Figure 2. PEPC is reversibly up-regulated and phosphorylated by −P_i Arabidopsis.

All values represent the mean PEPC activities±S.E.M. of replicate determinations of clarified extracts from n=4 different cultures (A) or seedlings (B). At 0 days, 10-ml aliquots of suspension cells (A) cultured for 7 days in 5 mM P_i were subcultured into 40 ml of fresh MS medium containing 0 or 5 mM P_i (−P_i and +P_i respectively). Flasks of 7-day-old −P_i cultures were resupplied with 2.5 mM P_i (P_i refed) and cultured for an additional 2 days as shown. (A, inset) Immunological detection of PEPC from the 7-day-old (d-7) +P_i and −P_i cells. Clarified extracts were subjected to SDS/PAGE (5 μg of protein/lane) and blot-transferred on to a PVDF membrane. Blots were probed with affinity-purified anti-RcPEPC Ab [26] and immunoreactive polypeptides were detected using an alkaline-phosphatase-linked secondary Ab. Seedlings (B) were germinated and cultivated aseptically in liquid 0.5× MS medium containing 0.2 mM P_i for 7 days then transferred into fresh MS media containing 20 μM P_i (−P_i) or 3 mM P_i (+P_i) and cultivated for an additional 14 days as described in the Experimental section. At 21 days, replicate −P_i seedlings were resupplied with 3 mM P_i and cultivated for an additional 3 days (P_i refed). (B, inset) Immunological detection of PEPC from +P_i compared with −P_i roots and shoots (5 μg of protein/lane) using anti-RcPEPC Ab as described above. (C) Clarified extracts from the −P_i, +P_i and P_i-refed suspension cells or seedlings were subjected to SDS/PAGE and electroblotted on to a PVDF membrane. Immunoblots were probed with an anti-pSer-11 Ab raised against the conserved N-terminal Ser-11 phosphorylation domain of a plant-type RcPEPC isoenzyme, in the presence of 10 μg · ml⁻¹ of the corresponding dephosphopeptide [28]. Each lane of (C) was loaded according to the PEPC activity measured in the corresponding clarified extracts, as determined using the optimal assay conditions outlined in the Experimental section (0.2 m-units/lane for suspension cells; 0.7 m-units/lane for roots and shoots).