Photosynthetic and Other Phosphoenolpyruvate Carboxylase Isoforms in the Single-Cell, Facultative C4System of Hydrilla verticillata  

Abstract

The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C 4 plant. It typically exhibits C 3 photosynthetic characteristics, but exposure to low [CO 2 ] induces a C 4 system in which the C 4 and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C 4 induction. This and enzyme kinetic data were consistent with it being the C 4 photosynthesis isoform. However, the C 4 signature serine of terrestrial plant C 4 isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C 3 sequences, was present. Western analyses of C 3 and C 4 leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C 3 , non-graminaceous C 4 , and Crassulacean acid metabolism PEPCs but not with the graminaceous C 4 , and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C 4 angiosperms.Phosphoenolpyruvate (PEP) carboxylase (PEPC; EC 4.1.1.31) occurs in eubacteria, cyanobacteria, green algae, and all higher plants. In the latter, it is encoded by a small multigene family (Lepiniec et al., 1993(Lepiniec et al., , 1994Ernst and Westhoff, 1997). A major function of the enzyme in higher plants is anapleurotic, providing carbon skeletons for the synthesis of compounds that serve in processes such as C/N partitioning, guard cell movements, and nitrogen fixation in legumes (Chollet et al., 1996). In C 4 and Crassulacean acid metabolism (CAM) photosynthesis, alternate forms of PEPC catalyze the initial carboxylation step in a C 4 acid cycle that functions as a CO 2 concentrating mechanism. In terrestrial C 4 plants, PEPC and Rubisco fixation events are separated between mesophyll and bundle sheath cells, and PEPC expression is in the cytosol of the former (Matsuoka and Sanada, 1991). In CAM plants, the fixation events are separated temporally with the CAM photosynthetic PEPC expressed in the cytosol of chloroplastic cells (Cushman and Bohnert, 1999).Investigations on the origins of the C 4 syndrome indicate that it arose independently in a number of angiosperm taxa and included changes in the genes controlling anatomical and chloroplastic development and in those orchestrating photosynthetic biochemistry (Hermans and Westhoff, 1992;Kellogg, 1999). PEPC has played an important role in these studies, and the structure, function, and phylogenetic relationships of its sequences have been used to better understand the evolution of C 4 and CAM photosynthetic systems (Lepiniec et al., 1994).The aquatic monocot Hydrilla verticillata (L.f.) Royle is the best documented case ...

Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features

“…However, genes putatively encoding all of the enzymes required for C 4 photosynthesis were identified (see Table 10). Whereas C 4 photosynthesis has yet to be unequivocally shown in unicellular organisms (24,25,37,38), C 4 in the absence of Kranz anatomy is now well documented, especially in Hydrilla verticillata, a facultative C 4 aquatic monocot (39). Unlike T. pseudonana, which appears to lack plastid-localized NADP-dependent malic enzymes (NADP-ME), O. tauri has two NADP-ME orthologs most similar to H. verticillata (40) with at least one apparently targeted to the chloroplast based on ChloroP and TargetP predictions.…”

CO 2 concentrating mechanisms and environmental change

“…This was followed by a very detailed analysis of the single-cell C 4 metabolism in H. verticillata including studies of the biochemical pathways, enzymes and molecular genetics and its relationship to the acid-base polarity of leaf surfaces Magnin et al, 1997;van Ginkel et al, 2001;Rao et al, 2002Rao et al, , 2006aRao et al, , 2006bRao et al, , 2008Estavillo et al, 2007). Reiskind et al (1988) and Reiskind and Bowes (1991) demonstrated C 4 biochemistry in photosynthesis of the one ulvophycean green macroalga, Udotea flabellum, but not in another, Codium decorticatum.…”

De novo transcriptome sequencing and analysis of Hydrilla verticillata (L.f.) Royle

“…verticillata is an aquatic macrophyte that can shift from C 3 to C 4 photosynthesis under low CO 2 conditions, without its leaf cells undergoing any structural modifications (Rao et al, 2006a;Rao et al, 2002;Estavillo et al, 2007). During this transition, genes encoding C 4 -specific isozymes of phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), NADP-malic enzyme (NADP-ME) and carbonic anhydrase (CA) are up-regulated (Rao et al, 2006a;Rao et al, 2002;Estavillo et al, 2007;Brautigam et al, 2014;Reiskind and Maberly, 2014). In H. verticillata there are three isoforms of PEPC, of which only one isoform is up regulated in C 4 leaves and has C 4 -like isoform kinetics (Rao et al, 2008).…”