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.
Cultivated apple (Malus domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has ...undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome-wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom super( registered ) genotyping array for apple and discuss its potential applications. The array has been built from the high-depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non-genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom super( registered )Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom super( registered )Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple. Significance Statement Single nucleotide polymorphism (SNP) array-based genotyping has revolutionized the study of genome-wide genetic variation. Here we describe a SNP array for cultivated apple, developed from 63 apple cultivars, with more than 487K SNPs well distributed among the 17 chromosomes. We describe the filtering pipeline and validate a large subset of highly reliable markers that can be used for germplasm surveys.
Pectin was extracted from apple (
'Fălticeni') pomace with Celluclast 1.5L, at doses of 20, 40, and 60 µL/g of material. The temperature and time of extraction were varied on three ...levels-temperature-40, 50, and 60 °C; time-12, 18, and 24 h. For each experiment, the extraction yield (R
= 0.8905), the galacturonic acid content (R
= 0.9866), and the degree of esterification (R
= 0.9520) of pectin was determined. Response surface methodology (RSM) was implemented via a Box-Behnken design, to optimize pectin extraction. In the optimum extraction conditions (temperature of 48.3 °C, extraction time of 18 h 14 min, and enzyme dose of 42.5 µL/g of pomace), the design predicted a 6.76% yield with a galacturonic acid content of 97.46 g/100 g of pectin and a degree of esterification of 96.02%. FT-IR analysis of the pectin sample obtained in these conditions showed a chemical structure similar to that of commercial apple and citrus pectin.
Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood.
We used a ...homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles.
MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives.
Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis.
•Organic, organo-mineral and mineral fertilizers were used for fertilization of apple orchard with ‘Golden Delicious Reinders’ cultivar.•Manure improved yield per tree and yield efficiency, whereas ...the highest tree size was observed in control treatment.•Fertilizers had less impact on the fruit physical properties compared to chemical ones.•Compound NPK, organo-mineral ITP fertilizer and manure induced ideal balanced leaf nutritional values.•PCA can be a very useful tool for segregating fertilizers which induced the best tree growth, fruit physico-chemical traits and antioxidant activity of apple.
This work was carried out to obtain data about the vegetative growth, productivity, fruit external and internal quality and leaf nutrient composition of apple cv. ‘Golden Delicious Reinders’ from different fertilization regimes, with reference to their role in tree and fruit growth and development, especially for their specific recommendations. Organic, organo-mineral and mineral fertilizers were applied to soil during three consecutive years. Results showed that organo-mineral fertilizer called Italpolina (ITP) and N fertilizer called calcium ammonium nitrate (CAN) increased TPC, antioxidant activity, acidity, FW and D. Compound NPK induced the best values of SSC, TS and IS contents and TFC. Urea as a physiologically acidic N fertilizer mostly affected TS/TA and SSC/TA ratios. Interestingly, manure conditioned the best values of yield per tree and unit area and YE, while the greatest tree vigor was found in the control treatment (without fertilization). In paradox, the lowest tree growth was observed by CAN and compound NPK, i.e. fertilizers with high N contents. Compound NPK, organo-mineral ITP fertilizer and manure induced ideal balanced leaf nutritional values, followed by CAN and urea. Besides, it is highly important that experts and growers are encouraging the application and use of organic and organo-mineral fertilizers to increase soil fertility and its quality. Thus, the applied fertilizers behaved differently in terms of the impact on the tested properties. One improved agronomic properties, while the other improved pomological traits, nutritional value of fruits and the amount of elements in the leaf. However, cattle manure, organo-mineral Italpolina and compound NPK fertilizer could be recommended generally for fertilizing the ‘Golden Delicious Reinders’ apple on M.9 T337 in similar soil and climatic conditions. PCA can be a useful tool for segregation fertilizers that have the best influence on tree growth, productivity and apple fruit quality.
•Photosynthetic traits were more affected by drought (DT) than by waterlogging (WT).•Leaf water potential, sap flow and photosynthetic traits had similar trends.•Stomatal conductance was more reduced ...with increasing abscisic acid under DT.•Plant hydraulic traits recovered earlier than photosynthetic traits in DT and WT.•Photosynthetic and plant hydraulic traits performed better under waterlogging.
Drought and waterlogging are major abiotic stresses in fruit cultivation that should both be considered when establishing an orchard as the frequency and severity of extreme climate events continue to rise. This study investigated the photosynthetic traits and plant–water relations of Gamhong/M.9 apple cultivar during and after drought (DT) and waterlogging (WT) treatments. Both stresses adversely affected the photosynthetic and plant hydraulic traits, with stress-specific effects. Drought significantly increased the leaf mass per area, indicating that the cultivar allocated more resources to structural investment than to photosynthesis upregulation. The net photosynthetic rate and stomatal conductance were reduced at an earlier stage under DT (day 3) than under WT (day 6). The recovery of these photosynthetic traits was slower under DT than that under WT, with the net photosynthetic rate recovering 12 days after DT and 9 days after WT, whereas the stomatal conductance recovered 11 days after DT and 7 days after WT. Additionally, the progressive increase in xylem sap abscisic acid concentration resulted in a linear decrease in stomatal conductance, with a greater under DT than under WT. The leaf water potential and sap flow followed similar decreasing trends as that of the photosynthetic traits; however, hydraulic traits recovered earlier than photosynthetic traits under both stress conditions. The leaf water potential was reduced on day 4 of DT and recovered 6 days after watering was resumed. Contrastingly, under WT, this trait was reduced on day 7 of the treatment and recovered 4 days after its end. Our findings highlight that the Gamhong cultivar has higher acclimation potential to waterlogging and can alleviate the adverse effects of excess water while maintaining its physiological traits and improving plant–water relations.
Summary
Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous ...and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome‐wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom® genotyping array for apple and discuss its potential applications. The array has been built from the high‐depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non‐genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom®Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom®Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.
Significance Statement
Single nucleotide polymorphism (SNP) array‐based genotyping has revolutionized the study of genome‐wide genetic variation. Here we describe a SNP array for cultivated apple, developed from 63 apple cultivars, with more than 487K SNPs well distributed among the 17 chromosomes. We describe the filtering pipeline and validate a large subset of highly reliable markers that can be used for germplasm surveys.
Summary
The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein ...MdDIPM4. In this work, MdDIPM4 knockout has been produced in two Malus × domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty‐seven transgenic lines were screened to identify CRISPR/Cas9‐induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss‐of‐function mutation were inoculated with the pathogen. Highly significant reduction in susceptibility was observed compared to control plants. Sequencing of five potential off‐target sites revealed no mutation event. Moreover, our construct contained a heat‐shock inducible FLP/FRT recombination system designed specifically to remove the T‐DNA harbouring the expression cassettes for CRISPR/Cas9, the marker gene and the FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat‐treated and screened by real‐time PCR to quantify the exogenous DNA elimination. The T‐DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9‐FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA.
•Pre- and post-harvest aroma profiles in peel and pulp of Honeycrisp were determined.•Postharvest fruit accumulated abundant esters, alcohols, and sesquiterpenoids.•More sesquiterpenoids, aldehydes, ...and esters were in the fruit skin than the pulp.•AEDA-GC-O, omission tests, and sensory description analysis were used in the study.•Hexyl 2-methylbutyrate with a strong fruity note is the key aroma of Honeycrisp.
‘Honeycrisp’ is a popular apple cultivar because of its superior appearance and flavor. We investigated its aroma profiles and characteristic aroma. Whereas the aroma profiles of developing fruits were dominated by aldehydes, postharvest fruit accumulated abundant esters, alcohols, and sesquiterpenoids. Most of these components showed maxima at the fruit’s climacteric peak. There were more types and higher contents of sesquiterpenoids, aldehydes, and esters in the fruit skin than in the pulp, while alcohol contents and types were comparable between the pulp and skin. Aroma extract dilution and gas chromatography–olfactometry analyses revealed that hexyl 2-methylbutyrate, α-farnesene, 1,3-octanediol, hexanal, (E)-2-hexenal, hexanol, butanol, and 2-methyl-butanol are the most potent odor compounds in ‘Honeycrisp’ apple. Aroma reconstruction and omission tests combined with sensory analyses suggested that hexyl 2-methylbutyrate is responsible for the strong fruity note of ‘Honeycrisp’ apple, and that other alcohols, aldehydes, and α-farnesene are essential for its background aroma notes.
Summary
The molecular genetic mechanisms underlying fruit size remain poorly understood in perennial crops, despite size being an important agronomic trait. Here we show that the expression level of ...a microRNA gene (miRNA172) influences fruit size in apple. A transposon insertional allele of miRNA172 showing reduced expression associates with large fruit in an apple breeding population, whereas over‐expression of miRNA172 in transgenic apple significantly reduces fruit size. The transposon insertional allele was found to be co‐located with a major fruit size quantitative trait locus, fixed in cultivated apples and their wild progenitor species with relatively large fruit. This finding supports the view that the selection for large size in apple fruit was initiated prior to apple domestication, likely by large mammals, before being subsequently strengthened by humans, and also helps to explain why signatures of genetic bottlenecks and selective sweeps are normally weaker in perennial crops than in annual crops.
Significance Statement
The molecular genetics underlying fruit size is poorly understood in perennial species. Here we show how a transposable element insertion in a microRNA (miRNA172) gene enhanced fruit size in apple. The fixation this allele in domesticated apples and in large‐fruited progenitor species supports the view that selection for this key ‘domestication’ trait predates human civilization.
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