美人蕉作植物解剖实验材料

美人蕉(Canna indica),为美人蕉科植物,常见的花卉品种,各地均有栽培。具有取材方便,根尖粗,结构明显,细胞大,容易观察等优点。一、观察表皮细胞、气孔和叶绿体 1、用镊子撕下叶片下表皮,制成水装片,镜检:可看到表皮细胞壁不规则,明显的细胞核;气孔器有二个绿色,肾形保卫细胞组成,气孔呈椭圆形。 2、用刀片刮取叶肉少许,制成水装片,镜检:清楚可见二种形态(球型和长柱型)叶肉细胞内,分布明显的椭圆形或近球     

H+参与茉莉酸调控蚕豆气孔运动的信号转导

以BCECF-AM为pH的荧光探针,结合激光共聚焦扫描显微技术,研究H 可能参与茉莉酸(JA)调控气孔运动信号转导途径的结果表明,0.1~100μmol·L~(-1)浓度的(-)JA可诱导蚕豆气孔关闭,在引起气孔孔径改变之前,(-)JA能引起蚕豆保卫细胞胞质的碱化;而(±)JA可诱导气孔适当开放,它未引起蚕豆保卫细胞胞质中pH的明显改变。药理学实验证明,质膜上质子泵的抑制剂矾酸钠能减弱(-)JA诱导气孔关闭的作用;而质膜上质子泵的激活剂壳梭孢菌素(fusicoccin)基本上未改变(±)JA的作用趋势。(-)JA和(±)JA刺激保卫细胞胞质Ca2 变化则表现出不同趋势。说明不同异构体形式的JA在调节气孔运动中的作用和信号转导途径有所不同。     

植物体表面的通道

植物在生长发育的过程中，要不断与外界进行气体和水分的交换。而植物体各器官表面的气孔是这一交换的主要通道，气孔是如何起调控作用的呢？ 首先，让我们先看一看气孔的结构，气孔的结构因植物种类而异，双子叶植物表皮的气孔是由两个半月形的保卫细胞围合而成，保卫细胞内含有叶绿体，它的细胞壁在靠近气孔器的一面较厚，其它面较薄。而单子叶植物上的气孔的保卫细胞为哑铃形，两端膨大、壁较薄，中部的细胞壁较厚，保卫细胞两边还有一对副卫细胞。不论是哪一类植物上的气孔，当保卫细胞从邻近表皮细胞吸水而膨胀，气孔就张开；而当保卫…     

保卫细胞的光合作用在光调节的气孔运动中的功能

本文介绍植物叶片上保卫细胞中叶绿体在光诱导气孔开放过程中的作用等研究进展,并对叶肉细胞中的光合作用与气孔运动之间的关系也作简要分析和讨论。     

Ca2+参与茉莉酸诱导蚕豆气孔关闭的信号转导

以Fluo-3AM为Ca~(2 )荧光探针,结合激光共聚焦扫描显微技术,观察到在处理后数十秒内,气孔关闭之前,茉莉酸(JA)可引起[Ca~(2 )]cyt的迅速上升;叶照和JA的前体物亚麻酸(LA)几乎不能引起[Ca~(2 )]cyt的明显变化;钙的螯合剂EGTA预处理可完全阻断JA诱导气孔关闭的效应,并且JA不再引起保卫细胞[Ca~(2 )]cyt增加;质膜Ca~(2 )通道的抑制剂硝苯吡啶(nifedipine,NIF)可减弱JA诱导气孔关闭的效应,也使JA诱导保卫细胞[Ca~(2 )]cyt增加的幅度有所下降;胞内Ca~(2 )释放的抑制剂钌红不能明显改变JA诱导气孔关闭的趋势,但使JA引起的保卫细胞[Ca~(2 )]cyt增加有所降低。实验结果表明:Ca~(2 )参与JA诱导气孔关闭的信号转导;推测JA引起的[Ca~(2 )]cyt升高可能主要来源于胞外,但不能完全排除胞内Ca~(2 )的释放。     

Ca^2＋参与茉莉酸诱导蚕豆气孔关闭的信号转导

以Fluo-3 AM为Ca^2＋荧光探针,结合激光共聚焦扫描显微技术,观察到在处理后数十秒内,气孔关闭之前,茉莉酸（JA）可引起[Ca^2＋]cyt的迅速上升;对照和JA的前体物亚麻酸（LA）几乎不能引起[Ca^2＋]cyt的明显变化;钙的螯合剂EGTA预处理可完全阻断JA诱导气孔关闭的效应,并且JA不再引起保卫细胞[Ca^2＋]cyt增加;质膜Cah通道的抑制剂硝苯吡啶（nifedipine,NIF）可减弱JA诱导气孔关闭的效应,也使JA诱导保卫细胞[Ca^2＋]cyt增加的幅度有所下降;胞内Ca^2＋释放的抑制剂钌红不能明显改变JA诱导气孔关闲的趋势,但使JA引起的保卫细胞[Ca^2＋]cyt增加有所降低。实验结果表明：Ca^2＋参与JA诱导气孔关闭的信号转导;推测JA引起的[Ca^2＋]cyt升高可能主要来源于胞外,但不能完全排除胞内Ca^2＋的释放。     

盾叶薯蓣四倍体与二倍体叶表皮及气孔器显微结构的比较

对盾叶薯蓣四倍体和二倍体阴叶和阳叶表皮及气孔器的显微形态结构进行了比较。结果显示:叶片表皮细胞及气孔器的形态结构因染色体倍性及光照的不同而存在明显差异。表皮细胞有规则多边形与不规则细胞壁内褶2种类型;气孔器为典型的双子叶植物无规则型气孔器,仅分布在下表皮。四倍体表皮细胞密度、气孔器密度及气孔指数平均值分别为476.28±6.87个.mm-2、78.22±3.1个.mm-2、14.11,较二倍体的分别小4.60%、17.95%和11.98%。前者气孔器长、宽及保卫细胞宽度平均值分别为32.78±2.09μm、26.07±1.55μm、9.63±1.14μm,较后者的分别大10.73%、3.90%和18.01%,差异极显著。前者阴叶的较其阳叶的分别大5.77%、6.00%、8.72%;后者的则分别大1.72%、1.74%、2.41%。前者叶片保卫细胞中的叶绿体数目为23.93±3.19个,较后者的多74.00%。表皮细胞及气孔器密度、气孔指数,气孔器大小,保卫细胞叶绿体数目、尤其是保卫细胞宽度,可作为倍性鉴定的参考依据。四倍体高产有其良好的叶片结构基础。     

蚕豆叶片细胞中IAA的胶体金免疫电镜定位

利用胶体金免疫电镜技术对蚕豆(Vicia faba L.)叶片细胞中的IAA定位进行了研究。幼嫩叶片的叶肉细胞中金颗粒主要分布在细胞核和叶绿体中,细胞质及细胞壁也有金颗粒标记。成熟叶片的叶肉细胞中金颗粒主要分布在叶绿体和细胞质,细胞壁也有少量金颗粒标记,液泡中没有发现金颗粒标记。成熟叶片小叶脉的韧皮细胞发现有大量的金颗粒标记,金颗粒主要标记在传递细胞的细胞壁中。小叶脉的维管束鞘细胞中也有很多的金颗粒标记,金颗粒主要分布在叶绿体、细胞质及细胞壁中。幼嫩叶片组织不进行IAA的固定或用正常兔IgG代替IAA抗体染色的对照,很难发现金颗粒标记。对IAA在组织及亚细胞中的定位及其生理意义进行了讨论。     

蚕豆保卫细胞中钙调素的免疫电镜定位

以蚕豆横切和平切气孔为材料,对钙调素进行了免疫胶体金电镜定位的结果表明:在蚕豆保卫细胞的细胞核、细胞质、细胞膜、叶绿体、液泡、高尔基体、细胞壁中都有金颗粒分布,在线粒体上的分布较少.     

落羽杉属(杉科)叶表皮结构及气孔参数

落羽杉属Taxodium Rich.现生3种植物——落羽杉T. distichum (L.) Rich.、池杉T. ascendens Brongn.和墨西哥落羽杉T. mucronatum Tenore.的条形叶为双面气孔型或单面气孔型。叶片远轴面气孔分布于中脉两侧,每侧各有4-8列气孔。叶片中部气孔数量稳定,顶部和基部气孔数量比中部略少。近轴面气孔在中脉两侧各有1-4行,有时仅少数几个气孔或没有气孔分布。非气孔分布区内,表皮细胞长方形,细胞壁直或稍微呈波状,细胞长轴与叶片长轴一致。气孔分布区内的表皮细胞有时为多边形。气孔器椭圆形,长轴与叶片长轴垂直或成一定的角度。保卫细胞壁加厚明显,极端联合形成极层结构。落羽杉属3种现生植物的气孔密度和气孔指数差异显著,不同采集地的落羽杉气孔密度和气孔指数差异不显著。这3种植物的气孔指数的变异系数均小于气孔密度的变异系数,用气孔指数指示大气CO2浓度比用气孔密度可靠。     

Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Mechanical stress was one of stresses with whichplants often met. With the development of fruit andvegetable finish machining in food industry, artificialinjury also appeared. As response to other stresses,plants have evolved with some adaptive mechanismsto cope with wounding[1]. Jasmonic acid (JA) andmethyl jasmonate (MeJA), as important signal mole-cules in plant response to wounding, have attracted agreat deal of attention. The studies on some crops, suchas potato[2], rice[3], and tomato[…     

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.     

Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Pea seedlings (Pisum sativum L.) were used as materials to test the timings and compartments of hydrogen peroxide (H₂O₂) triggered by wounding and exogenous jasmonic acid (JA). The results showed that H₂O₂ could be systemically induced by wounding and exogenous JA. H₂O₂ increased within 1 h and reached the peak 3–5 h after wounding in either the wounded leaves or the unwounded leaves adjacent to the wounded ones and the inferior leaves far from the wounded ones. After this, H₂O₂ decreased and recovered to the control level 12 h after wounding. The activities of antioxidant enzymes, however, were rapidly increased by wounding. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, could significantly inhibit H₂O₂ burst that was mediated by wounding and exogenous JA. Assay of H₂O₂ subcellular location showed that H₂O₂ in response to wounding and exogenous JA was predominantly accumulated in plasma membrane, cell wall and apoplasmic space. Numerous JA (gold particles) was found via immunogold electron microscopy to be located in cell wall and phloem zones of mesophyll cell after wounding.     

Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua: Analysis of Fluorescence Kinetics as a Function of Excitation Intensity

The chlorophyll fluorescence induction curves from mesophyll and guard cell chloroplasts of Saxifraga cernua, including both the fast (O to P, the transients involved in the rise in variable fluorescence) and slow (P to steady state fluorescence due to quenching) components, were characterized over a range of excitation intensities using microspectrophotometry (with epi-lumination) equipped with apertures designed to eliminate cross contamination of the fluorescence signal between the two chloroplast types. At low excitation intensities, the fast fluorescence kinetics from guard cell plastids showed an extended I to D phase and a more rapid appearance of P while minimal quenching from P to steady state fluorescence was observed compared to the transients from mesophyll chloroplasts suggesting a lower activity of photochemical (electron movement via carriers between donor and acceptor sites) and nonphotochemical (such as membrane conformational changes) events which regulate the fluorescence induction curve kinetics. As the excitation intensity was increased, the quenching rates of guard cells were faster at initiating conditions for photophosphorylation and the fast and slow fluorescence kinetics from guard cells resembled those of the mesophyll cells.

Guard cell chloroplasts of S. cernua from intact epidermal peels showed a low temperature (77 K) fluorescence emission spectrum having three major peaks (at 685, 695, and 730 nanometers when excited at 440 nanometers) which were qualitatively similar to those in the spectrum obtained from mesophyll tissue.

These data suggest that S. cernua guard cell chloroplast photosystems I and II contribute to light-dependent stomatal activity only at high light intensities.

     

Guard cell wall: immunocytochemical detection of polysaccharide components

The composition of guard cell walls in sugar beet leaves (Beta vulgaris L.) was studied by using histochemical staining and immunocytochemical detection of cell wall antigens. The findings were compared with those in the walls of epidermal and mesophyll cells. Probing of leaf sections with monoclonal antibodies against pectins, terminal fucosyl residues linked alpha-(1-->2) to galactose, beta-(1-->3)-glucans and arabinogalactan-proteins revealed several specific features of guard cells. Pectic epitopes recognized by JIM7 were homogeneously distributed in the wall, whereas pectins recognized by JIM5 were not found in the walls themselves, but were abundant in the cuticular layer. Large amounts of molecules bearing terminal fucose were located predominantly in ventral and lateral guard cell walls. Much smaller amounts were detected in dorsal walls of these cells, as well as in the walls of pavement and mesophyll cells. Conspicuous accumulation of these compounds was observed in the vicinity of the guard cell plasmalemma, whereas labelling was scarce in the areas of the wall adjacent to the cell surface. The presence of callose clearly marked the ventral wall between the recently formed, very young guard cells. Callose also appeared in some mature walls, where it was seen as punctate deposits that probably reflected a specific physiological state of the guard cells. Large amounts of arabinogalactan-proteins were deposited within the cuticle, and smaller amounts of these proteoglycans were also detected in other tissues of the leaf. The histochemical and immunocytochemical structure of the guard cell wall is discussed in the light of its multiple functions, most of which involve changes in cell size and shape.     

Malate Dehydrogenases in Guard Cells of Pisum sativum

Guard cell protoplasts of Pisum sativum show considerable NADP-dependent malate dehydrogenase (MDH) activity in darkness which can be enhanced severalfold by illumination or treatment with dithiothreitol (DTT). The question arose whether guard cells possess an NADP-MDH different from that present in the chloroplasts of the mesophyll (which is inactive in darkness or in the absence of DTT). MDH activities were determined in extracts of isolated protoplasts from mesophyll and epidermis, and in mechanically prepared epidermal pieces (with guard cells as the only living cells and no interference from proteases originating from the cell wall digesting enzymes). Guard cells possessed NAD-dependent MDHs of high activity and incomplete exclusion of NADP as a coenzyme. This NADP-dependent activity of the NAD-MDH(s) could not be stimulated by DTT or, inferentially, by light. The DTT- (and light-) dependent NADP-MDH represented 0.05% of the total protein of the guard cells and had a specific activity of 0.1 unit per milligram protein; both values are in the same range as the corresponding ones of the mesophyll cells. Agreement was also found in the extent of light activation, in subunit molecular weight, immunological cross-reactions, and in the behavior on an ion exchange column. The activity of the chloroplastic NADP-MDH in guard cells barely suffices to meet the malate requirement for stomatal opening in the light. It is therefore likely that NAD-MDHs residing in other compartments of the guard cells supplement the activity of the chloroplastic NADP-MDH particularly during stomatal opening in darkness.     

The kinetics of in vivo state transitions in mesophyll and guard cell chloroplasts monitored by 77 k fluorescence emission spectra

Fluorescence emission spectral peaks at 685, 695 and 730 nanometers (F685, F695, and F730) were recorded 77 K from diluted leaf tissue and epidermal powders prepared from Saxifraga cernua. The time course for state 1 to state 2 transitions was monitored as changes in the ratios of the three emission peaks. During illumination with light 2 (580 nm) the F730/F695 and F730/F685 ratios increased within minutes to establish a condition characteristic of state 2. A major difference between the two chloroplast types was the more rapid establishment of state 2 by mesophyll chloroplasts. An increase in light 2 intensity caused an increase in the magnitude of the F730/F695 ratio for both chloroplast types and, for guard cell chloroplasts, a decrease in the time required to establish the new ratio. The role of reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex in regulating state transitions for both mesophyll and guard cell chloroplasts was assessed using DCMU and sodium fluoride, a specific phosphatase inhibitor. DCMU-treated mesophyll and epidermal tissues failed to show a state 1-state 2 transition. NaF-treated tissues attained state 2 but lacked the ability to revert back to state 1.     

Nitric oxide production in tobacco leaf cells: a generalized stress response?

The function of nitric oxide (NO), a gaseous free radical emitted by many plants, is incompletely understood. In the present study the hypothesis that NO generation, like that of the reactive oxygen species, occurs as a general response to different environmental cues was tested. Leaf peels and mesophyll cell suspensions of Nicotiana tabacum cv. Xanthi were loaded with the NO‐specific fluorophore, diaminofluorescein, and subjected to an abiotic stressor. Light stress and mechanical injury had no apparent effect on NO production. In contrast, high temperatures, hyperosmotic stress, salinity and epi‐illumination in a microscope all led to rapid surges in NO‐induced fluorescence. The fluorescence originated from cells of the palisade mesophyll and across all epidermal cell types, including guard cells, subsidiary cells, and long and short trichomes. Fluorescence was evident first in the plastids, then in the nucleus and finally throughout the cytosol. Nicotiana plumbaginifolia cell suspensions expressing the calcium reporter aequorin provided evidence that, under hyperosmotic stress, NO participates in the elevation of free Ca²⁺ in the cytoplasm. The physiological significance of NO production in response to abiotic stressors is discussed.     

Metabolomics of red‐light‐induced stomatal opening in Arabidopsis thaliana: Coupling with abscisic acid and jasmonic acid metabolism

Environmental stimuli‐triggered stomatal movement is a key physiological process that regulates CO₂ uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red‐light‐induced stomatal opening arises from direct guard cell sensing of red light versus indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red‐light‐stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry and liquid chromatography mass spectrometry for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red‐light‐modulated metabolites participate in the tricarboxylic acid cycle, carbon balance, phytohormone biosynthesis and redox homeostasis. We next analyzed selected Arabidopsis mutants, and discovered that stomatal opening response to red light is correlated with a decrease in guard cell abscisic acid content and an increase in jasmonic acid content. The red‐light‐modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells.     

Guard cell zeaxanthin tracks photosynthetically active radiation and stomatal apertures in Vicia faba leaves*

Zeaxanthin, antheraxanthin and violaxanthin concentrations in guard cells from sonicated abaxial epidermal peels of Vicia faba were measured from dawn to dusk, and compared with concentrations in mesophyll tissue of the same leaves. Measured changes in guard cell zeaxanthin and violaxanthin concentrations indicate that guard cells operate the xanthophyll cycle throughout the day. Mesophyll tissue had no detectable zeaxanthin at dawn, whereas guard cells had 30–50 mmol mol^?1 chlorophyll a+b. On a chlorophyll basis, maximal zeaxanthin levels were 3–4 fold higher in guard cells than in mesophyll cells. Zeaxanthin concentrations tracked levels of photosynthetically active radiation (PAR) in both mesophyll and guard cells. In the mesophyll, most of the zeaxanthin changes occurred in mid-morning and mid-afternoon. In guard cells, zeaxanthin concentrations changed nearly linearly with PAR in the early morning and late afternoon, and closely tracked PAR levels throughout the day. Guard cell zeaxanthin concentrations were also closely correlated with stomatal apertures. The close relationship between zeaxanthin concentrations and PAR levels in guard cells indicates that zeaxanthin is well suited to function as a molecular photosensor in stomatal movements.