丛枝菌根真菌根外菌丝跨膜H +和Ca 2+流对干旱胁迫的响应

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徐丽娇¹^,², 郝志鹏¹, 谢伟¹^,², 李芳¹^,², 陈保冬^,¹^,²^,^*

1 中国科学院生态环境研究中心, 城市与区域生态国家重点实验室, 北京 100085

2 中国科学院大学, 北京 100049

Transmembrane H ⁺ and Ca ²⁺ fluxes through extraradical hyphae of arbuscular mycorrhizal fungi in response to drought stress

XU Li-Jiao¹^,², HAO Zhi-Peng¹, XIE Wei¹^,², LI Fang¹^,², CHEN Bao-Dong^,¹^,²^,^*

1 State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

2 University of Chinese Academy of Sciences, Beijing 100049, China

通讯作者: *( bdchen@rcees.ac.cn)

编委: 郭良栋
责任编辑: 李敏

基金资助:

中国科学院战略性先导科技专项(XDB15030102)
国家自然基金项目(41371264)
国家自然基金项目(41401281)

Online:2018-07-20

Supported by:

SupportedbytheStrategicPriorityResearchProgramoftheChineseAcademyofSciences(XDB15030102)
the National Natural Science Foundation of China(41371264)
the National Natural Science Foundation of China(41401281)

摘要
丛枝菌根真菌(AMF)能够和大多数陆地植物形成共生体系, 对于植物生长发育和适应各种逆境胁迫具有重要作用。很多研究表明干旱胁迫下AMF能够促进宿主植物对水分的吸收从而增强植物抗旱能力, 但目前针对AMF根外菌丝响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究并不多。该研究利用AMF Rhizophagus irregularis和胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净根外菌丝, 向培养基添加聚乙二醇(PEG)模拟干旱胁迫, 运用场发射扫描电子显微镜(FE-SEM-EDS)观察干旱胁迫对AMF根外菌丝形态的影响, 同时采用非损伤微测技术(NMT)观测根外菌丝跨膜H ⁺和Ca ²⁺离子流变化。结果发现, PEG处理1 h后菌丝尖端和侧面发生H ⁺外流和强烈的Ca ²⁺内流, 荧光探针分析也显示菌丝胞内pH值显著上升、Ca ²⁺浓度增加; PEG处理24 h后菌丝形态发生明显变化, 培养基pH值降低, P、Ca、Fe等元素在菌丝际积累。这些试验结果表明, 干旱胁迫下AMF根外菌丝跨膜H ⁺和Ca ²⁺流发生变化, 促进了菌丝与环境之间的物质交换。菌丝酸化生长环境有利于养分吸收, 并促进AMF与宿主植物之间的信号交流以增强植物的耐旱性。
关键词： 丛枝菌根真菌;干旱胁迫;氢离子;钙离子;非损伤微测技术

Abstract

Aims Arbuscular mycorrhizal fungi (AMF) can form symbiotic associations with most terrestrial plants to improve plant growth and stress resistance. It has been well demonstrated that AMF can promote plant acquisition of water and enhance plant tolerance to drought. In this study, AMF extraradical hyphae were obtained from in vitro culture of AMF Rhirophagus irregularis with hairy carrot (Daucus carota var. sativa) root to investigate the morphological and physiological changes of hyphae in response to drought stress induced by polyethylene glycol (PEG).Methods The influence of drought stress on the hyphal morphology was observed by using the field emission-scanning electron microscope-energy dispersive X-ray spectroscopy (FE-SEM-EDS), while H ⁺ and Ca ²⁺ion fluxes through living hyphae were monitored by non-invasive micro-test technique (NMT). Important findings The results showed that significant H⁺ efflux and Ca²⁺ influx through the tip and side of the extraradical hyphae were detected in response to drought stress induced by PEG for 1 h. Fluorescence probing confirmed that the intracellular pH value and Ca²⁺ concentration of hyphae significantly increased under PEG treatment. The morphology of hyphae changed and the pH value of the growth medium decreased after treatment with PEG for 24 h. The P, Ca, and Fe elements accumulated at the hyphosphere to enhance the nutrient absorption by hyphae. The study confirmed that AMF regulated the transmembrane H⁺ and Ca²⁺ flux to promote the material exchange between hyphae and environment under drought stress. The acidification of the hyphosphere environment potentially promoted the absorption of nutrients and also the signal exchange between AMF and the host plant to enhance plant drought tolerance.

Keywords：arbuscular mycorrhizal fungi;drought stress;H⁺;Ca²⁺;non-invasive micro-test technology

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引用本文
徐丽娇, 郝志鹏, 谢伟, 李芳, 陈保冬. 丛枝菌根真菌根外菌丝跨膜H ⁺和Ca ²⁺流对干旱胁迫的响应 . 植物生态学报, 2018, 42(7): 764-773 doi:10.17521/cjpe.2018.0089
XU Li-Jiao, HAO Zhi-Peng, XIE Wei, LI Fang, CHEN Bao-Dong. Transmembrane H ⁺ and Ca ²⁺ fluxes through extraradical hyphae of arbuscular mycorrhizal fungi in response to drought stress . Chinese Journal of Plant Ecology, 2018, 42(7): 764-773 doi:10.17521/cjpe.2018.0089

丛枝菌根真菌(AMF)是一类专性共生真菌, 可以与大多数陆地植物形成共生体系, 并通过多种途径促进植物的生长发育, 增强植物对各种逆境胁迫的适应能力(Smith & Read, 2008)。AMF根外菌丝在根系与土壤之间建立起物质传输通道, 将吸收的水分和营养物质快速输送给宿主植物(Chitarra et al., 2016)。由于菌丝直径远小于植物根系, 因此根外菌丝能进入细小的土壤空隙获取养分和水分资源(Liu & Chen, 2007), 这对于植物适应养分缺乏和干旱胁迫具有重要意义。Rhizophagus irregularis (原名Glomus intraradices)是分布最广泛的一种AMF。Tisserant等(2013)通过全基因测序方法获得了R. irregularis DAOM 197198菌株的基因组数据, 为AMF真菌的生理和分子研究提供了重要基础, 也使得该菌株成为AMF生理研究的模式菌株。干旱情况下接种R. irregularis的加杨(Populus × canadensis)具有更高的水分利用效率和抗氧化酶活性, 此外AMF还上调根系水孔蛋白基因的表达提高植物抗旱性(Liu et al., 2016)。Li等(2013)从R. intraradices中克隆了两个水通道蛋白基因GintAQPF1和GintAQPF2, 并通过异源表达验证了水孔蛋白的输水功能, 为干旱胁迫下AMF向宿主植物输送水分,从而增强植物抗旱性提供了直接的分子证据。

细胞膜质子泵, 即H⁺-ATPase的功能是水解ATP并将H⁺泵出细胞(Santi et al., 2003)。H⁺通过细胞内外的电化学梯度进出细胞膜, 为包括阳离子、阴离子、氨基酸和糖在内的各种小分子物质的跨膜运输提供驱动力(Gaxiola et al., 2007)。干旱胁迫、盐胁迫、缺磷等环境胁迫会导致质子泵发生改变(Yan et al., 2002; Gévaudant et al., 2007)。细胞膜质子泵活性不仅反映物质跨膜运输的能力, 还在一定程度上反映生长环境对植物生理活性的影响。玉米(Zea mays)可以通过提高根系细胞膜质子泵的活性来适应低pH环境, 燕麦(Avena sativa)在感受到干旱胁迫的早期就可以激活根系H⁺-ATPase来增加渗透物质的合成(Gong et al., 2010)。对AMF H⁺-ATPase的研究表明, 真菌质子泵在控制共生体营养元素和水分转运过程中起到重要作用(Ferrol et al., 2000)。Gianinazzi- Pearson等(1991)通过测试ATPases水解作用过程中释放的磷酸对ATP酶进行细胞化学研究, 证明在AMF根内、根外菌丝的质膜上均存在H⁺-ATPase。

Ca²⁺是生物细胞内重要的信号物质, 在植物抵抗干旱胁迫方面具有重要作用(Ma et al., 2005)。胞质内游离Ca²⁺的分布以及跨膜转移是Ca²⁺信号的转移和传导的基本途径, 而Ca²⁺进入和流出细胞速率的变化是产生Ca²⁺振荡的基础(Bunney et al., 2000)。质膜Ca²⁺-ATPases和H⁺-ATPase共同实现了质膜H⁺/Ca²⁺的逆向转运和逆境信号的传递(Rudd & Franklin-Tong, 2001)。干旱胁迫可以导致细胞内游离Ca²⁺浓度产生短暂而快速的升高, 胞内Ca²⁺的波动对钙调蛋白、激酶和离子通道等下游调控元件产生影响(Campo et al., 2014)。由钙信号传导的干旱胁迫信息会导致相关基因表达和代谢过程的改变(如渗透调节物质合成和营养元素运输的变化), 从而减轻干旱胁迫对生物机体的损害(Ludwig et al., 2004; Zou et al., 2015)。

细胞膜是生物遭受逆境胁迫以后受损的初始位点, 其结构及功能的改变势必会影响生物体内水分代谢以及各种无机离子的运输, 而无机离子的跨膜运输对维持植物正常生长发育起着至关重要的作用(Yoshida, 1991)。干旱胁迫下细胞会通过特定的调节机制, 改变离子和小分子的跨膜运输来调节离子平衡, 从而维持细胞的渗透压, 以抵御干旱胁迫。因此, 研究关键离子的跨膜转运过程对揭示植物在干旱胁迫下通过自我调节以维持生命活动的机制具有重要意义。Kühtreiber和Jaffe (1990)首次利用非损伤微测技术(non-invasive micro-test technology, NMT)在活体原位条件下测量了Ca²⁺进出细胞的流速和方向, 该方法被广泛应用于生物体逆境应答机制研究。目前, NMT技术可以实时记录活体植物细胞各种离子的跨膜运输, 为研究植物应答环境变化提供了重要技术平台。应用这种技术, Mak等(2014)发现在不同时间长度的干旱胁迫下大豆(Glycine max)叶肉细胞K⁺、H⁺和Ca²⁺转运情况出现显著变化, 而各种离子的转运能力可作为重要的生理指标来衡量大豆的耐旱性。

很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015)。干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少。本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制。

1 材料和方法

1.1 植物材料

参照St-Arnaud等(1996)的方法, 建立AMF-胡萝卜毛状根双重无菌培养体系。供试AMF菌株为R. irregularis DAOM 197198, 真菌孢子先经过吐温80和氯胺T进行表面消毒, 然后再用质量浓度1%的硫酸链霉素和0.5%的硫酸庆大霉素清洗, 最后在1.5%的水琼脂上面萌发。将转移Ri T-DNA并且能够连续继代培养的胡萝卜毛状根接种于固体M培养基(Bécard & Fortin, 1988, 液体M培养基组分参见表1。液体培养基加入1%的蔗糖和0.4%的植物凝胶即得到固体M培养基), 然后在根尖部位接入10-15个萌发的AMF孢子, 在25 ℃下黑暗培养。大概3个月后, 菌根生长铺满整个培养皿后转接。

Table 1
表1
表1M培养基成分
Table 1Formulation of the M medium

离子/化合物 Ion/Compound	含量 Content (mg·L^-1)	离子/化合物 Ion/Compound	含量 Content (mg·L^-1)
Mg²⁺	70.8	Fe²⁺	0.1
SO₄^2-	280.1	Mn²⁺	1.6
K⁺	61	Zn²⁺	0.6
NO₃^-	230	BO₃^3-	0.24
Cl^-	37.2	甘氨酸 Glycine	3
H₂PO₄^-	4.8	维生素B₁ Vitamin B₁	0.1
Ca²⁺	49	维生素B₆ Vitamin B₆	0.1
I^-	0.58	烟酸 Nicotinic acid	0.5
Na⁺	0.43	肌醇 Inositol	50

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1.2 试验处理

试验在分室培养系统(分为菌根室和菌丝室)中进行(图1)。先在菌根室加入30 mL固体M培养基, 然后在其中接入已被AMF侵染的胡萝卜毛状根, 在25 ℃下黑暗倒置培养8周, 待根系铺满菌根室后正置培养, 并在菌丝室加入15 mL液体M培养基。继续培养4-6周直到菌丝长满菌丝室, 挑选菌丝量大致相同的培养皿进行后续试验。

图1

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图1丛枝菌根真菌-胡萝卜毛状根分室培养系统示意图。左边是菌根室(mycorrhizal compartment), 右边是菌丝室(hyphal compartment)。菌根室中加入用质量浓度0.4%的植物凝胶固化后的M培养基, 并接入菌根化的转移Ri T-DNA胡萝卜毛状根; 菌丝室加入液体M培养基, 以供根外菌丝生长(参考St-Arnaud et al., 1996)。

Fig. 1Diagram of the two-compartments in vitro culture system of arbuscular mycorrhizal fungi with hairy carrot root. Mycorrhizal compartment was filled with solid M medium gelled with 0.4% phytagel, allowing development of mycorrhizal roots; extraradical mycelium ramified into hyphal compartment filled with liquid M medium without sucrose and phytagel, and the roots that crossed the central wall were trimmed to prevent their growth in hyphal compartment (referred to St-Arnaud et al., 1996).

菌丝室中干旱处理的培养基由液体M培养基加入15%的PEG制成, 对照为正常液体M培养基。处理前调节培养基pH值至6.0, 每个菌丝室加入15 mL处理液, 分别处理1 h和24 h, 总共有4组样品: 对照1 h (CK1h)、PEG处理1 h (PEG1h)、对照24 h (CK24h)和PEG处理24 h (PEG24h)。每组处理有12个培养皿(试验重复), 其中6个培养皿收获菌丝作为扫描电子显微镜和荧光光谱分析的样品, 同时收集液体培养基测定pH值, 另外6个培养皿用于离子流分析。

1.3 培养基pH测定

将菌丝室的液体培养基收集到15 mL离心管中保存, pH值测定采用PHS-3C型酸度计(雷克斯仪器厂, 上海)进行, 每个样品重复测量3次。

1.4 场发射扫描电子显微镜(FE-SEM-EDS)分析

新鲜菌丝收获后, 取适量的样品用超纯水清洗3次去除处理液, 然后置入2.5%戊二醛(溶解于pH值7.2的PIPES缓冲液中)中于4 ℃过夜固定, 再用相同的PIPES缓冲液清洗3次, 每次15-30 min。固定后的样品分别用30%、50%、70%、80%、90%、95%、100%的乙醇清洗(每一级浓度梯度清洗2次, 每次停留15-30 min)。将脱水后的样品放在乙酸异戊酯中过夜以置换样品中的脱水剂(Wu et al., 2015)。而后再将样品置于临界点干燥仪(HC P-2, Hitachi, Tokyo, Japan)中, 通入CO₂进行干燥。将干燥后的菌丝粘在导电胶布上经自然干燥后, 喷金粉50 s, 然后在连接有能谱分析仪的场发射扫描电子显微镜(FE-SEM-EDS, SU-8020, Hitachi, Tokyo, Janpan)下观察分析。

1.5 菌丝内H⁺和Ca²⁺荧光光谱分析

采用pH荧光探针(BCECF AM, 碧云天, 上海)或Ca²⁺荧光探针(Fluo-4 AM, 碧云天, 上海)在原位条件下分别染色菌丝室中的根外菌丝, 将菌丝置于含有5 μmol·L^-1染料的PBS工作液中, 37 ℃黑暗孵育60 min进行荧光探针装载, 随后用PBS洗涤3次, 再孵育20 min保证荧光探针在菌丝内完全转化。使用激光扫描共聚焦显微镜(Leica T CS SP5II, Wetzlar, Germany)扫描样品获取荧光图像(激发波长488 nm, 发射波长512-520 nm)。

1.6 离子流测定

利用北京旭月公司的NMT测试平台(BIO-001A, YOUNGER, Barnstable, USA)测定菌丝尖端和侧面的H⁺和Ca²⁺离子流。用石英片轻轻压住待测菌丝防止测量时电极震动使菌丝飘动。通过电脑控制将选择性离子电极尖端直接伸到菌丝室中菌丝附近且在电极尖端不触碰菌丝的情况下尽量靠近菌丝进行测定。

选择性离子电极沿x轴垂直菌丝方向进行往返测试。电极运动一次间距(从近菌丝表面一端到远菌丝表面一端)为30 μm, 运动频率为0.3-0.5 Hz。供NMT数据采集的每个测量点需测试10 min。利用ASET 2.0软件进行数据显示、图像获取、数据的预处理、电极三维位置调试和显微镜精细聚焦的步进控制等操作。

1.7 数据分析

测试离子流速可通过Fick’s扩散法则计算得出, 公式如下:

J = -D(dc/dx)

式中: J为x方向的离子流, D为在特定介质中离子或分子扩散常数, dc/dx为离子浓度梯度。离子流数据根据北京旭月公司提供的JCal V3. 0软件计算得出。本试验中阳离子外流表示为正值, 内流为负值, 且所得数据是净离子流速, 即内流与外流相抵消后的离子流速。

培养基pH值、菌丝离子流、菌丝元素能谱分析结果等数据采用SPSS 18.0软件进行统计分析(SPSS, Chicago, USA)。培养基pH值采用双因素方差分析检验PEG处理、处理时间及其交互作用的显著性。元素能谱分析、离子流结果采用三因素方差分析检验PEG处理、处理时间和不同菌丝部位及其交互作用的显著性。Duncan’s多重比较检验各处理之间的差异显著性(p < 0.05; Duncan, 1955)。柱形图由Microsoft Excel 2010生成。

2 结果

2.1 PEG处理下菌丝室培养基的pH值

菌丝室添加PEG处理1 h后, 菌丝室培养基pH值显著下降至4.39 (p < 0.05), 而对照组没有发生明显变化。PEG处理24 h后, 菌丝室培养基pH为4.13, 而对照组pH值仍无明显变化(图2)。

图2

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图2PEG处理下菌丝室培养基的pH值(平均值±标准偏差, n = 6)。不同小写字母表示处理间差异显著 (Duncan’s多重比较, p < 0.05)。

Fig. 2The pH value of culture medium in hyphal compartment after treatment by PEG (mean ± SD, n = 6). Different lowercase letters indicate significant difference between treatments (Duncan’s multiple range test, p < 0.05).

2.2 PEG处理对菌丝形态及元素含量变化

在扫描电子显微镜下可以看到对照处理的菌丝尖端完整, 粗细均匀, 菌丝饱满且光滑。PEG处理1 h后, 菌丝尖端出现了损伤, 在细胞壁上分布有微小的不规则孔洞, 并且尖端发生扭曲折叠; 菌丝出现脱水状萎缩, 并伴有点状损伤。在PEG处理24 h后, 菌丝形态发生了显著变化, 菌丝尖端出现较大的损伤, 细胞壁破损, 形成大小不一的凸起, 粗细变得不均匀, 呈结状分布; 同时, 菌丝出现明显皱缩, 有突起的褶皱和损伤(图3)。

图3

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图3PEG处理1 h (PEG 1 h)和24 h (PEG 24 h)后丛枝菌根真菌根外菌丝FE-SEM图像和能谱分析图。CK 1 h和CK 24 h为空白对照。

Fig. 3FE-SEM images and selective elemental analysis (by EDS) of arbuscular mycorrhizal fungi hyphae after treatment by PEG for 1 h (PEG 1 h) and 24 h (PEG 24 h). CK 1 h and CK 24 h are corresponding controls.

与对照相比, PEG处理菌丝表面P的原子百分比(at.%)增加, 且处理24 h显著高于处理1 h (p < 0.05), 而菌丝尖端和侧面两个部位之间P的at.%值无明显差异(图3; 表2)。类似地, PEG处理菌丝尖端P的质量分数显著高于对照, 且处理24 h高于处理1 h的情况。

Table 2
表2
表2PEG处理1 h和24 h后丛枝菌根真菌菌丝尖端和侧面P、Ca和Fe的浓度值(能谱分析结果)(平均值±标准偏差, n = 6)
Table 2Concentrations of P, Ca and Fe at the tip and side of arbuscular mycorrhizal fungi hyphae after treatment by PEG for 1 h and 24 h (determined by energy-dispersive X-ray spectroscopy) (mean ± SD, n = 6)

			P		Ca		Fe
			原子百分比 Atom percentage content (%)	质量分数 Mass percentage content (%)	原子百分比 Atom percentage content (%)	质量分数 Mass percentage content (%)	原子百分比 Atom percentage content (%)	质量分数 Mass percentage content (%)
尖端 Tip	CK	1 h	29.53 ± 2.03^cd	30.03 ± 0.52^d	5.97 ± 0.68^c	6.14 ± 0.72^c	15.86 ± 0.26^c	24.64 ± 2.07^cd
	CK	24 h	31.34 ± 2.34^c	32.85 ± 0.61^d	4.49 ± 0.86^c	5.39 ± 1.27^c	14.60 ± 1.26^c	26.53 ± 1.91^c
	PEG	1 h	38.80 ± 1.07^b	39.62 ± 0.41^c	16.15 ± 0.84^a	18.00 ± 0.87^a	37.15 ± 0.69^a	37.44 ± 1.13^b
	PEG	24 h	51.49 ± 1.63^a	50.46 ± 0.39^a	5.32 ± 1.45^c	4.91 ± 0.66^c	32.48 ± 1.70^ab	52.43 ± 0.49^a
侧面 Side	CK	1 h	23.51 ± 1.12^d	39.01 ± 0.34^c	7.83 ± 1.3^bc	7.88 ± 1.40^bc	12.03 ± 1.11^d	19.51 ± 1.39^d
	CK	24 h	28.00 ± 1.67^cd	43.47 ± 1.07^bc	9.84 ± 0.83^b	10.32 ± 0.92^b	12.07 ± 0.78^d	19.17 ± 0.87^d
	PEG	1 h	37.69 ± 0.84^b	37.51 ± 1.43^c	10.34 ± 1.12^b	12.03 ± 0.75^b	28.54 ± 0.76^b	33.28 ± 1.13^b
	PEG	24 h	56.12 ± 2.21^a	45.04 ± 0.47^b	14.99 ± 0.73^a	16.21 ± 0.40^a	35.09 ± 1.43^a	41.70 ± 0.60^ab

同列不同小写字母表示处理间差异显著(Duncan’s多重比较, p < 0.05)。
Different lowercase letters in the same column indicate significant difference between treatments (Duncan’s multiple range test, p < 0.05).

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PEG处理1 h菌丝尖端Ca的at.%和质量分数均显著高于对照(p < 0.05), 但是菌丝侧面的Ca信号没有明显变化; 处理24 h后菌丝尖端的Ca没有明显变化, 而菌丝侧面Ca的at.%和质量分数均显著高于对照(p < 0.05)(图3; 表2)。

PEG处理菌丝尖端和侧面Fe的at.%显著增高(p < 0.05), 而1 h菌丝尖端和侧面两个部位之间Fe的at.%差异显著(图3; 表2); PEG处理菌丝尖端Fe的质量分数显著高于对照, 而处理24 h菌丝尖端的质量分数值高于处理1 h的值(p < 0.05)。

2.3 PEG处理对菌丝胞内pH、Ca²⁺荧光光谱的影响

利用LSCM进行荧光光谱分析, 发现PEG处理引起了AMF菌丝胞内pH值变化。与对照相比, PEG处理1 h的菌丝尖端荧光信号亮度明显增强, 而菌丝侧面的胞内荧光亮度急剧增强, 菌丝内呈现明亮的绿色荧光, 表明胞内pH值显著升高。PEG处理24 h后菌丝尖端和侧面细胞内荧光信号亮度高于对照组, 但弱于PEG处理1 h后胞内的荧光强度(图4)。

图4

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图4PEG处理1 h和24 h对丛枝菌根真菌菌丝尖端和侧面细胞内pH值的影响。荧光图像显示激发波长为488 nm条件下菌丝内pH值变化。

Fig. 4The effect of PEG treatment for 1 h and 24 h on the pH value at the tip and side of arbuscular mycorrhizal fungi hypha. Fluorescence image at 488 nm excitation shows the pH variation in the hyphal cell.

对照组中, 菌丝内Ca²⁺荧光信号较弱, 无论是尖端还是侧面细胞内均只能观察到不连续的微弱荧光。PEG处理1 h后菌丝尖端的Ca²⁺荧光信号强度显著高于对照, 菌丝侧面细胞内呈现了强烈的绿色荧光; PEG处理24 h后菌丝尖端Ca²⁺荧光信号强度减弱, 而菌丝侧面荧光信号减弱到与对照组基本相同的程度(图5)。

图5

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图5PEG处理1 h和24 h对丛枝菌根真菌菌丝尖端和侧面细胞内Ca²⁺浓度的影响。荧光图像显示激发波长为488 nm条件下菌丝内Ca²⁺浓度变化。

Fig. 5The effect of PEG treatment for 1 h and 24 h on the Ca²⁺concentration at the tip and side of arbuscular mycorrhizal fungi hypha. Fluorescence image at 488 nm excitation shows the Ca²⁺variation in the hyphal cell.

2.4 PEG处理对菌丝跨膜H⁺和Ca²⁺离子流的影响

对照处理菌丝尖端H⁺表现为微弱外流, PEG处理1 h后H⁺加速外流(流速为0.813 pmol·cm^-2·s^-1)、24 h后H⁺流速达到2.53 pmol·cm^-2·s^-1 (是处理1 h的3.1倍)。对照处理菌丝侧面的H⁺外流速度为1.27 pmol·cm^-2·s^-1, PEG处理1 h后菌丝侧面H⁺流速达到3.32 pmol·cm^-2·s^-1, PEG处理24 h后H⁺仍然保持强烈的外流状态(图6A)。对照处理菌丝尖端和侧面的Ca²⁺表现为外流, 且尖端的流速高于侧面。PEG处理1 h后菌丝尖端和侧面的Ca²⁺变为强烈的内流, 而PEG处理24 h后菌丝尖端Ca²⁺内流的强度减弱(流速为-12.45 pmol·cm^-2·s^-1), 菌丝侧面表现为Ca²⁺外流(流速为27.21 pmol·cm^-2·s^-1)(图6B)。

图6

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图6PEG处理1 h和24 h对菌丝尖端和侧面H⁺ (A), 和Ca²⁺ (B)离子流的影响(平均值±标准偏差, n = 6)。tip表示菌丝尖端, side表示菌丝侧面。柱形上方标示不同字母代表相应处理之间有显著性差异(Duncan’s多重比较, p < 0.05)。

Fig. 6The effects of PEG treatment on H⁺ (A) and Ca²⁺(B) flux at the tip and side of arbuscular mycorrhizal hyphae (mean ± SD, n = 6). tip stands for hyphal tip, and side stands for lateral hypha. Columns marked by different letters are significantly different according to Duncan’s multiple range test (p < 0.05).

3 讨论

本研究利用AMF-胡萝卜毛状根双重无菌培养体系, 在菌丝室获得纯净AMF根外菌丝, 排除了根系分泌物以及复杂土壤环境的影响。采用PEG模拟干旱胁迫, 在菌丝非离体条件下利用NMT技术测试菌丝跨膜离子流变化情况, 结合荧光探针检测和FE-SEM-EDS分析, 探讨干旱胁迫对菌丝离子吸收的影响。结果表明, 干旱胁迫下AMF菌丝H⁺外流速率增大, 而Ca²⁺迅速内流, 培养介质中pH值降低, AMF响应干旱胁迫而增加了对水分和养分的吸收。

PEG处理后菌丝的形态发生了明显变化, 并随着胁迫时间的延长出现了不同程度的萎蔫状态。菌丝室培养基的pH值显著下降, 而对照处理没有明显变化, 表明干旱胁迫促进了菌丝中的H⁺外排, 使培养基酸化。干旱处理很可能激活了AMF质膜上的质子泵, 使菌丝内H⁺持续外排, 使培养基维持了较低的pH值。有研究表明, 干旱胁迫下植物根际和非根际土壤的pH值上升, 降低了土壤无机离子的活性, 影响根系对无机离子的吸收(杨培志, 2012)。干旱条件下菌丝中质子泵的激活改变了根际微环境, 使更多的无机离子溶解于土壤溶液中。无机离子被菌丝吸收并进一步传输到植物中, 从而增强植物的抗旱性(Sun et al., 1999)。

pH值变化是生物对环境变化的最初响应, 该变化直接引起菌丝质膜的信号传导, 激活下游受体的信号识别过程(Ayling et al., 2000)。本试验利用LSCM观察了PEG处理对菌丝胞内pH的影响, 结果表明PEG处理1 h后菌丝尖端和侧面的荧光信号亮度明显增强。pH荧光探针在pH越高的环境中荧光强度越高, 因此该现象说明PEG处理使胞内pH值升高,

细胞环境碱化。真菌胞内的碱化与胁迫信号的长距离传递有关, pH变化可能引起真菌-植物之间的信号传递发生改变(Harrison, 2005)。离子动力学研究表明, 真菌和植物的信号交流与细胞跨膜H⁺离子梯度的调节有关, 该过程受到细胞膜上P型H⁺-ATPases的调控(Ramos et al., 2008a)。为了进一步验证PEG模拟水分胁迫下菌丝H⁺的跨膜运输过程, 利用NMT技术验证了菌丝尖端和侧面两个部位H⁺的流动情况。对照处理下, 两个部位的H⁺都呈现微弱外流的趋势, 而且菌丝侧面的H⁺外流流速大于尖端。该结果与前人研究结果相似, Ramos等(2008b)证明从菌丝尖端到菌丝侧面不同位置上H⁺流动规律不同, 而且菌丝侧面的H⁺外流速率大于尖端。该现象可能和真菌的信号识别机制以及菌丝尖端生长有关(Bartnicki-Garcia et al., 2000)。在PEG处理下, 菌丝H⁺显著外流。随着处理时间的延长, 菌丝尖端H⁺外流强度增大, 外流的H⁺使菌丝际环境酸化。该结论与液体培养基的pH值变化结果相符, 表明在模拟干旱处理1 h后, 菌丝对于外部环境的pH值进行了调节。菌丝尖端的H⁺外流调节了真菌对营养物质(如蔗糖、氨基酸和P)的吸收和交换(Requena et al., 2003)。能谱分析显示, PEG处理增加了菌丝表面P信号强度, 说明在干旱处理下更多的P积累到菌丝界面。Hijikata等(2010)研究证实, 干旱情况下根外菌丝的磷转运蛋白活性提高, 将更多的P从土壤中通过菌丝吸收运输到植物中, 而AMF提高植物的营养元素吸收是提高植物耐旱性的重要机制(Zhao et al., 2015a)。

质膜H⁺外流不仅是营养物质吸收的动力, 还作为信号传导的中间体, 促进逆境信号的传导和响应元件的激活(Fromm & Lautner, 2007)。H⁺的外流可以促进Ca²⁺、K⁺等阳离子的跨膜运输, 提高胞内的离子浓度(Isfort et al., 1993)。本研究中, PEG处理1 h后, 菌丝尖端和表面的Ca²⁺表现了强烈的内流现象。当生物受到干旱胁迫的时候, 细胞内Ca²⁺瞬时产生快速的增高, 引发钙振荡(Zhao et al., 2015b)。Ca²⁺作为第二信使, 在胁迫信号的传递过程中起到重要作用, 胞内游离Ca²⁺的分布与转移是Ca²⁺信号产生和传递的基础。使用LSCM观测菌丝细胞内Ca²⁺浓度的结果与离子流测定结果相符, PEG处理1 h后菌丝尖端与侧面均呈现强烈的绿色荧光, 说明菌丝内Ca²⁺浓度显著上升。干旱胁迫诱导的胞内Ca²⁺浓度增加能够激活下游的逆境信号响应元件, Ca依赖的蛋白激酶(CDPK)等, 而CDPK可以通过磷酸化质膜水通道蛋白促进细胞膜对水分的吸收和扩散(Azad et al., 2004)。Li等(2013)证实, 干旱胁迫情况下AMF中两个水通道蛋白基因GintAQPF1和GintAQPF2的表达量会显著上调, 为植物输送水分以缓解干旱胁迫。

干旱条件下水分由根外菌丝向根内菌丝运输, 微量元素、氨基酸和其他低分子量的代谢产物也被AMF菌丝选择吸收并运送到植物中, 由此增强植物的耐旱性(Querejeta et al., 2003; van Hees et al., 2006)。菌丝中元素能谱分析显示, PEG处理促进了菌丝表面P、Ca和Fe的积累。各种元素在菌丝表面的增多很可能是由于PEG诱导激活了质子泵, 而细胞对离子的吸收通常依赖于H⁺外流产生的电位梯度, 质膜H⁺-ATPase产生的质子动势为营养物质的吸收和转运提供了能量(Riquelme & Bartnicki- Garcia, 2004)。干旱情况下AMF根外菌丝能够将更多的P和Ca等矿质养分从土壤中通过菌丝通道运输到植物中, 同时调控相关基因的表达以促进根系对水分的吸收, 使植物具有较强的耐旱性。Bárzana等(2012)的研究表明在充分供水和干旱胁迫下, AMF能够调控植物质外体和细胞间水分运输的途径。AMF这种调节植物水分吸收和运输的能力, 可以帮助植物更灵活地适应水分胁迫(Sánchez-Romera et al., 2016), 但AMF这一功能的生理和分子机制还需要系统深入的研究。

4 结论

干旱胁迫下, AMF根外菌丝感知水势的变化, 激活质膜质子泵引起H⁺外流和Ca²⁺迅速内流, 从而引起钙振荡和信号传导。一方面菌丝通过胞间通信促进水分的运输和重新分配, 另一方面菌丝际pH值的降低促进了菌丝P、Ca和Fe等营养元素的吸收, 并将这些物质通过菌丝通道运输到植物根细胞中。本研究证实菌丝通过调节H⁺和Ca²⁺的跨膜运输应答干旱胁迫, 不仅增强了菌丝与环境之间的物质交换, 同时也加强了AMF与宿主之间的信号交流, 从而增强植物的抗旱能力。

参考文献原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子

[1]

Augé

, Duan

( 1991). Mycorrhizal fungi and nonhydraulic root signals of soil drying
Plant Physiology, 97, 821-824.

DOI:10.1104/pp.97.2.821 URL [本文引用: 1]

We propose that mycorrhizal colonization of roots alters nonhydraulic root to shoot communication of soil drying. Split-root rose (Rosa hybrida L. cv Samantha) plants--one side of the root system colonized by Glomus intraradices Schenck & Smith, the other side nonmycorrhizal-displayed different stomatal conductances upon partial drying, depending upon whether mycorrhizal or nonmycorrhizal roots were dried. No differences in leaf water status were observed among control plants and those whose mycorrhizal or nonmycorrhizal roots were dried.

[2]

Augé

, Toler

, Saxton

( 2015). Arbuscular mycorrhizal symbiosis alters stomatal conductance of host plants more under drought than under amply watered conditions: A meta-analysis
Mycorrhiza, 25, 13-24.

DOI:10.1007/s00572-014-0585-4 URLPMID:24831020 [本文引用: 1]

Stomata regulate rates of carbon assimilation and water loss. Arbuscular mycorrhizal (AM) symbioses often modify stomatal behavior and therefore play pivotal roles in plant productivity. The size of the AM effect on stomatal conductance to water vapor ( g s ) has varied widely, has not always been apparent, and is unpredictable. We conducted a meta-analysis of 460 studies to determine the size of the AM effect under ample watering and drought and to examine how experimental conditions have influenced the AM effect. Across all host and symbiont combinations under all soil moisture conditions, AM plants have shown 24 % higher g s than nonmycorrhizal (NM) controls. The promotion of g s has been over twice as great during moderate drought than under amply watered conditions. The AM influence on g s has been even more pronounced under severe drought, with over four times the promotion observed with ample water. Members of the Claroideoglomeraceae, Glomeraceae, and other AM families stimulated g s by about the same average amount. Colonization by native AM fungi has produced the largest promotion. Among single-AM symbionts, Glomus deserticola , Claroideoglomus etunicatum , and Funneliformis mosseae have had the largest average effects on g s across studies. Dicotyledonous hosts, especially legumes, have been slightly more responsive to AM symbiosis than monocotyledonous hosts, and C3 plants have shown over twice the AM-induced promotion of C4 plants. The extent of root colonization is important, with heavily colonized plants showing 脳10 the g s promotion of lightly colonized plants. AM promotion of g s has been larger in growth chambers and in the field than in greenhouse studies, almost 3 as large when plants were grown under high light than low light, and 2.5 as large in purely mineral soils than in soils having an organic component. When AM plants have been compared with NM controls given NM pot culture, they have shown only half the promotion of g s as NM plants not given anything at inoculation to control for associated soil organisms. The AM effect has been much greater when AM plants were larger or had more phosphorus than NM controls. These findings should assist in further investigations of predictions and mechanisms of the AM influence on host g s .

[3]

Ayling

, Smith

( 2000). Transmembrane electric potential difference of germ tubes of arbuscular mycorrhizal fungi responds to external stimuli
New Phytologist, 147, 631-639.

DOI:10.1046/j.1469-8137.2000.00723.x URL [本文引用: 1]

Measurements of the electric potential difference across the hyphal wall and the cell membrane were made on external hyphae of three species of arbuscular mycorrhizal fungus Gigaspora margarita , Scutellospora calospora and Glomus coronatum and on germ tubes of Gi. margarita . The values of transmembrane electric potential difference recorded (09080409000940 mV) are less negative than those previously reported from hyphae of arbuscular mycorrhizal fungi closely associated with roots and from filamentous fungi. The external hyphae of arbuscular mycorrhizal fungi grown in soil had similar values of electric potential difference to those grown in soil-less culture, and to germ tubes. Thermodynamic calculations showed that despite these low values of electric potential difference, efficient high-affinity uptake of phosphate is possible. The transmembrane electric potential difference of germ tubes of Gi. margarita became more negative when plant root extract was added to the medium, showing for the first time that the early stages of interaction between plant and fungus occur via direct effects on the plasma membrane rather than via effects on gene expression. Addition of K + reversibly depolarized the transmembrane electric potential difference of germ tubes of Gi. margarita , indicating that despite the low electric potential difference the fungus has control over the permeability of the plasmamembrane to K + .

[4]

Azad

, Sawa

, Ishikawa

, Shibata

( 2004). Phosphorylation of plasma membrane aquaporin regulates temperature-?dependent opening of tulip petals
Plant and Cell Physiology, 45, 608-617.

DOI:10.1093/pcp/pch069 URLPMID:15169943 [本文引用: 1]

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

[5]

Bárzana

, Aroca

, Paz

, Chaumont

, Martinez-Ballesta

, Carvajal

, Ruiz-Lozano

( 2012). Arbuscular mycorrhizal symbiosis increases relative apoplastic water flow in roots of the host plant under both well-watered and drought stress conditions
Annals of Botany, 109, 1009-1017.

DOI:10.1093/aob/mcs007 URLPMID:22294476 [本文引用: 1]

Background and Aims The movement of water through mycorrhizal fungal tissues and between the fungus and roots is little understood. It has been demonstrated that arbuscular mycorrhizal (AM) symbiosis regulates root hydraulic properties, including root hydraulic conductivity. However, it is not clear whether this effect is due to a regulation of root aquaporins (cell-to-cell pathway) or to enhanced apoplastic water flow. Here we measured the relative contributions of the apoplastic versus the cell-to-cell pathway for water movement in roots of AM and non-AM plants. Methods We used a combination of two experiments using the apoplastic tracer dye light green SF yellowish and sodium azide as an inhibitor of aquaporin activity. Plant water and physiological status, root hydraulic conductivity and apoplastic water flow were measured. Key Results Roots of AM plants enhanced significantly relative apoplastic water flow as compared with non-AM plants and this increase was evident under both well-watered and drought stress conditions. The presence of the AM fungus in the roots of the host plants was able to modulate the switching between apoplastic and cell-to-cell water transport pathways. Conclusions The ability of AM plants to switch between water transport pathways could allow a higher flexibility in the response of these plants to water shortage according to the demand from the shoot.

[6]

Bartnicki-Garcia

, Bracker

, Gierz

, López-Franco

, Lu

( 2000). Mapping the growth of fungal hyphae: Orthogonal cell wall expansion during tip growth and the role of turgor
Biophysical Journal, 79, 2382-2390.

DOI:10.1016/S0006-3495(00)76483-6 URLPMID:11053117 [本文引用: 1]

By computer-enhanced videomicroscopy, we mapped the trajectory of external and internal cell surface markers in growing fungal hyphae to determine the pattern of cell wall expansion during apical growth. Carbon particles (India ink) were chosen as external markers for tip expansion of Rhizoctonia solani hyphae. Irregularities in the growing apical walls of R. solani served as internal markers. Marker movement was traced in captured frames from the videotaped sequences. External and internal markers both followed orthogonal trajectories; i.e., they moved perpendicular to the cell surface regardless of their initial position in the hyphal apex. We found no evidence that the tip rotates during elongation. The discovery that the cell wall of a growing hypha expands orthogonally has major repercussions on two fronts: 1) It supports the long-held view that turgor pressure is the main force driving cell wall expansion. 2) It provides crucial information to complete the mathematical derivation of a three-dimensional model of hyphal morphogenesis based on the vesicle supply center concept. In three dimensions, the vesicle gradient generated by the vesicle supply center is insufficient to explain shape; it is also necessary to know the manner in which the existing surface is displaced during wall expansion.

[7]

Bécard

, Fortin

( 1988). Early events of vesicular?-arbuscular mycorrhiza formation on Ri T-DNA transformed roots
New Phytologist, 108, 211-218.

DOI:10.1111/nph.1988.108.issue-2 URL [本文引用: 1]

[8]

Bunney

, Shaw

, Watkins

, Taylor

, Beven

, Wells

, Calder

, Dr?bak

( 2000). ATP-dependent regulation of nuclear Ca ²⁺ levels in plant cells
FEBS Letters, 476, 145-149.

DOI:10.1016/S0014-5793(00)01709-9 URLPMID:10913602 [本文引用: 1]

Localised alterations in cytoplasmic Ca 2+ levels are an integral part of the response of eukaryotic cells to a plethora of external stimuli. Due to the large size of nuclear pores, it has generally been assumed that intranuclear Ca 2+ levels reflect the prevailing cytoplasmic Ca 2+ levels. Using nuclei prepared from carrot ( Daucus carota L.) cells, we now show that Ca 2+ can be transported across nuclear membranes in an ATP-dependent manner and that over 95% of Ca 2+ is accumulated into a pool releasable by the Ca 2+ ionophore A.23187. ATP-dependent nuclear Ca 2+ uptake did not occur in the presence of ADP or ADP S and was abolished by orthovanadate. Confocal microscopy of nuclei loaded with dextran-linked Indo-1 showed that the initial ATP-induced rise in [Ca 2+] occurs in the nuclear periphery. The occurrence of ATP-dependent Ca 2+ uptake in plant nuclei suggests that alterations of intranuclear Ca 2+ levels may occur independently of cytoplasmic [Ca 2+] changes.

[9]

Campo

, Baldrich

, Messeguer

, Lalanne

, Coca

, San Segundo

( 2014). Overexpression of a calcium-dependent protein kinase confers salt and drought tolerance in rice by preventing membrane lipid peroxidation
Plant Physiology, 165, 688-704.

DOI:10.1104/pp.113.230268 URLPMID:24784760 [本文引用: 1]

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage.

[10]

Chitarra

, Pagliarani

, Maserti

, Lumini

, Siciliano

, Cascone

, Schubert

, Gambino

, Balestrini

, Guerrieri

( 2016). Insights on the impact of arbuscular mycorrhizal symbiosis on tomato tolerance to water stress
Plant Physiology, 171, 1009-1023.

DOI:10.1104/pp.16.00307 URLPMID:27208301 [本文引用: 1]

Arbuscular mycorrhizal (AM) fungi, which form symbioses with the roots of the most important crop species, are usually considered biofertilizers, whose exploitation could represent a promising avenue for the development in the future of a more sustainable next-generation agriculture. The best understood function in symbiosis is an improvement in plant mineral nutrient acquisition, as exchange for carbon compounds derived from the photosynthetic process: this can enhance host growth and tolerance to environmental stresses, such as water stress (WS). However, physiological and molecular mechanisms occurring in arbuscular mycorrhiza-colonized plants and directly involved in the mitigation of WS effects need to be further investigated. The main goal of this work is to verify the potential impact of AM symbiosis on the plant response to WS. To this aim, the effect of two AM fungi (Funneliformis mosseae and Rhizophagus intraradices) on tomato (Solanum lycopersicum) under the WS condition was studied. A combined approach, involving ecophysiological, morphometric, biochemical, and molecular analyses, has been used to highlight the mechanisms involved in plant response to WS during AM symbiosis. Gene expression analyses focused on a set of target genes putatively involved in the plant response to drought, and in parallel, we considered the expression changes induced by the imposed stress on a group of fungal genes playing a key role in the water-transport process. Taken together, the results show that AM symbiosis positively affects the tolerance to WS in tomato, with a different plant response depending on the AM fungi species involved.

[11]

Duncan

( 1955). Multiple range and multiple
F tests. Biometrics, 11, 1-42.

[本文引用: 1]

[12]

Ferrol

, Barea

, Azcón-Aguilar

( 2000). The plasma membrane H +-ATPase gene family in the arbuscular mycorrhizal fungus Glomus mosseae
Current Genetics, 37, 112-118.

[本文引用: 1]

[13]

Fromm

, Lautner

( 2007). Electrical signals and their physiological significance in plants
Plant, Cell & Environment, 30, 249-257.

DOI:10.1111/j.1365-3040.2006.01614.x URLPMID:17263772 [本文引用: 1]

Electrical excitability and signalling, frequently associated with rapid responses to environmental stimuli, are well known in some algae and higher plants. The presence of electrical signals, such as action potentials (AP), in both animal and plant cells suggested that plant cells, too, make use of ion channels to transmit information over long distances. In the light of rapid progress in plant biology during the past decade, the assumption that electrical signals do not only trigger rapid leaf movements in 'sensitive' plants such as Mimosa pudica or Dionaea muscipula , but also physiological processes in ordinary plants proved to be correct. Summarizing recent progress in the field of electrical signalling in plants, the present review will focus on the generation and propagation of various electrical signals, their ways of transmission within the plant body and various physiological effects.

[14]

Gaxiola

, Palmgren

, Schumacher

( 2007). Plant proton pumps
FEBS Letters, 581, 2204-2214.

DOI:10.1016/j.febslet.2007.03.050 URL [本文引用: 1]

[15]

Gévaudant

, Duby

, von Stedingk

, Zhao

, Morsomme

, Boutry

( 2007). Expression of a constitutively activated plasma membrane H⁺-ATPase alters plant development and increases salt tolerance
Plant Physiology, 144, 1763-1776.

DOI:10.1104/pp.107.103762 URL [本文引用: 1]

[16]

Gianinazzi-Pearson

, Smith

, Gianinazzi

, Smith

( 1991). Enzymatic studies on the metabolism of vesicular-?arbuscular mycorrhizas
New Phytologist, 117, 61-74.

DOI:10.1111/nph.1991.117.issue-1 URL [本文引用: 1]

[17]

Gong

, Xiong

, Ma

, Wang

, Ge

, Qin

, Li

, Kong

, Li

( 2010). Early activation of plasma membrane H +-ATPase and its relation to drought adaptation in two contrasting oat ( Avena sativa L.) genotypes
Environmental and Experimental Botany, 69, 1-8.

[本文引用: 1]

[18]

Harrison

( 2005). Signaling in the arbuscular mycorrhizal symbiosis
Annual Review Microbiology, 59, 19-42.

DOI:10.1146/annurev.micro.58.030603.123749 URL [本文引用: 1]

[19]

Hijikata

, Murase

, Tani

, Ohtomo

, Osaki

, Ezawa

( 2010). Polyphosphate has a central role in the rapid and massive accumulation of phosphorus in extraradical mycelium of an arbuscular mycorrhizal fungus
New Phytologist, 186, 285-289.

DOI:10.1111/j.1469-8137.2009.03168.x URLPMID:20409186 [本文引用: 1]

This study evaluated the dynamics of polyP, total P and Pi in an arbuscular mycorrhizal (AM) fungus grown under P-starvation conditions to clarify the significance of polyP in P storage/translocation in AM fungal associations. Lotus japonicus cv Miyakojima MG-20 were sown on moistened filter paper in a Petri dish and germinated at 25 degrees C for 2 days in the dark. Three seedlings were transplanted to the mycorrhizal compartment (MC) of a dual mesh bag culture system in a 230 ml plastic pot (7.6 cm diameter) and inoculated with Glomus sp. HR1 (MAFF 520076) at 500 spores per pot. The time course analysis (from 0 to 9 h after Pi application) of total P and that of polyP in the HC were conducted separately using different batches of plant/fungal material. For these analyses, 22 pots were prepared as one batch, and mycelial samples collected from two pots grown in the same batch were combined as one sample (5-30 mg FW per sample). One set of time course experiments (from 0 to 9 h after Pi application) was conducted using one batch (without replication) and triplicated using three independent batches. The total P content of extraradical mycelium in the HC was 3.6 micro mol mg-1 protein at time zero, which increased to 8.2 micro mol mg-1 protein 5 h after Pi application and then decreased to 5.2 micro mol mg-1 protein 9 h after Pi application. Results showed that the polyP content of extraradical mycelium in the HC increased from 0.5 to 7.1 micro mol mg-1 protein from 0 to 6 h after Pi application and decreased to 2.8 micro mol mg-1 protein by 9 h after Pi application. The apparent accumulation rates of total P and polyP from 0 to 5 h after Pi application were 1.03 and 1.14 micro mol mg-1 protein h-1, respectively, and were not significantly different (P-1 protein and from 0.2 to 4.3 micro mol mg-1 protein, respectively, from 0 to 5 h after Pi application. This study demonstrated the significance of polyP as the largest P storage and a mediator of long-distance P translocation in AM fungi.

[20]

Isfort

, Cody

, Asquith

, Ridder

, Stuard

, Leboeuf

( 1993). Induction of protein phosphorylation, protein synthesis, immediate-early-gene expression and cellular proliferation by intracellular pH modulation
FEBS Journal, 213, 349-357.

[本文引用: 1]

[21]

Kühtreiber

, Jaffe

( 1990). Detection of extracellular calcium gradients with a calcium-specific vibrating electrode
The Journal of Cell Biology, 110, 1565-1573.

DOI:10.1083/jcb.110.5.1565 URL [本文引用: 1]

[22]

, Du

, Hao

, Zhang

, Chen

( 2012). Molecular basis for enhancement of plant drought tolerance by arbuscular mycorrhizal symbiosis: A mini-review
Acta Ecologica Sinica, 32, 7169-7176.

DOI:10.5846/stxb201110141518 URL [本文引用: 1]

Arbuscular mycorrhizal (AM) symbiosis, a ubiquitous symbiotic association established between AM fungi and roots of higher plants in most terrestrial ecosystems, is essentially important for plant adaptation to various environmental stresses, such as nutrient deficiency, environmental pollution and drought, etc. Many studies proved the positive influences of AM on plant drought tolerance and made efforts to uncover the underlying mechanisms: for plant individuals, AM fungi could stimulate plant physiological responses to drought stress; at the ecosystem level, AM fungi could interact with host plant to adapt to an adverse environment. However, systematic study is still necessary to reveal the fundamental role of AM fungi in improving plant drought tolerance. -pyrroline-5-carboxylate synthetase, MIPs encoding major intrinsic proteins, and NCED encoding 9--epoxycarotenoid dioxygenase. As seen in reports, AM fungi could up- or down-regulate these genes under drought stresses, however, there were conflicting results as for the mycorrhizal effects on gene expressions in different experiments. In most cases, this could be attributed to incomparable experimental conditions, considering that 1) not all members in a gene family had been examined in each experiment; 2) different symbiotic associations (plant-AM fungus combinations) might exert different strategies to resist drought stresses, and each fungal species/strain might exhibit different capacity to assist host plant against environmental adversities; 3) gene expression varies in different plant tissues at different plant developmental stages. Obviously, it is still necessary to carry out further research for a better understanding of AM regulation of drought tolerance related genes in host plants, and model plants and AM fungal strains might be ideal choices to make sure of comparable results from different experiments. In addition to a full discussion on the insufficiency of previous studies, we also introduced the advances of proteomics in AM physiology and proposed perspectives for future research by the end of this review.

[ 李涛, 杜娟, 郝志鹏, 张莘, 陈保冬 ( 2012). 丛枝菌根提高宿主植物抗旱性分子机制研究进展
生态学报, 32, 7169-7176.]

DOI:10.5846/stxb201110141518 URL [本文引用: 1]

, Hu

, Hao

, Li

, Wang

, Chen

( 2013). First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus
Glomus intraradices. New Phytologist, 197, 617-630.

DOI:10.1111/nph.12011 URLPMID:23157494 [本文引用: 2]

Arbuscular mycorrhizal (AM) symbiosis is known to stimulate plant drought tolerance. However, the molecular basis for the direct involvement of AM fungi (AMF) in plant water relations has not been established.Two full-length aquaporin genes, namely GintAQPF1 and GintAQPF2, were cloned by rapid amplification of cDNA 5090005- and 3090005-ends from an AMF, Glomus intraradices. Aquaporin localization, activities and water permeability were examined by heterologous expression in yeast. Gene expression during symbiosis was also analyzed by quantitative real-time polymerase chain reaction.GintAQPF1 was localized to the plasma membrane of yeast, whereas GintAQPF2 was localized to both plasma and intracellular membranes. Transformed yeast cells exhibited a significant decrease in cell volume on hyperosmotic shock and faster protoplast bursting on hypo-osmotic shock. Polyethylene glycol (PEG) stimulated, but glycerol inhibited, the aquaporin activities. Furthermore, the expression of the two genes in arbuscule-enriched cortical cells and extraradical mycelia of maize roots was also enhanced significantly under drought stress.GintAQPF1 and GintAQPF2 are the first two functional aquaporin genes from AMF reported to date. Our data strongly support potential water transport via AMF to host plants, which leads to a better understanding of the important role of AMF in plant drought tolerance.

[24]

Liu

, Li

, Hui

, Tang

, Zhang

( 2016). Effect of Rhizophagus irregularis on osmotic adjustment, antioxidation and aquaporin PIP genes expression of Populus × canadensis ‘Neva’ under drought stress
Acta Physiologiae Plantarum, 38, 191. DOI: 10.1007/s11738-016-2207-6.

[本文引用: 1]

[25]

Liu

, Chen

( 2007). Mycorrhizology. Science Press, Beijing. 447-448.

[本文引用: 1]

[26]

Ludwig

, Romeis

, Jones

( 2004). CDPK-mediated signalling pathways: Specificity and cross-talk
Journal of Experimental Botany, 55, 181-188.

DOI:10.1093/jxb/erh008 URLPMID:14623901 [本文引用: 1]

Abstract Plants are constantly exposed to environmental changes and have to integrate a variety of biotic and abiotic stress stimuli. Calcium-dependent protein kinases (CDPKs) are implicated as important sensors of Ca2+ flux in plants in response to these stresses. CDPKs are encoded by multigene families, and expression levels of these genes are spatially and temporally controlled throughout development. In addition, a subset of CDPK genes responds to external stimuli. Biochemical evidence supports the idea that CDPKs are involved in signal transduction during stress conditions. Furthermore, loss-of-function and gain-of-function studies revealed that signalling pathways leading to cold, salt, drought or pathogen resistance are mediated by specific CDPK isoforms

[27]

, Zhang

, Li

, Du

, Wang

, Chen

( 2005). The effects of exogenous Ca ²⁺ on endogenous polyamine levels and drought-resistant traits of spring wheat grown under arid conditions
Journal of Arid Environments, 63, 177-190.

DOI:10.1016/j.jaridenv.2005.01.021 URL [本文引用: 1]

This study investigates the effects of exogenous Ca 2+ on physiological and phenotypic characteristics of spring wheat ( Triticum aestivum L.) in an arid region of China. These traits, which are closely linked to drought tolerance in plants, include alteration in endogenous levels of polyamines (PAs), root-to-stem ratios and relative water content (RWC). Exogenous Ca 2+ was applied to the seeds by soaking in a 0.5% CaCl 2 solution before sowing and also by spraying the leaves with the same solution. It was determined that the foliar RWC increased at all stages of development following the Ca 2+ application. The levels of endogenous PAs in the leaves were altered: the putrescine (Put) content decreased whereas the spermidine (Spd) and spermine (Spm) content increased. The ratio of Put to PAs titers was reduced. At the same time, phenological events of wheat were also changed in response to the application of Ca 2+. In particular, reproductive maturity was significantly delayed. Moreover, the application of Ca 2+ increased both the root-to-shoot ratios of wheat at the early stages of growth and the final grain yield. The results obtained from this study show that the exogenous Ca 2+ could not only enhance the tolerance of spring wheat to drought but also delay its senescence. Finally, the role of Ca 2+ in the signal regulation of endogenous PA metabolism is discussed. We suggest that exogenous Ca 2+ may have a role in modifying endogenous PAs levels under drought stress leading to increased drought tolerance.

[28]

Mak

, Babla

, Xu

, O’Carrigan

, Liu

, Gong

, Holford

, Chen

( 2014). Leaf mesophyll K⁺, H⁺and Ca²⁺ fluxes are involved in drought-induced decrease in photosynthesis and stomatal closure in soybean
Environmental and Experimental Botany, 98, 1-12.

DOI:10.1016/j.envexpbot.2013.10.003 URL [本文引用: 1]

Understanding the roles of chemical signals for drought tolerance is important for improving plant water use efficiency. Microelectrode ion flux measurement (MIFE), leaf gas exchange, and stomatal imaging were employed to assess the impact of short-term, PEG-induced and prolonged drought stress on soybean plants. We developed a new method to record steady-state K+, H+ and Ca2+ fluxes from leaf mesophyll of soybean plants grown in a glasshouse over a long time period. Long-term K+, H+ and Ca2+ fluxes under drought condition differed significantly from short-term PEG-induced drought stress. Moreover, the magnitude of changes differed between the ion fluxes and the physiological and growth traits. For instance, in the severe drought treatment, differences in the magnitude of Ca2+ efflux between the drought-stressed plants and the control were greater than the changes in aperture width, guard cell width and leaf temperature. In addition, H+ influx and K+ and Ca2+ efflux of leaf mesophyll were highly significantly (P<0.01) correlated with many physiological traits. In summary, our results suggest that a large K+ efflux, alkalisation of apoplastic pH (H+ influx), and an early response of Ca2+ efflux from leaf mesophyll are likely to serve as chemical signals and significant indicators for levels of drought stress in soybean.

[29]

Porcel

, Ruiz-Lozano

( 2004). Arbuscular mycorrhizal influence on leaf water potential, solute accumulation, and oxidative stress in soybean plants subjected to drought stress
Journal of Experimental Botany, 55, 1743-1750.

DOI:10.1093/jxb/erh188 URLPMID:15208335 [本文引用: 1]

This study investigated several aspects related to drought tolerance in arbuscular mycorrhizal (AM) soybean plants. The investigation included both shoot and root tissues in order to reveal the preferred target tissue for AM effects against drought stress. Non-AM and AM soybean plants were grown under well-watered or drought-stressed conditions, and leaf water status, solute accumulation, oxidative damage to lipids, and other parameters were determined. Results showed that AM plants were protected against drought, as shown by their significantly higher shoot-biomass production. The leaf water potential was also higher in stressed AM plants (-1.9 MPa) than in non-AM plants (-2.5 MPa). The AM roots had accumulated more proline than non-AM roots, while the opposite was observed in shoots. Lipid peroxides were 55% lower in shoots of droughted AM plants than in droughted non-AM plants. Since there was no correlation between the lower oxidative damage to lipids in AM plants and the activity of antioxidant enzymes, it seems that first the AM symbiosis enhanced osmotic adjustment in roots, which could contribute to maintaining a water potential gradient favourable to the water entrance from soil into the roots. This enabled higher leaf water potential in AM plants during drought and kep the plants protected against oxidative stress, and these cumulative effects increased the plant tolerance to drought.

[30]

Querejeta

, Egerton-Warburton

, Allen

( 2003). Direct nocturnal water transfer from oaks to their mycorrhizal symbionts during severe soil drying
Oecologia, 134, 55-64.

DOI:10.1007/s00442-002-1078-2 URL [本文引用: 1]

[31]

Ramos

, Fa?anha

, Feijó

( 2008a). Ion dynamics during the polarized growth of arbuscular mycorrhizal fungi: From presymbiosis to symbiosis
In: Varma A ed. Mycorrhiza. Springer, Berlin, Heidelberg.

DOI:10.1007/978-3-540-78826-3_12 URL [本文引用: 1]

The 450-million-year-old symbiosis between the majority of land plants and arbuscular mycorrhizal (AM) fungi is one of the most ancient, abundant, and ecologically important symbioses on earth (Remy e

[32]

Ramos

, Fa?anha

, Feijó

( 2008b). Proton (H⁺) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi
. New Phytologist, 178, 177-188.

DOI:10.1111/j.1469-8137.2007.02344.x URL [本文引用: 1]

[33]

Requena

, Breuninger

, Franken

, Ocón

( 2003). Symbiotic status, phosphate, and sucrose regulate the expression of two plasma membrane H⁺-ATPase genes from the mycorrhizal fungus
Glomus mosseae. Plant Physiology, 132, 1540-1549.

DOI:10.1016/j.colsurfa.2004.09.019 URLPMID:12857834 [本文引用: 1]

The establishment of the arbuscular mycorrhizal symbiosis results in a modification of the gene expression pattern in both plant and fungus to accomplish the morphological and physiological changes necessary for the bidirectional transfer of nutrients between symbionts. H+-ATPase enzymes play a key role establishing the electrochemical gradient required for the transfer of nutrients across the plasma membrane in both fungi and plants. Molecular analysis of the genetic changes in arbuscular mycorrhizal fungi during symbiosis allowed us to isolate a fungal cDNA clone encoding a H+-ATPase, GmPMA1, from Glomus mosseae (BEG12). Despite the high conservation of the catalytic domain from H+-ATPases, detailed analyses showed that GmPMA1 was strongly related only to a previously identified G. mosseae ATPase gene, GmHA5, and not to the other four ATPase genes known from this fungus. A developmentally regulated expression pattern could be shown for both genes, GmPMA1 and GmHA5. GmPMA1 was highly expressed during asymbiotic development, and its expression did not change when entering into symbiosis, whereas the GmHA5 transcript was induced upon plant recognition at the appressorium stage. Both genes maintained high levels of expression during intraradical development, but their expression was reduced in the extraradical mycelium. Phosphate, a key nutrient to the symbiosis, also induced the expression of GmHA5 during asymbiotic growth, whereas sucrose had a negative effect. Our results indicate that different fungal H+-ATPases isoforms might be recruited at different developmental stages possibly responding to the different requirements of the life in symbiosis.

[34]

Riquelme

, Bartnicki-Garcia

( 2004). Key differences between lateral and apical branching in hyphae of
Neurospora crassa. Fungal Genetics and Biology, 41, 842-851.

[本文引用: 1]

[35]

Rudd

, Franklin-Tong

( 2001). Unravelling response-?specificity in Ca ²⁺ signalling pathways in plant cells
New Phytologist, 151, 7-33.

DOI:10.1046/j.1469-8137.2001.00173.x URL [本文引用: 1]

[36]

Sánchez-Romera

, Ruiz-Lozano

, Zamarre?o

áM

, García-?Mina

, Aroca

( 2016). Arbuscular mycorrhizal symbiosis and methyl jasmonate avoid the inhibition of root hydraulic conductivity caused by drought
Mycorrhiza, 26, 111-122.

DOI:10.1007/s00572-015-0650-7 URLPMID:26070449 [本文引用: 1]

Abstract Hormonal regulation and symbiotic relationships provide benefits for plants to overcome stress conditions. The aim of this study was to elucidate the effects of exogenous methyl jasmonate (MeJA) application on root hydraulic conductivity (L) of Phaseolus vulgaris plants which established arbuscular mycorrhizal (AM) symbiosis under two water regimes (well-watered and drought conditions). The variation in endogenous contents of several hormones (MeJA, JA, abscisic acid (ABA), indol-3-acetic acid (IAA), salicylic acid (SA)) and the changes in aquaporin gene expression, protein abundance and phosphorylation state were analyzed. AM symbiosis decreased L under well-watered conditions, which was partially reverted by the MeJA treatment, apparently by a drop in root IAA contents. Also, AM symbiosis and MeJA prevented inhibition of L under drought conditions, most probably by a reduction in root SA contents. Additionally, the gene expression of two fungal aquaporins was upregulated under drought conditions, independently of the MeJA treatment. Plant aquaporin gene expression could not explain the behaviour of L. Conversely, evidence was found for the control of L by phosphorylation of aquaporins. Hence, MeJA addition modified the response of L to both AM symbiosis and drought, presumably by regulating the root contents of IAA and SA and the phosphorylation state of aquaporins.

[37]

Santi

, Locci

, Monte

, Pinton

, Varanini

( 2003). Induction of nitrate uptake in maize roots: Expression of a putative high-affinity nitrate transporter and plasma membrane H⁺- ATPase isoforms
Journal of Experimental Botany, 54, 1851-1864.

DOI:10.1093/jxb/erg208 URLPMID:12869520 [本文引用: 1]

An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.

[38]

Shi

, Zhang

, Han

, Feng

, Hu

, Guo

, Gong

( 2016). Silicon enhances water stress tolerance by improving root hydraulic conductance in
Solanum lycopersicum L. Frontiers in Plant Science, 7, 196. DOI: 10.3389/fpls.?2016.?00196.

[本文引用: 1]

[39]

Smith

, Read

( 2008). Mycorrhizal Symbiosis
Academic Press, New York.

[本文引用: 1]

[40]

St-Arnaud

, Hamel

, Vimard

, Caron

, Fortin

( 1996). Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus
Glomus intraradices in an in vitro system in the absence of host roots. Mycological Research, 100, 328-332.

[本文引用: 3]

[41]

Sun

, Unestam

, Lucas

, Johanson

, Kenne

, Finlay

( 1999). Exudation-reabsorption in a mycorrhizal fungus, the dynamic interface for interaction with soil and soil microorganisms
Mycorrhiza, 9, 137-144.

DOI:10.1007/s005720050298 URL [本文引用: 1]

[42]

Tisserant

, Malbreil

, Kuo

, Kohler

, Symeonidi

, Balestrini

, Charron

, Duensing

, Frey

, Gianinazzi-?Pearson

, Gilbert

, Handa

, Herr

, Hijri

, Koul

, Kawaguchi

, Krajinski

, Lammers

, Masclaux

, Murat

, Morin

, Ndikumana

, Pagni

, Petitpierre

, Requena

, Rosikiewicz

, Riley

, Saito

, Clemente

, Shapiro

, van Tuinen

, Bécard

, Bonfante

, Paszkowski

, Shachar-Hill

, Tuskan

, Young

, Sanders

, Henrissat

, Rensing

, Grigoriev

, Corradi

, Roux

, Martin

( 2013). Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis
Proceedings of the National Academy of Sciences of the United States of America, 110, 20117-20122.

DOI:10.1073/pnas.1313452110 URLPMID:24277808 [本文引用: 1]

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

[43]

van Hees

, Rosling

, Essén

, Godbold

, Jones

, Finlay

( 2006). Oxalate and ferricrocin exudation by the extramatrical mycelium of an ectomycorrhizal fungus in symbiosis withPinus sylvestris
New Phytologist, 169, 367-378.

[本文引用: 1]

[44]

, Zhang

, Sun

, Wu

, Li

, Hu

, Su

, Lü

, Li

, Zhang

, Zheng

, Zhang

, Chen

( 2015). Transformation and immobilization of chromium by arbuscular mycorrhizal fungi as revealed by SEM-EDS, TEM-EDS, and XAFS
Environmental Science & Technology, 49, 14036-14047.

[本文引用: 1]

[45]

Xiong

, Li

, Zhang

, Xia

( 2007). Evolution mechanism of non-hydraulic root-to-shoot signal during the anti-drought genetic breeding of spring wheat
Environmental and Experimental Botany, 59, 193-205.

DOI:10.1016/j.envexpbot.2005.12.003 URL [本文引用: 1]

The objectives of this study were to: (1) characterize the evolutional tendency of the non-hydraulic root-sourced signal (NRS) from wheat wild relatives to its modern hexaploid species, and (2) test whether species sensitivity to the NRS was allied with their drought tolerance profiles. The NRS was judged to begin when there was a significant lowering of stomatal conductance without change in leaf relative water content (RWC). The lethal soil water content (LSWC) was operationally characterized as the soil water content (SWC) at the drying lethal point of wheat plants. The threshold of soil water content (TSWC) at which the NRS was triggered, and the LSWC differed amongst six wheat species. For “ MO1” and “ MO4” representing ‘diploid’ species, the TSWC and the LSWC were initiated successively at about 51% FWC (field water capacity) and about 30% FWC, respectively. Conversely, “ Plateau 602” and “ Longchun 8139-2” (modern hexaploid species) exhibited the TSWC and the LSWC between about 68% FWC and less than 14% FWC, a much wider threshold range (TR). Increasing TSWC was significantly correlated with decreasing LSWC ( r = 0.9464 **). The widened TR from the TSWC to the LSWC was also significantly correlated with longer survival days (SD) and higher maintenance ratio of grain yield (MRGY), respectively ( r = 0.9411 ** and 0.8068 *, respectively). Meanwhile, those species having higher TSWC had the least reduction ratio of stomatal conductance under the decreasing soil moisture from 610.2 to 611 MPa. This suggests that advances in yield performance and drought tolerance would be made evolutionally by targeted selection for an earlier onset of NRS.

[46]

, Li

, Wu

, Feng

, Yu

, Zhang

, Chen

( 2018). Arbuscular mycorrhiza enhances drought tolerance of tomato plants by regulating the 14-3-3 genes in the ABA signaling pathway
Applied Soil Ecology, 125, 213-221.

DOI:10.1016/j.apsoil.2018.01.012 URL [本文引用: 1]

Arbuscular mycorrhizal (AM) fungi can substantially contribute to plant water uptake and drought tolerance. However, few studies examined whether AM symbiosis regulates the key functional genes in plant signaling under drought stress. The objective of present study was to investigate the influence of AM symbiosis on the expression of 14-3-3 genes (key genes in ABA signaling pathway) and plant adaptation to water deficit. The ABA-deficient tomato ( Solanum lycopersicum ) mutant notabilis ( not ) and its wild-type ( wt ) were cultivated with or without AM fungus Rhizophagus intraradices under well-watered or drought-stressed conditions. Plant phosphorus (P) concentration, transpiration rate (Tr), water use efficiency (WUE) and expression of 14-3-3 genes ( TFT1 - TFT12 ) were determined. The results indicated that mycorrhizal inoculation significantly increased shoot P concentrations of wt plants regardless of water regimes, and increased root P concentrations of the two genotypes under well-watered conditions. AM significantly up-regulated the expression of TFT2 and TFT3 in wt plants under drought stress. The decreased Tr led to increase of WUE and enhanced drought tolerance of wt plants. For not plants, AM significantly up-regulated the expression of TFT5 , TFT7 , TFT9 and TFT10 under drought stress, which subsequently altered the response of Tr to drought and prevented the decrease in WUE. The study supported that AM symbiosis could modulate the stomatal behavior and maintain WUE to improve plant drought tolerance possibly through regulation of the 14-3-3 genes in the ABA signaling pathway.

[47]

Yan

, Zhu

, Müller

, Z?rb

, Schubert

( 2002). Adaptation of H⁺-pumping and plasma membrane H⁺-ATPase activity in proteoid roots of white lupin under phosphate deficiency
Plant Physiology, 129, 50-63.

DOI:10.1104/pp.010869 URL [本文引用: 1]

[48]

Yang

( 2012). Mechanism Involved in Drought/Salt Tolerance Improvement in Alfalfa Due to Symbiotic Interaction with Rhizobium
PhD dissertation, Northwest A&F University,Yangling, Shaanxi. 4-6.

URL [本文引用: 1]

干旱、盐碱等逆境是全世界农业生产所面临的重要问题，我国更为严重。紫花苜蓿是我国乃至全世界最重要的牧草，以收获营养器官为主的苜蓿生产，更适应于干旱、盐碱区域，是开发旱区及盐碱地的重要选择。其抗逆机理研究，已成为草学研究重要内容，有关苜蓿根瘤菌共生对干旱及盐胁迫的响应机制研究，鲜见文献报道。本研究分别在干旱、盐及高温处理的条件下，应用根瘤菌接种温室培养（盆、钵）苜蓿，精确控制逆境的手段，测定了逆境存活率；采用常规方法测定了相关生理指标；非损伤微测技术测定了钙、钾、氢离子的进出根细胞情况；采用基因芯片技术筛选了差异基因；并对芯片筛选的3个基因进行半定量表达与分析。旨在研究探索苜蓿根瘤菌共生对干旱及盐胁迫下的响应机制。试验研究结论如下： 1.通过干旱、盐、高温处理及去除根瘤后存活率研究表明，接种根瘤菌可大幅度提高紫花苜蓿的抗旱、耐盐、抗高温及根部机械损伤等非生物胁迫能力，抗逆能力表现为激活根瘤菌苜蓿＞不激活根瘤菌苜蓿＞无根瘤菌苜蓿，且激活根瘤菌苜蓿抗逆性远远高于不激活根瘤菌苜蓿。苜蓿接种根瘤菌激活后，其抗非生物胁迫能力的提高来源于三个方面：根瘤菌的固氮作用、根瘤菌和苜蓿共生的免疫作用、根瘤菌固氮对植物自身组成变化或其生理应答机制的改变。根瘤菌对于苜蓿的抗逆性提高，在失去根瘤菌的固氮作用后，只要曾经接种根瘤菌，无论激活与否，依旧比未曾接种过根瘤菌的苜蓿具有更强的抗性，表明根瘤菌提高苜蓿的抗逆能力，不是完全依赖于根瘤菌的固氮作用，且其对抗逆能力的提高是长期的。 2.干旱和盐处理下不同组织相关生理指标测试表明，干旱和盐胁迫下，接种根瘤菌后，苜蓿具有更强的渗透调节能力，根瘤在抵御干旱/盐胁迫上具有重要作用。具有活性的根瘤能减缓植物受到干旱/盐胁迫的时间，并能维持植物细胞稳定性，细胞内环境平衡，减少氧化胁迫。 3.在干旱胁迫后复水和NaCl胁迫下，随着干旱程度的逐步增加和盐胁迫时间的延长，紫花苜蓿通过改变根部细胞离子浓度及进出细胞膜的方向来保持细胞内环境的平衡，维持渗透压。相对于不接种根瘤菌的紫花苜蓿，接种根瘤菌激活的紫花苜蓿在干旱胁迫和盐胁迫下，具有较强的渗透调节能力，能更好的维持植株根系活力、保持细胞内环境的平衡，提高紫花苜蓿的抗逆性。 4.通过基因芯片分析发现，干旱胁迫是一个多基因参与的过程。轻度干旱与充足给水相比，差异基因数量较少，差异基因中上调基因数量多，而下调基因数量少；重度干旱阶段与充足给水相比，差异表达基因数量多，上调下调基因相数目接近。接种根瘤菌不激活和无根瘤菌处理在轻度阶段下调基因多于上调基因；在充足给水、重度干旱阶段，接种根瘤菌激活、接种根瘤菌不激活和不接种根瘤菌互相之间比较，上调基因多于下调基因。从三个干旱处理阶段来看，有5类基因有规律性的表达，有5006个基因在接种根瘤菌激活、接种根瘤菌不激活和不接种根瘤菌中表现相同相似的变化规律；从根瘤菌处理来看，有三类基因（446个）在接种根瘤菌激活中表达量上调，接种根瘤菌不激活和不接种根瘤菌中下调。有一类基因（79个）在干旱后在接种根瘤菌激活时下调，而在干旱后期接种根瘤菌不激活和不接种根瘤菌时上调。从苯丙素途径（合成木质素）通路分析，发现25个差异基因参与了EC4.3.1.24、EC2.3.1.91、EC2.3.1.92、EC1.14.13.11、EC6.2.1.2、EC3.2.1.21、EC1.11.1.7等代谢步骤。 5.通过半定量PCR表达，对基因芯片筛选后的TC81048、TC83565和TC87183三个基因进行进一步的验证试验表明，这三个基因均对干旱有响应，不接种根瘤菌三个基因在苜蓿干旱时的表达呈现规律性升高。在接种根瘤菌后干旱时，TC83565表达量下降，而TC87183表达量增加，TC81048无变化。三个基因在干旱敏感的品种Semip中，表达量均显著升高，进一步证实这三个基因和干旱应答有关。综上，本文已基本明确接种根瘤菌的苜蓿，可在一定程度上抵御干旱和盐胁迫。这一过程有多个基因参与，涉及细胞稳定性、细胞内环境平衡、细胞氧化代谢、根际离子(Ca~(2+)、K~+、H~+)浓度和进出方向的渗透调节等。这为今后多视角系统研究苜蓿干旱和盐胁迫机制奠定了一定的基础。

[ 杨培志 ( 2012). 紫花苜蓿根瘤菌共生对干旱及盐胁迫的响应机制研究
博士学位论文, 西北农林科技大学, 陕西杨凌. 4-6.]

URL [本文引用: 1]

Yoshida

( 1991). Chilling-induced inactivation and its recovery of tonoplast H⁺-ATPase in mung bean cell suspension cultures
Plant Physiology, 95, 456-460.

DOI:10.1104/pp.95.2.456 URL [本文引用: 1]

[50]

Zhao

, Guo

, Bi

, Guo

, Wang

, Zhao

, Zhang

( 2015a). Arbuscular mycorrhizal fungi affect the growth, nutrient uptake and water status of maize (Zea mays L.) grown in two types of coal mine spoils under drought stress
Applied Soil Ecology, 88, 41-49.

DOI:10.1016/j.apsoil.2014.11.016 URL [本文引用: 1]

Drought stress greatly affects the growth and development of plants in coal mine spoils located in the Inner Mongolia grassland ecosystem. Arbuscular mycorrhizal fungi (AMF) can increase plant tolerance to drought. However, little is known regarding the contribution of AMF to plants that are grown in different types of coal mine spoils under drought stress. To evaluate the mycorrhizal effects on the drought tolerance of maize (Zea mays L.) grown in weathered (S1) and spontaneously combusted (S2) coal mine spoils, a greenhouse pot experiment was conducted to investigate the effects of inoculation with Rhizophagus intraradices on the growth, nutrient uptake, carbon:nitrogen:phosphorus (C:N:P) stoichiometry and water status of maize under well-watered, moderate and severe drought stress conditions. The results indicated that drought stress increased mycorrhizal colonization and decreased plant dry weights, nutrient contents, leaf moisture percentage of fresh weight (LMP), water use efficiency (WUE) and rehydration rate. A high level of AMF colonization ranging from 65 to 90% was observed, and the mean root colonization rates in S1 were lower than those in S2. In both substrates, inoculation with R. intraradices significantly improved the plant growth, P contents, LMP and WUE and decreased the C:P and N:P ratios of plants under drought stress. In addition, maize grown in S1 and S2 exhibited different wilting properties in response to AMF inoculation, and plant rehydration after drought stress occurred faster in mycorrhizal plants. The results suggested that inoculation with R. intraradices played a more positive role in improving the drought stress resistance of plants grown in S2 than those grown in S1. AMF inoculation has a beneficial effect on plant tolerance to drought and effectively facilitates the development of plants in different coal mine spoils.

[51]

Zhao

, Xu

, Wei

, Liu

( 2015b). Expression OsCAS (calcium-sensing receptor) in an Arabidopsis mutant increases drought tolerance
PLOS ONE, 10, e0131272. DOI: 10.1371/journal.pone.0131272.

DOI:10.1371/journal.pone.0131272 URLPMID:26098425 [本文引用: 1]

Abstract The calcium-sensing receptor (CaS), which is localized in the chloroplasts, is a crucial regulator of extracellular calcium-induced stomatal closure in Arabidopsis. It has homologs in Oryza sativa and other plants. These sequences all have a rhodanese-like protein domain, which has been demonstrated to be associated with specific stress conditions. In this study, we cloned the Oryza sativa calcium-sensing receptor gene (OsCAS) and demonstrated that OsCAS could sense an increase of extracellular Ca2+ concentration and mediate an increase in cytosolic Ca2+ concentration. The OsCAS gene was transformed into an Arabidopsis CaS knockout mutant (Salk) and overexpressed in the transgenic plants. OsCAS promoted stomatal closure. We screened homozygous transgenic Arabidopsis plants and determined physiological indices such as the oxidative damage biomarker malondialdehyde (MDA), relative membrane permeability (RMP), proline content, and chlorophyll fluorescence parameters, after 21 days of drought treatment. Our results revealed lower RMP and MDA contents and a higher Proline content in transgenic Arabidopsis plants after drought stress, whereas the opposite was observed in Salk plants. With respect to chlorophyll fluorescence, the electron transport rate and effective PSII quantum yield decreased in all lines under drought stress; however, in the transgenic plants these two parameters changed fewer and were higher than those in wild-type and Salk plants. The quantum yield of regulated energy dissipation and nonregulated energy dissipation in PSII were higher in Salk plants, whereas these values were lower in the transgenic plants than in the wild type under drought stress. The above results suggest that the transgenic plants showed better resistance to drought stress by decreasing damage to the cell membrane, increasing the amount of osmoprotectants, and maintaining a relatively high photosynthetic capacity. In conclusion, OsCAS is an extracellular calcium-sensing receptor that helps to compensate for the absence of CaS in Arabidopsis and increases the drought stress tolerance of transgenic plants.

[52]

Zou

, Li

, Ratnasekera

, Wang

, Liu

, Song

, Zhang

, Wu

( 2015). Arabidopsis calcium-dependent protein kinase 8 and catalase 3 function in abscisic acid-mediated signaling and H₂O₂ homeostasis in stomatal guard cells under drought stress
The Plant Cell, 27, 1445-1460.

[本文引用: 1]

[53]

Zhou

, Han

, Chen

, Kong

, Wang

( 2015). The involvement of expansins in response to water stress during leaf development in wheat
Journal of Plant Physiology, 183, 64-74.

DOI:10.1016/j.jplph.2015.05.012 URLPMID:26092364 [本文引用: 1]

Expansins are cell wall proteins that are generally considered to be the key regulator of cell wall extension during plant growth. In this study, we used two different wheat (Triticum aestivum L.) cultivars to demonstrate that expansins are involved in wheat leaf growth and response to water stress, by regulating the expansin activity and cell wall susceptibility to expansins. Expansin activity was associated with the relative elongation rate of leaves during leaf development, suggesting their involvement in leaf elongation. Moreover, cell wall extension characteristics and expansin gene transcription were closely involved in the leaf cell elongation region. Water stress restrains leaf growth, but the growth rate of leaves was changed after rehydration, which is consistent with the response of expansin activity to water stress. Meanwhile, increased cell wall susceptibility to expansin by water deficit played an important role in maintaining cell wall extension. Furthermore, the expansin activity in drought-resistant cultivar HF9703 was always higher than that in drought-sensitive cultivar 921842 under water stress condition, which may be correlated with the higher expansin gene expression in HF9703 versus 921842. These data provide evidence for a role of expansins in the growth and response of wheat leaves to water stress.
Mycorrhizal fungi and nonhydraulic root signals of soil drying
1
1991

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

Arbuscular mycorrhizal symbiosis alters stomatal conductance of host plants more under drought than under amply watered conditions: A meta-analysis
1
2015

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

Transmembrane electric potential difference of germ tubes of arbuscular mycorrhizal fungi responds to external stimuli
1
2000

... pH值变化是生物对环境变化的最初响应, 该变化直接引起菌丝质膜的信号传导, 激活下游受体的信号识别过程(Ayling et al., 2000).本试验利用LSCM观察了PEG处理对菌丝胞内pH的影响, 结果表明PEG处理1 h后菌丝尖端和侧面的荧光信号亮度明显增强.pH荧光探针在pH越高的环境中荧光强度越高, 因此该现象说明PEG处理使胞内pH值升高, ...

Phosphorylation of plasma membrane aquaporin regulates temperature-?dependent opening of tulip petals
1
2004

... 质膜H⁺外流不仅是营养物质吸收的动力, 还作为信号传导的中间体, 促进逆境信号的传导和响应元件的激活(Fromm & Lautner, 2007).H⁺的外流可以促进Ca²⁺、K⁺等阳离子的跨膜运输, 提高胞内的离子浓度(Isfort et al., 1993).本研究中, PEG处理1 h后, 菌丝尖端和表面的Ca²⁺表现了强烈的内流现象.当生物受到干旱胁迫的时候, 细胞内Ca²⁺瞬时产生快速的增高, 引发钙振荡(Zhao et al., 2015b).Ca²⁺作为第二信使, 在胁迫信号的传递过程中起到重要作用, 胞内游离Ca²⁺的分布与转移是Ca²⁺信号产生和传递的基础.使用LSCM观测菌丝细胞内Ca²⁺浓度的结果与离子流测定结果相符, PEG处理1 h后菌丝尖端与侧面均呈现强烈的绿色荧光, 说明菌丝内Ca²⁺浓度显著上升.干旱胁迫诱导的胞内Ca²⁺浓度增加能够激活下游的逆境信号响应元件, Ca依赖的蛋白激酶(CDPK)等, 而CDPK可以通过磷酸化质膜水通道蛋白促进细胞膜对水分的吸收和扩散(Azad et al., 2004).Li等(2013)证实, 干旱胁迫情况下AMF中两个水通道蛋白基因GintAQPF1和GintAQPF2的表达量会显著上调, 为植物输送水分以缓解干旱胁迫. ...

Arbuscular mycorrhizal symbiosis increases relative apoplastic water flow in roots of the host plant under both well-watered and drought stress conditions
1
2012

... 干旱条件下水分由根外菌丝向根内菌丝运输, 微量元素、氨基酸和其他低分子量的代谢产物也被AMF菌丝选择吸收并运送到植物中, 由此增强植物的耐旱性(Querejeta et al., 2003; van Hees et al., 2006).菌丝中元素能谱分析显示, PEG处理促进了菌丝表面P、Ca和Fe的积累.各种元素在菌丝表面的增多很可能是由于PEG诱导激活了质子泵, 而细胞对离子的吸收通常依赖于H⁺外流产生的电位梯度, 质膜H⁺-ATPase产生的质子动势为营养物质的吸收和转运提供了能量(Riquelme & Bartnicki- Garcia, 2004).干旱情况下AMF根外菌丝能够将更多的P和Ca等矿质养分从土壤中通过菌丝通道运输到植物中, 同时调控相关基因的表达以促进根系对水分的吸收, 使植物具有较强的耐旱性.Bárzana等(2012)的研究表明在充分供水和干旱胁迫下, AMF能够调控植物质外体和细胞间水分运输的途径.AMF这种调节植物水分吸收和运输的能力, 可以帮助植物更灵活地适应水分胁迫(Sánchez-Romera et al., 2016), 但AMF这一功能的生理和分子机制还需要系统深入的研究. ...

Mapping the growth of fungal hyphae: Orthogonal cell wall expansion during tip growth and the role of turgor
1
2000

... 细胞环境碱化.真菌胞内的碱化与胁迫信号的长距离传递有关, pH变化可能引起真菌-植物之间的信号传递发生改变(Harrison, 2005).离子动力学研究表明, 真菌和植物的信号交流与细胞跨膜H⁺离子梯度的调节有关, 该过程受到细胞膜上P型H⁺-ATPases的调控(Ramos et al., 2008a).为了进一步验证PEG模拟水分胁迫下菌丝H⁺的跨膜运输过程, 利用NMT技术验证了菌丝尖端和侧面两个部位H⁺的流动情况.对照处理下, 两个部位的H⁺都呈现微弱外流的趋势, 而且菌丝侧面的H⁺外流流速大于尖端.该结果与前人研究结果相似, Ramos等(2008b)证明从菌丝尖端到菌丝侧面不同位置上H⁺流动规律不同, 而且菌丝侧面的H⁺外流速率大于尖端.该现象可能和真菌的信号识别机制以及菌丝尖端生长有关(Bartnicki-Garcia et al., 2000).在PEG处理下, 菌丝H⁺显著外流.随着处理时间的延长, 菌丝尖端H⁺外流强度增大, 外流的H⁺使菌丝际环境酸化.该结论与液体培养基的pH值变化结果相符, 表明在模拟干旱处理1 h后, 菌丝对于外部环境的pH值进行了调节.菌丝尖端的H⁺外流调节了真菌对营养物质(如蔗糖、氨基酸和P)的吸收和交换(Requena et al., 2003).能谱分析显示, PEG处理增加了菌丝表面P信号强度, 说明在干旱处理下更多的P积累到菌丝界面.Hijikata等(2010)研究证实, 干旱情况下根外菌丝的磷转运蛋白活性提高, 将更多的P从土壤中通过菌丝吸收运输到植物中, 而AMF提高植物的营养元素吸收是提高植物耐旱性的重要机制(Zhao et al., 2015a). ...

Early events of vesicular?-arbuscular mycorrhiza formation on Ri T-DNA transformed roots
1
1988

... 参照St-Arnaud等(1996)的方法, 建立AMF-胡萝卜毛状根双重无菌培养体系.供试AMF菌株为R. irregularis DAOM 197198, 真菌孢子先经过吐温80和氯胺T进行表面消毒, 然后再用质量浓度1%的硫酸链霉素和0.5%的硫酸庆大霉素清洗, 最后在1.5%的水琼脂上面萌发.将转移Ri T-DNA并且能够连续继代培养的胡萝卜毛状根接种于固体M培养基(Bécard & Fortin, 1988, 液体M培养基组分参见表1.液体培养基加入1%的蔗糖和0.4%的植物凝胶即得到固体M培养基), 然后在根尖部位接入10-15个萌发的AMF孢子, 在25 ℃下黑暗培养.大概3个月后, 菌根生长铺满整个培养皿后转接. ...

ATP-dependent regulation of nuclear Ca ²⁺ levels in plant cells
1
2000

... Ca²⁺是生物细胞内重要的信号物质, 在植物抵抗干旱胁迫方面具有重要作用(Ma et al., 2005).胞质内游离Ca²⁺的分布以及跨膜转移是Ca²⁺信号的转移和传导的基本途径, 而Ca²⁺进入和流出细胞速率的变化是产生Ca²⁺振荡的基础(Bunney et al., 2000).质膜Ca²⁺-ATPases和H⁺-ATPase共同实现了质膜H⁺/Ca²⁺的逆向转运和逆境信号的传递(Rudd & Franklin-Tong, 2001).干旱胁迫可以导致细胞内游离Ca²⁺浓度产生短暂而快速的升高, 胞内Ca²⁺的波动对钙调蛋白、激酶和离子通道等下游调控元件产生影响(Campo et al., 2014).由钙信号传导的干旱胁迫信息会导致相关基因表达和代谢过程的改变(如渗透调节物质合成和营养元素运输的变化), 从而减轻干旱胁迫对生物机体的损害(Ludwig et al., 2004; Zou et al., 2015). ...

Overexpression of a calcium-dependent protein kinase confers salt and drought tolerance in rice by preventing membrane lipid peroxidation
1
2014

... Ca²⁺是生物细胞内重要的信号物质, 在植物抵抗干旱胁迫方面具有重要作用(Ma et al., 2005).胞质内游离Ca²⁺的分布以及跨膜转移是Ca²⁺信号的转移和传导的基本途径, 而Ca²⁺进入和流出细胞速率的变化是产生Ca²⁺振荡的基础(Bunney et al., 2000).质膜Ca²⁺-ATPases和H⁺-ATPase共同实现了质膜H⁺/Ca²⁺的逆向转运和逆境信号的传递(Rudd & Franklin-Tong, 2001).干旱胁迫可以导致细胞内游离Ca²⁺浓度产生短暂而快速的升高, 胞内Ca²⁺的波动对钙调蛋白、激酶和离子通道等下游调控元件产生影响(Campo et al., 2014).由钙信号传导的干旱胁迫信息会导致相关基因表达和代谢过程的改变(如渗透调节物质合成和营养元素运输的变化), 从而减轻干旱胁迫对生物机体的损害(Ludwig et al., 2004; Zou et al., 2015). ...

Insights on the impact of arbuscular mycorrhizal symbiosis on tomato tolerance to water stress
1
2016

... 丛枝菌根真菌(AMF)是一类专性共生真菌, 可以与大多数陆地植物形成共生体系, 并通过多种途径促进植物的生长发育, 增强植物对各种逆境胁迫的适应能力(Smith & Read, 2008).AMF根外菌丝在根系与土壤之间建立起物质传输通道, 将吸收的水分和营养物质快速输送给宿主植物(Chitarra et al., 2016).由于菌丝直径远小于植物根系, 因此根外菌丝能进入细小的土壤空隙获取养分和水分资源(Liu & Chen, 2007), 这对于植物适应养分缺乏和干旱胁迫具有重要意义.Rhizophagus irregularis (原名Glomus intraradices)是分布最广泛的一种AMF.Tisserant等(2013)通过全基因测序方法获得了R. irregularis DAOM 197198菌株的基因组数据, 为AMF真菌的生理和分子研究提供了重要基础, 也使得该菌株成为AMF生理研究的模式菌株.干旱情况下接种R. irregularis的加杨(Populus × canadensis)具有更高的水分利用效率和抗氧化酶活性, 此外AMF还上调根系水孔蛋白基因的表达提高植物抗旱性(Liu et al., 2016).Li等(2013)从R. intraradices中克隆了两个水通道蛋白基因GintAQPF1和GintAQPF2, 并通过异源表达验证了水孔蛋白的输水功能, 为干旱胁迫下AMF向宿主植物输送水分,从而增强植物抗旱性提供了直接的分子证据. ...

Multiple range and multiple
1
1955

... 培养基pH值、菌丝离子流、菌丝元素能谱分析结果等数据采用SPSS 18.0软件进行统计分析(SPSS, Chicago, USA).培养基pH值采用双因素方差分析检验PEG处理、处理时间及其交互作用的显著性.元素能谱分析、离子流结果采用三因素方差分析检验PEG处理、处理时间和不同菌丝部位及其交互作用的显著性.Duncan’s多重比较检验各处理之间的差异显著性(p < 0.05; Duncan, 1955).柱形图由Microsoft Excel 2010生成. ...

The plasma membrane H +-ATPase gene family in the arbuscular mycorrhizal fungus Glomus mosseae
1
2000

... 细胞膜质子泵, 即H⁺-ATPase的功能是水解ATP并将H⁺泵出细胞(Santi et al., 2003).H⁺通过细胞内外的电化学梯度进出细胞膜, 为包括阳离子、阴离子、氨基酸和糖在内的各种小分子物质的跨膜运输提供驱动力(Gaxiola et al., 2007).干旱胁迫、盐胁迫、缺磷等环境胁迫会导致质子泵发生改变(Yan et al., 2002; Gévaudant et al., 2007).细胞膜质子泵活性不仅反映物质跨膜运输的能力, 还在一定程度上反映生长环境对植物生理活性的影响.玉米(Zea mays)可以通过提高根系细胞膜质子泵的活性来适应低pH环境, 燕麦(Avena sativa)在感受到干旱胁迫的早期就可以激活根系H⁺-ATPase来增加渗透物质的合成(Gong et al., 2010).对AMF H⁺-ATPase的研究表明, 真菌质子泵在控制共生体营养元素和水分转运过程中起到重要作用(Ferrol et al., 2000).Gianinazzi- Pearson等(1991)通过测试ATPases水解作用过程中释放的磷酸对ATP酶进行细胞化学研究, 证明在AMF根内、根外菌丝的质膜上均存在H⁺-ATPase. ...

Electrical signals and their physiological significance in plants
1
2007

... 质膜H⁺外流不仅是营养物质吸收的动力, 还作为信号传导的中间体, 促进逆境信号的传导和响应元件的激活(Fromm & Lautner, 2007).H⁺的外流可以促进Ca²⁺、K⁺等阳离子的跨膜运输, 提高胞内的离子浓度(Isfort et al., 1993).本研究中, PEG处理1 h后, 菌丝尖端和表面的Ca²⁺表现了强烈的内流现象.当生物受到干旱胁迫的时候, 细胞内Ca²⁺瞬时产生快速的增高, 引发钙振荡(Zhao et al., 2015b).Ca²⁺作为第二信使, 在胁迫信号的传递过程中起到重要作用, 胞内游离Ca²⁺的分布与转移是Ca²⁺信号产生和传递的基础.使用LSCM观测菌丝细胞内Ca²⁺浓度的结果与离子流测定结果相符, PEG处理1 h后菌丝尖端与侧面均呈现强烈的绿色荧光, 说明菌丝内Ca²⁺浓度显著上升.干旱胁迫诱导的胞内Ca²⁺浓度增加能够激活下游的逆境信号响应元件, Ca依赖的蛋白激酶(CDPK)等, 而CDPK可以通过磷酸化质膜水通道蛋白促进细胞膜对水分的吸收和扩散(Azad et al., 2004).Li等(2013)证实, 干旱胁迫情况下AMF中两个水通道蛋白基因GintAQPF1和GintAQPF2的表达量会显著上调, 为植物输送水分以缓解干旱胁迫. ...

Plant proton pumps
1
2007

... 细胞膜质子泵, 即H⁺-ATPase的功能是水解ATP并将H⁺泵出细胞(Santi et al., 2003).H⁺通过细胞内外的电化学梯度进出细胞膜, 为包括阳离子、阴离子、氨基酸和糖在内的各种小分子物质的跨膜运输提供驱动力(Gaxiola et al., 2007).干旱胁迫、盐胁迫、缺磷等环境胁迫会导致质子泵发生改变(Yan et al., 2002; Gévaudant et al., 2007).细胞膜质子泵活性不仅反映物质跨膜运输的能力, 还在一定程度上反映生长环境对植物生理活性的影响.玉米(Zea mays)可以通过提高根系细胞膜质子泵的活性来适应低pH环境, 燕麦(Avena sativa)在感受到干旱胁迫的早期就可以激活根系H⁺-ATPase来增加渗透物质的合成(Gong et al., 2010).对AMF H⁺-ATPase的研究表明, 真菌质子泵在控制共生体营养元素和水分转运过程中起到重要作用(Ferrol et al., 2000).Gianinazzi- Pearson等(1991)通过测试ATPases水解作用过程中释放的磷酸对ATP酶进行细胞化学研究, 证明在AMF根内、根外菌丝的质膜上均存在H⁺-ATPase. ...

Expression of a constitutively activated plasma membrane H⁺-ATPase alters plant development and increases salt tolerance
1
2007

... 细胞膜质子泵, 即H⁺-ATPase的功能是水解ATP并将H⁺泵出细胞(Santi et al., 2003).H⁺通过细胞内外的电化学梯度进出细胞膜, 为包括阳离子、阴离子、氨基酸和糖在内的各种小分子物质的跨膜运输提供驱动力(Gaxiola et al., 2007).干旱胁迫、盐胁迫、缺磷等环境胁迫会导致质子泵发生改变(Yan et al., 2002; Gévaudant et al., 2007).细胞膜质子泵活性不仅反映物质跨膜运输的能力, 还在一定程度上反映生长环境对植物生理活性的影响.玉米(Zea mays)可以通过提高根系细胞膜质子泵的活性来适应低pH环境, 燕麦(Avena sativa)在感受到干旱胁迫的早期就可以激活根系H⁺-ATPase来增加渗透物质的合成(Gong et al., 2010).对AMF H⁺-ATPase的研究表明, 真菌质子泵在控制共生体营养元素和水分转运过程中起到重要作用(Ferrol et al., 2000).Gianinazzi- Pearson等(1991)通过测试ATPases水解作用过程中释放的磷酸对ATP酶进行细胞化学研究, 证明在AMF根内、根外菌丝的质膜上均存在H⁺-ATPase. ...

Enzymatic studies on the metabolism of vesicular-?arbuscular mycorrhizas
1
1991

... 细胞膜质子泵, 即H⁺-ATPase的功能是水解ATP并将H⁺泵出细胞(Santi et al., 2003).H⁺通过细胞内外的电化学梯度进出细胞膜, 为包括阳离子、阴离子、氨基酸和糖在内的各种小分子物质的跨膜运输提供驱动力(Gaxiola et al., 2007).干旱胁迫、盐胁迫、缺磷等环境胁迫会导致质子泵发生改变(Yan et al., 2002; Gévaudant et al., 2007).细胞膜质子泵活性不仅反映物质跨膜运输的能力, 还在一定程度上反映生长环境对植物生理活性的影响.玉米(Zea mays)可以通过提高根系细胞膜质子泵的活性来适应低pH环境, 燕麦(Avena sativa)在感受到干旱胁迫的早期就可以激活根系H⁺-ATPase来增加渗透物质的合成(Gong et al., 2010).对AMF H⁺-ATPase的研究表明, 真菌质子泵在控制共生体营养元素和水分转运过程中起到重要作用(Ferrol et al., 2000).Gianinazzi- Pearson等(1991)通过测试ATPases水解作用过程中释放的磷酸对ATP酶进行细胞化学研究, 证明在AMF根内、根外菌丝的质膜上均存在H⁺-ATPase. ...

Early activation of plasma membrane H +-ATPase and its relation to drought adaptation in two contrasting oat ( Avena sativa L.) genotypes
1
2010

... 细胞膜质子泵, 即H⁺-ATPase的功能是水解ATP并将H⁺泵出细胞(Santi et al., 2003).H⁺通过细胞内外的电化学梯度进出细胞膜, 为包括阳离子、阴离子、氨基酸和糖在内的各种小分子物质的跨膜运输提供驱动力(Gaxiola et al., 2007).干旱胁迫、盐胁迫、缺磷等环境胁迫会导致质子泵发生改变(Yan et al., 2002; Gévaudant et al., 2007).细胞膜质子泵活性不仅反映物质跨膜运输的能力, 还在一定程度上反映生长环境对植物生理活性的影响.玉米(Zea mays)可以通过提高根系细胞膜质子泵的活性来适应低pH环境, 燕麦(Avena sativa)在感受到干旱胁迫的早期就可以激活根系H⁺-ATPase来增加渗透物质的合成(Gong et al., 2010).对AMF H⁺-ATPase的研究表明, 真菌质子泵在控制共生体营养元素和水分转运过程中起到重要作用(Ferrol et al., 2000).Gianinazzi- Pearson等(1991)通过测试ATPases水解作用过程中释放的磷酸对ATP酶进行细胞化学研究, 证明在AMF根内、根外菌丝的质膜上均存在H⁺-ATPase. ...

Signaling in the arbuscular mycorrhizal symbiosis
1
2005

... 细胞环境碱化.真菌胞内的碱化与胁迫信号的长距离传递有关, pH变化可能引起真菌-植物之间的信号传递发生改变(Harrison, 2005).离子动力学研究表明, 真菌和植物的信号交流与细胞跨膜H⁺离子梯度的调节有关, 该过程受到细胞膜上P型H⁺-ATPases的调控(Ramos et al., 2008a).为了进一步验证PEG模拟水分胁迫下菌丝H⁺的跨膜运输过程, 利用NMT技术验证了菌丝尖端和侧面两个部位H⁺的流动情况.对照处理下, 两个部位的H⁺都呈现微弱外流的趋势, 而且菌丝侧面的H⁺外流流速大于尖端.该结果与前人研究结果相似, Ramos等(2008b)证明从菌丝尖端到菌丝侧面不同位置上H⁺流动规律不同, 而且菌丝侧面的H⁺外流速率大于尖端.该现象可能和真菌的信号识别机制以及菌丝尖端生长有关(Bartnicki-Garcia et al., 2000).在PEG处理下, 菌丝H⁺显著外流.随着处理时间的延长, 菌丝尖端H⁺外流强度增大, 外流的H⁺使菌丝际环境酸化.该结论与液体培养基的pH值变化结果相符, 表明在模拟干旱处理1 h后, 菌丝对于外部环境的pH值进行了调节.菌丝尖端的H⁺外流调节了真菌对营养物质(如蔗糖、氨基酸和P)的吸收和交换(Requena et al., 2003).能谱分析显示, PEG处理增加了菌丝表面P信号强度, 说明在干旱处理下更多的P积累到菌丝界面.Hijikata等(2010)研究证实, 干旱情况下根外菌丝的磷转运蛋白活性提高, 将更多的P从土壤中通过菌丝吸收运输到植物中, 而AMF提高植物的营养元素吸收是提高植物耐旱性的重要机制(Zhao et al., 2015a). ...

Polyphosphate has a central role in the rapid and massive accumulation of phosphorus in extraradical mycelium of an arbuscular mycorrhizal fungus
1
2010

... 细胞环境碱化.真菌胞内的碱化与胁迫信号的长距离传递有关, pH变化可能引起真菌-植物之间的信号传递发生改变(Harrison, 2005).离子动力学研究表明, 真菌和植物的信号交流与细胞跨膜H⁺离子梯度的调节有关, 该过程受到细胞膜上P型H⁺-ATPases的调控(Ramos et al., 2008a).为了进一步验证PEG模拟水分胁迫下菌丝H⁺的跨膜运输过程, 利用NMT技术验证了菌丝尖端和侧面两个部位H⁺的流动情况.对照处理下, 两个部位的H⁺都呈现微弱外流的趋势, 而且菌丝侧面的H⁺外流流速大于尖端.该结果与前人研究结果相似, Ramos等(2008b)证明从菌丝尖端到菌丝侧面不同位置上H⁺流动规律不同, 而且菌丝侧面的H⁺外流速率大于尖端.该现象可能和真菌的信号识别机制以及菌丝尖端生长有关(Bartnicki-Garcia et al., 2000).在PEG处理下, 菌丝H⁺显著外流.随着处理时间的延长, 菌丝尖端H⁺外流强度增大, 外流的H⁺使菌丝际环境酸化.该结论与液体培养基的pH值变化结果相符, 表明在模拟干旱处理1 h后, 菌丝对于外部环境的pH值进行了调节.菌丝尖端的H⁺外流调节了真菌对营养物质(如蔗糖、氨基酸和P)的吸收和交换(Requena et al., 2003).能谱分析显示, PEG处理增加了菌丝表面P信号强度, 说明在干旱处理下更多的P积累到菌丝界面.Hijikata等(2010)研究证实, 干旱情况下根外菌丝的磷转运蛋白活性提高, 将更多的P从土壤中通过菌丝吸收运输到植物中, 而AMF提高植物的营养元素吸收是提高植物耐旱性的重要机制(Zhao et al., 2015a). ...

Induction of protein phosphorylation, protein synthesis, immediate-early-gene expression and cellular proliferation by intracellular pH modulation
1
1993

... 质膜H⁺外流不仅是营养物质吸收的动力, 还作为信号传导的中间体, 促进逆境信号的传导和响应元件的激活(Fromm & Lautner, 2007).H⁺的外流可以促进Ca²⁺、K⁺等阳离子的跨膜运输, 提高胞内的离子浓度(Isfort et al., 1993).本研究中, PEG处理1 h后, 菌丝尖端和表面的Ca²⁺表现了强烈的内流现象.当生物受到干旱胁迫的时候, 细胞内Ca²⁺瞬时产生快速的增高, 引发钙振荡(Zhao et al., 2015b).Ca²⁺作为第二信使, 在胁迫信号的传递过程中起到重要作用, 胞内游离Ca²⁺的分布与转移是Ca²⁺信号产生和传递的基础.使用LSCM观测菌丝细胞内Ca²⁺浓度的结果与离子流测定结果相符, PEG处理1 h后菌丝尖端与侧面均呈现强烈的绿色荧光, 说明菌丝内Ca²⁺浓度显著上升.干旱胁迫诱导的胞内Ca²⁺浓度增加能够激活下游的逆境信号响应元件, Ca依赖的蛋白激酶(CDPK)等, 而CDPK可以通过磷酸化质膜水通道蛋白促进细胞膜对水分的吸收和扩散(Azad et al., 2004).Li等(2013)证实, 干旱胁迫情况下AMF中两个水通道蛋白基因GintAQPF1和GintAQPF2的表达量会显著上调, 为植物输送水分以缓解干旱胁迫. ...

Detection of extracellular calcium gradients with a calcium-specific vibrating electrode
1
1990

... 细胞膜是生物遭受逆境胁迫以后受损的初始位点, 其结构及功能的改变势必会影响生物体内水分代谢以及各种无机离子的运输, 而无机离子的跨膜运输对维持植物正常生长发育起着至关重要的作用(Yoshida, 1991).干旱胁迫下细胞会通过特定的调节机制, 改变离子和小分子的跨膜运输来调节离子平衡, 从而维持细胞的渗透压, 以抵御干旱胁迫.因此, 研究关键离子的跨膜转运过程对揭示植物在干旱胁迫下通过自我调节以维持生命活动的机制具有重要意义.Kühtreiber和Jaffe (1990)首次利用非损伤微测技术(non-invasive micro-test technology, NMT)在活体原位条件下测量了Ca²⁺进出细胞的流速和方向, 该方法被广泛应用于生物体逆境应答机制研究.目前, NMT技术可以实时记录活体植物细胞各种离子的跨膜运输, 为研究植物应答环境变化提供了重要技术平台.应用这种技术, Mak等(2014)发现在不同时间长度的干旱胁迫下大豆(Glycine max)叶肉细胞K⁺、H⁺和Ca²⁺转运情况出现显著变化, 而各种离子的转运能力可作为重要的生理指标来衡量大豆的耐旱性. ...

丛枝菌根提高宿主植物抗旱性分子机制研究进展
1
2012

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

丛枝菌根提高宿主植物抗旱性分子机制研究进展
1
2012

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus
2
2013

... 丛枝菌根真菌(AMF)是一类专性共生真菌, 可以与大多数陆地植物形成共生体系, 并通过多种途径促进植物的生长发育, 增强植物对各种逆境胁迫的适应能力(Smith & Read, 2008).AMF根外菌丝在根系与土壤之间建立起物质传输通道, 将吸收的水分和营养物质快速输送给宿主植物(Chitarra et al., 2016).由于菌丝直径远小于植物根系, 因此根外菌丝能进入细小的土壤空隙获取养分和水分资源(Liu & Chen, 2007), 这对于植物适应养分缺乏和干旱胁迫具有重要意义.Rhizophagus irregularis (原名Glomus intraradices)是分布最广泛的一种AMF.Tisserant等(2013)通过全基因测序方法获得了R. irregularis DAOM 197198菌株的基因组数据, 为AMF真菌的生理和分子研究提供了重要基础, 也使得该菌株成为AMF生理研究的模式菌株.干旱情况下接种R. irregularis的加杨(Populus × canadensis)具有更高的水分利用效率和抗氧化酶活性, 此外AMF还上调根系水孔蛋白基因的表达提高植物抗旱性(Liu et al., 2016).Li等(2013)从R. intraradices中克隆了两个水通道蛋白基因GintAQPF1和GintAQPF2, 并通过异源表达验证了水孔蛋白的输水功能, 为干旱胁迫下AMF向宿主植物输送水分,从而增强植物抗旱性提供了直接的分子证据. ...

... 质膜H⁺外流不仅是营养物质吸收的动力, 还作为信号传导的中间体, 促进逆境信号的传导和响应元件的激活(Fromm & Lautner, 2007).H⁺的外流可以促进Ca²⁺、K⁺等阳离子的跨膜运输, 提高胞内的离子浓度(Isfort et al., 1993).本研究中, PEG处理1 h后, 菌丝尖端和表面的Ca²⁺表现了强烈的内流现象.当生物受到干旱胁迫的时候, 细胞内Ca²⁺瞬时产生快速的增高, 引发钙振荡(Zhao et al., 2015b).Ca²⁺作为第二信使, 在胁迫信号的传递过程中起到重要作用, 胞内游离Ca²⁺的分布与转移是Ca²⁺信号产生和传递的基础.使用LSCM观测菌丝细胞内Ca²⁺浓度的结果与离子流测定结果相符, PEG处理1 h后菌丝尖端与侧面均呈现强烈的绿色荧光, 说明菌丝内Ca²⁺浓度显著上升.干旱胁迫诱导的胞内Ca²⁺浓度增加能够激活下游的逆境信号响应元件, Ca依赖的蛋白激酶(CDPK)等, 而CDPK可以通过磷酸化质膜水通道蛋白促进细胞膜对水分的吸收和扩散(Azad et al., 2004).Li等(2013)证实, 干旱胁迫情况下AMF中两个水通道蛋白基因GintAQPF1和GintAQPF2的表达量会显著上调, 为植物输送水分以缓解干旱胁迫. ...

Effect of Rhizophagus irregularis on osmotic adjustment, antioxidation and aquaporin PIP genes expression of Populus × canadensis ‘Neva’ under drought stress
1
2016

... 丛枝菌根真菌(AMF)是一类专性共生真菌, 可以与大多数陆地植物形成共生体系, 并通过多种途径促进植物的生长发育, 增强植物对各种逆境胁迫的适应能力(Smith & Read, 2008).AMF根外菌丝在根系与土壤之间建立起物质传输通道, 将吸收的水分和营养物质快速输送给宿主植物(Chitarra et al., 2016).由于菌丝直径远小于植物根系, 因此根外菌丝能进入细小的土壤空隙获取养分和水分资源(Liu & Chen, 2007), 这对于植物适应养分缺乏和干旱胁迫具有重要意义.Rhizophagus irregularis (原名Glomus intraradices)是分布最广泛的一种AMF.Tisserant等(2013)通过全基因测序方法获得了R. irregularis DAOM 197198菌株的基因组数据, 为AMF真菌的生理和分子研究提供了重要基础, 也使得该菌株成为AMF生理研究的模式菌株.干旱情况下接种R. irregularis的加杨(Populus × canadensis)具有更高的水分利用效率和抗氧化酶活性, 此外AMF还上调根系水孔蛋白基因的表达提高植物抗旱性(Liu et al., 2016).Li等(2013)从R. intraradices中克隆了两个水通道蛋白基因GintAQPF1和GintAQPF2, 并通过异源表达验证了水孔蛋白的输水功能, 为干旱胁迫下AMF向宿主植物输送水分,从而增强植物抗旱性提供了直接的分子证据. ...

1
2007

... 丛枝菌根真菌(AMF)是一类专性共生真菌, 可以与大多数陆地植物形成共生体系, 并通过多种途径促进植物的生长发育, 增强植物对各种逆境胁迫的适应能力(Smith & Read, 2008).AMF根外菌丝在根系与土壤之间建立起物质传输通道, 将吸收的水分和营养物质快速输送给宿主植物(Chitarra et al., 2016).由于菌丝直径远小于植物根系, 因此根外菌丝能进入细小的土壤空隙获取养分和水分资源(Liu & Chen, 2007), 这对于植物适应养分缺乏和干旱胁迫具有重要意义.Rhizophagus irregularis (原名Glomus intraradices)是分布最广泛的一种AMF.Tisserant等(2013)通过全基因测序方法获得了R. irregularis DAOM 197198菌株的基因组数据, 为AMF真菌的生理和分子研究提供了重要基础, 也使得该菌株成为AMF生理研究的模式菌株.干旱情况下接种R. irregularis的加杨(Populus × canadensis)具有更高的水分利用效率和抗氧化酶活性, 此外AMF还上调根系水孔蛋白基因的表达提高植物抗旱性(Liu et al., 2016).Li等(2013)从R. intraradices中克隆了两个水通道蛋白基因GintAQPF1和GintAQPF2, 并通过异源表达验证了水孔蛋白的输水功能, 为干旱胁迫下AMF向宿主植物输送水分,从而增强植物抗旱性提供了直接的分子证据. ...

CDPK-mediated signalling pathways: Specificity and cross-talk
1
2004

... Ca²⁺是生物细胞内重要的信号物质, 在植物抵抗干旱胁迫方面具有重要作用(Ma et al., 2005).胞质内游离Ca²⁺的分布以及跨膜转移是Ca²⁺信号的转移和传导的基本途径, 而Ca²⁺进入和流出细胞速率的变化是产生Ca²⁺振荡的基础(Bunney et al., 2000).质膜Ca²⁺-ATPases和H⁺-ATPase共同实现了质膜H⁺/Ca²⁺的逆向转运和逆境信号的传递(Rudd & Franklin-Tong, 2001).干旱胁迫可以导致细胞内游离Ca²⁺浓度产生短暂而快速的升高, 胞内Ca²⁺的波动对钙调蛋白、激酶和离子通道等下游调控元件产生影响(Campo et al., 2014).由钙信号传导的干旱胁迫信息会导致相关基因表达和代谢过程的改变(如渗透调节物质合成和营养元素运输的变化), 从而减轻干旱胁迫对生物机体的损害(Ludwig et al., 2004; Zou et al., 2015). ...

The effects of exogenous Ca ²⁺ on endogenous polyamine levels and drought-resistant traits of spring wheat grown under arid conditions
1
2005

... Ca²⁺是生物细胞内重要的信号物质, 在植物抵抗干旱胁迫方面具有重要作用(Ma et al., 2005).胞质内游离Ca²⁺的分布以及跨膜转移是Ca²⁺信号的转移和传导的基本途径, 而Ca²⁺进入和流出细胞速率的变化是产生Ca²⁺振荡的基础(Bunney et al., 2000).质膜Ca²⁺-ATPases和H⁺-ATPase共同实现了质膜H⁺/Ca²⁺的逆向转运和逆境信号的传递(Rudd & Franklin-Tong, 2001).干旱胁迫可以导致细胞内游离Ca²⁺浓度产生短暂而快速的升高, 胞内Ca²⁺的波动对钙调蛋白、激酶和离子通道等下游调控元件产生影响(Campo et al., 2014).由钙信号传导的干旱胁迫信息会导致相关基因表达和代谢过程的改变(如渗透调节物质合成和营养元素运输的变化), 从而减轻干旱胁迫对生物机体的损害(Ludwig et al., 2004; Zou et al., 2015). ...

Leaf mesophyll K⁺, H⁺and Ca²⁺ fluxes are involved in drought-induced decrease in photosynthesis and stomatal closure in soybean
1
2014

... 细胞膜是生物遭受逆境胁迫以后受损的初始位点, 其结构及功能的改变势必会影响生物体内水分代谢以及各种无机离子的运输, 而无机离子的跨膜运输对维持植物正常生长发育起着至关重要的作用(Yoshida, 1991).干旱胁迫下细胞会通过特定的调节机制, 改变离子和小分子的跨膜运输来调节离子平衡, 从而维持细胞的渗透压, 以抵御干旱胁迫.因此, 研究关键离子的跨膜转运过程对揭示植物在干旱胁迫下通过自我调节以维持生命活动的机制具有重要意义.Kühtreiber和Jaffe (1990)首次利用非损伤微测技术(non-invasive micro-test technology, NMT)在活体原位条件下测量了Ca²⁺进出细胞的流速和方向, 该方法被广泛应用于生物体逆境应答机制研究.目前, NMT技术可以实时记录活体植物细胞各种离子的跨膜运输, 为研究植物应答环境变化提供了重要技术平台.应用这种技术, Mak等(2014)发现在不同时间长度的干旱胁迫下大豆(Glycine max)叶肉细胞K⁺、H⁺和Ca²⁺转运情况出现显著变化, 而各种离子的转运能力可作为重要的生理指标来衡量大豆的耐旱性. ...

Arbuscular mycorrhizal influence on leaf water potential, solute accumulation, and oxidative stress in soybean plants subjected to drought stress
1
2004

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

Direct nocturnal water transfer from oaks to their mycorrhizal symbionts during severe soil drying
1
2003

... 干旱条件下水分由根外菌丝向根内菌丝运输, 微量元素、氨基酸和其他低分子量的代谢产物也被AMF菌丝选择吸收并运送到植物中, 由此增强植物的耐旱性(Querejeta et al., 2003; van Hees et al., 2006).菌丝中元素能谱分析显示, PEG处理促进了菌丝表面P、Ca和Fe的积累.各种元素在菌丝表面的增多很可能是由于PEG诱导激活了质子泵, 而细胞对离子的吸收通常依赖于H⁺外流产生的电位梯度, 质膜H⁺-ATPase产生的质子动势为营养物质的吸收和转运提供了能量(Riquelme & Bartnicki- Garcia, 2004).干旱情况下AMF根外菌丝能够将更多的P和Ca等矿质养分从土壤中通过菌丝通道运输到植物中, 同时调控相关基因的表达以促进根系对水分的吸收, 使植物具有较强的耐旱性.Bárzana等(2012)的研究表明在充分供水和干旱胁迫下, AMF能够调控植物质外体和细胞间水分运输的途径.AMF这种调节植物水分吸收和运输的能力, 可以帮助植物更灵活地适应水分胁迫(Sánchez-Romera et al., 2016), 但AMF这一功能的生理和分子机制还需要系统深入的研究. ...

Ion dynamics during the polarized growth of arbuscular mycorrhizal fungi: From presymbiosis to symbiosis
1
2008a

... 细胞环境碱化.真菌胞内的碱化与胁迫信号的长距离传递有关, pH变化可能引起真菌-植物之间的信号传递发生改变(Harrison, 2005).离子动力学研究表明, 真菌和植物的信号交流与细胞跨膜H⁺离子梯度的调节有关, 该过程受到细胞膜上P型H⁺-ATPases的调控(Ramos et al., 2008a).为了进一步验证PEG模拟水分胁迫下菌丝H⁺的跨膜运输过程, 利用NMT技术验证了菌丝尖端和侧面两个部位H⁺的流动情况.对照处理下, 两个部位的H⁺都呈现微弱外流的趋势, 而且菌丝侧面的H⁺外流流速大于尖端.该结果与前人研究结果相似, Ramos等(2008b)证明从菌丝尖端到菌丝侧面不同位置上H⁺流动规律不同, 而且菌丝侧面的H⁺外流速率大于尖端.该现象可能和真菌的信号识别机制以及菌丝尖端生长有关(Bartnicki-Garcia et al., 2000).在PEG处理下, 菌丝H⁺显著外流.随着处理时间的延长, 菌丝尖端H⁺外流强度增大, 外流的H⁺使菌丝际环境酸化.该结论与液体培养基的pH值变化结果相符, 表明在模拟干旱处理1 h后, 菌丝对于外部环境的pH值进行了调节.菌丝尖端的H⁺外流调节了真菌对营养物质(如蔗糖、氨基酸和P)的吸收和交换(Requena et al., 2003).能谱分析显示, PEG处理增加了菌丝表面P信号强度, 说明在干旱处理下更多的P积累到菌丝界面.Hijikata等(2010)研究证实, 干旱情况下根外菌丝的磷转运蛋白活性提高, 将更多的P从土壤中通过菌丝吸收运输到植物中, 而AMF提高植物的营养元素吸收是提高植物耐旱性的重要机制(Zhao et al., 2015a). ...

Proton (H⁺) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi
1
2008b

... 细胞环境碱化.真菌胞内的碱化与胁迫信号的长距离传递有关, pH变化可能引起真菌-植物之间的信号传递发生改变(Harrison, 2005).离子动力学研究表明, 真菌和植物的信号交流与细胞跨膜H⁺离子梯度的调节有关, 该过程受到细胞膜上P型H⁺-ATPases的调控(Ramos et al., 2008a).为了进一步验证PEG模拟水分胁迫下菌丝H⁺的跨膜运输过程, 利用NMT技术验证了菌丝尖端和侧面两个部位H⁺的流动情况.对照处理下, 两个部位的H⁺都呈现微弱外流的趋势, 而且菌丝侧面的H⁺外流流速大于尖端.该结果与前人研究结果相似, Ramos等(2008b)证明从菌丝尖端到菌丝侧面不同位置上H⁺流动规律不同, 而且菌丝侧面的H⁺外流速率大于尖端.该现象可能和真菌的信号识别机制以及菌丝尖端生长有关(Bartnicki-Garcia et al., 2000).在PEG处理下, 菌丝H⁺显著外流.随着处理时间的延长, 菌丝尖端H⁺外流强度增大, 外流的H⁺使菌丝际环境酸化.该结论与液体培养基的pH值变化结果相符, 表明在模拟干旱处理1 h后, 菌丝对于外部环境的pH值进行了调节.菌丝尖端的H⁺外流调节了真菌对营养物质(如蔗糖、氨基酸和P)的吸收和交换(Requena et al., 2003).能谱分析显示, PEG处理增加了菌丝表面P信号强度, 说明在干旱处理下更多的P积累到菌丝界面.Hijikata等(2010)研究证实, 干旱情况下根外菌丝的磷转运蛋白活性提高, 将更多的P从土壤中通过菌丝吸收运输到植物中, 而AMF提高植物的营养元素吸收是提高植物耐旱性的重要机制(Zhao et al., 2015a). ...

Symbiotic status, phosphate, and sucrose regulate the expression of two plasma membrane H⁺-ATPase genes from the mycorrhizal fungus
1
2003

... 细胞环境碱化.真菌胞内的碱化与胁迫信号的长距离传递有关, pH变化可能引起真菌-植物之间的信号传递发生改变(Harrison, 2005).离子动力学研究表明, 真菌和植物的信号交流与细胞跨膜H⁺离子梯度的调节有关, 该过程受到细胞膜上P型H⁺-ATPases的调控(Ramos et al., 2008a).为了进一步验证PEG模拟水分胁迫下菌丝H⁺的跨膜运输过程, 利用NMT技术验证了菌丝尖端和侧面两个部位H⁺的流动情况.对照处理下, 两个部位的H⁺都呈现微弱外流的趋势, 而且菌丝侧面的H⁺外流流速大于尖端.该结果与前人研究结果相似, Ramos等(2008b)证明从菌丝尖端到菌丝侧面不同位置上H⁺流动规律不同, 而且菌丝侧面的H⁺外流速率大于尖端.该现象可能和真菌的信号识别机制以及菌丝尖端生长有关(Bartnicki-Garcia et al., 2000).在PEG处理下, 菌丝H⁺显著外流.随着处理时间的延长, 菌丝尖端H⁺外流强度增大, 外流的H⁺使菌丝际环境酸化.该结论与液体培养基的pH值变化结果相符, 表明在模拟干旱处理1 h后, 菌丝对于外部环境的pH值进行了调节.菌丝尖端的H⁺外流调节了真菌对营养物质(如蔗糖、氨基酸和P)的吸收和交换(Requena et al., 2003).能谱分析显示, PEG处理增加了菌丝表面P信号强度, 说明在干旱处理下更多的P积累到菌丝界面.Hijikata等(2010)研究证实, 干旱情况下根外菌丝的磷转运蛋白活性提高, 将更多的P从土壤中通过菌丝吸收运输到植物中, 而AMF提高植物的营养元素吸收是提高植物耐旱性的重要机制(Zhao et al., 2015a). ...

Key differences between lateral and apical branching in hyphae of
1
2004

... 干旱条件下水分由根外菌丝向根内菌丝运输, 微量元素、氨基酸和其他低分子量的代谢产物也被AMF菌丝选择吸收并运送到植物中, 由此增强植物的耐旱性(Querejeta et al., 2003; van Hees et al., 2006).菌丝中元素能谱分析显示, PEG处理促进了菌丝表面P、Ca和Fe的积累.各种元素在菌丝表面的增多很可能是由于PEG诱导激活了质子泵, 而细胞对离子的吸收通常依赖于H⁺外流产生的电位梯度, 质膜H⁺-ATPase产生的质子动势为营养物质的吸收和转运提供了能量(Riquelme & Bartnicki- Garcia, 2004).干旱情况下AMF根外菌丝能够将更多的P和Ca等矿质养分从土壤中通过菌丝通道运输到植物中, 同时调控相关基因的表达以促进根系对水分的吸收, 使植物具有较强的耐旱性.Bárzana等(2012)的研究表明在充分供水和干旱胁迫下, AMF能够调控植物质外体和细胞间水分运输的途径.AMF这种调节植物水分吸收和运输的能力, 可以帮助植物更灵活地适应水分胁迫(Sánchez-Romera et al., 2016), 但AMF这一功能的生理和分子机制还需要系统深入的研究. ...

Unravelling response-?specificity in Ca ²⁺ signalling pathways in plant cells
1
2001

... Ca²⁺是生物细胞内重要的信号物质, 在植物抵抗干旱胁迫方面具有重要作用(Ma et al., 2005).胞质内游离Ca²⁺的分布以及跨膜转移是Ca²⁺信号的转移和传导的基本途径, 而Ca²⁺进入和流出细胞速率的变化是产生Ca²⁺振荡的基础(Bunney et al., 2000).质膜Ca²⁺-ATPases和H⁺-ATPase共同实现了质膜H⁺/Ca²⁺的逆向转运和逆境信号的传递(Rudd & Franklin-Tong, 2001).干旱胁迫可以导致细胞内游离Ca²⁺浓度产生短暂而快速的升高, 胞内Ca²⁺的波动对钙调蛋白、激酶和离子通道等下游调控元件产生影响(Campo et al., 2014).由钙信号传导的干旱胁迫信息会导致相关基因表达和代谢过程的改变(如渗透调节物质合成和营养元素运输的变化), 从而减轻干旱胁迫对生物机体的损害(Ludwig et al., 2004; Zou et al., 2015). ...

Arbuscular mycorrhizal symbiosis and methyl jasmonate avoid the inhibition of root hydraulic conductivity caused by drought
1
2016

... 干旱条件下水分由根外菌丝向根内菌丝运输, 微量元素、氨基酸和其他低分子量的代谢产物也被AMF菌丝选择吸收并运送到植物中, 由此增强植物的耐旱性(Querejeta et al., 2003; van Hees et al., 2006).菌丝中元素能谱分析显示, PEG处理促进了菌丝表面P、Ca和Fe的积累.各种元素在菌丝表面的增多很可能是由于PEG诱导激活了质子泵, 而细胞对离子的吸收通常依赖于H⁺外流产生的电位梯度, 质膜H⁺-ATPase产生的质子动势为营养物质的吸收和转运提供了能量(Riquelme & Bartnicki- Garcia, 2004).干旱情况下AMF根外菌丝能够将更多的P和Ca等矿质养分从土壤中通过菌丝通道运输到植物中, 同时调控相关基因的表达以促进根系对水分的吸收, 使植物具有较强的耐旱性.Bárzana等(2012)的研究表明在充分供水和干旱胁迫下, AMF能够调控植物质外体和细胞间水分运输的途径.AMF这种调节植物水分吸收和运输的能力, 可以帮助植物更灵活地适应水分胁迫(Sánchez-Romera et al., 2016), 但AMF这一功能的生理和分子机制还需要系统深入的研究. ...

Induction of nitrate uptake in maize roots: Expression of a putative high-affinity nitrate transporter and plasma membrane H⁺- ATPase isoforms
1
2003

... 细胞膜质子泵, 即H⁺-ATPase的功能是水解ATP并将H⁺泵出细胞(Santi et al., 2003).H⁺通过细胞内外的电化学梯度进出细胞膜, 为包括阳离子、阴离子、氨基酸和糖在内的各种小分子物质的跨膜运输提供驱动力(Gaxiola et al., 2007).干旱胁迫、盐胁迫、缺磷等环境胁迫会导致质子泵发生改变(Yan et al., 2002; Gévaudant et al., 2007).细胞膜质子泵活性不仅反映物质跨膜运输的能力, 还在一定程度上反映生长环境对植物生理活性的影响.玉米(Zea mays)可以通过提高根系细胞膜质子泵的活性来适应低pH环境, 燕麦(Avena sativa)在感受到干旱胁迫的早期就可以激活根系H⁺-ATPase来增加渗透物质的合成(Gong et al., 2010).对AMF H⁺-ATPase的研究表明, 真菌质子泵在控制共生体营养元素和水分转运过程中起到重要作用(Ferrol et al., 2000).Gianinazzi- Pearson等(1991)通过测试ATPases水解作用过程中释放的磷酸对ATP酶进行细胞化学研究, 证明在AMF根内、根外菌丝的质膜上均存在H⁺-ATPase. ...

Silicon enhances water stress tolerance by improving root hydraulic conductance in
1
2016

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

Mycorrhizal Symbiosis
1
2008

... 丛枝菌根真菌(AMF)是一类专性共生真菌, 可以与大多数陆地植物形成共生体系, 并通过多种途径促进植物的生长发育, 增强植物对各种逆境胁迫的适应能力(Smith & Read, 2008).AMF根外菌丝在根系与土壤之间建立起物质传输通道, 将吸收的水分和营养物质快速输送给宿主植物(Chitarra et al., 2016).由于菌丝直径远小于植物根系, 因此根外菌丝能进入细小的土壤空隙获取养分和水分资源(Liu & Chen, 2007), 这对于植物适应养分缺乏和干旱胁迫具有重要意义.Rhizophagus irregularis (原名Glomus intraradices)是分布最广泛的一种AMF.Tisserant等(2013)通过全基因测序方法获得了R. irregularis DAOM 197198菌株的基因组数据, 为AMF真菌的生理和分子研究提供了重要基础, 也使得该菌株成为AMF生理研究的模式菌株.干旱情况下接种R. irregularis的加杨(Populus × canadensis)具有更高的水分利用效率和抗氧化酶活性, 此外AMF还上调根系水孔蛋白基因的表达提高植物抗旱性(Liu et al., 2016).Li等(2013)从R. intraradices中克隆了两个水通道蛋白基因GintAQPF1和GintAQPF2, 并通过异源表达验证了水孔蛋白的输水功能, 为干旱胁迫下AMF向宿主植物输送水分,从而增强植物抗旱性提供了直接的分子证据. ...

Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus
3
1996

... 参照St-Arnaud等(1996)的方法, 建立AMF-胡萝卜毛状根双重无菌培养体系.供试AMF菌株为R. irregularis DAOM 197198, 真菌孢子先经过吐温80和氯胺T进行表面消毒, 然后再用质量浓度1%的硫酸链霉素和0.5%的硫酸庆大霉素清洗, 最后在1.5%的水琼脂上面萌发.将转移Ri T-DNA并且能够连续继代培养的胡萝卜毛状根接种于固体M培养基(Bécard & Fortin, 1988, 液体M培养基组分参见表1.液体培养基加入1%的蔗糖和0.4%的植物凝胶即得到固体M培养基), 然后在根尖部位接入10-15个萌发的AMF孢子, 在25 ℃下黑暗培养.大概3个月后, 菌根生长铺满整个培养皿后转接. ...

... 试验在分室培养系统(分为菌根室和菌丝室)中进行(图1).先在菌根室加入30 mL固体M培养基, 然后在其中接入已被AMF侵染的胡萝卜毛状根, 在25 ℃下黑暗倒置培养8周, 待根系铺满菌根室后正置培养, 并在菌丝室加入15 mL液体M培养基.继续培养4-6周直到菌丝长满菌丝室, 挑选菌丝量大致相同的培养皿进行后续试验.
图1 丛枝菌根真菌-胡萝卜毛状根分室培养系统示意图.左边是菌根室(mycorrhizal compartment), 右边是菌丝室(hyphal compartment).菌根室中加入用质量浓度0.4%的植物凝胶固化后的M培养基, 并接入菌根化的转移Ri T-DNA胡萝卜毛状根; 菌丝室加入液体M培养基, 以供根外菌丝生长(<xref ref-type="bibr" rid="b40">参考St-Arnaud et al., 1996</xref>). Diagram of the two-compartments in vitro culture system of arbuscular mycorrhizal fungi with hairy carrot root. Mycorrhizal compartment was filled with solid M medium gelled with 0.4% phytagel, allowing development of mycorrhizal roots; extraradical mycelium ramified into hyphal compartment filled with liquid M medium without sucrose and phytagel, and the roots that crossed the central wall were trimmed to prevent their growth in hyphal compartment (referred to <xref ref-type="bibr" rid="b40">St-Arnaud et al., 1996</xref>).

Fig. 1

菌丝室中干旱处理的培养基由液体M培养基加入15%的PEG制成, 对照为正常液体M培养基.处理前调节培养基pH值至6.0, 每个菌丝室加入15 mL处理液, 分别处理1 h和24 h, 总共有4组样品: 对照1 h (CK1h)、PEG处理1 h (PEG1h)、对照24 h (CK24h)和PEG处理24 h (PEG24h).每组处理有12个培养皿(试验重复), 其中6个培养皿收获菌丝作为扫描电子显微镜和荧光光谱分析的样品, 同时收集液体培养基测定pH值, 另外6个培养皿用于离子流分析. ...

... culture system of arbuscular mycorrhizal fungi with hairy carrot root. Mycorrhizal compartment was filled with solid M medium gelled with 0.4% phytagel, allowing development of mycorrhizal roots; extraradical mycelium ramified into hyphal compartment filled with liquid M medium without sucrose and phytagel, and the roots that crossed the central wall were trimmed to prevent their growth in hyphal compartment (referred to St-Arnaud et al., 1996). Fig. 1 菌丝室中干旱处理的培养基由液体M培养基加入15%的PEG制成, 对照为正常液体M培养基.处理前调节培养基pH值至6.0, 每个菌丝室加入15 mL处理液, 分别处理1 h和24 h, 总共有4组样品: 对照1 h (CK1h)、PEG处理1 h (PEG1h)、对照24 h (CK24h)和PEG处理24 h (PEG24h).每组处理有12个培养皿(试验重复), 其中6个培养皿收获菌丝作为扫描电子显微镜和荧光光谱分析的样品, 同时收集液体培养基测定pH值, 另外6个培养皿用于离子流分析. ...

Exudation-reabsorption in a mycorrhizal fungus, the dynamic interface for interaction with soil and soil microorganisms
1
1999

... PEG处理后菌丝的形态发生了明显变化, 并随着胁迫时间的延长出现了不同程度的萎蔫状态.菌丝室培养基的pH值显著下降, 而对照处理没有明显变化, 表明干旱胁迫促进了菌丝中的H⁺外排, 使培养基酸化.干旱处理很可能激活了AMF质膜上的质子泵, 使菌丝内H⁺持续外排, 使培养基维持了较低的pH值.有研究表明, 干旱胁迫下植物根际和非根际土壤的pH值上升, 降低了土壤无机离子的活性, 影响根系对无机离子的吸收(杨培志, 2012).干旱条件下菌丝中质子泵的激活改变了根际微环境, 使更多的无机离子溶解于土壤溶液中.无机离子被菌丝吸收并进一步传输到植物中, 从而增强植物的抗旱性(Sun et al., 1999). ...

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis
1
2013

... 丛枝菌根真菌(AMF)是一类专性共生真菌, 可以与大多数陆地植物形成共生体系, 并通过多种途径促进植物的生长发育, 增强植物对各种逆境胁迫的适应能力(Smith & Read, 2008).AMF根外菌丝在根系与土壤之间建立起物质传输通道, 将吸收的水分和营养物质快速输送给宿主植物(Chitarra et al., 2016).由于菌丝直径远小于植物根系, 因此根外菌丝能进入细小的土壤空隙获取养分和水分资源(Liu & Chen, 2007), 这对于植物适应养分缺乏和干旱胁迫具有重要意义.Rhizophagus irregularis (原名Glomus intraradices)是分布最广泛的一种AMF.Tisserant等(2013)通过全基因测序方法获得了R. irregularis DAOM 197198菌株的基因组数据, 为AMF真菌的生理和分子研究提供了重要基础, 也使得该菌株成为AMF生理研究的模式菌株.干旱情况下接种R. irregularis的加杨(Populus × canadensis)具有更高的水分利用效率和抗氧化酶活性, 此外AMF还上调根系水孔蛋白基因的表达提高植物抗旱性(Liu et al., 2016).Li等(2013)从R. intraradices中克隆了两个水通道蛋白基因GintAQPF1和GintAQPF2, 并通过异源表达验证了水孔蛋白的输水功能, 为干旱胁迫下AMF向宿主植物输送水分,从而增强植物抗旱性提供了直接的分子证据. ...

Oxalate and ferricrocin exudation by the extramatrical mycelium of an ectomycorrhizal fungus in symbiosis withPinus sylvestris
1
2006

... 干旱条件下水分由根外菌丝向根内菌丝运输, 微量元素、氨基酸和其他低分子量的代谢产物也被AMF菌丝选择吸收并运送到植物中, 由此增强植物的耐旱性(Querejeta et al., 2003; van Hees et al., 2006).菌丝中元素能谱分析显示, PEG处理促进了菌丝表面P、Ca和Fe的积累.各种元素在菌丝表面的增多很可能是由于PEG诱导激活了质子泵, 而细胞对离子的吸收通常依赖于H⁺外流产生的电位梯度, 质膜H⁺-ATPase产生的质子动势为营养物质的吸收和转运提供了能量(Riquelme & Bartnicki- Garcia, 2004).干旱情况下AMF根外菌丝能够将更多的P和Ca等矿质养分从土壤中通过菌丝通道运输到植物中, 同时调控相关基因的表达以促进根系对水分的吸收, 使植物具有较强的耐旱性.Bárzana等(2012)的研究表明在充分供水和干旱胁迫下, AMF能够调控植物质外体和细胞间水分运输的途径.AMF这种调节植物水分吸收和运输的能力, 可以帮助植物更灵活地适应水分胁迫(Sánchez-Romera et al., 2016), 但AMF这一功能的生理和分子机制还需要系统深入的研究. ...

Transformation and immobilization of chromium by arbuscular mycorrhizal fungi as revealed by SEM-EDS, TEM-EDS, and XAFS
1
2015

... 新鲜菌丝收获后, 取适量的样品用超纯水清洗3次去除处理液, 然后置入2.5%戊二醛(溶解于pH值7.2的PIPES缓冲液中)中于4 ℃过夜固定, 再用相同的PIPES缓冲液清洗3次, 每次15-30 min.固定后的样品分别用30%、50%、70%、80%、90%、95%、100%的乙醇清洗(每一级浓度梯度清洗2次, 每次停留15-30 min).将脱水后的样品放在乙酸异戊酯中过夜以置换样品中的脱水剂(Wu et al., 2015).而后再将样品置于临界点干燥仪(HC P-2, Hitachi, Tokyo, Japan)中, 通入CO₂进行干燥.将干燥后的菌丝粘在导电胶布上经自然干燥后, 喷金粉50 s, 然后在连接有能谱分析仪的场发射扫描电子显微镜(FE-SEM-EDS, SU-8020, Hitachi, Tokyo, Janpan)下观察分析. ...

Evolution mechanism of non-hydraulic root-to-shoot signal during the anti-drought genetic breeding of spring wheat
1
2007

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

Arbuscular mycorrhiza enhances drought tolerance of tomato plants by regulating the 14-3-3 genes in the ABA signaling pathway
1
2018

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

Adaptation of H⁺-pumping and plasma membrane H⁺-ATPase activity in proteoid roots of white lupin under phosphate deficiency
1
2002

... 细胞膜质子泵, 即H⁺-ATPase的功能是水解ATP并将H⁺泵出细胞(Santi et al., 2003).H⁺通过细胞内外的电化学梯度进出细胞膜, 为包括阳离子、阴离子、氨基酸和糖在内的各种小分子物质的跨膜运输提供驱动力(Gaxiola et al., 2007).干旱胁迫、盐胁迫、缺磷等环境胁迫会导致质子泵发生改变(Yan et al., 2002; Gévaudant et al., 2007).细胞膜质子泵活性不仅反映物质跨膜运输的能力, 还在一定程度上反映生长环境对植物生理活性的影响.玉米(Zea mays)可以通过提高根系细胞膜质子泵的活性来适应低pH环境, 燕麦(Avena sativa)在感受到干旱胁迫的早期就可以激活根系H⁺-ATPase来增加渗透物质的合成(Gong et al., 2010).对AMF H⁺-ATPase的研究表明, 真菌质子泵在控制共生体营养元素和水分转运过程中起到重要作用(Ferrol et al., 2000).Gianinazzi- Pearson等(1991)通过测试ATPases水解作用过程中释放的磷酸对ATP酶进行细胞化学研究, 证明在AMF根内、根外菌丝的质膜上均存在H⁺-ATPase. ...

紫花苜蓿根瘤菌共生对干旱及盐胁迫的响应机制研究
1
2012

... PEG处理后菌丝的形态发生了明显变化, 并随着胁迫时间的延长出现了不同程度的萎蔫状态.菌丝室培养基的pH值显著下降, 而对照处理没有明显变化, 表明干旱胁迫促进了菌丝中的H⁺外排, 使培养基酸化.干旱处理很可能激活了AMF质膜上的质子泵, 使菌丝内H⁺持续外排, 使培养基维持了较低的pH值.有研究表明, 干旱胁迫下植物根际和非根际土壤的pH值上升, 降低了土壤无机离子的活性, 影响根系对无机离子的吸收(杨培志, 2012).干旱条件下菌丝中质子泵的激活改变了根际微环境, 使更多的无机离子溶解于土壤溶液中.无机离子被菌丝吸收并进一步传输到植物中, 从而增强植物的抗旱性(Sun et al., 1999). ...

紫花苜蓿根瘤菌共生对干旱及盐胁迫的响应机制研究
1
2012

... PEG处理后菌丝的形态发生了明显变化, 并随着胁迫时间的延长出现了不同程度的萎蔫状态.菌丝室培养基的pH值显著下降, 而对照处理没有明显变化, 表明干旱胁迫促进了菌丝中的H⁺外排, 使培养基酸化.干旱处理很可能激活了AMF质膜上的质子泵, 使菌丝内H⁺持续外排, 使培养基维持了较低的pH值.有研究表明, 干旱胁迫下植物根际和非根际土壤的pH值上升, 降低了土壤无机离子的活性, 影响根系对无机离子的吸收(杨培志, 2012).干旱条件下菌丝中质子泵的激活改变了根际微环境, 使更多的无机离子溶解于土壤溶液中.无机离子被菌丝吸收并进一步传输到植物中, 从而增强植物的抗旱性(Sun et al., 1999). ...

Chilling-induced inactivation and its recovery of tonoplast H⁺-ATPase in mung bean cell suspension cultures
1
1991

... 细胞膜是生物遭受逆境胁迫以后受损的初始位点, 其结构及功能的改变势必会影响生物体内水分代谢以及各种无机离子的运输, 而无机离子的跨膜运输对维持植物正常生长发育起着至关重要的作用(Yoshida, 1991).干旱胁迫下细胞会通过特定的调节机制, 改变离子和小分子的跨膜运输来调节离子平衡, 从而维持细胞的渗透压, 以抵御干旱胁迫.因此, 研究关键离子的跨膜转运过程对揭示植物在干旱胁迫下通过自我调节以维持生命活动的机制具有重要意义.Kühtreiber和Jaffe (1990)首次利用非损伤微测技术(non-invasive micro-test technology, NMT)在活体原位条件下测量了Ca²⁺进出细胞的流速和方向, 该方法被广泛应用于生物体逆境应答机制研究.目前, NMT技术可以实时记录活体植物细胞各种离子的跨膜运输, 为研究植物应答环境变化提供了重要技术平台.应用这种技术, Mak等(2014)发现在不同时间长度的干旱胁迫下大豆(Glycine max)叶肉细胞K⁺、H⁺和Ca²⁺转运情况出现显著变化, 而各种离子的转运能力可作为重要的生理指标来衡量大豆的耐旱性. ...

Arbuscular mycorrhizal fungi affect the growth, nutrient uptake and water status of maize (Zea mays L.) grown in two types of coal mine spoils under drought stress
1
2015a

... 细胞环境碱化.真菌胞内的碱化与胁迫信号的长距离传递有关, pH变化可能引起真菌-植物之间的信号传递发生改变(Harrison, 2005).离子动力学研究表明, 真菌和植物的信号交流与细胞跨膜H⁺离子梯度的调节有关, 该过程受到细胞膜上P型H⁺-ATPases的调控(Ramos et al., 2008a).为了进一步验证PEG模拟水分胁迫下菌丝H⁺的跨膜运输过程, 利用NMT技术验证了菌丝尖端和侧面两个部位H⁺的流动情况.对照处理下, 两个部位的H⁺都呈现微弱外流的趋势, 而且菌丝侧面的H⁺外流流速大于尖端.该结果与前人研究结果相似, Ramos等(2008b)证明从菌丝尖端到菌丝侧面不同位置上H⁺流动规律不同, 而且菌丝侧面的H⁺外流速率大于尖端.该现象可能和真菌的信号识别机制以及菌丝尖端生长有关(Bartnicki-Garcia et al., 2000).在PEG处理下, 菌丝H⁺显著外流.随着处理时间的延长, 菌丝尖端H⁺外流强度增大, 外流的H⁺使菌丝际环境酸化.该结论与液体培养基的pH值变化结果相符, 表明在模拟干旱处理1 h后, 菌丝对于外部环境的pH值进行了调节.菌丝尖端的H⁺外流调节了真菌对营养物质(如蔗糖、氨基酸和P)的吸收和交换(Requena et al., 2003).能谱分析显示, PEG处理增加了菌丝表面P信号强度, 说明在干旱处理下更多的P积累到菌丝界面.Hijikata等(2010)研究证实, 干旱情况下根外菌丝的磷转运蛋白活性提高, 将更多的P从土壤中通过菌丝吸收运输到植物中, 而AMF提高植物的营养元素吸收是提高植物耐旱性的重要机制(Zhao et al., 2015a). ...

Expression OsCAS (calcium-sensing receptor) in an Arabidopsis mutant increases drought tolerance
1
2015b

... 质膜H⁺外流不仅是营养物质吸收的动力, 还作为信号传导的中间体, 促进逆境信号的传导和响应元件的激活(Fromm & Lautner, 2007).H⁺的外流可以促进Ca²⁺、K⁺等阳离子的跨膜运输, 提高胞内的离子浓度(Isfort et al., 1993).本研究中, PEG处理1 h后, 菌丝尖端和表面的Ca²⁺表现了强烈的内流现象.当生物受到干旱胁迫的时候, 细胞内Ca²⁺瞬时产生快速的增高, 引发钙振荡(Zhao et al., 2015b).Ca²⁺作为第二信使, 在胁迫信号的传递过程中起到重要作用, 胞内游离Ca²⁺的分布与转移是Ca²⁺信号产生和传递的基础.使用LSCM观测菌丝细胞内Ca²⁺浓度的结果与离子流测定结果相符, PEG处理1 h后菌丝尖端与侧面均呈现强烈的绿色荧光, 说明菌丝内Ca²⁺浓度显著上升.干旱胁迫诱导的胞内Ca²⁺浓度增加能够激活下游的逆境信号响应元件, Ca依赖的蛋白激酶(CDPK)等, 而CDPK可以通过磷酸化质膜水通道蛋白促进细胞膜对水分的吸收和扩散(Azad et al., 2004).Li等(2013)证实, 干旱胁迫情况下AMF中两个水通道蛋白基因GintAQPF1和GintAQPF2的表达量会显著上调, 为植物输送水分以缓解干旱胁迫. ...

Arabidopsis calcium-dependent protein kinase 8 and catalase 3 function in abscisic acid-mediated signaling and H₂O₂ homeostasis in stomatal guard cells under drought stress
1
2015

... Ca²⁺是生物细胞内重要的信号物质, 在植物抵抗干旱胁迫方面具有重要作用(Ma et al., 2005).胞质内游离Ca²⁺的分布以及跨膜转移是Ca²⁺信号的转移和传导的基本途径, 而Ca²⁺进入和流出细胞速率的变化是产生Ca²⁺振荡的基础(Bunney et al., 2000).质膜Ca²⁺-ATPases和H⁺-ATPase共同实现了质膜H⁺/Ca²⁺的逆向转运和逆境信号的传递(Rudd & Franklin-Tong, 2001).干旱胁迫可以导致细胞内游离Ca²⁺浓度产生短暂而快速的升高, 胞内Ca²⁺的波动对钙调蛋白、激酶和离子通道等下游调控元件产生影响(Campo et al., 2014).由钙信号传导的干旱胁迫信息会导致相关基因表达和代谢过程的改变(如渗透调节物质合成和营养元素运输的变化), 从而减轻干旱胁迫对生物机体的损害(Ludwig et al., 2004; Zou et al., 2015). ...

The involvement of expansins in response to water stress during leaf development in wheat
1
2015

... 很多研究证实菌根化植物在干旱时有更高的蒸腾速率、水分利用效率及抗氧化能力(Porcel & Ruiz-Lozano, 2004; Xiong et al., 2007; Augé et al., 2015).干旱条件下AMF能够通过多种途径调控植物新陈代谢并调节抗旱相关基因, 以增强植物的耐旱性(Augé & Duan, 1991; 李涛等, 2012; Xu et al., 2018), 然而目前对AMF自身响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究却很少.本研究利用AMF-胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净AMF根外菌丝, 应用聚乙二醇(PEG 6000)模拟干旱胁迫(PEG 6000 是一种惰性的非离子型的渗透调节剂, 广泛应用于模拟干旱胁迫(Zhou et al., 2015; Shi et al., 2016), 结合场发射扫描电子显微镜能谱分析(FE-SEM- EDS)和NMT技术, 在原位条件下研究AMF根外菌丝形态和跨膜离子流对干旱胁迫的响应, 期望进一步揭示AMF与植物协同抗旱的生理机制. ...

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