Moritz Knoche

Rain cracking limits the production of sweet cherries wherever they are grown. Cracking occurs by the separation of neighboring cells along their cell walls (by cell:cell separation). The objectives were to identify the cell wall components involved in cell:cell adhesion using immunolabeling and monoclonal antibodies (mAbs) and digestion assays employing pectinases, hemicellulases, and a cellulase. The mAbs identified homogalacturonans (LM19 and LM20), arabinans (LM6), and (to a lesser extent) xyloglucans (LM25) in skin, parenchyma, xylem, and phloem. Galactan (LM5) occurred only in the phloem, and xylan/arabinoxylan (LM11) occurred only in the xylem. Digestion of parenchyma by polygalacturonase (PGase) increased with time and concentration. Throughout development, digestion was highest by galactanase (GALase), followed by PGase and pectate lyase (PLase). Digestion by the hemicellulases xylanase (XYLase), xyloglucanase (XGase), and mannanase (MANase) was lower. The lowest digestion was due to cellulase (CELase). Incubation in pectinases released more cells, protoplasts, and cell wall fragments than incubation in hemicellulases or CELase. Storage duration had no effect on the digestion of cell walls. Digestion of parenchyma by native enzymes from fruit juice was similar to that by purified enzymes. Boiling juice to destroy enzyme decreased digestion. Adding Ca reduced digestion by PGase. Extracting and complexing Ca by EGTA increased digestion. Across eight cultivars, GALase, PGase, and PLase digested more cell walls than hemicellulases or cellulase. Within pectinases, GALase, PGase, and PLase were equally effective in “Adriana”, whereas in “Kordia”, PGase was more effective than GALase and PLase. In “Flamengo Srim”, “Regina”, and “Staccato”, digestion by GALase, PGase, and PLase was low. We conclude that pectins are primarily responsible for cell:cell adhesion in sweet cherry fruit. Further studies should therefore explore the relationship between cell wall chemistry, Ca status, and cell:cell adhesion.

For plant growth regulators, uptake and physiological performance must be consistent and predictable. The objectives were to establish the uptake behavior of ¹⁴ C labeled 1-aminocyclopropane-[1- ¹⁴ C]carboxylic acid (ACC) into leaves and fruit of apple from simulated spray droplets (1 µl). Uptake into leaves and fruit increased with time. Uptake through the abaxial leaf surface exceeded fruit uptake two-fold, and adaxial leaf uptake seven-fold. Abaxial leaf and fruit uptakes were log-normally distributed. Uptake into fruit increased up to 17% of the applied dose at 32 days after full bloom, but declined thereafter. Along a shoot, uptake increased continuously from the tip to the tenth leaf. Higher ACC concentrations increased the amount of ACC taken up into leaves but not into fruit. The pH of the solution had no effect. Adding salts having a deliquescence point up to 75%, such as CaCl ₂ , enhanced ACC uptake. Tween 20 increased ACC uptake by fruit 9-fold, into young leaves 6-fold and old leaves 2-fold. Uptake of ¹⁴ C-ACC into fruit was higher from commercial formulations than from technical grade ACC. Temperature had no effect on ACC uptake, but higher humidity increased uptake. Penetration of ¹⁴ C-ACC through isolated cuticles of apple fruit was equally low. Increasing the humidity above the droplet residue increased penetration, particularly when droplets were applied on to lenticels. Removal of cuticular wax increased ACC penetration. Our data demonstrate that the mobility of ACC in the droplet deposit limits uptake and that deposit hydration may overcome this limitation.

High levels of fruit calcium (Ca) in strawberry are associated with low levels of surface disorders, including water soaking. Because of the dysfunctional xylem in developing strawberry, fruit Ca can only be increased by spray application. The objectives were to quantify ⁴⁵Ca uptake through the fruit surface from drying spray droplets. Uptake of ⁴⁵CaCl₂ was initially rapid but slowed down after droplet drying. There was no difference in Ca uptake through the rim between achene depressions and that through the achene depressions. The uptake of ⁴⁵CaCl₂ increased with fruit development and with increasing spray concentration. Uptake was highest for CaCl₂ and Ca(NO₃)₂, but was lower for organic salts. The surfactants Glucopon (0.01%) and Triton X-100 (0.1%) increased Ca uptake. Increased temperature and increased relative humidity increased Ca uptake. Incubating ripe strawberries for 1 h in deionized water increased microcracking. Despite the increased microcracking ⁴⁵CaCl₂ uptake did not increase. Spray applications of 30 mM in the greenhouse increased fruit Ca content and decreased susceptibility to water soaking. Our study establishes that strawberry fruit take up Ca very readily from surface sprays of either CaCl₂ or Ca(NO₃)₂. Increases in fruit Ca decrease susceptibility to water soaking.

Thinning reduces biennial bearing in apple. ACC (1-aminocyclopropane-1-carboxylic acid, a precursor of ethylene) is a recent thinning agent. The objective was to identify factors affecting ethylene evolution and fruit abscission following ACC application. Long term, time-course studies reveal that an ACC spray (200 mg l⁻¹) applied 21 days after full bloom (DAFB) induces ethylene evolution which peaks about 1 d after application. From 32 days after application onwards, ethylene evolution did not differ from the un-treated control. ACC induced fruit abscission from 16 days after application onwards. Abscission ceased about 21 days after application. By this time, 27% of the fruit remained on the tree, compared to 61% in the un-treated control. Ethylene evolution per spur peaked, when ACC was applied between 16 and 47 DAFB (equiv. to fruit diameters from 8 to 32 mm). Later ACC applications had little effect on ethylene evolution. Ethylene evolution following ACC application was localized. Selective application to a fruit resulted in increased ethylene evolution by the fruit, but not by leaves and vice versa. ACC effects on ethylene evolution and thinning were similar across six apple cultivars. Ethylene evolution increased linearly with increasing ACC concentration. Most of the ethylene released from spurs was accounted for by ethylene from fruits. Less ethylene was released from leaves. Thinning occurred for ACC concentrations of 200 to 400 mg l⁻¹, lower concentrations were ineffective. Across all experiments, the percentage of fruit remaining on the spur was negatively related to ethylene evolution. Our study shows that ACC (200 to 400 mg l⁻¹), applied when fruit are 6 to 20 mm diameter, induces ethylene evolution and abscission.

Water soaking is a common disorder of field-grown strawberries, and its incidence can be severe. Calcium (Ca) sprays reduce susceptibility to water soaking. Vascular import of Ca to developing strawberry fruit decreases during development as a result of progressive xylem dysfunction. Calcium sprays can raise Ca levels in late-stage strawberry fruit. The objective of this study was to quantify Ca uptake through the fruit skin. Submerging fruit in calcium chloride (CaCl ₂ ) solutions increased both the mass of Ca per fruit and the mass of Ca per fruit dry mass. There was no effect of CaCl ₂ solution concentration on fruit water uptake. Fruit mass increases exponentially during development. When in a CaCl ₂ solution, water soaking was increased, but less severely, rather than when in pure water. Fruit Ca content and fruit size were related positively, but the Ca/dry mass ratio was independent of fruit size. Both were consistently higher for fruit incubated in CaCl ₂ than for control fruit incubated in water. There was no preferential Ca uptake through the calyx or through the abscission scars from petals or stamens. Both fruit Ca content and the fruit Ca/dry mass ratio were related positively to fruit CaCl ₂ concentration and were increased by incubation in solutions of Ca salts. There was no effect of temperature on fruit Ca content or the Ca/dry mass ratio. At all temperatures, fruit incubated in CaCl ₂ had greater Ca contents and Ca/dry mass ratios than fruit incubated in water. Inducing microcracking in the cuticle had no significant effect on the water uptake rate, on the Ca content, or on the Ca/dry mass ratio. Across all experiments, Ca content and the Ca/dry mass ratio correlated negatively with water soaking. In summary, Ca is taken up from a Ca salt solution in contact with the fruit skin, and fruit Ca content and susceptibility to water soaking are related negatively.

The mango cultivar ‘Apple’ is commercially important in Kenya but highly susceptible to russeting. Russeting refers to an area of fruit skin where the primary (epidermal) surface has been replaced by a secondary (peridermal) surface. The objective was to establish histologies, gene expressions and chemical compositions of a natural periderm, a wound-induced periderm and of cuticles of an un-russeted skin. Fluorescence microscopy revealed a suberized phellem in natural and wound-induced periderms. Wound-induced periderms had more cell layers and a higher mass than natural periderms. Compared with cuticles, periderms showed decreased expressions of cuticle-related genes (MiSHN1, MiGPAT6, MiCUS1, MiCER1, MiWCB11) and increased expressions of periderm-related genes (MiMYB93, MiNAC058, MiCYP86A1, MiCYP86B1, MiGPAT5, MiABCG20). Natural periderms and wound-induced periderms contained cutin and suberin monomers (C16-C28), including carboxylic, dicarboxylic, and ω-hydroxy acids and primary alcohols. Cuticles of the primary skin contained cutin monomers of similar chemistry but shorter chain lengths (C16-C22). The wax composition in natural and wound-induced periderms was similar to that in cuticles. Lignin monomers in natural and wound-induced periderms contained p-hydroxyphenyl, guaiacyl and syringyl units, but only traces of these occurred in cuticles. In ‘Apple’ mango, the histologies, gene expressions and compositions of natural and wound-induced periderms are largely the same.

Rain cracking compromises quality and quantity of sweet cherries worldwide. Cracking susceptibility differs among genotypes. The objective was to (1) phenotype the progeny of a cross between a tolerant and a susceptible sweet cherry cultivar for cuticle mass per unit area, strain release on cuticle isolation, cuticular microcracking and calcium/dry mass ratio and (2) relate these characteristics to cracking susceptibilities evaluated in laboratory immersion assays and published multiyear field observations. Mass of the dewaxed cuticle per unit area and strain release upon cuticle isolation were significantly related to cracking susceptibility in lab or field. Cuticular microcracking in the stylar end region as indexed by infiltration with acridine orange was more severe in susceptible than in tolerant genotypes and significantly correlated with susceptibility to cracking in lab and field. The Ca/dry mass ratio was lower (-8%) for susceptible than for tolerant genotypes. Fruit that cracked early had less Ca than those that cracked later. Only the Ca/dry mass ratio of the stylar end region was significantly correlated with cracking susceptibility in the field. Based on stepwise regression analyses microcracking of the cuticle accounted for most of the cracking susceptibilities in field and lab (partial r² = 0.331 to 0.338 for field vs. r² = 0.326 to 0.453 for lab). The variability in cracking susceptibility accounted for increased to a r² = 0.571 (lab) when adding mass of dewaxed cuticle, up to r² = 0.421 (field) when adding the Ca/dry mass ratio in the stylar end region or up to r² = 0.478 (field) when entering the strain release on isolation into the model. A protocol for phenotyping is suggested that allows larger progenies to be phenotyped for microcracking, DCM mass and strain release.

Water soaking is a commercially important disorder of field-grown strawberries that is exacerbated by surface wetness and high humidity. The objective was to establish the effect of genotype on susceptibility to water soaking. Three greenhouse-grown model ‘collections’ were used comprising a total of 172 different genotypes: (1) a segregating F2 population, (2) a collection of strawberry cultivars and breeding clones, and (3) a collection of wild Fragaria species. A standardized immersion assay was used to induce water soaking. Potential relationships between water soaking and water uptake characteristics, depth of the achene depressions, fruit firmness, cuticle mass and strain relaxation and microcracking were investigated. Further, the effect of downregulating the polygalacturonase genes (FaPG1 and FaPG2) on the susceptibility to water soaking was investigated. The collection of wild species was most susceptible to water soaking. This was followed by the collection of cultivars and breeding clones, and by the F2 population. Susceptibility to water soaking was strongly correlated with water uptake rate (mass of water, per fruit, per time). For the pooled dataset of 172 genotypes, 46% of the variability in water soaking was accounted for by the permeance of the skin to osmotic water uptake. Susceptibility to water soaking was not, or was only poorly correlated with measurements of fruit surface area or of the osmotic potential of the expressed fruit juice. The only exceptions were the wild Fragaria species which were highly variable in fruit size and also in fruit osmotic potential. For genotypes from the F2 and the wild species collections, firmer fruit were less susceptible to water soaking than softer fruit. There were no relationships between fruit firmness and susceptibility to water soaking in transgenic plants in which FaPG1 and FaPG2 were down-regulated. Susceptibility to water soaking was not related to cuticle mass per unit fruit surface area, nor to strain relaxation of the cuticle upon isolation, nor to achene position. In summary, strawberry’s susceptibility to water soaking has a significant genetic component and is closely and consistently related to the skin’s permeance to osmotic water uptake.

Background: Ideally, the barrier properties of a fruit’s cuticle persist throughout its development. This presents a challenge for strawberry fruit, with their rapid development and thin cuticles. The objective was to establish the developmental time course of cuticle deposition in strawberry fruit. Results: Fruit mass and surface area increase rapidly, with peak growth rate coinciding with the onset of ripening. On a whole-fruit basis, the masses of cutin and wax increase but on a unit surface-area basis, they decrease. The decrease is associated with marked increases in elastic strain. The expressions of cuticle-associated genes involved in transcriptional regulation (FaSHN1, FaSHN2, FaSHN3), synthesis of cutin (FaLACS2, FaGPAT3) and wax (FaCER1, FaKCS10, FaKCR1), and those involved in transport of cutin monomers and wax constituents (FaABCG11, FaABCG32) decreased until maturity. The only exceptions were FaLACS6 and FaGPAT6 that are presumably involved in cutin synthesis, and FaCER1 involved in wax synthesis. This result was consistent across five strawberry cultivars. Strawberry cutin consists mainly of C16 and C18 monomers, plus minor amounts of C19, C20, C22 and C24 monomers, ω-hydroxy acids, dihydroxy acids, epoxy acids, primary alcohols, carboxylic acids and dicarboxylic acids. The most abundant monomer is 10,16-dihydroxyhexadecanoic acid. Waxes comprise mainly long-chain fatty acids C29 to C46, with smaller amounts of C16 to C28. Wax constituents are carboxylic acids, primary alcohols, alkanes, aldehydes, sterols and esters. Conclusion: The downregulation of cuticle deposition during development accounts for the marked cuticular strain, for the associated microcracking, and for their high susceptibility to the disorders of water soaking and cracking.

Rain cracking (hereinafter referred to as macrocracking) severely impacts the production of sweet cherry ( Prunus avium ). Calcium (Ca) sprays can reduce macrocracking, but the reported responses to Ca sprays are variable and inconsistent. The objective of this study was to establish the physiological mechanism through which Ca reduces macrocracking in sweet cherry fruit. Six spray applications of 50 mM CaCl ₂ had no effect on macrocracking (assessed using a standardized immersion assay) despite a 28% increase in the Ca-to-dry mass ratio. Similarly, during another experiment, there was no effect of up to nine Ca sprays on macrocracking, although the Ca-to-dry mass ratio increased as the number of applications increased. In contrast, CaCl ₂ spray applications during simulated rain (in a fog chamber) significantly reduced the proportion of macrocracked fruit. Additionally, immersion of fruit in CaCl ₂ decreased macrocracking in a concentration-dependent manner. Monitoring macrocrack extension using image analysis revealed that the rate of macrocrack extension decreased markedly as the CaCl ₂ concentration increased. This effect was significant at concentrations as low as 1 mM CaCl ₂ . Decreased anthocyanin leakage, decreased epidermal cell wall swelling, and increased fruit skin stiffness and fracture force contributed to the decrease in macrocracking. There was no effect of CaCl ₂ on the cuticle deposition rate. Our results demonstrated that Ca decreased macrocracking when applied to a wet fruit surface either by spraying on wet fruit or by incubation in solutions containing CaCl ₂ . Under these circumstances, Ca had direct access to the cell wall of an extending macrocrack. The mode of action of Ca in reducing macrocracking is primarily decreasing the rate of crack extension at the tip of a macrocrack.