An apple (Malus domestica) AP2/ERF transcription factor modulates carotenoid accumulation

Color is an important trait for horticultural crops. Carotenoids are one of the main pigments for coloration and have important implications for photosynthesis in plants and benefits for human health. Here, we identified an APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factor named MdAP2-34 in apple (Malus domestica Borkh.). MdAP2-34 expression exhibited a close correlation with carotenoid content in ‘Benin Shogun’ and ‘Yanfu 3’ fruit flesh. MdAP2-34 promotes carotenoid accumulation in MdAP2-34-OVX transgenic apple calli and fruits by participating in the carotenoid biosynthesis pathway. The major carotenoid contents of phytoene and β-carotene were much higher in overexpressing MdAP2-34 transgenic calli and fruit skin, yet the predominant compound of lutein showed no obvious difference, indicating that MdAP2-34 regulates phytoene and β-carotene accumulation but not lutein. MdPSY2-1 (phytoene synthase 2) is a major gene in the carotenoid biosynthesis pathway in apple fruit, and the MdPSY2-1 gene is directly bound and transcriptionally activated by MdAP2-34. In addition, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. Our findings will advance and extend our understanding of the complex molecular mechanisms of carotenoid biosynthesis in apple, and this research is valuable for accelerating the apple breeding process.


Introduction
Color is an important trait for horticultural crops, such as fruits, vegetables, and flowers 1 . Carotenoids are one of the main pigments for coloration, showing yellow, orange, red, or others in horticultural reproductive organs 2 . Carotenoids are important components for photosynthesis and are involved in light harvesting and photoprotection, plant growth, and development 3 . Carotenoids are valuable molecules that are beneficial for human health and animal survival, and carotenoids are also precursors of provitamin A and are antioxidants for reducing various chronic diseases 4,5 .
The biosynthetic pathway of carotenoids is well known in a variety of plant species, and it is modulated by many factors, such as limiting and metabolic enzyme steps, the availability of storage organs and structures, or transcription factors (TFs) 2,6,7 . The structural genes encoding metabolic enzymes in the carotenoid pathway, including phytoene synthase (PSY), phytoene desaturase (PDS), ζcarotene isomerase (ZISO), zeta-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), lycopene β-cyclase (LCY-β), lycopene ε-cyclase (LCY-ε), beta-carotene hydroxylase (CHY-β), zeaxanthin epoxidase (ZEP), carotenoid cleavage dioxygenases (CCD), and 9-cisepoxycarotenoid dioxygenase (NCED), have previously been characterized 2 . Previous studies have shown that PSYs play a significant role in carotenoid accumulation 8,9 . Transgenic tomato fruits and potato tubers overexpressing PSY show increased levels of total carotenoids 10,11 . Multiple PSY genes modulate carotenoid accumulation in many species, such as maize, rice, loquat, and apple [12][13][14][15] . CaPSY2 can increase carotenoid content and compensate for the absence of PSY1 in pepper (Capsicum spp.) fruit 16 . OR/OR-like proteins influence carotenoid content by affecting PSY protein levels through posttranscriptional regulation in Arabidopsis and sweet potato 17,18 . For the other limiting genes, the downregulation of LCY-ε increases the β-carotene content in potato 19 . The expression of the NCED gene results in a low β-carotenoid content in the white apricot cultivar 20 . Inhibition of BoaCRTISO expression in Chinese kale affected carotenoid pathways and led to decreased carotenoid concentrations 21 . The Orange gene CmOr regulates β-carotene accumulation in melon fruit by repressing β-carotene turnover and posttranslationally enhancing carotenogenesis in chromoplasts 22 .
A number of TFs have been identified to be involved in the carotenoid pathway. AGAMOUS-like 1 and FRUIT-FULL, which belong to MADS-box TFs, regulate carotenoid content in tomato 23 , and CsMADS6 modulates carotenoid metabolism by directly regulating LCYb1 in sweet orange 24 . The NAC TFs SlNAC1, SlNAC4, and CpNAC1 regulate carotenoid accumulation during tomato and papaya fruit ripening [25][26][27] . MYB TFs, RCP1 in Mimulus lewisii flowers 28 , CrMYB68 in citrus 29 , and AdMYB7 in kiwifruit 30 , are all involved in carotenoid regulation. The R2R3-MYB TF WP1 together with MtTT8 and MtWD40-1 promotes floral carotenoid accumulation by directly activating the expression of MtLYCe and MtLYCb in Medicago petals 31 . CpbHLH1/2 regulates carotenoid biosynthesis and is related to carotenoid content during fruit ripening of papaya 32 . Sl_H2A. Z plays an essential role in the modulation of carotenoid content in tomato 33 .
The APETALA2 (AP2)/ETHYLENE RESPONSE FAC-TOR (ERF) superfamily contains four subfamilies according to the number of AP2/ERF domains and sequences, including AP2, CBF/DREB, ERF, and RAV 34 . AP2 subfamily proteins contain single or two AP2/ERF domains that are involved in the regulation of development 35 . AP2/ERF proteins can bind to DNA cis-acting elements, including DRE (CCGAC), GCC box (AGCCGCC), (A/G)CC(G/C)AC, and AA(T)TTCAAA 36 . The TFs of the AP2/ERF superfamily play important roles in growth, development, and stress response processes in higher plants [34][35][36][37] . PpERF3 activates ABA biosynthesis by positively regulating PpNCED2/3 transcription in peach 38 . AP2/ERF TFs have been reported to modulate carotenoid accumulation. SIERF6 negatively regulates carotenoid accumulation in tomato 39 . In Arabidopsis, ERFs and RAP2.2 promote carotenoid biosynthesis by binding to the PSY promoter 40 . However, whether and how AP2/ERF TFs modulate carotenoid metabolism in apple fruit are currently unknown.
Apple (Malus domestica Borkh.), which has high nutrient value, is one of the most economical fruit crops worldwide. Fruit flavor and color are major apple traits that determine consumer preferences. Because of differences in carotenoid accumulation, the flesh color varies from white to yellow in different apple genotypes 41 . Previous studies indicated that MdPSY2 played a dominant role in carotenoid accumulation in apple fruits and was activated by AP2/ERF TFs 15,41 . There was a strong positive correlation between the expression of the TF AP2D26 and PSY2 in apple fruit 15 . In this study, we found that the total carotenoid contents of 'Benin Shogun' fruits were significantly higher than those of 'Yanfu 3' fruits during the developmental period at 150 and 170 days after full bloom (DAFB). We characterized an AP2 TF named MdAP2-34 and found that it induced increases in carotenoid accumulation. MdAP2-34 was able to promote carotenoid levels by enhancing MdPSY2-1 promoter activity. Moreover, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. Our findings will advance and extend our understanding of the complex molecular mechanisms of carotenoid biosynthesis in apple.

Plant materials
Apple fruits of 'Benin Shogun' and 'Yanfu 3' were collected at 120, 150, and 170 days after full bloom (DAFB). 'Benin Shogun' and 'Yanfu 3' are bud mutations of 'Red Fuji'. Every treatment included six fruits. Leaves of 'Benin Shogun' were collected in late spring and used for DNA extraction. Apple fruits of Granny Smith used for the transient transformation were collected at 150 DAFB. The fruits and leaves were stored at -78°C until use.

Subcellular localization
The subcellular localization of MdAP2-34 was conducted as previously described 42 . The open reading frame (ORF) sequence of MdAP2-34 without the stop codon was cloned and inserted into the pRI101:GFP expression vector. The fusion construct 35S:MdAP2-34:GFP was transformed into N. benthamiana leaf epidermal cells. Fluorescence was detected by confocal microscopy after 2 −3 days of transfection. DAPI (4′,6-Diamidino-2-phenylin-dole; Invitrogen) was used to stain the nucleus.

Total carotenoids extraction and measurement
The extraction and determination of total carotenoids were performed using a method as described previously 43 . Each sample was extracted with acetone-hexane (1:1, vol/ vol). The total carotenoid content of the extracts was determined using a UV-2550 UV−vis spectrophotometer.
RNA extraction, qRT-PCR assay, and extraction of genomic DNA Total RNA was isolated from fruit flesh of 'Benin Shogun' and 'Yanfu 3', fruit skin of transiently transformed 'Granny Smith' and transgenic apple calli (TAC), and qRT-PCR (reverse transcription-quantitative PCR) was carried out as previously described 44 . EF1α was used to serve as the reference gene. The qRT-PCR assays were conducted with three biological replicates, and each biological replicate was conducted with three technical replicates. Genomic DNA was isolated form 'Granny Smith' leaves. All primers are used in Supplemental Table S1.

Construction of plasmids and genetic transformation
The overexpression and interference plasmids were constructed as previously described 45 . Each ORF sequence of MdAP2-34 and MdPSY2-1 was cloned and inserted into pRI101-FLAG (overexpression vector). A 728 bp fragment (located between +5 and +732 bp of the ORF) of MdAP2-34 was cloned and inserted into pFGC5941 (RNAi vector). Every recombinant plasmid of 35S:MdAP2-34, RNAi:MdAP2-34, and 35 S:MdPSY2-1 was then transformed into Granny Smith apple fruit skin and Orin apple calli 45 . All primers are used in Supplemental Table S1.

Proteins extraction and western blotting
Protein extraction and western blotting assays were performed as previously described 42,45 . Anti-ACTIN antibody and anti-FLAG antibody were used, which were obtained from Beyotime Biotech Company.
Yeast one-hybrid assay Y1H (yeast one-hybrid) assays were conducted as previously described 45 Table S1.

EMSAs
The EMSA was conducted as previously described 45 .
The ORF sequence of MdAP2-34 was cloned into the vector pGEX-4T-1 (with GST-tag) and then transformed into BL21 (DE3) Escherichia coli cells for MdAP2-34 protein expression. The 5ʹ biotin end-labeled probes were synthesized by Sangon Biotech. The EMSA was performed using the LightShift™ Chemiluminescent EMSA Kit (Thermo Fisher Scientific, USA). The primers were used, and the biotin-labeled sequences of the MdPSY2-1 promoter are shown in Supplemental Table S1.

ChIP-PCR assay
The ChIP-PCR assay was performed following Jia et al. 45 . The ORF of MdAP2-34 was inserted into the pRI101-FLAG vector (with 3×FLAG), and the fusion MdAP2-34-FLAG was generated. Recombinant pRI101-FLAG-MdAP2-34 was transformed into apple calli, and the MdAP2-34-FLAG protein was extracted from transgenic calli. ChIP-PCR analysis was conducted using the EpiQuik™ Plant ChIP Kit (Epigentek, USA) and anti-DDDDK tag (Binds to FLAG® tag sequence) antibody (ab125243, Abcam, UK). Three promoter regions of MdPSY2-1 were used to assess their enrichment. Every ChIP assay was conducted three replicates. The primers used are in Supplemental Table S1.

LUC assay
The LUC analysis was conducted as previously described 45 . The ORF of MdAP2-34 was inserted into pGreenII 62-SK (the effector vector) under the control of the CaMV35S promoter. The promoter fragment of MdPSY2-1 was inserted into pGreenII 0800-LUC (the reporter vector). The vectors of the effector and reporter were cotransformed into 4-week-old N. benthamiana leaves.
The luciferase signals of Firefly and Renilla were observed and measured using an Infinite M200 (Tecan, Switzerland). Every LUC analysis was conducted six replicates. The primers used are in Supplemental Table S1.

Statistical analysis
In the experimental design used, the data were tested and analyzed for three biological replicates, and each biological replicate was analyzed three technical repetitions. Significant differences were determined using GraphPad Prism 5 software 42,45 (* represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001).

Results
Characterization of MdAP2-34 associated with carotenoid content in 'Benin Shogun' and 'Yanfu 3' apple fruits A previous study indicated that the AP2 subfamily consists of 51 genes in apple, which contain single or double AP2 domain 34 . Based on the annotation of the website (http://bioinformatics.cau.edu.cn/AppleMDO/) 46 , 12 AP2 TFs were identified that could be expressed in apple fruits (Supplemental Table S2). In the 'Benin Shogun' and 'Yanfu 3' fruit flesh (Fig. 1a), the total carotenoid content increased during the developmental period at 120, 150, and 170 DAFB (Fig. 1b). The total carotenoid content of 'Benin Shogun' fruits was significantly higher than that of 'Yanfu 3' fruits at 150 and 170 DAFB (Fig. 1b). Pearson's correlation (r) between each AP2 TF transcript levels and total carotenoid content in apple flesh of 'Benin Shogun' and 'Yanfu 3' indicated that the expression level of MdAP2-34 was significantly positively associated with carotenoid contents (Supplemental Table S2). However, the MdAP2-24 expression level was inconsistent with the total carotenoid content in 'Yanfu 3' fruit flesh, and the MdAP2-44 expression level was negatively correlated with the total carotenoid content, so the two genes were filtered out. The transcript level of MdAP2-34 increased in both 'Benin Shogun' and 'Yanfu 3' fruit flesh during the developmental period at 120, 150, and 170 DAFB (Fig. 1c). The expression level of MdAP2-34 in 'Benin Shogun' fruits was significantly higher than that in 'Yanfu 3' fruits at 150 and 170 DAFB (Fig. 1c). Furthermore, the expression of MdAP2-34 showed a close correlation (r = 0.9844, P < 0.001) with carotenoid content in 'Benin Shogun' and 'Yanfu 3' fruit flesh (Fig. 1d) S1a). Phylogenetic analysis showed that MdAP2-34 and AtAIL5 have high homology (Supplemental Fig. S1b), and previous results indicated that AtAIL5 could induce a larger floral organ phenotype 47 . To investigate the subcellular localization of the MdAP2-34 protein, the ORF of MdAP2-34 was fused to EGFP (MdAP2-34:GFP) and transiently expressed in Nicotiana benthamiana leaves. The vector pRI101-GFP (35S:GFP) was used as a control. The results indicated that fusion protein signals were identified in the nucleus (Fig. 1e).
A transient expression assay based on Agrobacterium tumefaciens infiltration was used to overexpress or silence MdAP2-34 in the fruit skin of Granny Smith (Fig. 2e). After 14 days of the transient transformation of Granny Smith, the MdAP2-34-OVX Granny Smith fruit skin was harvested (Fig. 2e). The expression level of MdAP2-34 was remarkably higher in MdAP2-34-OVX fruit skin than in P101F fruit skin but much lower in MdAP2-34-RNAi fruit skin than in P5941 fruit skin (Fig. 2f). The total carotenoid content was significantly higher in MdAP2-34-OVX fruit skin (24.33 µg/g FW) than in P101F fruit skin (15.15 µg/g FW), with an increase of 60.59% (Fig. 2g; Table 1). The total carotenoid content was lower in MdAP2-34-RNAi fruit skin (12.33 µg/g FW) than in P5941 fruit skin (17.25 µg/g FW), with a reduction of 28.52% (Fig. 2g; Table 1). For the carotenoid compounds, in total, 19 carotenoids were isolated from transgenic Granny Smith fruit skin (Table 1). Among these carotenoids, β-carotene, phytoene, and lutein were the major carotenoids present. The ratios of β-carotene, phytoene, and lutein to total carotenoids in P101F 'Granny Smith' fruit skin were 16.63, 13.86, and 58.42%, respectively ( Table 1). The contents of four specific carotenoids (β-carotene, β-cryptoxanthin, phytoene, and violaxanthin) were remarkably higher in MdAP2-34-OVX fruit skin than in P101F fruit skin and were remarkably lower in MdAP2-34-RNAi calli than in P5941 calli (Table 1). These findings suggest that MdAP2-34 plays an important role in the carotenoid accumulation and positively regulates carotenoid accumulation in the fruit skin of apple trees.

Discussion
Carotenoids are important pigments for coloration and nutrition in horticultural plants and play important roles in plant growth and development. Apples with healthful metabolites and high nutritional value are consumed worldwide. Recently, carotenoid content and components have become important breeding objectives in apple 30,41 . In this study, we characterized the AP2-type TF MdAP2-34 in Benin Shogun apple, in which ripened fruits appeared orange. Through experiments, we developed a model that explains the molecular basis for MdAP2-34. We found that MdAP2-34 enhances carotenoid accumulation. MdAP2-34 was able to promote carotenoid levels by activating MdPSY2-1 promoter activity (Fig. 6).
Apples are consumed in large quantities worldwide because of their healthy metabolites. Carotenoid pigments contribute to the skin coloration of apple fruit; however, their concentrations are very low in the apple flesh. A previous study suggested that they have low concentrations of carotenoids in the fruit of commercial apple cultivars (<2.5 µg/g fresh weight) but high fruit carotenoid concentrations in the rootstock cultivar 'Aotea' 41 . The study indicated that the concentration of total carotenoids in the fruit of the commercial cultivar 'Benin Shogun' (with flesh color of orange, Fig. 1a) was up to 9.4 µg/g fresh weight at 170 DAFB, which was much higher than that of 'Yanfu 3' and the carotenoid concentrations reported previously. The results suggest that the commercial cultivar 'Benin Shogun' may be better for research on the carotenoid metabolic mechanism. Our future study is intended to focus on the mechanisms of carotenoid biosynthesis in different apple cultivars, including the full transcriptome, genetics, and epigenetics. The cause of different genetic variations in carotenoid accumulation in Malus will help us to understand the mechanisms of carotenoid accumulation in apple and will be beneficial for the breeding of new apple cultivars with high carotenoid concentrations.
Many TFs play important roles in carotenoid biosynthesis. It is well known that AP2/ERFs participate in many plant development processes and play important roles in regulating fruit quality and pigment biosynthesis 37,50 . However, AP2/ERF TFs have rarely been reported to regulate carotenoid biosynthesis in apple fruits. In this study, based on the previous analysis of the AP2/ERF family 34 and the annotation of websites (http:// bioinformatics.cau.edu.cn/AppleMDO/) 46 , we identified 12 AP2 TFs from the AP2 subfamily expressed in apple fruits. By correlation analysis, we characterized MdAP2-34 as a candidate AP2 TF associated with carotenoid accumulation (Supplemental Table S2). The study only identified the AP2 subfamily, and other AP2/ERF subfamilies may also modulate carotenoid accumulation in apple fruit and requires further research. MdAP2-34 contains two conserved AP2 domains (Fig. 1e); however, the other reported carotenoid-related AP2/ERF TFs  Fig. S1a). MdAP2-34 is a member of the AP2 subfamily, which has high homology with AtAIL5 in Arabidopsis, and AtAIL5 could induce a larger floral organ phenotype 47 . MdAP2-34 may have a similar function, and further studies are required to test its function.
It has been reported that AP2/ERF superfamily TFs can modulate carotenoid biosynthesis. With regard to carotenoid regulation, RAP2.2 promotes carotenoid accumulation by binding to the PSY promoter in Arabidopsis 40 . However, SIERF6 represses carotenoid accumulation in tomato, and reduced SlERF6 expression results in increased carotenoid concentration 39 . This study indicated that overexpressing MdAP2-34 in transgenic calli and fruits enhanced carotenoid accumulation ( Fig. 2 and Table 1). The contents of four specific carotenoids (β-carotene, β-cryptoxanthin, phytoene, and violaxanthin) were significantly higher in MdAP2-34-OVX fruit skin of transgenic Granny Smith than in P101F fruit skin and were remarkably lower in MdAP2-34-RNAi calli than in P5941 calli ( Table 1). The predominant carotenoid compounds differed among different apple cultivars 41 . In ripening apple fruit flesh, the predominant compounds in 'Royal Gala' were violaxanthin and neoxanthin, those in 'Aotea' flesh were β-cryptoxanthin and β-carotene, and those in 'Granny Smith' were lutein. The study also indicated that lutein was the predominant compound in Granny Smith fruit skin, followed closely by β-carotene and phytoene ( Table 1). The β-carotene and phytoene contents were much higher in MdAP2-34overexpressing transgenic calli and fruit skin, yet lutein showed no obvious difference, indicating that MdAP2-34 regulates β-carotene and phytoene accumulation but not lutein. MdAP2-34 could not modulate lutein-related genes, and other regulators or proteins may contribute to lutein accumulation in apple, which needs further study.
The structural genes encoding enzymes of carotenoid biosynthesis included MdPSY2-1, MdPDS, MdZDS1, MdLCY-β, and MdCHY-β2-1, which were markedly upregulated in both MdAP2-34-OVX TAC and fruits and were markedly downregulated in both MdAP2-34-RNAi TAC and fruits (Fig. 3). MdAP2-34 could enhance carotenoid accumulation by activating MdPSY2-1 promoter activity. However, MdAP2-34 only directly binds to the MdPSY2-1 promoter but not the other carotenoid-related structural genes (Fig. 4a). There may be other mechanisms by which MdAP2-34 interacts with other TFs or genes to regulate carotenoid biosynthesis. Taken together, carotenoid accumulation in apple fruit is probably controlled by a complex regulatory network and warrants further study.
The genes encoding enzymes of carotenoid biosynthesis include PSY, PDS, ZISO, ZDS, CRTISO, LCY-β, CHY-β, ZEP, VDE, CCS, CCD, and NCED, which have previously been characterized 2 . Multiple PSY genes deserve functional diversity, some of which play important roles in carotenoid accumulation 8,9 . Previous studies indicated that PSYs modulate carotenoid accumulation in many fruits of plant species, such as tomato 8 , potato 11 , loquat 14 , apple 15 , and peppers 16 . MdPSY2 exhibited higher transcript levels than MdPSY1 in diverse apple cultivars, indicating that MdPSY2 could be mainly responsible for the first carotenoid pathway step and plays a dominant role in carotenoid accumulation in apple fruits 15,41 . The different expression levels of MdPSY1 and MdPSY2 in apple were influenced by their promoters and activated by AP2/ERF TFs. There was a strong positive correlation between the expression of the TF AP2D26 and PSY2 in apple fruit, revealing a potential regulatory relationship 15 . The role of PSYs in the carotenoid pathway was prominent in Arabidopsis, and the PSY promoter was implicated and activated by AtRAP2.2 40 . The results indicated that MdPSY2-1 increases carotenoid content and was directly activated by 5). In addition, posttranscriptional regulation of PSY in controlling carotenoid biosynthesis has been reported, and PSYs are verified as the rate-limiting step in carotenogenesis 17 . In this study, by overexpressing the MdPSY2-1 gene in apple calli, the total carotenoid and phytoene contents were markedly increased (Fig. 5). The results show that MdPSY2-1 is a major gene in the carotenoid biosynthesis pathway in apple fruit.
Herein, we found that MdAP2-34 promotes carotenoid accumulation by participating in the carotenoid biosynthesis pathway. MdPSY2-1 is a major gene in the carotenoid biosynthesis pathway in apple fruit, and the MdPSY2-1 gene is directly bound and transcriptionally activated by MdAP2-34. Furthermore, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. This research is valuable for accelerating the apple breeding process and for further understanding the complex mechanisms of carotenoid synthesis in horticultural plants.