盐胁迫对玉米叶片叶肉细胞生物膜超微结构的影响

研究了NaCl胁迫对玉米叶肉细胞生物膜超微结构的影响. 结果表明：NaCl胁迫破坏了玉米叶片叶肉细胞生物膜的正常结构,50 mmol·L^-1 NaCl处理胁迫下,玉米叶肉细胞核膜,线粒体膜,细胞膜,叶绿体膜,液泡膜都受到不同程度的破坏,叶绿体基粒类囊体膨胀,间质片层空间增大,片层紊乱。100 mmol·L^-1 NaCl处理胁迫下,质膜,液泡膜,线粒体,叶绿体都受到严重的破坏。细胞质膜破坏,破损的叶绿体充斥在细胞间隙中;叶绿体外膜破坏,甚至解体消失,叶肉细胞中充满膜结构,基粒排列方向改变,垛叠层数减少,基粒和基质片层界限模糊不清,有的基粒解体消失,甚至叶绿体完全解体;核膜破坏、解体,核中的染色质高度凝缩;线粒体的数量增多,线粒体膜破坏,脊的数量减少,甚至整个线粒体破损解体;液泡膜破坏;由于各种生物膜的破坏,使细胞内充满许多囊状小泡、多泡体或斑层小体;叶肉细胞发生严重的质壁分离,严重时发生细胞壁断裂;甚至整个细胞溶解。     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化。结果表明：随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化。(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双层膜破坏,基粒片层松散排列,杂乱无章,出现膨胀和空泡现象,淀粉粒和嗜锇颗粒增多,叶肉细胞中线粒体发生轻微变化;幼根中皮层薄壁细胞线粒体形状发生改变,结构破坏,内膜和外膜模糊甚至破裂,大多数嵴模糊,出现空泡现象;细胞核解体,基质外溢。研究表明, 不同浓度的NaCl胁迫对宁夏枸杞叶片和幼根细胞的显微及超微结构影响不同,NaCl浓度大于200 mmol/L时,宁夏枸杞叶片和幼根细胞的显微及超微结构发生了明显变化,且叶肉细胞中线粒体的变化没有叶绿体的变化显著,推测叶肉细胞中线粒体的耐盐性比叶绿体强。     

水分胁迫下小麦叶肉细胞超微结构变化与抗旱性的关系

本文用电子显微镜观察研究了抗旱性不同的6个小麦品种在不同程度水分胁迫下叶肉细胞超微结构的变化。结果表明:轻度水分胁迫(-0.5MPa)对参试的6个小麦品种叶肉细胞超微结构几乎没有影响。中度(-1.0MPa)和严重(-1.5MPa)水分胁迫下的叶肉细胞超微结构发生了程度不同的变化,且这种变化与品种抗旱性相一致。抗旱性愈弱的品种,对水分胁迫反应愈敏感。但表现在叶肉细胞结构上的变化过程基本一致。胁迫导致叶肉细胞质壁分离,液泡膜破裂。叶绿体变成球形挤入细胞中央,类囊体肿胀。线粒体基质变稀,脊减少。最终叶绿体、线粒体解体。其它细胞器消失,细胞中出现大量的小泡。     

环境胁迫对库拉索芦荟叶片超微结构影响研究

对1年生库拉索芦荟分别用盐(1.8%的NaCl)、低温(10℃)、干旱[25%(w/v)的聚乙二醇-6000]3种胁迫条件处理7d后,对其叶肉细胞超微结构进行观察.结果发现:3种胁迫处理均可使库拉索芦荟细胞膜系统、叶绿体、线粒体、细胞核等结构受到不同程度的破坏,叶绿体周围出现许多小泡,导致细胞内膜系统紊乱,细胞器结构稳定性降低;盐胁迫下高尔基体在细胞质中解体;盐和低温胁迫下均可见线粒体膜与叶绿体膜发生融合、线粒体嵌在叶绿体当中的现象.另外,本研究发现,盐胁迫、低温胁迫比干旱胁迫对库拉索芦荟细胞膜的损伤更严重,而水分胁迫对其的伤害程度较小,表明库拉索芦荟的抗旱性较其抗盐性更强.     

干旱胁迫对不同耐旱性大麦品种叶片超微结构的影响

选用耐旱性不同的3个大麦（Hordeum sativum）品种作为研究对象,分析干旱胁迫对其叶肉细胞叶绿体、线粒体和细胞核超微结构的影响。结果表明,3个大麦品种在非胁迫条件下其超微结构无明显差异。遭受干旱胁迫后,不耐旱大麦品种Moroc9-75叶片细胞核中染色质的凝聚程度高,叶绿体变形,外被膜出现较大程度的波浪状和膨胀,同时基粒出现弯曲、膨胀、排列混乱的现象;线粒体外形及膜受到破坏、内部嵴部分消失等。耐旱大麦品种HS41-1叶片细胞中染色质虽出现凝聚,但凝聚程度低;其叶绿体及线粒体与非胁迫条件下基本相似,多数未见明显损伤。耐旱中等的大麦品种Martin叶片超微结构的变化则介于二者之间。因此,干旱胁迫下叶绿体外形、基粒和基质类囊体膜结构的完整性与基粒的排列次序、染色质的凝聚度和线粒体膜及嵴的完整性与大麦的耐旱性相关,这些特性可作为评价大麦耐旱性强弱的形态结构指标。     

干旱胁迫对不同耐旱性大麦品种叶片超微结构的影响

选用耐旱性不同的3个大麦(Hordeum sativum)品种作为研究对象, 分析干旱胁迫对其叶肉细胞叶绿体、线粒体和细胞核超微结构的影响。结果表明, 3个大麦品种在非胁迫条件下其超微结构无明显差异。遭受干旱胁迫后, 不耐旱大麦品种Moroc9-75叶片细胞核中染色质的凝聚程度高, 叶绿体变形, 外被膜出现较大程度的波浪状和膨胀, 同时基粒出现弯曲、膨胀、排列混乱的现象; 线粒体外形及膜受到破坏、内部嵴部分消失等。耐旱大麦品种HS41-1叶片细胞中染色质虽出现凝聚, 但凝聚程度低; 其叶绿体及线粒体与非胁迫条件下基本相似, 多数未见明显损伤。耐旱中等的大麦品种Martin叶片超微结构的变化则介于二者之间。因此, 干旱胁迫下叶绿体外形、基粒和基质类囊体膜结构的完整性与基粒的排列次序、染色质的凝聚度和线粒体膜及嵴的完整性与大麦的耐旱性相关, 这些特性可作为评价大麦耐旱性强弱的形态结构指标。     

低温对桂花‘状元红’叶肉细胞超微结构的影响

为探讨北引桂花(Osmanthus fragrans)在低温胁迫下叶肉细胞超微结构的变化,揭示桂花于低温胁迫下细胞结构变化规律,该研究以3年生桂花品种‘状元红’(O.fragrans‘Zhuangyuan Hong’)为试材,分别于一系列低温下处理,经制样切片后,用透射电子显微镜观察叶肉细胞超微结构的变化。结果表明:常温(20~25°C)处理时,各细胞器超微结构正常;5°C低温处理时,叶绿体有轻微膨大现象,线粒体结构正常;0°C处理时叶绿体内嗜锇体增多,叶绿体肿胀加剧,线粒体数量增加,淀粉粒出现亮暗相间的轮纹;–10°C处理时,细胞器降解。在同一低温胁迫下不同细胞的叶绿体敏感程度不同,这为遭受低温后植株的恢复生长提供了细胞学基础。叶肉细胞中叶绿体、线粒体、细胞核的稳定性可作为桂花对低温响应的重要参考指标。     

Na2CO3胁迫对星星草叶肉细胞超微结构的影响

利用透射电镜技术对Na2CO3胁迫下星星草叶肉细胞超微结构进行了观察。结果表明：未胁迫的叶肉细胞排列疏松,各种细胞器结构完整,叶绿体含少量淀粉粒和脂质球。轻度盐胁迫（2g/L,4g／LNa2CO3）对叶肉细胞超微结构影响较小。中度盐胁迫（6g／L,8g／L Na2CO3）引起叶肉细胞超微结构的变化,叶绿体类囊体肿胀,基粒紊乱,不含淀粉粒,脂质球数量增加,叶绿体由原来的梭形或椭球形变成圆球状;部分线粒体嵴消失,出现晶体结构;中央大液泡破裂;核逐渐降解。高度盐胁迫（10g／L,12g／LNa2CO3）下,叶绿体片层结构消失,脂质球数量增加,体积变大,被大量的膜片层所包围,叶绿体内、外膜消失,叶肉细胞中看不到叶绿体的存在;膜片层包围线粒体;叶肉细胞中可见大量的泡状结构和膜片层,叶肉细胞死亡。上述结果表明,细胞器特别是叶绿体膜结构的破坏与盐胁迫叶肉细胞最终死亡密切相关。     

Na+和Cl- 在小麦叶肉细胞超微结构中的分布

小麦经200mmol NaCl溶液培养3天后,采用改进的焦锑酸钾方法对叶肉细胞中Na~+及Cl~-进行超微结构定位。电镜观察及电子探针X-射线显微分析表明,Cl~-主要分布在细胞间隙、细胞壁及细胞质膜中。用电子探针X~-射线能谱仪在这些部位中未探测出Na~+,提示Cl~-比Na~+更多地进入小麦的叶肉细胞。此外,在叶肉细胞的细胞核、线粒体及叶绿体中也可见到离子沉淀颗粒。经氯化钠溶液培养的小麦幼苗,其叶肉细胞的叶绿体、线粒体的超微结构受损,植株生长受到抑制。     

NaCl胁迫对5个桂花品种叶片超微结构的影响

该研究以5个桂花品种为材料,以Hoagland培养液为对照,设计2个NaCl含量(70、100 mmol/L)处理10 d后利用透射电镜和扫描电镜观察各处理不同品种的叶片超微结构特征,以明确桂花品种对耐NaCl胁迫的解剖结构响应机制。结果显示：(1)透射电镜观察发现：随NaCl 胁迫程度的加强,5个桂花品种叶肉细胞中叶绿体结构受到不同程度地破坏;70 mmol/L NaCl处理后,5个品种的细胞核基本保持正常,而100 mmol/L NaCl处理后核内染色质发生降解;随着NaCl胁迫程度的加强,5个桂花品种的类囊体片层结构中嗜锇颗粒明显增多;在膜结构方面,大叶银桂的叶肉细胞被破坏程度最为严重,叶绿体膜被破坏,叶绿体形状基本不能辨认。(2)扫描电镜观察结果显示：随着NaCl浓度的增大,5个品种叶片表面的气孔密度不断增大,而张开气孔的密度却不断减小,且叶肉细胞体积均缩小;‘大叶银桂’、‘笑秋风’、‘晚籽银桂’的栅栏组织占叶厚的比重随NaCl胁迫浓度的增大而升高,‘潢川金桂’和‘紫梗籽银桂’的栅栏组织占叶厚的比重则随NaCl胁迫浓度的增大而呈先升高后降低的趋势。研究表明,NaCl胁迫对桂花叶片细胞叶绿体、细胞核等的超微结构会造成损伤,且NaCl胁迫浓度越高损伤越明显。该试验可初步判断‘大叶银桂’、‘笑秋风’、‘晚籽银桂’的耐盐性略高于‘潢川金桂’和‘紫梗籽银桂’。     

NaCl对齿肋赤藓叶肉细胞超微结构的影响

齿肋赤藓(Syntrichia caninervis)是古尔班通古特沙漠苔藓结皮层中的优势物种,对荒漠生态系统的稳定性及功能多样性具有十分重要的意义。利用透射电镜技术对不同浓度Na Cl胁迫下齿肋赤藓叶肉细胞超微结构进行了观察。结果表明:齿肋赤藓叶肉细胞在未胁迫(0 mmol/L)处理下排列疏松,各种细胞结构完整,叶绿体基质排列均匀且叶绿体内含少量淀粉粒和脂质球。在轻度盐Na Cl胁迫(100 mmol/L)下,齿肋赤藓叶肉细胞结构依然保持完整,叶绿体基质均匀,叶肉细胞超微结构仅有较小变化。在中度盐Na Cl胁迫(200、300 mmol/L)下,齿肋赤藓叶肉细胞发生质壁分离,出现晶体结构,且中央大液泡发生破裂;叶绿体由梭形变成椭球形或圆球状,出现空泡化并伴随有轻微的解体;叶绿体类囊体肿胀,脂质球数量增加。在高度Na Cl胁迫(400、500 mmol/L)下,齿肋赤藓细胞的质壁分离加剧,叶肉细胞出现大量泡状结构和膜片层,叶肉细胞死亡;叶绿体片层结构消失,空泡化加重,脂质球数量增加且体积变大,叶绿体内外膜消失,叶绿体大部分解体,在叶肉细胞中几乎看不到叶绿体的存在。上述结果表明,叶绿体膜结构的损伤与盐胁迫下叶肉细胞死亡有密切关系。     

Ultrastructure of Mesophyll Cells and Pigment Content in Senescing Leaves of Maize and Barley

Leaf senescence is a genetically regulated stage in the plant life cycle leading to death. Ultrastructural analysis of a particular region of the leaf and even of a particular mesophyll cell can give a clear picture of the time development of the process. In this study we found relations between changes in mesophyll cell ultrastructure and pigment concentration in every region of the leaf during leaf senescence in maize and barley. Our observations demonstrated that each mesophyll cell undergoes a similar senescence sequence of events: a) chromatin condensation, b) degradation of thylakoid membranes and an increase in the number of plastoglobules, c) damage to internal mitochondrial membrane and chloroplast destruction. Degradation of chloroplast structure is not fully correlated with changes in photosynthetic pigment content; chlorophyll and carotenoid content remained at a rather high level in the final stage of chloroplast destruction. We also compared the dynamics of leaf senescence between maize and barley. We showed that changes to the mesophyll cells do not occur at the same time in different parts of the leaf. The senescence damage begins at the base and moves to the top of the leaf. The dynamics of mesophyll cell senescence is different in leaves of both analyzed plant species; in the initial stages, the process was faster in barley whereas in the later stages the process occurred more quickly in maize. At the final stage, the oldest barley mesophyll cells were more damaged than maize cells of the same age.     

香蒲对不同浓度Pb~(2+)胁迫的生理应答及其细胞超微结构变化

通过室内水培试验,研究了不同浓度Pb2+(0、0.25、0.50、1.00和2.00mmol·L-1)胁迫对东方香蒲根和叶中Pb含量、叶绿素含量、丙二醛(MDA)含量、抗氧化酶(SOD、CAT和POD)活性以及亚细胞结构的影响。结果显示:(1)随着外源Pb2+浓度的增加,Pb在香蒲根和叶中的积累量均显著高于对照,且Pb在根中的含量明显高于叶中,并与外源Pb2+浓度呈显著正相关关系。(2)香蒲叶片中的叶绿素a和叶绿素b含量随着外源Pb2+浓度的增加呈先升后降趋势,均在处理浓度为0.50mmol·L-1时达到峰值。(3)胁迫处理叶片的MDA含量与对照相比变化不显著,但根中MDA含量呈显著下降趋势。(4)叶片中SOD活性在1.00mmol·L-1 Pb2+处理时达到峰值,然后下降,但始终高于对照,CAT和POD活性则均低于对照组;根中SOD活性除1.00mmol·L-1 Pb2+处理组外均显著低于对照组,CAT和POD活性分别在0.25和0.50mmol·L-1 Pb2+处理时达到峰值,然后随处理Pb2+浓度升高而下降。(5)电镜观察发现,Pb2+胁迫使香蒲叶细胞中叶绿体被膜破裂,类囊体膨胀、破损;根和叶细胞中的线粒体被膜均破裂、内腔空泡化,细胞核核膜破损、核仁消失、染色质凝集。研究表明,Pb2+胁迫致使东方香蒲根、叶生理代谢失衡,亚细胞结构出现不可逆的损伤,这为从分子水平研究Pb2+作用的具体机理以及香蒲在重金属污染修复中的应用提供了依据。     

Hg2+和Cd2+胁迫对满江红生理和细胞超微结构的影响

研究了在梯度浓度Hg2+和Cd2+胁迫下,满江红(Azolla imbricata (Roxb.) Nakai)的叶绿素含量、叶绿素a/b比值、光合放氧速率、呼吸速率、抗氧化酶系(超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD))和细胞超微结构受Hg2+和Cd2+的毒害影响.结果显示:随着胁迫程度的增大,叶绿素含量、叶绿素a/b比值、光合放氧速率明显下降,呼吸速率均在2 mg/L浓度下达到峰值,尔后下降; SOD、CAT、POD的活性均出现不同程度的应激性升高(除POD在Cd2+处理时下降),尔后下降.电镜观察发现,随着污染物浓度的增加和胁迫时间的延长,叶绿体出现膨大、破损和解体;线粒体嵴突膨胀和线粒体变形及空泡化;核染色质凝集,核仁消失,核膜破裂.实验结果表明: Hg2+和Cd2+污染不仅损害植物的生理活性,而且也破坏细胞的超微结构,最终导致植物死亡;随着Hg2+和Cd2+胁迫的增大,细胞超微结构的损伤程度和植物的生理变化是同步的;植物受毒害的程度表现出明显的剂量效应关系;在同一处理时间和浓度下,Cd2+对满江红的毒性大于Hg2+.Hg2+对满江红的致死浓度为3.5～4.0 mg/L,Cd2+为3.0～3.5 mg/L.对满江红鱼腥藻(Anabaena azollae Strasburger)细胞的超微结构变化观察表明,满江红鱼腥藻对Hg2+和Cd2+的耐受性明显高于满江红.     

Hg^2＋和Cd^2＋胁迫对满江红生理和细胞超微结构的影响

研究了在梯度浓度Hg^2 和Cd^2 胁迫下,满江红（Azolla imbricata(Roxb.)Nakai)的叶绿素含量,叶绿素a/b比值,光合放氧速率,呼吸速率,抗氧化酶系（超氧化物歧化酶（SOD）,过氧化氢酶（CAT）,过氧化物酶（POD）和细胞超微结构受He^2 和Cd^2 的毒害影响。结果显示：随着胁迫程度的增大,叶绿素含量,叶绿素a/b比值,光合放氧速率明显下降,呼吸速率均在2mg/L浓度下达到峰值,尔后下降;SOD,CAT,POD的活性均出现不同程度的应激性升高（除POD在Cd^2 处理时下降）,尔后下降,电镜观察发现,随着污染物浓度的增加和胁迫时间的延长,叶绿体出现膨大,破损和解体;线粒体嵴突膨胀和线粒体变形及空泡化;核染色质凝集,核仁消失。核膜破裂,实验结果表明：Hg^2 和Cd^2 污染不仅损害植物的生理活性,而且也破坏细胞的超微结构,最终导致植物死亡,随着Hg^2 和Cd^2 为3.0-3.5mg/L。对满江红鱼腥藻（Anabaena azollae Strasburger)细胞的超微结构变化观察表明,满江红鱼腥藻对Hg^2 和Cd^2 的耐受性明显高于满江红。     

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca²⁺ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca²⁺ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca²⁺ overload. However, exposure of yeast mitochondria to 50–100 μM Ca²⁺ in the presence of the Ca²⁺ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca²⁺/nH⁺-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca²⁺- ETH129-induced activation of the Ca²⁺/H⁺-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca²⁺ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Changes in photosynthetic apparatus of mustard (<Emphasis Type="Italic">Sinapis alba</Emphasis>) cotyledons following cycloheximide and kinetin treatments

Biochemical and accompanying structural characteristics of the photosynthetic process were studied in mustard seedlings cultivated on medium with increasing concentrations of cycloheximide alone as well as in combination with various kinetin concentrations. After 7 days of cultivation the contents of total chlorophyll, carotenoids and content of Rubisco in mustard cotyledons were determined. The content of chlorophyll pigments and carotenoids decreased in dependence of cycloheximide concentration. Following antibiotic treatment the content of both Rubisco subunits markedly decreased. In addition cycloheximide caused disturbance in mesophyll organization and chloroplast ultrastructure. Kinetin applied with cycloheximide increased the amount of photosynthetic pigments as well as of Rubisco, compared to the cycloheximide alone. In the seedlings treated with cycloheximide+kinetin the structure of leaf mesophyll and chloroplast membrane system was similar to control. Our results indicate that kinetin diminished the negative effects of cycloheximide on photosynthetic pigments and Rubisco as well as on the structural traits of the cotyledons.     

汞、镉、铜污染对鱼草细胞膜系统的毒害作用

通过生理生化反应测定、激光共聚焦扫描显微镜及透射电镜观察等实验手段,研究了汞（Hg²⁺）、镉（Cd²⁺）、铜（Cu2⁺）对高等水生植物鱼草(Cabomba caroliniana A. Gray)细胞膜系统的毒害作用.结果表明：在3种重金属离子作用下,鱼草叶细胞活性氧（ROS）与丙二醛（MDA）含量上升,保护酶系统活性紊乱,膜脂过氧化程度加剧;质膜受损,膜透性增加,质壁分离;叶绿体膨胀至解体,类囊体膜上的光合色素光激发过程受阻,平均自发荧光强度降低;线粒体嵴突膨胀、减少,膜破损;核膜破裂.Hg²⁺、Cd²⁺、Cu²⁺对鱼草细胞膜系统的影响存在着一定的剂量效应关系.膜系统的稳定性在植物抗重金属胁迫的过程中起着关键性的作用.鱼草对Hg²⁺污染较为敏感,致死浓度为0.3～0.5 mg·L^-1,而对Cd²⁺、Cu²⁺具有较强的抗性,可用作生物防治中的抗性植物.     

Possible involvement of protein phosphorylation/dephosphorylation in the modulation of Ca2+ channel in tonoplast-free cells ofNitellopsis

Summary The regulation of voltage-dependent Ca²⁺ channels by protein phosphorylation and dephosphorylation was studied using tonoplast-free cells ofNitellopsis. Since the Ca²⁺-channel activation has a dominant role in the membrane excitation of tonoplast-free cells (T. Shiina and M. Tazawa,J. Membrane Biol. 96:263–276, 1987), it seems to be reasonable to assume that any change of the membrane excitability reflects a modulation of the Ca²⁺ channel. When agents that enhance phosphoprotein dephosphorylation (protein kinase, inhibitor, phosphoprotein phosphatase-1, -2A) were introduced to the intracellular surface of the plasmalemma (twice-perfused tonoplast-free cells), the membrane potential depolarized and the membrane resistance decreased under current-clamp experiments. By contrast, when cells were challenged with agents that enhance protein phosphorylation (phosphoprotein phosphatase inhibitor-1, -naphthylphosphate), the membrane potential hyperpolarized, and the membrane resistance increased. When phosphoprotein phosphatase-1 or -2A was perfused, the current-voltage (I–V) curve which was obtained under ramp voltage-clamp condition exhibited the so-called N-shaped characteristic, indicating an acceleration of the Ca²⁺-channel activation. This effect was suppressed by the addition of phosphoprotein phosphatase inhibitors. ATP--S, which is assumed to stimulate protein phosphorylation, decreased the inward current in theI–V curve. The dependence of the Ca²⁺-channel activation on intracellular ATP was different between the once-perfused and twice-perfused cells. In once-perfused cells, the membrane excitability was reduced by low intracellular ATP concentration. By contrast, in twice-perfused cells, excitability was enhanced by ATP.