Metagenomic analysis of rhizosphere microbiome provides insights into occurrence of iron deficiency chlorosis in field of Asian pears.

BACKGROUND: Fe-deficiency chlorosis (FDC) of Asian pear plants is widespread, but little is known about the association between the microbial communities in the rhizosphere soil and leaf chlorosis. The leaf mineral concentration, leaf subcellular structure, soil physiochemical properties, and bacterial species community and distribution had been analysed to gain insights into the FDC in Asian pear plant. RESULTS: The total Fe in leaves with Fe-deficiency was positively correlated with total K, Mg, S, Cu, Zn, Mo and Cl contents, but no differences of available Fe (AFe) were detected between the rhizosphere soil of chlorotic and normal plants. Degraded ribosomes and degraded thylakloid stacks in chloroplast were observed in chlorotic leaves. The annotated microbiome indicated that there were 5 kingdoms, 52 phyla, 94 classes, 206 orders, 404 families, 1,161 genera, and 3,043 species in the rhizosphere soil of chlorotic plants; it was one phylum less and one order, 11 families, 59 genera, and 313 species more than in that of normal plant. Bacterial community and distribution patterns in the rhizosphere soil of chlorotic plants were distinct from those of normal plants and the relative abundance and microbiome diversity were more stable in the rhizosphere soils of normal than in chlorotic plants. Three (Nitrospira defluvii, Gemmatirosa kalamazoonesis, and Sulfuricella denitrificans) of the top five species (N. defluvii, G. kalamazoonesis, S. denitrificans, Candidatus Nitrosoarchaeum koreensis, and Candidatus Koribacter versatilis). were the identical and aerobic in both rhizosphere soils, but their relative abundance decreased by 48, 37, and 22%, respectively, and two of them (G. aurantiaca and Ca. S. usitatus) were substituted by an ammonia-oxidizing soil archaeon, Ca. N. koreensis and a nitrite and nitrate reduction related species, Ca. K. versatilis in that of chlorotic plants, which indicated the adverse soil aeration in the rhizosphere soil of chlorotic plants. A water-impermeable tables was found to reduce the soil aeration, inhibit root growth, and cause some absorption root death from infection by Fusarium solani. CONCLUSIONS: It was waterlogging or/and poor drainage of the soil may inhibit Fe uptake not the amounts of AFe in the rhizosphere soil of chlorotic plants that caused FDC in this study.

A mutation in the promoter of the arabinogalactan protein 7-like gene PcAGP7-1 affects cell morphogenesis and brassinolide content in pear (Pyrus communis L.) stems.

Dwarfing rootstocks and dwarf cultivars are urgently needed for modern pear cultivation. However, germplasm resources for dwarfing pear are limited, and the underlying mechanisms remain unclear. We previously showed that dwarfism in pear is controlled by the single dominant gene PcDw (Dwarf). We report here that the expression of PcAGP7-1 (ARABINOGALACTAN PROTEIN 7-1), a key candidate gene for PcDw, is significantly higher in dwarf-type pear plants because of a mutation in an E-box in the promoter. Electrophoretic mobility shift assays and transient infiltration showed that the transcription factors PcBZR1 and PcBZR2 could directly bind to the E-box of the PcAGP7-1 promoter and repress transcription. Moreover, transgenic pear lines overexpressing PcAGP7-1 exhibited obvious dwarf phenotypes, whereas RNA interference pear lines for PcAGP7-1 were taller than controls. PcAGP7-1 overexpression also enhanced cell wall thickness, affected cell morphogenesis, and reduced brassinolide (BL) content, which inhibited BR signaling via a negative feedback loop, resulting in further dwarfing. Overall, we identified a dwarfing mechanism in perennial woody plants involving the BL-BZR/BES-AGP-BL regulatory module. Our findings provide insight into the molecular mechanism of plant dwarfism and suggest strategies for the molecular breeding of dwarf pear cultivars.

Transcriptome and metabolomic analysis to reveal the browning spot formation of 'Huangguan' pear.

BACKGROUND: Browning spot (BS) disorders seriously affect the appearance quality of 'Huangguan' pear and cause economic losses. Many studies on BS have mainly focused on physiological and biochemical aspects, and the molecular mechanism remains unclear. RESULTS: In the present study, the structural characteristics of 'Huangguan' pear with BS were observed via scanning electron microscopy (SEM), the water loss and brown spots were evaluated, and transcriptomic and metabolomics analyses were conducted to reveal the molecular mechanism underlying 'Huangguan' pear skin browning disorder. The results showed that the occurrence of BS was accompanied by a decrease in the wax layer and an increase in lignified cells. Genes related to wax biosynthesis were downregulated in BS, resulting in a decrease in the wax layer in BS. Genes related to lignin were upregulated at the transcriptional level, resulting in upregulation of metabolites related to phenylpropanoid biosynthesis. Expression of calcium-related genes were upregulated in BS. Cold-induced genes may represent the key genes that induce the formation of BS. In addition, the results demonstrated that exogenous NaH2PO4·2H2O and ABA treatment could inhibit the incidence of BS during harvest and storage time by increasing wax-related genes and calcium-related genes expression and increasing plant resistance, whereas the transcriptomics results indicated that GA3 may accelerate the incidence and index of BS. CONCLUSIONS: The results of this study indicate a molecular mechanism that could explain BS formation and elucidate the effects of different treatments on the incidence and molecular regulation of BS.

Transcriptome and Metabolome Analyses Provide Insights into the Watercore Disorder on "Akibae" Pear Fruit.

Watercore is a physiological disorder that commonly occurs in sand pear cultivars. The typical symptom of watercore tissue is transparency, and it is often accompanied by browning, breakdown and a bitter taste during fruit ripening. To better understand the molecular mechanisms of watercore affecting fruit quality, this study performed transcriptome and metabolome analyses on watercore pulp from "Akibae" fruit 125 days after flowering. The present study found that the "Akibae" pear watercore pulp contained higher sorbitol and sucrose than healthy fruit. Moreover, the structure of the cell wall was destroyed, and the content of pectin, cellulose and hemicellulose was significantly decreased. In addition, the content of ethanol and acetaldehyde was significantly increased, and the content of polyphenol was significantly decreased. Watercore induced up-regulated expression levels of sorbitol synthesis-related (sorbitol-6-phosphate dehydrogenase, S6PDH) and sucrose synthesis-related genes (sucrose synthesis, SS), whereas it inhibited the expression of sorbitol decomposition-related genes (sorbitol dehydrogenase, SDH) and sorbitol transport genes (sorbitol transporter, SOT). Watercore also strongly induced increased expression levels of cell wall-degrading enzymes (polygalactosidase, PG; ellulase, CX; pectin methylesterase, PME), as well as ethanol synthesis-related (alcohol dehydrogenase, ADH), acetaldehyde synthesis-related (pyruvate decarboxylase, PDC) and polyphenol decomposition-related genes (polyphenol oxidase, PPO). Moreover, the genes that are involved in ethylene (1-aminocyclopropane- 1-carboxylate oxidase, ACO; 1-aminocyclopropane- 1-carboxylate synthase, ACS) and abscisic acid (short-chain alcohol dehydrogenase, SDR; aldehyde oxidase, AAO) synthesis were significantly up-regulated. In addition, the bitter tasting amino acids, alkaloids and polyphenols were significantly increased in watercore tissue. Above all, these findings suggested that the metabolic disorder of sorbitol and sucrose can lead to an increase in plant hormones (abscisic acid and ethylene) and anaerobic respiration, resulting in aggravated fruit rot and the formation of bitter substances.

Multiscale Localization of Procyanidins in Ripe and Overripe Perry Pears by Light and Transmission Electron Microscopy.

Histochemical staining with 4-dimethylaminocinnamaldehyde (DMACA), light microscopy, and transmission electron microscopy (TEM) were applied to characterize procyanidin localization at ripe and overripe stages in perry pear flesh (cv. 'De Cloche'). Pear flesh contained stone cell clusters surrounded by very large parenchyma cells. DMACA staining showed procyanidins mainly located in parenchyma cells from the fruit mesocarp. Under light microscopy and TEM, procyanidins appeared in the vacuole of parenchyma cells as uniformly stained granules, probably tannosomes. They were differently dispersed in ripe and overripe perry pears, as the granules remained free inside the vacuole in ripe pears and mostly attached to the tonoplast in overripe pears.

Methyl salicylate delays peel yellowing of 'Zaosu' pear (Pyrus bretschneideri) during storage by regulating chlorophyll metabolism and maintaining chloroplast ultrastructure.

BACKGROUND: In some cultivars, yellowing resulting from chlorophyll breakdown has a direct and negative effect on food supply and health. The 'Zaosu' pear (Pyrus bretschneideri Rehd.), a commercial Asian pear cultivar in China, rapidly turns yellow when stored at room temperature after harvest. To develop techniques that delay or suppress chlorophyll degradation, the effects of methyl salicylate (MeSA) on yellowing in 'Zaosu' pear fruit during storage were evaluated. RESULTS: Compared with the untreated fruit, the application of 0.05 mmol L-1 MeSA delayed the decline of the total chlorophyll, chlorophyll a and chlorophyll b content, and maintained more intact chloroplasts with fewer and smaller plastoglobuli. Methyl salicylate suppressed enzyme activities, including chlorophyllase, chlorophyll-degrading peroxidase, Mg dechelatase, and pheophytinase, and the expression levels of NYC, NOL, CLH, SGR, PPH, PAO and RCCR in treated fruit. CONCLUSION: Methyl salicylate could delay chlorophyll breakdown in the fruit. The results also suggested that the conversion from chlorophyll a to pheophorbide a could proceed via two pathways, and that alternative pathways for the breakdown of chlorophyll a exist in 'Zaosu' pears. © 2019 Society of Chemical Industry.

The change in microstructure of petioles and peduncles and transporter gene expression by potassium influences the distribution of nutrients and sugars in pear leaves and fruit.

Potassium (K) is one of the most important mineral nutrients required for fruit growth and development and is known as a 'quality element'. To investigate the role of K in more detail, we performed experiments in which seven-year-old pot-grown 'Huangguan' pear trees were treated with three levels of K (0, 0.4, or 0.8 g K2O kg-1 soil). K supply improved the development of vascular bundles in pear petioles and fruit peduncles and enhanced expression of genes involved in nutrients and sugar transport. Different from K and calcium (Ca), magnesium (Mg) concentrations in the leaves, petioles, and fruit peduncles were significantly higher under low K but lower under high K. However, the concentrations of K, Ca, and Mg in fruit all increased as more K was applied. Correspondingly, the expression of leaf Mg transporters (MRS2-1 and MRS2-3) increased under low K, indicating that Mg had an obvious compensation effect on K, while their expression decreased under medium and high K, showing that K had an obvious antagonistic effect on Mg. Except for NIPA2, the expressions of fruit K, Ca, and Mg transporters increased under high K, implying a synergistic effect among them in fruit. The concentration of sorbitol, sucrose, and total sugar in leaves and fruit at maturity significantly increased in response to the supply of K. The increase in sugar concentration was closely related to the up-regulated expression of sucrose transporter (SUT) and sorbitol transporter (SOT) genes. Together, these effects may promote the transport of nutrients and sugar from sources (leaves) to sinks (fruit) and increase the accumulation of sugar in the fruit.

Automatic analysis of the 3-D microstructure of fruit parenchyma tissue using X-ray micro-CT explains differences in aeration.

BACKGROUND: 3D high-resolution X-ray imaging methods have emerged over the last years for visualising the anatomy of tissue samples without substantial sample preparation. Quantitative analysis of cells and intercellular spaces in these images has, however, been difficult and was largely based on manual image processing. We present here an automated procedure for processing high-resolution X-ray images of parenchyma tissues of apple (Malus × domestica Borkh.) and pear (Pyrus communis L.) as a rapid objective method for characterizing 3D plant tissue anatomy at the level of single cells and intercellular spaces. RESULTS: We isolated neighboring cells in 3D images of apple and pear cortex tissues, and constructed a virtual sieve to discard incorrectly segmented cell particles or unseparated clumps of cells. Void networks were stripped down until their essential connectivity features remained. Statistical analysis of structural parameters showed significant differences between genotypes in the void and cell networks that relate to differences in aeration properties of the tissues. CONCLUSIONS: A new model for effective oxygen diffusivity of parenchyma tissue is proposed that not only accounts for the tortuosity of interconnected voids, but also for significant diffusion across cells where the void network is not connected. This will significantly aid interpretation and analysis of future tissue aeration studies. The automated image analysis methodology will also support pheno- and genotyping studies where the 3D tissue anatomy plays a role.

Mitochondrial dysfunction mediated by cytoplasmic acidification results in pollen tube growth cessation in Pyrus pyrifolia.

The length of pollen tubes grown in synthetic media is normally shorter than those grown in vivo. However, the mechanism(s) underlying the cessation of pollen tube growth under culture conditions remain(s) largely unknown. Here, we report a previously unknown correlation between vacuolar function and the cell's ability to sustain mitochondrial functions in pear pollen tubes. The pear pollen tubes in vitro grew slowly after 15 hours post-cultured (HPC) and nearly ceased growth at 18 HPC. There was increased malondialdehyde content and membrane ion leakage at 15 HPC compared with 12 HPC. Furthermore, cytoplasmic acidification mainly mediated by decreased vacuolar H(+)-ATPase [V-ATPase, Enzyme Commission (EC) 3.6.1.3] activity was observed in pollen tubes after 15 HPC, and this further resulted in mitochondrial dysfunction, including mitochondrial structure disruption, mitochondrial membrane potential collapse and decreases in both oxygen consumption and ATP production. Our findings suggest that vacuoles and mitochondria intimately linked in regulating pollen tube elongation.

Ultrastructure of plant leaf cuticles in relation to sample preparation as observed by transmission electron microscopy.

The leaf cuticular ultrastructure of some plant species has been examined by transmission electron microscopy (TEM) in only few studies. Attending to the different cuticle layers and inner structure, plant cuticles have been grouped into six general morphological types. With the aim of critically examining the effect of cuticle isolation and preparation for TEM analysis on cuticular ultrastructure, adaxial leaf cuticles of blue-gum eucalypt, grey poplar, and European pear were assessed, following a membrane science approach. The embedding and staining protocols affected the ultrastructure of the cuticles analysed. The solubility parameter, surface tension, and contact angles with water of pure Spurr's and LR-White resins were within a similar range. Differences were however estimated for resin : solvent mixtures, since Spurr's resin is combined with acetone and LR-White resin is mixed with ethanol. Given the composite hydrophilic and lipophilic nature of plant cuticles, the particular TEM tissue embedding and staining procedures employed may affect sample ultrastructure and the interpretation of the results in physicochemical and biological terms. It is concluded that tissue preparation procedures may be optimised to facilitate the observation of the micro- and nanostructure of cuticular layers and components with different degrees of polarity and hydrophobicity.

Distribution of transglutaminase in pear pollen tubes in relation to cytoskeleton and membrane dynamics.

Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca(2+) dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.

Effects of 1-MCP on chlorophyll degradation pathway-associated genes expression and chloroplast ultrastructure during the peel yellowing of Chinese pear fruits in storage.

The peel yellowing is an important pigment physiological process of green fruit ripening, which mainly results from chlorophyll degradation in the fruit peel. In this work, two typical cultivars with different ripening speed, a slow ripening pear 'Emerald' (Pyrus bretschneideri Rehd. cv. Emerald) and a fast ripening 'Jingbai' (Pyrus ussuriensis Maxim. cv. Jingbai) were used to investigate the molecular mechanism of chlorophyll degradation in pear yellowing/ripening during postharvest storage. The fruits after harvest were treated with 1-methylcyclopropene (1-MCP), an ethylene action inhibitor at 1.0 µLl(-1) to determine its effect on chloroplast ultrastructure and the expression of chlorophyll degradation associated genes in peel tissues. Our results show that the pears treated with 1-MCP had a lower ethylene production rate and higher chlorophyll content compared to those of untreated fruit. The more intact chloroplasts with well-organised grana thylakoids and small plastoglobuli were maintained in the peel of 1-MCP treated fruit for up to 30 and 15 d in 'Emerald' and 'Jingbai', respectively. The expression of chlorophyll degradation associated genes: pheophorbide a oxygenase (PAO), non-yellow colouring (NYC), NYC1-like (NOL), stay-green 1(SGR1), was suppressed, while no significant change was found in chlorophyllase 1 (CHL1) and red chlorophyll catabolite reductase (RCCR) in both cultivar fruits treated with 1-MCP. These results suggest that 1-MCP can delay chlorophyll degradation by inhibiting ethylene production and suppressing the gene expression of PAO, NYC, NOL and SGR1, which are closely associated with chlorophyll catabolic pathway.

Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia.

Loading of Ca(2+)-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca(2+) ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca(2+) indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca(2+) distribution increased in the three apertures of pollen after hydration and showed a Ca(2+) gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca(2+) activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pH(i) regulated pollen tube growth.

S-RNase disrupts tip-localized reactive oxygen species and induces nuclear DNA degradation in incompatible pollen tubes of Pyrus pyrifolia.

Pear (Pyrus pyrifolia L.) has an S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. However, RNA degradation might be only the beginning of the SI response, not the end. Recent in vitro studies suggest that S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia, and it seems that a relationship exists between self S-RNase, actin depolymerization and DNA degradation. To further uncover the SI response in pear, the relationship between self S-RNase and tip-localized reactive oxygen species (ROS) was evaluated. Our results show that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest of ROS formation in mitochondria and cell walls. The mitochondrial ROS disruption was related to mitochondrial alteration, whereas cell wall ROS disruption was related to a decrease in NADPH. Tip-localized ROS disruption not only decreased the Ca(2+) current and depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. These results indicate that tip-localized ROS disruption occurs in Pyrus pyrifolia SI. Importantly, we demonstrated nuclear DNA degradation in the incompatible pollen tube after pollination in vivo. This result validates our in vitro system in vivo.

Ultrastructural study on acibenzolar-S-methyl-induced scab resistance in epidermal pectin layers of Japanese pear leaves.

The infection behavior of Japanese pear scab pathogen Venturia nashicola race 1 was studied ultrastructurally in acibenzolar-S-methyl (ASM)-pretreated susceptible Japanese pear (cv. Kousui) leaves to determine the mechanism of ASM-induced scab resistance. On ASM-pretreated leaf surfaces, the infection behavior (conidial germination and appressorial formation) was similar to that on distilled water (DW)-pretreated leaves prior to cuticle penetration by the pathogen. However, after penetration, differentiated behavior was found in epidermal pectin layers and middle lamellae of the ASM-pretreated leaves. Subcuticular hyphae in epidermal pectin layers and middle lamellae of ASM-pretreated pear leaves were observed at lower frequency than in DW-treated leaves. The results indicated that fungal growth was suppressed in ASM-pretreated pear leaves. In the pectin layers of ASM- and DW-pretreated leaves, some hyphae showed morphological modifications, which were used as criteria to judge collapse of hyphal cells, including plasmolysis, necrotic cytoplasm, and cell wall destruction. More hyphae had collapsed in ASM-pretreated leaves than in DW-treated ones. In addition, the cell walls of collapsed hyphae broke into numerous fibrous and amorphous pieces, suggesting that ASM-induced scab resistance might be associated with cell-wall-degrading enzymes from pear plants. In addition, results from morphometrical analysis suggested that the activity or production of pectin-degrading enzyme from hyphae were inhibited by ASM application when compared with DW treatment.

Accelerators increase permeability of cuticles for the lipophilic solutes metribuzin and iprovalicarb but not for hydrophilic methyl glucose.

Effects of diethylsuberate (DESU), tributyl phosphate (TBP), and monodisperse ethoxylated alcohols (EAs) on rate constants of penetration (k) of model solutes across astomatous cuticular membranes isolated from Madagascar ivy (Stephanotis floribunda) and pear (Pyrus communis) leaves were studied. Model solutes (selected on the basis of their octanol/water partition coefficients, K(ow)) were iprovalicarb (log K(ow) = 3.18), metribuzin (log K(ow) = 1.60), and methyl glucose (MG) (log K(ow) = -3.0). K(ow) varied by more than 6 orders of magnitude. Accelerators had wax/water partition coefficients (log K(ww)) ranging from 1.75 (DESU) to 4.32 (C(12)E(2)), and their equilibrium concentrations in Stephanotis wax varied from 0 to about 160 g kg(-)(1). Accelerators increase solute mobility in cuticles by increasing fluidity of cutin and waxes. This effect was quantified by plotting log k versus the accelerator concentration in wax. With the lipophilic solutes metribuzin and iprovalicarb, these plots were linear. Slopes of these plots characterize the intrinsic activities of the accelerators, and they decreased in the order DESU (0.029) > TBP (0.015) > EAs (0.01). Using these intrinsic activities, the effects of accelerators on rate constants of penetration can be calculated for any accelerator concentration in wax. For instance, at 50 g kg(-)(1), rate constants for lipophilic solutes increased by factors of 28 (DESU), 5.6 (TBP), and 3.2 (C(12)E(n)()), respectively. Permeability of cuticles for the hydrophilic MG was not increased by DESU, TBP, C(12)E(2), and C(12)E(4), while C(12)E(6) and C(12)E(8) increased it. Small hydrophilic solutes such as MG can access aqueous pores in cuticles, and this pathway is not affected by changes in fluidity of amorphous waxes. After rate constants of penetration of ionic CaCl(2) were compared with those for nonionic MG, it was concluded that 60% of the MG diffused across aqueous pores, while 40% used an alternative pathway. Because the solubility of MG in wax is extremely low, it is unlikely that MG diffused along the lipophilic pathway used by metribuzin and iprovalicarb. This agrees with the observation that DESU and TBP had no effect on rate constants for MG. An alternative pathway of unknown properties is suggested. It is speculated that C(12)E(6) and C(12)E(8) sorbed in cuticles might have generated a polar pathway for MG.

Fluorescence shell: a novel view of sclereid morphology with the Confocal Laser Scanning Microscope.

Confocal Laser Scanning Microscopy (CLSM) was used to observe sclereids from stems of Avicennia germinans and from fruits of two species of pear (Pyrus calleryana "Bradford" and P. communis "Red Bartlett"). The images obtained from thick (25 to 100 microm) free-hand sections were, in certain respects, far superior to those obtained by other, more invasive and time-consuming microscopic techniques upon which previous reports of sclereid morphology were based. The cell wall surfaces, including the "internal" surfaces of the branched pit canals and cell lumens, were much accentuated with the techniques we describe, resulting in a "fluorescence shell" image, meaning the cell wall did not stain all the way through but instead only at the inner and outer wall surfaces, including the edges of ramiform pits. By controlling the time of staining with 1% aqueous Safranin O, or by changing the number of optical sections used in extended focus images, it was possible to get either a conventional view of the cell wall structure or a unique, three-dimensional view of the elaborate cell interconnections. Similar fluorescence shell images of sclereids were also obtained using a periodic-Schiff (PAS) staining system, but the stain was not as specific to sclereid cell walls as was the Safranin O stain. Particularly with the use of a narrow range band pass emission filter of 505-530 nm, the Safranin O staining may be more specific to lignin than reported in the literature.