Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single-cell, facultative C(4) system 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.

Figure 1

Multiple alignment of the deduced amino acid sequences of three H. verticillata PEPCs and those of maize (Zea mays; C₃ and C₄), Vanilla planifolia (CAM), and Flaveria trinervia (C₄). Only residues that differ among the sequences are shown. Gaps (-) and identical (.) bp are indicated. Boxed residues indicate the most conserved regions among prokaryotes and eukaryotes. Putative regulatory and catalytic sites are also shown. , The Ser residue that is common to all plant PEPCs and that is the target for phosphorylation; ●, the unique Hvpepc4 Met residue; , the F. trinervia putative C₄ Lys site; ○, the unique Hvpepc5 Val; and ★, the position of the C₄ signature Ser.

Figure 2

Northern analyses of PEPC isoform expression in H. verticillata leaves in the C₃ state and during induction of the C₄ state. One microgram per lane of total RNA from H. verticillata C₃ and C₄ leaves was separated on a 1.2% (w/v) denaturing agarose gel and blotted onto a positively charged nylon membrane. DIG-labeled 3′-end RNA probes from Hvpepc3 and Hvpepc4, and a consensus probe were used to hybridize the membranes for transcript identification. The ethidium bromide-stained gel shows uniform loading of RNA samples. The size (kb) of the full-length cDNAs encoding PEPC isoforms is indicated on the right.

Figure 3

Specific activities of PEPC in desalted extracts of H. verticillata leaves in the C₃ state and during induction of the C₄ state. The PEPC was extracted from leaves harvested during the light period, and activity was measured with saturating substrates at pH 8. The arrows indicate sampling times for the northern analyses. Data are means ± se, n = 3.

Figure 4

The effect of PEP concentration on the specific activity of PEPC in the C₃ and C₄ leaf peak fractions from anion-exchange chromatography (see Table II). The PEPC was extracted from the H. verticillata leaves harvested during the light period. The C₄ sample was taken 288 h into the induction period. The assay was run at pH 7.3 in the absence of dithiothreitol. The apparent K_0.5 PEP values were determined from the curves. Data are means ± se, n = 3.

Figure 5

Western analyses of PEPC from leaves of H. verticillata. Leaves of H. verticillata were harvested midway through their light cycle, C₃ at 0 h and C₄ at 288 h into the induction period. Six micrograms of protein from the C₃ and C₄ leaf peak fractions from anion-exchange chromatography (see Table II) was resolved by 5% (w/v) SDS-PAGE and transblotted to a nitrocellulose membrane. The membrane was probed with antibodies raised against maize PEPC. The PEPC signals from C₃ and C₄ leaves are shown. The kilodalton value of the prominent PEPC band is indicated at the right.

Figure 6

Phylogenetic analysis of deduced amino acid PEPC sequences. The PHYLIP package was used to construct a consensus tree with 100 bootstrap replications using the parsimony method. The four prokaryotic species served as the outgroup. The stars at the fork of the tree represent >85% support. The three groupings are: I, C₄ graminaceous; II, graminaceous roots; and III, sequences of other higher plants. Deduced amino acid sequences other than H. verticillata were obtained from GenBank/SwissProt.