Research progress on the mechanism of apple aroma formation and the mapping of key genes

As China's most significant fruit crop, apple leads the world in both cultivation area and production, and have long been the country’s top fruit export. Aroma serves as a crucial indicator for sensory evaluation and a key determinant of intrinsic fruit quality. The interplay between aromatic components and fruit characteristics including sugar contents and acidity collectively determines apple flavor profiles. Currently, China has developed numerous apple cultivars, yet few exhibit comprehensive excellent traits and are widely cultivated. The field still faces challenges such as monotonous breeding objectives, and severe lag in high-efficiency breeding technologies like molecular marker-assisted selection and genetic engineering. This paper systematically elaborates on the composition of volatile compounds in apple fruit, their biosynthetic pathways, genetic characteristics, and localization of key genes, aiming to provide a theoretical foundation for molecular marker-assisted selection of new apple cultivars with distinctive aromas and superior comprehensive traits. Since the mid- 20th century, Chinese scientists have systematically explored apple aroma. Early studies primarily focused on identifying volatile compounds produced during apple ripening. After the 1990s, advancements in analytical instruments and scientific technologies enabled investigations into the biosynthesis and regulatory mechanisms of apple aroma compounds. Over 300 volatile compounds have been identified in apples, with 20-30 key compounds primarily contributing to the characteristic fragrance, predominantly consisting of esters, alcohols, aldehydes, ketones, and terpenes. Characteristic aroma compounds are defined by their significant contribution to fragrance, which is determined by their specific composition, concentration, detection thresholds, and synergistic interactions. The predominant characteristic aroma compounds in apples in- clude butyl acetate, hexyl acetate, butyl butyrate, 2-methylbutyl butyrate, hexyl butyrate, 1-butanol, 1- hexanol, hexanal, α-farnesene, etc. Distinct molecular structures confer unique aromatic properties. Esters typically exhibit“sweet and fruity”notes, while aldehydes, alkenes, and alcohols predominantly contribute“green and grassy”aromas characteristics. Apple aroma biosynthesis primarily occurs through three metabolic pathways, such as fatty acid metabolism pathway, amino acid metabolism pathway, and terpenoid metabolism pathway. The major unsaturated fatty acids involved in the synthesis of volatile substances via fatty acid pathway include stearic acid, oleic acid (OA), linoleic acid (LA) and α- linolenic acid (ALA). Most aroma compounds in apples are synthesized through fatty acid metabolism, which mainly occurs via two pathways, β-oxidation and the lipoxygenase (LOXs) pathways. The β-oxidation pathway primarily degrades stearic acid, OA, and ALA, at the same time forming substantial amounts of acetyl-CoA, which is a crucial precursor for ester biosynthesis from alcohols. Following β- oxidation, the LOX pathway produces most aroma compounds in fruits, and is closely associated with the formation of ester compounds. In the amino acid metabolic pathway, the amino acids involved in aroma synthesis include valine, leucine, isoleucine, alanine, cysteine, and phenylalanine. These contribute to the characteristic apple flavor by forming branched-chain esters. Terpene compounds are the largest class of floral volatiles in plants. The terpenes contained in apple volatile substances include α-farnesene, trans-β-farnesene and α-acacene. The synthesis of terpenoids in apple mainly occurs through two pathways: the mevalonic acid pathway (MVA pathway) and the methyl-D-erythritol-4-phosphate pathway (MEP pathway). The precursors of the two pathways are isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), respectively. In MVA pathway, the precursor IPP undergoes enzymatic reaction to produce the intermediate product farnesyl pyrophosphate (FPP), which finally synthesizes sesquiterpenoids or triterpenoids under the action of various terpenoid synthases. The MEP pathway is precursors of pyruvate and glyceraldehyde 3-phosphate. IPP and DMAPP are produced through 7 enzymatic reactions. DMAPP produces hemiterpenes, monopterpenes and multiple polyterpenes through different pathways. Among the volatile substances in the hybrid offspring of Fuji × Pink Lady, the characteristic aroma substances in apple such as hexyl acetate, 2-methylhexyl butyrate, hexyl caproate, hexal, 2-hexenal, and α -farnene have typical quantitative trait genetic characteristics. In the F1 generation, the mean values of these six aroma substances are all higher than the mid-parent value, dislaying an addtive genetic effect. And more than half of the esters show superherophilic phenomena. In the progeny, hexaldehyde, 2-hexenal and α - farnene showed positive transgressive segregation, displaying obvious heterosis. The mean values of hexyl acetate, butyl butyrate, hexyl butyrate, butyl caproate and n-hexanol in the offspring of F1 generation of Fuji × Golden Crown are all higher than the parent values, showing a relatively high transgressive inheritance. The mean values of the progeny of 2-methylbutylhexanoate, hexal, 2-hexenal and ɑ -farnene were higher than the mid-parent value. In F1 progeny fruit of Honeycrisp × Qinguan, most ester substances such as hexyl acetate, butyl butyrate, hexyl caproate, and hexyl butyrate, as well as e- 2- hexenal and ɑ - farnene, showed an negative transgressive segregation. Hexaldehyde exhibits a moderately negative transgressive inheritance. The same aroma substance in apple fruit shows different separation pattern in the offspring of different hybrid combinations. It may be affected by environmental factors or have gene interactions or recessive synergistic genes, resulting in the occurrence of positive transgressive segregation in the F1 generation. Currently, through the construction of QTL genetic mapping, candidate genes of LOXs, HPL, ADH and AAT, which are key genes for aroma synthesis, have been identified in apples, grapes and jujubes. Notably, MdLOX1a and MdAAT are functionally linked to the biosynthesis of characteristic aroma esters in apples. Dunemann F.mapped a candidate gene of MdLOX on LG9. HPL has been proved to be involved in the synthesis of aldehydes in fruits, and ZjHPL in jujube may be the key gene involved in (E) -2-hexenal synthesis in jujube. The candidate genes of MdADH and MdAAT have been identified on LG2 and MdAAT6 on LG6. This review systematically elucidates the composition, biosynthetic pathways, genetic characteristics, and key gene localization of apple volatile compounds, in order to provide insights into precise aroma phenotyping and genotyping, and to provide reference for the marker-assisted selection of new apple varieties with characteristic aroma and excellent comprehensive characteristics.