Note: Colonization and invasion of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria

The colonization of leaves of the aquatic macrophyteCeratophyllum demersum L. by epiphytic bacteria, and the hypothesis that bacterial invasion causes leaf senescence, was studied using transmission and scanning electron microscopy and light microscopy. Population densities of epiphytic bacterial communities onCeratophyllum leaves were positively correlated with leaf age. Initial settlement of bacteria on young leaves appeared to favour the boundaries between epidermal cells. On older leaves, large populations of bacteria were present over the whole surface. One third of senescentCeratophyllum leaves examined by transmission electron microscopy showed signs of bacterial invasion. Of these, up to 54% of the leaf's epidermal cells contained bacteria. Areas of cell wall degradation were associated with invasive bacteria in senescent leaves. In healthy, nonsenescent leaves, no bacterial invasion was observed. These results suggest that epiphytic bacteria did not cause leaf senescence but probably colonized the internal tissues of leaves once senescence had occurred.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

ANATOMY OF SOME LEAF GALLS OF ROSA WOODSII (ROSACEAE)

Infection of Rosa woodsii by some members of the order Hymenoptera results in neoplasmic outgrowths on the leaves. One type of outgrowth produces a spherical swelling (leaf gall) while the other has extensive hair-like proliferations (hairy gall). The anatomy and ultrastructure of these galls were examined by light microscopy and transmission electron microscopy. The leaf gall cells were considerably larger than normal cells, lacked well-developed chloroplasts and were loosely arranged with prominent intercellular spaces. Vascular bundles were scattered throughout the gall tissue. The upper three cell layers of the leaf gall tissue resembles a periderm, having many suberin lamellae. The suberin lamellae were often traversed by pores which may represent incomplete plasmodesmata. Phenolic compounds were commonly seen both in the normal and gall cells. A layer of internal cells of the hairy galls have remarkably thickened cell walls, presumably due to the deposition of cellulosic substances. Unlike leaf galls, the epidermal cells of the hairy galls were not heavily cuticularized and no periderm was found. The hair-like outgrowths present on the outer surface of these galls had a central vascular bundle. The epidermis of the outgrowths also had thickened cell walls, and trichomes occurred on the outer surface. The structural modifications brought about by the insect invasion in these two galls are compared and their roles in gall formation are discussed.     

Mode of Attack on Orchardgrass Leaf Blades by Rumen Protozoa

Leaf blade sections of orchardgrass were incubated with rumen fluid and examined by scanning and transmission electron microscopy for the mode of attack on tissues by rumen protozoa. Rumen protozoa resembling Epidinium ecaudatum from caudatum degraded forage tissue in diluted, whole rumen fluid suspensions of microbes containing 1.6 mg of streptomycin per ml, which inhibited bacterial fiber-digesting activity. Cell walls of mesophyll, parenchyma bundle sheath, and epidermis became swollen and frayed to reveal a microfibrillar network and loss of electron density, indicating partial degradation. Then the protozoa ingested whole cells and fragments of cell walls with the aid of their cilia. Plant cells with partially degraded walls as well as chloroplasts without walls were present within the protozoa. These entodiniomorphs digested orchardgrass leaves by partially degrading the plant cell walls apparently by extracellular enzymes and then ingestion of the plant cells and cell wall fragments.     

Characterization of microstructure and cell wall components of Arabidopsis thaliana overexpressing cyclin-dependent kinase inhibitor 2

Overexpression of a cyclin-dependent kinase inhibitor (KRP2) caused changes in the general morphology in the leaves of Arabidopsis thaliana. The wild type plant had obovate leaves with entire margins whereas the transgenic line had leaves with denticulate margins. The epidermal cells and stomata of the adult transgenic leaves were significantly larger than those of the wild-type plants and the number of stomata was in proportion to the number of epidermal cells. No apparent differences in thickness and structure of cell walls of the mesophyll cells between the two samples were observed. The smaller amount of cell wall material in the transgenic leaves caused by the larger cell size was also apparent in the lower dry weight of the transgenic leaves. The chemical analysis revealed the main differences to be in pectin and neutral sugar contents, and especially in the amounts of glucose, all being higher in the leaves of the KRP2 transgenic plants. p-Coumaric acid content varied more in the transgenic leaf material than in the control one reflecting possibly fewer cross-links in the cell walls of transgenic plants.     

Quantitative ion localization within Suaeda maritima leaf mesophyll cells

Grown under saline conditions, Suaeda maritima accumulates Na⁺ and Cl^- into its leaves, where individual mesophyll cells behave differently in their compartmentation of these ions. Measurements of ion concentrations within selected subcellular compartments show that freeze-substitution with dry sectioning is a valuable preparative technique for analytical electron microscopy of highly vacuolate plant material. Using this approach, absolute estimates were made of Na⁺, K⁺ and Cl^- concentrations in the cytoplasm, cell walls, chloroplasts and vacuoles of leaf mesophyll cells.Abbreviation TAEM transmission analytical electron microscopy     

Rumen bacterial interrelationships with plant tissue during degradation revealed by transmission electron microscopy

The mode of rumen bacterial degradation of cell walls in coastal bermudagrass [Cynodon dactylon (L) Pers.] differed with the plant tissue type. Bacteria degraded thin, primary cell walls of mesophyll and phloem apparently by extracellular enzymes and without prior attachment; thick-walled bundle sheath and epidermal cells apparently were degraded after bacterial attachment, in some types by an extracellular substance, to the plant cell walls. Rumen bacteria split the nondegraded cuticle from the epidermis by preferentially attacking the cell just underneath the cuticle. The propensity for bacterial attachment to lignified cells of the vascular tissue was low, and bacterial degradation of these cells did not occur after 72 h of incubation.     

苜蓿假盘菌侵染苜蓿叶片的细胞学研究

采用微分干涉相差显微镜、扫描和透射电镜技术系统研究了苜蓿假盘菌Pseudopeziza medicaginis在苜蓿叶片的侵染过程及超微结构特征。结果表明,接种4h后,子囊孢子萌发产生芽管;12h后,芽管以直接侵入的方式进入表皮细胞形成侵染菌丝;24h后,表皮细胞中侵染菌丝向相邻表皮细胞扩展,同时侵入到叶肉细胞以胞内生长方式扩展;接种72h后,侵染菌丝在表皮细胞下的叶肉组织中形成初始菌落;第5d后,菌丝扩展至整个叶片组织,大量菌丝聚集形成子座组织,并进一步形成子囊盘与子囊。病菌菌丝在侵入寄主细胞初期,并不     

苜蓿假盘菌侵染苜蓿叶片的细胞学研究

采用微分干涉相差显微镜、扫描和透射电镜技术系统研究了苜蓿假盘菌Pseudopeziza medicaginis在苜蓿叶片的侵染过程及超微结构特征。结果表明,接种4h后,子囊孢子萌发产生芽管;12h后,芽管以直接侵入的方式进入表皮细胞形成侵染菌丝;24h后,表皮细胞中侵染菌丝向相邻表皮细胞扩展,同时侵入到叶肉细胞以胞内生长方式扩展;接种72h后,侵染菌丝在表皮细胞下的叶肉组织中形成初始菌落;第5d后,菌丝扩展至整个叶片组织,大量菌丝聚集形成子座组织,并进一步形成子囊盘与子囊。病菌菌丝在侵入寄主细胞初期,并不穿透寄主质膜与原生质,而是被其所包围。但随着菌丝进一步扩展,叶片组织发生了一系列的病理变化,其中包括叶肉细胞肿胀、细胞质消解、叶绿体等细胞器解体以及寄主细胞坏死塌陷,并最终在叶表面产生典型的褐斑病症状。     

Immunocytochemical localisation of auxin-binding proteins in coleoptiles and embryos ofZea mays L.

Summary The auxin-binding protein ABP-1 was localised immunocytochemically in coleoptiles and immature embryos ofZea mays. Two primary polyclonal antibodies raised against ABP-1 and secondary antibodies were either labelled with FITC or 10 nm gold particles for light microscopy, and with 10 nm gold particles for transmission electron microscopy. Light microscopy revealed that ABP-1 was localised in the epidermal cells of etiolated maize coleoptiles, in subepidermal parenchymatic mesophyll cells of the coleoptile and in the companion cells of the vascular bundles. Most labelling was found in the cytoplasm, less in nuclei and vacuoles and cell walls appeared negative. The region of the plasma membrane exhibited prominent labelling. Embryos showed low labelling throughout their tissues just after excision, but after culture for 7 days intensive labelling was found in the epidermis of the scutellum. Quantitative electron microscopy confirmed that ABP-1 was present in the cytoplasm of epidermal, mesophyll, and companion cells of coleoptiles. Gold particles were neither found in cell walls nor in the cuticle. Areas with ER and dictyosomes within epidermal and mesophyll cells of coleoptiles had a denser labelling with gold particles than elsewhere. Labelling at the plasma membrane, being the site where the auxin binds to the ABP, was observed at low levels in all cells examined, which is due to the method applied. Epidermal cells of embryos cultured for 5 days exhibited high levels of gold particles in ER and nuclei, and lower levels in the cytoplasm. The distribution is only partly in accordance with the model in which ABP is thought to cycle through the plant cell from the ER via the Golgi system towards the plasma membrane.Abbreviations ABP-1 auxin-binding protein 1 - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - LM light microscopy - LR Write London resin white - PBS phosphate-buffered saline - PEG polyethylene glycol - TEM transmission electron microscopy     

Distribution of cadmium in leaves of Thlaspi caerulescens

Knowledge of the intracellular distribution of Cd in leaves is necessary in order to understand the mechanisms of hyperaccumulation in Thlaspi caerulescens. Ganges and Prayon, two ecotypes accumulating Cd to different levels, were grown in nutrient medium containing varying concentrations (0, 5, 10, 50, and 100 microM) of Cd. Several different approaches were combined in this study to (i) validate the results obtained by a specific method and (ii) establish the link between observations and measurements performed at different scales. In both ecotypes, Cd, localized by autoradiography, was found mainly at the edges of the leaves, but also in points of higher concentration spread over the whole limb surface. This localization was clearly correlated with the necrotic spots observed on Prayon leaves. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (cryo-SEM-EDXMA) and tissue fractionation (apoplasm, cell walls, mesophyll protoplasts, and lower epidermis) showed that Cd had similar patterns of distribution in leaf cells of both ecotypes. Cadmium was found both inside the cells and in the cell walls, mainly in the large epidermal cells but also in small epidermal cells. All the methods used agreed well and the results indicated that metal storage in the plants studied involves more than one compartment and that Cd is stored principally in the less metabolically active parts of leaf cells.     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.     

Studies on turfgrass snow mold caused byTyphula ishikariensis. II. Microscopical observation of infected bentgrass leaves

Light and transmission electron microscopy revealed thatTyphula ishikariensis penetrated into bentgrass leaves either through cuticles or stomata either by single hyphae or infection cushions formed on host surfaces. Time course study on infected leaves showed that penetration through stomatal subsidiary cells and their adjacent cells seemed to occur earlier than that through epidermal cells located farther from stomata. More than 30% of epidermal cells were infected by 10 days after inoculation. When hyphae penetrated through an intact cuticle of epidermal cells, they seemed to dissolve host cell walls enzymatically at penetration sites. Physical pressure also seemed to be involved in penetration.     

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。     

Ultrastructural aspects of leaves inFestuca ovina andPoa sphondylodes (C-3 Poaceae)

Structural aspects of the leaves of two common festucoids,Festuca ovina andPoa sphondylodes, have been examined employing the electron microscopy. The nature of vascular bundles and of sheaths that surround vascular tissues was discussed in the study. The festucoids exhibited a non-Kranz C-3 anatomy with more than four mesophyll cells separating the bundle sheaths of a leaf blade. Vascular tissues in theseFestuca andPoa leaves were surrounded by a double sheath: an inner distinct mestome sheath (MST) and an outer indistinctive layer of parenchymatous bundle sheath (PBS) cells. The PBS cells were much larger than the MST and had thin walls. The MST cells were relatively small and rectangular inP. sphondylodes and more or less hexangular in transverse sections ofF. ovina. InP. sphondylodes, MST had conspicuously thickened inner tangential walls with asymmetrically uninterrupted suberized lamellae in radial and tangential walls. In most differentiated MST cells, all walls were highly suberized. During suberin deposition, MST cells were quite vacuolated and most of the cytoplasm was present as a thin peripheral layer. However, MST walls inF. ovina revealed very thin suberized lamellae with translucent striations. No chloroplasts were detected inP. sphondylodes, whereas the MST inF. ovina contained small chloroplasts. Plasmodesmata were well developed in the primary pit fields of walls between MST and vascular cells, and between adjacent MST cells. Plasmodesmata were less frequent in the walls between the inner and outer sheath cells. Suberized lamellae were totally absent from the PBS cell walls in all veins. External to the PBS, the mesophyll comprised thin walled cells with abundant intercellular spaces. Peripherally arranged chloroplasts in the mesophyll were numerous and often larger than those of PBS and MST cells. Characteristics associated with C-3 and other ultrastructural features were also discussed in the study.     

Leaf expansion and carbon assimilation in cotton leaves grown at two photosynthetic photon flux densities

Increasing photosynthetic photon flux density (PPFD) received during development from 5.5 to 31.2 mol m^-2 d^-1 resulted in greater leaf and mesophyll cell surface areas in cotton (Gossypium hirsutum L.). The relationships between the amounts of these surface areas and potential CO₂ assimilation by these leaves were evaluated. Leaf area (epidermal surface area of one side of a leaf), mesophyll cell surface area, and net rate of CO₂ uptake (P_n) were measured from the time leaves first unfolded until P., was substantially reduced. At the higher PPFD, leaf and mesophyll surface areas increased more rapidly during expansion, and P_n per unit leaf area was greater than at the lower PPFD. Although leaves at the higher PPFD reached the maximum P., per unit mesophyll cell surface area 4 to 5 days earlier than leaves at the lower PPFD, the maxima for these P., were similar. Leaves grown at the higher PPFD had the potential to assimilate 2.2, 3.5, or 5.8 times the amount of CO₂ as leaves from the lower PPFD when P., was expressed per unit mesophyll surface, per unit leaf surface, or per whole leaf, respectively. Greater and earlier development of both P., and mesophyll cell surface area at higher PPFD apparently had a compounding effect on the potential for carbon assimilation by a leaf.     

In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5-6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.     

The Hypersensitive Reaction in Tobacco Leaf Tissue infiltrated with Pseudomonas pisi 4. Scanning Electron Microscope Studies on fractured Leaf Tissue

Leaves of tobacco infiltrated with Pseudomonas pisi were fractured at various times during the course of the hypersensitive reaction to expose cell surfaces within the tissue and mesophyll cell contents. Scanning electron microscopy of cross-fractured mesophyll cells did not reveal any gross change in internal structure during the reaction induction period (0—2 h), but breakdown of tonoplast and collapse of chloroplasts commenced at about 5 h, during the latent period. Death of the mesophyll cells was followed by condensation of cell contents, and pronounced stretching of cell walls, due to desiccation and shrinkage.Between 0—6 h after infiltration, bacteria were largely confined to cell junctions, frequently within droplets. With collapse of the host cells and release of cell fluid, numbers of bacteria increased considerably (many dividing cells), and there was a shift of bacterial distribution to the whole mesophyll cell surface. The progressive desiccation that occurred between 10—20 h prevented further bacterial increase, but numbers of bacteria remained stable. Death of bacteria commenced at about 15 h, and was accompanied by the formation of numerous surface protrusions, which detached and deposited over the whole mesophyll surface.     

Infection of Subterranean Clover (Trifolium subterraneum) by Kabatiella caulivora

Kabatiella caulivora is a serious pathogen of clover ( Trifolium ) spp. Subterranean clover ( T. subterraneum ) cv. Woogenellup was inoculated with K. caulivora , to study the attachment and germination of conidia, germ-tube penetration of the plant surface, and histochemistry and ultrastructure of changes in the host associated with lesion development. The foliar architecture caused the conidia to concentrate at the base of leaflets and on the petiolules (between the leaflets and petioles). Epidermal cells immediately beneath conidia and, occasionally, also adjacent cells developed a yellow-brown discoloration 1 day post-inoculation. Penetration appeared to be directly through the cuticle, characterized by constricted hyphae at the point of entry. No appressoria were observed. In leaves, invasion was restricted to the area proximal to the petiolule and leaf mid-rib. In petioles and petiolules, the hyphae initially remained between the epidermal cells and first layer of mesophyll cells before moving intercellularly through the mesophyll tissue towards phloem tissues. The cuticle was occasionally degraded in petiole and petiolule infections, the loss of epidermal and mesophyll cell wall components was detected, and chloroplasts and starch grains were disrupted. Plants developed macroscopic symptoms 10–11 days post-inoculation with necrotic lesions occurring on leaves, petioles and petiolules. Sporulation occurred approximately 15–18 days post-inoculation when affected plants collapsed. This information may be useful for breeding programmes aimed at selecting varieties with improved resistance to the clover scorch disease.     

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。