In vivo regulatory phosphorylation of novel phosphoenolpyruvate carboxylase isoforms in endosperm of developing castor oil seeds.

TRIPODI, KARINA; TURNER, W.; GENIDAKIS, S.; PLAXTON, W. C.

Plant Physiology

American Society of Plant Biologists

Año: 2005 vol. 139 p. 969 - 978

0032-0889

Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) fromdeveloping castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterialPEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer andPEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conservedp107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated inCOS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purifiedPEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion.Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 bydecreasing its Km(PEP) and sensitivity to L-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107phosphorylation increased PEPC2’s Km(PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylationof p107 was promoted duringCOSdevelopment (coincident with a.5-fold increase in the I50 [malate] value for total PEPCactivity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylatedin planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylationstatus of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.