MdSCL8 as a Negative Regulator Participates in ALA-Induced FLS1 to Promote Flavonol Accumulation in Apples

Apples (Malus domestica) are rich in flavonols, and 5-aminolevulinic acid (ALA) plays an important role in the regulation of plant flavonoid metabolism. To date, the underlying mechanism of ALA promoting flavonol accumulation is unclear. Flavonol synthase (FLS) is a key enzyme in flavonol biosynthesis. In this study, we found that ALA could enhance the promoter activity of MdFLS1 in the ‘Fuji’ apple and improve its expression. With MdFLS1 as bait, we screened a novel transcription factor MdSCL8 by the Yeast One-Hybrid (Y1H) system from the apple cDNA library which we previously constructed. Using luciferase reporter assay and transient GUS activity assay, we verified that MdSCL8 inhibits the activity of MdFLS1 promoter and hinders MdFLS1 expression, thus reducing flavonol accumulation in apple. ALA significantly inhibited MdSCL8 expression. Therefore, ALA promoted the expression of MdFLS1 and the consequent flavonol accumulation probably by down-regulating MdSCL8. We also found that ALA significantly enhanced the gene expression of MdMYB22 and MdHY5, two positive regulators of MdFLS. We further demonstrated that MdMYB22 interacts with MdHY5, but neither of them interacts with MdSCL8. Taken together, our data suggest MdSCL8 as a novel regulator of MdFLS1 and provide important insights into mechanisms of ALA-induced flavonol accumulation in apples.


Introduction
Flavonol, a key class of secondary metabolites, plays important roles in preventing and treating human tumors, preventing cardiovascular diseases, lowering blood sugar and blood lipid, and resisting oxidation, viruses and allergies [1]. For plants, flavonol participates in auxin transportation [2], root growth [3], and the adaptation of plants to the ever-changing environment [4]. The apple (Malus domestica) is cultivated and consumed worldwide. Apples are rich in flavonol, the content of which accounts for 9.3% to 70.6% of total polyphenols [5]. Understanding flavonol biosynthesis regulation is crucial to improv fruit quality, increase nutritional value, and enhance environmental adaptability of apples.
Transcription factors regulating the accumulation of flavonols were continuously identified. MYB transcription factors were the most frequently reported, such as MYB111 and MYB12 in Arabidopsis thaliana [13], CsMYB60 in cucumbers [14], VvMYBF1 in grapes [15], PbMYB12b in pears [16], and MdMYB22 [17] and MdMYB8 [18] in apples. They were reported to positively regulate flavonol biosynthesis. As an exception, FaMYB1 in strawberries negatively regulates gene expression and enzyme activity at a late stage of flavonoid biosynthesis [19]. These results indicated that the synthesis of flavonol was strictly regulated by positive and negative transcription factors. Excepting MYBs, other transcription factors were increasingly proved to participate in flavonol regulation. For example, in apples, except for MdMYB22 [17] and MdMYB8 [18], MdHY5 [20] and MdNAC9 [21] were also reported to enhance the activity of the FLS promoter and promote the flavonol accumulation. These results provide a theoretical basis for the regulatory mechanism of fruit flavonol synthesis.
5-Aminolevulinic acid (ALA), a natural and environmentally friendly multifunctional plant growth regulator [22,23], has been extensively verified for its efficacy in improving plant resistance to abiotic stress [24,25], leaf photosynthetic performance [26], and anthocyanin accumulation [27,28]. Importantly, ALA has been proven to significantly promote the accumulation of flavonol in guard cells of Arabidopsis thaliana [29], 'Fuji' apple fruits [30], and apple calli [31]. Using ALA is undoubtedly a safe and effective method to improve the content of phytoflavonol. ALA promoted the expression of structural genes related to the flavonoid pathway to influence flavonol accumulation in plants [27,[31][32][33]. In addition, ALA can participate in the regulation of gene expression of transcription factors to promote the accumulation of plant anthocyanin [28,30,33]. So far, ALA has been proven to enhance flavonol accumulation in plants; however, the mechanism still remains largely unknown.
MdFLS1 is the key enzyme in apple flavonol biosynthesis [6]. In this study, we found that the activity of proMdFLS1 and expression level of MdFLS1 was enhanced by ALA. A novel transcription factor, MdSCL8 as a negative regulator, and two known positive regulators of MdFLS1, MdMYB22 and MdHY5, were screened and verified to participate in ALA-promoted expression of MdFLS1 and flavonol accumulation. This study aimed to provide a theoretical basis for the application of ALA in improving the nutritional and health care quality of fruits. The results will be helpful to enrich the regulatory theory of flavonol biosynthesis.

ALA Promotes Flavonol Accumulation and Enhances MdFLS1 Expression
The significantly higher level of flavonol content in apple calli treated by 50 mg L −1 ALA ( Figure 1A) confirmed the promotive effect of ALA on flavonol accumulation. Since MdFLS1 is the key biosynthetic enzyme in apple flavonol biosynthesis, we investigated the effect of ALA on MdFLS1 expression to explore how ALA regulates flavonol accumulation. We found that ALA significantly up-regulated MdFLS1 expression (by about 2.5-20 times, Figure 1B). To reveal the regulatory effect of ALA on MdFLS1, the promoter of MdFLS1 upstream of the β-glucuronidase reporter gene (GUS) was constructed. Transient expression assays in apple calli showed that the proMdFLS1::GUS transgenic staining was significantly deepened after ALA treatment, and GUS enzyme activity was significantly increased ( Figure 1C,D). These results indicate that ALA-improved flavonol accumulation is closely related to the higher proMdFLS1 activity and MdFLS1 expression induced by ALA.

MdSCL8 Binds to MdFLS1 Promoter and Inhibits Its Activity
To excavate the specific regulators in ALA-induced flavonol accumulation, we constructed bait yeast [proMdFLS1-AbAi] to screen the cDNA library of ALA-treated apple calli ( Figure S1). The results showed that the proteins screened were mainly involved in the regulation of photosynthesis, carbon and glucose metabolism, response to oxidative/osmotic stress, hormone signal transduction, lignin biosynthesis, fruit maturation and development, and some proteins with unknown functions (Table 1). Among them was transcription factor MdSCL8, the expression of whose homologous gene in strawberries was previously reported to be related to synthesis of flavonoid compounds [34,35]. Yeast one-hybrid assays confirmed that MdSCL8 interacts with proMdFLS1. In the Y1H assay, the positive control (p53-AbAi) grew normally on SD/-Leu or SD/-Leu (AbA) medium, while the negative control (AD+pFLS1-AbAi) did not. The transformation of the PGADT7-MdSCL8 plasmid into pMdFLS1-AbAi yeast competence resulted in normal growth on either SD/-Leu or SD/-Leu (AbA) media, indicating that MdSCL8 activates the expression of the reporter gene in pMdFLS1-AbAi to allow normal growth on AbA-containing plates (Figure 2A). To investigate whether and how MdSCL8 regulates MdFLS1, we determined the promoter activity of MdFLS1 by dual-luciferase assays. The results showed that the promoter activity of MdFLS1 was strongly inhibited by MdSCL8 ( Figure  2B). Transient expression assays in tobacco leaves further showed that when MdSCL8 and proMdFLS1 were co-transferred, the GUS activity was only 15% of that in the single-transferred leaves of proMdFLS1 ( Figure 2C,D). These results indicate that MdSCL8 negatively regulates the activity of MdFLS1 promoter.

MdSCL8 Binds to MdFLS1 Promoter and Inhibits Its Activity
To excavate the specific regulators in ALA-induced flavonol accumulation, we constructed bait yeast [proMdFLS1-AbAi] to screen the cDNA library of ALA-treated apple calli ( Figure S1). The results showed that the proteins screened were mainly involved in the regulation of photosynthesis, carbon and glucose metabolism, response to oxidative/osmotic stress, hormone signal transduction, lignin biosynthesis, fruit maturation and development, and some proteins with unknown functions (Table 1). Among them was transcription factor MdSCL8, the expression of whose homologous gene in strawberries was previously reported to be related to synthesis of flavonoid compounds [34,35]. Yeast one-hybrid assays confirmed that MdSCL8 interacts with proMdFLS1. In the Y1H assay, the positive control (p53-AbAi) grew normally on SD/-Leu or SD/-Leu (AbA) medium, while the negative control (AD+pFLS1-AbAi) did not. The transformation of the PGADT7-MdSCL8 plasmid into pMdFLS1-AbAi yeast competence resulted in normal growth on either SD/-Leu or SD/-Leu (AbA) media, indicating that MdSCL8 activates the expression of the reporter gene in pMdFLS1-AbAi to allow normal growth on AbA-containing plates ( Figure 2A). To investigate whether and how MdSCL8 regulates MdFLS1, we determined the promoter activity of MdFLS1 by dual-luciferase assays. The results showed that the promoter activity of MdFLS1 was strongly inhibited by MdSCL8 ( Figure 2B). Transient expression assays in tobacco leaves further showed that when MdSCL8 and proMdFLS1 were co-transferred, the GUS activity was only 15% of that in the single-transferred leaves of proMdFLS1 ( Figure 2C,D). These results indicate that MdSCL8 negatively regulates the activity of MdFLS1 promoter.

Identification and Molecular Characterization of MdSCL8
The full-length coding sequence (CDS) of MdSCL8 was cloned from a 'Fuji' apple. The length of the protein encoded by MdSCL8 contains 653 amino acids. The protein MdSCL8 is weakly acidic, and MdSCL8 is located on chromosome 7 with a molecular weight of 71.38 kDa and an isoelectric point of 6.42. The extinction coefficients are 0.545 and 0.538, and the stability coefficient is 51.56. In amino acid composition, Serine (Ser) and Alanine (Ala) are the most abundant, accounting for 11.3% and 7.8%, respectively. Total mean hydrophilicity index is −0.506, suggesting it is a hydrophilic protein. In addition, conserved domains prediction reveals a typical conservative GRAS domain at the C-terminal of MdSCL8 ( Figure 3A). In the tobacco leaves which were injected with a vector of 35S:MdSCL8-GFP, we found that the GFP signal of MdSCL8 was located in the nucleus, while the GFP signal of 35S::GFP was detected throughout the cells ( Figure 3B).

Identification and Molecular Characterization of MdSCL8
The full-length coding sequence (CDS) of MdSCL8 was cloned from a 'Fuji' apple. The length of the protein encoded by MdSCL8 contains 653 amino acids. The protein MdSCL8 is weakly acidic, and MdSCL8 is located on chromosome 7 with a molecular weight of 71.38 kDa and an isoelectric point of 6.42. The extinction coefficients are 0.545 and 0.538, and the stability coefficient is 51.56. In amino acid composition, Serine (Ser) and Alanine (Ala) are the most abundant, accounting for 11.3% and 7.8%, respectively. Total mean hydrophilicity index is −0.506, suggesting it is a hydrophilic protein. In addition, conserved domains prediction reveals a typical conservative GRAS domain at the C-terminal of MdSCL8 ( Figure 3A). In the tobacco leaves which were injected with a vector of 35S:MdSCL8-GFP, we found that the GFP signal of MdSCL8 was located in the nucleus, while the GFP signal of 35S::GFP was detected throughout the cells ( Figure 3B).

MdSCL8 Negatively Regulates Flavonol Accumulation and ALA Suppresses This Process
To verify the role of MdSCL8 in the accumulation of flavonol, apple calli were treated with ALA under light for 0 h, 24 h, 48 h and 72 h, respectively. As shown in Figure 4A, the expression of MdSCL8 was up-regulated after illumination, which was significantly inhibited by ALA treatment. We further established an overexpressing MdSCL8 transgenic apple callus and an RNA interference transgenic line, MdSCL8(i). Compared with their corresponding control, the expression of MdSCL8 was increased by 150% in MdSCL8(OE) and decreased by 50% in MdSCL8(i) (Figure 4B,C). The expression of MdFLS1 was reduced by 90% in MdSCL8 (OE) and increased by 130% in MdSCL8 (i), compared with their control. Consequently, the flavonol content in MdSCL8(OE) transgenic calli was significantly reduced, and that in MdSCL8(i) was significantly increased ( Figure  4D-I). These results suggest a negative role of MdSCL8 in apple flavonol accumulation. ALA treatment decreased MdSCL8 expression by 36% in the control group, and by 62% in

MdSCL8 Negatively Regulates Flavonol Accumulation and ALA Suppresses This Process
To verify the role of MdSCL8 in the accumulation of flavonol, apple calli were treated with ALA under light for 0 h, 24 h, 48 h and 72 h, respectively. As shown in Figure 4A, the expression of MdSCL8 was up-regulated after illumination, which was significantly inhibited by ALA treatment. We further established an overexpressing MdSCL8 transgenic apple callus and an RNA interference transgenic line, MdSCL8(i). Compared with their corresponding control, the expression of MdSCL8 was increased by 150% in MdSCL8(OE) and decreased by 50% in MdSCL8(i) (Figure 4B,C). The expression of MdFLS1 was reduced by 90% in MdSCL8 (OE) and increased by 130% in MdSCL8 (i), compared with their control. Consequently, the flavonol content in MdSCL8(OE) transgenic calli was significantly reduced, and that in MdSCL8(i) was significantly increased ( Figure 4D-I). These results suggest a negative role of MdSCL8 in apple flavonol accumulation. ALA treatment decreased MdSCL8 expression by 36% in the control group, and by 62% in the MdSCL8(OE) strains.
Meanwhile, MdFLS1 increased by 30% and 450% in the control group and MdSCL8(OE) strains, respectively ( Figure 4B). This result implies that ALA can strengthen its inhibitory effect on MdSCL8 in MdSCL8(OE) line to release more FLS1. Consistently, ALA increased relatively more flavonol accumulation in MdSCL8(OE) line (54%) than in control (21%) ( Figure 4F). In the MdSCL8 interfering experiment, ALA inhibited MdSCL8 expression and increased MdFLS1 expression in both the control group and MdSCL8(i) line ( Figure 4C), thus significantly enhancing their flavonol accumulation ( Figure 4H,I). These results indicate that MdSCL8 plays a negative regulatory role in flavonol biosynthesis, and may participate in ALA-regulated flavonol accumulation.

Regulatory Effects of MdHY5 and MdMYB22 in ALA-Induced Flavonol Accumulation
MdHY5 [20], MdMYB8 [18], MdMYB22 [17] and MdNAC9 [21] are positive regulators in apple flavonol accumulation. Hence, we verified the relationship between ALA and these regulatory factors. The expression levels of MdHY5 and MdMYB22 were upregulated by ALA ( Figure S2), while that of MdNAC9 and MdMYB8 were not significantly affected (data not shown). This result indicates that ALA may stimulate flavonol accumulation by improving the expression of MdMYB22 and MdHY5. Next, MdMYB22 and

Regulatory Effects of MdHY5 and MdMYB22 in ALA-Induced Flavonol Accumulation
MdHY5 [20], MdMYB8 [18], MdMYB22 [17] and MdNAC9 [21] are positive regulators in apple flavonol accumulation. Hence, we verified the relationship between ALA and these regulatory factors. The expression levels of MdHY5 and MdMYB22 were up-regulated by ALA ( Figure S2), while that of MdNAC9 and MdMYB8 were not significantly affected (data not shown). This result indicates that ALA may stimulate flavonol accumulation by improving the expression of MdMYB22 and MdHY5. Next, MdMYB22 and MdHY5 were RNA-interfered in apple calli to do further analysis. The expression levels of MdMYB22 and MdHY5 were both reduced by 50% in interference lines, and MdFLS1 expression was reduced by 77% and 59%, respectively. ALA increased the expression of MdMYB22 and MdHY5 both by 52% in the control group, and by 70% and 120%, respectively, in the corresponding interfering strains. MdFLS1 was increased by ALA by 33% in the control group and by 150% and 120%in the MdMYB22(i) and MdHY5(i) strains, respectively ( Figure 5A,B). Flavonol content significantly decreased in the two interfering transgenic lines compared to the control ( Figure 5C-E). ALA increased the flavonol content by 72% in the control group, and by 108% and 450% in the MdMYB22(i) and MdHY5(i) lines, respectively ( Figure 5E). These results indicate that ALA displays a compensating effect on flavonol accumulation when the MdMYB22 or MdHY5 pathway was suppressed. In other words, MdMYB22 and MdHY5 may be involved in the accumulation of flavonol induced by ALA, and some other pathways also exist. MdHY5 were RNA-interfered in apple calli to do further analysis. The expression levels of MdMYB22 and MdHY5 were both reduced by 50% in interference lines, and MdFLS1 expression was reduced by 77% and 59%, respectively. ALA increased the expression of MdMYB22 and MdHY5 both by 52% in the control group, and by 70% and 120%, respectively, in the corresponding interfering strains. MdFLS1 was increased by ALA by 33% in the control group and by 150% and 120%in the MdMYB22(i) and MdHY5(i) strains, respectively ( Figure 5A,B). Flavonol content significantly decreased in the two interfering transgenic lines compared to the control ( Figure 5C-E). ALA increased the flavonol content by 72% in the control group, and by 108% and 450% in the MdMYB22(i) and MdHY5(i) lines, respectively ( Figure 5E). These results indicate that ALA displays a compensating effect on flavonol accumulation when the MdMYB22 or MdHY5 pathway was suppressed. In other words, MdMYB22 and MdHY5 may be involved in the accumulation of flavonol induced by ALA, and some other pathways also exist.

MdMYB22 and MdHY5 Interact, and Neither of Them Interacts with MdSCL8
Since MdSCL8, MdMYB22 and MdHY5 all participate in ALA-induced flavonol accumulation, it is interesting to investigate whether they interact with each other. In the Yeast two-hybrid (Y2H) assay, the negative control will not grow on SD/-Leu/-Trp/-His/-Ade (X-α-gal) medium, while the positive control and the yeast transformed with the interacting proteins grew normally and turned blue. When the two proteins interacted, the AD and BD domains to which they were connected were close in space, thus activating the expression of the reporter gene. The Y2H results showed that BD-MdHY5 interacted with AD-MdMYB22, and BD-MdMSCL8 interacted with AD-MdMYB22, but MdHY5 did not interact with MdMSCL8 ( Figure 6A). To confirm this result, we performed bimolecular fluorescence complementarity assay (BIFC). Results showed that MdHY5-YNE and

MdMYB22 and MdHY5 Interact, and Neither of Them Interacts with MdSCL8
Since MdSCL8, MdMYB22 and MdHY5 all participate in ALA-induced flavonol accumulation, it is interesting to investigate whether they interact with each other. In the Yeast two-hybrid (Y2H) assay, the negative control will not grow on SD/-Leu/-Trp/-His/-Ade (X-α-gal) medium, while the positive control and the yeast transformed with the interacting proteins grew normally and turned blue. When the two proteins interacted, the AD and BD domains to which they were connected were close in space, thus activating the expression of the reporter gene. The Y2H results showed that BD-MdHY5 interacted with AD-MdMYB22, and BD-MdMSCL8 interacted with AD-MdMYB22, but MdHY5 did not interact with MdMSCL8 ( Figure 6A). To confirm this result, we performed bimolecular fluorescence complementarity assay (BIFC). Results showed that MdHY5-YNE and MdMYB22-YCE interacted with each other, consistent with the Y2H result ( Figure 6B). However, in contrast to the Y2H results, there was no fluorescence between MdSCL8-YNE and MdMYB22-YCE. To further validate the interaction, we performed a double luciferase in vivo imaging experiment in tobacco leaves. Results showed that the fluorescence occurred only in the regions of co-transNLUC-MdHY5 and cLUC-MdMYB22, suggesting that only MdHY5 interacts with MdMYB22 ( Figure 6C). These results indicate that there is an interaction between MdMYB22 and MdHY5, without the involvement of MdSCL8.
MdMYB22-YCE interacted with each other, consistent with the Y2H result ( Figure 6B). However, in contrast to the Y2H results, there was no fluorescence between MdSCL8-YNE and MdMYB22-YCE. To further validate the interaction, we performed a double luciferase in vivo imaging experiment in tobacco leaves. Results showed that the fluorescence occurred only in the regions of co-transNLUC-MdHY5 and cLUC-MdMYB22, suggesting that only MdHY5 interacts with MdMYB22 ( Figure 6C). These results indicate that there is an interaction between MdMYB22 and MdHY5, without the involvement of MdSCL8. Figure 6. Analyses of the interactions between MdHY5, MdMYB22, and MdSCL8. (A) Yeast twohybrid analysis. SD/-TL is SD/-Leu/-Trp solid medium, SD/-TLHA (X-α-gal) is SD/-Leu/-Trp/-His/-Ade/X-α-gal solid medium. (B) BiFC assay analysis of the interactions in tobacco leaves. YFP fluorescence signal was observed by confocal microscope at 510 nm. Bright, YFP, and Merged correspond to bright field, yellow fluorescence, and merged field, respectively. (C) Luciferase assay analysis in vivo imaging system. Three biological replicates were performed for each experiment.

ALA Positively Regulates MdFLS1
Flavonol is an important class of polyphenols which plays a crucial role in the oxidation resistance and prevention of oxidative stress-related diseases, such as cardiovascular disease [36] and cancer health protection [37]. Apples are one of the most important fruits that consumers often intake as one source of flavonol [38]. Therefore, increasing the content of flavonol is of great significance for improving the nutrition and health benefits of apple fruits. It was reported that the flavonol content was affected by many factors, such as light [39], temperature [40], plant growth regulator [41], and cultivation factors [42]. ALA, a natural and non-toxic plant growth regulator [23], has attracted the attention of many scholars due to its important roles in improving plant resistance [24,25] and fruit quality [27]. ALA promoting flavonol accumulation was first proved in guard cells of apple [43] and Arabidopsis [29], which scavenged reactive oxygen species and induced stomatal opening. In apple fruits [30] and calli [31], ALA was found to promote the content

ALA Positively Regulates MdFLS1
Flavonol is an important class of polyphenols which plays a crucial role in the oxidation resistance and prevention of oxidative stress-related diseases, such as cardiovascular disease [36] and cancer health protection [37]. Apples are one of the most important fruits that consumers often intake as one source of flavonol [38]. Therefore, increasing the content of flavonol is of great significance for improving the nutrition and health benefits of apple fruits. It was reported that the flavonol content was affected by many factors, such as light [39], temperature [40], plant growth regulator [41], and cultivation factors [42]. ALA, a natural and non-toxic plant growth regulator [23], has attracted the attention of many scholars due to its important roles in improving plant resistance [24,25] and fruit quality [27]. ALA promoting flavonol accumulation was first proved in guard cells of apple [43] and Arabidopsis [29], which scavenged reactive oxygen species and induced stomatal opening. In apple fruits [30] and calli [31], ALA was found to promote the content of quercetin and kaempferol. In this study, we also found that ALA promoted the accumulation of flavonol in the apple calli using both DPBA and UPLC methods ( Figure 1A). Therefore, ALA-promoted phytoflavonol accumulation is universal, which provides a safer and more effective way to improve phytoflavonol accumulation.
FLS is a key enzyme in the branch of flavonol biosynthesis. It plays a key role in the biosynthesis of flavonol [6]. FLS genes have been cloned and their functions have been verified in a variety of plants, such as Arabidopsis thaliana [44], tobacco [45], tartary buckwheat [46], and corn [47]. The expression of FLS is affected by a variety of external environments, and it is positively correlated with the content of flavonol. There have been many reports that plant growth regulators participated in the regulation of flavonol biosynthesis by affecting the expression of FLS. In Ginkgo biloba, GbFLS1 expression was induced by abscisic acid (ABA), ethylene (ETH), and salicylic acid (SA), with a concomitant increase in flavonol content [48]. SA induced up-regulation of VcFLS expression in Vaccinium myrtillus [11]. ABA induced up-regulation of MdFLS1 expression in plantlets of 'Gala' apple in vitro [12]. Treatments of 'Fuji' apples with 24-epibrassinolide and ALA promoted the accumulation of quercetin and kaempferol [31]. ALA has been shown to promote flavonol accumulation and up-regulate MdFLS [30]. In the present study, we further found that ALA significantly increased the activity of MdFLS1 promoter, which might be the direct reason for ALA increasing the expression abundance of MdFLS1 ( Figure 1C,D). These results indicate that ALA positively regulated MdFLS1 expression, and ALA-improved flavonol accumulation is probably achieved by regulating MdFLS1 expression.

ALA-Inhibited MdSCL8 Negatively Regulates MdFLS1 and Flavonol Accumulation in Apples
Flavonol synthesis is regulated by various transcription factors, such as HY5 [49] and MYB22 [17]. In this study, the promoter sequence of MdFLS1 was used as a bait to screen the functional proteins that may interact with the promoter ( Figure S1, Table 1). Interestingly, MdSCL8, a new transcript factor, was screened first, which has never been mentioned in the regulation of MdFLS1 expression and accumulation of flavonoids before.
SCL8 belongs to the GRAS family, which plays an important role in regulating transcription and plant signal transduction pathway [50,51]. The apple GRAS gene family contains 127 genes, which are divided into eight subtribes according to phylogenetic tree: DELLA, SCL3, SCR, LS, LISCL, PAT1, SHR, and HAM [52]. MdSCL8 belongs to the PAT1 subfamily. The PAT1 subfamily plays a role in light-sensitive signal transduction and response to abiotic stress [53]. The transcription factors in the PAT1 subfamily reported in Arabidopsis are PAT1, SCL21, and SCL13. PAT1 and SCL21 are positive regulatory factors for the signal transduction of phytochrome A pathway [53,54]. In this study, MdSCL8 was proved to interact with MdFLS1, and overexpression of MdSCL8 significantly inhibited the activity of FLS1 promoter (Figure 2). Genetic data further suggest that MdSCL8 is a negative regulator of MdFLS1 expression and flavonol accumulation in apples, while ALA negatively regulates MdSCL8 expression (Figure 4). Interestingly, when MdSCL8 was overexpressed, ALA strengthened its inhibitory effect on MdSCL8 (Figure 4). This is the first report that a GRAS transcription factor is involved in the regulation of fruit flavonol biosynthesis.

MdHY5 and MdMYB22 Are Involved in ALA-Induced Flavonol Accumulation in Apples
Many transcription factors have been reported to be involved in the regulation of flavonol accumulation, among which the MYB family is the most important. VvMYBF1 in grapes [15], MdMYB22 [17] and MdMYB8 [18] in apples, and PpMYB17 [55] and Pb-MYB12b [16] in pears, are all suggested to bind and activate FLS promoter, leading to the accumulation of flavonols. The bZIP family, such as AtHY5 in Arabidopsis [49], VvbZIPC22 in grapes [56], CsHY5 in tea [57], and MdHY5 in apples [20], have also been suggested to indirectly regulate FLS by regulating other transcription factors, and promote the accumulation of flavonols. In addition, MdNAC9 in apple has also been shown to promote flavonol accumulation [21]. Here, we found that the expression levels of MdMYB22 and MdHY5 were significantly increased in ALA-treated calli ( Figure S2). MdMYB22 and MdHY5 still respond to ALA treatment after interference ( Figure 5). ALA increased the expression levels of MdMYB22 and MdHY5 in the MdMYB22(i) and MdHY5(i) strains much more obviously than in the control group, which in turn increased the expression of MdFLS1 and improved the content of flavonol. This result is similar to the effect of ALA on MdSCL8. It seems that multiple pathways are involved in ALA-induced flavonol accumulation, and other pathways will compensate when one pathway is suppressed. How these pathways work synergistically is worthy to be further studied. HY5 has been considered as the center of a transcriptional network hub, which promotes flavonol biosynthesis through combinatorial action of MYB and bHLH transcription factors [58]. In this study, we proved the interaction of MdMYB22 with MdHY5, but neither of them interacted with the negative regulator MdSCL8 (Figure 6). Our data suggest three pathways for ALA-induced flavonol accumulation, providing new insights to the regulation of flavonol biosynthesis, but also indicate that some other pathways are involved in this process. We will continue to explore the molecular mechanism of ALA-induced accumulation of flavonol in apples.

Experimental Materials and Treatments
The calli of apples were cultured in the Fruit Physiology and Ecology Laboratory of College of Horticulture, Nanjing Agricultural University according to the method described by An et al. [59]. Apple calli were treated by ALA according to previous methods in our lab [30].

DNA Extraction, RNA Extraction, and qRT-PCR
Total DNA/RNA of apple calli was isolated using the plant total DNA/RNA extraction kit (Tiangen, Beijing, China). First-strand cDNA was synthesized using a RevertAid TM First Strand cDNA Synthesis Kit (Transgen, Beijing, China). The primers (Table S1) were designed with Primer Premier software (Premier, Canada) and synthesized by Generay Biotechnology (Shanghai, China). MdUBQ was used as the internal control. Taking cDNA as template, the qRT-PCR reactions were performed on a Quantitative real-time PCR instrument (ABI, Los Angeles, CA, USA) according to the instructions of Chamq SYBR qPCR master mix (Vazyme, Nanjing, China), and 2 −∆∆CT method was used for calculation [60].

Construction and Infection of MdFLS1 Promoter Vector
MdFLS1(MDP0000260404) of 'Fuji' apple was obtained in Genome Database for Rosaceae (GDR). Taking 'Fuji' apple DNA as template, the promoter of MdFLS1 was cloned by 2 × Phanta Max Master Mix (Vazyme, Nanjing, China). The PBI121 vector was linearized with Flycut ® mix (Transgen, Beijing, China) and the 35S promoter was excised for recombinant transformation.
Agrobacterium tumefaciens GV3101 with recombinant plasmid was propagated to 0.5 OD 600 nm , and the cells were re-suspended by using suspension solution (100 mM MES, 100 mM MgCl 2 , 150 mM AS) to 0.5 OD 600 nm . Apple calli were infected by the above suspension solution for 15-30 min and then the calli were transferred to solid MS medium containing 50 mg L −1 ALA in darkness at 22 • C for 48 h. After another 48 h of light culture, samples were collected for qualitative and quantitative GUS experiment.

Bioinformatics Analysis
The amino acid sequence of MdSCL8 was downloaded from the NCBI database; meanwhile, the chromosomal location, CDS sequence, and the number of exons and introns were found in NCBI. The physicochemical properties of protein, such as the number of amino acids, isoelectric point, molecular weight, and other information on protein were analyzed by ExPASY online. The hydrophobicity of protein was analyzed using the online tool Protscale. The NCBI conserved domains tool was used for protein conservative functional domain prediction. Amino acid sequences were aligned using DNAMAN software (Lynnon Biosoft, San Ramon, CA, USA).

Yeast One-Hybrid Assay
The expression vector of proMdFLS1-AbAi was constructed using proMdFLS1 as the bait sequence. The CDS region of MdSCL8 was cloned into pGADT7 vector to form PGADT7-MdSCL8. The proMdFLS1-AbAi plasmid linearized by BstBI restriction endonuclease (NEB, Beijing, China) was transformed into yeast competence to obtain bait yeast [proMdFLS1-AbAi]. The library screening experiment was conducted according to the yeast one-hybrid instruction of Shanghai OE Biotech Co., Ltd. (Shanghai, China). The screened genes were subjected to yeast colony PCR, sequencing analysis, and point-to-point verification.

MdSCL8, MdHY5, and MdMYB22 Cloning and Transformation into Apple Calli
For the construction of the overexpression vector, the CDS region of MdSCL8 was amplified and connected to the PBI121 vector. For the construction of the interference vector, the conservative region was searched on the SMART online software according to the amino acid sequence information of MdSCL8, MdMYB22, and MdHY5, and 200-400 bp sequence fragments were selected. After gene cloning and recovery, the fragments were recombined onto PHG2 (pHELLSGATE2) vector by using the Gateway BP™ LR Clonase™ Enzyme Mix (Thermo Fisher, Waltham, MA, USA).
Infection was carried out according to the method of Zheng [28]. The recombinant plasmids were inserted into A. tumefaciens GV3101 cells and then transformed in apple calli. The calli were then co-cultured on solid MS medium for 48 h at 22 • C in darkness. The calli were treated with 50 mg L −1 ALA or deionized water (Control) for 3 h in darkness and then cultured in solid MS medium under light of 100 µmol m −2 s −1 photon flux density at 22 • C for 48 h.

Subcellular Localization
The full-length MdSCL8 CDS was amplified (with the terminator codon removed) and inserted into pCambia1300 vector for recombinant plasmid. The empty pCambia1300 was used as control. The A. tumefaciens GV3101 containing the plasmid was injected into the 5-week-old tobacco (Nicotiana benthamiana) leaves. The infiltrated tobacco was placed for 2-3 days in dark conditions. The fluorescence signal of tobacco was detected using ultra-high-resolution confocal microscope (Zeiss, Oberkochen, Germany) with an excitation wave length 488 nm.

Luciferase Reporter Assay
The CDS of MdSCL8 was ligated to pCambia1300 effector, and the proMdFLS1 was inserted into pGreenII 0800-LUCreporter. Tobacco leaves were used as the materials for transient transfection. The 'reporter' was set as the control, and 'effector' and 'reporter' were uniformly mixed in a proportion and jointly converted to tobacco leaves. Fifty milligram leaves were ground and operated according to the instruction of the double luciferase reporter gene detection kit (Transgen, Beijing, China). LUC and REN activities were then measured on a nite200Pro microplate reader (Tecan, Männedorf, Switzerland). The luciferase activity was expressed by the ratio of LUC/REN. The control group LUC/REN value was set to 1.

Transient GUS Activity Assays
The ORF of MdSCL8 was cloned into the pCambia1300 effector, and the promoter of MdFLS1 was inserted into the PBI121 reporter. The 'effector' and 'reporter' were mixed and injected into 5-week-old tobacco leaves. After reaction at 22 • C for 48 h, the GUS histochemical assays were measured as previously [14]. X-β-D-glucuronide (X-Gluc) was adsorbed 50 µL into 20 mg mL −1 DMF and added to 450 µL of buffer (50 mM phosphate buffer pH 7.0, 10 mM EDTA pH = 8.0, 10% Triton X-100, 50 mM potassium ferricyanide, 50 mM potassium ferrocyanide). The treated tobacco leaves were beaten into disk-shaped leaves, and then 500 µL GUS staining solution was added into the disk-shaped leaves at 37 • C for 12 h. The samples were rinsed with 80% alcohol, and the staining of the leaves was observed. GUS activity was measured using a microplate reader according to the instructions of the GUS enzyme activity detection kit (FCNCS, Nanjing, China).

Analysis of DPBA Staining in Transgenic Calli
The flavonol of apple calli was detected by diphenylboric acid 2-amino-ethyl ester (DPBA) staining. DPBA can bind to flavonols such as quercetin and cause fluorescence under laser light [61]. Twenty-five milligram DPBA powder was weighed and prepared into a 0.25% (w/v) solution, which was then diluted to 0.005% (v/v) by adding TritonX-100. After being normally illuminated for 48 h, the transgenic apple calli was treated with DPBA in darkness for 30-60 min. Afterwards, the tissue was observed under GFP light of a stereo fluorescence microscope (Leica, Wetzlar, Germany). The fluorescence intensity of DPBA was analyzed by ImageJ software.

Determination of Luciferase Transient Expression in Tobacco Leaves
MdHY5, MdSCL8, and MdMYB22 were constructed into N terminal of pCambia1300-35S-nLUC vector and the C terminal of pCambia1300-35S-cLUC vector, respectively. The recombinant plasmids were transferred into GV3101, infected tobacco leaves, which were soaked for 48 h. One mM firefly substrate D-luciferin (Yeasen, Shanghai, China) was sprayed on the backs of the leaves, and stood for 7 min under dark conditions. The back of the leaf was placed upward on the plant in vivo imaging system (Princeton Instruments, Trenton, NJ, USA) to detect the luminescence.

Data Analysis
All the data have at least three biological repetitions, and SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The analysis of variance (ANOVA) and Duncan multiple range test were used to compare the significant differences at p ≤ 0.05 level.

Conclusions
In summary, ALA improves flavonol accumulation in apples by regulating MdFLS1. In this process, a novel negative regulator, MdSCL8, which belongs to GRAS family, and two positive regulators, MdMYB22 and MdHY5, were confirmed to play important roles. Based on these results, we proposed a working model for how ALA regulates flavonol biosynthesis (Figure 7). It is the first report to explore the mechanism underlying ALAinduced flavonol accumulation in apples, providing new insights into the regulation of plant flavonol biosynthesis and beneficitting improvement of fruit nutrition for the health of human beings.