Expression characteristics of ZmARF1.

Relative expression of ZmARF1 in roots of low-Pi tolerant (A) and low-Pi sensitive (B) maize inbred lines after low-Pi treatment. (C) Phosphate stress increased ZmARF1 protein abundance in maize. Two-week-old maize overexpression lines of ZmARF1 were subjected to high Pi (HP) and low Pi (LP) treatment for 14 days and ZmARF1 protein levels were revealed by immunoblotting using anti-Flag antibody. The experiment was repeated three times with the similar results. (D) Subcellular localization of ZmARF1-GFP fusion protein in maize protoplasts. The top and down panel represent CaMV35S::GFP and CaMV35S::ZmARF1-GFP expression, respectively. The extreme left is the GFP fluorescence, next to the right is the RFP nuclear localization signal of the nuclear marker, followed by the bright field signal and to the extreme right an overlap of the three images. Bar = 50 μm. (E) The abundance of ZmARF1 in total cell extracts, cytoplasmic, or nuclear fractions of roots from both WT and OE2# were assessed under HP and after 48 hours of LP treatment. The abundance of ZmARF1-3×Flag was determined through western blot using an anti-Flag antibody. Simultaneously, the abundance of cytoplasmic and nuclear fractions was determined using anti-Actin and anti-Histone (H3) as internal control, respectively.