Plant materials and growing conditions
In the present study, ‘Red Zaosu’ (P. bretschneideri Rehd.) and ‘Zaosu’ (P. bretschneideri Rehd.) were collected from the Horticultural Research Base of Northwest A&F University in Yangling Distinct, Shaanxi Province, China. The pear fruit were harvested and sampled at four representative points during the whole-fruit growth period, including 5 d before pollination (BP), 15 d after pollination (AP), 50 d after pollination (DP) and 110 d after pollination (fruit ripen period, RP). Moreover, the pear fruit in DP and three-day-old leaves of tissue cultured pear plants were selected to transiently verify the bio-funtion of PbMYB12b. The detailed experimental design was illustrated in Additional file 1: Figure S1.
Fresh plant tissues for later use were immediately frozen, ground to a powder in liquid nitrogen and stored at − 80 °C.
DNA and RNA extraction and purification
Total DNA and RNA were extracted and purified by SDS solubilization and phenol extraction .
RNA-seq and analysis
During the DP, 3 μg total RNA were extracted from receptacles and fruit skins of ‘Red Zaosu’ and ‘Zaosu’, with three biological replicates. These were used for sequencing. Briefly, total RNA was randomly fragmented, reversed-transcribed, amplified and purified to form cDNA libraries. After the assessment of the cDNA libraries using an Agilent Bioanalyzer 2100 system, the library preparations were paired-end sequenced (100 bp) on an Illumina HiSeq2500 platform. Clean reads were screened by removing reads containing adapters and poly-Ns, and low-quality reads, from the raw data. Paired-end clean reads were aligned to the pear genome using TopHat . Genes with q-values < 0.05, as assessed by the DESeq R package, and fold changes > 2 were assigned as differentially expressed genes between ‘Red Zaosu’ and ‘Zaosu’ .
In total, 2 μg purified RNA was reverse-transcribed to cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). The primer pairs of selected genes and PbActin (internal control) are listed in Additional file 1: Table S2. PCR reactions were performed on a StepOnePlus PCR system (Applied Biosystems) with SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer’s instructions. Expression data of three biological replicates were analyzed according to the cycle threshold 2−ΔΔCt method.
The transient expression assays were performed as previously described, with a modified infiltration method . For the overexpression of PbMYB12b, the full-length PbMYB12b CDS was PCR-amplified from ‘Red Zaosu’ cDNA sources and then replaced the GFP-coding sequence in the pCambia 1301 binary vector using ClonExpress One Step Cloning Kit (Vazyme) based on homologous recombination technology. For the RNAi of PbMYB12b using VIGS, a 479-bp fragment of the PbMYB12b CDS was PCR-amplified from ‘Red Zaosu’ cDNA sources and then inserted into the MCS region in the pTRV2 vector using a ClonExpress One Step Cloning Kit (Vazyme) based on homologous recombination technology. For the record, the non-specific silencing of PbMYB12a is inevitable. The mRNA sequence homology between PbMYB12a and PbMYB12b were 89%, and for the 479-bp fragment were 92%. The plasmid was transferred into the Agrobacterium tumefaciens strain GV3101. The Agrobacterium cells containing pCambia 1301-GUS, pCambia 1301-PbMYB12b, pTRV2-PbMYB12b, original pTRV2 and pTRV1 were suspended at 28 °C in LB medium containing 10 mM MES and 20 μM acetosyringone with appropriate antibiotics, respectively. Agrobacterium cells were harvested and resuspended for 4 h in infiltration buffer (10 mM MgCl2, 10 mM MES (pH 5.6) and 100 μM acetosyringone) to a final OD600 = 0.8 at room temperature before infiltration. Fruit infiltration was performed by injecting 1 mL Agrobacterium cells. Leaf infiltration was performed by vacuuming instead of injection. Young leaves (three-day-old) of ‘Zaosu’ and ‘Red Zaosu’ explants that cultured in 1/2 MS solid medium (pH = 5.5) were totally immersed and vacuumed in 2 mL Agrobacterium suspensions at 25 kPa for 5 min. For transient overexpression, the DP fruit and young leaves of ‘Zaosu’ infiltrated by pCambia 1301-PbMYB12b were sampled at 48 h after infiltration, the DP fruit and young leaves of ‘Zaosu’ infiltrated by pCambia 1301-GUS and the un-infiltrated DP fruit and leaves were also sampled as controls. For transient RNAi, the DP fruit and young leaves of ‘Red Zaosu’ co-infiltrated by pTRV2-PbMYB12b and pTRV1 were sampled at 48 h after infiltration, the DP fruit and young leaves of ‘Red Zaosu’ co-infiltrated by pTRV2 and pTRV1 and the un-infiltrated DP fruit and leaves were also sampled as controls. For GUS staining, the plant materials were stained with 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) at 37 °C for 12 as described . The primer pairs are listed in Additional file 1: Table S2.
Analysis of flavonoid compounds
The extraction and analysis of flavonoid compounds were carried out as previously described . Briefly, the flavonoids were extracted with 70% methanol containing 2% formic acid at 0–4 °C. The supernatant was filtered through a 0.45-μm syringe filter prior to HPLC analysis. Phenolic compounds were analyzed using a HP1200 Liquid Chromatograph equipped with a diode array detector (Agilent Technology, Palo Alto, CA, USA). An Inertsil ODS-3 column (5.0 μm, 4.0 × 250 mm, GL Sciences Inc., Tokyo, Japan) was used in the separation, preceded by an Inertsil ODS-3 Guard Column (5.0 μm, 4.0 × 10 mm). Solvent A consisted of 10% formic acid (11.36% of 88% formic acid) dissolved in water, and solvent B was 10% formic acid (11.36% of 88% formic acid) and 1.36% water in acetonitrile (HPLC grade, purity: 99.9%). The gradient was 95% solvent A (0 min), 85% solvent A (25 min), 78% solvent A (42 min), 64% solvent A (60 min) and 95% solvent A (65 min). The post-run-time was 10 min. The flow rate was 1.0 mL min− 1 at 30 °C. Simultaneous monitoring was performed at 365 nm for quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-arabinoside, quercetin-3-rutinoside, isrohamnetin-3-galactoside and isrohamnetin-3-gluctoside, and at 520 nm for cyanidin-3-galactoside. Peaks were identified by a comparison of retention times and UV spectra with those of authentic standards. The concentrations of individual phenolic compounds were determined based on peak areas and calibration curves that were derived from the corresponding authentic phenolic compounds. All of the phenolic standards were obtained from Sigma Aldrich (St Louis, MO, USA), Extrasynthese (Genay Cedex, France) and AApin Chemicals (Abingdon, Oxon, UK).
Sub-cellular localization of the PbMYB12b protein in the onion epidermis
The full-length PbMYB12b CDS was amplified and fused into pCambia 2300 to generate the transgene construct p35S::GFP-PbMYB12b using a ClonExpress One Step Cloning Kit (Vazyme) based on homologous recombination technology. The construct was transiently introduced into onion epidermal cells by injection, and control cells were transformed in the same way with p35S::GFP. After their transformation, the onion epidermal samples were held for 16 h at 25 °C in the dark, after which GFP fluorescence was imaged using a BX51 + PD72 + IX71 microscopic imaging system (OLYMPUS).
Dual-LUC reporter assays in tobacco leaves
To assay the effects of PbMYB12b on PbCHSb and PbFLS, their promoters were amplified and inserted into pGreenII 0800-LUC double-reporter plasmid as reporters (primer pairs are listed in Additional file 1: Table S2) . The effector plasmid was constructed by inserting PbMYB12b into the p62-SK vector. The constructed effector and reporter plasmids, in different combinations, were co-transformed into tobacco leaves.
The activities of LUC and REN were quantified 48 h after infiltration with a dual LUC assay kit (Promega) using a Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific). The transcriptional capability of PbMYB12 was assessed by the LUC/REN ratio. Five biological repeats were included for each pair.
GUS reporter assays in tobacco leaves
To assay the effects of PbMYB12b on PbCHSb and PbFLS, their promoters were individually inserted into the pBI121-GUS plasmid as reporters. An effector plasmid was constructed by inserting PbMYB12b into the p62-SK vector. The constructed effector and reporter plasmids, in different combinations, were co-transformed into tobacco leaves. The GUS activity was measured as described previously .
To determine significant differences among the data, Student’s t test was conducted using SPSS 16.0.