A multidisciplinary approach providing new insight into fruit flesh browning physiology in apple (Malus x domestica Borkh.).
ABSTRACT: In terms of the quality of minimally processed fruit, flesh browning is fundamentally important in the development of an aesthetically unpleasant appearance, with consequent off-flavours. The development of browning depends on the enzymatic action of the polyphenol oxidase (PPO). In the 'Golden Delicious' apple genome ten PPO genes were initially identified and located on three main chromosomes (2, 5 and 10). Of these genes, one element in particular, here called Md-PPO, located on chromosome 10, was further investigated and genetically mapped in two apple progenies ('Fuji x Pink Lady' and 'Golden Delicious x Braeburn'). Both linkage maps, made up of 481 and 608 markers respectively, were then employed to find QTL regions associated with fruit flesh browning, allowing the detection of 25 QTLs related to several browning parameters. These were distributed over six linkage groups with LOD values spanning from 3.08 to 4.99 and showed a rate of phenotypic variance from 26.1 to 38.6%. Anchoring of these intervals to the apple genome led to the identification of several genes involved in polyphenol synthesis and cell wall metabolism. Finally, the expression profile of two specific candidate genes, up and downstream of the polyphenolic pathway, namely phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), provided insight into flesh browning physiology. Md-PPO was further analyzed and two haplotypes were characterised and associated with fruit flesh browning in apple.
Project description:Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten <i>PPO</i> genes (named <i>SmelPPO1</i>-<i>10</i>) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target <i>PPO</i> genes (<i>SmelPPO4, SmelPPO5</i>, and <i>SmelPPO6)</i>, which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three <i>PPO</i> genes. The successful editing of the <i>SmelPPO4, SmelPPO5</i>, and <i>SmelPPO6</i> loci of <i>in vitro</i> regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified <i>in silico</i> proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T<sub>1</sub> and T<sub>2</sub> progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.
Project description:The elevation of anthocyanin contents in fruits and vegetables is a breeding target for many crops. In some fruit, such as tomato, higher anthocyanin concentrations enhance storage and shelf life. In contrast, highly anthocyanic red-fleshed apples (Malus x domestica) have an increased incidence of internal browning flesh disorder (IBFD). To determine the mechanisms underlying this, 'Royal Gala' cultivar apples over-expressing the anthocyanin-related transcription factor (TF) MYB10 (35S:MYB10), which produces fruit with highly pigmented flesh, were compared with standard 'Royal Gala' Wild Type (WT) grown under the same conditions. We saw no incidence of IBFD in WT 'Royal Gala' but the over-expression of MYB10 in the same genetic background resulted in a high rate of IBDF. We assessed concentrations of potential substrates for IBDF and a comparison of metabolites in these apples showed that anthocyanins, chlorogenic acid, pro-cyanidins, flavon-3-ols, and quercetin were all higher in the MYB10 lines. For the flavol-3-ols sub-group, epicatechin rather than catechin was elevated in MYB10 lines compared with the control fruit. Internal ethylene concentrations were measured throughout fruit development and were significantly higher in 35S:MYB10 lines, and ethylene was detected at an earlier developmental stage pre-harvest. Expression analysis of key genes associated with ethylene biosynthesis (aminocyclopropane-1-carboxylic acid synthase and oxidase; ACS and ACO) and polyphenol oxidase (PPO) showed the potential for increased ethylene production and the mechanism for enhanced PPO-mediated browning. The expression of a transcription factor of the ethylene response factor (ERF) class, ERF106, was elevated in red flesh. Analysis of transcriptional activation by MYB10 showed that this transcription factor could activate the expression of apple ACS, ACO, and ERF106 genes. Our data show a link between the elevation of anthocyanin-related transcription factors and an undesirable fruit disorder. The accelerated advancement of maturity via premature ethylene induction has implications for the breeding and storage of these more highly pigmented plant products.
Project description:Direct cold plasma treatment has been investigated as an alternative non-thermal technology as a means of maintaining and improving quality of fresh cloudy apple juice. Process variables studied included type of plasma discharge, input voltage and treatment time on polyphenol oxidase (PPO) inactivation. Spark discharge plasma at 10.5?kV for 5?min was the best treatment, with near total inactivation of PPO achieved, although good PPO inactivation was also recorded using shorter treatment times. Residual activity (RA) of PPO was 16 and 27.6% after 5 and 4?min of treatment respectively. This PPO inactivation was maintained throughout the storage trials, but decreased with samples treated for a shorter time. Plasma treatment improved key quality parameters of Golden delicious cloudy apple juice, with retention of critical quality parameters during extended storage trials. Color was the most noticeable change, which was enhanced with retention of a greener color. An increase of 69 and 64% was obtained in the total phenolic content after 4 and 5?min of treatment, respectively. Therefore, cold plasma was demonstrated to be a good alternative to traditional heat treatments for enhanced quality retention of fresh cloudy apple juice and over its storage.
Project description:This study examined the effect of debagging time on color and flavor / taste compounds in the non-red apple cultivar 'Golden Delicious' and red cultivar 'Qinguan' at mid and late stages of fruit development. Debagging briefly improved the red color in both cultivars, the peel of 'Golden Delicious' presenting pale-pink hue. However, rapid anthocyanin accumulation occurred in apple peel at a specific time (after 179 days after flowering (DAF) in 'Qinguan') and was unaltered by debagging time in the red cultivar 'Qinguan'. Furthermore, untimely debagging had a detrimental effect on the content of anthocyanin. All sugars increased and organic acids decreased in apple pulp at mid to late stages of development. Bagging treatment reduced the content of most sugars and organic acids, as well as, the overall total. However, glucose and citric acid contents were higher in bagged fruit than non-bagged fruit; the maximum occurred in T7 treatment that was no-debagging at DAF 159 / 196 ('Golden delicious' / 'Qinguan'), i.e., 24.35 and 0.07 mg g-1 FW in 'Golden delicious', and 38.86 and 0.06 mg g-1 FW in 'Qinguan', respectively. In a word, bagging treatment can alter the pattern of peel color development in apple fruit; however, it remains difficult to alter the timing of rapid anthocyanin accumulation as it is regulated solely by development. Moreover, bagging treatment reduced the total accumulation of sugars and organic acids, and even the over total in pulp, but increased the glucose and citric acid contents in apple pulp.
Project description:Optimisation of processing time and pre-treatments are crucial factors prior to apple drying to produce a high-quality product. The purpose of the present study was to test the utility of physical (hot-water, HWB and steam blanching, SB) and chemical (1% ascorbic acid, AA; and 1% citric acid, CA) treatments, alone or in combination in reducing surface discolouration as well as oxidative enzyme activity in apple slices (cv. Golden Delicious and Elstar) exposed to air at room temperature for 0, 30 and 60 min. The total colour change (?E) for Golden Delicious was equal to 2.38, 2.68, and 4.05 after 0, 30 and 60 min of air exposure, respectively. Dipping in AA solution (1% w/v) was found to be the best treatment to limit surface discolouration of both apple cultivars. The best heat treatments to inhibit polyphenol oxidase/peroxidase enzymes activity were 70 °C HWB for Golden Delicious and 60 °C HWB for Elstar slices, both in combination with a solution of 1% AA and 1% CA. The tested apple cultivars were found to require different treatments at minimum ambient air exposure to obtain the best surface colour condition.
Project description:To learn how the endogenous polyphenols may play a role in fruit ripening and senescence, apple pulp discs were used as a model to study the influences of chlorogenic acid (CHA, a major polyphenol in apple pulp) on fruit ripening and senescence. Apple ('Golden Delicious') pulp discs prepared from pre-climacteric fruit were treated with 50 mg L(-1) CHA and incubated in flasks with 10 mM MES buffer (pH 6.0, 11% sorbitol). Compared to the control samples, treatment with CHA significantly reduced ethylene production and respiration rate, and enhanced levels of firmness and soluble solids content of the pulp discs during incubation at 25°C. These results suggested that CHA could retard senescence of the apple pulp discs. Proteomics analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry (MALDI-TOF/TOF) revealed that the expressions of several key proteins correlated to fruit ripening and senescence were affected by the treatment with CHA. Further study showed that treating the pulp discs with CHA remarkably reduced levels of lipoxygenase, ?-galactosidase, NADP-malic enzyme, and enzymatic activities of lipoxygenase and UDP-glucose pyrophosphorylase, all of which are known as promoters of fruit ripening and senescence. These results could provide new insights into the functions of endogenous phenolic compounds in fruit ripening and senescence.
Project description:BACKGROUND:Fruit color in apple (Malus domestica Borkh.) is ascribed mainly to the accumulation of anthocyanin pigments, and is an important trait for determining fruit market acceptance. Bagging is a commonly used treatment to enhance the red pigmentation in apple skin. The MdMYB1 transcription factor gene plays an important role in the biosynthesis of anthocyanin in apple after bag removal, but little is known about how MdMYB1 transcription is regulated. RESULTS:In this study, we investigated pigmentation in the non-red skinned cultivars 'Granny Smith' and 'Golden Delicious' after bag removal. The fruit skins of the two cultivars showed red/pink pigmentation after bag treatment. Transcript levels of MdMYB1, the master regulator of anthocyanin biosynthesis in apple, increased, and showed a correlation with anthocyanin content in both cultivars after bag removal. The MdMYB1 genomic sequences were compared in the two cultivars, which showed that the green-fruited cultivar 'Granny Smith' harbors the MdMYB1-1 and MdMYB1-2 alleles, while the yellow-fruited cultivar 'Golden Delicious' harbors only MdMYB1-2. A comparison of methylation levels in the 2 kb region upstream of the MdMYB1 ATG between the bag-treated fruits after removal from the bags and the unbagged fruits showed a correlation between hypomethylation and the red-skin phenotype in 'Granny Smith'. Moreover, 'Granny Smith' fruits responded to treatment with 5-aza-2'-deoxycytidine, an inducer of DNA demethylation. An investigation of the MdMYB1 promoter in 'Granny Smith' showed reduced methylation in the regions -?2026 to -?1870 bp, -?1898 to -?1633 bp, and?-?541 to -?435 bp after bag removal and 5-aza-2'-deoxycytidine treatments. CONCLUSIONS:Differences in anthocyanin levels between 'Granny Smith' and 'Golden Delicious' can be explained by differential accumulation of MdMYB1-specific mRNA. Different levels of MdMYB1 transcripts in the two cultivars are associated with methylation levels in the promoter region. Hypomethylation of the MdMYB1 promoter is correlated with the formation of red pigmentation in 'Granny Smith' fruit skins. As a result, red pigmentation in Granny Smith' was more intense than in 'Golden Delicious' fruits after bag removal.
Project description:BACKGROUND: Apple is a widely cultivated fruit crop for its quality properties and extended storability. Among the several quality factors, texture is the most important and appreciated, and within the apple variety panorama the cortex texture shows a broad range of variability. Anatomically these variations depend on degradation events occurring in both fruit primary cell wall and middle lamella. This physiological process is regulated by an enzymatic network generally encoded by large gene families, among which polygalacturonase is devoted to the depolymerization of pectin. In apple, Md-PG1, a key gene belonging to the polygalacturonase gene family, was mapped on chromosome 10 and co-localized within the statistical interval of a major hot spot QTL associated to several fruit texture sub-phenotypes. RESULTS: In this work, a QTL corresponding to the position of Md-PG1 was validated and new functional alleles associated to the fruit texture properties in 77 apple cultivars were discovered. 38 SNPs genotyped by gene full length resequencing and 2 SSR markers ad hoc targeted in the gene metacontig were employed. Out of this SNP set, eleven were used to define three significant haplotypes statistically associated to several texture components. The impact of Md-PG1 in the fruit cell wall disassembly was further confirmed by the cortex structure electron microscope scanning in two apple varieties characterized by opposite texture performance, such as 'Golden Delicious' and 'Granny Smith'. CONCLUSIONS: The results here presented step forward into the genetic dissection of fruit texture in apple. This new set of haplotypes, and microsatellite alleles, can represent a valuable toolbox for a more efficient parental selection as well as the identification of new apple accessions distinguished by superior fruit quality features.
Project description:Polyphenol oxidase (PPO) plays a key role in the postharvest pericarp browning of litchi fruit, but its underlying mechanism remains unclear. In this study, we cloned the litchi PPO gene (LcPPO, JF926153), and described its expression patterns. The LcPPO cDNA sequence was 2120 bps in length with an open reading frame (ORF) of 1800 bps. The ORF encoded a polypeptide with 599 amino acid residues, sharing high similarities with other plant PPO. The DNA sequence of the ORF contained a 215-bp intron. After carrying out quantitative RT-PCR, we proved that the LcPPO expression was tissue-specific, exhibiting the highest level in the flower and leaf. In the pericarp of newly-harvested litchi fruits, the LcPPO expression level was relatively high compared with developing fruits. Regardless of the litchi cultivar and treatment conditions, the LcPPO expression level and the PPO activity in pericarp of postharvest fruits exhibited the similar variations. When the fruits were stored at room temperature without packaging, all the pericarp browning index, PPO activity and the LcPPO expression level of litchi pericarps were reaching the highest in Nandaowuhe (the most rapid browning cultivar), but the lowest in Ziniangxi (the slowest browning cultivar) within 2 d postharvest. Preserving the fruits of Feizixiao in 0.2-?m plastic bag at room temperature would decrease the rate of pericarp water loss, delay the pericarp browning, and also cause the reduction of the pericarp PPO activity and LcPPO expression level within 3 d postharvest. In addition, postharvest storage of Feizixiao fruit stored at 4°C delayed the pericarp browning while decreasing the pericarp PPO activity and LcPPO expression level within 2 d after harvest. Thus, we concluded that the up-regulation of LcPPO expression in pericarp at early stage of postharvest storage likely enhanced the PPO activity and further accelerated the postharvest pericarp browning of litchi fruit.