Minisymposium 8: Photosynthesis: Carbon

Abs # 18003: PEPC expression and regulation: studies with a C₄ model system for C₃ crop transformation

Presenter:	Bowes, George , gbowes@botany.ufl.edu
Authors	Bowes, George  (A)   Rao, Srinath K. (A)   Reiskind, Julia B. (A)
Affiliations:	(A): Department of Botany, University of Florida

Hydrilla verticillata leaves exposed to low [CO₂] change from C₃ to C₄ photosynthesis. The C₄ and Calvin cycles function in the same cell to concentrate CO₂ in the chloroplasts, without using the cellular segregation found in Kranz anatomy. As such, this system provides a model for a "minimalist" C₄ process within a C₃ plant, such as rice, which also lacks Kranz anatomy. We have sequenced two PEPC isoforms (Hvpepc3 and Hvpepc4), both expressed in leaves. Northern analyses indicated that expression of both isoforms was induced during the C₄ shift and that both were upregulated in the light; however, both regulation and signal were stronger for Hvpepc4. Western analyses with a maize anti-PEPC showed two bands in both C₃ and C₄ extracts, but the lower molecular mass band, putatively Hvpepc4, dominated in the C₄ extract. Antibodies against a phosphorylated Ser/Thr Akt substrate gave a stronger positive signal in the lower band, the reputed Hvpepc4, of light and dark C₄ extracts, suggesting phosphorylation of both light and dark forms. Malate inhibition of PEPC activity from C₄ extracts subjected to dephosphorylation indicated that the light form was the more highly phosphorylated. From these studies Hvpepc3 appeared largely non-phosphorylated. PEPC kinetics, using C₃ and C₄ leaf extracts assayed at cytosolic pH values, showed differences. C₄ leaf activity was high and showed substantial malate inhibition, both regulated with light/dark transitions. Activity of PEPC from C₃ leaves was low, as was malate inhibition; there was no evidence of diel regulation. The effector glucose-6-P increased activities and reduced malate inhibition, but in C₄ extracts it also reduced the K_0.5PEP values. C₄ and C₃ extracts exhibited sigmoidal and Michaelis-Menten [PEP] responses, respectively. A number of laboratories are attempting to produce C₄ transformants from C₃ crop species, but a balanced C₄ system has yet to be generated. There is a need to understand the characteristics of each introduced gene product and how it is integrated into the target C₃ environment. The Hydrilla system enables us to investigate the individual components and their coordination in a C₃ milieu. It is clear from these studies with Hydrilla that, to optimize a transgenic C₄ system, careful consideration should be made of the expression, kinetic and regulatory characteristics of the isoforms used to introduce into a C₃ background. Supported by USDA NRICGP 98-35306-6449 and 2002-35318-12540.