Transformation of Salicylic Acid and Its Distribution in Tea Plants (Camellia sinensis) at the Tissue and Subcellular Levels

Salicylic acid (SA) is a well-known immune-related hormone that has been well studied in model plants. However, less attention has been paid to the presence of SA and its derivatives in economic plants, such as tea plants (Camellia sinensis). This study showed that tea plants were rich in SA and responded differently to different pathogens. Feeding experiments in tea tissues further confirmed the transformation of SA into salicylic acid 2-O-β-glucoside (SAG) and methyl salicylate. Nonaqueous fractionation techniques confirmed that SA and SAG were mostly distributed in the cytosol of tea leaves, consistent with distributions in other plant species. Furthermore, the stem epidermis contained more SA than the stem core both in C. sinensis cv. “Jinxuan” (small-leaf species) and “Yinghong No. 9” (large-leaf species). Compared with cv. “Yinghong No. 9”, cv. “Jinxuan” contained more SAG in the stem epidermis, which might explain its lower incidence rate of wilt disease. This information will improve understanding of SA occurrence in tea plants and provide a basis for investigating the relationship between SA and disease resistance in tea plants.


1.. Extraction and Analysis of Salicylic Acid (SA) 23
The extraction and analysis of SA in tea samples were referred to the previous study 24 [1]. Finely powdered sample (300 mg, fresh weight) was extracted with 3 mL ethyl acetate 25 by vortexing for 30 s followed by ultrasonic extraction in ice-cold water for 20 min. After 26 centrifuging at 10000×g for 5 min at 4 °C, 2.5 mL supernatants were collected. [ 2 H4]SA as 27 an internal standard was added to supernatants before dried under a stream of nitrogen.
33 Solvent A was Milli-Q water with 0.1% (v/v) formic acid. Solvent B was acetonitrile with 34 0.1% (v/v) formic acid. The solvent gradient was started at 20% B, then linearly increased 35 to 35% within 10 min, later increased to 95% B in 0.1 min and kept for 3 min. In that mo-36 ment, it suddenly dropped to 20% in 0.1 min and maintain for 3 min. The flow rate was 37 0.4 mL/min. The column temperature was 30 °C. The electrospray ionization operated on 38 negative mode. The MS conditions were capillary voltage: 1.5 kV; source temperature: 100 39 °C; desolvation temperature: 300 °C; cone gas flow: 50 L/ h; and desolvation gas flow: 600 40 L/ h. The quantitative analysis of SA in tea samples was based on the authentic standard. 41

Extraction and Analysis of Methyl Salicylate (MeSA) and [ 2 H4]MeSA 42
Extraction and analysis of volatile compounds were referred to the previous study 43 [2]. Finely powdered sample (200 mg) was extracted with dichloromethane (1.8 mL) con-44 taining ethyl decanoate (0.5 nmol) as an internal standard using a shaker at room temper-45 ature for 5-6 h. The extraction solution was collected, dried using anhydrous sodium sul-46 fate, and concentrated to 50-100 μL under a stream of nitrogen. The extract (1 μL) was 47 then subjected to gas chromatography-mass spectrometry (GC-MS) analysis carried on a 48  GC-MS QP2010 SE (Shimadzu Corporation, Kyoto, Japan) equipped with GCMS Solution 49 software (Version 2.72, Shimadzu Corporation, Japan). Samples were injected into the GC 50 injection port held at 230 °C for 1 min, with all injections made in splitless mode. Volatile 51 compounds were separated on a SUPELCOWAX 10 column (30 m × 0.25 mm × 0.25 μm, 52 Supelco Inc., Bellefonte, PA, USA). Helium was used as the carrier gas, with a velocity of 53 1 mL/min. The initial GC oven temperature was 60 °C for 3 min, which was ramped up to 54 240 °C at a rate of 4 °C/min, and then held at 240 °C for 20 min. Mass spectrometry was 55 operated in full scan mode (mass range, m/z 40-200). The characteristic ions of MeSA and 56 [ 2 H4]MeSA are m/z 120 and 124, respectively. The quantitative analyses of MeSA and 57 [ 2 H4]MeSA in tea samples were based on the unlabeled MeSA standard. 58

Extraction and Analysis of Salicylic Acid 2-O--Glucoside (SAG) and [ 2 H4]SAG 59
Sample was extracted with 1 mL 70% methanol and vortexed for 30 s. The mixture 60 followed by ultrasonic extraction in ice-cold water for 10 min. After centrifuging at 10000 61 g for 10 min at 4 °C, supernatants were collected and filtered through a 0.22 μm mem-

Determination of Subcellular Distribution of SA and SAG 77
The tea leaves from C. sinensis cv. Jinxuan plucked in March 2019 were fractionated 78 using a nonaqueous procedure according to the published studies with a slightly modi-79 fied [3][4][5][6]. The finely powered tea leaves (4 g, fresh weight) was placed into the lyophilizer 80 at 0.02 bar and -50 °C for 3 days. The dry powder was resuspended in tetrachlorethylene-81 heptane mixture (20 mL, 66:34 (v/v); density = 1.3 g/cm 3 ; the solvents were stored with 3 82 Å molecular sieve) and ultrasonicated for 2 min with 6 cycles of 10 s pules and 10 s breaks 83 at 65% power. The suspension was filtered through nylon net with a pore size at 20 μm, 84 washed the net 3 times with 10 mL of heptane, and centrifuged for 10 min at 3,200 g and 85 4 °C. After centrifugation, the organic supernatant was removed and the pellet was resus-86 pended in C2Cl4/C7H16 mixture (3 mL, 66:34 (v/v)). Aliquots (500 μL with 10×50 μL) were 87 withdrawn for determination of metabolites and enzyme activity in the unfractionated 88 material, and the remaining 2.5 mL of the suspension was loaded on the top of the gradi-89 ent. A linear gradient (25 mL, for leaf tissue between 1.43 and 1.50 g/cm 3 ) was made using 90 a gradient former connected to a peristaltic pump. The gradients were centrifuged for 1 h 91 at 3,800 g at 4 °C. The fractions (F1 to F5, 4-6 mL for each fraction) was carefully removed 92 from the top using Pasteur pipettes into a clean 50 mL tube. Three volumes of C7H16 were 93 added to each tube and the mixture was mixed well. The suspensions were centrifuged 94 for 10 min at 3,200 g at 4 °C in a swing-out-rotor centrifuge. The supernatants were dis-95 carded and the pellet was resuspended in 5 mL C7H16, and 10 aliquots of 500 μL of the 96 suspension were transferred into 2 mL tubes. The sample in tube was dried by N2 for 1 97 hour, and then extracted for assay of metabolites (SA and SAG) and enzymes (proteins). 98 The metabolites (SA and SAG) from the dried samples were resolved in 1 mL ethyl 99 acetate, and subjected to ultrasonic treatment for 20 min. After centrifugation, 800 μL su-100 pernatant was collected and dried using under a stream of nitrogen. The residue was re-101 solved in 100 μL methanol. The solutions were filtered through a 0.22 μm membrane, and 102 analyzed by UHPLC (Ultimate 3000, Thermo Scientific, Waltham, MA, USA) connected 103 with a triple quadrupole mass spectrometer (TSQ Endura, Thermo Scientific, Waltham, 104 U.S.A.). The sample (2 μL) was injected and separated on a Hypersil GOLD C18 column 105 (2.1 mm × 100 mm i.d., 1.9 μm). The flow rate was 0.3 mL/min. The column temperature 106 was 40 °C. The mobile phase contained acetonitrile solution (A) and water with 0.2% (v/v) 107 formic acid (B) with an initial condition of 95% of mobile phase B. The linear gradient 108 elution was carried out as follows: 0-3 min, 95% B; 3-20 min, 95%-5% B; 20-23 min, 5% B; 109 23-23.1 min, 5%-95% B; 23.1-26 min, 95% B. The electrospray ionization (ESI) was oper-110 ated in the negative ion mode, and the optimal conditions were peak width resolution set 111 at 0.7 m/z, spray voltage set at 2500 V, sheath gas pressure set at 35 units, auxiliary gas 112 pressure set at 15 units, and vaporizer temperature set at 300 °C. Selected reaction moni-113 toring (SRM) mode was used in MS/MS instrument to obtain the optimized selectivity and 114 sensitivity. The SRM transitions of SA and SAG are listed in Table 1. The quantitative 115 analyses of SA and SAG were based on calibration curves obtained from the authentic 116 standards.