Plant Materials and Sample Preparation
The chimeric leaves of two-year-old A. comosus var. bracteatus from an experimental field at Sichuan Agricultural University were used in this study. Two types of samples were collected: the green central parts (GR) and the red edges (RE) from chimeric leaves (Fig. 1). After being washed completely, the two types of samples were collected for color estimation, pigment content determination and anatomical cross sectioning. Additionally, samples were cut into small pieces and immediately frozen with liquid nitrogen. The frozen samples were stored at -80°C for further transcriptome and metabolome analyses.
Leaf Color Estimation
In this assay, the two types of samples were measured by a CM-2600d color analyzer (Konica Minolta CM-2600d, Japan) for color digitization. The measurement of leaf color was based on the Commission Internationale de l’Éclairage (CIE D65/10°) scale[53, 54].CIE color indexes include the lightness component L* and two chromatic components a* and b*. L* values from 0 to 100 indicate the range from black to white; values from -a* to +a* represent a decrease in green and an increase in red; values from -b* to +b* represent a decrease in blue and an increase in yellow. In addition, C* represents chroma, which is calculated by the following equation: C*=(a*2+b*2)1/2[55]. The color data were finally arranged and visualized by Excel 2019 and Photoshop 5.0.
Anatomical Cross Sectioning
To obtain a better understanding of the anthocyanin distribution in the tissues of GR and RE samples, the samples were cut into blocks of an appropriate size for freehand cross sectioning. Blocks were further cut by a pair of overlapping blades, and a section from the gap between the blades was immediately transferred onto a glass slide sealed with a cover glass. Sections were observed by a microscope (XSP-15CA, Shanghai CSOIF Co., Ltd) and photographed by Cellsens Standard software.
Chlorophyll and Carotenoid Content Determination
The chlorophyll and carotenoid contents of GR and RE samples were determined. A sample of 0.1 g was cut into slices and transferred into a 15 mL centrifuge tube with 5 mL 95% ethanol for extraction. The tube was kept in the dark until the samples became colorless. Chlorophyll and carotenoid absorption was measured at 665, 649 and 470 nm with a UV-Vis spectrophotometer (UV1901 s/UV1901PCS, Shanghai Youke Instrument Co., Ltd.) equipped with 1.0 cm quartz cells, and the concentration was calculated following the method of Arnon [56].
Anthocyanin Content Determination
The total anthocyanin contents of GR and RE samples were measured and calculated by using a pH differential protocol[57, 58]
Sample Extraction for Ultra‑Performance Liquid Chromatography (UPLC)/MS Analysis
Freeze-dried leaves stored at -80℃ were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. Then, 100 mg of the powder was weighed and extracted overnight at 4℃ with 0.6 ml 70% aqueous methanol. Following centrifugation at 10,000 g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 ml; ANPEL, Shanghai, China, www.anpel.com.cn/cnw) and filtrated (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) before the UPLC-MS/MS analysis.
UPLC Conditions
The sample extracts were analyzed using a UPLC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/). The analytical conditions for the UPLC analysis included an Agilent SB-C18 column (1.8 µm, 2.1 mm*100 mm), and the mobile phase for solvent A consisted of pure water with 0.1% formic acid and for solvent B consisted of acetonitrile. Sample measurements were performed with a gradient program that employed the starting conditions of 95% A and 5% B. A linear gradient to 5% A and 95% B over 9 min was programmed, and a composition of 5% A and 95% B was maintained for 1 min. Subsequently, a composition of 95% A and 5.0% B was adjusted within 1.1 min and maintained for 2.9 min. The column oven was set to 40°C, and the injection volume was 4 μl. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.
ESI-QTRAP-MS/MS
LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP) and API 4500 Q TRAP UPLC/MS/MS System equipped with an ESI Turbo Ion-Spray interface. The system was operated in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature, 550℃; ion spray voltage (IS), 5500 V (positive ion mode)/-4500 V (negative ion mode); ion source gas I (GSI), gas II(GSII), curtain gas (CUR), 50, 60, and 30.0 psi, respectively; collision gas (CAD), high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired via MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions were performed with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.
RNA Library preparation for transcriptome sequencing
A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via the exonuclease/polymerase activities. After adenylation of the 3’ ends of DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments with lengths of 150~200 bp (preferentially), the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq platform and 125 bp/150 bp paired-end reads were generated. Quality-filtered Illumina reads of the reference data are available in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJEB33121)[59].
Gene Annotation and Differential Gene Expression Analysis
All expressed genes were functionally annotated using the following databases: KEGG, Nr, Swiss-Prot, Tremble, KOG, GO, and Pfam. Gene expression levels were measured using FPKM (fragments per kilobase of transcript per million mapped reads) values based on the number of uniquely mapped reads[60]. For genes with more than one alternative transcript, the longest transcript was selected to calculate the FPKM value. Differential expression analysis of the three groups was performed using the DESeq R package (1.22.2). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini-Hochberg approach for controlling the false discovery rate (FDR). Genes with an adjusted P value < 0.05 found by DESeq were considered differentially expressed.
Validation of RNA‑seq Data by qPCR
Total RNA was reverse transcribed to generate first strand cDNA using an Evo M-MLV RT for PCR kit (Accurate Biotechnology, Hunan, Co., Ltd.). The α-tubulin was used as an internal control for normalization[61]. qPCR was performed on a qPCR instrument (BIORAD CFX96 real-time PCR detection system), and real-time PCRs were performed using a SYBR® Green Premix Pro TaqHS qPCR kit (Accurate Biotechnology, Hunan, Co., Ltd.). The 2−ΔΔCT method[62] was used for quantitative analysis of the relative expression levels. The primers used in the qPCR analysis are shown in Table 3.
Correlation Analysis between Metabolome and Transcriptome Data
Pearson correlation coefficients were calculated for metabolome and transcriptome data integration. In this study, log conversion of data was performed uniformly before analysis. For the joint analysis between the metabolome and transcriptome, the cor program from R was used, and the screening criterion was a Pearson correlation coefficient (PCC) > 0.8. The relationships between metabolomic and transcriptomic data were visualized by using Cytoscape.