This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture ...and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.
Apples are seasonal fruits, and thus after harvesting apples of optimal picking maturity, it is important to prepare them properly for storage and to ensure proper storage conditions in order to ...minimize changes in the chemical composition and commercial quality of the apples. We studied the quantitative composition of triterpenic compounds in the whole apple, apple peel and apple flesh samples before placing them in the controlled atmosphere (CA) chambers, and at the end of the experiment, 8 months later. HPLC analysis showed that highest total amount of triterpenic compounds (1.99 ± 0.01 mg g−1) was found in the whole apple samples of the ‘Spartan’ cultivar stored under variant VIII (O2—20%, CO2—3%, N2—77%) conditions. Meanwhile, the highest amount of triterpenic compounds (11.66 ± 0.72 mg g−1) was determined in the apple peel samples of the ‘Auksis’ cultivar stored under variant II (O2—5%, CO2—1%, N2—94%) conditions. In the apple peel samples of the ‘Auksis’ cultivar stored under variant I (O2—21%, CO2—0.03%, N2—78.97%) conditions, the amount of individual triterpenic compounds (ursolic, oleanolic, corosolic, and betulinic acids) significantly decreased compared with amount determined before the storage. Therefore, in the apple flesh samples determined triterpenic compounds are less stable during the storage under controlled atmosphere conditions compared with triterpenic compounds determined in the whole apple and apple peel samples.
Pectin is a group of structurally diverse dietary fibers, very abundant in agri-food waste and by-products such as those generated during apple cider manufacturing. In recent years, pectin and ...pectinoligosaccharides have demonstrated good fermentation properties and prominent health promoting traits, particularly ameliorating certain inflammatory conditions. In previous investigations apple pomace derived from production of monovarietal Asturian ciders was demonstrated to represent a source of pectin with varied structural characteristics. In this work we investigated in vitro the modulatory effect of pectin and pomace fractions derived from the production of selected monovarietal ciders on human microbiota from healthy subjects and inflammatory bowel disease (IBD) patients through fecal batch fermentations and 16S rRNA gene sequencing. Overall, these fractions promoted the growth of Akkermansia, Lachnospiraceae UCG-010, Prevotella, Sucinivibrio and Turicibacter on samples from healthy donors, while Blautia, Lachnospiraceae CAG-56, Dialister, Eubacterium eligens and Intestinimonas were stimulated in fermentations from IBD patients. The growth of Akkermansia, Blautia, E. eligens group, Intestinimonas and Succinivibrio only occurred with pomace and pectin derived from the tested by-products, and not with other non-pectic prebiotics/substrates. Galactose content and (Arabinose + Galactose)/Rhamnose ratio in apple pomace, and galactose and rhamnose content in pectin, were positively associated to the promotion of most of these genera. This work comprehensively characterize the gut microbiota modulation of apple pectin and pomace fractions derived from cider by-products, demonstrating diverse modulatory capacity of structurally distinct pectin and apple pomace fractions. This diversifies the opportunities to achieve cider by-products valorization through formulation of novel prebiotics for particular population groups.
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•Apple pomace and pectin modulated beneficial gut bacteria including Eubacterium and Lachnospiraceae in fecal fermentations.•Apple pomace promoted short-chain fatty acid producers in Chron's disease microbiota.•Galactose and rhamnose contents of pectin and apple pomace determine their modulatory activity.•Similar composition-activity relationships were found for apple pomace and pectin.
Apple russet ring and apple green crinkle are graft-transmitted diseases first reported more than 60 years ago, but at present, no association between a specific virus (variant) and the disease has ...been clearly demonstrated. In this study, we conducted the following series of experiments to identify the causal viruses (variants) of these apple diseases; (1) comprehensive analysis by next-generation sequencing of all viruses in each apple tree affected with russet ring or green crinkle disease, (2) amplification of full-length genomic cDNA of viruses using primers containing the T3 promoter and the
in vitro
transcription of infectious viral RNAs, (3) inoculation of viral RNA transcripts to both herbaceous and apple plants, (4) analysis of sequence variants of viruses present in infected plants, (5) back-inoculation of sequence variants of candidate viruses to apple seedlings combined with the virus-induced flowering technology using the apple latent spherical virus vector to reproduce the symptom on the fruit as soon as possible, and (6) reproduction of symptoms on the fruits of apple trees inoculated with sequence variants and the re-isolation of each virus variant from apples showing fruit symptoms. The results showed that one of the sequence variants of the apple chlorotic leaf spot virus causes a characteristic ring-shaped rust on the fruits of infected apple trees and that a sequence variant of the apple stem pitting virus probably causes green crinkle symptoms on an infected apple fruit. Thus, we were able to fulfill Koch’s postulates to prove the viral etiology of both the apple russet ring and green crinkle diseases. We also propose an experimental system that can prove whether a virus found in diseased tissues is the pathogen responsible for the diseases when the etiology is undetermined.
Apple (Malus × domestica) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold-hardy cultivars. This lack of progress is due at least partly ...to lack of understanding of the molecular mechanisms of freezing tolerance in apple.
In this study, we evaluated the potential roles for two R2R3 MYB transcription factors (TFs), MYB88 and the paralogous FLP (MYB124), in cold stress in apple and Arabidopsis. We found that MYB88 and MYB124 positively regulate freezing tolerance and cold-responsive gene expression in both apple and Arabidopsis.
Chromatin-Immunoprecipitation-qPCR and electrophoretic mobility shift assays showed that MdMYB88/MdMYB124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 (MdCSP3) and CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1) genes. Dual luciferase reporter assay indicated that MdCCA1 but not MdCSP3 activated the expression of MdCBF3 under cold stress. Moreover, MdMYB88 and MdMYB124 promoted anthocyanin accumulation and H2O2 detoxification in response to cold.
Taken together, our results suggest that MdMYB88 and MdMYB124 positively regulate cold hardiness and cold-responsive gene expression under cold stress by C-REPEAT BINDING FACTOR (CBF)-dependent and CBF-independent pathways.
•Virally transmitted diseases in apple cause economic losses as a result of their direct effect on production and yield and by causing an increased susceptibility to other phytopathogenic agents.•In ...this research, an efficient droplet-vitrification cryotherapy method for the eradication of Malus latent viruses was demonstrated.•The droplet-vitrification cryotherapy protocol successfully removed cells harbouring ACLSV, ASPV and ASGV viruses from in vitro apple ‘Monalisa’.•This promising cryotherapy procedure may facilitate the production of virus-free apple plants.
In apple, virally transmitted diseases cause economic losses as a result of their direct effect on production and yield and by causing an increased susceptibility to other phytopathogenic agents. This study evaluated the effectiveness of a droplet-vitrification cryopreservation technique in eradicating latent viruses: Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) from in vitro axillary shoot tips excised from ‘SC417 Monalisa’ apple cultivar shoot tips. In vitro stock cultures, four weeks after subculture, infected with ASGV, ASPV and ACLSV were used as source material. Axillary shoot tips were excised from stock cultures, precultured in MS + 2 M glycerol + 0.8 M sucrose, and then exposed to plant vitrification solution 2 (PVS2) for between 0 and 80 min (six treatments) at 0 °C or room temperature prior to liquid nitrogen (LN) exposure. Shoot tips were then warmed and recovered on growth medium. The survival and regrowth of cryopreserved (+LN) and non-cryopreserved (-LN) 'Monalisa' shoot tips were evaluated and the efficiency of virus eradication was determined using RT-PCR after recovered plants were grown in a greenhouse for 6 months. With this protocol, the highest degree of regrowth (45%) were obtained after 20 to 40 min PVS2 exposure at room temperature. After 6 months of growth in a greenhouse, all of the plants regenerated after cryopreservation were free of ASPV, 95% were free of ACLSV, and 35% were free of ASGV. This promising cryotherapy procedure may facilitate the production of virus-free plants.
An analytic procedure based on high-performance liquid chromatography with solid-phase extraction and UV detection (SPE–HPLC–UV) was validated and used to analyse 144 apple-based-foods, including 68 ...apple juices (32 clear and 36 cloudy) and 76 homogenised apple purees marketed in Portugal. Patulin was detected in 33 samples (23%) with values ranging from 1.2
μg/kg to 42
μg/kg. Patulin was not detected in the analysed infant drinks but its presence was quantified in five homogenised apple puree (7%) labelled as being intended for infants and young children consumption. A higher incidence of positive samples was detected in cloudy juices (67%) when compared with clear ones (13%). When the production mode is considered, the incidence of positive samples was 20% and 24% for products of organic and conventional origin, respectively. This is the first study on the occurrence of patulin in apple-based-foods in Portugal, including baby food.
SUMMARY
Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought ...resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole‐3‐acetic acid (IAA) conjugating enzyme, to be a negative regulator of water‐deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water‐deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water‐deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water‐deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water‐deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water‐deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.
Significance Statement
We characterized MdGH3.6 in the response of apple to water‐deficit stress. We found that MdGH3.6 was a direct target of MdMYB94 and a negative regulator of water‐deficit stress tolerance in apple. The negative regulation of water‐deficit stress tolerance may stem from the combined roles of MdGH3.6 in the negative regulation of ROS detoxification and cuticular wax. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.
Fresh-cut apples, which offer consumers health benefits and convenience, have become popular in recent years. One of the main challenges for processing fresh-cut apples is rapid development of cut ...surface browning, immediately after fruits are cut. Browning, a physiological response that impacts organoleptic properties and deters consumer purchase of fresh-cut fresh produce, is mainly a result of enzymatic reaction of phenolic compounds with oxygen catalyzed by polyphenol oxidase (PPO), a decapper enzyme. Many antibrowning agents have been developed and evaluated to inhibit PPO activities by using reducing agents (antioxidants), chelating agents, acidulants, etc. The present manuscript reviews the diverse characteristics of PPO (such as optimum pH and temperature, and molecular weight) in apples reported in the literature and the enzyme's latency, multiplicity and copper states in the active site. It also summarizes the latest development in the investigation and formulations of antibrowning compounds, and discusses future research needs. This review should stimulate further research to discover more effective, low cost, and natural antibrowning compounds to meet the demand of consumers as well as the food industry for clean label and long shelf-life of fresh-cut apples.
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