Phosphate availability regulates ethylene biosynthesis gene expression and protein accumulation in white clover (Trifolium repens L.) roots

The expression and accumulation of members of the 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO) gene families was examined in white clover roots grown in either P(i) (phosphate) sufficient or P(i)-deprived defined media. The accumulation of one ACO isoform, TR-ACO1, was positively influenced after only 1 h of exposure to low P(i), and this was maintained over a 7-day time-course. Up-regulation of TR-ACS1, TR-ACS2 and TR-ACS3 transcript abundance was also observed within 1 h of exposure to low P(i) in different tissue regions of the roots, followed by a second increase in abundance of TR-ACS2 after 5–7 days of exposure. An increase in transcript abundance of TR-ACO1 and TR-ACO3, but not TR-ACO2, was observed after 1 h of exposure to low P(i), with a second increase in TR-ACO1 transcripts occurring after 2–5 days. These initial increases of the TR-ACS and TR-ACO transcript abundance occurred before the induction of Trifolium repens PHOSPHATE TRANSPORTER 1 (TR-PT1), and the addition of sodium phosphite did not up-regulate TR-ACS1 expression over 24 h. In situ hybridization revealed some overlap of TR-ACO mRNA accumulation, with TR-ACO1 and TR-ACO2 in the root tip regions, and TR-ACO1 and TR-ACO3 mRNA predominantly in the lateral root primordia. TR-ACO1p-driven GFP expression showed that activation of the TR-ACO1 promoter was initiated within 24 h of exposure to low P(i) (as determined by GFP protein accumulation). These results suggest that the regulation of ethylene biosynthesis in white clover roots is biphasic in response to low P(i) supply.