Changes in strain and deposition of cuticle in developing sweet cherry fruit

Changes in surface area, deposition and elastic strain of the cuticular membrane (CM) were monitored during development of sweet cherry (Prunus avium L.) fruit. Fruit mass and surface area ('Sam') increased in a sigmoidal pattern between 16 and 85 days after full bloom (DAFB) with maximum rates of 0.35 g day(-1) and 0.62 cm(2) day(-1), respectively. Rates of total area strain, namely the sum of elastic plus plastic strain, were highest in cheek and stem cavity regions followed by stylar and suture regions. Rates of total uniaxial strain were higher in transverse, namely perpendicular to the stem/stylar axis, than in longitudinal direction, namely parallel to the stem/stylar axis. On a whole fruit basis CM mass remained essentially constant during fruit development. Mass of CM, dewaxed CM and wax per unit surface area decreased during development, particularly between 43 and 71 DAFB. There was no change in wax content of isolated CM. Up to 43 DAFB the surface area of isolated CM was similar to the area prior to excision indicating little elastic strain, but markedly decreased thereafter. Calculating elastic and plastic components of total strain of the CM revealed, that initial deformation up to 22 to 43 DAFB was mostly plastic. Thereafter, elastic strain was evident and both, elastic and plastic deformation, increased linearly with an increase in total strain. There was no consistent difference in the relative contribution of elastic strain to total strain between transverse and longitudinal directions, but both total and elastic strain were larger in the transverse direction. Abrading the CM had only little effect on fruit turgor. However, turgor decreased when the exocarp was cut indicating that the exocarp provided a significant structural shell of a mature sweet cherry fruit ('Regina'). Our data demonstrate, that (1) surface area expansion in sweet cherry fruit causes elastic and plastic strain of the CM, and (2) the onset of elastic strain coincided with the cessation of CM formation.     

Expression of a sweet cherry DREB1/CBF ortholog in Arabidopsis confers salt and freezing tolerance

Dehydration responsive element binding protein 1 (DREB1)/C-repeat binding factor (CBF) induces the expression of many stress-inducible genes in Arabidopsis. We have previously reported the identification of three DREB1/ICBF homologs from sweet cherry (Prunus avium). To identify the function of these homologs, one of the genes, CIG-B, was transformed into Arabidopsis. In one of the transgenic plant lines, the DREB1/CBF target gene cor15a was induced in the absence of stress treatment. The cor15a-overexpressing transgenic plant exhibited mild growth retardation and had greater salt and freezing tolerance than did the wild-type and the transgenic lines in which cor15a was not induced. These results suggest that this sweet cherry DREB1/CBF homolog has a function similar to that of DREB1/CBF.     

Isozyme diversity in sour,sweet, and ground cherry

Thirty-six sour (Prunus cerasus L.), sweet (P. avium L.), and ground cherry (P. fruticosa Pall.) selections were evaluated for seven enzyme systems and principal coordinate analysis was used to examine isozyme divergence among these cherry species. The enzyme systems studied were phosphoglucose isomerase (PGI), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGD), leucine aminopeptidase (LAP), shikimate dehydrogenase (SKDH), and malate dehydrogenase (MDH). The first principal coordinate, which accounted for 41% of the total variation, separated the diploid sweet cherry selections from the sour, ground, and sour x ground cherry tetraploids. An additional 86 selections were evaluated for up to six of the enzyme systems to determine the polymorphisms at the enzyme loci and the level of heterozygosity between the diploid sweet cherry and the tetraploid species and interspecific hybrids. 6-PGD was the most polymorphic enzyme exhibiting 16 patterns. The tetraploid cherry species were more heterozygous than the diploid sweet cherry with an average heterozygosity of 78% compared to 19% for the diploids.     

Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development

Pseudomonas syringae relies on type III secretion system to deliver effector proteins into the host cell for parasitism. Type III genes are induced in planta, but host factors affecting the induction are poorly understood. Here we report on the identification of an Arabidopsis mutant, att1 (for aberrant induction of type three genes), that greatly enhances the expression of bacterial type III genes avrPto and hrpL. att1 plants display enhanced disease severity to a virulent strain of P. syringae, suggesting a role of ATT1 in disease resistance. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase catalyzing fatty acid oxidation. The cutin content is reduced to 30% in att1, indicating that CYP86A2 plays a major role in the biosynthesis of extracellular lipids. att1 has a loose cuticle membrane ultrastructure and shows increased permeability to water vapor, demonstrating the importance of the cuticle membrane in controlling water loss. The enhanced avrPto-luc expression is specific to att1, but not another cuticle mutant, wax2. The results suggest that certain cutin-related fatty acids synthesized by CYP86A2 may repress bacterial type III gene expression in the intercellular spaces.     

Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.     

Studies on water transport through the sweet cherry fruit surface: II. Conductance of the cuticle in relation to fruit development

Water conductance of the cuticular membrane (CM) of sweet cherry (Prunus avium L. cv. Sam) fruit during stages II and III (31-78 days after full bloom, DAFB) was investigated by gravimetrically monitoring water loss through segments of the exocarp. Segments were mounted in stainless-steel diffusion cells, filled with 0.5 ml of deionized water and incubated for 8 h at 25 +/- 2 degrees C over dry silica. Conductance was calculated by dividing the amount of water transpired per unit surface area and time by the difference in water vapor concentration across the segment (23.07 g m(-3) at 25 degrees C). Fruit mass and fruit surface area increased 4.9- and 2.8-fold between 31 and 78 DAFB, respectively. However, CM mass per unit area decreased from 3.9 to 1.5 g m(-2) and percentage of total wax content remained constant at about 31%. Stomatal density decreased from 0.8 to 0.2 mm(-2) (31-78 DAFB). Total conductance of the CM on the fruit cheek (gtot.) remained constant during stage II of development (approx. 1.38 x 10(-4) m s(-1) from 31 to 37 DAFB), increased to 1.73 x 10(-4) m s(-1) during early stage III of fruit growth (43-64 DAFB) then decreased to 0.95 x 10(-4) m s(-1) at maturity (78 DAFB). Partitioning gtot. into cuticular (gcut.) and stomatal conductance (gsto.) revealed that the relative contribution of gcut. to gtot. increased linearly from 30% to 87% of gtot. between 31 and 78 DAFB. respectively. On a whole-fruit basis, g,tot. and gcut. consistently increased up to 64 DAFB, and decreased thereafter. A significant negative linear relationship was obtained between gcut. and CM thickness, but not between the permeability coefficient (p) and CM thickness. Further, p was positively related to strain rate, suggesting that strain associated with expansion of the fruit surface increased p.     

Studies on water transport through the sweet cherry fruit surface: III. Conductance of the cuticle in relation to fruit size

Rain-cracking of sweet cherry fruit has been related to water absorption through the fruit surface and large fruit has been reported to be more susceptible to cracking than small fruit. Therefore, the effect of fruit size on water conductance of the cuticular membrane (CM) of exocarp segments excised from cheek, suture or stylar end region of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated. Segments consisting of epidermis, hypodermis and several layers of mesocarp cells were mounted in diffusion cells filled with deionized water. Mass loss due to transpiration was monitored gravimetrically during an 8-h incubation period (25 +/- 2 degrees C) over dry silica in the dark. Conductance was calculated from the amount of water transpired per unit surface area and time divided by the difference in water vapour concentration across the segment. For an average size cv. Sam sweet cherry CM conductance was 1.06 x 10-4, 0.91 x 10-4 and 2.09 x 10-4 m s-1 in cheek, suture and stylar end region, respectively. Fruit size had no significant effect on conductance in cheek or suture regions, but for the stylar end region conductance was positively related to fruit size. Stomatal density in the cheek, but not the suture or stylar end region increased as fruit size increased. The area of the stylar scar was positively related to fruit size. Conductance of the stylar scar averaged 37.6 +/- 4.0 x 10-4 m s-1 and was 54-fold higher than that of the CM between stomata in the cheek region (mean 0.69 x 10-4 m s-1). Conductance calculated on a whole fruit basis is estimated to increase by 108% as fruit size increases from 6 to 12 g. Increased conductance on a whole fruit basis may be attributed to increased fruit surface area and increased conductance per unit fruit surface area, particularly in the stylar end region.     

Linkage disequilibrium in French wild cherry germplasm and worldwide sweet cherry germplasm

A basic knowledge on linkage disequilibrium (LD) is necessary in order to determine resolution of association studies. We investigated the extent and patterns of LD in a self-incompatible species (Prunus avium L.), in 3 groups (wild cherry, sweet cherry landraces and sweet cherry modern varieties), using a set of 35 microsatellite markers and the gametophytic self-incompatibility locus. Since population structure might create spurious LD, we thus used the information provided by a structure analysis published in a previous study to perform the LD analysis. In the current study, we detected a greater LD extent in sweet cherry than in wild cherry, which is plausibly due to the bottleneck associated with domestication and breeding. Higher LD values in sweet cherry sub-groups may be explained by smaller sample sizes. We also showed that the remaining structure in the groups of sweet cherry, in particular landraces, is responsible for a part of the LD extent. Intra-group relatedness may also account for extensive LD in two sub-groups. These results demonstrate, if ever necessary, the importance of controlling the genetic structure and relatedness when estimating LD. Moreover, LD decays very rapidly with genetic linkage distance in both wild and sweet cherries, which seems promising for future association studies.     

Effect of temperature on pollen tube kinetics and dynamics in sweet cherry, Prunus avium (Rosaceae)

Prevailing ambient temperature during the reproductive phase is one of several important factors for seed and fruit set in different plant species, and its consequences on reproductive success may increase with global warming. The effect of temperature on pollen performance was evaluated in sweet cherry (Prunus avium L.), comparing as pollen donors two cultivars that differ in their adaptation to temperature. 'Sunburst' is a cultivar that originated in Canada with a pedigree of cultivars from Northern Europe, while 'Cristobalina' is a cultivar native to southeast Spain, adapted to warmer conditions. Temperature effects were tested either in controlled-temperature chambers or in the field in a plastic cage. In both genotypes, an increase in temperature reduced pollen germination, but accelerated pollen tube growth. However, a different genotypic response, which reflected the overall adaptation of the pollen donor, was obtained for pollen tube dynamics, expressed as the census of the microgametophyte population that successfully reached the base of the style. While both cultivars performed similarly at 20°C, the microgametophyte population was reduced at 30°C for Sunburst and at 10°C for Cristobalina. These results indicate a differential genotypic response to temperature during the reproductive phase, which could be important in terms of the time needed for a plant species to adapt to rapid temperature changes.     

Cell number regulator genes in Prunus provide candidate genes for the control of fruit size in sweet and sour cherry

Striking increases in fruit size distinguish cultivated descendants from small-fruited wild progenitors for fleshy fruited species such as Solanum lycopersicum (tomato) and Prunus spp. (peach, cherry, plum, and apricot). The first fruit weight gene identified as a result of domestication and selection was the tomato FW2.2 gene. Members of the FW2.2 gene family in corn (Zea mays) have been named CNR (Cell Number Regulator) and two of them exert their effect on organ size by modulating cell number. Due to the critical roles of FW2.2/CNR genes in regulating cell number and organ size, this family provides an excellent source of candidates for fruit size genes in other domesticated species, such as those found in the Prunus genus. A total of 23 FW2.2/CNR family members were identified in the peach genome, spanning the eight Prunus chromosomes. Two of these CNRs were located within confidence intervals of major quantitative trait loci (QTL) previously discovered on linkage groups 2 and 6 in sweet cherry (Prunus avium), named PavCNR12 and PavCNR20, respectively. An analysis of haplotype, sequence, segregation and association with fruit size strongly supports a role of PavCNR12 in the sweet cherry linkage group 2 fruit size QTL, and this QTL is also likely present in sour cherry (P. cerasus). The finding that the increase in fleshy fruit size in both tomato and cherry associated with domestication may be due to changes in members of a common ancestral gene family supports the notion that similar phenotypic changes exhibited by independently domesticated taxa may have a common genetic basis.     

Effect of ethephon on sweet cherry pedicel-fruit retention force and quality is cultivar dependent

Ethephon (2-chloroethyl phosphonic acid) is effectively used to promote development of the pedicel-fruit abscission zone in tart cherry (Prunus cerasus) for mechanical harvest. Our research program is investigating the use of ethephon to promote pedicel-fruit retention force (PFRF) reduction on new sweet cherry (P. avium) cultivars to assist mechanized harvest and its affect upon fresh market quality fruit. Ethephon treatments were made at different timings and rates to ‘Bing’ and ‘Chelan’ during the 2006 season. Ethephon applications to ‘Bing’ trees more than 10 days prior to harvest were effective at reducing PFRF and facilitating mechanical harvest, irrespective of rate (1.2, 3.5, 5.8 L ha⁻¹ [1, 3, 5 pt A⁻¹]). Ethephon applied fewer than 10 days prior to harvest did not reduce PFRF sufficiently. In contrast, no rate or timing of ethephon studied induced a reduction in ‘Chelan’ PFRF sufficient for mechanical harvest. Accompanying PFRF analyses, fruit quality was assessed by measuring firmness (g mm⁻¹), soluble solids (oBrix), weight (g) and color (CTIFL, scale 1–7). Ethephon applied 22 days before harvest at a rate of 3.5 L ha⁻¹ enhanced exocarp color in ‘Bing’ by 27%, while reducing firmness in both ‘Bing’ (−19%, 22 days prior to harvest) and ‘Chelan’ (−15%, 20 days prior to harvest). We observed a significant natural decline in ‘Skeena’ PFRF to levels acceptable for mechanical harvest. This research documents genotypic-specific pedicel-fruit abscission characteristics and the potential to facilitate mechanical harvest of fresh market quality sweet cherry fruit using ethephon.     

Chloroplast inheritance and DNA variation in sweet, sour, and ground cherry

Sour cherry (Prunus cerasus L.) is an allotetraploid and both sweet cherry (P avium L.) and ground cherry (P. fruticosa Pall.) are the proposed progenitor species. The study investigated the maternal species origin(s) of sour cherry using chloroplast DNA (cpDNA) markers and a diverse set of 22 sweet, 25 sour, and 7 ground cherry selections. Two cpDNA restriction fragment length polymorphisms (RFLPs) and one polymerase chain reaction (PCR) fragment length polymorphism were identified among the 54 selections. The three polymorphisms considered together resolved four haplotypes. Analysis of sour cherry progeny indicated that the chloroplast genome is maternally inherited and therefore appropriate to use in determining maternal phylogenetic relationships. Ground cherry was found more likely than sweet cherry to be the maternal progenitor species of sour cherry since 23 of 25 of the sour cherry selections had the most prevalent ground cherry haplotype. However, the other two sour cherry selections tested had the most prevalent sweet cherry haplotype and a wild French sweet cherry selection had the most prevalent ground cherry haplotype. The results underscore the importance of using diverse Prunus germplasm to investigate phylogenetic relationships.     

Expression pattern of ABA metabolic and signalling genes during floral development and fruit set in sweet cherry

Abscisic acid plays a crucial role in the regulation of fruit development and ripening, however, its role in the floral development and the fruit set is still unclear. In the present study, the ABA accumulation and the expression patterns of genes related to ABA metabolism and signalling in sweet cherry were investigated. The results showed that ABA accumulation increased and peaked at stage V in ovary, at stage VI in stamen, and in young fruit it peaked at 7 days after full bloom. The expression pattern of ABA synthetase PaNCED1 was consistent with the changes of ABA accumulation. Among four ABA degradation enzymes PaCYP707As, PaCYP707A4 was highly expressed in ovary, PaCYP707A1 was mainly in stamen, and PaCYP707A2 was in young fruit, and their expressions were reversed to the trend of PaNCED1. With regard to ABA signalling genes, among three ABA receptors PaPYLs, PaPYL2 and PaPYL3 were high expression genes in ovary and in young fruit with similar expression patterns, while PaPYL3 was the high expression gene in stamen. Within six PaPP2Cs, PaPP2C1/2/3 were highly expressed in ovary and young fruit, while PaPP2C3/4 were mainly in stamen. The six PaSnRK2s showed different expression patterns: PaSnRK2.1/2.2/2.4 were highly expressed in ovary and young fruit, while PaSnRK2.1/2.3 were highly expressed in stamen. In situ hybridization results showed that PaPYL3, PaPP2C3 and PaSnRK2.4 were expressed in seed, pulp and fruit peel during fruit set. In conclusion, ABA and its signaling may play an important role in the regulation of floral development and fruit set.     

Studies on water transport through the sweet cherry fruit surface: IX. Comparing permeability in water uptake and transpiration

Water uptake and transpiration were studied through the surface of intact sweet cherry (Prunus avium L.) fruit, exocarp segments (ES) and cuticular membranes (CM) excised from the cheek of sweet cherry fruit and astomatous CM isolated from Schefflera arboricola (Hayata) Hayata, Citrus aurantium L., and Stephanotis floribunda Brongn. leaves or from Lycopersicon esculentum Mill. and Capsicum annuum L. var. annuum Fasciculatum Group fruit. ES and CM were mounted in diffusion cells. Water (deionized) uptake into intact sweet cherry fruit, through ES or CM interfacing water as a donor and a polyethyleneglycol (PEG 6000, osmotic pressure 2.83 MPa)-containing receiver was determined gravimetrically. Transpiration was quantified by monitoring weight loss of a PEG 6000-containing donor (2.83 MPa) against dry silica as a receiver. The permeability coefficients for osmotic water uptake and transpiration were calculated from the amount of water taken up or transpired per unit surface area and time, and the driving force for transport. Permeability during osmotic water uptake was markedly higher than during transpiration in intact sweet cherry fruit (40.2-fold), excised ES of sweet cherry fruit (12.5- to 53.7-fold) and isolated astomatous fruit and leaf CM of a range of species (on average 23.0-fold). Partitioning water transport into stomatal and cuticular components revealed that permeability of the sweet cherry fruit cuticle for water uptake was 11.9-fold higher and that of stomata 56.8-fold higher than the respective permeability during transpiration. Increasing water vapor activity in the receiver from 0 to 1 increased permeability during transpiration across isolated sweet cherry fruit CM about 2.1-fold. Permeability for vapor uptake from saturated water vapor into a PEG 6000 receiver solution was markedly lower than from liquid water, but of similar magnitude to the permeability during self-diffusion of ³H₂O in the absence of osmotica. The energy of activation for self-diffusion of water across ES or CM was higher than for osmotic water uptake and decreased with increasing stomatal density. The data indicate that viscous flow along an aqueous continuum across the sweet cherry fruit exocarp and across the astomatous CM of selected species accounted for the higher permeability during water uptake as compared to self-diffusion or transpiration.     

Polymorphisms in sweet taste genes (TAS1R2 and GLUT2), sweet liking,and dental caries prevalence in an adult Italian population

The aim of the study was to assess the relationship between sweet taste genes and dental caries prevalence in a large sample of adults. In addition, the association between sweet liking and sugar intake with dental caries was investigated. Caries was measured by the decayed, missing, filled teeth (DMFT) index in 647 Caucasian subjects (285 males and 362 females, aged 18–65 years), coming from six villages in northeastern Italy. Sweet liking was assessed using a 9-point scale, and the mean of the liking given by each individual to specific sweet food and beverages was used to create a sweet liking score. Simple sugar consumption was estimated by a dietary history interview, considering both added sugars and sugar present naturally in foods. Our study confirmed that polymorphisms in TAS1R2 and GLUT2 genes are related to DMFT index. In particular, GG homozygous individuals for rs3935570 in TAS1R2 gene (p value = 0.0117) and GG homozygous individuals for rs1499821 in GLUT2 gene (p value = 0.0273) showed higher DMFT levels compared to both heterozygous and homozygous for the alternative allele. Furthermore, while the relationship sugar intake–DMFT did not achieve statistical significance (p value = 0.075), a significant association was identified between sweet liking and DMFT (p value = 0.004), independent of other variables. Our study showed that sweet taste genetic factors contribute to caries prevalence and highlighted the role of sweet liking as a predictor of caries risk. Therefore, these results may open new perspectives for individual risk identification and implementation of target preventive strategies, such as identifying high-risk patients before caries development.

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