No abstract

Maximum photosynthesis rates in ferns are generally lower than those of seed plants, but little is known about the limiting factors, which are crucial to understand the evolution of photosynthesis in land plants. To address this issue, a gas exchange/chlorophyll fluorescence analysis was performed in three fern species spanning high phylogenetic range within Polypodiopsida (Osmunda regalis, Blechnum gibbum and Nephrolepis exaltata) to determine their maximum net photosynthesis (AN ), stomatal (gs ) and mesophyll (gm ) conductances to CO2 , and the maximum velocity of carboxylation (Vc,max ). The in vitro Rubisco specificity factor (SC /O ) was also determined. All three species had values for SC /O similar to those typical of seed plants, but values of AN , gs , gm and Vc,max were within the lowest range of those observed in seed plants. In addition, gs was unresponsive to light and CO2 , as already described in other fern species. On the contrary, gm varied with changes CO2 . A quantitative photosynthesis limitation analysis suggested that early land plants (ferns) presented not only stomatal limitations-which were less adjustable to the environment-but also restricted gm and Vc,max , resulting in limited maximum photosynthesis rates.

Systematic deletions and point mutations in the C-terminal extension of mammalian PFK (phosphofructokinase) led us to identify Leu-767 and Glu-768 of the M-type isoform (PFK-M) as the motifs responsible for the role of this region in inhibition by MgATP. These amino acids are the only residues of the C-terminus that are conserved in all mammalian isoforms, and were found to have a similar function in the C-type isoenzyme. Both residues in PFK-C and Leu-767 in PFK-M were also observed to be critical for inhibition by citrate, which is synergistic with that by MgATP. Binding studies utilizing titration of intrinsic protein fluorescence indicated that the C-terminal part of the enzyme participates in the signal transduction route from the MgATP inhibitory site to the catalytic site, but does not contribute to the binding of this inhibitor, whereas it is essential for the binding of citrate. Mutations of the identified structural motifs did not alter either the action of other allosteric effectors that also interact with MgATP, such as the inhibitor phosphoenolpyruvate and the strong activator fructose 2,6-bisphosphate, or the co-operative effect of fructose 6-phosphate. The latter data provide evidence that activation by fructose 2,6-bisphosphate and fructose 6-phosphate co-operativity are not linked to the same allosteric transition as that mediating inhibition by MgATP.