5.1 Plant materials
The tea plants (Camellia sinensis L. O. Kuntze cv. ‘Yanlinghuayecha’) were cultivated at the Tea Research Institute of Hunan Province, China. On April 4, 2019, tea leaves with one bud and two leaves were randomly plucked, washed using ultrapure watern and wiped dry. Then, the green sector (G), albino sector (A), and mosaic sector (M) were cut with scissors, fixed with liquid nitrogen immediately and brought back to the lab for storage in a -80 ℃ refrigerator.
5.2 Measurement of chlorophyll abundance
An aliquot of the ground samples (0.02 g) was mixed with 95% ethanol and incubated in darkness for 12 hours. The chlorophyll extract was filtered and analysed with a NanoDrop 2000 ultraviolet spectrophotometer (Thermo Fisher Scientific, USA). The ultraviolet absorption wavelengths of chlorophyll a and b and total carotenoids were recorded as follows: absorption peaks of 665 nm for chlorophyll a, 649 nm for chlorophyll b, and 470 nm for total carotenoids. The abundances of chlorophyll a, chlorophyll b, and total carotenoids were calculated.
5.3 Transmission electron microscopy (TEM)
Small pieces of fresh leaf tissue (1.0 mm × 1.0 mm) were obtained from green, albino and mosaic sectors of each individual and then cut and fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) at 4℃ for 12 hours. After removing OsO4, the tissues were rinsed in 0.1 M sodium phosphate buffer (pH 7.4) 3 times for 15 min each. These samples were then fixed in pure acetone for 20 minutes following ethanol-series dehydration. These materials were then embedded in Spurr’s resin (DER = 6.0) (Spurr, 1969) and polymerized at 70 °C for 12 h. These samples were then cut into semithin sections (60-80 nm) with an ultramicrotome (Leica UC7, Leica Microsystems, Wetzlar, Germany), and the tissues were removed onto 150 mesh cuprum grids with a formvar film. For further transmission electron microscopy (TEM) examination, sections were stained with 2% uranium acetate saturated alcohol solution for 8 min, rinsed in 70% ethanol 3 times, and then rinsed in ultrapure water 3 times. Then, 2.6% lead citrate was added to avoid CO2 staining for 8 min, and then samples were rinsed with ultra-pure water 3 times. After the sections were dried by filter paper, the cuprum grids were put into the grid board and dried overnight at room temperature. Then, the cells were examined using TEM (HT7800, Hitachi, Tokyo, Japan).
5.4 Determination of the free amino acid content
The free amino acid content was determined by using an HPLC (Agilent Technology, San Diego, CA, USA) with an ASB C18 analytical column (250 mm × 4.6 mm, 5 μm) according to the following setup: injection volume, 2.0 μL; column temperature, 30 °C; mobile phase A, 97% sodium acetate (0.1 M) and 3% acetonitrile, pH 6.5; solvent B, 80% acetic acid and 20% water; mobile phase flow rate, 1.0 mL/min; detection wavelength, 254 nm. Gradient conditions were such that 93% of solution A was maintained for 5 min; this was then changed to 62% solution A and 38% solution B until 25 min, then to 100% solution B from 30 to 40 min, both in a linear manner; finally, solution B decreased to 7% for 5 min and this was held for 10 min. Individual amino acids were identified and quantified by comparing their retention times and peaks with those of authentic standards. Their contents were expressed as percent dry weight of tea samples. Standard samples of free amino acids were purchased from Sigma-Aldrich Chemical Reagent Co., Ltd. (SigmaAldrich, St. Louis, MO, USA). All chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, People’s Republic of China). The total amount of free amino acids was determined by weighing 3 g of the sample and 450 ml of boiling distilled water into a 500 ml Erlenmeyer flask, immediately heating it in water for 45 minutes, and shaking it several times during this period. Then, the hot solution was filtered, and the filtrate was diluted to 500 ml. After the solution was cooled, 1 ml of the solution, 0.5 ml of phosphate buffer solution, and 0.5 mL of ninhydrin were absorbed in a 25 ml test tube and heated at 100 °C for 15 minutes. After cooling the solution, the volume was brought up to 25 ml with distilled water and a spectrophotometer (NanoDrop 2000 spectrophotometer) was used to measure the absorbance at 570 nm.
5.5 RNA extraction, cDNA library preparation, and sequencing
Extraction, cDNA library preparation, and sequencing of libraries were performed using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations. The RNA concentration was measured using a Qubit® RNAAssay Kit in a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA), and RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyser 2100 system (Agilent Technologies, CA, USA).
A total of 3 μg of mixed RNA from each sample was used for cDNA library construction. Sequencing libraries were prepared using the NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, MA, USA) following the manufacturer’s instructions. Briefly, the mRNA was purified using Oligo (dT). The purified mRNA was fragmented using NEBNext First Strand Synthesis Reaction Buffer. The first-strand cDNA was then synthesized using random hexamer primers, and then the second strand was synthesized using RNaseH, DNA Polymerase I, and dNTPs. After adenylation of the 3′ end and purification of the cDNA library, polymerase chain reaction (PCR) was performed using Phusion HighFidelity DNA polymerase. Finally, the PCR products were purified using the AMPure XP system (Beckman Coulter, Indianapolis, IN, USA), and the library quality was assessed using the Agilent 2100 Bioanalyser. After clustering with TruSeq PE Cluster Kit v3-cBot-HS (Illumina), the generated cDNA library was sequenced on the Illumina HiSeqTM 2500 platform (Biomarker Biotech, Beijing, China), and paired-end reads were generated. Three biological replicates were conducted for each sample.
5.6 Quality control and transcriptome analysis
The clean data were produced by removing low-quality reads, adapters, and reads with poly-A. The clean reads were aligned to the tea plant genome by HISAT2 v2.0.5. FeatureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. Then, the FPKM value of each gene was calculated based on the length of the gene and read counts mapped to this gene. Differential expression analysis of three groups (three biological replicates per condition) was performed using the DESeq2 R package (1.16.1). Genes with an adjusted P-value <0.05 found by DESeq2 were considered differentially expressed. Gene Ontology (GO) enrichment analysis of DEGs was implemented by the cluster profile R package, in which gene length bias was corrected. GO terms with a corrected p-value less than 0.05 were considered significantly enriched by DEGs. We used the cluster profile R package to test the statistical enrichment of DEGs in KEGG pathways (https://www.genome.jp/kegg/). New gene prediction was performed with StringTie differential expression analysis of fragments.
5.7 Phylogenetic analysis
To better understand the possible functions of the above-identified thea-related TF genes, we focused on the MYB, bHLH, WRKY, and WD40 genes involved in theanine biosynthesis. Phylogenetic analysis was conducted based on the protein sequences of the screened MYB genes in tea plants and their counterpart MYB genes in five plant species, including Arabidopsis thaliana (https://www.arabidopsis.org/), Actinidia chinensis, Vitis vinifera, Prunus persica, and Theobroma cacao (https://www.ncbi.nlm.nih.gov/). Multisequence alignments were performed using ClustalX32 (version 1.8). A phylogenetic tree was then constructed using MEGA33 (version 6.0) through the neighbour-joining method and tested using the bootstrap method with 1000 replicates.
5.8 qRT-PCR validation
16 DEGs were validated by real-time quantitative reverse transcription PCR (qRT-PCR). Total RNA was reverse transcribed into cDNA using a PrimeScript RT-PCR Kit (TaKaRa, Otsu, Shiga, Japan). Quantitative PCR amplification was conducted on the ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). A 20 μL portion of the PCR system and TB Green Premix Ex Taq (TaKaRa, Otsu, Shiga, Japan) were used for PCRs. The PCR amplification conditions were as follows: 90 s of degeneration at 95 °C, followed by 30 s of degeneration at 95 °C, 60 s of annealing at 55 °C, for a total of 40 reaction cycles, and finally 1 min of extension at 60 °C. Dissociation curves were collected at temperatures from 60 to 95 °C. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The sequences of differential gene primers were listed (Table S2). The relative expression levels of the genes were normalized by the 2−ΔΔCT method.
5.9 Statistical analysis
Analysis of variance was performed with SPSS software (v22.0, SPSS Inc., Chicago, IL) to determine the least significant differences between different treatments (p < 0.05), and Duncan’s multiple range test was used to compare the averages. The mean and standard deviation (SD) were calculated based on three independent biological replicates. GraphPad Prism (v8.0.1) was used to process the data and generate the figures. TBtools and pseudoQC were used to analyse correlations.