Physiological characteristics of ‘Ruixue’ and its parents apples during fruit development
The ‘Ruixue’ apple, a yellow cultivar, was selected from a cross between ‘Pink Lady’ and ‘Fuji’ (Fig.1A). A total of six developmental stages were collected from 120 DAFB to the ripened stages. The colour of the ‘Ruixue’ apple peel changed from green to yellow and the colour of the pulp from green to milky white (Fig.1B). Fruit firmness, total soluble solids and titrable acidity are critical factors for fruit quality. Fruit firmness of the three cultivars showed a significant decline before 150 DAFB (Fig.1C), and then declined continually during latter development. There were no significant differences in fruit firmness between ‘Ruixue’ and ‘Pink Lady’ at 200 DAFB. However, the fruit firmness of ‘Ruixue’ was greater, approximately 1.3-fold that of ‘Fuji’. The total soluble solids of the three cultivars showed a significant increase during fruit development. The total soluble solid content of ‘Ruixue’ was 17.6%, greater than those of ‘Pink Lady’ and ‘Fuji’ at 200 DAFB (Fig.1D). The titrable acidity of the three cultivars showed a first increase, and then slowly declined during fruit development (Fig.1D). The titrable acidity of ‘Ruixue’ was far less than that of ‘Pink Lady’, while greater than that of ‘Fuji’. Generally, ‘Ruixue’ apple fruit has stronger fruit firmness,higher sugar and lower acidity.
Aroma volatiles profile of ‘Ruixue’ and its parents apples during fruit development
Fruit aroma is a crucial quality attribute, playing a vital role in consumer selection and acceptance. The volatile aroma compounds and relative changes in content of ‘Ruixue’ and its parents apples were identified during different fruit developmental stages. A total of 54 aroma volatiles were monitored from ‘Ruixue’,‘Fuji’ and ‘Pink Lady’ during fruit development (Supplementary Table S1), including 35 esters (the most abundant volatiles), 9 aldehydes, 5 alcohols, 4 terpenoids and 1 acid. ‘Ruixue’ contained 40 aroma volatiles: 25 esters, 8 aldehydes, 3 alcohols, 3 terpenoids and 1 acid. ‘Fuji’ contained 46 aroma volatiles: 29 esters, 9 aldehydes, 4 alcohols, 3 terpenoids and 1 acid. ‘Pink Lady’ contained only 32 aroma volatiles: 22 esters, 4 aldehydes, 3 alcohols, 2 terpenoids and 1 acid.
The total aroma volatile contents of ‘Ruixue’ and its parents apples increased slowly from 120 DAFB to 150 DAFB then increased rapidly from 150 DAFB to 200 DAFB. In contrast to ‘Fuji’ (190 DAFB), the total aroma volatiles content of ‘Ruixue’ and ‘Pink Lady’ reached maximum levels (4033.05944 μg/kg and 27845.15664 μg/kg) at 200 DAFB (Fig.2A). The relative content of aroma volatiles (esters, aldehydes, alcohols, terpenoids and acids) during fruit development exhibited dynamic change (Fig.2B, C and D). For ‘Ruixue’, aldehydes were the main aroma volatiles and account for more than 96% of total aroma volatiles prior to 170 DFAB (immature fruit), then declined to a minimum (32.19%)at 200 DAFB (mature fruit). Esters began to accumulate after 170 DAFB, reaching a maximum (29.91%) at 200 DAFB. Similarly the relative terpenoid content exhibited a significant increase after 170 DAFB, reaching its maximum (32.91%) at 200 DAFB, becoming the most prominent aroma volatiles. Few alcohols and acids were detected during fruit development, and the results for ‘Pink Lady’ and ‘Fuji’ were similar. For ‘Pink Lady’, aldehydes were the predominant aroma volatiles and account for more than 90% of total aroma volatile content before 170 DFAB (immature fruit) then rapidly decreased to a minimum(4.28%)at 200 DAFB. Conversely, terpenoids began to accumulate after 170 DAFB, reaching a maximum (53.37%) at 200 DAFB. For ‘Fuji’, the relative content of aldehydes continued to decrease during fruit development and reached a minimum (14.95%) at 200 DAFB; however, the relative content of esters increased to a maximum level (63.92%) at 190 DAFB and then declined.
To visualise the apple fruit aroma volatiles profile of the maturation stage, aroma volatiles of ‘Ruixue’,‘Fuji’ and ‘Pink Lady’ at 200 DAFB were analysed for hierarchical clustering (Fig. 2E). A total of 29 volatiles were detected in ‘Ruixue’ apple fruit, including 17 esters, 6 aldehydes, 2 alcohols, 3 terpenoids and 1 acid. For ‘Pink Lady’ a total of 28 aroma volatiles were identified, including 21 esters, 3 aldehydes, 1 alcohol, 2 terpenoids and 1 acid. In ‘Fuji’ apples, 31 aroma volatiles were identified, including 22 esters, 5 aldehydes, 2 alcohols, 1 terpenoid and 1 acid. Interestingly, in ‘Ruixue’ apples, aldehydes (2-hexenal, 2-hexenal and (E)-2-octenal), esters (butanoic acid, propyl ester, butanoic acid, 2-methylbutyl ester, propanoic acid, 2-methyl-, pentyl ester), alcohols (alcohol) and terpenoids (5-hepten-2-one, 6-methyl-) were significantly higher than in ‘Fuji’ and ‘Pink Lady’. Conversely, esters (butyl 2-methylbutanoate, propanoic acid, hexyl ester, butanoic acid, butyl ester, heptanoic acid, butyl ester, hexanoic acid, butyl ester and hexanoic acid, and hexyl ester) were significantly lower than in ‘Fuji’ and ‘Pink Lady’. In summary, there were differences in aroma volatile composition and content in different apple cultivars.
Odour activity value (OAV)evaluation and aroma descriptions of the volatile compounds
To further evaluate the contribution of all detected volatile compounds to aroma in ‘Ruixue’,‘Fuji’ and ‘Pink Lady’ during the mature fruit period (200 DAFB), OAVs were calculated using thresholds of volatile compounds from the literature. Volatile aroma compounds with OAVs greater than 1 have been termed aroma-active compounds [18]. As shown in Supplementary Table S2, crucial volatile aroma compounds (OAVs ≥ 1) contributed to aroma in ‘Ruixue’,‘Fuji’ and ‘Pink Lady’, with 10, 16, and 16 volatile compounds exceeding their aroma thresholds, respectively. The OAVs of hexanal and 2-hexenal in ‘Ruixue ’were greater than that of ‘Fuji’ and ‘Pink Lady’. These volatile compounds manifested as green and grassy notes. However, the OAVs of propanoic acid, butyl ester, butyl 2-methylbutanoate and propanoic acid, and hexyl ester in ‘Ruixue’ were lower than in ‘Fuji’ and ‘Pink Lady’. These volatile compounds were responsible for apple, strawberry and sweet notes. Although the amount of hexanoic acid, hexyl ester was quite high, it has a very high aroma threshold (64000μg/kg). It is noteworthy that 1-octen-3-one exhibited a very high OAV, making it a likely vital aroma contributor to ‘Ruixue ’and ‘Fuji’ fruit (none detected in ‘Pink Lady’), imparting sweet and mushroom notes, although the concentration of 1-octen-3-one was very low. In a word, it is especially important to understand that the impact of aroma-active volatile compounds is not only related to their content but also their aroma threshold.
Transcriptome analysis of ‘Ruixue’ and its parents apples during fruit development
RNA-sequencing (RNA-Seq) was utilized to obtain genome-wide gene expression profiles during fruit development with same samples used for volatile compound analysis. A total of 54 samples (three cultivars × six developmental stages × three biological replicates) were subjected to RNA-seq analysis in order to identify differentially expressed genes related to volatile compound biosynthesis and transcriptional regulation responsible for the diverse flavours exhibited by the three cultivars. After filtering, RNA-Seq produced 1073298706, 979721018 and 1015631116 clean reads from ‘Ruixue’, ‘Fuji’ and ‘Pink Lady’. A total of 460.76 GB nucleotides were obtained with an average GC content of 47.28%. Q30 percentage (error rates lower than 0.3%) was over 90% (Supplementary Table S3).
Principal component analysis was based on the transcriptome profiles from 18 samples. The first two principal components explain 41.63% (PC1) and 21.20% (PC2) of the variance among the samples. Similarities and differences among the apple transcriptomes were mostly driven by fruit developmental stage. Additionally, ‘Ruixue’, ‘Fuji’ and ‘Pink Lady’ samples were separated by PC1 (Fig. 3A). The number of differentially expressed genes (DEGs) showed first an increase and then a decrease during fruit development (Fig. 3B). The number of DEGs was most abundant in the middle and later stages of development (150-170 DAFB).
KEGG pathway analysis was used to ascertain the potential involvement of metabolic pathways in the regulation of fruit aroma volatile synthesis. Since the majority of volatile aroma compounds were synthesized, and the numbers of DEGs most abundant, in the middle and later stages of fruit development, we selected comparisons of X3 versus F3 and X3 versus P3 (‘Ruixue’, ‘Fuji’ and ‘Pink Lady’ fruit at 170 DAFB) for KEGG pathway analysis (Supplementary Table 1; Fig. 3B). The DEGs mapped to 110 KEGG pathways in the pairwise comparison of X3 versus F3, with the greatest number of DEGs mapped to phenylpropanoid biosynthesis (mdm00940) (Fig. 3C). Volatile aroma compound synthesis pathways included sesquiterpenoid and triterpenoid biosynthesis, and fatty acid degradation was also significantly enriched in KEGG. A comparison (X3 versus P3)of DEGs resulted in the identification of 114 KEGG pathways, with significant enriched pathways identified as plant-pathogen interaction (mdm04626), phenylpropanoid biosynthesis (mdm00940) and sesquiterpenoid and triterpenoid biosynthesis (mdm00909) (Fig. 3D). In general, the KEGG enrichment analyses indicated that fatty acid metabolism, phenylpropanoid metabolism and sesquiterpenoid metabolism play a critical role in fruit aroma volatile synthesis in ‘Ruixue’, ‘Fuji’ and ‘Pink Lady’.
Expression changes in aroma-related genes involved in fatty acid,isoleucine and sesquiterpenoid metabolism pathways
Esters (straight and branched chain esters) are among the most abundant volatile aroma compounds in apple fruits. Straight chain esters are produced by the fatty acid metabolism pathway while branched chain esters are produced by the isoleucine metabolism pathway (Fig. 4A). The expression levels of numerous genes showed significant increases, but were differently expressed in the different cultivars during fruit development (Fig. 4B). DH-1(3-hydroxyacyl ACP dehydratase) exhibited a very low expression level in ‘Ruixue’, significantly lower than in ‘Fuji’ during fruit development. Interestingly, ER-1 and ER-2 (2, 3-trans-enoyl ACP reductase) had an opposite expression pattern. Lipoxygenases (LOX) are involved in the initial steps of ester biosynthesis in fatty acid degradation, including six genes (LOX-1 to LOX-6). Four lipoxygenases (LOX-1 to LOX-4) dramatically increased during fruit development, transcribed at high levels in ‘Ruixue’ but at low levels in ‘Pink Lady’. However, LOX-5 and LOX-6 showed high expression levels prior to 170 DAFB then decreased rapidly. Hydroperoxidelyase (HPL) is the final step toward biosynthesis of aldehydes (hexanal) with an expression level that first increased then decreased. Alcohol dehydrogenase reduced aldehydes from the fatty acid and isoleucine degradation pathways to alcohols. Transcript abundance of ADH-1, ADH-2 and ADH-3 continuously deceased during fruit development. Alcohol acyl transferases (AAT) are the rate limiting enzymes for ester biosynthesis. Transcript abundance of AAT-1, AAT-2 and AAT-3 continuously increased during fruit development. It is noteworthy that the transcript abundance of AAT in ‘Ruixue’ was lower than in ‘Fuji’ and ‘Pink Lady’. Aldehyde dehydrogenase (ALDH-1 to ALDH-3) and carboxylesterase (CXE-1 to CXE-2) are important enzymes in biosynthesis of branched chain esters by the isoleucine metabolism pathway and were differentially expressed during fruit development.
Sesquiterpenes are the most prominent terpenoids in apples. The main sesquiterpene that accumulated was (Z, E)-α-farnesene, the most abundant of any aroma volatile compounds (Supplementary Table S1). Sesquiterpenes are produced by the mevalonate (MVA) pathway in the cytoplasm in nine enzymatic steps (Fig. 5A). Expression levels of numerous genes showed significant increases during fruit development. There were different expressions of sesquiterpene biosynthetic genes in the different cultivars during fruit development. Notably, the expression levels of α-farnesene synthase (AFS) exhibited drastic increases, higher in ‘Pink Lady’ than in ‘Fuji’ and ‘Ruixue’. Similarly, α-Farnesene exhibited rapid increases during fruit development with the same expression pattern as AFS (Fig. 5 B).
Expression changes in potential transcriptional factors
Transcriptional factors play a crucial role in regulating aroma volatile synthesis. To clarify potential transcription factors that may be involved, we further analysed expression patterns of transcription factors during fruit development, including MYC2, ERF, WRKY, MYB, BZIP and MADS-box TFs. The expression patterns of numerous transcription factors increased during fruit development, but were differently expressed in the different cultivars (Supplementary Fig. S1).
Quantitative reverse transcriptase-PCR validation of the transcriptome data
To validate the reliability and repeatability of the transcriptome data, 6 aroma biosynthesis and 3 transcription factor genes were selected for analysis of their expression levels using qRT-PCR. Gene-specific primers used in this analysis are listed in Supplementary Table S4. The expression profiles of 9 candidate genes generated using qPCR were very similar to the RNA-Seq results (RPKM values), which had high a Pearson correlation coefficient (R2 > 0.9). These results indicate that transcriptomic data were accurate and reproducible.