BN-PAGE实验操作骤BN-PAGE实验操作步骤.doc

PAGE  PAGE 7 Supplemental Material Appendix S1. Experimental procedures Pigment analysis Prior to pigment extraction, each sample was weighed. Seedlings were frozen in liquid nitrogen and ground to powder with a high-speed mixer mill (model MM301, Retsch, ). Total chlorophyll and carotenoid were extracted from 30 seedlings with 100% acetone. The debris was removed by centrifugation at 10,000 rpm for 10 min. Absorbances of the supernatant were measured with a Beckman DU?650 spectrophotometer (), and the contents of the pigments were determined according to Lichtenthaler and Wellburn (1983). Anthocyanin extraction and quantification was performed as described previously (Kim et al., 2003). In brief, anthocyanin was extracted from 40 seedlings with 300 μL of acidified (1% HCl) methanol overnight at 4°C in dark conditions. The extract was separated by the addition of 200 μL of water and 200 μL of chloroform, followed by centrifugation for 5 min at 3,000 rpm. The absorbance of the upper phase was determined spectrophotometrically at 530 nm and 657 nm, and the anthocyanin content was calculated as A530 ? 0.33A657. For Pchlide analysis, wild-type and heterozygous atterC-1 seeds were sown onto MS medium and grown in the dark for 4 d at 22°C. At the end of the dark treatment, plates were exposed to light for 20 h to select the pigment-deficient homozygous atterC-1 seedlings. The selected mutant seedlings were then transferred to fresh MS medium on a clean bench. The wild-type and mutant seedlings were kept in darkness again for an additional two days. Seedlings were harvested under a dim-green light, and Pchlide extraction was performed as described previously (Terry and Kendrick, 1999). Seedlings were homogenized as described above. Extracts were obtained from 80 seedlings. After extraction with 0.5 mL of cold acetone (0.1 M NH4OH (90/10, v/v)), samples were centrifuged at 30,000 g for 10 min. The pellet was then re-extracted in 150 μL solvent an