## 4 Materials and Methods  
### Plant Materials and Treatment  
For phenotype difference experiment, mint (_Mentha canadensis_) plants were grown on the field in Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, Jiangsu Province, China (28\({}^{\circ}\)05\({}^{\prime}\) N, 113\({}^{\circ}\)37\({}^{\prime}\) E). Low-light treatment group was performed by covering plots with sunshade nets at a height of 2.5m above the ground, which caused a reduction of 50% light. The control group grown at normal Sunshine. Two groups both grown in the same soil with the same 12 h photoperiod. In total, three replications were made for the control and the low light groups, respectively, each replication containing 10 plants. After 30 d low light treatment, leaves were harvest to statistic of peltate glandular trichomes densities and analysis essential oil composition. For transcriptome analysis, mint (_Mentha Canadensis_) were propagated from rhizomes and were planted in plastic pots containing a mixture of soil and vermiculite (3:1, _v/v_). Plants were cultured in growth chamber under control condition at 1000 \(\mu\)mol\(\cdot\)m\({}^{-2}\cdot\)s\({}^{-1}\), with the 14 h photoperiod and temperature maintained at 25 \({}^{\circ}\)C/22 \({}^{\circ}\)C. For darkness treatment, black paper bags was used to cover the whole plants for 24h, then move the bags and exposure the plants to light 24 h. Leaves at the same developing stage were collection at 24h darkness treatment time point and at 24 h illumination time point. Control materials were collected at the same time point as darkness treatment. Three plants were selected as biological replicates. Samples were frozen in liquid nitrogen and stored at \(-80\)\({}^{\circ}\)C before RNA extraction. Statistics of Peltate Glandular Trichomes Densities and Measurement of Essential Oil Composition in Mint Leaves under Darkness Treatment  
Leaves at the same development stage were divided into five sampling zones from bottom to top. The number of peltate glandular trichomes in each 1 mm\({}^{2}\) were countedafter taking a photograph using fluorescence stereomicroscope MVX 10. The analysis was performed in fifth repetition. Sampling of volatiles from 0.5 g dry weight leaves were collected by headspace solid-phase microextraction (HS-SPME), followed by gas chromatography/mass spectrometry (GC/MS) determination. The polydimethylsiloxane (PDMS) 65 \(\upmu\)m fiber (65 \(\upmu\)m Carboxen(tm)-PDMS StableFlex, Supelco) was then exposed to the headspace of the sample for 3 min at 60 \({}^{\circ}\)C to adsorption. Then volatiles absorbed were desorbed in the injection port of GC at 250 \({}^{\circ}\)C for 3 min. Each sample was spiked with 300 ng bormanone internal standard. The analysis was performed in triplicate. GC analyses were carried out using a Trace 1300 gas chromatograph equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. GC setting were as follow: inlet 250 \({}^{\circ}\)C, the carrier gas was N2 (U) with a split flow 50.0 mL/min and the split ratio was 33:1. Analyses were performed using the following temperature program: oven temperature isotherm at 60 \({}^{\circ}\)C for 1 min, from 60 \({}^{\circ}\)C to 220 \({}^{\circ}\)C at the rate of 3 \({}^{\circ}\)C min\({}^{-1}\). GC-MS analysis was performed on a gas chromatograph Trace 1300 HP interfaced with a HP 5972 mass spectrometer with electron impact ionization (70 eV). The column temperature was programmed as follows: initial temperature was 50 \({}^{\circ}\)C, increased to 240 \({}^{\circ}\)C at 5 \({}^{\circ}\)C min-1, and isotherm at 205 \({}^{\circ}\)C during 10 min. Helium was used as the carrier gas with a flow rate of 1.0 mL/min\({}^{-1}\). The split ratio was 33:1. Scan time and mass range were 0.2 s and 33-450 \(m/z\), respectively.