Comparative Physiological and Transcriptomic Analysis Reveal MdWRKY75 Associated With Sucrose Accumulation in Postharvest Apples With Bitter Pits

Background: Calcium (Ca) deciency can cause apple bitter pits, reduce the quality and shelf life. WRKY Transcription factors play essential role in plant response to multiple diseases. However, the underlying mechanisms causing bitter pits in apple fruit due to Ca deciency during storage is extremely limited. Results: In the present study, the nutritional metabolites and reactive oxygen species (ROS) were compared in Ca-decient and healthy apple fruit (CK) during storage. Results showed that Ca-decient apples sustained signicantly higher production of ROS, PPO activity, avonoids, total phenol, total soluble solids (TSS), and sucrose contents, but the contents of Ca, H 2 O 2 , titratable acids (TA), glucose and fructose were signicantly lower than those of CK during storage. Principal component analysis (PCA) showed that TSS, •O 2− , PPO, MDA and Ca were the main factors, and TSS had a positive correlation with sucrose. Furthermore, transcriptome analysis revealed that WRKYs were co-expressed with sucrose metabolism-related enzymes (SWEETs, SS, SPS). RT-qPCR and correlation analysis indicated that MdWRKY75 were signicantly positively correlated with MdSWEET1. Moreover, transient overexpression of MdWRKY75 could signicantly increase the sucrose content and promote the expression of MdSWEET1 in apple fruit. Conclusions: Calcium deciency could decrease antioxidant capacity, accelerate nutritional metabolism and up-regulate the expression of WRKYs in apple with bitter pits. Overexpression of MdWRKY75 signicantly increased sucrose accumulation and the expression of MdSWEET1. These ndings further strengthened knowledge of the basic molecular mechanisms in calcium-decient apple esh and contributed to improving the nutritional quality of apple fruit.

indicators to measure the senescence of fruits. And malondialdehyde (MDA) is an important product of membrane lipid oxidation [8]. It was reported that exogenous calcium ions delayed fruit ripening and maintained nutritional quality and appearance. With an increase in the calcium concentration, papaya fruit ripening and the senescence process were inhibited, slowing down softening and prolonging storage life [9,10]; rmness and TSS increased and postharvest decay was reduced in strawberries; total phenolics and total antioxidant capacity increased and TA, ascorbic acid and decay decreased in sweet cherry [11]; protein, ascorbic acid, TA and carbohydrates decreased in banana [12]; and calcium sprays decreased ascorbic acid and sugar content and stimulated catalase enzyme activity and pathogen defense genes during storage in grape berries [13,14]. In apples, low levels of calcium cause faster ripening, and ascorbic acid and rmness are lower [15]. Furthermore, spraying calcium chloride on preharvest apples can effectively reduce the occurrence of bitter pit, and it can signi cantly improve the rmness and appearance of apple to enhance shelf time [16][17][18]. Calcium can reduce fruit cracking and promote the healing of mechanical injury [19]. Thus, calcium de ciency can cause many diseases that in uence the edible quality and postharvest storage of fruit.
Sugar content is an important determinant of fruit edible quality, and bitter pits increase TSS and sugar contents, such as sucrose content. Sucrose synthesis (SS) can reversibly catalyze sucrose to fructose, glucose and UDP-glucose [20]. SPS is an important enzyme in the irreversible reaction that catalyzes UDPG and 6-phosphate-fructose to sucrose [21]. In peaches, nitric oxide (NO) enhances gene expression and the activities of SPS and SS and leads to an increase in sucrose content [22]. In apple fruit, it was reported that sodium nitroprusside (SNP) treatment delayed loss of quality by enhancing MdSPS and MdSS expression and then increasing the sucrose content [23], and the sucrose transporter MdSUT4.1 participates in the regulation of fruit sugar accumulation [24]. SWEETs were identi ed as sugar transporters responsible for fruit sugar accumulation, and MdSWEET9b and MdSWEER15a were involved in regulating fruit sugar accumulation in apple [25]. In pear fruit, PuWRKY31 can accelerate the synthesis of sucrose by binding to PuSWEET15 [26]. However, whether WRKY TFs regulate the sugar transporter SWEETs in calcium-de cient apple fruit remains unclear.
To explore the molecular mechanisms of postharvest quality in apple fruit calcium de ciency caused by bitter pit, the content of nutrients and antioxidant capacity in apple fruit during the storage period were determined. The main factors involved in bitter pits (Ca, TSS, TA and MDA) were screened by PCA and correlation analysis, and the sugar transporter-related enzyme SWEET1 and WRKYs TF were identi ed by bioinformatics and RT-qPCR analysis. Furthermore, WRKY75 was transformed and identi ed by apple transient expression system. These results will provide new insights into candidate genes for sugar accumulation in calcium-de cient apples and the improvement of fruit quality.

Plant Materials and Treatment
The healthy and calcium-de cient 'Honeycrisp' apple fruit used in this study were provided by Shandong Academy of Agricultural Sciences (Tai'an, Shandong Province). During the ripening period in 2018, 60 calcium de cient apple fruits were harvested from the tree, and 60 healthy fruits without pests or mechanical damage were harvested at the ripening stage as the controls (CK). Every ten healthy fruits/ calcium de cient were randomly divided into one group, and they were respectively placed in six 350 mm glass-vacuum-dryer with the appropriate amount of distilled water to maintain 75~85 % relative humidity at room temperature (25 ℃). Ten apple fruits (without seeds and skins) were sampled every seven days until 21 days after storage (DAS). The samples were frozen in liquid N 2 quickly and stored in a -80°C refrigerator for subsequent experiments.

Determination of Ca content in apple esh
The Ca content of apple esh was determined by ame atomic absorption spectrometry (FAAS) according to the methods described by Barea-Álvarez et al. [29]. Apple esh (1 g, dry weight) was carbonized on a crucible. Then, samples were transferred to a high-temperature mu e furnace, and the temperature gradient was raised to 500 ℃ (50 ℃/30 min) until samples were burnt to white gray. When the samples cooled, they were mixed with 15 mL of a mixture of HNO 3 and HClO 4 (5:1 v/v). The best parameters for determination were λ = 422.7, current = 10 mA, and spectral resolution = 1.2 nm, and the gases were C 2 H 2 and air (C 2 H 2 3.0 L·min -1 ; atmospheric air 13 L·min -1 ) (Hitachi Z2000). CaCl 2 solution was used as the standard for calibration.
Determination of avonoid and total phenol contents and polyphenol oxidase activity The avonoid contents were assayed as described by Li et al. [30], and the absorbance was determined at 510 nm. Rutin was used as the standard for calibration.
Total phenols were assayed following the method of Pirie and Mullins [31]. The total phenols were determined by spectrophotometry at 280 nm. Gallic acid was used as the standard to make a calibration curve.
The activity of polyphenol oxidase (PPO) was determined by procedures described by Benjamin and Montgomery [32]. Apple esh (5.0±0.01 g) was ground in 5 ml of ice-cold extraction buffer [1 mM PEG, 4% polyvinylpyrrolidone (PPVP), 1% Triton X-100, pH 5.5 acetic acid-sodium acetate buffer]. The homogenate was centrifuged, and then the supernatant was collected for protease activity determination.
Absorbance was recorded at 540 nm, and the protease activity was quanti ed as U/g FW. Corrections were made for the background absorbance in the presence of 50 units of superoxide dismutase (SOD) and presented as nmol/min/g.

Detection of soluble protein and dry matter contents
For soluble protein, the supernatant was mixed with Coomassie brilliant blue, and the absorbance was determined at 595 nm after 5 min according to the method described by Bradford [34]. The results are expressed as milligrams per gram of FW (fresh weight).
For dry matter, samples (5.00±0.05 g) were placed in an oven at 80 °C until the weight at the third weighing remained unchanged. Then, the proportion relative to the initial weight was calculated.
Determination of total soluble solid contents, ascorbic acid and titratable acids Total soluble solids (TSS) were determined in samples of apple fruit (5.00±0.05 g). After grinding and centrifugation (4000 r/min, 10 min), the juice was measured by an Abbe Refractometer (JH-WYA2S, Jiahang Instrument Co., Ltd, Shanghai, China). The titratable acid (TA) content was determined by acidbase neutralization with NaOH [35]. TSS and TA contents were presented as mg/g. The extraction and determination of ascorbic acid in apples followed the method of Nath et al. [36], and the assessment was performed by indophenol titration with minor changes, with values expressed as mg/100 g.

Measurements of the soluble sugar content in apple eshes
The soluble sugars in apple esh were extracted following the method of Li et al. [26]. Brie y, two grams of the fruit esh was homogenized, mixed with 5 ml of sterile deionized water, incubated in a water bath at 80 ℃ for 30 minutes, and then extracted by ultrasonic for 30min at 50W. Finally, the supernatant was collected by centrifugation (12000×g, 5 min) and ltration through a 0.22 μm membrane. The soluble sugars were measured by HPLC (Agilent Technologies 1260 Series) following the method of Jia et al. [37]. HPLC (Agilent 1260) was performed with a 7.8 × 300 mm Carbomix Ca-NP column (Sepax); the mobile phase was ultrapure water with a ow rate of 1 ml min -1 ; the column temperature at 80 °C; the refractive index detector temperature at 35 °C, and the injection volume was 20 μl. At each sampling point, at least ve fruits were randomly selected and divided into three groups as three biological replicates.

Quantitative reverse transcription PCR analysis
Total RNA from 0.1 g frozen apple fruit samples was extracted by an RNA Extraction Kit (Tiangen, Beijing, China). Then, cDNA was synthesized by a reverse transcription kit (PrimeScript RT Master Mix, Takara, Kyoto, Japan) and further used for quantitative PCR. The speci c primers used for qPCR are listed in Supplementary Table S4. MdTUB (TUB, accession number GO562615) and MdUBQ (UBQ, accession number MDU74358) were used as housekeeping genes for the normalization of data. All data are expressed as the means and standard deviations of the values obtained from three biological replicates.

Gene cloning and transient transformation of MdWRKY75 in apple fruit
The full Coding DNA Sequence (CDS) of MdWRKY75 (MD13G1122100) in apple was obtained by GDR (https://www.rosaceae.org/), and PCR ampli cation was conducted using Phanta Super-Fidelity DNA Polymerase (P501-d1, Vazyme Biotech Co. Ltd., China) and the primer sequences listed in Table S4. The full CDS fragment of WRKY75 was inserted into pSAK277 vector under the control of the 35S promoter with EcoRI and XhoI. The recombinant expression vector WRKY75-pSAK277 was transformed into Agrobacterium tumefaciens (GV3101), it was cultured at 37°C, and then collected, subsequently resuspended in a solution (included 10 mm MES, 10 mm MgCl 2 , and 200 μm acetosyringone) to a nal optical density of 0.8~1.0 at OD 600 , and then incubated at room temperature for 3−4 h. The in ltration protocol and culture methods for transient expression were adapted from previously described methods [38,39]. The infected apples were placed at 23°C for 3 days. All fruit samples were frozen in liquid nitrogen upon collection, and stored at −80 °C.

Statistical Analysis
Statistical analysis was performed using a t-test in SPSS 22.0. PCA was performed using factor analysis in dimension reduction, and the rotation method was carried out by varimax with Kaiser normalization. Correlation analysis and heatmap analysis were performed by R studio software.

Results
Low calcium content caused bitter pit disease, which shortened the shelf life of apple fruit In our experiments, calcium-de cient and healthy apple fruit (CK) were analyzed during the storage period. As shown in Fig. 1A, calcium-de cient 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-de cient apple fruit was signi cantly 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-de cient apples. The results showed that calcium-de cient apple fruit maintained lower levels of H 2 O 2 during postharvest storage. Signi cantly lower levels of H 2 O 2 were observed in calcium-de cient 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-de cient apple fruit. Thereafter, the H 2 O 2 content in the control fruit decreased gradually due to senescence and rot with prolonged storage time. The production of H 2 O 2 in calcium-de cient fruit was accelerated from 0 to 7 DAS and reduced rapidly thereafter.
As shown in Figure 2B, the rate of •O 2 − production remained high during storage irrespective of disease.
Compared with the control fruit, signi cantly lower •O 2 − production was observed in calcium-de cient apple fruit during the entire storage period (P<0.01). Figure 2C shows that the MDA content in calcium-de cient fruit was enhanced rapidly during the entire storage period. From 0 to 7 DAS, the levels of MDA in calcium-de cient apple fruit were signi cantly lower than those in CK. From 7 to 14 DAS, the levels of MDA in calcium-de cient 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-de cient apple fruit was nearly 2.5-fold that in CK. Then, the activity of PPO in calcium-de cient 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 rst and declined slightly from 14 to 21 DAS.
Comparison of avonoids and total phenols in calcium-de cient apple fruit with CK fruit during the storage period To better understand the improved appearance quality in calcium-de cient apple fruit relative to CK, we determined the contents of avonoids and total phenols. Figure 3A shows that the avonoid contents in calcium-de cient apple fruit were enhanced at 14 DAS and reduced thereafter. The trend of avonoids in control apple fruit was similar to that of calcium-de cient apple fruit except at 0 DAS. Until 21 DAS, the avonoid contents tended to be consistent between calcium-de cient apples and control apple fruit.
In Figure 3B, there was a signi cant difference in total phenols between the two kinds of apples at 0 DAS. In the next 7 days, the total phenols in calcium-de cient 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 avonoid content, the total phenols in calcium-de cient 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-de cient apple fruit It was shown that dry matter and soluble protein increased in calcium-de cient apple fruit. During calcium-de cient 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 calciumde cient apple fruit (P<0.01).
Identi cation of TA, TSS, ascorbate acid, ratio of TSS/TA and soluble sugars in calcium-de cient 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-de cient apple fruit and control apple fruit. During the whole storage period, TA showed a downward trend in apples regardless of calcium de ciency (Fig. 4A). The TA content in control apple fruit was always higher than that in calcium-de cient 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 signi cant difference in ascorbate acid between the calcium-de cient 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-de cient apple fruit.
The change of TSS is shown in Figure 4C. During the entire storage time, TSS increased slightly in calcium-de cient apple fruit and was always higher than that in control apple fruit (P<0.05 or P<0.01). This means that calcium-de cient apple fruit sugar accumulates faster than that in control apple fruit.
The ratio of TSS/TA is an important index for evaluating the avor of apples. During the storage time, the ratio of TSS/TA in calcium-de cient apples was always higher than that of control apples (Fig. 4D). In particular, the ratio of TSS to TA in calcium-de cient apple fruit was signi cantly 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 signi cantly higher in calcium-de cient apple fruit than in control apple fruit (P<0.01) (Fig. 4E).
The glucose contents in calcium-de cient and control fruit shared the same trends. The glucose contents increased from 0 to 7 DAS, decreased from 7 to 14 DAS, and nally increased from 14 to 21 DAS (Fig. 4F). During the whole storage time, the glucose contents of calcium-de cient apple fruit were always signi cantly lower than those of control apple fruit (P<0.01).
During storage, the fructose content was always lower in calcium-de cient apple fruit than in the control fruit ( Fig. 4G). At 14 DAS, the fructose contents of calcium-de cient apple fruit and control apple fruit tended to be consistent. However, the fructose content of control apple fruit was signi cantly higher than that of calcium-de cient 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.

MdWRKY75 was related to MdSWEET1 by RT-qPCR and correlation analysis
In order to further con rm the expression pattern of the above candidate genes in calcium-de cient apple fruit and healthy apple fruit at 0, 14, and 21 DAS, we determined the expression levels of MdWRKY75

Transient transformation of MdWRKY75 in apple fruit
Because of MdWRKY75 have a high expression level in calcium de cient 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).

Discussion
Calcium-de cient apples signi cantly stimulated the activity of ROS and decreased antioxidant capacity In this study, compared with healthy apples, calcium-de cient apples showed stronger senescence appearance during storage (Fig. 1A), and ·O 2 − and MDA contents and PPO activity were higher than those of healthy apples (Fig. 2B, C, D), while H 2 O 2 content was lower in calcium-de cient apples than in healthy apples ( Fig. 2A). Thus, the antioxidant capacity of calcium-de cient apples was lower than that of healthy apples. MDA is one of the main products of membrane lipid peroxidation and can be used to re ect the degree of membrane peroxidation. In tomato, MDA content increased sharply in plants with low calcium contents [27]. Moreover, ascorbic acid can deoxidize H 2 O 2 to H 2 O via its own oxidation. Our results were similar to a report showing that calcium can reduce the degradation of ascorbic acid and enhance the total antioxidant capacity in sweet cherry and 'Royal delicious' [12,15]. Thus, the antioxidant capacity of 'Honey Crisp' apple with calcium de ciency is lower than that of healthy apple.
The deterioration of calcium-de cient apples was faster than that of healthy apples Calcium was reported to play a critical role in fruit development and ripening. Calcium de ciency can accelerate the senescence of apples. In our study, there were signi cant differences in the contents of ascorbic acid, soluble protein, avonoids, total phenols, TSS and TA between calcium-de cient apples and healthy apples during postharvest storage, which strongly suggested that calcium played a key role in regulating apple fruit senescence. The contents of avonoids and total phenols in calcium-de cient apples were higher than those in healthy apples (Fig. 3A, B). A previous report suggested a strong correlation between total antioxidant activity and total phenolic content [28]. Soluble protein, as an important component of enzymes in fruit and vegetables, is also one of the important evaluation indexes of fruit and vegetable quality and nutrition. The content of soluble protein in calcium-de cient apples was higher than that in healthy apples (Fig. 3D). Similarly, the TSS content and the ratio of TSS to TA were higher in calcium-de cient apples than in healthy apples (Fig. 4C, D), but the trends of TA and ascorbate content were the opposite (Fig. 4A, B). Thus, it was inferred that the taste of calcium-de cient apples is sweeter than that of healthy apples. The sucrose content of calcium-de cient apple fruit was higher than that of control apple fruit (Fig. 4E), providing direct evidence that calcium de ciency accelerates the accumulation of sweet substances. The TA and carbohydrate contents of banana without exogenous calcium treatment were consistent with our results [12]. Meanwhile, the PCA results showed that Ca was the main factor for bitter pits in postharvest apple fruit (Fig. 5A). The correlation analysis showed that Ca was positively correlated with TA and negatively correlated with TSS accumulation. In addition, TSS was positively correlated with sucrose accumulation (Pearson: 0.71) ( Table. S2). This result indicated that sugar accumulation is related to calcium de ciency in apples.
MdWRKY75-MdSWEET1 is a potential regulatory model of sucrose transport in calcium-de cient apples Sugar content was an important criterion for evaluating fruit maturity. The sugar/acid ratio is an index that affects fruit nutritional quality. In this study, we found that the TSS, ratio of TSS to TA and sucrose content of calcium-de cient apples were higher than those of healthy apples (Fig. 4C, D, E). Furthermore, MdSS, MdSSL and MdSWEET1 had signi cantly higher transcript abundance in calcium-de cient apples than healthy apple fruit by transcriptome data mining. This suggested that MdSS, MdSSL, MdSWEET1 and twenty-four WRKYs were activated by calcium de ciency. In the other hand, MdAmmonium transporter, MdU-box 21 and MdU-box 21-like had similar expression pro les with MdSWEET1, MdSS, MdSSL and WRKYs (Fig. 5C). This suggested calcium de ciency also activated apoptosis. The results of RT-qPCR analysis further showed that the expression patterns of MdSS, MdSSL, and MdSWEET1 were consistent with seven WRKY TFs during storage (Fig. 6). Meanwhile, MdSS and MdSWEET1 had a positive correlation with TSS content and a negative correlation with Ca content (Fig. 7, Table S3), and MdWRKY75 had a strongly positive correlation with MdSWEET1 by correlation analysis. Recently, it was reported that PuWRKY31 can bind to the promoter of PuSWEET15 to regulate sugar accumulation in pear fruit [26]. Thus, we overexpressed MdWRKY75 in apple fruit by injecting pSAK277-MdWRKY75, resulting in sucrose content and expression level of MdSWEET1 increased (Fig. 8A, B). This suggests that MdWRKY75 can activate the expression of MdSWEET1 to increase the accumulation of sucrose in calcium de cient apple. Furthermore, MdAmmonium transporter, MdU-box 21, MdU-box 21-like, MdCal1 and MdCal4 have no obvious change in apple with over-expressing MdWRKY75 (Fig. 8B). It implied that MdWRKY75 cannot regulate the expression of calcium signaling and apoptosis related genes in apple, but apoptosis related genes may be involved in the sucrose metabolic pathway of apple fruit and affect the accumulation of sucrose. This study further strengthened the regulatory mechanism of calciumde cient apple esh and contributed to improving the appearance quality of apple fruit.

Conclusions
In summary, this study found that the deterioration of calcium-de cient apples, including nutrients and antioxidant capacity, was faster than that of healthy apples. The results also indicated that the TSS and sucrose contents of calcium apples were higher than those of CK during storage. TSS, sucrose, ROS and   and 21days after storage (DAS). * and ** indicate signi cance at P < 0.01 and P < 0.05, respectively.

Figure 2
The changes of reactive oxygen species (ROS) in postharvest healthy apples and calcium-de cient apples. (A) hydrogen peroxide, (B) superoxide anion, (C) malondialdehyde and (D) activity of polyphenol oxidase. Data are presented as means ± SD (n = 3). * and ** indicate signi cance at P < 0.01 and P < 0.05, respectively.     Correlation analysis of the relationship between sucrose content and the expression levels of candidate genes.

Figure 8
The changes of sucrose, glucose, fructose and related genes expression in apples infected pSAK277-  A proposed model of the mechanism of sucrose accumulation, which in apples with Ca de ciency.

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