Low calcium content caused bitter pit disease, which shortened the shelf life of apple fruit
In our experiments, calcium-deficient and healthy apple fruit (CK) were analyzed during the storage period. As shown in Fig. 1A, calcium-deficient apple fruit exhibited bitter pit disease 7 days after storage (DAS), while CK did not show disease characteristics during the storage period. The apple peels of bitter pit disease turned dark yellow compared to CK at 21 DAS. Furthermore, the calcium content of apple fruit was determined, and the results showed that the calcium content in calcium-deficient apple fruit was significantly lower than that of the CK fruit during the storage period (P<0.01) (Fig. 1B). This showed the reliability of bitter pit disease in calcium-deficient apples.
Comparison of H 2 O 2 , •O2–, MDA and activity of PPO in calcium-deficient and CK apple fruit during the storage period
The results showed that calcium-deficient apple fruit maintained lower levels of H2O2 during postharvest storage. Significantly lower levels of H2O2 were observed in calcium-deficient fruit than in the CK fruit during the storage period until 14 DAS (P<0.01) (Fig. 2A). From 7 to 14 DAS, the control fruit increased as much as 4-fold compared to calcium-deficient apple fruit. Thereafter, the H2O2 content in the control fruit decreased gradually due to senescence and rot with prolonged storage time. The production of H2O2 in calcium-deficient fruit was accelerated from 0 to 7 DAS and reduced rapidly thereafter.
As shown in Figure 2B, the rate of •O2− production remained high during storage irrespective of disease. Compared with the control fruit, significantly lower •O2− production was observed in calcium-deficient apple fruit during the entire storage period (P<0.01).
Figure 2C shows that the MDA content in calcium-deficient fruit was enhanced rapidly during the entire storage period. From 0 to 7 DAS, the levels of MDA in calcium-deficient apple fruit were significantly lower than those in CK. From 7 to 14 DAS, the levels of MDA in calcium-deficient apple fruit were higher than those in the control. Finally, at 21 DAS, the MDA content was approximately the same regardless of disease.
Figure 2D shows the change in polyphenol oxidase (PPO) activity in apple fruit throughout storage. At 0 DAS, the activity of PPO in calcium-deficient apple fruit was nearly 2.5-fold that in CK. Then, the activity of PPO in calcium-deficient apple fruit decreased rapidly and was maintained at a stable state but was always higher than that in control apple fruit during the entire storage time (P<0.01). The activity of PPO in control apple fruit rose slowly at first and declined slightly from 14 to 21 DAS.
Comparison of flavonoids and total phenols in calcium-deficient apple fruit with CK fruit during the storage period
To better understand the improved appearance quality in calcium-deficient apple fruit relative to CK, we determined the contents of flavonoids and total phenols. Figure 3A shows that the flavonoid contents in calcium-deficient apple fruit were enhanced at 14 DAS and reduced thereafter. The trend of flavonoids in control apple fruit was similar to that of calcium-deficient apple fruit except at 0 DAS. Until 21 DAS, the flavonoid contents tended to be consistent between calcium-deficient apples and control apple fruit.
In Figure 3B, there was a significant difference in total phenols between the two kinds of apples at 0 DAS. In the next 7 days, the total phenols in calcium-deficient apples increased and then dropped from 14 to 21 DAS. The total phenols of the control apple fruit increased from 7 to 14 DAS and dropped at 21 DAS. Similar to the change trend of flavonoid content, the total phenols in calcium-deficient apple fruit were always lower than those of the control apple fruit (except at 7 DAS).
Analysis of dry matter and soluble protein content in calcium-deficient apple fruit
It was shown that dry matter and soluble protein increased in calcium-deficient apple fruit. During calcium-deficient apple fruit storage for 21 days, the dry matter content increased slightly and was always higher than that of the control apple fruit (P<0.01). However, dry matter in control apple fruit did not obviously change during the entire storage time (Fig. 3C). Similarly, the change trend of soluble protein content was the same trend as dry matter content. During 0 to 7 DAS, the content of soluble protein increased rapidly and then was maintained at a high level (Fig. 3D). In contrast, it was reduced slightly in control apple fruit at the beginning of storage, and then there was a slight increase within 14 to 21 DAS. The content of soluble protein in control apple fruit was always lower than that in calcium-deficient apple fruit (P<0.01).
Identification of TA, TSS, ascorbate acid, ratio of TSS/TA and soluble sugars in calcium-deficient apple fruit
TA and ascorbic acid play a sour taste role in apple fruit, while TSS play a sweet taste role. Figure 4 shows the changes in TA, ascorbate, TSS, ratio of TSS/TA and soluble sugars in calcium-deficient apple fruit and control apple fruit. During the whole storage period, TA showed a downward trend in apples regardless of calcium deficiency (Fig. 4A). The TA content in control apple fruit was always higher than that in calcium-deficient apple fruit (P<0.05).
The content of ascorbate acid always decreased during the full storage period in the two kinds of apple fruit, and it was the lowest at 21 DAS (Fig. 4B). There was no significant difference in ascorbate acid between the calcium-deficient and control apple fruit mid-storage. However, at the beginning and late stage of storage, ascorbate acid in control apple fruit was higher than that in calcium-deficient apple fruit.
The change of TSS is shown in Figure 4C. During the entire storage time, TSS increased slightly in calcium-deficient apple fruit and was always higher than that in control apple fruit (P<0.05 or P<0.01). This means that calcium-deficient apple fruit sugar accumulates faster than that in control apple fruit.
The ratio of TSS/TA is an important index for evaluating the flavor of apples. During the storage time, the ratio of TSS/TA in calcium-deficient apples was always higher than that of control apples (Fig. 4D). In particular, the ratio of TSS to TA in calcium-deficient apple fruit was significantly higher than that in control apple fruit (except at 14 DAS) (P<0.01).
During the storage time, the sucrose content presented a declining trend in apple fruit, and it was always significantly higher in calcium-deficient apple fruit than in control apple fruit (P<0.01) (Fig. 4E).
The glucose contents in calcium-deficient and control fruit shared the same trends. The glucose contents increased from 0 to 7 DAS, decreased from 7 to 14 DAS, and finally increased from 14 to 21 DAS (Fig. 4F). During the whole storage time, the glucose contents of calcium-deficient apple fruit were always significantly lower than those of control apple fruit (P<0.01).
During storage, the fructose content was always lower in calcium-deficient apple fruit than in the control fruit (Fig. 4G). At 14 DAS, the fructose contents of calcium-deficient apple fruit and control apple fruit tended to be consistent. However, the fructose content of control apple fruit was significantly higher than that of calcium-deficient apple fruit (except at 14 DAS) (P<0.01).
PCA and correlation analysis of the changes in bioactive substances in apple fruit
The PCA results showed that the contribution rates of PC1 and PC2 were 79.8% and 20.2%, respectively. In PC1, Ca and MDA contents were the main factors. In PC2, TA and TSS were the main factors (Fig. 5, Table S1). Correlation analysis indicated that Ca content showed a negative correlation with TSS (person: -0.345), sucrose (person: -0.4), •O2− (person: -0.42) and MDA (person: -0.928), and it had a positive correlation with ascorbate (person: 0.576), glucose (person: 0.405), fructose (person: 0.709), H2O2 (person: 0.386), and TA (person: 0.719). In addition, TSS had a positive correlation with sucrose (person: 0.713), TSS (person: -0.239) and sucrose (person: -0.125) and a negative correlation with TA (Fig. S1, Table. S2). The results showed that Ca content was positively correlated with antioxidant capacity and the accumulation of acidic substances and negatively correlated with the accumulation of sweet substances.
Identification Of The Candidate Genes Related Calcium-deficient Metabolism
According to transcriptome data of calcium-deficient “Fuji” apple fruit (T01), calcium-deficient apple healthy flesh (T02) and healthy apple fruit (T03) at fruit ripening period , eight expression patterns of all differentially expressed genes (DEGs) were obtained. We selected a total of 42 DEGs from profiles 0, 1, 6 and 7 (Fig. 5B). Among of them, 24 WRKY transcription factors, eleven genes of sugar metabolism (SS, SSL, SWEET and SPS), five genes of apoptosis and two genes of calcium signal have the same or opposite expression trend (Fig. 5C). Furthermore, promoters of genes encoding sucrose synthesis and transport enzymes, specifically MdSWEET1-like (MD10G101220), MdSWEET2a-like(MD10G1269300༉, MdSWEET15 (MD16G1125300), MdSPS2 (MD04G1013500), MdSS2 (MD02G1100600), MdSPS4 (MD05G1006400), MdSS (MD15G1223500), MdSSL (MD02G1100500) and MdSWEET1 (MD10G1012200), were predicted by PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). We found that there were W-box cis-elements in their 2000 bp upstream promoters. This suggested that WRKY TFs may be involved in the regulation of fruit sugar accumulation by binding to genes encoding sucrose synthesis and transport enzymes.
MdWRKY75 was related to MdSWEET1 by RT-qPCR and correlation analysis
In order to further confirm the expression pattern of the above candidate genes in calcium-deficient apple fruit and healthy apple fruit at 0, 14, and 21 DAS, we determined the expression levels of MdWRKY75 (MD13G1122100), MdWRKY65 (MD05G1295700), MdWRKY23 (MD17G1278100), MdWRKY31 (MD05G1349800), MdWRKY48 (MD13G1150700), MdWRKY26 (MD03G1057400), MdWRKY40 (MD00G1143500), MdSSL (MD02G1100500), MdSS (MD15G1223500), MdSWEET1 (MD10G1012200) MdAmmonium transporter (MD12G1174700), MdU-box 21 (MD13G1017300) and MdU-box 21-like (MD16G1015400) by RT-qPCR. As shown in Fig. 6, the expression levels of MdSS, MdSSL, MdSWEET1, MdAmmonium transporter, MdU-box 21 and MdU-box 21-like were higher in calcium-deficient apple fruit than those of the CK fruit (P<0.01). The expression levels of WRKYs in calcium-deficient apple fruit were always higher than those in the CK fruit. In particular, the expression patterns of MdWRKY75 and MdWRKY31 were similar to those of MdSWEET1.
Furthermore, correlation analysis showed that sucrose had positive correlation with MdWRKY75 (Pearson: 0.815), MdWRKY23 (Pearson: 0.802). Meanwhile, MdWRKY75 (Pearson: 0.959) and MdWRKY31 (Pearson: 0.987) had positive correlations with MdSWEET1 (Fig. 7, Table. S3). Thus, sucrose accumulation was significantly positively correlated with MdWRKY75 and MdWRKY23, the TFs MdWRKY75 and MdWRKY31 were related to MdSWEET1. Moreover, MdWRKY75 had a positive correlation with MdAmmonium transporter (Pearson: 0.88), MdU-box 21 (Pearson: 0.92) and MdU-box 21-like (Pearson: 0.95). These results indicated that MdWRKY75 might be regulate the expression of MdSWEET1 and result in accelerating sucrose accumulation, and might be related to apoptosis in calcium-deficient apple fruit.
Transient transformation of MdWRKY75 in apple fruit
Because of MdWRKY75 have a high expression level in calcium deficient apples, and have positive correlation with sucrose content and the expression of apoptosis related genes, we injected MdWRKY75 into apple fruit and measured the sugar content and the expression level of sugar-, Ca- and apoptosis related genes. As shown in Fig. 8A, the content of sucrose, glucose and fructose in apple fruit were higher than those of the empty vector (pSAK277). Especially, the sucrose content of MdWRKY75-oe in apple fruit is 5-fold higher than those of the empty vector. RT-qPCR analysis also showed that the expression levels of MdWRKY75 and MdSWEET1 were higher in MdWRKY75 induced apple fruit than those of the empty vector (Fig. 8B). However, MdCal1, MdCal4, MdAmmonium transporter, MdU-box 21 and MdU-box 21-like don’t change obviously (Fig. 8B).