## 4 Materials and Methods
### RNA Extraction, Sequencing, and Annotation  
Leaf samples were fully ground with liquid nitrogen, and 200 mg sample was used for total RNA extraction with an EASYspin Plus CompleSx Plant RNA Kit following the manufacturer's instruction (Aidlab Biotech, Beijing, China). Then the integrity and purity of the total RNA quality was determined by a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA) and quantified using the NanoDrop-2000 (Thermo Scientific, Wilmington, DE, USA). Only a high-quality RNA sample (OD260/280 = 1.8-2.2, OD260/230 \(\geq\) 2.0, RIN \(\geq\) 8.5, 28S:18S \(\geq\) 1.0, \(>\)2 \(\upmu\)g) was used to construct the sequencing library. The NEBsNext(r) Ultra(tm) RNA Library Prep Kit for Illumina(r)(r)E7530L, NEB, Ipswich, MA, USA) was used for the sequencing library construction [46]. Three biological replicates of each group were used for RNA-seq analysis. Poly(A) mRNA was purified from total RNA using oligo-dT-attached magnetic beads and then fragmented by using the fragmentation buffer. Taking these short fragments as templates, double-stranded cDNA was synthesized by using a SuperScript double-stranded cDNA synthesis kit with random hexamer primers. Then the synthesized cDNA was subjected to end-repair, phosphorylation, and \"A\" base addition, according to NEB's library construction protocol. Libraries were size selected for cDNA target fragments of 200-300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB, USA) for 15 PCR cycles.  
After the index-coded samples were clustered, the libraries were sequenced on a HiSeq X Ten platform, and 150 bp paired-end reads were generated. The raw RNA-seq reads were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA, study accession number: SRR8399985-SRR8399999). The raw paired end reads were trimmed and quality controlled by SeqPrep ([[https://github.com/jstjohn/SeqPrep](https://github.com/jstjohn/SeqPrep)]([https://github.com/jstjohn/SeqPrep](https://github.com/jstjohn/SeqPrep))) and Sickle ([[https://github.com/najoshi/sickle](https://github.com/najoshi/sickle)]([https://github.com/najoshi/sickle](https://github.com/najoshi/sickle))) with default parameters. Then clean data from the samples were used to complete _de novo_ assembly with Trinity ([[http://trinityrnaseq.sourceforge.net/](http://trinityrnaseq.sourceforge.net/)]([http://trinityrnaseq.sourceforge.net/](http://trinityrnaseq.sourceforge.net/))) [50]. All the assembled transcripts were searched against the NCBI protein non-redundant, String, and KEGG databases using BLASTX to identify the proteins that had the highest sequence similarity with the given transcripts to retrieve their function annotations and a typical cut-off E-values less than 1.0\(\times\)10\({}^{-5}\) was set. The BLAST2 GO ([[http://www.blast2](http://www.blast2) go.com/b2 ghome]([http://www.blast2](http://www.blast2) go.com/b2 ghome)) [51] program was used to get GO annotations of unique assembled transcripts for describing biological processes, molecular functions, and cellular components. Metabolic pathway analysis was performed by using the KEGG database ([[http://www.genome.jp/kegg/](http://www.genome.jp/kegg/)]([http://www.genome.jp/kegg/](http://www.genome.jp/kegg/))) [52].