Apple Fruit Skin Research Articles

Hypermethylation in the promoter regions of flavonoid pathway genes is associated with skin color fading during 'Daihong' apple fruit development.

Apple fruit skin color fading is not well understood although the molecular mechanism of skin color formation is well known. The red-fleshed apple cultivar 'Daihong' (DH) exhibited fading skin color during fruit development despite having a heterozygous R6 allele but lacking Red-TE for red fruit skin. In this study, transcriptomic analysis revealed the expression level of MdMYB10 increased with fruit development whereas reduced expression levels of MdMYBPA1, MdCHS, MdANS, MdUFGT, MdLAR, and MdANR were observed, consistent with decreased levels of chalcone, anthocyanin, catechin, epicatechin, and procyanidin B2. Whole-genome bisulfite sequencing (WGBS) indicated a global gain in cytosine methylation levels and increased methylation in 5' and 3' flanking regions of genes and transposable elements (TEs), and in TE bodies in all CG, CHG and CHH contexts, especially the mCHH context, during fruit development. The increased DNA methylation was attributed to reduced expression levels of DNA demethylase genes, including MdDME1, MdROS1, and MdROS2. Association analysis revealed a significant negative correlation between promoter methylation levels of MdCHS, MdCHI, MdMYBPA1, and their respective transcript levels, as well as a negative correlation between promoter methylation levels of MdCHS, MdCHI, MdANR, and MdFLS, and the content of chalcones, naringenin-7-glucoside, epicatechin, and quercetin. Treatment with the DNA demethylation agent 5-aza-2'-deoxycytidine verified the negative correlation between DNA methylation and gene expression within the flavonoid pathway. These findings suggest that hypermethylation in promoter regions of genes of the flavonoid biosynthesis pathway is associated with the reduction of gene expression and flavonoid content, and fruit skin color fading during DH apple development.

Identification of Apple Fruit-Skin Constitutive Laws by Full-Field Methods Using Uniaxial Tensile Loading.

The protective and preservative role of apple skin in maintaining the integrity of the fruit is well-known, with its mechanical behaviour playing a pivotal role in determining fruit storage capacity. This study employs a combination of experimental and numerical methodologies, specifically utilising the digital image correlation (DIC) technique. A specially devised inverse strategy is applied to evaluate the mechanical behaviour of apple skin under uniaxial tensile loading. Three apple cultivars were tested in this work: Malus domestica Starking Delicious, Malus pumila Rennet, and Malus domestica Golden Delicious. Stress-strain curves were reconstructed, revealing distinct variations in the mechanical responses among these cultivars. Yeoh's hyperelastic model was fitted to the experimental data to identify the coefficients capable of reproducing the non-linear deformation. The results suggest that apple skin varies significantly in composition and structure among the tested cultivars, as evidenced by differences in elastic properties and non-linear behaviour. These differences can significantly affect how fruit is handled, stored, and transported. Thus, the insights resulting from this research enable the development of mathematical models based on the mechanical behaviour of apple tissue, constituting important data for improvements in the economics of the agri-food industry.

Characterization of MdMYB68, a suberin master regulator in russeted apples.

Apple russeting is mainly due to the accumulation of suberin in the cell wall in response to defects and damages in the cuticle layer. Over the last decades, massive efforts have been done to better understand the complex interplay between pathways involved in the suberization process in model plants. However, the regulation mechanisms which orchestrate this complex process are still under investigation. Our previous studies highlighted a number of transcription factor candidates from the Myeloblastosis (MYB) transcription factor family which might regulate suberization in russeted or suberized apple fruit skin. Among these, we identified MdMYB68, which was co-expressed with number of well-known key suberin biosynthesis genes. To validate the MdMYB68 function, we conducted an heterologous transient expression in Nicotiana benthamiana combined with whole gene expression profiling analysis (RNA-Seq), quantification of lipids and cell wall monosaccharides, and microscopy. MdMYB68 overexpression is able to trigger the expression of the whole suberin biosynthesis pathway. The lipid content analysis confirmed that MdMYB68 regulates the deposition of suberin in cell walls. Furthermore, we also investigated the alteration of the non-lipid cell wall components and showed that MdMYB68 triggers a massive modification of hemicelluloses and pectins. These results were finally supported by the microscopy. Once again, we demonstrated that the heterologous transient expression in N. benthamiana coupled with RNA-seq is a powerful and efficient tool to investigate the function of suberin related transcription factors. Here, we suggest MdMYB68 as a new regulator of the aliphatic and aromatic suberin deposition in apple fruit, and further describe, for the first time, rearrangements occurring in the carbohydrate cell wall matrix, preparing this suberin deposition.

Comprehensive genomic identification and expression analysis 4CL gene family in apple

To clarify the structural characteristics, phylogeny, biological function and regulation of 4-coumarate-CoAligase (4CL) in anthocyanin synthesis, the 4CL gene family members in apples were identified and bioinformatic analysis was performed. qRT-PCR was used to analyze the expression levels of this gene family members in different apple varieties, and the role of the 4CL gene in apple anthocyanin synthesis was preliminaries clarified, which provided a certain theoretical basis for the regulatory network of apple anthocyanin synthesis. The results showed that a total of 69 members of the 4CL gene family were identified in the apple (Malus domestica Brokh.), encoding amino acids ranging from 97 to 2310 with theoretical isoelectric points ranging from 5.28 to 9.84. The 69 4CL family members were distributed on 17 chromosomes in the apple, among which chromosome 17 had the largest distribution (9 members), followed by chromosome 9 (7 members), chromosomes 16 and 14 (6 members each), and chromosomes 15 and 13 (5 members each). The subcellular localization prediction showed that apple 4CL gene family members were mainly expressed in cytoplasm, chloroplast, nucleus and cell membrane, with a small amount of expression in mitochondria, vacuoles, peroxisomes, cytoskeleton, golgi and cell matrix, but not in endoplasmic reticulum. The secondary structures are mainly α-helices and irregular coils. Microarray expression profile analysis showed that the expression levels of each member in apple were related to fruit variety and tissue structure, and the expression levels were mainly higher in fruit, flower and leaf. Real-time PCR analysis showed that the expression level of each member was directly proportional to the degree of fruit coloring and anthocyanin accumulation. The expression levels of Md4CL10 and Md4CL23 in ‘Astar’ (G4) apple fruit skin with the highest anthocyanin content were 516, 20 and 2 times higher than those in ‘Chengji NO.1′ (G1), ‘Golden Delicious’ (G2) and ‘Ruixue’ (G3), respectively.

Multi-omics analyses reveal MdMYB10 hypermethylation being responsible for a bud sport of apple fruit color.

Apple bud sports offer a rich resource for clonal selection of numerous elite cultivars. The accumulation of somatic mutations as plants develop may potentially impact the emergence of bud sports. Previous studies focused on somatic mutation in the essential genes associated with bud sports. However, the rate and function of genome-wide somatic mutations that accumulate when a bud sport arises remain unclear. In this study, we identified a branch from a 10-year-old tree of the apple cultivar 'Oregon SpurII' as a bud sport. The mutant branch showed reduced red coloration on fruit skin. Using this plant material, we assembled a high-quality haplotype reference genome consisting of 649.61Mb sequences with a contig N50 value of 2.04Mb. We then estimated the somatic mutation rate of the apple tree to be 4.56 × 10 -8 per base per year, and further identified 253 somatic single-nucleotide polymorphisms (SNPs), including five non-synonymous SNPs, between the original type and mutant samples. Transcriptome analyses showed that 69 differentially expressed genes between the original type and mutant fruit skin were highly correlated with anthocyanin content. DNA methylation in the promoter of five anthocyanin-associated genes was increased in the mutant compared with the original type as determined using DNA methylation profiling. Among the genetic and epigenetic factors that directly and indirectly influence anthocyanin content in the mutant apple fruit skin, the hypermethylated promoter of MdMYB10 is important. This study indicated that numerous somatic mutations accumulated at the emergence of a bud sport from a genome-wide perspective, some of which contribute to the low coloration of the bud sport.

Expression analysis of MYB genes in different apple cultivars with distinct coloration pattern during fruit maturation

Abstract Anthocyanins are water-soluble pigments responsible for red, pink, purple or blue coloration in the flesh and/or skin of apple fruit. Since consumers prefer anthocyanin rich fruits due to their health benefits, anthocyanin content is an important trait for marketability of apples. Synthesis and accumulation of anthocyanins are controlled by environmental and genetic factors such as transcription factors (TFs). Two-repeat (R2R3) MYB TFs are involved in the regulation of anthocyanin biosynthesis in plants. In this study, the expression of the MdMYBA and MdMYB3 genes encoding R2R3 type MYB TFs were analyzed in apple cultivars with fruit skin color from green to dark red at different growth stages. Fruit samples were collected from “Scarlet Spur”, “Galaxy Gala”, Golden Delicious” “Granny Smith” apple cultivars with dark red, light red, yellow and green fruits, respectively, at four different time periods after full-bloom. Total RNA was isolated from the peel of the collected fruits and the expression of the MdMYBA and MdMYB3 genes was analyzed by real-time RT-PCR. The expression of the MdMYBA gene started to increase at 92 DAFB and thereafter in red-skin apples; however, no expression was observed at any time points in yellow or green-skin apple cultivars. On the other hand, the MdMYB3 gene was expressed in red-skin and yellow-skin apple cultivars starting at 55 DAFB, but no expression was observed in green apple cultivar at any time of fruit maturation. The expression levels of the MdMYBA and MdMYB3 genes varied in apple fruits with different skin coloration indicating that they play a role regulation of the anthocyanin biosynthesis in apple cultivars during fruit maturation.

MdMYB114 regulates anthocyanin biosynthesis and functions downstream of MdbZIP4-like in apple fruit

Anthocyanins, a major class of compounds derived from the flavonoid pathway, are important pigments of apple fruit. They can also prevent certain diseases and are beneficial to human health. Fruit pigmentation is a key quality trait that influences consumer preference; therefore, it is of great importance to investigate its regulatory mechanism. Here, we identified a MYB transcription factor (TF), MdMYB114, whose transcript level increased in the skin of the deep red apple fruit. It was determined to belong to the R2R3-MYB TF family and was localized in the nucleus. MdMYB114 overexpression led to anthocyanin accumulation in apple calli. MdMYB114 was not able to form an MBW complex but could enhance anthocyanin biosynthesis and transport by directly binding to the promoters of MdANS, MdUFGT, and MdGST to promote their expression. In addition, multiple assays revealed that MdbZIP4-like, a basic leucine-zipper TF, could directly bind to the MdMYB114 promoter to enhance its expression. Taken collectively, our results provide evidence that MdMYB114 is a positive regulator of anthocyanin biosynthesis and transport and it functions downstream of MdbZIP4-like in apple fruit.

Structural Studies of the Cutin from Two Apple Varieties: Golden Delicious and Red Delicious (Malus domestica).

The cuticle, a protective cuticular barrier present in almost all primary aerial plant organs, has a composition that varies between plant species. As a part of the apple peel, cuticle and epicuticular waxes have an important role in the skin appearance and quality characteristic in fresh fruits destined for human consumption. The specific composition and structural characteristics of cutin from two apple varieties, “golden delicious” and “red delicious”, were obtained by enzymatic protocols and studied by means of cross polarization magic angle spinning nuclear magnetic resonance (CP-MAS 13C NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and mass spectrometry, and were morphologically characterized by specialized microscopy techniques (atomic force microscopy (AFM), confocal laser scanning microscopy (CLMS), and scanning electron microscopy (SEM)). According to CP-MAS 13C NMR and ATR-FTIR analysis, cutins from both varieties are mainly composed of aliphatics and a small difference is shown between them. This was corroborated from the hydrolyzed cutins analysis by mass spectrometry, where 9,10,18-trihydroxy-octadecanoic acid; 10,20-Dihydroxy-icosanoic acid; 10,16-dihydroxy hexadecenoic acid (10,16-DHPA); 9,10-epoxy-12-octadecenoic acid; and 9,10-epoxy-18-hydroxy-12-octadecenoic acid were the main monomers isolated. The low presence of polysaccharides and phenolics in the cutins obtained could be related to the low elastic behavior of this biocomposite and the presence of cracks in the apple cutin’s surface. These cracks have an average depth of 1.57 µm ± 0.57 in the golden apple, and 1.77 µm ± 0.64 in those found in the red apple. The results obtained in this work may facilitate a better understanding that mechanical properties of the apple fruit skin are mainly related to the specific aliphatic composition of cutin and help to much better investigate the formation of microcracks, an important symptom of russet formation.

MYB_SH[AL]QKY[RF] transcription factors MdLUX and MdPCL-like promote anthocyanin accumulation through DNA hypomethylation and MdF3H activation in apple.

Heritable DNA methylation is a highly conserved epigenetic mark that is important for many biological processes. In a previous transcriptomic study on the fruit skin pigmentation of apple (Malus domestica Borkh.) cv. 'Red Delicious' (G0) and its four continuous-generation bud sport mutants including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4), we identified MYB transcription factors (TFs) MdLUX and MdPCL-like involved in regulating anthocyanin synthesis. However, how these TFs ultimately determine the fruit skin color traits remains elusive. Here, bioinformatics analysis revealed that MdLUX and MdPCL-like contained a well-conserved motif SH[AL]QKY[RF] in their C-terminal region and were located in the nucleus of onion epidermal cells. Overexpression of MdLUX and MdPCL-like in 'Golden Delicious' fruits, 'Gala' calli and Arabidopsis thaliana promoted the accumulation of anthocyanin, whereas MdLUX and MdPCL-like suppression inhibited anthocyanin accumulation in 'Red Fuji' apple fruit skin. Yeast one-hybrid assays revealed that MdLUX and MdPCL-like may bind to the promoter region of the anthocyanin biosynthesis gene MdF3H. Dual-luciferase assays indicated that MdLUX and MdPCL-like activated MdF3H. The whole-genome DNA methylation study revealed that the methylation levels of the mCG context at the upstream (i.e., promoter region) of MdLUX and MdPCL-like were inversely correlated with their mRNA levels and anthocyanin accumulation. Hence, the data suggest that MYB_SH[AL]QKY[RF] TFs MdLUX and MdPCL-like promote anthocyanin biosynthesis in apple fruit skins through the DNA hypomethylation of their promoter regions and the activation of the structural flavonoid gene MdF3H.

Identification of gene co-expression networks and key genes regulating flavonoid accumulation in apple (Malus × domestica) fruit skin

Anthocyanin provides a red color for apple and health benefit for human. To better understand the molecular mechanisms of regulating apple color formation, we analyzed 27 transcriptomes of fruit skin from three cultivars ‘Huashuo’ (red-skinned), ‘Hongcuibao’ (red-skinned), and ‘Golden Delicious’ (yellow-skinned) at 0, 2, and 6 days after bag removal. Using pairwise comparisons and weighted gene co-expression network analyses (WGCNA), we constructed 17 co-expression modules. Among them, a specific module was negatively correlated to anthocyanin accumulation. The genes in the module are enriched in flavonoid biosynthesis pathways. These pathway genes were used to construct gene co-expression network of anthocyanin accumulation. Finally, a R2R3-MYB repressor designated MdMYB28 was identified as a key hub gene in the anthocyanin metabolism network. During the anthocyanin accumulation of apple fruit skin reaching a peak, MdMYB28 expression level was negatively correlated with the anthocyanin content. MdMYB28 was shown to directly bind to the promoter of MdMYB10 in yeast one-hybrid analyses. Over-expression of MdMYB28 decreased the anthocyanin biosynthesis in tobacco flower petals, suggesting that MdMYB28 acts as a negatively regulator of anthocyanin biosynthesis.

Surface moisture increases microcracking and water vapour permeance of apple fruit skin.

Surface moisture induces microcracking in the cuticle of fruit skins. Our objective was to study the effects of surface moisture on cuticular microcracking, the permeance to water vapour and russeting in developing 'Pinova' apple fruit. Surface moisture was applied by fixing to the fruit a plastic tube containing deionized water. Microcracking was quantified by fluorescence microscopy and image analysis following infiltration with acridine orange. Water vapour permeance was determined gravimetrically using skin segments (ES) mounted in diffusion cells. Cumulative water loss through the ES increased linearly with time. Throughout development, surface moisture significantly increased skin permeance. The effect was largest during early development and decreased towards maturity. Recovery time courses revealed that following moisture treatment of young fruit for 12days, skin permeance continued to increase until about 14days after terminating the moisture treatment. Thereafter, skin permeance decreased over the next 28days, then approaching the control level. This behaviour indicates gradual healing of the impaired cuticular barrier. Nevertheless, permeance still remained significantly higher compared with the untreated control. Similar patterns of permeance change were observed following moisture treatments at later stages of development. The early moisture treatment beginning at 23 DAFB resulted in russeting of the exposed surfaces. There was no russet in control fruit without a tube or in control fruit with a tube mounted for 12days without water. The data demonstrate that surface moisture increases microcracking and water vapour permeance. This may lead to the formation of a periderm and, hence, a russeted fruit surface.

Shading affects fracture force and fracture strain of apple fruit skins

Simple observation shows that naturally shaded apple fruit are more prone to russeting than sun-exposed fruit. The formation of microcracks in the fruit skin is the first visible sign of russeting. Our objective was to establish the effects of different levels of shading on the mechanical properties of apple (Malus × domestica Borkh.) fruit skins.Exposure of developing ‘Idared’ apples to visible and UV light was varied by shade-bagging and by sampling fruit from naturally shaded and exposed positions in the canopy. Fruit exposure to UV light was also varied using UV-absorbing film. Epidermal skin segments were excised, the cuticular membranes were isolated enzymatically and both were subjected to uniaxial tensile testing.Partial shading or blocking of UV light decreased the frequency of fruit having a red surface and also reduced the intensity of the red coloration, as indexed by increases in hue angle. The stiffness of epidermal skin segments was not affected by partial shading but fracture force and fracture strain were decreased. Blockage of UV slightly increased stiffness and decreased fracture force and fracture strain. The stiffness, fracture force and fracture strain of isolated cuticle samples were all markedly less than of epidermal skin segments. This indicates that, regardless of light treatment (visible and/or UV), it is the cellular layers of the skin, not the cuticle, that represent the load-bearing structure.There were no effects of shading or of UV blockage on cell size, cell shape or cell aspect ratio of either epidermal or hypodermal cells, or on sizes of parenchyma cells.There were also no detectable effects of shading or of UV blockage on thickness or wax content of the cuticle.We conclude that the effect of shading (visible and UV light) on the mechanical properties of the apple fruit skin was small, but was nevertheless consistent with an increase in the russet susceptibility of shaded fruit.

High-resolution melting (HRM) analysis with SNP or SSR markers related to apple skin color or rootstock identification

Anthocyanin biosynthesis is reported to be controlled by one of the transcription factors, called MdMYB1 in apple fruit skin. MdMYB1-1, one allele of MdMYB1 gene, is the only allele associated with the red skin color and other alleles produce yellow or green skin apples. In this study, five single-nucleotide polymorphism (SNP) markers were developed to distinguish which is red-skinned or non-red apples based on the sequences of MdMYB1 alleles. The SNP markers were assessed by high-resolution melting (HRM) analysis according to the melting curves patterns. The “M.9” and “M.26” are the widely used dwarfing rootstocks in South Korea. The discrimination of rootstocks using morphological traits is almost impossible as apple trees get older. HRM analysis has been used to identify “M.9” and “M.26” with simple sequence repeat (SSR) markers. Two SSR markers were highly informative and generated distinguishable curve profiles from HRM analysis. These markers would be efficiently and accurately utilized in breeding program targeting fruit skin color and cultivar identification with HRM analysis.

Control of Fruit Cracking by Shaping Skin Traits – Apple as a Model

Skin cracking limits fruit quality and marketability. Suggested causes are environmental conditions, orchard management, and failure of the skin to resist surface tension due to fruit expansion. Fruit skin is made up of epidermis cells and cuticular matrix. Theoretical and experimental studies of skin mechanics, together with anatomical and molecular comparisons of cracking-susceptible vs. tolerant genotypes, suggest that increased cuticle thickness, high epidermal cell density and strong adhesion between neighboring cells are associated with cracking resistance. Calyx-end cracking disorder in apple is treated with a mixture of gibberellic acids 4 and 7 (GA4 + 7) and the cytokinin 6-benzyladenine (BA) early in fruit development. The treatment not only significantly reduces cracking incidence in the orchard, it also provides information on the cellular and molecular factors determining fruit-skin resistance to growth strain. BA + GA4 + 7 application results in an immediate increase in epidermal cell density that is maintained until fruit maturation. Moreover, the epidermal cells form clusters within the cuticular matrix, which may strengthen the cuticle by adding more cell-wall components and may enhance crack repair. Skin anatomical modifications are complemented by the expression of genes associated with epidermal cell patterning and cuticle formation. Gene-networking analysis supports the interaction between cell-wall synthesis, cuticle-formation, and GA-signaling gene clusters. Overall, data suggest that BA + GA4 + 7 treatment does not modify developmental cues, but promotes or enhances the innate developmental program. This review presents data on BA- and GA4 + 7-induced skin modifications that complement previously suggested models for cracking resistance in fruit. Knowledge gained on apple fruit skin traits may be applied to control cracking in other fruit as well.

Whole-genome DNA methylation patterns and complex associations with gene expression associated with anthocyanin biosynthesis in apple fruit skin.

DNA methylation of anthocyanin biosynthesis-related genes and MYB/bHLH transcription factors was associated with apple fruit skin color revealed by whole-genome bisulfite sequencing. DNA methylation is a common feature of epigenetic regulation and is associated with various biological processes. Anthocyanins are among the secondary metabolites that contribute to fruit colour, which is a key appearance and nutrition quality attribute of apple fruit. Although few studies reported that DNA methylation in the promoter of MYB transcription factor was associated with fruit skin color, there is a general lack of understanding of the dynamics of global and genic DNA methylation in apple fruit. Here, whole-genome bisulfite sequencing was carried out in fruit skin of apple (Malus domestica Borkh.) cv. 'Red Delicious' (G0) and its four-generation bud sport mutants, including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4) at color break stage. Correlation and linear-regression analysis between DNA methylation level and anthocyanin content, as well as the transcription levels of genes related to anthocyanin biosynthesis were carried out. The results showed that the number of differentially methylated regions (DMRs) and differentially methylated genes (DMGs) was considerably increased from G1 to G4 versus the number observed in G0. The mCHH context was dominant in apple, but the levels of mCG and mCHG of DMGs were significantly higher than that of the mCHH. Genetic variation of bud sport mutants from 'Red Delicious' was associated with differential DNA methylation. Additionally, hypomethylation of mCG and mCHG contexts in flavonoid biosynthesis pathway genes (PAL, 4CL, CYP98A, PER, CCoAOMT, CHS, and F3'H), mCHG context in MYB10 at upstream, led to transcriptional activation and was conductive to anthocyanin accumulation. However, hypermethylation of mCG context in bHLH74 at upstream led to transcriptional inhibition, inhibiting anthocyanin accumulation.

Biochemical changes in diabetic rats treated with ethanolic extract of Chysophyllum albidum fruit-skin

BackgroundDiabetes mellitus (DM), also known as diabetes, involves a group of metabolic disorders in which blood sugar levels are high over a prolonged period. The present study was designed to evaluate the effect of 21 days administration of 100 and 200 mg per kg bodyweight of ethanolic extract of Chrysophyllum albidum (African star apple) fruit-skin on streptozotocin (STZ)-diabetic Wistar albino rats. A randomized control trial was used. Diabetic state of test rats was induced with streptozotocin at a dose of 60 mg streptozotocin/kg body using saline (0.9% w/v NaCl) as vehicle. Upon confirmation of diabetic state, crude ethanolic extract of C. albidum was dissolved in citrate buffer at pH 4.2 before administration to the rats at specific doses.ResultsResults show a significant (p < 0.05) decreased in the level of plasma creatinine and insulin but increased level of glucose in diabetic rats relative to control. Induction of diabetes significantly increased the activities of alanine aminotransferases and aspartate aminotransferases in the liver and kidney and activities of both enzymes were also correspondingly increased in the plasma. There was significant increase in the level of total cholesterol, low-density lipoprotein-cholesterol, very low-density lipoprotein-cholesterol and triglyceride, but decreased high-density lipoprotein-cholesterol in the test rats. Results also show alterations in the antioxidant status of experimental rats. The diabetic-induced changes were reversed in diabetic rats upon administration of C. albidum extract at a dose of 200 mg/kg body weight to levels comparable with control.ConclusionThe findings obtained revealed that ethanolic extract of C. albidum fruit-skin at a dose of 200 mg/kg body weight reversed the effect of diabetes due to its antihyperglycemic, anti-hyperlipidemic and antioxidant properties. The antidiabetic effect of the extract may be due to its abundant flavonoid and alkaloid contents.

Combined use of cutinase and high-resolution mass-spectrometry to query the molecular architecture of cutin

BackgroundCutin is a complex, highly cross-linked polyester consisting of hydroxylated and epoxidated acyl lipid monomers. Because of the complexity of the polymer it has been difficult to define the chemical architecture of the polymer, which has further limited the ability to identify the catalytic components that assemble the polymer. Analogous to methods that define the structure of oligosaccharides, we demonstrate a strategy that utilizes cutinase to generate cutin subfragments consisting of up to four monomeric units, whose structure and spatial distribution in the polymer is revealed by high-resolution mass spectrometry. Moreover, the application of mass-spectrometric fragmentation and labelling of the end of the oligomers, one is able to define the order of monomers in the oligomer. The systematic application of this strategy can greatly facilitate understanding the chemical architecture of this complex polymer.ResultsThe chemical architecture of plant cutin is dissected by coupling an enzymatic system that deconstructs the polymer into subfragments consisting of dimers, trimers and tetramers of cutin monomers, with group-specific labeling and mass spectrometry. These subfragments can be generated with one of over 1200 of cutinases identified from diverse biological sources. The parallel chemical labeling of the polymer with dansyl, alkyl or p-dimethylaminophenacyl reagents can identify the chemical distribution of non-esterified hydroxyl- and carboxyl-groups among the monomers. This combined strategy is applied to cutin isolated from with apple fruit skins, and a combination of gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–quadrupole time-of-flight (Q-TOF) MS is used to determine the order of the monomers in the cutinase-generated subfragments. Finally, we demonstrate the use of matrix-assisted laser desorption-ionization-MS to determine the spatial distribution of the cutinase-generated subfragments.ConclusionOur experimental results demonstrate an advancement to overcome the current limitations in identifying cutin oligomeric structure and allows one to more efficiently address new biological questions about cutin biosynthesis. We submit that the systematic application of these methods will enable the construction of more accurate architectural models of cutin, which is a prerequisite to identifying cutin-biosynthetic components.

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

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 20kPa, strains were low and microcracks shallow, but at > 40kPa, 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.

Effects of epigallocatechin-3-gallate (EGCG) on skin greasiness and related gene expression in ‘Jonagold’ apple fruit during ambient storage

Some apple (Malus × domestica) cultivars can become greasy during ripening due to changes in their surface wax composition. Epicuticular waxes are derived by the de novo synthesis of fatty acids, catalyzed by fatty acid synthase (FAS). Epigallocatechin-3-gallate (EGCG) has previously been shown to be an inhibitor of FAS in both animals and bacteria. Therefore, we hypothesize it may also affect the development of postharvest skin greasiness in apples. In this study, ‘Jonagold’ apples were treated with EGCG and then stored at 20 °C. The composition of the surface waxes was analyzed and the expressions of genes related to wax synthesis were recorded. The results show that at these postharvest storage temperatures, the level of skin greasiness rises rapidly. This is consistent with measured increases in the content of the main fluid waxes - linoleate and oleate esters. Compared with the controls, the accumulation of fluid waxes was suppressed by EGCG at most stages during ambient storage. A number of genes related to fluid wax biosynthesis, including MdKASIII, MdKAR, MdFAD2 and MdWSD11, showed significant increases in expression during the development of skin greasiness. Meanwhile, the expressions of MdKASII and MdENR, the transport gene MdLTPG1 and the regulation gene MdSHN3 declined during storage. The expressions of all these genes were down-regulated by EGCG, while the fruit respiration and firmness were similar between the EGCG-treated and control fruit throughout the storage period. We suppose that the inhibitory effects of EGCG are to down-regulate expression levels of genes involved in wax biosynthesis. This results in reduced accumulation of the fluid wax components and delays and lowers the development of skin greasiness. Our results also indicate that MdFAD2 and MdWSD11 may be critical for formation of greasy esters in apple skins. This work is the first evaluation of the effects of EGCG on apple skin greasiness. The results supply useful information elucidating the mechanisms through which apple fruit skins can become greasy during postharvest storage.

Pre-harvest spray application of phenylpropanoids influences accumulation of anthocyanin and flavonoids in ‘Cripps Pink’ apple skin

Abstract Poor colour development on ‘Cripps Pink’ apple fruit causes serious economic losses to the growers in the warmer regions of the world. Effects of pre-harvest spray application of different concentrations (0, 50, 100, or 200 mg L−1) of cinnamic acid, p-coumaric acid, d -phenylalanine, l -phenylalanine, and naringenin, on red blush development on the fruit surface and accumulation of anthocyanins, dihydrochalcones, hydroxycinnamic acid, flavanols, and flavonols, in the skin of ‘Cripps Pink’ apple at commercial fruit maturity were investigated. Skin of apple fruit harvested from the trees sprayed with l -phenylalanine or cinnamic acid contained significantly higher concentrations of total anthocyanins, cyanidin 3-galactoside, and flavonols, compared to control and to those treated with other phenylpropanoids, in a dose- independent pattern. Based on tristimulus system of colour perception {i.e. chromaticity values such as L*, a*, b*, chroma (C*), and hue angle (h°)}, a similar trend of red blush development was observed on fruit’s exterior at commercial maturity, in response to pre-harvest spray application of phenylpropanoids. The apple fruit harvested from the trees sprayed with l -phenylalanine or cinnamic acid had the highest values of a* and the lowest values of h° for colour perception on the exposed side of apple fruit, irrespective of their doses applied, indicating a substantial trend of red blush intensification on the fruit surface. In conclusion, a single pre-harvest spray application of l -phenylalanine (100 mg L−1), applied 3–4 weeks prior to the anticipated commercial fruit maturity was most effective in improving red blush development, increased production of export-grade fruit, accumulation of total anthocyanins, cyanidin 3-galactoside, flavanols, chlorogenic acid and flavonols in the fruit skin, without adversely affecting the fruit quality.