利用激光扫描共聚焦显微镜研究植物细胞发育形态学变化

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究植物细胞发育形态学变化。结果表明,利用紫外激光(351 nm)扫描可以清楚地观察到拟南芥叶片表皮细胞的形态及其变化,在已分化的叶片表皮上可观察到包括“铺垫”表皮细胞(epidermal pavement cells)、气孔保卫细胞(guard cell)、气孔伴胞(subsidiarycells)、表皮毛细胞(trichomes)和表皮毛的足细胞(socket cells)等多种形态不同的细胞种类;利用蓝光激光(488nm)辅助曙红浅染,可清晰地显示出拟南芥根生长区内部的各种原始细胞,包括静止区(quiescent center)细胞、皮层/内皮层原始细胞(cortex/endodermal initial cell)、表皮/根冠原始细胞(epidermal/root cap initial cell)和中柱/根冠原始细胞(columella/root cap initial cell)等。利用双光子激光(800 nm)连续扫描30 s可以诱发叶绿体产生自发荧光,并可观察到叶绿体在叶肉细胞中的运动轨迹。结果说明激光扫描共聚焦显微镜在植物细胞形态及发育研究上具有独特的功能。     

狭基巢蕨叶表皮的结构和气孔器发育的观察

狭基巢蕨Ｎｅｏｔｏｐｔｅｒｉｓａｎｔｒｏｐｈｙｏｉｄｅｓ（Ｃｈｒｉｓｔ）Ｃｈｉｎｇ叶片的上表皮无气孔器,仅具表皮细胞,下表皮由表皮细胞和气孔器组成,气孔指数为２．５。上下表皮细胞和气孔器的细胞中均含有叶绿体。每个气孔器由２个肾形的保卫细胞和２～６个副卫细胞组成,其中以３个和４个副卫细胞的占绝大多数（３细胞的占４５．１％,４细胞的占４３．５％）。从发育上看,气孔器原始细胞进行２次分裂,产生２个保卫细胞和１个同源的副卫细胞。气孔器的发育过程大体可分为４个时期：（１）气孔器原始细胞的分化和分裂期;（２）保卫细胞母细胞成熟期;（３）保卫细胞母细胞分裂和气孔器幼期;（４）气孔器成熟期。狭基巢蕨的气孔器属于中周型     

海南莎草属植物叶下表皮微形态的研究

利用光学显微镜对海南莎草属(Cyperus Linnaeus)15个种1个变种的叶下表皮微形态进行了研究。结果显示:(1)该属叶下表皮微形态基本特征如下:长细胞为长筒形、短筒形,少数为近方形,边缘波状、深波状;无短细胞的存在;副卫细胞为高圆屋顶形、圆屋顶形、圆屋顶至三角形或三角形;(2)根据保卫细胞的形态将海南莎草属分为两大类型即保卫细胞两端明显加厚型和保卫细胞两端不明显加厚型;(3)海南莎草属叶下表皮微形态一致,表明莎草属是一个自然类群,但在莎草属种间叶下表皮的长细胞形态、保卫细胞及副卫细胞形态、气孔长宽等存在一定的差异,可作为本属种间分类鉴别的参考依据。     

银杏(Ginkgo biloba)叶表皮特征及其气孔的发育

通过光学显微镜和扫描电子显微镜,对离析和未离析的、成熟的和刚开始发育的银杏叶表皮进行了研究.表皮基本特征:两面生气孔型,但气孔主要分布在下表皮,上表皮气孔的数目和分布因个体而异;气孔器为单唇单环式,围绕每个气孔的副卫细胞数目多为5～6个;副卫细胞的平周壁明显加厚,具乳突状突起,拱盖着下陷的保卫细胞;偶有双气孔发生;上表皮厚于下表皮;表皮细胞可明显分为叶脉区和脉间区.角质外蜡质层:在高倍扫描电子显微镜下,管状的蜡质晶体交错覆盖在叶片的外表面,还观察到有些管状的蜡质晶体呈分叉状态.气孔的发育:通过我们的观察,认为银杏气孔的发生属于典型的周源型, 即副卫细胞与保卫细胞各自发生于不同的母细胞,而不是前人报道的中--周型起源.     

玉米花粉单倍体植株异常气孔的观察

研究了玉米花粉小植株和长期继代再生的单倍体小植株气孔的结构,发现有些小植株叶表皮上存在着各种异常气孔,它们可分为四组十个类型:(1)无卫型,没有保卫细胞,但有不同数量的副卫细胞;(2)无副单副和多副型,有保卫细胞和不同数量的副卫细胞;(3)双孔或多孔型,两个或多个气孔连在一起,有共同的副卫细胞;(4)败育型,保卫细胞母细胞     

UV-B对拟南芥叶片不同来源H2O2的活化和气孔关闭的诱导

在UV-B调控植物许多生理过程中过氧化氢(H2O2)作为第二信使发挥着重要作用,但H2O2来源途径并不清楚。该研究借助气孔开度分析和激光扫描共聚焦显微镜技术,探讨H2O2在介导不同剂量UV-B诱导拟南芥叶片气孔关闭过程中的酶学来源途径。结果发现:0.5W.m-2 UV-B能诱导野生型拟南芥叶片保卫细胞的H2O2产生和气孔关闭,且该效应能被NADPH氧化酶抑制剂二苯基碘(DPI)抑制,而不能被细胞壁过氧化物酶抑制剂水杨基氧肟酸(SHAM)抑制,同时该剂量UV-B也不能诱导NADPH氧化酶功能缺失单突变体AtrbohD和AtrbohF以及双突变体AtrbohD/F保卫细胞的H2O2产生和气孔关闭;相反,0.65 W.m-2 UV-B既能诱导野生型也能诱导NADPH氧化酶突变体保卫细胞的H2O2产生和气孔关闭,且该效应能被SHAM抑制,却不能被DPI抑制。结果表明,不同剂量UV-B通过活化不同生成途径的H2O2来诱导拟南芥叶片气孔关闭,即低剂量UV-B主要诱导NADPH氧化酶AtrbohD和AtrbohF途径来源的H2O2生成,而高剂量UV-B主要活化细胞壁过氧化酶途径来源的H2O2。     

合意非洲铁羽片表皮及气孔器的发育研究

用光学显微镜和扫描电镜对合意非洲铁羽片表皮及气孔器的发育过程进行观察研究。结果显示,(1)在羽片发育过程中表皮细胞变化不明显,主要是细胞伸长和壁增厚的过程;表皮的角质层连续出现。(2)气孔器发育可大致分3个阶段:保卫细胞母细胞阶段、幼保卫细胞阶段和成熟保卫细胞阶段;气孔器发育方式为单唇型或周源型;成熟气孔器属单环型。(3)在羽片发育过程中,气孔密度起初很低,后急剧升高并达到峰值,之后逐渐下降并趋于稳定。(4)有早熟气孔器。(5)羽片表皮发育成熟的方式是由基部向顶部逐步推进。     

杜鹃花属照山白叶表皮及花粉形态的研究

利用光学显微镜和扫描电子显微镜对杜鹃花属植物照山白的叶表皮及花粉形态进行了观察,该种的叶表皮细胞表面光滑,胞间界限明显;叶上下表皮均有盾状鳞,上表皮仅有针状毛;气孔器仅见于下表皮,分布稀疏,副卫细胞和保卫细胞表面光滑。扫描电镜下可见该种的花粉为四合花粉,呈正四面体排列,具三沟,表面纹饰为负网状雕纹,局部区域有大颗粒状突起。     

安徽黄精属(Polygonatum)植物叶表皮研究

利用光学显微镜及扫描电子显微镜观察了安徽省黄精属９种植物的叶表皮;统计并测量了气孔类型、气孔大小、气孔密度及气孔指数等;描述了表皮角质膜、蜡质纹饰、气孔的形状及气孔外拱盖等有关特征。结果表明：黄精属植物叶表皮气孔器类型属只有２个保卫细胞而无任何副卫细胞的单子叶植物气孔器类型。气孔的某些特征,如气孔器类型、气孔器分布特征、表皮细胞形状及垂周壁式样、角质层及蜡质纹饰等在种间差异不大,可作种间区别的次级特征用。     

激光扫描共聚焦显微镜在植物学中的应用

激光扫描共聚焦显微镜(LSCM)是普通光学显微镜与激光和计算机及其相应的软件技术组合的产物,实现了连续光学切片,能在亚细胞水平观察细胞骨架的动态变化、细胞内特异蛋白、钙等离子的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。并广泛应用于生物三维结构重组及动态分析。本文综述了应用激光扫描共聚焦显微镜技术在植物细胞学、植物发育、组织化学以及基因表达、检测等领域取得的进展。     

Direct Measurements of Turgor Pressure Potentials of Guard Cells, I.

Measurements were made of pressures applied in subsidiary andguard cells which caused closure and opening of stomatal pores.These pressures were lower than would be expected from plasmolyticallydetermined osmotic potentials of guard cell saps. The pressuresneeded in guard cells to open almost closed stomata were practicallythe same as those required to close open stomata when appliedin adjacent subsidiary cells. Completely closed stomata couldnot be opened by this technique. The implications for the understandingof the mechanism of guard cell deformation, the Spannungsphase,and the pressure potentials of guard cells are discussed.     

Elevated carbon dioxide affects the patterning of subsidiary cells in Tradescantia stomatal complexes

The influence of elevated CO₂ concentration (670 ppm) on thestructure, distribution, and patterning of stomata in Tradescantialeaves was studied by making comparisons with plants grown atambient CO₂. Extra subsidiary cells, beyond the normal complementof four per stoma, were associated with nearly half the stomatalcomplexes on leaves grown in elevated CO₂. The extra cells sharedcharacteristics, such as pigmentation and expansion, with thetypical subsidiary cells. The position and shape of the extrasubsidiary cells in face view differed in the green and purplevarieties of Tradescantia. Substomatal cavities of complexeswith extra subsidiary cells appeared larger than those foundin control leaves. Stomatal frequency expressed on the basisof leaf area did not differ from the control. Stomatal frequencybased on cell counts (stomatal index) was greater in leavesgrown in CO₂-enriched air when all subsidiary cells were countedas part of the stomatal complex. This difference was eliminatedwhen subsidiary cells were included in the count of epidermalcells, thereby evaluating the frequency of guard cell pairs.The extra subsidiary cells were, therefore, recruited from theepidermal cell population during development. Stomatal frequencyin plants grown at elevated temperature (29 C) was not significantlydifferent from that of the control (24 C). The linear aggregationsof stomata were similar in plants grown in ambient and elevatedCO₂. Since enriched CO₂ had no effect on the structure or patterningof guard cells, but resulted in the formation of additionalsubsidiary cells, it is likely that separate and independentevents pattern the two cell types. Plants grown at enrichedCO₂ levels had significantly greater internode lengths, butleaf area and the time interval between the appearance of successiveleaves were similar to that of control plants. Porometric measurementsrevealed that stomatal conductance of plants grown under elevatedCO₂ was lower than that of control leaves and those grown atelevated temperature. Tradescantia was capable of regulatingstomatal conductance in response to elevated CO₂ without changingthe relative number of stomata present on the leaf. Key words: Elevated CO₂, stomata, subsidiary cells, patterning     

GFP-mTn融合蛋白对蓝猪耳生活细胞微丝骨架的标记

用农杆菌介导法将嵌合基因GFP-mTn(mTn是微丝结合蛋白Talin的微丝结合域,可以显示活体细胞中微丝的结构)导入蓝猪耳.经激光共聚焦显微镜观察了转基因植株的各种不同组织中融合蛋白的表达和分布情况.在叶片的表皮细胞、保卫细胞、根部的皮层细胞中有融合蛋白的不同程度表达.但仅在保卫细胞中微丝标记状况良好,显示基因表达的组织特异性.经光诱导处于开放态的气孔的保卫细胞微丝呈网状结构,在细胞内无规则分布;经黑暗诱导处于关闭态的气孔保卫细胞中微丝束沿保卫细胞纵轴排列,呈卷曲状分布,并观察到螺旋和环状的微丝结构.在转基因植株的其他部位,例如茎表皮细胞、根毛细胞和花粉粒中,未检测到目的基因的表达.本研究获得的转基因植株为研究气孔运动过程中微丝动态变化提供了有用的材料.     

GFP-mTn融合蛋白对蓝猪耳生活细胞微丝骨架的标记

用农杆菌介导法将嵌合基因GFP-mTn(mTn是微丝结合蛋白Talin的微丝结合域,可以显示活体细胞中微丝的结构)导入蓝猪耳。经激光共聚焦显微镜观察了转基因植株的各种不同组织中融合蛋白的表达和分布情况。在叶片的表皮细胞、保卫细胞、根部的皮层细胞中有融合蛋白的不同程度表达。但仅在保卫细胞中微丝标记状况良好,显示基因表达的组织特异性。经光诱导处于开放态的气孔的保卫细胞微丝呈网状结构,在细胞内无规则分布;经黑暗诱导处于关闭态的气孔保卫细胞中微丝束沿保卫细胞纵轴排列,呈卷曲状分布,并观察到螺旋和环状的微丝结构。在转基因植株的其他部位,例如茎表皮细胞、根毛细胞和花粉粒中,未检测到目的基因的表达。本研究获得的转基因植株为研究气孔运动过程中微丝动态变化提供了有用的材料。     

Variation in the structure and development of foliar stomata in the Euphorbiaceae

The structure and ontogeny of foliar stomata were studied in 50 species of 28 genera belonging to 17 tribes of the family Euphorbiaceae. The epidermal cells are either polygonal, trapezoidal, or variously elongated in different directions and diffusely arranged. The epidermal anticlinal walls are either straight, arched or sinuous. The architecture of cuticular striations varies with species. The mature stomata are paracytic (most common), anisocytic, anomocytic and diacytic. Occasionally a stoma may be tetracytic, cyclocytic or with a single subsidiary cell. The ontogeny of paracytic stomata is mesogenous dolabrate or trilabrate, mesoperigenous dolabrate; that of diacytic stomata is mesogenous dolabrate, whereas that of anisocytic stomata is mesogenous trilabrate; rarely an anisocytic stoma may be mesoperigenous. Hemiparacytic stomata are mesoperigenous unilabrate; tetracytic stomata are mesoperigenous dolabrate and anomocytic stomata perigenous. Abnormalities encountered include four types of contiguous stomata, stomata with a single or both guard cells aborted and persistent stomatal initials. Cytoplasmic connections between the guard cells of two adjacent stomata or the guard cell of a stoma and an adjacent epidermal/subsidiary cell, or both types occurring in a species, were noticed. The stomatal development, distribution, diversity and basic stomatal type with reference to systematics are discussed.     

Epidermal Structure and Development of Stomata in some Annonaceae

Epidermal structure and development of stomata are studied in20 species of the Annonaceae. Epidermal cells are polygonalor irregular in outline, isodiametnc or elongated with straight,arched, or sinuous anticlinal walls. Cuticular striations radiatingfrom guard cells or hair bases are noticed. Four types of trichomesand secretory cells are seen. The mature stomata are paracyticwith 2–6 subsidiary cells, non-contiguous at both poles,or contiguous at one or both the poles. The increase in numberof subsidiary cells is due to their divisions. Contiguous stomata,arrested development, single guard cell, and degeneration ofguard cells are observed. The development is either syndetocheilicor mesogenous.     

Some new observations and interpretations on the vital staining of leaf epidermal tissue by neutral red

Summary The lower leaf epidermis from 5 plant species was stained with neutral red at 2 pH's (7.1 and 5.6) in the light and dark when the stomata were open or closed. At pH 5.6 no globule (= droplet) formation was observed in the guard cells whether the stomata were open or closed and cell walls possessed a high affinity for the stain. At pH 7.1 globules appeared in guard cells of open stomata, but not closed stomata, within 15 minutes. Anaerobic conditions prevented this globule formation. InZea mays, globules also appeared in subsidiary cells when the stomata were closed and in certain epidermal cells. Where globule formation did not occur increased diffuse staining of certain epidermal cells was considered to be the indication of cell integrity. In old leaf material very large numbers of dark blue globules appeared in epidermal cells ofCommelina diffusa, C. communis andSenecio odoris and this was associated with cell senescence.The staining characteristics were discussed in terms of cellular K⁺, Cl^–, tannin and flavonoid content and vacuolar pH.     

Stomatal Features in the Leaf of Aganosma dichotoma (Roth) K. Schum

Paracytic and anisocytic types of mature stomata are found inthe leaf of Aganosma dichotoma. Stomata with one guard cell,stomata with degenerated guard cells, and contiguous stomataare common. Stomata with arrested pore development are alsofound in certain cases. A single guard cell without any porehas not been designated as a stoma with one guard cell in thepresent investigation. Ontogeny of contiguous stomata have beentraced. Subsidiary cells are, morphologically, just like theircontiguous guard cells. Subsidiary cells may retain their shapeand contents even when their contiguous stoma becomes mature,or may change their shape and lose their contents. They mayor may not divide. Subsidiary cells form a whorl of more thantwo subsidiary cells around a stoma by their divisions. Degenerationof guard cell(s)— their contents and nuclei—havebeen traced. In certain cases guard cells divide forming morethan two guard cells associated to a single pore. Cytoplasmicconnections are found between two guard cells of nearby stomata,and between a guard cell and an epidermal cell. Near the wound,the epidermal cells over the veins become meristermatic givingrise to new epidermal cells but no meristemoid.     

Epidermal Structure and Stomatal Development in Some Malvaceae and Bombacaceae

Epidermal structure and development of stomata in 15 speciesof the Malvaceae and two of Bombacaceae are described. The cellsof the epidermis are polygonal, isodiametric, or elongated withthick, straight, arched, or sinuous anticlinal walls and containchloroplasts and abundant druses of calcium oxalate. Cuticularstriations radiating from the guard cells or hair bases arenoticed. Six types of glandular and non-glandular trichomesare seen. The mature stomata are anomocytic, anisocytic, andparacytic in the members investigated of both the families.The ontogeny of anisocytic and paracytic stomata is syndetochelicor mesogenous, while that of anomocytic is haplocheilic or perigenous.An abnormal stoma with a single guard cell is also observed.An increase in number of subsidiary cells in anisocytic stomatais due to the division of the subsidiary cells.