Although the aromatic quality of apples is a primary driver of consumer choice, the molecular mechanisms controlling its postharvest dynamics are not well defined. In this study, we conducted a comprehensive analysis of ‘Ruixue’ apples during storage and discovered that aroma biosynthesis is mediated by the lipoxygenase (LOX) pathway. Through correlation and expression analyses, we identified the MdLOX5a as a positive regulator of key volatile organic compounds, including (E)-2-hexenal and hexanal, a role we subsequently validated through transient overexpression and silencing. We further show that this regulation is directly controlled by the transcription factor MdDof1.2, which binds to and activates the MdLOX5a promoter. Our findings thus establish a novel regulatory module for postharvest aroma biosynthesis, offering a promising genetic target for breeding programs focused on enhancing fruit flavor and extending postharvest quality.

Apple (Malus × domestica Borkh.) fruit firmness is a crucial determinant of fruit quality and postharvest storage capacity. The DNA-binding one-finger (Dof) transcription factor family has been demonstrated to play a significant role in regulating fruit ripening and softening; however, the genome-wide role of Dofs in determining apple firmness remains unclear. Here, we examined the expression patterns of 50 MdDofs in two apple varieties, ‘Fuji’ and ‘Cripp's Pink’, known for their distinct fruit firmness and cell wall components during fruit development and ripening. We also clarified the function and regulatory network of the MdDof43 in regulating apple fruit firmness. Functional validation indicated that MdDof43 enhanced the water-soluble pectin content, increased neutral sugar loss, decreased hemicellulose content and fruit firmness in transgenic apple, calli and tomato. RNA sequencing analysis revealed that MdDof43 overexpression induced the expression levels of many genes involved in the cell wall degradation in apple calli. DNA affinity purification and sequencing (DAP seq) demonstrated that MdDof43 directly binds to the promoters of Mdβ-Gal2 and Mdα-AF3, two genes encoding pectin side-chain regulating enzymes. In vitro assays confirmed that MdDof43 positively regulates the expression of Mdβ-Gal2 and Mdα-AF3, facilitating the degradation of cell wall components and promoting fruit softening. These findings provide insights into the regulatory mechanisms of cell wall structures that contribute to apple fruit firmness, which has practical significance for fruit quality in breeding.

Aromatic esters are key determinants of apple fruit aroma and consumer preference. Here, we investigated the molecular mechanisms underlying ester biosynthesis by characterizing MdAAT2-like, a critical ester-synthesizing gene in apple. MdAAT2-like expressions were significantly upregulated during fruit ripening and positively correlated with ester accumulation. Functional validation in apple and tomato demonstrated that MdAAT2-like overexpression enhanced ester contents, while silencing or knockout reduced ester production. Enzymatic assays revealed that MdAAT2-like exhibits higher catalytic efficiency for medium- and short-chain acyl-CoAs compared to other AAT family members. Two transcription factors, MdMYB98-like and MdWRKY21, were identified as direct activators of MdAAT2-like. Both factors bind its promoter and synergistically enhance transcription through protein interaction. Notably, we uncovered a positive feedback model wherein MdMYB98-like and MdWRKY21 reciprocally activate each other's expression, reinforcing rapid ester synthesis during ripening. This study reveals a MYB-WRKY regulatory module controlling ester biosynthesis in apple.

Volatile esters are critical for apple fruit aroma, yet the regulatory mechanism underlying their degradation remains poorly understood. In this study, we identified the transcription factor MdWRKY40 as a direct activator of MdCXE1, a key carboxylesterase gene responsible for ester hydrolysis. Through comparative analysis of 10 apple cultivars, we observed a significant negative correlation between MdCXE1 expression and ester content. Transient overexpression and silencing assays confirmed that MdCXE1 negatively regulates ester levels. Combined in vitro and in vivo binding assays demonstrated that MdWRKY40 binds to the W-box motif in the MdCXE1 promoter. Functional studies in apple calli and tomato fruits showed that MdWRKY40 overexpression reduces ester accumulation, while its knockout enhances it. Furthermore, dual silencing of MdWRKY40 and MdCXE1 attenuated ester degradation. Our findings complement the established biosynthetic machinery by demonstrating that the MdWRKY40-MdCXE1 module functions as a transcriptional regulator of ester catabolism, thereby revealing a potential target for molecular breeding of apple varieties with tailored aroma profiles.

Apple (Malus u00D7 domestica Borkh.) fruit firmness is a crucial determinant of fruit quality and postharvest storage capacity. The DNA-binding one-finger (Dof) transcription factor family has been demonstrated to play a significant role in regulating fruit ripening and softening; however, the genome-wide role of Dofs in determining apple firmness remains unclear. Here, we examined the expression patterns of 50 MdDofs in two apple varieties, u2018Fujiu2019 and u2018Cripp's Pinku2019, known for their distinct fruit firmness and cell wall components during fruit development and ripening. We also clarified the function and regulatory network of the MdDof43 in regulating apple fruit firmness. Functional validation indicated that MdDof43 enhanced the water-soluble pectin content, increased neutral sugar loss, decreased hemicellulose content and fruit firmness in transgenic apple, calli and tomato. RNA sequencing analysis revealed that MdDof43 overexpression induced the expression levels of many genes involved in the cell wall degradation in apple calli. DNA affinity purification and sequencing (DAP seq) demonstrated that MdDof43 directly binds to the promoters of Mdu03B2-Gal2 and Mdu03B1-AF3, two genes encoding pectin side-chain regulating enzymes. In vitro assays confirmed that MdDof43 positively regulates the expression of Mdu03B2-Gal2 and Mdu03B1-AF3, facilitating the degradation of cell wall components and promoting fruit softening. These findings provide insights into the regulatory mechanisms of cell wall structures that contribute to apple fruit firmness, which has practical significance for fruit quality in breeding.

Bagging apples can enhance fruit appearance and quality by reducing pest and disease damage. However, upon removal of the fruit bag, apples become susceptible to photodamage and heat stress, potentially resulting in sunburn symptoms. In this study, we examined ‘Ruixiang Hong’ apples post-bag removal and observed that mild photodamage preserved the cellular structure of pericarp tissue and showed low antioxidant enzyme activity. In contrast, severe injury led to cellular disorganization and dehydration in the pericarp, triggering increased antioxidant enzyme activity as a protective response. We identified a transcription factor (TF), MdGATA15, from previous transcriptomic and metabolomic datasets that appeared relevant to sunburn mitigation. To elucidate the role of MdGATA15 in sunburn response, we introduced this TF into plants through both transient and stable transformation methods. Subsequently, we measured flavonoid content after exposing the plants to intense light and high temperature. All three sets of transgenic materials exhibited increased content of most flavonoids in the overexpression plants compared with the control, with a significant increase in procyanidin content. Further analysis revealed that MdGATA15 bound to the promoter of MdANR, a key gene involved in procyanidin biosynthesis, thereby upregulating anthocyanin expression. These findings suggest that MdGATA15 may alleviate sunburn symptoms in apple peels.

Apple fruit firmness is a crucial index for measuring the internal quality of apples, which influences palatability, storage capacity and transportability. The primary cause of reduced firmness during fruit development is the hydrolysis of cell wall polysaccharides. Xyloglucan endotransglycosylase/hydrolase (XTH) is a key enzyme involved in the depolymerization of cell wall polysaccharides, but the mechanism of its involvement in the formation of fruit firmness remains unclear. Here, we identified the gene MdXTH2 by integrating metabolomic and transcriptomic data, and analyzed its function and molecular mechanism in the formation of apple fruit firmness. The results showed downward trends in both fruit firmness and cell wall components throughout fruit development. The contents of cell wall material, cellulose, and hemicellulose in various apple varieties exhibited significant positive correlations with firmness, with total correlation coefficients of 0.862, 0.884, and 0.891, respectively. Overexpression of MdXTH2 significantly increased fruit firmness in apple and tomato, inhibited fruit ripening, and significantly suppressed the growth of calli. The upstream transcription factor MdNAC72 of the MdXTH2 gene can promote the expression of fruit ripening-related genes. Furthermore, dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assay (EMSA) demonstrated that MdNAC72 down-regulates the transcription of MdXTH2 by binding to its promoter. In summary, the results of this study provide a strategy for examining fruit quality regulation and a theoretical basis for breeding apple varieties with moderate firmness through genetic improvement.

China is the largest producer and consumer of apples and plays a vital role in the fruit sector worldwide. However, few studies have examined the environmental impacts of apples from production to consumption in China concerning apple losses. Adopting reasonable initiatives in the apple industry from a whole life-cycle perspective can guide sustainable decision-making and support the formulation of policies for achieving carbon neutrality. This study utilized the ReCiPe2016 methodology because it covers a broad spectrum of possible environmental impacts. The aim of the paper was to conduct a comprehensive life cycle assessment of the environmental impacts of apples from production to consumption in China and the associated apple losses along the supply chain. The results showed that producing 1 kg of apples produced the global warming potential of 0.842 kg CO2-eq. Nitrogen fertilizer was the most critical environmental hotspot in the apple production phase and contributed more than 60 % in 11 of the 18 environmental impact categories. The environmental impacts in the apple consumption phase generally increased with transport distance. However, short-distance air transport (domestic market) had a higher impact than long-distance truck transport (international market). Among the five consumption scenarios in the domestic market, packaging had a higher environmental impact (1.767-17.926 mPt), exceeding transportation (0.729-14.949 mPt); conversely, in the international market, packaging (10.968 mPt) had a lower impact than transportation (12.181 mPt). Additionally, the study found that more complex sales models resulted in higher apple loss rates, requiring more adequate apple production to meet demand and thereby increasing environmental pollutant emissions. China can take measures such as reducing nitrogen fertilizer usage, designing sustainable packaging, discouraging air transportation, and concerning apple losses to address these challenges. These actions are essential for improving the environmental performance of the apple supply chain in China. Therefore, there is an urgent need to make some recommendations for different stakeholders in the whole life cycle, from production to consumption, to achieve sustainable development. Our work can contribute to offering a blueprint for unlocking unknown knowledge for the apple industry, ultimately catalyzing the realization of sustainable production and consumption in apple systems.

Fruit senescence negatively affects apple quality and postharvest storage. γ-Aminobutyric acid (GABA) accumulates rapidly following stress. Here, we found that the HD-ZIP transcription factor (TF) MdHD14 induces GABA accumulation by upregulating MdGAD2 and MdGAD4. This subsequently increases antioxidant enzyme activity in apple fruits and delays fruit senescence. The overexpression of MdGAD2/4 enhances GABA levels and decreases reactive oxygen species levels. Conversely, MdGAD2/4 silencing revealed that MdGAD2/4 mediates GABA synthesis and delays the senescence of apple fruit. We also found that MdHD14 can activate the transcription of MdGAD2 and MdGAD4. Both the overexpression of MdHD14 and its editing indicate that MdHD14 alters MdGAD2 and MdGAD4 expression to regulate GABA synthesis. The expression of MdHD14 was regulated by the TF MdWRKY50. Antioxidant enzyme activity, transcript levels of MdGAD2/4, and the GABA content were increased in plants overexpressing MdWRKY50. The molecular mechanism by which MdWRKY50, MdHD14, and MdGAD2/4 collectively regulate GABA homeostasis and fruit senescence in apple was clarified. These findings clarify the molecular mechanisms of senescence resistance in apple.

Sunburn in apple peel significantly affects fruit appearance and reduces its commercial value. Previous research has shown that apple peel reduces sunburn by increasing the accumulation of proanthocyanidins (PAs) and other protective compounds. However, the precise molecular regulatory mechanism remains unclear. In this study, we systematically investigated MdANR, a key gene involved in PAs biosynthesis. We found that MdANR expression in apple peel is responsive to temperature and light fluctuations, with higher expression levels observed under increased temperature and light exposure. Functional analysis revealed that MdANR overexpression in apple peel and callus enhanced resistance to high-temperature and -light-intensity stress, accompanied by a corresponding increase in PAs and chlorogenic acid contents. In addition, we demonstrated that MdMYBR9 can activate MdANR promoter activity and promote its expression through yeast one-hybrid, dual-luciferase, and electrophoretic mobility transfer experiments. The results indicated that MdMYBR9 was an upstream regulator of MdANR. Based on these findings, this study proposes the MdMYBR9-MdANR-PAs regulatory model for apple sunburn resistance, providing a molecular framework for enhancing sunburn tolerance in apple breeding programs.

Malic acid is a crucial determinant of apple ( ) fruit quality, influencing acidity and flavor. While transcriptional regulation of malic acid metabolism is well-studied, post-transcriptional control and the role of jasmonate (JA) remain largely unexplored. We identify a novel regulatory pathway involving JA signaling, a microRNA (miRNA), and vacuolar transport regulators that control malic acid accumulation in apple fruit. We show that mdm-miR858, which increases during fruit maturation, directly targets and cleaves transcripts. MdMYB73 is a known positive regulator of vacuolar H -pumping and malate transport, activating genes like , , and . Overexpression of mdm-miR858 suppressed , thereby reducing , , and expression and malic acid content in apple calli, fruits, and GL-3 plantlets, while silencing mdm-miR858 had opposite effects. Crucially, the JA-responsive transcription factor MdMYC2, the expression of which increases during fruit maturation, directly binds the mdm-miR858 promoter and activates its expression. Furthermore, the Mediator complex subunit MdMED25 interacts with MdMYC2, enhancing this activation. Manipulating or expression altered mdm-miR858 levels, expression, and malic acid accumulation, mirroring exogenous methyl jasmonate (MeJA) treatment effects. A miR858-resistant variant confirmed the miRNA-target interaction's specificity and functional significance. Our findings reveal a novel JA–MdMYC2/MdMED25–miR858– regulatory cascade controlling malic acid accumulation in apple, providing a mechanistic link between hormonal signaling and post-transcriptional regulation of fruit acidity. This discovery offers new targets for manipulating fruit quality.

Malic acid is a crucial determinant of apple (Malus domestica) fruit quality, influencing acidity and flavor. While transcriptional regulation of malic acid metabolism is well-studied, post-transcriptional control and the role of jasmonate (JA) remain largely unexplored. We identify a novel regulatory pathway involving JA signaling, a microRNA (miRNA), and vacuolar transport regulators that control malic acid accumulation in apple fruit. We show that mdm-miR858, which increases during fruit maturation, directly targets and cleaves MdMYB73 transcripts. MdMYB73 is a known positive regulator of vacuolar H+-pumping and malate transport, activating genes like MdVHA-A, MdVHP, and MdALMT9. Overexpression of mdm-miR858 suppressed MdMYB73, thereby reducing MdVHA-A, MdVHP, and MdALMT9 expression and malic acid content in apple calli, fruits, and GL-3 plantlets, while silencing mdm-miR858 had opposite effects. Crucially, the JA-responsive transcription factor MdMYC2, the expression of which increases during fruit maturation, directly binds the mdm-miR858 promoter and activates its expression. Furthermore, the Mediator complex subunit MdMED25 interacts with MdMYC2, enhancing this activation. Manipulating MdMYC2 or MdMED25 expression altered mdm-miR858 levels, MdMYB73 expression, and malic acid accumulation, mirroring exogenous methyl jasmonate (MeJA) treatment effects. A miR858-resistant MdMYB73 variant confirmed the miRNA-target interaction's specificity and functional significance. Our findings reveal a novel JA-MdMYC2/MdMED25-miR858-MdMYB73 regulatory cascade controlling malic acid accumulation in apple, providing a mechanistic link between hormonal signaling and post-transcriptional regulation of fruit acidity. This discovery offers new targets for manipulating fruit quality.

Flesh firmness is a core quality trait in apple breeding because of its correlation with ripening and storage. Quantitative trait loci (QTLs) were analyzed through bulked segregant analysis sequence (BSA-seq) and comparative transcriptome analysis (RNA-seq) to explore the genetic basis of firmness formation. In this study, phenotypic data were collected at harvest from 251 F1 hybrids derived from ‘Ruiyang’ and ‘Scilate’, the phenotype values of flesh firmness at harvest were extensively segregated for two consecutive years. A total of 11 candidate intervals were identified on chromosomes 03, 05, 06, 07, 13, and 16 via BSA-seq analysis. We characterized a major QTL on chromosome 16 and selected a candidate gene encoding expansin MdEXP-A1 by combining RNA-seq analysis. Furthermore, the genotype of Del-1166 (homozygous deletion) in the MdEXP-A1 promoter was closely associated with the super-hard phenotype of F1 hybrids, which could be used as a functional marker for marker-assisted selection (MAS) in apple. Functional identification revealed that MdEXP-A1 positively expedited fruit softening in both apple fruits and tomatoes that overexpressed MdEXP-A1. Moreover, the promoter sequence of TE-1166 was experimentally validated containing two binding motifs of MdNAC1, and the absence of the MdEXP-A1 promoter fragment reduced its transcription activity. MdNAC1 also promotes the expression of MdEXP-A1, indicating its potential modulatory role in quality breeding. These findings provide novel insight into the genetic control of flesh firmness by MdEXP-A1.

Apple (Malus domestica Borkh.) is a globally significant crop and a vital dietary component worldwide. During ripening, apples exhibit a longitudinal gradient, ripening first at the stalk cavity and extending towards the calyx concave. When the fruit is harvested at the right time or later, the stalk cavity of many varieties often shows over-ripening, that is, premature senescence such as peel browning, which diminishes fruit quality. This study examines the natural senescence process in 6-year-old 'Ruixue' apples by screening transcriptome data to uncover senescence-related genes and validate their molecular functions. Our analysis of antioxidant capacity and reactive oxygen species (ROS) in different peel regions revealed that malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion ( O 2 - ⋅ ) levels increased with senescence, where ROS-scavenging enzyme activity was notably reduced, especially in the stalk cavity (compared with the fruits in the stalk cavity at 120 days, the activities of SOD, POD, and CAT in stalk cavity of fruits at 205 days were significantly decreased in 65.4%, 82.7%, and 91.1%, respectively). Transcriptome clustering and enrichment analyses across developmental stages revealed MdWRKY70L, MdSAG101, and MdZAT12 as key regulators of peel senescence. MdWRKY70L could interact with MdSAG101/MdZAT12 both in vivo and in vitro, thereby mediating ROS accumulation in the peel and accelerating the fruit senescence process. Further in vitro and in vivo studies demonstrated that MdWRKY70L is phosphorylated at Ser199 by MdMPK6/02G, enhancing MdWRKY70L protein stability and promoting peel senescence. These findings offer insights for developing strategies to delay fruit senescence and improve postharvest quality control.

Fruit firmness is an important trait for characterizing the quality and value of apple. It also serves as an indicator of fruit maturity, as it is a complex trait regulated by multiple genes. Resequencing techniques can be employed to elucidate variations in such complex fruit traits. Here, the whole genomes of 294 F1 hybrids of 'Fuji' and 'Cripp's Pink' were resequenced, and a high-density binmap was constructed using 5014 bin markers with a total map distance of 2213.23 cM and an average map distance of 0.44 cM. Quantitative trait loci (QTLs) of traits related to fruit were mapped, and an A-T allele variant identified in the coding region of MdNAC5 was found to potentially regulate fruit firmness and ripening. The overexpression of MdNAC5A resulted in higher production of methionine and 1-aminocyclopropanecarboxylic acid compared to MdNAC5T, leading to reduced fruit firmness and accelerated ripening in apples and tomatoes. Furthermore, the activities of MdNAC5A and MdNAC5T were enhanced through their differential binding to the promoter regions of MdACS1 and MdERF3. Spatial variations in MdNAC5A and MdNAC5T caused changes in MdACS1 expression following their interaction with MdERF3. Ultimately, utilizing different MdNAC5 alleles offers a strategy to manipulate fruit firmness in apple breeding.

Fruit texture and storage properties of various apple varieties exhibit significant variation. The rate of fruit softening post-harvest plays a crucial role in determining fruit quality and shelf life. This research utilized four apple varieties as test subjects to investigate the internal factors influencing fruit texture changes among different varieties. By monitoring changes in relevant physiological indicators during the post-harvest texture softening process, the study examined fruit quality, cell wall material content, hydrolase activity, and gene transcription levels during storage of ‘Orin’, ‘RX’, ‘RXH’, and ‘Envy’ apples. Initial fruit softening was primarily linked to heightened post-harvest fruit respiration intensity, ethylene production, and rapid amylase activity. Subsequent softening was associated with increased activity of water-soluble pectin (WSP), cellulose (CEL), and other hydrolases. With the extension of the storage period, the fruit cells of the four varieties became more loosely arranged, resulting in larger intercellular gaps. Variations in WSP and cellulose content, CEL activity, and relative expression of Mdβ-gal were observed among the different apple varieties, potentially accounting for the disparities in fruit texture.

‘Ruixue’ apples were used as the test material to study the effect of 10 μM methyl jasmonate (MeJA) on the quality and cell wall metabolism of apples after 18 d of storage. The results showed that MeJA significantly decreased the respiratory rate, reduced the titratable acid content and maintained a high soluble solids content. MeJA has been shown to suppress the activities and gene expressions of WSP, CSP, ISP, and cellulose in contrast to the control group, thereby maintaining a lower cell permeability and higher exocarp firmness. MeJA significantly decreased the expression of MdACS, MdACO, MdPL, Mdgal, and MdPG genes in the apple exocarp when compared to the control group. In addition, the overexpression of MdPL18 increased the content of cell wall polysaccharides such as WSP and CSP, enhanced cell wall-degrading enzyme activities, and accelerated fruit ripening and softening, whereas silencing MdPL18 did the opposite. Together, these results demonstrate that exogenous MeJA maintains the Ruixue apple fruit quality by regulating the metabolism of cell wall substances.

Somatic variations can give rise to bud sports with advantageous traits, serving as the foundation for bud sport breeding in perennial plants. Here, we report a fully phased genome assembly of 'Fuji' apple, enabling comprehensive identification of somatic variants across 74 clonally propagated 'Fuji' varieties. Phylogenetic analysis indicates that spur-type and early-maturation traits in 'Fuji' sport varieties arise from multiple independent events. Several putative functional somatic variants have been identified, including a spur-type-specific deletion in the promoter of the TCP transcription factor gene MdTCP11. DNA methylation level of the deletion-associated miniature inverted-repeat transposable element is lower in spur-type varieties compared to standard-type varieties, while the expression of MdTCP11 is significantly higher. Overexpression of MdTCP11 in apple decreases plant height, highlighting its important role in the development of spur-type apple varieties. This study sheds light on the cloning history of 'Fuji' and provides valuable resources for apple breeding. Spur-type of growth is preferred for apple production. Here, the authors employ the trio-based phasing approach to assemble a fully phased Fuji genome and reveal the association of a 167-bp deletion in the promoter region of the MdTCP11 gene with the spur-type varieties.

Apples are one of the most important economic crops worldwide.Because of global warming and an aggravation of environmental,abnormally high temperatures occur frequently in fruit-growing season and seriously affect normal fruit growth and reduce fruit quality and yield.We took five-year-old Ruixue’（Qinfu 1×Pink Lady;CNA20151469.1） fruits as test materials,and the ambient temperature during fruit development was monitored.The results showed that during the fruit-growing season,especially during the rapid growth stage （July to August）,the maximum daily temperature exceeded 30℃ and lasted for more than 40 days.To determine the effects of high temperature stress on the apple fruit resistance,we treated expanding,veraison,and maturity-period fruits at different temperatures.It was found that the fruits of the expanding period showed strong resistance to high temperature stress,whereas during veraison and maturity,fruit resistance to high temperature stress decreased,and the fruit peel browning phenotype appeared.Meanwhile,the content of malonaldehyde （MDA）,hydrogen peroxide （H 2 O 2 ）,and superoxide anion （O. 2 - ） in the peel gradually increased with increasing temperature.The content of total phenols,flavanol,and flavonoids in the peel decreased substantially at 45℃.Moreover,it was found that polyphenol oxidase gene （MdPPO1） was most sensitive to high temperature stress in apple.Furthermore,transient and stable MdPPO1 overexpression significantly promoted peel browning.The transgenic materials were more sensitive to high temperatures,and browning was more severe compared to non-genetically modified organism （WT）.Stable MdPPO1 knockout calli obtained via clustered regularly interspersed short palindromic repeats （CRISPR/Cas9） gene knockout technology reduced the browning phenotype,and the resultant fruits were not sensitive to the effects of high temperature stress.Thus,MdPPO1 expression may be a key factor of high temperature-related changes observed in the browning phenotype that provides a scientific theoretical basis for the selection of high temperature-resistant varieties and apple cultivation and management in the future.

Flesh firmness is a critical breeding trait that determines consumer selection, shelf life, and transportation. The genetic basis controlling firmness in apple (Malus x domestica Borkh.) remains to be fully elucidated. We aimed to decipher genetic variance for firmness at harvest and develop potential molecular markers for marker-assisted breeding. Maturity firmness for 439 F1 hybrids from a cross of "Cripps Pink" and "Fuji" was determined in 2016 and 2017. The phenotype segregated extensively, with a Gaussian distribution. In a combined bulked segregant analysis (BSA) and RNA-sequencing analysis, 84 differentially expressed genes were screened from the 10 quantitative trait loci regions. Interestingly, next-generation re-sequencing analysis revealed a Harbinger-like transposon element insertion upstream of the candidate gene PECTATE LYASE5 (MdPL5); the genotype was associated with flesh firmness at harvest. The presence of this transposon repressed MdPL5 expression and was closely linked to the extra-hard phenotype. MdPL5 was demonstrated to promote softening in apples and tomatoes. Subsequently, using the MdPL5 promoter as bait, MdNAC1-L was identified as a transcription activator that positively regulates ripening and softening in the developing fruit. We also demonstrated that MdNAC1-L could induce the up-regulation of MdPL5, MdPG1, and the ethylene-related genes MdACS1 and MdACO1. Our findings provide insight into TE-related genetic variation and the PL-mediated regulatory network for the firmness of apple fruit. A series of genome resequencing-based methods reveal a transposable element insertion that influences flesh firmness in apples.