Nano md uv vis

Total RNA extraction was performed using Pure link^TM RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the RNA quality was vitalized by agarose gel loading (Figure S3). The extracted total RNA was quantified using a spectrophotometer (Nano-MD UV-Vis, Scinco, Seoul, Korea). RevertAid Reverse transcriptase (Thermo, Waltham, MA, USA) was used in a 20 μL reaction volume to synthesize the complementary DNA (cDNA). Real-time quantitative PCR (qPCR) was performed using TB Green™ Premix Ex Taq™ (Takara, Shiga, Japan) and Thermal Cycle Dice real-time PCR system (Takara, Shiga, Japan) as previously described [4 (link)]. To determine the relative fold-differences in template abundance for each sample, the Ct value for each of the analyzed genes was normalized to the Ct value for β-actin (At5g09810) and was calculated relative to a calibrator using the formula 2-ΔΔCt. Three independent experiments were performed for each primer set (Table S1). The primer efficiency was determined according to the method of Livak and Schmittgen [25 (link)] in order to validate the ΔΔCt method. The gene-specific primer sequences for the target genes are listed in Supplementary Table S1.

Cellulose quantification in Arabidopsis was conducted according to a previously reported protocol [28 (link)]. Specifically, 50 mm sized stem segments were collected from 8-week-old plants and sequentially treated with 70% (v/v) EtOH and acetone followed by air-drying at 37 °C. The mass of the samples, which is the weight of cell wall materials, was measured before further processing. The samples were boiled in acetic/nitric reagent (acetic acid:nitric acid:water = 8:1:2) for 30 min to remove hemicellulose and lignin. They were then washed with water before being treated with acetone and then air-dried at 37 °C. Thereafter, samples were treated with 67% (w/v) H₂SO₄ for cellulose breakdown to produce monomeric sugars, which were measured by spectrophotometer (Nano-MD UV-Vis, Scinco, Seoul, Korea) at 620 nm using 0.3% anthrone (w/w, in concentrated H₂SO₄) as a dye. The amount of glucose in the samples was calculated based on the standard curve of D-glucose. Cellulose content (% cell wall) was calculated using the formula: Cellulose content (% cell wall) = amount of glucose in the sample (µg)/cell wall weight (µg) × total volume of H₂SO₄ used in the anthrone assay (µL).

Acetyl bromide assay to determine lignin content was performed according to a previously reported protocol [26 (link)]. The whole primary stems from 7-week-old plants were ground with liquid nitrogen and lyophilized for 48 h. Lyophilized powders were then filtered through a 425 μm screen. Each sample (10 mg) was washed four times with 95% (v/v) EtOH and twice with distilled water to remove soluble components. After air-drying at 60 °C for 12 h, products were dissolved in 2 mL of 25% (v/v, in glacial acetic acid) acetyl bromide and incubated at 70 °C for 30 min, to prevent excessive carbohydrate degradation that can distort the absorption spectrum. After incubation, 0.9 mL of 2 M NaOH, 3 mL of acetic acid, and 0.1 mL of 7.5 M hydroxylamine HCl were sequentially added, followed by centrifugation at 4000× g for 10 min. The supernatant was diluted 20-fold with glacial acetic acid, and absorbance was measured at 280 nm using a spectrophotometer (Nano-MD UV-Vis, Scinco, Seoul, Korea). Acetyl bromide soluble lignin (%) was calculated according to the previous report [27 (link)].