝时,液泡明显增大增多,细胞壁局部遭到破坏,发生了明显的质壁分离,线粒体数量进一步增加,细胞核膜被破坏,细胞核形状不规则化,核仁解散消失,核内染色质凝集。
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如图5所示,在马尾松幼苗根尖中,随着外源Al3+浓度的提高,SL和NE组的根尖铝含量均呈上升趋势,同一铝浓度,SL组的根系铝含量都高于NE组。低铝下,菌根苗根尖吸收的铝含量显著高于非菌根苗,高出33.44%,中铝水平下,菌根苗根尖吸收的铝含量也高于非菌根苗,但差异不显著,到高铝时,菌根苗吸收的铝含量又显著高于非菌根苗,高出38.55%。表明接种褐环乳牛肝菌能够显著提高马尾松根系吸收铝的能力。
图 5 不同Al3+水平下未接菌/接菌幼苗根尖铝含量
Figure 5. Aluminium content in the root of inoculated and non-inoculated seedlings at different aluminum levels
从根尖亚细胞组分的铝含量结果(表1) 可以看出,不同外源Al3+浓度处理下,铝在马尾松幼苗根尖的细胞壁组分(F1) 中含量最高,有铝处理时,平均铝含量在240.89~451.72 mg·kg−1 之间,在线粒体组分(F3) 中分布最少,表明铝在马尾松幼苗根系内主要分布在细胞壁,这可能是由于细胞壁对铝的滞留作用强,而使得其在线粒体内分布较少,而且由于菌根的吸附作用,使菌根苗的细胞壁吸收的铝更多。随Al3+浓度升高,铝在细胞壁组分(F1) 、细胞核组分(F2)和线粒体组分(F3)中富集均呈上升趋势,但在未接菌NE组中,铝在以液泡为主的可溶性组分(F4) 中的含量随铝浓度的升高反而减少,这可能是由于在高浓度的铝处理下,NE组的细胞核膜被破坏,核仁解体,染色质溶解在胞质中,在提取细胞核组分时,大部分的铝被提取至细胞核(F2)组分,而使可溶性组分中的铝含量下降。结果表明,铝胁迫使得马尾松幼苗根系将大部分铝吸附在细胞壁上,随着外源Al3+浓度的升高,马尾松幼苗根系亚细胞组分对铝的吸收总体呈增加趋势,随着铝胁迫的加强,由于细胞器受损,亚细胞组分中铝含量出现动态变化。
表 1 不同Al3+水平下马尾松幼苗根尖Al亚细胞组分分布
Table 1. The distribution of Al in subcellular components in the root tips of Pinus massoniana seedlings at different aluminum levels
不同处理
Different treatments细胞壁组分
Cell wall (F1) /(mg·kg−1)细胞核组分
Nuclear (F2)/(mg·kg−1)线粒体组分
Mitochondrial (F3) /(mg·kg−1)可溶组分
Soluble component (F4) /(mg·kg−1)NE0 76.52 ± 6.51 Ce 31.86 ± 4.41 Cd 11.36 ± 0.76 Ce 17.00 ± 0.76 Dd NE02 240.89 ± 1.64 Bd 143.84 ± 7.65 Bb 75.63 ± 6.13 Bc 173.47 ± 8.65 Aa NE04 287.67 ± 10.25 Ac 233.85 ± 11.28 Aa 98.98 ± 4.78 Bb 126.98 ± 16.68 Bb NE08 302.72 ± 6.10 Ac 212.51 ± 12.03 Aa 125.46 ± 13.81 Aa 61.49 ± 0.68 Cc SL0 81.72 ± 8.50 De 27.83 ± 3.37 Dd 12.32 ± 1.53 De 13.70 ± 1.78 Dd SL02 263.03 ± 7.07 Ccd 86.75 ± 34.65 Cc 47.88 ± 1.76 Cd 93.36 ± 1.10 Cbc SL04 369.68 ± 34.23 Bb 150.31 ± 10.48 Bb 94.52 ± 0.17 Bb 124.24 ± 1.91 Bb SL08 451.72 ± 14.78 Aa 189.66 ± 13.59 Aab 102.94 ± 1.69 Ab 169.28 ± 11.17 Aa 注:平均值 ± 标准误差(n=3),不同大写字母表示NE和SL组内不同铝浓度下差异显著,不同小写字母表示不同处理间差异显著(P<0.05)
Notes: mean ± SE(n=3). Different capital letters indicated that there were significant differences in NE or SL groups under different aluminum concentrations, different small letters indicated significant differences in the different treatments (P < 0.05)从铝在根尖亚细胞组分的分布(图6)可以看出,接菌和未接菌组在不同铝浓度处理下,铝的亚细胞分布的比例总体差异不大,其中,细胞壁(F1) 吸附的铝占据的比例最大,接菌SL组细胞壁(F1)所占比例在45.95%~59.98%之间,明显高于未接菌NE组。同时,SL组的细胞核组分(F2)和线粒体组分(F3)中所占比例则低于NE组。SL组的可溶性组分所占比在10.25%~20.80%之间,NE组所占比例8.77%~27.35%之间,差异较大,这可能与细胞器的结构破坏而导致的分布失调有很大关系。
铝对马尾松菌根化幼苗抗逆生理和根尖细胞超微结构的影响
Effects of Aluminum on Stress Resistance Physiology and Root Tip Cell Ultrastructure of Mycorrhizal Seedlings of Pinus massoniana
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摘要:
目的 不同Al3+水平下,研究马尾松菌根/非菌根幼苗的生理、根尖细胞超微结构的变化以及Al的亚细胞分布,分析菌根化苗木对铝的响应及其耐铝性,为外生菌根真菌提高寄主植物耐铝性和育苗造林应用提供理论依据。 方法 以半年生菌根和非菌根马尾松苗为材料,采用砂培盆栽浇铝法,分别设置 0、0.2、0.4、0.8 mmol·L−1 Al3+ (AlCl3) 处理,分析其根系抗氧化酶活性和MDA含量等生理指标变化,Al的亚细胞分布,通过组织染色观察根尖ROS和MDA分布,并观察根尖超微结构变化。 结果 (1)随外源[Al3+]的升高,马尾松菌根/非菌根苗根系SOD、CAT、POD活性和MDA含量均呈总体上升趋势,在高铝(0.8 mmol·L−1)水平时,抗氧化酶活性和MDA含量最大,且非菌根苗受到铝的影响程度更大;(2)随外源[Al3+]的升高,马尾松根尖吸收的铝含量显著增加,且菌根苗吸收的铝含量显著高于非菌根苗;(3)从细胞超微结构和Al的亚细胞分布看,大量Al3+首先与细胞壁结合,细胞内的Al3+与生物膜强烈结合,使细胞器物质向外渗漏作用加强,干扰细胞核和线粒体等的各种调节过程。而由于菌根对铝的吸附作用,降低了侵入细胞内的铝离子含量,保护亚细胞器结构的同时也维持了细胞的基本功能,从而缓解铝毒性。 结论 铝处理使马尾松根系抗氧化酶活性和MDA含量增加,0.4 mmol·L−1以上的铝浓度产生明显铝毒害症状。而菌根可以通过吸收更多的铝降低铝毒害,并提高植物耐铝能力,这很可能是马尾松菌根苗的一个重要外部抗性机制。 Abstract:Objective To provide theoretical basis for the application of ectomycorrhizal fungi in improving the aluminum tolerance of host plants and afforestation, we studied the changes of physiology, root tip cell ultrastructure and subcellular distribution of Al in mycorrhizal / non mycorrhizal seedlings of Pinus massoniana under different Al3+ levels. Method Semi annual mycorrhizal and non mycorrhizal P. massoniana seedlings were treated with 0, 0.2, 0.4 and 0.8 mmol·L−1 Al3+ (AlCl3) respectively by sand culture and pot irrigation with aluminum. We analyzed the changes of antioxidant enzyme activity, MDA content and subcellular distribution of Al in roots, the distribution of ROS and MDA in roots, and the ultrastructure of root tips. Results (1) With the increase of exogenous aluminum concentration, the activities of SOD, CAT, POD and the content of MDA in the roots of mycorrhizal / non mycorrhizal seedlings of masson pine showed an overall upward trend. At high aluminum level (0.8 mmol·L−1), the activities of antioxidant enzymes and the content of MDA were the highest, and the non mycorrhizal seedlings were more affected by aluminum. (2) With the increase of exogenous aluminum concentration, the aluminum content absorbed by root tips of P. massoniana increased significantly, and the aluminum content absorbed by mycorrhizal seedlings was significantly higher than that of non mycorrhizal seedlings. (3) From the cell ultrastructure and subcellular distribution of Al, a large number of Al3+ first combined with the cell wall, and the intracellular Al3+ strongly combined with the biofilm, which strengthened the leakage of organelles and interfered with various regulatory processes of nucleus and mitochondria. Because of the adsorption of aluminum by mycorrhiza, the content of aluminum ions in cells reduced, the structure of subcellular organelles was protected, the basic function of cells was maintained, and the toxicity of aluminum was alleviated. Conclusion The antioxidant enzyme activity and MDA content of root of P. massoniana are increased by aluminum treatment, and aluminum with 0.4 mmol·L−1 produces obvious aluminum toxicity symptoms. Mycorrhizal fungi can reduce aluminum toxicity and improve plant aluminum tolerance by absorbing more aluminum, which is probably an important external resistance mechanism of root seedlings mycorrhiza of P. massoniana. -
表 1 不同Al3+水平下马尾松幼苗根尖Al亚细胞组分分布
Table 1. The distribution of Al in subcellular components in the root tips of Pinus massoniana seedlings at different aluminum levels
不同处理
Different treatments细胞壁组分
Cell wall (F1) /(mg·kg−1)细胞核组分
Nuclear (F2)/(mg·kg−1)线粒体组分
Mitochondrial (F3) /(mg·kg−1)可溶组分
Soluble component (F4) /(mg·kg−1)NE0 76.52 ± 6.51 Ce 31.86 ± 4.41 Cd 11.36 ± 0.76 Ce 17.00 ± 0.76 Dd NE02 240.89 ± 1.64 Bd 143.84 ± 7.65 Bb 75.63 ± 6.13 Bc 173.47 ± 8.65 Aa NE04 287.67 ± 10.25 Ac 233.85 ± 11.28 Aa 98.98 ± 4.78 Bb 126.98 ± 16.68 Bb NE08 302.72 ± 6.10 Ac 212.51 ± 12.03 Aa 125.46 ± 13.81 Aa 61.49 ± 0.68 Cc SL0 81.72 ± 8.50 De 27.83 ± 3.37 Dd 12.32 ± 1.53 De 13.70 ± 1.78 Dd SL02 263.03 ± 7.07 Ccd 86.75 ± 34.65 Cc 47.88 ± 1.76 Cd 93.36 ± 1.10 Cbc SL04 369.68 ± 34.23 Bb 150.31 ± 10.48 Bb 94.52 ± 0.17 Bb 124.24 ± 1.91 Bb SL08 451.72 ± 14.78 Aa 189.66 ± 13.59 Aab 102.94 ± 1.69 Ab 169.28 ± 11.17 Aa 注:平均值 ± 标准误差(n=3),不同大写字母表示NE和SL组内不同铝浓度下差异显著,不同小写字母表示不同处理间差异显著(P<0.05)
Notes: mean ± SE(n=3). Different capital letters indicated that there were significant differences in NE or SL groups under different aluminum concentrations, different small letters indicated significant differences in the different treatments (P < 0.05) -
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