Assay of ATP-sulfurylase activity For the determination of ATP-sulfurylase activity in plant 0.3g fresh samples (leaf and root) were ground with chilled mortar and pestle using ice cool buffer consisting of 10 mm Na2EDTA, 20 mm Tris–HCl (pH 8.0), 2 mm dithiothreitol and approximately 0.01 g ml-1 insoluble polyvinylpyrollidone, using 1 : 4 (w⁄ v) tissue to buffer ratio. The homogenate was strained through gauze and centrifuged at 20 000 g for 10 min at 4 °C. The supernatant was used for the in vitro ATP-sulfurylase assay. ATP-sulfurylase activity was measured using molybdate-dependent formation of pyrophosphate. The reaction was started by adding 0.1 ml of the crude extract to 0.5 ml of the reaction mixture, which contained 7 mm MgCl2, 5 mm Na2MoO4, 2mm Na2-ATP and 0.032 U ml-1 of sulphate-free inorganic pyrophosphate in 80 mm Tris–HCl buffer (pH 8.0). Another aliquot from the same extract was added to the …show more content…
A 1 µg aliquot of total RNA was reverse-transcribed to generate cDNA using a ReverTra Ace qPCR RT Kit (Toyobo, Japan), following the manufacturer’s instructions. qRT-PCR was performed with SYBR Green PCR Master Mix (Takara, Tokyo, Japan) using an ABI Step One Plus real-time machine (Eppendorf, USA) under the default thermal cycling conditions with an added melting curve. Gene-specific primers for qRT-PCR were designed based on the published sequences and the primers corresponding to the genes ASMT, SUT1:1, SUT1:2, ATPS, APS, SiR, OASTL, CP1, and CP2; the marker gene UBI3 and actin were used as internal control. Gene-specific primer pairs are presented in Supplementary Table S1. The PCR conditions consisted of denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. Relative transcript levels were calculated according to Livak and