PbLAC4-like, activated by PbMYB26, related to the degradation of anthocyanin during color fading in pear

doi:10.21203/rs.3.rs-225654/v1

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Research Article

PbLAC4-like, activated by PbMYB26, related to the degradation of anthocyanin during color fading in pear

https://doi.org/10.21203/rs.3.rs-225654/v1

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Background

Anthocyanin degradation results in the loss of red color in leaves, petals and receptacles during development. But the degradation mechanism is not fully investigated. It is vital to study the degradation mechanism of anthocyanin in pear for promoting the accumulation of anthocyanin and inhibiting the red fading in pear.

Results

Here, we reported that laccase encoded by PbLAC4-like was associated with anthocyanin degradation in pear. The expression pattern of PbLAC4-like was negatively correlated with the content of anthocyanin during the color fading process of pear leaves, petals and receptacles. Phylogenetic analysis and sequence alignment revealed that PbLAC4-like played a vital role in anthocyanin degradation. Thus, the degradation of anthocyanin induced by PbLAC4-like was further verified by transient assays and prokaryotic expression. More than 80% of anthocyanin compounds were degraded by transiently over-expressed PbLAC4-like in pear fruitlet peel. The activity of crude enzyme to degrade anthocyanin in leaves at different stages was basically consistent with the expression of PbLAC4-like. The anthocyanin degradation ability of prokaryotic induced PbLAC4-like protein was also verified by enzyme activity assay. Besides, we also identified PbMYB26 as a positive regulator of PbLAC4-like. Yeast one-hybrid and dual luciferase assay results showed that PbMYB26 activated PbLAC4-like expression by directly binding to the PbLAC4-like promoter.

Conclusions

Taken together, the PbLAC4-like activated by PbMYB26, was involved in the degradation of anthocyanin, resulting in the redness fading in different pear tissues.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

anthocyanin degradation

PbLAC4-like

PbMYB26

pear

Anthocyanins are formed by glycosylation of anthocyanidins and glycosides and have a basic C6-C3-C6 skeleton [1, 2]. Anthocyanins are significant pigments that make plant tissues appear red, purple, blue, and black [3–5], so they can attract pollinators and seed carriers, thus expanding the area and scope of plant distribution [6]. Anthocyanins respond to various biotic and abiotic stresses to improve plant resistance and make plants better adapt to the environment [7]. Besides, anthocyanins are helpful for people to fight against diseases [8–10]. However, during the development process of some pear leaves, petals, receptacles, the anthocyanin content is reduced, resulting in the loss of color. The content of anthocyanin is affected not only by the anthocyanin biosynthesis but also by the anthocyanin degradation. Compared with the detailed study on anthocyanin biosynthesis, the knowledge of anthocyanin degradation is not clear enough.

In plants, the biosynthesis of anthocyanin is catalyzed by a series of enzymes, mainly including phenylalanine ammonia-lyase, chalcone synthase, chalcone isomerase, flavone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase/leucoanthocyanidin dioxygenase, and UDP glucose: flavonoid 3-O-glucosyl transferase (UFGT). And the expression of related structural genes is synergistically regulated by the MBW (MYB-bHLH-WD40) transcription complex [11, 12]. In pear, UFGT was identified as a key enzyme involved in anthocyanin biosynthesis [13]. MYB10, MYB10b, ERF22, REVEILLE promoted the biosynthesis of anthocyanin by activating the expression of anthocyanin biosynthesis genes in pear [14–16]. COP1.1 and MYB120 were identified as inhibitory regulators of anthocyanin biosynthesis in pear [17, 18]. bHLH3, MYB88, MYB124, NAC52 and in apple [19–21], MYB75, MYB90, MYB113 and MYB114 in Arabidopsis thaliana [22–24] also were shown to promote anthocyanin biosynthesis. MYB6 in apple [25] and MYB1 in Gerbera hybrid [26] were identified to inhibit anthocyanin biosynthesis.

At present, there have been some studies on the degradation mechanism of anthocyanin in plants. Anthocyanin degradation was associated with a variety of enzymes, including peroxidases (POD), polyphenol oxidases (PPO), β-glucosidases and laccases (LAC). During the browning of Litchi fruit pericarp, LAC degraded anthocyanin by the model of anthocyanin-LAC-epicatechin [27]. The browning of Litchi fruit pericarp was also related to POD and PPO [28–30]. The flower color change from dark purple to pure white was caused by POD (BcPrx01) and β-glycosidase (BcXyl) in Brunfelsia calycina [31–33]. Transcriptional analysis revealed the color fading process of ‘Red Bartlett’ might be related to POD and LAC [34]. The β-glucosidase was purified from blood orange juice and it was found that β-glucosidase was closely related to the degradation of anthocyanin in pericarp and juice [35].

LACs are copper-containing polyphenol oxidases that use molecular oxygen to oxidize various aromatic and non-aromatic compounds [36]. So far, the function of LAC was mostly related to lignin accumulation. LAC played a role in the formation of lignin by promoting oxidative conjugated monolignols in sycamore maple [37, 38]. In Arabidopsis and poplar, lignin content decreased when LAC genes were RNA interference [39–42]. Some studies have also shown that the LAC genes in pear were closely related to the formation of stone cells caused by lignin accumulation [43, 44]. Only a few studies have shown that LAC was associated with anthocyanin degradation. The degradation of anthocyanin by LAC resulted in the browning of Litchi fruit pericarp [27]. Many regulators of LAC have been identified, but all of them were related to lignin accumulation. MYB26, MYB46 and MYB83 in Arabidopsis [45–48], miR397a and MYB169 in pear [43, 44] were shown to involve in lignin accumulation by regulating LAC.

The leaves, petals and receptacles of some pear species lost color during development. According to the previous data on the differences between ‘Zaosu’ and ‘Red Zaosu’ leaves [49], we speculated PbLAC4-like probably related to the color fading of pear. In this study, there was a negative correlation between PbLAC4-like expression level and anthocyanin content during the color fading of pear leaves, petals and receptacles. Transiently overexpressed PbLAC4-like in pear fruitlet peel and the enzyme activity test for the degradation of anthocyanin further proved the role of PbLAC4-like in the degradation of anthocyanin. The regulator of PbLAC4-like has also been preliminarily analyzed and found that PbMYB26 could directly bind to the promoter of PbLAC4-like to up-regulate its expression. These results showed that PbLAC4-like, activated by PbMYB26, promoted the degradation of anthocyanin in pear. This provided a theoretical reference for regulating the degradation of anthocyanin.

Anthocyanin content and PbLAC4-like expression level during the color fading process

Some pear leaves, petals, receptacles exist color fading phenomenon during development (Fig. 1a). To study the relationship between color fading and PbLAC4-like, anthocyanin content and PbLAC4-like expression level in leaves of five pear varieties (‘Zaosu’, ‘Red Zaosu’, ‘2 hao’, ‘7 hao’ and ‘Cuiguan’) at three development stages (Red, Half Red, Green) were determined. During the color fading of leaves, the content of anthocyanin gradually decreased. The PbLAC4-like gene expression level was higher during Green and Half Red than that in Red (Fig. 1b). A correlation analysis revealed a negative correlation between anthocyanin content and PbLAC4-like expression level in leaves (Table 1). To further investigate the relationship between PbLAC4-like expression level and anthocyanin content, we also measured the PbLAC4-like expression level and anthocyanin content in ‘Zaosu’, ‘Red Zaosu’ petals of four stages and ‘Red Zaosu’ receptacles of four stages (S1 to S4; Fig. 1a). In general, the PbLAC4-like expression level increased in petals and receptacles during S1 to S4. The content of anthocyanin in petals and receptacles reduced from S1 to S4 (Fig. 1c, d). These results suggested that PbLAC4-like possibly played an important role in inhibiting the accumulation of anthocyanin during color fading progress.

Identification and analysis of PbLAC4-like

The complete coding sequence length of PbLAC4-like was 1680 bp，and the PbLAC4-like protein consisted of 559 amino acids. PbLAC4-like protein belonged to the LACs family, which was multicopper oxidase found in plants, fungi, and bacteria [36]. A phylogenetic tree containing PbLAC4-like protein and previously characterized LACs in plants was constructed, indicating high homologies of PbLAC4-like protein to LcLAC and AtLAC15 involved in the degradation of flavonoid [27, 50] (Fig. 2a). Besides, the protein sequence alignment results showed that their protein sequence similarity reached 56.58% (Fig. 2b). These results showed that the PbLAC4-like gene inhibited the accumulation of anthocyanin by degrading anthocyanin.

Transient overexpression of PbLAC4-like gene in the peel of pear fruitlet

To verify the function of PbLAC4-like in pear, PbLAC4-like was transiently overexpressed in 'Palacer' fruitlet peel with faded color after bagging, taking the empty vector containing GUS as the control. GUS staining of the control showed that this transformation method was feasible (Additional file 1: Fig. S1). After ten days of transient transformation, the control returned to red, but the 'Palacer' fruitlet peel of overexpressed PbLAC4-like remained unstained (Fig. 3a). Compared with the control, anthocyanin content in overexpressed PbLAC4-like pear fruitlet peel decreased significantly, which was consistent with the observed phenotype (Fig. 3b). The expression level of PbLAC4-like in the overexpressed PbLAC4-like pear fruitlet peel was about five times higher than that in the control group, with a significant difference (Fig. 3b). In other words, overexpressing PbLAC4-like caused a decrease of anthocyanin content in pear fruitlet peel. Together, these results indicated that PbLAC4-like played a major role in the degradation of anthocyanin in pear.

Detection of degrading anthocyanin activity of the PbLAC4-like protein

The crude enzyme activities of ‘Zaosu’, ‘Red Zaosu’, ‘2 hao’, ‘7 hao’ and ‘Cuiguan’ leaves were determined with anthocyanin as substrate. In ‘Zaosu’, ‘Red Zaosu’, ‘2 hao’ and ‘7 hao’ leaves, the crude enzyme activity in Green and Half Red leaves was higher than that in Red leaves. In ‘Cuiguan’ leaves, the crude enzyme activity of Green leaves was higher than that of Half Red and Red leaves (Fig. 4a). The activity of crude enzyme to degrade anthocyanin was basically consistent with the PbLAC4-like expression level in leaves at different stages. In addition, PbLAC4-like protein was induced by prokaryotic expression. The size of PbLAC4-like protein was predicted by the protein molecular calculator, which was 60.9 kDa. In addition, there were two 6×his tags on the pET28a (+) vector, each 6×his tag was 840.9 Da, so the size of the fusion vector after prokaryotic expression was about 62.6 kDa. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a band in the size of the his-PbLAC4-like fusion protein, indicating that the protein was successfully induced (Fig. 4b). The activity of induced PbLAC4-like protein was measured by using anthocyanin as the substrate and induced empty vector protein as the control. The results showed that the induced PbLAC4-like protein activity was five times as that of the control group (Fig. 4c). In summary, at the protein level, the PbLAC4-like protein has been proved to be involved in the degradation of anthocyanin, and it was an important enzyme in the color fading process of pear leaves, petals and receptacles.

Upstream transcriptional regulation of PbLAC4-like gene

MYB46 was verified to positively regulate the expression of LAC genes by binding to the AC elements in their promoter regions in Arabidopsis thaliana [48]. In the present study, the AC elements were also identified in the promoter region of PbLAC4-like. So the proteins similar to AtMYB46 in pear genome were screened using phylogenetic analysis. PbMYB26, PbMYB39, PbMYB46-44, PbMYB46-77, PbMYB46-97 and PbMYB86 showed high homologies with AtMYB46 (Additional file 2: Fig. S2a). To further screen out the candidate regulators of PbLAC4-like, the gene expression patterns of these MYB genes were verified in ‘Red Zaosu’ receptacles. The results showed that the expression patterns of PbMYB39, PbMYB46-44, PbMYB46-77, PbMYB46-97 and PbMYB86 were different from that of PbLAC4-like (Additional file 2: Fig. S2b), while only the expression pattern of PbMYB26 was consistent with that of PbLAC4-like, and the expression levels of both genes were increased and then decreased, reaching the highest value in the third stage, suggesting that PbMYB26 may take participate in regulating PbLAC4-like expression in pear (Fig. 5a). To confirm the possible interaction between PbLAC4-like and PbMYB26, yeast one-hybrid (Y1H) and dual luciferase assay were further conducted. The Y1H results showed that PbMYB26 could directly bind to the PbLAC4-like promoter (Fig. 5b). The effect of PbMYB26 on the transcriptional activity of the PbLAC4-like promoter was determined by the relative LUC / REN ratio. Compared to the control, the relative LUC / REN of transient expressing PbMYB26 was upregulated sixfold, indicating that PbMYB26 upregulated the expression of PbLAC4-like (Fig. 5c). Furthermore, the expression of PbLAC4-like was up-regulated in ‘Zaosu’ pear fruit with transient overexpression of PbMYB26 (Fig. 5d). These results confirmed that PbMYB26 activated PbLAC4-like expression by directly binding to the PbLAC4-like promoter.

Anthocyanin is a kind of plant pigment that can attract pollinators and promote plant pollination [6]. It can also help plants adapt to the environment in response to stresses [7–9]. The content of anthocyanin is affected by the biosynthesis and degradation of anthocyanin [27]. At present, the pathway of anthocyanin biosynthesis in pear is relatively clear. However, the degradation mechanism of anthocyanin has not been fully studied. In this study, an enzyme involved in the color fading of pear leaves, petals and receptacles was identified and characterized.

The PbLAC4-like gene was screened based on previous studies on the differences between ‘Zaosu’ and ‘Red Zaosu’ leaves [49], so the PbLAC4-like potentially involved in the accumulation of anthocyanin. There was a negative correlation between the PbLAC4-like expression level and the anthocyanin content during the color fading of pear leaves, petals and receptacles (Fig. 1). So we hypothesized that the PbLAC4-like inhibited the accumulation of anthocyanin and caused redness continuously lost in pear.

The phylogenetic analysis in this study revealed that PbLAC4-like protein had high homologies with LAC in Litchi chinensis and LAC4, LAC11, LAC15 in Arabidopsis thaliana (Fig. 2). In Litchi fruit pericarp, LAC was responsible for epicatechin-mediated anthocyanin degradation [27]. In Arabidopsis thaliana, LAC15 involved in lignin and proanthocyanidin biosynthesis by oxidizing their respective monomers [50, 51]. LAC11 and LAC4 have been shown to play a role in lignin polymerization in Arabidopsis thaliana [39, 41, 52]. The PbLAC4-like was screened out from the different genes between ‘Zaosu’ and ‘Red Zaosu’ leaves [49], so we speculated that the PbLAC4-like protein involved in the degradation of anthocyanin. Sequence alignment revealed that PbLAC4-like protein, LAC in Litchi chinensis and Arabidopsis thaliana had three Cu-oxidase domains that could oxidize substrates [53]. The structure indicated that PbLAC4-like protein probably participated in the degradation of anthocyanin by oxidizing anthocyanin.

The color fading of ‘Red Bartlett’ fruit might be related to LAC [34]. LAC involved in the degradation of anthocyanin during Litchi fruit pericarp browning [27]. LAC15 involved in the oxidation of flavonoids resulting in Arabidopsis thaliana seed coat browning [50]. In the present study, the function of the PbLAC4-like protein to degrade anthocyanin in pear was demonstrated. The content of anthocyanin in 'Palacer' fruitlet peel after overexpressing PbLAC4-like was less than that in control (Fig. 3). The activity to degrade anthocyanin of induced PbLAC4-like protein was significantly higher than that of control (Fig. 4). And we found that the color fading of pear leaves, petals, receptacles were all related to PbLAC4-like. However, in this study, PbLAC4-like protein directly degraded anthocyanin and did not need other substances to assist. This was different from the degradation of anthocyanin in Litchi fruit pericarp caused by LAC required the presence of epicatechin, and LAC played a role in anthocyanin degradation based on the oxidation of epicatechin [27]. In addition, which anthocyanin group did the PbLAC4-like protein act on and what were the degraded products were still unclear and need further study.

So far, there have been many studies on the regulators of LAC. MYB26, MYB46 and MYB83 in Arabidopsis thaliana [45–48], miR397a and MYB169 in pear [43, 44] regulated the expression of LAC. All of these studies were that the transcription factors involved in lignin accumulation by regulating LAC. However, there was no report that transcription factors involved in anthocyanin degradation by regulating LAC. Here, the expression pattern of MYB26 was consistent with that of PbLAC4-like in ‘Red Zaosu’ receptacles (Fig. 5a). Y1H and dual luciferase assay showed that PbMYB26 could directly bind to the PbLAC4-like promoter and activated its transcription, and this result was also verified by transient expression in pear (Fig. 5). However, whether PbMYB26 regulated PbLAC4-like by binding to the AC element in the PbLAC4-like promoter region remained to be further studied. Taken together, we hypothesized that PbMYB26 might involve in the degradation of anthocyanin by activating the PbLAC4-like promoter. These findings provide new ideas for further research on transcription factors that regulate the degradation of anthocyanin.

In this study, the effect of PbLAC4-like on the degradation of anthocyanin in pear was verified by transient transformation and enzyme activity determination. The upstream transcriptional regulators of PbLAC4-like have been preliminarily explored. PbMYB26 might relate to the degradation of anthocyanin by directly activating the PbLAC4-like promoter. Taken these results together, PbLAC4-like played an important role in the color fading process of pear leaves, petals and receptacles. Studying the degradation mechanism of anthocyanin can not only better understand the color fading process in pear, but also provide a new perspectiv for inhibiting the degradation of anthocyanin and promoting the accumulation of anthocyanin.

Plant materials and treatment methods

In this study, pear leaves, petals and receptacles were collected from the Horticultural Research Base of Northwest A&F University in Yangling, Shaanxi, China. The Red, Half Red and Green leaves of ‘Zaosu’ (Pyrus bretschneideri Rehd.), ‘Red Zaosu’ (Pyrus bretschneideri Rehd.), ‘2 hao’ (Inter specific Pyrus hybrid), ‘7 hao’ (Inter specific Pyrus hybrid), ‘Cuiguan’ (Pyrus pyrifolia Nakai.) were collected from the upper, middle and lower phyllotaxis of their branches respectively in mid-May. ‘Zaosu’, ‘Red Zaosu’ petals and ‘Red Zaosu’ receptacles of four different stages were collected from prophase to full bloom. All samples were collected and immediately frozen in liquid nitrogen and stored at -80℃. Moreover, 'Palacer' (Pyrus communis L.) and ‘Zaosu’ fruitlets grown in Meixian, Shaanxi, China, were selected for transient transformation to verify the function of PbLAC4-like and PbMYB26. Nicotiana benthamiana seedlings that had six leaves were used for dual luciferase activity assay.

The details of plant materials

‘Zaosu’ was identified formally by the Institute of Fruit Science, Chinese Academy of Agricultural Sciences. ‘Red Zaosu’, ‘2 hao’ and ‘7 hao’ were identified formally by our lab, while ‘Red Zaosu’ was a spontaneous bud sport of the ‘Zaosu’, ‘2 hao’ and ‘7 hao’ were the hybrid offspring of Pyrus pyrifolia, ‘Cuiguan’ was identified formally by Zhejiang Academy of Agricultural Sciences. These materials have been deposited in a publicly available herbarium. They were stored in the Pear Variety Resource Nursery of Northwest A&F University. The permissions were obtained to cultivate all the plants used in the study.

DNA and RNA extraction and purification

The total RNA was extracted using an RNAprep Pure Plant Kit (TIANGEN, Beijing, China) in accordance with the manufacturer’s instructions. The RNA concentration and quality were tested using Multiskan GO (Thermo, MA, USA). The first-strand cDNA was synthesized from 1 µg of total RNA using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China).

Sequence analysis of LAC

The protein sequences of LAC from Arabidopsis thaliana, Litchi chinensis, and Pyrus bretschneideri were used to construct a phylogenetic tree in the MEGA 5.0 software by Neighbor–Joining method and JTT+G model. Bootstrap values were calculated from 1000 replicate analyses. The protein sequences were aligned using DNAMAN. The GenBank accessions of related protein are listed in Additional file 3: Table S1.

Expression analysis using quantitative real-time PCR (qRT-PCR)

The primers for selected genes and PbActin (an internal control) were designed on NCBI web pages and were synthesized by AuGCT Biotech Company (Beijing, China). The primers are listed in Additional file 4: Table S2. The qRT-PCR reactions were performed on an Applied Biosystems StepOnePlus™ Real-Time PCR Systems (Applied Biosystems, Waltham, MA, USA) with TB Green Premix Ex Taq II (Tli RNaseH Plus; TaKaRa, Dalian, China) according to the manufacturer's instructions. Transcript levels of three biological replicates were analyzed using the cycle threshold (2^−ΔΔCt) method.

Determination of the anthocyanin content

The extraction and quantification of anthocyanin were performed using a previously reported method, with slight modifications [54]. The anthocyanin was extracted using homogenizing method with polyphenol extracting solution, which consisted of 50% methanol, 48% water and 2% formic acid at 4 °C. The supernatant was filtered by 0.22 μm organic filter for the determination of anthocyanin by high performance liquid chromatography (HPLC). Anthocyanin was analyzed using a LC-20A Liquid Chromatograph equipped with a diode array detector (Shimadzu Corporation, Tokyo, Japan). An Inertsil ODS-3 column (5.0 μm, 4.6 × 250 mm, GL Sciences Inc., Tokyo, Japan) was used in the separation. Solvent A consisted of 10% formic acid (HPLC grade, purity: 88%) and 90% water, and solvent B was 10% formic acid (HPLC grade, purity: 88%) dissolved in acetonitrile (HPLC grade, purity: 99.9%). The gradient elution procedure was 92% solvent A (0 min), 60% solvent A (10 min), 92% solvent A (24 min). The flow rate was 1.0 mL min⁻¹ at 30°C. Simultaneous monitoring was performed at 520 nm for anthocyanin (cyanidin-3-galactoside). Peaks were identified by a comparison of retention times and UV spectra with anthocyanin standard. The concentrations of anthocyanin in three biological replicates were determined according to the peak areas and calibration curves that were made with different concentrations of anthocyanin standard. The standard was obtained from Yuanye Bio-Technology (Shanghai, China).

Transient overexpression assay in pear fruitlet peel

The transient overexpression method referred to previous studies [16, 17]. The complete coding sequences (CDS) of PbLAC4-like and PbMYB26 were amplified by PCR from ‘Red Zaosu’ cDNA and then fused into the multiple cloning site (MCS) of pGreenⅡ 0029-62SK vector respectively to form 62SK-PbLAC4-like and 62SK-PbMYB26 plasmids. The CDS of GUS was PCR-amplified from pBI121 and then inserted into the MCS of pGreenⅡ 0029-62SK vector to form 62SK-GUS plasmid that as a control Empty. The primers for amplifying the sequences are described in Additional file 5: Table S3. The infusion vectors were transferred into Agrobacterium tumefaciens strain EHA105, and incubated in Luria-Bertani medium. After the activation of Agrobacterium, they were suspended with resuspension (10 μmol/L MES, 10 μmol/L MgCl₂, pH=5.6, and 200 μmol/L AS) and cultured in dark for 3 hours at room temperature, and then the OD₆₀₀ was adjusted to 0.6. The 62SK-GUS and 62SK-PbLAC4-like bacterial solutions were injected into 'Palacer' fruitlet peel with faded color after bagging. The 62SK-GUS and 62SK-PbMYB26 bacterial solutions were injected into 'Zausu' fruitlet peel. Each treatment consisted of 3 biological replicates and each biological replicate contained 5 fruitlets. Three days after injection, pericarp at the injection site was collected for quantitative analysis, and the phenotypic was analyzed ten days after injection. To prove the feasibility of this transformation method, GUS staining was performed on the transient GUS sites according to the previous method [55].

Crude enzyme extraction and PbLAC4-like protein activity assay

The crude enzyme was extracted by homogenizing the powder with polyvinylpyrrolidone (10% of the leaf by weight) and 2mL extracting solution which consisted of 0.02 M citrate buffer (pH 5.0), 0.02 M anhydrous calcium chloride, 0.005 M Dithiothreitol, 0.01 M thiourea. The homogenate was centrifuged for 20 min at 12,000 rpm and 4℃, and then the supernatant was collected after passing the PD-10 desalting column (GE Healthcare) as the crude enzyme.

The PbLAC4-like protein activity assay was performed according to the method of Zhang et al. [30] with minor changes. The enzyme was added to the 200 μL 0.2 M citrate buffer (pH 4.0) with 0.2 mM anthocyanin standard sample. The mixture was incubated for 30 minutes at 42℃. The reaction was terminated with 0.1 M hydrochloric methanol solution. The content of anthocyanin was measured by HPLC. The PbLAC4-like protein activity was expressed as the degradation of 1 μmol anthocyanin per minute at 42℃.

Prokaryotic expression of the PbLAC4-like

Prokaryotic expression of the PbLAC4-like was carried out according to the method of Kampatsikas et al. [56] with slight modifications. The CDS of PbLAC4-like was PCR-amplified and cloned into the pET-28a (+) vector. The primers are listed in Additional file 5: Table S3. The resulting construct was transformed into Escherichia coli strain BL21 (DE3). The E. coli BL21 (DE3) was cultured in 2×YT medium with ampicillin (100 µg/ml) at 37℃ until its OD₆₀₀ value was 0.6. Then 0.5 mM isopropyl β-D-1-thiogalactopyranoside and 0.5 mM CuSO₄ were added for protein induction. The expression culture was shaken for 8 hours at 28℃, centrifuged at 4℃, and the supernatant was discarded. The precipitation was suspended with 0.02 M citrate buffer (pH 5.0) for ultrasonic crushing. The broken expression culture was centrifuged at 12000 rpm for 30 minutes at 4℃, and the supernatant was taken for enzyme activity assay.

Yeast one-hybrid assay

The PbLAC4-like promoter sequence was cloned into the bait vector pAbAi, and the CDS of PbMYB26 was inserted into the prey vector pGADT7. The primers used to amplify the PbLAC4-like promoter and the CDS of PbMYB26 are listed in Additional file 5: Table S3. The pAbAi-bait vector was digested by BbsI and transferred into yeast strain Y1HGOLD to construct the bait yeast strain. To determine the minimum aureobasidin A (AbA) inhibitory concentration of the bait yeast strain, the bait yeast strain was cultured on the SD/-Ura medium containing 50-200 ng ml^-1 AbA. Then, transferring the prey plasmid into the bait yeast strain and culturing it on the SD/-Leu medium containing the minimum AbA inhibitory concentration that was screened out before to verify the interaction.

Dual luciferase assay in Nicotiana benthamiana leaves

To assay the effect of PbMYB26 on PbLAC4-like, the promoter of PbLAC4-like was amplified and inserted into the MCS of pGreenII 0800-LUC double-reporter plasmid as reporter. The effector plasmid was constructed by inserting the CDS of PbMYB26 into the MCS of pGreenⅡ 0029-62SK vector. The related primers are listed in Additional file 5: Table S3. Agrobacterium tumefaciens containing reporter plasmid and effector plasmid was injected into Nicotiana benthamiana leaves at a ratio of 1 to 4. The empty pGreenⅡ 0029-62SK plasmid and pGreenII 0800-LUC-proPbLAC4-like plasmid were injected into Nicotiana benthamiana leaves in the same proportion as control. After injection, Nicotiana benthamiana was cultured in dark for 12 hours and then cultured under the light. The activity of LUC and REN was measured three days after injection using a dual LUC assay kit (Promega, Madison, WI, USA) and an Infinite M200pro Full Wavelength Multifunctional Enzyme Standard Instrument (TECAN, Männedorf, Switzerland). Five biological repeats were included for each treatment.

Statistical analysis

Statistical analysis was performed using SPSS 20 software (SPSS, Chicago, IL, USA). One-way analysis of variance (ANOVA) with Tukey’s honestly significant difference test was conducted to determine the significant differences among the data. The correlation analysis among the date was carried out by SPSS 20 software. Each value represents the mean ± SEM of three biological replicates. Figures were made using GraphPad Prism 6 software (GraphPad Prism, San Diego, CA, USA).

UFGT: Flavonoid 3-O-glucosyl transferase; MBW: MYB-bHLH-WD40; POD: Peroxidases; PPO: Polyphenol oxidases ; LAC: Laccases; CDS: Complete coding sequences; MCS: Multiple cloning site; AbA: Aureobasidin A; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; Y1H: Yeast one-hybrid; OE: Overexpression; S: Stage; PCR: Polymerase chain reaction.

Acknowledgement

We are grateful to the College of Horticulture of Northwest A&F University for providing lab facilities to carry out the experiments.

Funding

This work was supported by the National Natural Science Foundation of China (31972372, 31572086, and 31401845) and the China Agriculture Research System (CARS 28-45).

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

Authors' contributions

Lingfei Xu and Rui Zhai: experiment design, review of the manuscript and financial support; Chengquan Yang, Zhigang Wang and Fengwang Ma: experiment design and review of the manuscript; Xieyu Li and Linyan Song: experiment design and implementation; Fangxin Xiang, Shichao Zhang and Junxing Song: experiment design, implementation and manuscript writing; Guangping Zhao: experiment design and implementation, data analysis, manuscript writing.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable.

Competing interests

All the authors declare that they have no competing interests.

Statement

The study complies with relevant institutional, national, and international guidelines and legislation. The permissions were obtained to cultivate all the plants used in the study.

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No competing interests reported.

Table1.xlsx
Correlation analysis of the anthocyanin content and the expression level of PbLAC4-like gene in leaves.
Additionalfile1Fig.S1.tif
GUS staining of the pear fruit after injecting pGreenⅡ0029 62SK-GUS.
Additionalfile2Fig.S2.tif
Screening of upstream regulators of PbLAC4-like. a Phylogenetic analysis of MYB46 in Arabidopsis thaliana and MYB transcription factors in pears. b Expression analysis of candidate upstream regulators in the receptacles of ‘Red Zaosu’.
Additionalfile3TableS1.xlsx
The GenBank accessions of related proteins.
Additionalfile4TableS2.xlsx
Sequences of primers used for qRT-PCR analysis.
Additionalfile5TableS3.xlsx
Sequences of primers used for vector construction.
Additionalfile6Titlepage.docx
Title page.
Additionalfile7TheoriginalgelofFig.4b.tif
The original gel of Fig. 4b.

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PbLAC4-like, activated by PbMYB26, related to the degradation of anthocyanin during color fading in pear

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