Growth strains cause vascular browning and cavities in ´Nicoter´ apples.

'Nicoter' apples (Malus × domestica Borkh.) occasionally develop a disorder referred to as vascular browning. Symptomatic fruit are perceived as being of low quality. The objective was to identify the mechanistic basis of this disorder. The frequency of symptomatic 'Nicoter' apples differed between growing sites and increased with delayed harvest. Typical symptoms are tissue browning and cavities in the ray parenchyma of the calyx region, and occasionally also of the stem end. Cavity size is positively correlated with the extent of tissue browning. Cavities were oriented radially in the direction of the bisecting line between the radii connecting the calyx/pedicel axis to the sepal and petal bundles. Microscopy revealed cell wall fragments in the cavities indicating physical rupture of cell walls. Immunolabelling of cell wall epitopes offered no evidence for separation of cells along cell walls. The growth pattern in 'Nicoter' is similar to that in its parents 'Gala' and 'Braeburn'. Allometric analyses revealed no differences in growth in fruit length among the three cultivars. However, the allometric analyses of growth in diameter revealed a marked increase in the distance between the surface of the calyx cavity and the vascular bundle in 'Nicoter', that was absent in 'Braeburn' and 'Gala'. This increase displaced the petal bundles in the ray parenchyma outwards and subjected the tissue between the petal and sepal bundles to tangential strain. Rupture of cells results in tissue browning and cavity formation. A timely harvest is a practicable countermeasure for decreasing the incidence of vascular browning.

Xylogenesis and phloemogenesis in the flesh of sweet cherry fruit are limited to early-stage development.

Water inflows into sweet cherry fruit occur via the xylem and the phloem vasculatures of the pedicel. The rates of these inflows are subject to marked changes during fruit development. The objective was to establish if, and when, xylogenesis and phloemogenesis occur in the fruit flesh (mesocarp) during fruit development. Fruit were cut in half and the median and the lateral bundles inspected by light microscopy. Fruit mass increased with time in a double sigmoid pattern. Xylogenesis and phloemogenesis were both limited to early fruit development (stage I). There were no consistent changes in the areas of either xylem or phloem after stage I until maturity (i.e., during stages II and III). The cross-sectional areas of xylem and of phloem in a bundle were both linearly related to total bundle area. Most of the increases (stage I) in bundle area (62%, r2 = 0.99***) were accounted for by increases in phloem area and about 35% (r2 = 0.97***) by increases in xylem area. A small percentage of the xylem area increase (about 4% of the total area of the bundle; r2 = 0.48***) was contributed by the appearance of intercellular spaces within the xylem. Our results suggest, that new xylem and phloem tissues are differentiated only during early development.

Sweet cherry flesh cells burst in non-random clusters along minor veins.

MAIN CONCLUSION: Sweet cherry flesh cells burst when exposed to water but they do so in clusters indicating heterogeneity with respect to osmotic concentration, which depends on proximity to a minor vein. Water plays a key role in cracking in sweet cherry fruit. Magnetic resonance imaging has previously indicated preferential partitioning of water along veins. A more negative osmotic potential along veins seems the likely explanation. Here we establish if cell bursting in mature sweet cherry fruit is also associated with the veins. Cell bursting was identified by a novel light microscope technique involving exposure of a cut fruit surface to water or to sucrose solutions. Upon exposure to water there was no bursting of skin cells but for cells of the flesh (mesocarp) bursting increased with time. When the cut surface was exposed to sucrose solutions of decreasing osmotic concentrations (increasing water potentials) the incidence of cell bursting increased from hypertonic (no bursting), to isotonic, to hypotonic. Cell bursting in the outer mesocarp occurred primarily in the vicinity of minor veins that in the inner mesocarp was primarily between radial veins. The median distance between a minor vein and a bursting cell (mean diameter 0.129 mm) was about 0.318 mm that between a radial vein and a bursting cell was about 0.497 mm. In contrast, the distance between adjacent minor veins averaged 2.57 mm, that between adjacent radial veins averaged 0.83 mm. Cell bursting tends to occur in clusters. Mapping of cell bursting indicates (1) that a seemingly uniform population of mesocarp cells actually represents a heterogeneous population with regard to their cell osmotic potentials and (2) cell bursting afflicts clusters of neighbouring cells in the vicinities of minor veins.

Strawberry fruit skins are far more permeable to osmotic water uptake than to transpirational water loss.

Water movements through the fruit skin play critical roles in many disorders of strawberry (Fragaria × ananassa Duch.) such as water soaking, cracking and shriveling. The objective was to identify the mechanisms of fruit water loss (dry skin, transpiration) and water uptake (wet skin, osmosis). Fruits were held above dried silica gel or incubated in deionized water. Water movements were quantified gravimetrically. Transpiration and osmotic uptake increased linearly with time. Abrading the thin cuticle (0.62 g m-2) increased rates of transpiration 2.6-fold, the rates of osmotic uptake 7.9-fold. The osmotic potential of the expressed juice was nearly the same for green and for white fruit but decreased in red fruit stages. Fruit turgor was low throughout development, except for green fruit. There was no relationship between the rates of water movement and fruit osmotic potential. The skin permeance for transpiration and for osmotic uptake were both high (relative to other fruit species) but were two orders of magnitude greater for osmotic uptake than for transpiration. Incubating fruit in isotonic solutions of osmolytes of different sizes resulted in increases in fruit mass that depended on the osmolyte. The rate of osmotic uptake decreased asymptotically as molecular size of the osmolyte increased. When transpiration and osmotic uptake experiments were conducted sequentially on the same fruit, the rates of transpiration were higher for fruit previously incubated in water. Fluorescence microscopy revealed considerable microcracking in a fruit previously incubated in water. Our findings indicate that the high permeance for osmotic uptake is accounted for by an extremely thin cuticle and by viscous water flow through microcracks and along polar pathways.

Spatial heterogeneity of flesh-cell osmotic potential in sweet cherry affects partitioning of absorbed water.

A fleshy fruit is commonly assumed to resemble a thin-walled pressure vessel containing a homogenous carbohydrate solution. Using sweet cherry (Prunus avium L.) as a model system, we investigate how local differences in cell water potential affect H2O and D2O (heavy water) partitioning. The partitioning of H2O and D2O was mapped non-destructively using magnetic resonance imaging (MRI). The change in size of mesocarp cells due to water movement was monitored by optical coherence tomography (OCT, non-destructive). Osmotic potential was mapped using micro-osmometry (destructive). Virtual sections through the fruit revealed that the H2O distribution followed a net pattern in the outer mesocarp and a radial pattern in the inner mesocarp. These patterns align with the disposition of the vascular bundles. D2O uptake through the skin paralleled the acropetal gradient in cell osmotic potential gradient (from less negative to more negative). Cells in the vicinity of a vascular bundle were of more negative osmotic potential than cells more distant from a vascular bundle. OCT revealed net H2O uptake was the result of some cells loosing volume and other cells increasing volume. H2O and D2O partitioning following uptake is non-uniform and related to the spatial heterogeneity in the osmotic potential of mesocarp cells.

Localized bursting of mesocarp cells triggers catastrophic fruit cracking.

The so-called rain-cracking of sweet cherry fruit severely threatens commercial production. Simple observation tells us that cuticular microcracking (invisible) always precedes skin macrocracking (visible). The objective here was to investigate how a macrocrack develops. Incubating detached sweet cherry fruit in deionized water induces microcracking. Incubating fruit in D2O and concurrent magnetic resonance imaging demonstrates that water penetration occurs only (principally) through the microcracks, with nondetectable amounts penetrating the intact cuticle. Optical coherence tomography of detached, whole fruit incubated in deionized water, allowed generation of virtual cross-sections through the zone of a developing macrocrack. Outer mesocarp cell volume increased before macrocracks developed but increased at a markedly higher rate thereafter. Little change in mesocarp cell volume occurred in a control zone distant from the crack. As water incubation continued, the cell volume in the crack zone decreased, indicating leaking/bursting of individual mesocarp cells. As incubation continued still longer, the crack propagated between cells both to form a long, deep macrocrack. Outer mesocarp cell turgor did not differ significantly before and after incubation between fruit with or without macrocracks; nor between cells within the crack zone and those in a control zone distant from the macrocrack. The cumulative frequency distribution of the log-transformed turgor pressure of a population of outer mesocarp cells reveals all cell turgor data followed a normal distribution. The results demonstrate that microcracks develop into macrocracks following the volume increase of a few outer mesocarp cells and is soon accompanied by cell bursting.

Sweet Cherry Fruit: Ideal Osmometers?

Osmotic water uptake through the skin is an important factor in rain cracking of sweet cherries. The objective was to establish whether a sweet cherry behaves like an ideal osmometer, where: (1) water uptake rates are negatively related to fruit osmotic potential, (2) a change in osmotic potential of the incubation solution results in a proportional change in water uptake rate, (3) the osmotic potential of the incubation solution yielding zero water uptake is numerically equal to the fruit water potential (in the absence of significant fruit turgor), and (4) the fruits' cuticular membrane is permeable only to water. The fruits' average osmotic potential and the rate of water uptake were related only weakly. Surprisingly, incubating a fruit in (a) the expressed juice from fruit of the same batch or (b) an isotonic artificial juice composed of the five major osmolytes of expressed juice or (c) an isotonic glucose solution-all resulted in significant water uptake. Decreasing the osmotic potential of the incubation solution decreased the rate of water uptake, while decreasing it still further resulted in water loss to the incubation solution. Throughout fruit development, the "apparent" fruit water potential was always more negative than the fruits' measured average osmotic potential. Plasmolysis of epidermal cells indicates the skin's osmotic potential was less negative than that of the flesh. When excised flesh discs were incubated in a concentration series of glucose solutions, the apparent water potential of the discs matched the osmotic potential of the expressed juice. Significant penetration of 14C-glucose and 14C-fructose occurred through excised fruit skins. These results indicate a sweet cherry is not an ideal osmometer. This is due in part to the cuticular membrane having a reflection coefficient for glucose and fructose less than unity. As a consequence, glucose and fructose were taken up by the fruit from the incubation solution. Furthermore, the osmotic potential of the expressed fruit juice is not uniform. The osmotic potential of juice taken from the stylar scar region is more negative than that from the pedicel region and that from the flesh more negative than that from the skin.

Physical rupture of the xylem in developing sweet cherry fruit causes progressive decline in xylem sap inflow rate.

MAIN CONCLUSION: Xylem flow is progressively shut down during maturation beginning with minor veins at the stylar end and progressing to major veins and finally to bundles at the stem end. This study investigates the functionality of the xylem vascular system in developing sweet cherry fruit (Prunus avium L.). The tracers acid fuchsin and gadoteric acid were fed to the pedicel of detached fruit. The tracer distribution was studied using light microscopy and magnetic resonance imaging. The vasculature of the sweet cherry comprises five major bundles. Three of these supply the flesh; two enter the pit to supply the ovules. All vascular bundles branch into major and minor veins that interconnect via numerous anastomoses. The flow in the xylem as indexed by the tracer distribution decreases continuously during development. The decrease is first evident at the stylar (distal) end of the fruit during pit hardening and progresses basipetally towards the pedicel (proximal) end of the fruit at maturity. That growth strains are the cause of the decreased conductance is indicated by: elastic strain relaxation after tissue excision, the presence of ruptured vessels in vivo, the presence of intrafascicular cavities, and the absence of tyloses.

Intracuticular wax fixes and restricts strain in leaf and fruit cuticles.

This paper investigates the effects of cuticular wax on the release of strain and on the tensile properties of enzymatically isolated cuticular membranes (CMs) taken from leaves of agave (Agave americana), bush lily (Clivia miniata), holly (Ilex aquifolium), and ivy (Hedera helix) and from fruit of apple (Malus × domestica), pear (Pyrus communis), and tomato (Lycopersicon esculentum). Biaxial strain release was quantified as the decrease in CM disc area following wax extraction. Stiffness, maximum strain and maximum force were determined in uniaxial tensile tests using strips of CM and dewaxed CMs (DCMs). Biaxial strain release, stiffness, and maximum strain, but not maximum force, were linearly related to the amount of wax extracted. Apple CM has the most wax and here the effect of wax extraction was substantially accounted for by the embedded cuticular wax. Heating apple CM to 80°C melted some wax constituents and produced an effect similar to, but smaller than, that resulting from wax extraction. Our results indicate that wax 'fixes' strain, effectively converting reversible elastic into irreversible plastic strain. A consequence of 'fixation' is increased cuticular stiffness.

Russeting in apple and pear: a plastic periderm replaces a stiff cuticle.

BACKGROUND AND AIMS: Russeting in apples (Malus × domestica Borkh.) and pears (Pyrus communis L.) is a disorder of the fruit skin that results from microscopic cracks in the cuticle and the subsequent formation of a periderm. To better understand russeting, rheological properties of cuticular membranes (CM) and periderm membranes (PM) were studied from the russet-sensitive apple 'Karmijn de Sonnaville' and from 'Conference' pear. METHODOLOGY: The CM and PM were isolated enzymatically, investigated by microscopy and subjected to tensile tests, creep/relaxation tests and to stepwise creep tests using a material testing machine. PRINCIPAL RESULTS: The isolated CM formed a continuous polymer, whereas the PM represented a cellular structure of stacked cork cells. Tensile tests revealed higher plasticity of the hydrated PM compared with the CM, as indicated by a higher strain at the maximum force (ɛ(max)) and a lower modulus of elasticity (E). In apple, the maximum force (F(max)) was higher in the CM than in the PM but in pear the higher F(max) value was found for the PM. In specimens obtained from the CM : PM transition zone, the weak point in apple was found to be at the CM : PM borderline but in pear it was within the CM. In both apple and pear, creep/relaxation tests revealed elastic strain, creep strain, viscoelastic strain and viscous strain components in both the PM and CM. For any particular force, strains were always greater in the PM than in the CM and were also greater in pear than in apple. The ɛ(max) and F(max) values of the CM and PM were lower than those of non-russeted and russeted whole-fruit skin segments, which included adhering tissue. CONCLUSIONS: In russeting, stiff CM are replaced by more plastic PM. Further, the cell layers underlying the CM and PM represent the load-bearing structure in the fruit skin in apple and pear.

Vascular connections between the receptacle and empty achenes in sunflower (Helianthus annuus L.).

Empty achenes in sunflower, particularly in the centre of the capitulum, may be caused by poor vascularization. This hypothesis was tested by microscopic examination and translocation experiments. Phloem and xylem were identified by fluorescence of aniline-blue-stained callose and autofluorescence, respectively. Vascular strands that extended from the receptacle into empty achenes were regularly found in longitudinal sections. The phloem-mobile probe, carboxyfluorescein, was translocated from the receptacle to the pericarp and the testa of empty achenes. Similarly, (14)CO(2)-derived (14)C-photoassimilates moved into empty achenes. The observations suggest that empty achenes are both structurally and functionally connected with the vascular system of the receptacle. Hence, deficient vascular connections do not prevent seed filling in sunflower.

Evidence for sectorial photoassimilate supply in the capitulum of sunflower (Helianthus annuus).

• Photoassimilate transport from source leaves to the capitulum was investigated in sunflower (Helianthus annuus) during anthesis and seed filling. • Following foliar application of a 13/14 CO2 -pulse, labelled photoassimilates were detected using mass spectrometry, phosphorimaging, HPTLC and HPLC. • The upper 10 (to 15) leaves exported photoassimilates into the capitulum. Photoassimilate distribution patterns were sectorial: each leaf supplied a defined 2/8-3/8 sector of the capitulum. Photoassimilates exported via the midvein accumulated in a 1/8 sector, which aligned exactly with the insertion site of the leaf. The two main lateral veins of the leaf exported photoassimilates into the two adjacent 1/8 sectors of the capitulum. During early and late stages of anthesis, strong sinks were staminate florets and young achenes, respectively. During seed filling, an import maximum and minimum appeared in the intermediate and central whorls, respectively. Sucrose was established as the only phloem transport sugar. Raffinose, although also 14 C-labelled in the path, is not transported in sunflower. • It is concluded that a single floret is typically connected with the leaves of three neighbouring ortostichies in sunflower. Photoassimilate distribution patterns demonstrated here generally may reflect the functional relationships between the phyllotaxy of source leaves and the position of sinks in developing inflorescences like those of Asteraceae.