PARENT SESSION
Posters P8C C4 and CAM. Abstracts (685-698)

Carbonic anhydrase isoforms in the C₄ CCM of Hydrilla. Vidya Rao¹, Srinath Rao¹, Julia Reiskind¹, George Bowes^*,1, ¹ Department of Botany, Gainesville, Florida, USA

ABSTRACT- Hydrilla verticillata is a facultative C₄ NADP-ME plant (aquatic monocot) that switches from C₃ to C₄ photosynthesis, and induces a CO₂ concentrating mechanism (CCM). The C₄ and Calvin cycles operate in the same cell, with the fixation events separated between cytosol and chloroplast. Thus it is an excellent model for transformation of C₃ crop species with a C₄ cycle. But the role of carbonic anhydrase (CA) in this system still requires elucidation. Terrestrial C₃ plants possess a chloroplastic CA. However, in the Hydrilla C₄ system a stromal CA at pH 8.2 could undermine the CCM by converting the CO₂ released by NADP-malic enzyme into bicarbonate. As such, it would compete with rubisco for CO₂. In contrast, because CO₂ is the species entering the leaf, a cytosolic CA could facilitate bicarbonate production for use by cytosolic phosphoenolpyruvate carboxylase. The data indicate that CA activity was higher in C₄ Hydrilla leaves as compared with C₃, as was the ratio of soluble to pellet-associated activities. Using degenerate primers, designed from published plant CA sequences, and RT-PCR, a partial cDNA was isolated, cloned and sequenced. It showed close identity to a tobacco chloroplastic isoform (80%) and a cytosolic isoform from a C₄ Flaveria (74%). Northern analysis showed that this CA was over-expressed in C₄ Hydrilla leaves. Preliminary data from western blots, using an antibody against a luminal CA from Chlamydomonas produced a stronger signal in the C₄ leaf sample as compared with the C₃. A second signal was seen in the C₄ sample that was weaker in the C₃. From a cell fractionation study using C₃ and C₄ leaves, these two bands appeared unique to the cytosolic fraction, and again the C₄ leaf had the greater signal strength. Supported by USDA NRICGP 2002-35318-12540.

KEY WORDS: carbonic anhydrase, co2 concentrating mechanism, c-4 photosynthesis, chloroplast