Kranz-less, C4-type photosynthesis was induced in the submersed monocot Hydrilla verticillata (L.f.) Royle. During a 12-d induction period the CO2 compensation point and O2 inhibition of photosynthesis declined linearly. Phosphoenolpyruvate carboxylase (PEPC) activity increased 16-fold, with the major increase occurring within 3 d. Asparagine and alanine aminotransferases were also induced rapidly. Pyruvate orthophosphate dikinase (PPDK) and NADP-malic enzyme (ME) activities increased 10-fold but slowly over 15 d. Total ribulose-1,5-bisphosphate carboxylase/oxygenase activity did not increase, and its activation declined from 82 to 50%. Western blots for PEPC, PPDK, and NADP-ME indicated that increased protein levels were involved in their induction. The H. verticillata NADP-ME polypeptide was larger (90 kD) than the maize C4 enzyme (62 kD). PEPC and PPDK exhibited up-regulation in the light. Subcellular fractionation of C4-type leaves showed that PEPC was cytosolic, whereas PPDK and NADP-ME were located in the chloroplasts. The O2 inhibition of photosynthesis was doubled when C4-type but not C3-type leaves were exposed to diethyl oxalacetate, a PEPC inhibitor. The data are consistent with a C4-cycle concentrating CO2 in H. verticillata chloroplasts and indicate that Kranz anatomy is not obligatory for C4-type photosynthesis. H. verticillata predates modern terrestrial C4 monocots; therefore, this inducible CO2-concentrating mechanism may represent an ancient form of C4 photosynthesis.

Udotea flabellum is a marine, macroscopic green alga with C4-like photosynthetic characteristics, including little 02 inhibition of photosynthesis, a low CO2 compensation point, and minimal photorespiration; but it lacks anatomical features analogous to the

Aquatic C4 photosynthesis probably arose in response to dissolved CO2 limitations, possibly before its advent in terrestrial plants. Of over 7600 C4 species, only about a dozen aquatic species are identified. Amphibious Eleocharis species (sedges) have C3–C4 photosynthesis and Kranz anatomy in aerial, but not submersed, leaves. Aquatic grasses have aerial and submersed leaves with C4 or C3–C4 photosynthesis and Kranz anatomy, but some lack Kranz anatomy in the submersed leaves. Two freshwater submersed monocots, Hydrilla verticillata and possibly Egeria densa, are C4 NADP-malic enzyme (NADP-ME) species. A marine macroalga, Udotea flabellum (Chlorophyta), and possibly a diatom, are C4, so it is not confined to angiosperms. Submersed C4 species differ from terrestrial in that β-carboxylation is cytosolic with chloroplastic decarboxylation and Rubisco carboxylation, so the C4 and Calvin cycles operate in the same cell without Kranz anatomy. Unlike terrestrial plants, Hydrilla is a facultative C4 that shifts from C3 to C4 in low [CO2]. It is well documented, with C4 gas exchange and pulse-chase characteristics, enzyme kinetics and localization, high internal [CO2], relative growth rate, and quantum yield studies. It has multiple phosphoenolpyruvate carboxylase isoforms with C3-like sequences. Hvpepc4 appears to be the photosynthetic form induced in C4 leaves, but it differs from terrestrial C4 isoforms in lacking a C4 signature Serine. The molecular mass of NADP-ME (72 kDa) also resembles a C3 isoform. Hydrilla belongs to the ancient Hydrocharitaceae family, and gives insight to early C4 development. Hydrilla is an excellent ‘minimalist’ system to study C4 photosynthesis regulation without anatomical complexities.

Two green macroalgae, Codium decorticatum and Udotea flabellum, differ photosynthetically. Codium had high O2-sensitive, and Udotea low 02-insensitive, CO2 compensation points; Codium showed a Warburg effect at seawater dissolved inorganic carbon levels and had photorespiratory C02 release, whereas Udotea did not. Seawater dissolved inorganic carbon levels did not saturate photosynthesis. For Codium, but not Udotea, the Warburg effect was increased by ethoxyzolamide, a carbonic anhydrase inhibitor, at high but not low pH. Isolated chloroplasts from both macroalgae showed a Warburg effect that was ethoxyzolamideinsensitive. In both macroalgae, chloroplastic and extrachloroplastic carbonic anhydrase activity was present. P-enolpyruvate carboxykinase (PEPCK) carboxylating activity in Udotea extracts was equivalent to that of ribulose bisphosphate carboxylase, and enzyme activities for C4 acid metabolism and P-enolpyruvate regeneration were sufficient to operate a limited C4-like system. In Udotea, malate and aspartate were earlylabeled photosynthetic products that turned over within 60 seconds. Photorespiratory compounds were much less labeled in Udotea. Low dark fixation rates ruled out Crassulacean acid metabolism. A limited C4-like system, based on PEPCK, is hypothesized to be the mechanism reducing photorespiration in Udotea. Codium showed no evidence of photosynthetic C4 acid metabolism. Marine macroalgae, like terrestrial angiosperms, seem to have diverse photosynthetic modes.In the three divisions of marine macroalgae, Rhodophyta (reds), Phaeophyta (browns), and Chlorophyta (greens), Ru-BPCO2 has been reported to be a major carboxylase, and labeling studies point to a functional PCR cycle (6,22,24). Photorespiration measurements and labeling of glycine and serine suggest that the PCO cycle also operates, and, furthermore, all three divisions contain species which exhibit 02 inhibition of photosynthesis or the Warburg effect and high r values (5-7, 22, 24, 28). This has led to the general conclusion that marine macroalgae are C3 (5) There is evidence that many, but not all, marine macroalgae use HCO3 ions for photosynthesis (4,6,10,22,24) and that CA is required (4, 10, 22). Thus, the possibility that they concentrate C02, in a manner comparable to unicellular organisms, has been broached (4, 6, 10); though there are no direct measurements of internal DIC levels to substantiate this hypothesis.Enzymes of C4 acid metabolism and C4 acid labeling during photosynthesis occur in some macroalgae (6,(22)(23)(24). In the case of brown macroalgae and marine diatoms, substantial ,8-carboxylation of PEP in the light and dark has been reported (20,24). The enzyme responsible is PEPCK acting in a carboxylating mode, not PEPC which occurs in terrestrial C4 plants (6,20,22,24). Dark fixation and diel titratable acidity changes associated with the PEPCK activity (22,24) are small, and a CAM-like system in brown macroalgae has been discounted (21). The operation of C4-like photosynthesis in marine macroalgae (23) is ...

Hydrilla verticillata (L.f.) Royle exhibits an inducible C4-type photosynthetic cycle, but lacks Kranz anatomy. Leaves in the C4-type state (but not C3-type) contained up to 5-fold higher internal dissolved inorganic carbon (DIC) concentrations than the medium, indicating that they possessed a CO2-concentrating mechanism (CCM). Several lines of evidence indicated that the chloroplast was the likely site of CO2 generation. From C4-type leaf [DIC] measurements, the estimated chloroplastic free [CO2] was 400 mmol m~^. This gave a calculated 2% O2 inhibition of photosynthesis, which was identical to the measured value, and provided independent evidence that the estimated [CO2] was close to the true value. A homogeneous distribution of DIC in the C4-type leaf could not account for such a high [CO2], or the resultant low O2 inhibition. For C3-type leaves the estimated chloroplastic [CO2] was only 7 mmol m~^, which gave high, and similar, calculated and measured O2 inhibition values of 22 and 26%, respectively. The CCM did not appear to be located at the plasma membrane, as it operated at low and high pH, indicating that it was independent of use of HCO3~ from the medium. Also, both C3-and C4-type Hydrilla leaves showed pH polarity in the light, with abaxial and adaxial boundary layer values of about pH 4*0 and 10-5, respectively. Thus, pH polarity was not a direct component of the CCM, though it probably improved access to HCO3. Additionally, iodoacetamide and methyl viologen greatly reduced abaxial acidification, but not the steady-state CCM. Inhibitor studies suggested that the CCM required photosynthetically generated ATP, but Calvin cycle activity was not essential. Both leaf types accumulated DIC in the dark by an ATP-requiring process, possibly respiration, and C4-type leaves fixed CO2 at 11-8% of the light rate. The operation of a CCM to minimize photorespiration, and the ability to recapture respiratory CO2 at night, would conserve DIC in a densely vegetated lake environ-