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Article

Functional Analysis of Cytochrome b5 in Regulating Anthocyanin Biosynthesis in Malus domestica

by
Fu-Jun Zhang
1,
Ning Ma
2,
Hao-Jian Li
2,
Lian-Zhen Li
2,
De-En Zhang
1,
Zhen-Lu Zhang
2,
Chun-Xiang You
2,* and
Xiao-Yan Lu
1,*
1
Key Laboratory of Special Fruits and Vegetables Cultivation Physiology and Germplasm Resources Utilization of Xinjiang Production and Construction Corps, Department of Horticulture, Agricultural College of Shihezi University, Shihezi 832003, China
2
Apple Technology Innovation Center of Shandong Province, Shandong Collaborative Innovation Center of Fruit & Vegetable Quality and Efficient Production, National Key Laboratory of Wheat Improvement, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An 271018, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(10), 1075; https://doi.org/10.3390/horticulturae10101075
Submission received: 25 August 2024 / Revised: 28 September 2024 / Accepted: 7 October 2024 / Published: 8 October 2024
(This article belongs to the Section Developmental Physiology, Biochemistry, and Molecular Biology)
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Abstract

:
Cytochrome b5 (CB5), a small heme-binding protein, plays an important role in plant biotic and abiotic stress. Anthocyanin is a critical determinant for fruit coloration, however, whether CB5 is involved in regulating anthocyanin biosynthesis has not yet been investigated in apple fruit (Malus domestica). In this study, we determined that MdCYB5, an apple CB5 gene, was a positive regulator for anthocyanin biosynthesis in apple fruit. We first found that MdCYB5 showed a high sequence and structural similarity with Arabidopsis cytochrome b5 isoform E (CB5E) at the protein level. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that MdCYB5 responds to light signals. Subcellular localization showed that MdCYB5 is localized to the cytoplasmin inthe epidermal cells of Nicotiana benthamiana leaves. Further investigation revealed that overexpressing MdCYB5 promoted anthocyanin biosynthesis in both apple calli and tissue-cultured apple seedlings. Furthermore, results of transient expression assay showed that overexpressing MdCYB5 promoted anthocyanin accumulation and fruit coloration in apple fruit. Taken together, this study suggests that MdCYB5 has a positive regulatory effect on anthocyanin biosynthesis in apple fruit.

1. Introduction

Anthocyanin is an important secondary metabolite involved in many aspects of plant growth and development [1]. In addition, anthocyanin has strong antioxidant and active oxygen scavenging properties [2,3]. Based on their important roles in biological functions and also in fruit coloration, anthocyanin biosynthetic pathways have been extensively studied [4]. Anthocyanins are synthesized in the cytoplasm via the flavonoid pathway and are stored in the vacuoles. Phenylalanine is a precursor of anthocyanin biosynthesis, which is converted to anthocyanin through a series of enzymes encoded by multiple genes. These include the early anthocyanin synthesis genes (EBGs), such as phenylalanine lysaminase (PAL), chalcone synthase (CHS), as well as the late anthocyanin synthesis genes (LBGs), dihydroflavonol 4-reductase (DFR), and anthocyanidin reductase (ANR) [5,6].
Anthocyanin biosynthesis is affected by various environmental stimuli such as nutrient deficiency, wounding, drought, pathogen infection, high temperature, and low temperature [7,8,9,10,11,12]. Light, including ultraviolet (UV) and visible light, is one of the major environmental factors regulating anthocyanin biosynthesis [13,14]. Many studies have shown that the transcription factorsHY5 and MYB1 play a central role in photo-induced anthocyanin synthesis in a variety of plants [15,16]. In addition, the plant hormones abscisic acid (ABA), gibberellins (GAs), and jasmonic acid (JA) also play an important role in regulating anthocyanin synthesis [6,17,18].
CB5 is a class of haem-binding proteins that are widely distributed in bacteria, fungi, mammals/humans, and plants. The plant CB5 protein family has multiple copies and various functions compared to that in yeast and mammals [19,20]. As a ubiquitous intercellular electron transporter, CB5 accepts electrons from either NADH-cytochrome b5 reductase (CBR)-CB5 or NADPH-cytochrome P450 reductase (CPR)-CB5 chains, transfers electrons to receptor proteins, and activates these proteins [21,22,23]. The protein-mediated electron transport system plays a key role in the survival and development of organisms, and CB5 protein plays an important role in plant growth and development and in regulating plant response to abiotic stress.
CB5 protein plays a role in the bioformation of metabolites such as fatty acids, flavonoids, and heteromeric lignin. In Arabidopsis, the cytochrome b5 gene is divided into five isoforms, AtCB5-A, AtCB5-B, AtCB5-C, AtCB5-D, AtCB5-E [19]. Yeast two-hybrid (Y2H) and luciferase experiments showed that four CB5 proteins (AtCB5-B, -C, -D and -E) can interact with eceriferum1 (CER1), which is involved in regulating synthesis of very long chain alkane [24,25]. Previous studies have shown that the AtCB5-C can act as an essential redox partner to ensure the proper function of CsF3′5′H [26]. AtCB5D interacts with single lignin biosynthesis P450 enzyme in Arabidopsis, affecting S-lignin formation and 5′-hydroxylated phenol biosynthesis [27,28,29].
In addition, CB5 has been shown to play a role in linking ethylene signals and interacting with sugar transporters to regulate sugar signaling. Y2H and bimolecular fluorescence complementation (BiFC) analysis showed that AtCB5-B, -C, -D and -E interacts with reversion to ethylene sensitivity1 (RTE1), which regulates the inhibitory function of the ethylene receptor ETR1. In this process, AtCB5 may activate RTE1 through redox modification, thereby linking the cellular redox state to ethylene signaling [30,31]. In apple fruit, MdCYB5 can interact with the sucrose transporter MdSUT1 and the sorbitan transporter MdSOT6 to increase the affinity of these two proteins for substrate sugars [32]. In Arabidopsis, all five CB5 isoforms interact with AtSUT4 (a homologue of MdSUT1) to regulate seed germination in response to sucrose and glucose [33].
The apple is one of the world’s most major fruits, and China is the largest country in the world apple industry, with its area and production ranking first in the world. Fruit color, as an important commodity characteristic of the apple, directly affects the market competitiveness of fruit. The function of MdCYB5 in sugar transport has been studied in the yeast system, but other functions of MdCYB5 have been less investigated [32]. In this study, MdCYB5 transgenic calli and apple tissue culture seedlings were obtained and their positive regulatory functions in anthocyanin synthesis was determined.

2. Materials and Methods

2.1. Plant Materials and Treatments

Apple tissue culture seedlings (Malus domestica, ‘Gala’) were grown in aseptic tissue culture (MS basal medium plus 0.8 mg L−1 6-BA, 0.2 mg L−1 NAA, 0.1 mg L−1 GA) under long day conditions (16 h light/8 h dark) at 25 °C, subculturing every 40 d. The apple calli (Malus domestica Borkh, ‘Orin’) was cultured in darkness at 25 °C and sub substituted every 15 d. medium conditions: MS basic medium + 1.5 mg L−1 2,4-D+ 0.4 mg L−1 6-BA. Tobacco (Nicotiana benthamiana): tobacco is cultivated in long day (16/8 h light/dark) conditions with a temperature of 24 °C. For photoinduced anthocyanin accumulation, wild-type (WT) calli and transgenic MdCYB5 calli, WT (GL3) and transgenic (MdCYB5-overexpressing) apple tissue culture seedlings, ‘Gala’ apple fruit (90 d after flowering) were placed under continuous 70 μmol·m−2·s−1 light at 22 °C. Fruit injection experiment: mixed plasmids (IL60-1:IL60-MdCYB5-2 = 1–3:10) were injected into the epidermis of apple fruits with 200 μL of infection solution using a needleless syringe and were kept under continuous light (22 °C, light intensity: 70 μmol·m−2·s−1) conditions for 5–7 d to observe the phenotype. Ten-year-old ‘Royal Gala’ apple tree roots, young stems, new leaves, flowers, and fruits were collected for RNA extraction and reverse transcriptional DNA for subsequent qRT-PCR. For the light response, the apple calli, which grew normally in darkness for about 14 d, were transferred to an incubator with continuous light (70 μmol·m−2·s−1) at 25 °C, and then samples were collected at 0 h, 1 h, 3 h, 6 h, and 12 h for qRT-PCR analysis.

2.2. Gene Identification, Cloning, and Biological Informational Analysis

Based on the sequence (MD01G1226000) retrieved from the apple GDDH13 (https://iris.angers.inra.fr/gddh13/, accessed on 12 July 2024) reference genome [34], a pair of primers MdCYB5-F 5′-ATGGCTTCAGATCCGAAAGT-3′ and MdCYB5-R 5′-CTCTTTCTTGGTGTAGTGACGGA-3′ were designed. PCR amplification was performed using the cDNA template obtained from the ‘Gala’ tissue culture seedlings. The reaction conditions were: predevaluation at 94 °C for 5 min, denatured at 95 °C for 25 s, annealed at 56 °C for 25 s, extended at 72 °C for 1 min, 35 cycles, and finally at 72 °C for 10 min. The sequence alignment of MdCYB5 and AtCB5-E was performed using DNAMAN software (Lynnon Biosoft, San Ramon, CA, USA). The SMART website (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1, accessed on 12 July 2024) was used for conservative motif analysis of AtCB5-E and MdCYB5. The upstream promoter region (2000 bp) of MdCYB5 was submitted to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 12 July 2024) for cis-element analysis [35].
The 3D structure prediction analysis of MdCYB5 and AtCB5-E proteins were performed using the homology modeling service platform Phyre2 (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index, accessed on 14 July 2024) [36].

2.3. Subcellular Localization of MdCYB5

The subcellular localization analysis of MdCYB5 is based on the method described by Zhang et al. [37]. Briefly, the open reading frame (ORF) of full-length MdCYB5 and the fluorescent signal green fluorescent protein (GFP) were fused to form 35S::MdCYB5-GFP. 35S::GFP and 35S::MdCYB5-GFP were individually transformed into Agrobacterium tumefaciens LBA4404, and the corresponding Agrobacterium was diluted with MMA (10 mM MES, 10 mM 6H2O.MgCI2, 150 mM Acetosyringone, ACE) solution to adjust the OD600 to 0.8–1.0, and then they were injected into tobacco leaves 3 d later. GFP signals were observed at 488 nm using confocal microscopy (Zeiss, LSM880, Jena, Germany).

2.4. Genetic Transformation of Apple Calli and Apple Tissue Culture Seedlings

For genetic transformation of apple calli, the pRI-MdCYB5 vector (the ORF of MdCYB5 was inserted into pRI to generate pRI-MdCYB5) was first constructed and transformed into Agrobacterium LBA4404. The Agrobacterium solution for apple calli infection was prepared and adjusted the OD600 to 0.6. The 14 d-old apple calli were then crushed on a super-clean workbench and were transferred to the above Agrobacterium solution, shaken in the dark at 120 r/min for 30 min. The excess Agrobacterium fluid was filtered and absorbed. Finally, the apple calli cultured in the dark for 2 d was plated on a medium containing 250 mg L−1 cephalosporin and 50 mg L−1 kanamycin. The positive transgenic apple calli were determined at both DNA and RNA levels.
For genetic transformation of apple tissue culture seedlings, 2–3 glossy, leathery leaves were selected from the stem tips of apple GL3 tissue culture seedlings. The leaves were cut evenly and divided into 2–3 pieces with a clean aseptic scalpel in the re-suspension solution. The leaves were similarly mixed in advance with the prepared Agrobacterium solution and then soaked by shaking for about 8 min. The excess bacterial liquid on the leaves was drained off, transferred to the co-medium and incubated in the dark for 3 d. After 3 d, the selected medium (leaf back down) was moved and continued dark culture for 3 d. Then, the selected medium (leaf back up) with kanamycin (75 mg L−1) was transferred to the selected medium (leaf back up). After 1 month of complete dark culture in the artificial culture room, the leaves with green calli at the leaf cut were selected and cultured under light at 25 °C. When the calli developed resistant buds, they were transferred to light culture and differentiated into apple plants. Transgenic seedlings were determined at both DNA and RNA levels.

2.5. DNA, RNA Extraction and qRT-PCR Analysis

DNA extraction was performed using the sodium dodecyl sulphate (SDS) method. Briefly, 400 µL EB extract (0.5 M EDTA, 2.5 mM NaCl, 1 M Tris-HCI, SDS) was added to the sample for thorough grinding. After centrifugation, 250 µL supernatant were absorbed and mixed with equal volume of isopropyl alcohol. The mixture was kept at −20 °C for 1 h, and then the DNA were precipitated by centrifugation. A total of 250 µL 70% ethanol was used to rinse the DNA once; the ethanol was completely removed by evaporation. Then, the total DNA were dissolved in 50 µL of ddH2O. All plant materials were extracted by RNA and analyzed by qRT-PCR as previously described by Zhang et al. [38]. All primers used in this study are shown in Table S1. Md18S rRNA serves as internal reference.

2.6. Determinationthe Accumulation of Measurement of Anthocyanin

Anthocyanin was extracted according to the ethanol–HCl method, and the formula of the extraction solution was as follows: anhydrous ethanol 95%, 1.5 M HCl 5%, plant tissue materials (apple calli, stem and leaves of apple tissue culture seedlings, apple fruit) were weighed, ground or cut, and were transferred into centrifuge tubes. Then 2–5 mL of extraction solution was kept in the dark overnight. The solutions were then centrifuged at 2348× g for 5 min, then the supernatant was collected and subjected to spectrophotometer analysis to determine the absorbance value at 530 nm, 620 nm, and 650 nm. The anthocyanin content was calculated as described by Zhang et al. [39].

2.7. Statistical Analysis

All experiments in this study were repeated 3 times and statistical analysis was performed using DPS software (http://www.dpsw.cn, accessed on 16 July 2023). The error bar represents the standard deviation (SD) of the three replicates, and the letters above the bars indicate significant difference between different treatments (p < 0.05). One-way analysis of variance (ANOVA) and least significant difference (LSD) tests were used for analysis.

3. Results

3.1. Cloning of MdCYB5 Gene and Protein Structure Analysis of MdCYB5

The total RNA was extracted from apple tissue culture seedlings, and the full-length cDNA sequence was obtained by reverse transcription. MdCYB5-F and MdCYB5-R were used for gene amplification, and a target band of approximately 400 bp (Figure S1a) was obtained by gel electrophoresis, which was confirmed as the MdCYB5 gene by sequencing, with a length of 402 bp (excluding stop codons) (Figure S1c). The protein sequence alignment showed that MdCYB5 and Arabidopsis AtCB5-E were highly homologous, with a sequence similarity of 78.36%. Protein domain analysis via SMART website showed that both MdCYB5 and AtCB5-E contained Cyt-b5 conserved domain (Figure S1b and Figure 1a). Furthermore, the three-dimensional structure prediction showed that MdCYB5 and AtCB5-E were highly consistent (Figure 1b). These results indicate a high degree of homology between MdCYB5 and AtCB5-E, which may imply functional consistency between them.

3.2. Subcellular Localization and Tissue Specificity Analysis of MdCYB5

To investigate the function of MdCYB5, subcellular localization tests were performed. The 35S::MdCYB5-GFP fusion construct and the control vector 35S::GFP were injected into appropriate tobacco leaves. Three days later, the GFP fluorescence signal was observed by LSM880 microscope. The results showed that MdCYB5-GFP localized in the cytoplasm of epidermal cells of N. benthamiana leaves, while the fluorescence signal of unloaded GFP was distributed in the nucleus, cell membrane, and the cytoplasm (Figure 2a). In addition, the expression pattern of MdCYB5 in different tissues, including roots, stems, leaves, flowers, and fruits, were investigated. The results indicated that MdCYB5 showed the highest expression level in fruits and the lowest in stems (Figure 2b).

3.3. MdCYB5 Responds to Light Signals

A promoter is a DNA sequence upstream of a gene that is closely associated with gene expression regulation. Thus we next analyzed the promoter of MdCYB5. The results showed that the MdCYB5 promoter contains several plant hormone-related response elements, such as ABA response elements, methyl jasmonate (MeJA) response elements, and salicylic (SA) acid response elements (Table S2). In addition, the MdCYB5 promoter contains a variety of environmental response elements, including anaerobic induction elements, drought response elements, and light response elements (Table S2). Among all the response elements, the optical response elements occupy the largest proportion, including three G-box-motifs, two GT1-motifs, one Box4-motifs, two TCCT-motifs, and two CTT-motifs (Table 1). Therefore, the results of promoter cis-acting element analysis showed that MdCYB5 may be involved in plant hormone signaling and environmental responses. Given the large number of MdCYB5 light response elements in promoters, our subsequent functional studies of MdCYB5 focused on its function in light response. In particular, the photoinduced synthesis of apple anthocyanin. Based on the above cis-element analysis, qRT-PCR was used to analyze the response of MdCYB5 to light, and the results showed that the expression level of MdCYB5 was increased under light induction, reaching the highest level at 3 h post treatment (Figure 3). The above results indicate that MdCYB5 is in response to light, and it might be involved in light-induced physiological processes.

3.4. MdCYB5 Is a Positive Regulator of Anthocyanin Accumulation in Apple Calli

In previous studies, light has been reported to promote and affect anthocyanin accumulation in apples, pears, and peaches [40,41,42]. To investigate whether MdCYB5 is involved in regulating anthocyanin biosynthesis, the recombinant overexpression vector pRI-MdCYB5 was constructed. Agrobacterium-mediated methods were then used to obtain transgenic apple calli and apple tissue culture seedlings overexpressing MdCYB5. The transgenic materials were identified at both DNA and RNA levels and MdCYB5-OE-1, 6, 9 transgenic apple calli were obtained (Figure S2). To investigate the role of MdCYB5 in anthocyanin accumulation, wild-type (WT) and MdCYB5-OE-1, 6, 9 apple calli were treated under continuous light and relative low temperature (22 °C, light intensity: 70 μmol·m−2·s−1). It was found that MdCYB5-OE-1, 6, 9 apple calli showed much redder than WT apple calli (Figure 4a). Meanwhile, MdCYB5-OE-1, 6, 9 apple calli accumulated much higher content of anthocyanin than WT (Figure 4b). This suggests that MdCYB5 overexpression promotes the accumulation of anthocyanin content in apple calli. Furthermore, qRT-PCR analysis showed that MdCYB5 overexpression promoted the expression of anthocyanin biosynthetic genes MdANR, MdCHI, MdDFR, and MdUF3GT (Figure 4c–f). The above results suggest that MdCYB5 can promote the accumulation of anthocyanin in apple calli by promoting the expression of anthocyanin synthesis-related genes.

3.5. Overexpression of MdCYB5 Increases Anthocyanin Accumulation in Leaves and Stems of Apple Tissue Culture Seedlings

To further elucidate the role of MdCYB5 in anthocyanin biosynthesis, transgenic apple seedlings overexpressing MdCYB5 were obtained. The transgenic lines were confirmed at both DNA and transcription levels (Figure S3). Because nitrogen is an important factor in anthocyanin accumulation, and nitrogen deficiency induces anthocyanin accumulation in apple fruit [43,44]. Therefore, the WT and transgenic apple seedlings were placed on a nitrogen-deficient medium to facilitate anthocyanin accumulation. The results showed that the stem base of transgenic plants of MdCYB5-31, 33, 35 showed deeper color accumulation in the nitrogen-deficient medium under the same light conditions compared to that of GL3 three days post treatment (Figure 5a). Determination of anthocyanin content also showed that overexpression of MdCYB5 promoted anthocyanin accumulation (Figure 5b). In addition, the leaves of transgenic seedling of MdCYB5-31, 33, 35 also accumulated more anthocyanins than GL3 (Figure 5g–h). Further qRT-PCR analysis showed that the expression of MdANS, MdCHS, MdDFR, and MdF3H in both the stems (Figure 5c–f) and leaves (Figure 5i–l) of transgenic apple tissue culture seedlings was higher than that of GL3. These results suggest MdCYB5 positively regulates anthocyanin biosynthesis in apple seedlings.

3.6. Transient Overexpression of MdCYB5 Increased Anthocyanin Accumulation in Apple Fruits

Finally, the role of MdCYB5 in regulating anthocyanin biosynthesis was verified in apple fruit (Figure 2b). The transient overexpression plasmid pIR-MdCYB5 (IL60-1 + MdCYB5-IL60-2) was constructed and injected into ‘Gala’ apple fruit to increase the expression level of MdCYB5 (Figure 6c), the infiltrated apple fruit were then kept in a growth chamber with continuous light and relative low temperature to facilitate anthocyanin accumulation. The results showed that overexpressing MdCYB5 resulted in deeper coloration at the injection site compared to control pIR, and anthocyanin content determination also indicated that MdCYB5 overexpression promoted anthocyanin accumulation in the infiltrated sites of apple peel (Figure 6a,b). qRT-PCR analysis revealed that the expression levels of MdF3H, MdANS, MdCHS, and MdDFR were significantly higher in the overexpressed apple peel (Figure 6d–g). These data indicate that MdCYB5 could promote anthocyanin accumulation and fruit coloration in apple fruit.

4. Discussion

CB5 is widely distributed in bacteria, fungi, mammals/humans, and plants [23]. In contrast to the single copy of CB5 in mammals and yeast, flowering plants, including Arabidopsis, have evolved multiple CB5 genes [19,20,45,46,47,48]. There are five CB5 genes in Arabidopsis (AtCB5-A, AtCB5-B, AtCB5-C, AtCB5-D, AtCB5-E) these isoforms isomer only 40–68% of the amino acid sequence. In this study, MdCYB5, which is homologous to Arabidopsis AtCB5-E (Similarity 78%), was cloned, and the secondary and tertiary structure of the protein showed a high degree of homology with AtCB5-E (Figure 1 and Figure S1). Considering the function of CB5s in secondary metabolism and the response of MdCYB5 to light signal (Figure 3), this study focused on the function of MdCYB5 in anthocyanin synthesis and carried out functional verification.
Flavonoids, also known as bioflavonoids, are one of the most important secondary metabolites in plants. Anthocyanin is a type of flavonoid that is widely found in flowers, fruits, leaves, stems, and the seeds of plants. Phenylalanine is a precursor of plant biosynthesis, such as anthocyanin and lignin [49]. Previous studies have shown that CB5s plays a critical role in some P450-catalysed reactions that synthesize a specific metabolite or cell wall structural component, lignin. AtCB5-D, as a specialized electron carrier, assists AtF5H1 in catalyzing the 5-hydroxylation of the benzene ring to synthesize S-lignin and 5-hydroxylated phenol [28]. Disruption of AtCB5D alone resulted in an approximately 70% reduction in 5-hydroxylated phenolic ester accumulation in leaves and a 69% reduction in stem S-lignin subunits compared to wild-type (WT) controls. It was also found that in stems, F5H1 mainly obtained reducing power through the NADPH–CPR–CB5 pathway to promote S-type lignin formation, whereas in seeds, F5H1 mainly obtained reducing power through the NADH–CBR–CB5 pathway to synthesize free sinapids in seeds and to bind to seed bark saponins [27]. In tea plant (Camellia sinensis), the CsCB5C electron carrier has an important influence on the catalytic activity of CsF3′5′H [26]. In addition, in petunia (Petunia hybrida Vilm.) and the disruption of DifF at the CB5 site leads to a decrease in F3′5′H activity, resulting in a change in flower color [50]. In this study, we demonstrated the role of MdCYB5 in enhancing anthocyanin accumulation in apple calli, tissue culture seedlings, and fruits (Figure 4, Figure 5 and Figure 6). Furthermore, overexpression of MdCYB5 affected the expression of anthocyanin biosynthetic genes, including MdANS, MdCHS, MdF3H, MdDFR and UF3GT (Figure 4, Figure 5 and Figure 6). Among them, we also found that the anthocyanin–synthetic gene MdF3H was significantly up-regulated with anthocyanin accumulation in stems and leaves of MdCYB5 apple tissue culture seedlings (Figure 5f,l), while the change was not obvious in apple calli and apple fruit. Does this indicate that MdCYB5 may also be directly involved in the anthocyanin synthesis pathway in different tissues as an electron carrier? Further biochemical experiments are needed to verify the relationship between MdCYB5 and anthocyanin synthesis genes (such as MdF3H).
Sugar can be used as a carbon source and metabolic substrate to participate in the biosynthesis of anthocyanin, and it can also participate in the regulation of the expression of genes related to anthocyanin synthesis in the form of signaling molecules [51]. As previously reported, MdCYB5 interacts with MdSUT1 and MdSOT6 to participate in sucrose and sorbitol transport [32]. Whether sugar signaling is involved in the regulation of anthocyanin by MdCYB5 needs to be further investigated.
Light is the most important factor affecting anthocyanin synthesis, which is necessary to induce anthocyanin synthesis in most plants. Under light conditions, phytochrome(PHY) and cryptochrome (CRYs) photoreceptors perceive light signals of different wavelengths and convert them into bioactive subtypes, thereby activating the expression of transcription factors such as bZIP, bHLH, MYB, Zinc-finger, GATA, and GT1, which further bind to light-responsive elements on downstream gene promoters, such as G-box, ACE, GT1-motif, GATA-motif and MRE, etc., regulate changes in transcription levels of downstream genes, thereby regulating anthocyanin synthesis [52]. MdBBX22 can enhance the binding ability of MdHY5 to the G-box present in the promoter of MdMYB10 and MdCHS, thereby regulating anthocyanin synthesis under light [53]. Light also promotes the accumulation of the PpbHLH64 and PbMYB10 proteins, which could bind to the MBS and G-box cis-acting elements of the promoters of anthocyanin synthesis-related genes, respectively, and activate their transcription, thereby promoting anthocyanin synthesis [54]. MdPIF1 can bind to promoters of MdPAL and MdF3H by recognizing and binding G-box motifs involved in the regulatory pathway of apple dimple fruit vidoid (ADFVd) affecting anthocyanin biosynthesis [55]. In this study, the cis-acting elements of the MdCYB5 promoter were examined, including the photoresponsive elements G-box, GT1 motif, and CTT motif (Table 1). Further qRT-PCR results also showed the response of MdCYB5 to the light signal (Figure 3). This suggests that there may be transcription factors that bind to these elements to regulate the effect of MdCYB5 on anthocyanin under light. In addition, the MdCYB5 promoter also contains cis-acting elements that may be bound by the transcription factors MYB and MYC (Table S2). Given that the MYB and MYC transcription factors play a role in the anthocyanin synthesis pathway [56,57,58,59], there are also mechanisms by which these transcription factors can bind directly to the corresponding elements to regulate anthocyanin synthesis.
There are other possible mechanisms by which MdCYB5 regulates anthocyanin accumulation. The biosynthesis of anthocyanin is influenced by plant hormones. In previous reports, the interaction of CB5 isoforms (AtCB5-B,-C,-D, and-E) and ETR1 ethylene receptor signaling through RTE1 was linked to the ethylene signal [31]. Ethylene signaling is involved in the regulation of fruit anthocyanin in a variety of fruit trees [60,61,62]. This may suggest that anthocyanin accumulation by MdCYB5 is related to ethylene signaling, which may be an interesting point for future work on MdCYB5. In addition, the cis-acting elements of the MdCYB5 promoter also have binding elements for the plant hormones ABA, SA, and JA (Table S2). Considering the functions of ABA, SA, and JA in anthocyanin synthesis [18,63,64], this may indicate that the mechanism of MdCYB5 regulation of anthocyanin may be related to various hormone signals.
In conclusion, we identified MdCYB5, an apple cytochrome b5 gene, as a positive regulator in regulating anthocyanin biosynthesis. We proved that overexpressing MdCYB5 promoted anthocyanin biosynthesis in apple calli, seedlings, and in apple fruit. Based on this, we provide a hypothesized model for the role of the MdCYB5 gene in anthocyanin biosynthesis in apple trees (Figure 7). Our work enriches research on the function of the cytochrome b5 gene in plants.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10101075/s1, Table S1: Primers for quantitative real-time PCR. Table S2: Cis-acting element analysis of MdCYB5 promoter. Figure S1: MdCYB5 gene amplification and protein secondary structure analysis. a. Agarose gel electrophoresis of PCR product of apple MdCYB5. DNA markers indicate the size of genes (from 100 bp to 2000 bp). b. The secondary structure of MdCYB5 and AtCB5-E proteins was analyzed via the SMART website. c. DNA sequencing results of MdCYB5 were amplified for a. Figure S2: Identification of overexpressed MdCYB5 transgenic calli. a. CaMV 35S plus MdCYB5-R gene for downstream DNA amplification of vector insertion fragments. 18S rRNA was used as a control. PC: pRI-MdCYB5 Plasmid positive control. NC: Wild type calli WL was used as negative control. b. Detection of MdCYB5 expression in transgenic and WT apple calli. The significance difference was analyzed by DPS (p < 0.05), note: different lowercase letters indicate significant differences according to the one-way ANOVA LSD test. Figure S3: Identification of MdCYB5 overexpressed apple tissue culture seedlings by DNA and RNA levels. a. DNA level identification of MdCYB5 transgenic apple seedlings. PC: pRI-MdCYB5 Plasmid positive control. NC: Wild type GL3was used as negative control. b. The expression of MdCYB5 in GL3 and transgenic apples was detected by qRT-PCR. The significance difference was analyzed by DPS (p < 0.05), note: different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.

Author Contributions

Z.-L.Z., F.-J.Z., C.-X.Y. and X.-Y.L. initiated and designed the research. F.-J.Z., N.M., H.-J.L. and Z.-L.Z. performed the experiments. F.-J.Z., D.-E.Z. and L.-Z.L. analyzed the data. F.-J.Z. and Z.-L.Z. wrote and revised the manuscript. C.-X.Y. and X.-Y.L. supervised the project. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Key Research and Development Program China (2022YFD1201700), the National Natural Science Foundation of China NSFC-Xinjiang Joint Fund (Cultivation) Project (U1703116), and the National Natural Science Foundation of China (32172538). We would like to thank Takaya Moriguchi of the National Institute of Fruit Tree Science, Japan, for the ‘Orin’ apple calli. We would like to thank Zhihong Zhang of Shenyang Agricultural University of China for the GL3 apple tissue culture seedlings.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Materials, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare that they have no conflicting interests.

References

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Figure 1. Sequence alignment of MdCYB5 and AtCB5-E and three-dimensional structure analysis of the protein. (a) The amino acid sequences of MdCYB5 and AtCB5-E were compared using DNAMAN software, and the conserved domain Cyt-b5 is highlighted in the red box. (b) Protein 3D structure prediction shows the protein structure of MdCYB5 and AtCB5-E, and Merge is the superposition of the two protein structures.
Figure 1. Sequence alignment of MdCYB5 and AtCB5-E and three-dimensional structure analysis of the protein. (a) The amino acid sequences of MdCYB5 and AtCB5-E were compared using DNAMAN software, and the conserved domain Cyt-b5 is highlighted in the red box. (b) Protein 3D structure prediction shows the protein structure of MdCYB5 and AtCB5-E, and Merge is the superposition of the two protein structures.
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Figure 2. Subcellular localization and tissue expression pattern analysis of MdCYB5. (a) Subcellular localization of MdCYB5. Tobacco epidermal cells were observed using the confocal microscopy (Zeiss, LSM880). Unfused 35S::GFP served as a control (Scale bar, 10 µm). (b) Tissue-specific expression of MdCYB5. Transcript levels of MdCYB5 in roots, stems, leaves, flowers, and fruits of ‘Royal Gala’. The significance difference was analyzed by DPS (p < 0.05), note: Different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
Figure 2. Subcellular localization and tissue expression pattern analysis of MdCYB5. (a) Subcellular localization of MdCYB5. Tobacco epidermal cells were observed using the confocal microscopy (Zeiss, LSM880). Unfused 35S::GFP served as a control (Scale bar, 10 µm). (b) Tissue-specific expression of MdCYB5. Transcript levels of MdCYB5 in roots, stems, leaves, flowers, and fruits of ‘Royal Gala’. The significance difference was analyzed by DPS (p < 0.05), note: Different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
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Figure 3. The response of MdCYB5 to light. MdCYB5 transcript levels with increasing light duration (0 h, 1 h, 3 h, 6 h, 12 h). The significance difference was analyzed by DPS (p < 0.05), note: Different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
Figure 3. The response of MdCYB5 to light. MdCYB5 transcript levels with increasing light duration (0 h, 1 h, 3 h, 6 h, 12 h). The significance difference was analyzed by DPS (p < 0.05), note: Different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
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Figure 4. Overexpression of MdCYB5 increases anthocyanin accumulation in apple calli. (a) Phenotype of anthocyanin accumulation in apple calli (WT and MdCYB5-OE-1, 6, 9) under light. (b) The anthocyanin content of WT apple calli and overexpressed MdCYB5 apple calli was determined, and the anthocyanin content of WL was calculated as 1. (c–f) Quantitative real-time PCR (qRT-PCR) analysis of the anthocyanin biosynthesis genes MdANR, MdCHI, MdDFR and MdUF3GT. The significance difference was analyzed by DPS (p < 0.05), note: Different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
Figure 4. Overexpression of MdCYB5 increases anthocyanin accumulation in apple calli. (a) Phenotype of anthocyanin accumulation in apple calli (WT and MdCYB5-OE-1, 6, 9) under light. (b) The anthocyanin content of WT apple calli and overexpressed MdCYB5 apple calli was determined, and the anthocyanin content of WL was calculated as 1. (c–f) Quantitative real-time PCR (qRT-PCR) analysis of the anthocyanin biosynthesis genes MdANR, MdCHI, MdDFR and MdUF3GT. The significance difference was analyzed by DPS (p < 0.05), note: Different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
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Figure 5. Overexpression of MdCYB5 increased anthocyanin accumulation in stems and leaves of apple tissue culture seedlings. (a,g) The anthocyanin accumulation phenotype was observed in the stems and leaves of tissue culture seedlings with GL3 and MdCYB5 overexpression, the scale in the figure represents 1 cm. (b,h) The relative anthocyanin content (GL3 and MdCYB5-31, 33, 35) in stems and leaves of apple tissue-cultured seedlings and the designated GL3 anthocyanin content was 1. The expression levels of GL3 and stem anthocyanin synthesis genes MdANS (c), MdCHS (d), MdDFR (e) and MdF3H (f) of overexpressed apple tissue culture seedlings were detected by qRT-PCR. (h–l) Expression of transcription level of anthocyanin synthesis gene in GL3 and overexpressed MdCYB5 apple tissue culture seedlings. The significance difference was analyzed by DPS (p < 0.05), note: Different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
Figure 5. Overexpression of MdCYB5 increased anthocyanin accumulation in stems and leaves of apple tissue culture seedlings. (a,g) The anthocyanin accumulation phenotype was observed in the stems and leaves of tissue culture seedlings with GL3 and MdCYB5 overexpression, the scale in the figure represents 1 cm. (b,h) The relative anthocyanin content (GL3 and MdCYB5-31, 33, 35) in stems and leaves of apple tissue-cultured seedlings and the designated GL3 anthocyanin content was 1. The expression levels of GL3 and stem anthocyanin synthesis genes MdANS (c), MdCHS (d), MdDFR (e) and MdF3H (f) of overexpressed apple tissue culture seedlings were detected by qRT-PCR. (h–l) Expression of transcription level of anthocyanin synthesis gene in GL3 and overexpressed MdCYB5 apple tissue culture seedlings. The significance difference was analyzed by DPS (p < 0.05), note: Different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
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Figure 6. Transient overexpression of MdCYB5 promotes anthocyanin accumulation in apple fruit. (a) Color accumulation of different degrees around injection site of the apple peel. MdCYB5 cDNA was cloned into the pIR vector for overexpression. Empty vector was used as control. (b) Relative anthocyanin content in apple peel after injection of pIR and pIR-MdCYB5 in (a). (c) The relative expression of MdCYB5 was in the injected area of the apple peel. (d–g) The expression levels of pIR and pIR-MdCYB5 anthocyanin synthesis-related genes (MdDFR, MdANS, MdF3H, and MdCHS) were detected. The significance difference was analyzed by DPS (p < 0.05), note: different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
Figure 6. Transient overexpression of MdCYB5 promotes anthocyanin accumulation in apple fruit. (a) Color accumulation of different degrees around injection site of the apple peel. MdCYB5 cDNA was cloned into the pIR vector for overexpression. Empty vector was used as control. (b) Relative anthocyanin content in apple peel after injection of pIR and pIR-MdCYB5 in (a). (c) The relative expression of MdCYB5 was in the injected area of the apple peel. (d–g) The expression levels of pIR and pIR-MdCYB5 anthocyanin synthesis-related genes (MdDFR, MdANS, MdF3H, and MdCHS) were detected. The significance difference was analyzed by DPS (p < 0.05), note: different lowercase letters indicate significant differences according to the one-way ANOVA LSD test.
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Figure 7. Hypothesized model of the role of MdCYB5 gene in anthocyanin biosynthesis in apple trees under light. The expression level of the MdCYB5 gene is high in apple fruit. During fruit growth and development, MdCYB5 can interact with sucrose transporter MdSUT1 and sorbitol transporter MdSOT6, which may regulate the mechanism of anthocyanin synthesis. In the stems and leaves of apple trees, on the one hand, MdCYB5 is likely to be regulated by some transcription factors such as MYB and MYC; on the other hand, MdCYB5 is likely to have a direct effect on MdF3H, thereby regulating anthocyanin synthesis.
Figure 7. Hypothesized model of the role of MdCYB5 gene in anthocyanin biosynthesis in apple trees under light. The expression level of the MdCYB5 gene is high in apple fruit. During fruit growth and development, MdCYB5 can interact with sucrose transporter MdSUT1 and sorbitol transporter MdSOT6, which may regulate the mechanism of anthocyanin synthesis. In the stems and leaves of apple trees, on the one hand, MdCYB5 is likely to be regulated by some transcription factors such as MYB and MYC; on the other hand, MdCYB5 is likely to have a direct effect on MdF3H, thereby regulating anthocyanin synthesis.
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Table 1. Promoter cis-acting element analysis of MdCYB5.
Table 1. Promoter cis-acting element analysis of MdCYB5.
Regulatory SequenceSequenceFunction of SiteLocation
G-BoxCACGTT
GACGAC
GACGTC		cis-acting regulatory element involved in light responsiveness+1962 +995 +1024
GT1-motifGGTTAAlight responsive element−360
−1742
Box 4ATTAATpart of a conserved DNA module involved in light responsiveness−1641
TCCC-motifTCTCCCTpart of a light responsive element−270
+1483
TCT-motifTCTTACpart of a light responsive element+370
+1090
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Zhang, F.-J.; Ma, N.; Li, H.-J.; Li, L.-Z.; Zhang, D.-E.; Zhang, Z.-L.; You, C.-X.; Lu, X.-Y. Functional Analysis of Cytochrome b5 in Regulating Anthocyanin Biosynthesis in Malus domestica. Horticulturae 2024, 10, 1075. https://doi.org/10.3390/horticulturae10101075

AMA Style

Zhang F-J, Ma N, Li H-J, Li L-Z, Zhang D-E, Zhang Z-L, You C-X, Lu X-Y. Functional Analysis of Cytochrome b5 in Regulating Anthocyanin Biosynthesis in Malus domestica. Horticulturae. 2024; 10(10):1075. https://doi.org/10.3390/horticulturae10101075

Chicago/Turabian Style

Zhang, Fu-Jun, Ning Ma, Hao-Jian Li, Lian-Zhen Li, De-En Zhang, Zhen-Lu Zhang, Chun-Xiang You, and Xiao-Yan Lu. 2024. "Functional Analysis of Cytochrome b5 in Regulating Anthocyanin Biosynthesis in Malus domestica" Horticulturae 10, no. 10: 1075. https://doi.org/10.3390/horticulturae10101075

APA Style

Zhang, F.-J., Ma, N., Li, H.-J., Li, L.-Z., Zhang, D.-E., Zhang, Z.-L., You, C.-X., & Lu, X.-Y. (2024). Functional Analysis of Cytochrome b5 in Regulating Anthocyanin Biosynthesis in Malus domestica. Horticulturae, 10(10), 1075. https://doi.org/10.3390/horticulturae10101075

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