Cutinsomes of Malus Mill. (Rosaceae) leaf and pericarp: genesis, localization, and transport.

New data were obtained on specific bionanostructures, cutinsomes, which are involved in the formation of cuticles on the surface of leaf blades and pericarp of Malus domestica Borkh (Malus Mill., Rosaceae)introduced to the mountains at the altitudes of 1200 and 1700 m above sea level. Cutinsomes, which are electron-dense structures of spherical shape, have been identified by transmission electron microscopy. It was demonstrated that plastids can be involved in the synthesis of their constituent nanocomponents. The greatest number of nanoparticles was observed in the granal thylakoid lumen of the chloroplasts in palisade mesophyll cells and pericarp hypodermal cells. The transmembrane transport of cutinsomes into the cell wall cuticle proper by exocytosis has been visualized for the first time. The plasma membrane is directly involved in the excretion of nanostructures from the cell. Nanoparticles of cutinsomes in the form of necklace-like formations line up in a chain near cell walls, merge into larger conglomerates and are loaded into plasmalemma invaginations, and then, in membrane packing, they move into the cuticle, which covers both outer and inner cell walls of external tissues. The original materials obtained by us supplement the ideas about the non-enzymatic synthesis of cuticle components available in the literature and expand the cell compartment geography involved in this process.

Effects of cell morphology on the textural attributes of fruit cubes in freeze-drying: Apples, strawberries, and mangoes as examples.

The influence of cell morphology on the textural characteristic of freeze-dried apple, strawberry, and mango cubes was evaluated. Corresponding restructured cube samples without intact cell morphology were prepared as controls. Results indicated that the presence of cell morphology strengthened the shrinkage and collapse of samples during freeze-drying, especially in mangoes due to the high content of sugar. Intact cell morphology was found in natural fruit cubes after freeze-drying by scanning electron microscopy (SEM) observation, making them exhibit a more regular microporous structure, further resulting in higher hardness than the restructured cubes. However, the intact cell morphology negatively affected the crispness of freeze-dried cubes since it enhanced structural collapse. The freeze-dried samples without cell morphology would destroy the cellulose structure and form a continuous open-pore structure under the concentration effect of ice crystals during freezing, which accelerates the escape of water molecules, increases the drying rate, and avoid collapse. Sensory experiments found that restructured cubes without intact cell morphology exhibited greater comprehensive acceptance, suggesting the potential application of cell morphology disruption in the future freeze-drying industry.

Improved laser capture microdissection (LCM)-based method for isolation of RNA, including miRNA and expression analysis in woody apple bud meristem.

MAIN CONCLUSION: Isolation of high-quality RNA, including miRNA, from microscopic woody apple bud meristem using laser capture microdissection-based method. It is often challenging to study the expression of microRNAs (miRNAs) or genes in less accessible inner tissues of tree species rich in polyphenols or polysaccharides. Here, we report a laser capture microdissection (LCM)-based method for efficient and cost-effective isolation and expression analysis of miRNAs and genes in the meristem tissue of woody apple bud. The tissue fixation, processing, infiltration, and sectioning steps were optimized for LCM-based excision and subsequent RNA isolation. Further, we have confirmed that RNA isolated from LCM-derived apple bud meristem contained miRNAs and was of good quantity and quality, sufficient for downstream expression analysis.

Direct Comparison of Leaf Plasmodesma Structure and Function in Relation to Phloem-Loading Type.

The export of photosynthetically produced sugars from leaves depends on plasmodesmatal transport of sugar molecules from mesophyll to phloem. Traditionally, the density of plasmodesmata (PD) along this phloem-loading pathway has been used as a defining feature of different phloem-loading types, with species proposed to have either many or few PD between the phloem and surrounding cells of the leaf. However, quantitative determination of PD density has rarely been performed. Moreover, the structure of PD has not been considered, even though it could impact permeability, and functional data are only available for very few species. Here, a comparison of PD density, structure, and function using data from transmission electron microscopy and live-cell microscopy was conducted for all relevant cell-cell interfaces in leaves of nine species. These species represent the three principal phloem-loading types currently discussed in literature. Results show that relative PD density among the different cell-cell interfaces in one species, but not absolute PD density, is indicative of phloem-loading type. PD density data of single interfaces, even combined with PD diameter and length data, did not correlate with the intercellular diffusion capacity measured by the fluorescence loss in photobleaching method. This means that PD substructure not visible on standard transmission electron micrographs may have a strong influence on permeability. Furthermore, the results support a proposed passive symplasmic loading mechanism in the tree species horse chestnut (Aesculus hippocastanum), white birch (Betula pubescens), orchard apple (Malus domestica), and gray poplar (Populus x canescens) as functional cell coupling and PD structure differed from active symplasmic and apoplasmic phloem-loading species.

Structural network of arabinogalactan proteins (AGPs) and pectins in apple fruit during ripening and senescence processes.

The cell wall is an essential framework determining the overall form of the plant cell. Our study was focused on the distribution of arabinogalactan proteins (AGPs), arabinan, and homogalacturonan in fruit cells during ripening and storage with emphasis on quantitative analysis of their presence in particular regions of the cell wall - plasma membrane. The localization of the examined compounds was determined with immunohistochemistry techniques and immunogold labelling. Spatio-temporal colocalization between AGPs epitopes - [ßGlcA(1→3)-αGalA(1→2)Rha] recognized by JIM13 and MAC207 antibodies, and arabinan labelled by the LM16 antibody was detected in the inner cell wall layer, in association with the plasma membrane. The specific arrangement of AGP and arabinan epitopes differentiated them from homogalacturonan epitopes, consisting of GalA residues recognized by LM19 and LM20 antibodies in all the examined fruit maturity stages. The disruption of cell wall - plasma membrane continuum, observed during ripening-associated softening process, was associated with both the substantial decrease of AGPs, pectins content and with remodeling of their arrangement. The results indicate that the textural properties of fruit during growth and postharvest storage, an attribute of fruit quality becoming selection criteria for consumers, depend on the existence of dynamic network organizing polysaccharides and glycoproteins in the extracellular matrix.

Patterns of microcracking in apple fruit skin reflect those of the cuticular ridges and of the epidermal cell walls.

MAIN CONCLUSION: Microcracks in the cuticle of developing apples are aligned with ridges on the inner cuticle surface and are indicative of stress-strain concentrations above the anticlinal cell walls. Microcracks occur in cuticles of most fruits. Growth strains are considered causal. In apples (Malus × domestica), microcracks usually form a mesh pattern similar to that formed by cuticular ridges. Ridge patterns are similar to those of the epidermal cells' anticlinal walls. Our aim was to identify the mechanistic bases for these pattern similarities. By quantifying ridge depth, ridge width, and the areas enclosed by ridges, we reveal the presence of major and minor ridges. Major ridges enclose two-to-four epidermal cells, minor ridges only one cell. There are similar and overlying patterns of microcracking on the cuticle's outer surface and of ridges on its inner surface-microcracks generally follow the outlines of the major ridges. In biaxial tensile tests at 20 kPa, strains were low and microcracks shallow, but at > 40 kPa, strains were higher and microcracks deeper. Microcracks traversing the cuticle are usually aligned with the anticlinal walls of the underlying epidermal cells. In general, increased skin strain is associated with increased skin transpiration. Transpiration increases are reversible for low strains but irreversible for high strains. The alignment of cuticular microcracks with the major ridges, and these with the anticlinal cell walls, indicates associated stress/strain concentrations.

Phloem Loading through Plasmodesmata: A Biophysical Analysis.

In many species, Suc en route out of the leaf migrates from photosynthetically active mesophyll cells into the phloem down its concentration gradient via plasmodesmata, i.e. symplastically. In some of these plants, the process is entirely passive, but in others phloem Suc is actively converted into larger sugars, raffinose and stachyose, and segregated (trapped), thus raising total phloem sugar concentration to a level higher than in the mesophyll. Questions remain regarding the mechanisms and selective advantages conferred by both of these symplastic-loading processes. Here, we present an integrated model-including local and global transport and kinetics of polymerization-for passive and active symplastic loading. We also propose a physical model of transport through the plasmodesmata. With these models, we predict that (1) relative to passive loading, polymerization of Suc in the phloem, even in the absence of segregation, lowers the sugar content in the leaf required to achieve a given export rate and accelerates export for a given concentration of Suc in the mesophyll and (2) segregation of oligomers and the inverted gradient of total sugar content can be achieved for physiologically reasonable parameter values, but even higher export rates can be accessed in scenarios in which polymers are allowed to diffuse back into the mesophyll. We discuss these predictions in relation to further studies aimed at the clarification of loading mechanisms, fitness of active and passive symplastic loading, and potential targets for engineering improved rates of export.

Molecular and genetic characterization of a self-compatible apple cultivar, 'CAU-1'.

In this study, we characterized a naturally occurring self-compatible apple cultivar, 'CAU-1' (S1S9), and studied the underlying mechanism that causes its compatibility. Analyses of both fruit set rate and seed number after self-pollination or cross-pollination with 'Fuji' (S1S9), and of pollen tube growth, demonstrated that 'CAU-1' is self-compatible. Genetic analysis by S-RNase PCR-typing of selfed progeny of 'CAU-1' revealed the presence of all progeny classes (S1S1, S1S9, and S9S9). Moreover, no evidence of S-allele duplication was found. These findings support the hypothesis that loss of function of an S-locus unlinked pollen-part mutation (PPM) expressed in pollen, rather than a natural mutation in the pollen-S gene (S1- and S9- haplotype), leads to SI breakdown in 'CAU-1'. In addition, there were no significant differences in pollen morphology or fertility between 'Fuji' and 'CAU-1'. However, we found that the effect of S1- and S9-RNase on the SI behavior of pollen could not be addressed better in 'CAU-1' than in 'Fuji'. Furthermore, we found that a pollen-expressed hexose transporter, MdHT1, interacted with S-RNases and showed significantly less expression in 'CAU-1' than in 'Fuji' pollen tubes. These findings support the hypothesis that MdHT1 may participate in S-RNase internalization during the SI process, and decrease of MdHT1 expression in 'CAU-1' hindered the release of self S-RNase into the cytoplasm of pollen tubes, thereby protecting pollen from the cytotoxicity of S-RNase, finally probably resulting in self-compatibility. Together, these findings indicate that S-locus external factors are required for gametophytic SI in the Rosaceae subtribe Pyrinae.

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.

Carotenoid accumulation affects redox status, starch metabolism, and flavonoid/anthocyanin accumulation in citrus.

BACKGROUND: Carotenoids are indispensable plant secondary metabolites that are involved in photosynthesis, antioxidation, and phytohormone biosynthesis. Carotenoids are likely involved in other biological functions that have yet to be discovered. In this study, we integrated genomic, biochemical, and cellular studies to gain deep insight into carotenoid-related biological processes in citrus calli overexpressing CrtB (phytoene synthase from Pantoea agglomerans). Fortunella hindsii Swingle (a citrus relative) and Malus hupehensis (a wild apple) calli were also utilized as supporting systems to investigate the effect of altered carotenoid accumulation on carotenoid-related biological processes. RESULTS: Transcriptomic analysis provided deep insight into the carotenoid-related biological processes of redox status, starch metabolism, and flavonoid/anthocyanin accumulation. By applying biochemical and cytological analyses, we determined that the altered redox status was associated with variations in O2 (-) and H2O2 levels. We also ascertained a decline in starch accumulation in carotenoid-rich calli. Furthermore, via an extensive cellular investigation of the newly constructed CrtB overexpressing Fortunella hindsii Swingle, we demonstrated that starch level reducation occurred in parallel with significant carotenoid accumulation. Moreover, studying anthocyanin-rich Malus hupehensis calli showed a negative effect of carotenoids on anthocyanin accumulation. CONCLUSIONS: In citrus, altered carotenoid accumulation resulted in dramatic effects on metabolic processes involved in redox modification, starch degradation, and flavonoid/anthocyanin biosynthesis. These findings provided new perspectives to understand the biological importance of carotenogenesis and of the developmental processes associated with the nutritional and sensory qualities of agricultural products that accumulate carotenoids.

Evidence for a radial strain gradient in apple fruit cuticles.

MAIN CONCLUSION: The morphological outer side of the apple fruit cuticle is markedly more strained than the inner side. This strain is released upon wax extraction. This paper investigates the effect of ablating outer and inner surfaces of isolated cuticular membranes (CM) of mature apple (Malus × domestica) fruit using cold atmospheric pressure plasma (CAPP) on the release of strain after extraction of waxes. Strain release was quantified as the decrease in area of CM discs following CAPP treatment and subsequent solvent extraction of wax. Increasing duration of CAPP treatment proportionally decreased CM mass per unit area. There was no difference in mass loss rate between CAPP treatments of outer or inner surfaces. Also, there was no difference in surface area of CMs before and after CAPP treatment. However, upon subsequent wax extraction, surface area of CMs decreased indicating the release of strain. Increasing the duration of CAPP treatment resulted in increasing strain release up to 47.7 ± 8.0 % at 20 min when CAPP was applied to the inner surface. In contrast, strain release was independent of CAPP duration averaging about 12.1 ± 0.6 % when applied to the outer surface of the CM. Our results provide evidence for a marked gradient of strain between the outer side (strained) and the inner side of the CM (not strained) of mature apple fruit.

X-ray micro-computer tomographic method to visualize the microstructure of different apple cultivars.

Apples are appreciated for their texture with firmness acting as an indicator of quality. During prolonged storage, apples can soften and their texture can become undesirably mealy. Using an X-ray microcomputer tomography (µ-CT) scanner, the porosity (ratio of intercellular space [IS] to total volume) and the structural arrangement of the parenchyma tissue of 4 apple cultivars (Braeburn, Fuji, Golden Delicious, Jazz) stored under similar conditions for 100 d were visualized via the development of 2D and 3D images. The texture of the apples was also measured using a puncture test. The morphometric and textural measurements revealed that firm Jazz apples (flesh firmness: 29.84N) had a lower porosity (17%) compared to soft Golden Delicious apples (flesh firmness: 14.16N; porosity: 29.8%). In general, firm apples had a higher dry matter (%) and a lower porosity (%), while the reverse was true for softer apples. However, this was not an absolute trend as cultivar specific differences in the microstructural organization and consequent mechanical strength of the parenchyma tissue also influenced firmness. For example, although Fuji apples were firm (28.42N), they had a high porosity (29.3%) due to the presence of numerous small and compact IS. In comparison, soft Golden Delicious apples had a high porosity (29.8%) due to the presence of large, interconnected IS. Imaging technologies have the potential to provide a pictorial or graphical database showing the size range distribution of IS corresponding to different parenchyma tissue types and how they relate to apple texture and eating quality.

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH.

The structure of the fruit peel in two varieties of Malus domestica Borkh. (Rosaceae) before and after storage.

The structure of fruit peel of two apple varieties 'Szampion' and 'Jonagold' was investigated using light microscopy as well as scanning and transmission electron microscopy. The samples were taken immediately after harvest and after 6-month controlled atmosphere storage. The Szampion and Jonagold fruit differed in terms of the surface type, number of lenticels, thickness of the cuticular epithelium, height of epidermal cells and thickness of the hypodermis as well as the amount of crystalline wax and the number of microcracks formed on the fruit surface. The 6-month storage resulted in fruit weight loss, increased numbers and depth of microcracks, thickening of the amorphous wax layer and enhanced production of platelet forms of crystalline wax, which filled the microcracks abundantly. Compared with Jonagold, the Szampion fruit exhibited a fewer lenticels, a bigger number of microcracks, smaller amounts of crystalline wax and more substantial weight loss. The apple varieties studied had a reticulate-lamellate cuticle, and at harvest, the epidermal and hypodermal cells contained numerous amyloplasts filled with starch grains, which were not found after the storage period. Additionally, after storage, the cell protoplasts in the apple peel displayed a disorganised structure, and their vacuoles contained fragments of cell membranes, intravacuolar precipitates and deposits, and spherical bodies. The results may facilitate better understanding of changes occurring in fruits of Szampion and Jonagold during storage and help choose the best storage conditions to reduce loss of weight and prevent impairment of fruit quality.

The relation of apple texture with cell wall nanostructure studied using an atomic force microscope.

In this study, the relation of the nanostructure of cell walls with their texture was investigated for six different apple cultivars. Cell wall material (CWM) and cellulose microfibrils were imaged by atomic force microscope (AFM). The mean diameter of cellulose microfibrils for each cultivar was estimated based on the AFM height topographs obtained using the tapping mode of dried specimens. Additionally, crystallinity of cellulose microfibrils and pectin content was determined. Texture of apple cultivars was evaluated by sensory and instrumental analysis. Differences in cellulose diameter as determined from the AFM height topographs of the nanostructure of cell walls of the apple cultivars are found to relate to the degree of crystallinity and pectin content. Cultivars with thicker cellulose microfibrils also revealed crisper, harder and juicier texture, and greater acoustic emission. The data suggest that microfibril thickness affects the mechanical strength of cell walls which has consequences for sensory and instrumental texture.

Down-regulation of POLYGALACTURONASE1 alters firmness, tensile strength and water loss in apple (Malus x domestica) fruit.

BACKGROUND: While there is now a significant body of research correlating apple (Malus x domestica) fruit softening with the cell wall hydrolase ENDO-POLYGALACTURONASE1 (PG1), there is currently little knowledge of its physiological effects in planta. This study examined the effect of down regulation of PG1 expression in 'Royal Gala' apples, a cultivar that typically has high levels of PG1, and softens during fruit ripening. RESULTS: PG1-suppressed 'Royal Gala' apples harvested from multiple seasons were firmer than controls after ripening, and intercellular adhesion was higher. Cell wall analyses indicated changes in yield and composition of pectin, and a higher molecular weight distribution of CDTA-soluble pectin. Structural analyses revealed more ruptured cells and free juice in pulled apart sections, suggesting improved integrity of intercellular connections and consequent cell rupture due to failure of the primary cell walls under stress. PG1-suppressed lines also had reduced expansion of cells in the hypodermis of ripe apples, resulting in more densely packed cells in this layer. This change in morphology appears to be linked with reduced transpirational water loss in the fruit. CONCLUSIONS: These findings confirm PG1's role in apple fruit softening and suggests that this is achieved in part by reducing cellular adhesion. This is consistent with previous studies carried out in strawberry but not with those performed in tomato. In apple PG1 also appears to influence other fruit texture characters such as juiciness and water loss.

Metabolic and gene expression analysis of apple (Malus x domestica) carotenogenesis.

Carotenoid accumulation confers distinct colouration to plant tissues, with effects on plant response to light and as well as health benefits for consumers of plant products. The carotenoid pathway is controlled by flux of metabolites, rate-limiting enzyme steps, feed-back inhibition, and the strength of sink organelles, the plastids, in the cell. In apple (Malus × domestica Borkh), fruit carotenoid concentrations are low in comparison with those in other fruit species. The apple fruit flesh, in particular, begins development with high amounts of chlorophylls and carotenoids, but in all commercial cultivars a large proportion of this is lost by fruit maturity. To understand the control of carotenoid concentrations in apple fruit, metabolic and gene expression analysis of the carotenoid pathway were measured in genotypes with varying flesh and skin colour. Considerable variation in both carotenoid concentrations and compound profile was observed between tissues and genotypes, with carotenes and xanthophylls being found only in fruit accumulating high carotenoid concentrations. The study identified potential rate-limiting steps in carotenogenesis, which suggested that the expression of ZISO, CRTISO, and LCY-ε, in particular, were significant in predicting final carotenoid accumulation in mature apple fruit.

Whole fruit staining with aniline blue at harvest is associated with superficial pathogenesis of "Fuji" apples after storage.

Whole "Fuji" apples (Malus domestica Borkh cv. Fuji) were treated with 0.2% aniline blue before storage in 2006, 2007 and 2008 to determine whether cuticular microcracking was associated with post-storage disorders. After storage for 7 months at 0° C and 90% relative humidity followed by 3 days at 20° C, a higher aniline blue staining scale value was associated with a higher peel browning and decay index. These results indicate that superficial disorders or diseases of apples may be related to cuticular microcracking that can be seen by aniline blue staining. Scanning electron microscopy was used to analyze the ultrastructure of stained portions of the cuticular complex. Disorders or diseases of the cuticle of epidermal tissue was associated with cracked lenticels, unhealed microcracks around the edge of the lenticel, and collapsed epicuticular wax; these areas stained more intensely. Our results indicated the potential of using an aniline blue staining prior to storing the fruit to predict the ultimate quality.

Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples.

Irrigation water and washing water have been inferred to be associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to determine whether apples experimentally contaminated with Cryptosporidium oocysts represent a food safety concern. Laser scanning confocal microscopy revealed no morphological changes in Cryptosporidium parvum oocysts attached to apples after 6 weeks of cold storage, suggesting that oocysts might remain viable and possibly infectious during prolonged storage. Mice were fed apple peels from experimentally contaminated apples to determine whether oocysts had remained infectious on apples stored for 4 weeks. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples, washing methods that have been reported to be helpful for recovery of oocysts from various foodstuffs were evaluated, except that the intensity of washing was increased in the present study. None of the tested washing methods succeeded in completely removing oocysts from the apple peel. The most efficient removal (37.5%) was achieved by rigorous manual washing in water with a detergent and by agitation in an orbital shaker with Tris-sodium dodecyl sulfate buffer. Glycine and phosphate-buffered saline buffers had no effect on oocyst removal. Scanning electron microscopy revealed that some oocysts were attached in deep natural crevices in the apple exocarp and others were attached to the smooth surface of the peel. Some oocysts were closely associated with what appeared to be an amorphous substance with which they might have been attached to the apple surface.

Evidence that prohexadione-calcium induces structural resistance to fire blight infection.

Mechanisms of fire blight control by the shoot-growth regulator prohexadione-calcium (ProCa) were investigated by comparing disease development in ProCa-treated potted apple trees (cv. Gala) to paclobutrazol (another shoot-growth regulator)-treated and nontreated trees and in ProCa-treated cv. McIntosh trees in the field. Twenty-eight days after inoculation with Erwinia amylovora Ea110, disease incidence on ProCa- and paclobutrazol-treated shoots was significantly reduced compared with that on nontreated shoots. Disease severity (percent shoot length infected) was also significantly lower on both ProCa- and paclobutrazol-treated shoots than on nontreated shoots. However, bacterial populations within inoculated shoots were high and bacterial growth occurred in all treatments. In addition, the mean cell wall width of the cortical parenchyma midvein tissue of the first and second youngest unfolded leaves of ProCa- and paclobutrazol-treated shoots was significantly wider both 0.5 and 2 cm from the leaf tips compared with the cell walls of the nontreated tissue. Taken together, these results suggest that reduction of fire blight symptoms by ProCa and paclobutrazol is not the result of reduced populations of E. amylovora in shoots. Moreover, because paclobutrazol also reduced disease severity and incidence, changes in flavonoid metabolism induced by ProCa but not paclobutrazol does not appear to be responsible for disease control as suggested in recent literature. Finally, although this study did not directly link disease control to the observed cell wall changes, the possibility that an increase in cell wall width impedes the spread of E. amylovora should be investigated in more depth.