蚕豆保卫细胞原生质体质膜ABA结合蛋白与气孔对ABA敏感性的关系

在测定蚕豆(Vicia faba L.)保卫细胞原生质体质膜ABA结合蛋白的解离常数(Kd)和最大结合容量的基础上,进一步研究了几种因子(pH、光)对Kd和最大结合容量的影响。结果显示:pH并不改变结合蛋白的Kd值,而仅影响每个原生质体结合的分子数;光、暗处理的结果表明,光可使结合蛋白的Kd值增大,每个原生质体结合的ABA分子数减少,而暗处理可使结合蛋白的Kd值减小及每个原生质体结合的分子数增多,说明黑暗可提高气孔保卫细胞对ABA的敏感性,而光可以降低气孔保卫细胞对ABA的敏感性。因此,光、暗可以通过调节ABA结合蛋白的Kd值来调节气孔对ABA的敏感性。     

Comparison of physiological responses of guard cell protoplasts of Nicotiana glauca isolated from leaves collected before dawn or at midday

We observed that guard cell protoplasts isolated from leaves collected at midday from Nicotiana glauca Graham (tree tobacco) did not give the same physiological responses to light as those isolated from leaves collected in early morning. Based on that observation, we attempted to determine whether there were significant differences between the physiological responses of guard cell protoplasts isolated from leaves collected before dawn (with closed stomata) and those isolated from leaves collected at midday (with open stomata). We isolated guard cell protoplasts from leaves collected before dawn and at midday and compared (1) rates of red and blue light-induced pH changes in weakly buffered media caused by changes in their metabolism, (2) their rates of oxygen consumption in darkness and oxygen evolution in light and (3) relative rates of decay of variable chlorophyll a fluorescence in their chloroplasts. Studies with the vital stain fluorescein diacetate failed to reveal any significant differences in the viabilities of protoplast preparations from leaves collected before dawn and at midday. Furthermore, protoplasts from leaves collected at these times swelled to similar extents in an osmotic medium containing 10 µM fusicoccin and 5 mM KCI. Nevertheless, rates of light-induced pH changes, rates of oxygen consumption and evolution and rates of decay of variable chlorophyll a fluorescence were all lower in preparations of guard cell protoplasts from leaves collected at midday than in preparations from leaves collected before dawn. Initial volumes of guard cell protoplasts isolated from leaves collected at midday were 150% of those of guard cell protoplasts isolated from leaves collected before dawn. We suggest that the differences in responses of guard cell protoplasts isolated from leaves collected before dawn and at midday may be caused by (1) nonoptimal isolation conditions for guard cell protoplasts prepared from leaves collected at midday, (2) the lower surface-to-volume ratio of guard cell protoplasts isolated from leaves collected at midday or (3) diurnal and/or circadian regulation of guard cell metabolism over the course of a day.     

蛙心包淋巴孔的发现

首次报道蛙心包淋巴孔 ,揭示心包腔淋巴转归途径。本实验应用扫描电镜和透射电镜观察心包淋巴孔的超微结构 ,并使用计算机图象处理技术对淋巴孔作定量分析。结果发现 ,正常蛙心包腔面有一些散在分布的心包淋巴孔和少量淋巴窦。构成淋巴孔的间皮细胞常出现粗大的胞质突起 ,伸入淋巴孔 ,形成瓣膜状结构。淋巴孔的平均直径为 0 72±0 33μm ,平均分布密度是 3 57± 2 0 7个 /0 0 1mm2 ;心包间皮淋巴窦的面积是 995 0 8±2 2 1 74μm2 /0 0 1mm2 。蛙心肌无血管 ,其血供仅由心腔内血液直接进入心肌的小梁间隙。心包脏层未发现有淋巴孔。结果表明 :间皮淋巴窦是心包膜正常“漏出”的形态依据。心包淋巴孔的发现 ,证明心包腔淋巴引流途径的存在。淋巴引流对于心肌组织间液的平衡 ,清除组织间液蛋白质 ,防止心肌间质水肿 ,有重要意义     

Flavonol content of guard cell and mesophyll cell protoplasts isolated from Vicia faba leaves

Guard cells of the lower epidermis of leaflets of Vicia faba L. cv. Weißkernige Hangdown contain several kaempferol 3,7-O-glycosides. This was demonstrated for the first time by the use of isolated, highly purified guard cell protoplasts for flavonol estimation and quantitation. From a total of ca 12 kaempferol glycosides, three were identified by comparative thin layer chromatography and high performance liquid chromatography as kaempferol 3-O-glucoside 7-O-rhamnoside (major component), 3-O-rhamnogalactoside 7-O-rhamnoside and 3,7-O-bisglucoside (minor components). On average, the total flavonol content was estimated to be 85 fmol protoplast⁻¹. From comparative investigations including alkaline-induced (green) fluorescence characteristics of flavonols and UV-microscopical studies we suggest that kaempferol glycosides are present in guard cells and epidermal cells in similar quantities, and that these compounds are in the vacuole.
By contrast, mesophyll protoplasts have a low flavonol content (one sixth that of guard cells). In spite of the different total flavonol contents, individual components of each cell-type are the same. However, they show differences in their quantitative distribution.     

裂叶沙参气孔行为与光合蒸腾特性通径分析

通过相关系数和通径系数分析方法,对不同海拔高度裂叶沙参(Adenophora lobophylla)气孔行为与光合、蒸腾特性的关系进行了相关性分析。气孔行为对光合、蒸腾均缺乏显著的相关性,说明裂叶沙参光合、蒸腾作用的气孔控制不显著;裂叶沙参叶片气孔开度直接影响光合速率和胞间CO₂浓度,气孔导度对裂叶沙参蒸腾速率影响较大。     

中国西北地区松科和柏科气孔器形态

现代针叶树气孔器的研究为鉴定化石气孔器奠定了基础,对第四纪植被变化和古气候的研究起着很重要的作用,是第四纪孢粉学的一个重要补充。本文运用常规的标准的孢粉分析方法,对中国西北地区常见的松科3属8种和柏科3属4种植物气孔器进行了观察分析。结果表明凭气孔器大小可以区分松科与柏科。利用气孔器大小、T型结构的形状?上部木质片与气孔器茎之间的夹角进行属的鉴别,再结合茎的长度、宽度、中间木质部的宽、T型外角度和上部木质片外缘形状等可以进行种的鉴别,并编制了一个初步的检索表。     

蚕豆保卫细胞原生质体质膜ABA结合蛋白的理化特性

以蚕豆(Vicia faba L.)保卫细胞原生质体为材料,证明在其质膜外侧存在着对ABA高亲和力的结合蛋白。这种蛋白与ABA作用的最适pH为6.5;在25℃时.其特异结合比高于0℃时的特异结合比;在温育30min时即达到其最大结合。它与ABA作用的解离常数为2.0×10~(-8)mol/L,每个原生质体上大约有3.2×10~6个结合位点。该结合蛋白内部具有维持其功能所必须的二硫键,而且其功能的发挥要求介质中有一定浓度的Ca~(2 )的存在。     

家兔肋胸膜淋巴孔的发现

为了研究成年家兔肋胸膜淋巴孔的超微结构与三维构形 ,作者应用扫描电镜和透射电镜对成年家兔肋胸膜淋巴孔进行观察 ,用计算机图像处理系统对胸膜淋巴孔作图像数据化处理 ;NaOH溶液消化间皮细胞 ,裸露间皮下结缔组织和筛斑 (maculacribriform)。发现肋胸膜立方形间皮细胞 (cuboidalmesothelialcell)之间有圆形或椭圆形的胸膜淋巴孔 (pleurallymphaticstomata) ,其平均面积和平均密度分别为 :7 2 0± 3 6 9μm2 和 1 2 1±0 72个 / 0 0 1mm2 。扁平形间皮细胞表面未见淋巴孔。胸膜淋巴孔籍胸膜下小管与毛细淋巴管相通。仅在立方形间皮下结缔组织中发现有筛斑。肋胸膜上还可见闭合淋巴孔 (closedlymphaticstomata)和由巨噬细胞组成的乳斑 (milkyspot)。覆盖在肋骨上的胸膜无淋巴孔分布。家兔胸膜淋巴孔通过胸膜下小管与淋巴管直接相连 ,形成从胸膜腔至脉管系的惟一直接通路。     

小鼠膈腹膜淋巴孔和膈淋巴管的发育

应用透射电镜、扫描电镜和酶组织化学方法,研究胚胎期和出生后不同时期小鼠膈腹膜淋巴孔（PLS）和膈淋巴管的发生和发育,并用Elescope计算机图像处理技术对PLS进行定量分析。结果发现：胚胎13天时,膈腹膜仅由扁平形间皮细胞（FMC）组成;胚胎15天时,FMC间出现立方形间皮细胞（CMC）和早期腹膜淋巴孔（NLS）;胚胎18天时,膈毛细淋巴管出现,台盼蓝吸收试验显示NLS无物质吸收功能;出生后1天（PND1）,膈毛细淋巴管内皮细胞向PLS伸出胞质突起,并横跨CMC下结缔组织纤维和基底膜,形成腹膜下小管。后者与PLS沟通,建立了腹膜腔内物质转归通路。台盼蓝吸收试验表明,出生后PLS具有物质吸收功能,即为成熟腹膜淋巴孔（MLS）。PND5,立方细胞嵴（CMCR）发生,膈毛细淋巴管数量增多。PND10,大量立方细胞嵴融合,形成条带状分布的立方细胞区域,其上分布有大量MLS。随着发育进展,MLS平均面积和平均分布密度逐渐增大,且随着膈淋巴管的发育,吸收功能逐渐增强。     

罗布麻属与白麻属叶表面气孔形态的比较观察——兼论Poacynum的归属

本文报道了罗布麻属和白麻属植物叶表面气孔形态的比较观察。结果表明,两者气孔形态的微观性状十分相似,同属一个类型。结合花部形态的比较分析,认为Poacynum Baill。独立成属的依据是不充分、不合理的,应将之归并回罗布麻属Apocynum L 中。列举了原白麻属所隶下2个种的正确学名。     

妊娠和激素干预对豚鼠卵巢囊淋巴孔的影响

本实验以台盼蓝为示踪剂,观察成年雌豚鼠在妊娠、注射促排卵激素和雄激素时,卵巢囊淋巴孔对示踪剂的吸收情况,并结合扫描电镜和计算机图像处理,对豚鼠卵巢囊内壁的淋巴孔进行观察和统计,以研究妊娠和激素干预对豚鼠卵巢囊淋巴孔开放和淋巴吸收功能的影响。结果表明,妊娠组卵巢囊吸收示踪剂的量最多,促排卵组次之,雄激素组最少,且组间两两比较均有显著性意义(P<0.05)。在每1000μm~2的视野中,妊娠组卵巢囊淋巴孔的开放面积为189.9±48.7μm~2,促排卵组为104.4±31.2μm~2,雄激素组为40.5±18.7μm~2,组间两两比较均有显著性意义(P<0.05)。妊娠组卵巢囊内层纤毛柱状上皮的分泌颗粒数量最少,而促排卵组的分泌颗粒较多,且纤毛最活跃。本实验研究结果表明,妊娠和激素干预能影响豚鼠卵巢囊淋巴孔的开放和吸收,囊内卵巢的功能状态与豚鼠卵巢囊淋巴孔的调控密切相关。     

Regulation of blue-light-induced proton pumping by Vicia faba L. guard-cell protoplasts: Energetic contributions by chloroplastic and mitochondrial activities

An initial response during signal transduction in guard cells, following absorption of blue light, is the extrusion of protons. Translocation of protons across the guard-cell plasmalemma is an energy-requiring activity. The present study has investigated the energetic contribution from guard-cell chloroplasts and mitochondria to blue-light-induced proton pumping by Vicia faba guard-cell protoplasts. The addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea to the protoplast suspension had a minimal effect on rates of acidification when oxygen concentrations of the medium were maintained close to near-saturating levels. Under the same conditions, oligomycin reduced both the rates of blue-light-induced acidification and total proton efflux. Lowering the oxygen concentration of the suspending medium to approximately 20 M resulted in complete inhibition of blue-light-induced acidification activity. Swelling of protoplasts induced by blue light was also inhibited by low oxygen levels. Levels of ATP from whole-protoplast extracts were reduced by about 64% when exposed to low levels of oxygen. Increasing oxygen levels to near-saturating levels restored both blue-light-induced acidification rates and swelling of the protoplasts within a 60-min recovery period. Levels of ATP also increased during the recovery period. Addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or oligomycin to the suspending medium prior to increasing the oxygen concentration caused a reduction in acidification rates after the recovery period by 40 and 80%, respectively. Levels of ATP in guard-cell protoplasts were also reduced by both inhibitors after a 60-min recovery period. The results demonstrate that both guard-cell chloroplasts and mitochondria contribute energetically to blue-light-induced proton pumping by guard-cell protoplasts. Furthermore, both energy sources are inhibited by low oxygen concentrations, suggesting coordinated metabolic regulation between photo- and oxidative phosphorylation in guard cells.Abbreviations BL blue light - Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - GCPs guard-cell protoplasts This research was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada and a University Research Grant from The University of Calgary. Dr. L. Gedamu (University of Calgary) is thanked for providing access to the bioluminometer. Technical assistance by C. Chmielewski, C. Turnnir, S. Ham and K. Meyer is gratefully acknowledged.     

豚鼠卵巢囊淋巴孔的发现及卵巢囊超微结构研究

本实验通过扫描电镜等方法研究豚鼠卵巢囊及卵巢囊淋巴孔的结构,并探讨了卵巢囊及其淋巴孔的种属差异.实验结果首次报道豚鼠卵巢囊内、外层上皮均存在淋巴孔;豚鼠卵巢囊结构在输卵管走行、囊闭合程度及囊表面超微结构等方面与金仓鼠存在差异.结果提示豚鼠卵巢囊内、外层淋巴孔是沟通卵巢囊腔、卵巢囊淋巴系及腹膜腔的形态基础,可能是三者间物质转运的重要途径.卵巢囊淋巴孔可能影响物种的繁殖并参与囊腔内局部免疫反应.卵巢囊结构和发育程度与对应的输卵管伞端等结构及其他生殖特点相适应,研究结果丰富了生殖形态学和比较解剖学资料.     

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent K _m (phosphoenolpyruvate) and K _i (malate) were compared. Malate sensitivity was much higher (K _i malate 0.4 mol m^–3) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (K _i malate 4.2 mol m^–3). The stimulation of activity by glucose-6-phosphate in the presence of malate (deinhibition) was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.Abbreviations Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase     

The chemiosmotic model of stomatal opening revisited

Stomata are light‐activated biological valves in the otherwise gas‐impermeable epidermis of aerial organs of higher plants. Stomata often regulate rates of photosynthesis and transpiration in ways that optimize whole‐plant carbon gain against water loss. Each stoma is flanked by a pair of opposing guard cells. Stomatal opening occurs by light‐activated increases in the turgor pressure of guard cells, which causes them to change shape so that the stomatal pore between them widens. These increases in turgor pressure oppose increases in cellular osmotic pressure that result from uptake of K⁺. K⁺ uptake occurs by a chemiosmotic mechanism in response to light‐activated extrusion of H⁺ outward across the plasma membrane of the guard cell. The initial changes in cellular membrane potential lead to the opening of inward‐rectifying K⁺ channels, after which K⁺ is taken up along its electrochemical gradient. Changes in membrane potential resulting from K⁺ uptake may be balanced by accumulation of Cl^?ions by guard cells and/or by synthesis of malic acid within each cell. Malic acid also acts to buffer increases in cytosolic pH caused by H⁺ extrusion. This review describes how the application of patch‐clamp technology to guard cell protoplasts has enabled investigators to elucidate the mechanisms by which H⁺ is extruded from guard cells, the types of ion channels present in the guard cell plasma membrane, how those ion channels are regulated, and the signal transduction processes that trigger stomatal opening and closing.     

Arabinogalactan-proteins stimulate the organogenesis of guard cell protoplasts-derived callus in sugar beet

Arabinogalactan proteins (AGPs) represent a class of proteoglycans implicated in the development and differentiation of cells and tissues both in planta and in vitro. Here we report that AGP-rich extracts isolated from media of embryogenic and non-embryogenic suspension cultures of sugar beet (Beta vulgaris L.) are able to enhance the organogenesis of guard protoplast-derived callus and to increase the number of shoots formed, in comparison to control cultures. Immunocytochemical detection of carbohydrate antigens in the extracts revealed the presence of epitopes that typify both AGP and pectin, the latter being frequently bound to AGPs or, in some cases, even contributing to the polysaccharide structure of proteoglycan molecules. The most abundant epitopes proved to be those recognized by the JIM13, LM2, and MAC207 antibodies, whereas some others could be found only in relatively small or trace amounts--these included epitopes recognized by JIM16, JIM5, and LM6. Surprisingly, the JIM4- and JIM8-binding epitopes that are expressed in the course of in vitro morphogenetic processes of many species could not be detected at all in sugar beet AGPs. This is the first report of the improvement of sugar beet protoplast-derived callus organogenesis by exogenous AGP-rich extracts, an achievement that will have great impact on the biotechnological applications of protoplast technology in this species.     

DEVELOPMENTAL STUDIES IN PORPHYRA (RHODOPHYCEAE). III. EFFECT OF CULTURE CONDITIONS ON WALL REGENERATION AND DIFFERENTIATION OF PROTOPLASTS1

Protoplasts isolated from thalli of four Porphyra species regenerated successfully into differentiated plantlets. The efficiency of protoplast isolation and the developmental patterns of the regenerating protoplasts depended on the type of tissues from which they were isolated. However, culture conditions greatly influenced the patterns of development at the cellular and organismal levels. Sorbitol, nitrogen, and agar concentration in the medium controlled rates of cell division, thickening of cell walls, development of rhizoids, and formation of calluses or differentiated blades. Agitation disturbed the attachment of the protoplasts to a substrate. Cells in agitated cultures produced suspensions of single cells and non-polarized small calluses. Calluses which developed from protoplasts survived in storage for over two years. The stored calluses, and cells and protoplasts that were isolated from them, were subcultured successfully. We forsee extensive use of Porphyra cell suspensions for strain selection and vegetative propagation of cultivars. This technology, which makes vegetative cloning of selected Porphyra plants possible, may eliminate the need for cultivation and storage of the conchocelis phase. Protoplasts are also being used as tools for studies in genetic engineering of these commercial species.