Julia B. Reiskind

Regulation and Localization of Key Enzymes during the Induction of Kranz-Less, C4-Type Photosynthesis in Hydrilla verticillata

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.

Photosynthesis, photorespiration and ecophysiological interactions in marine macroalgae

Abstract Although macroalgae play a significant role in the productivity of marine ecosystems, the marine environment presents constraints to the achievement of maximum photosynthesis and productivity. Many macroalgae have developed strategies to minimize these constraints, which include low saturating irradiance requirements and bicarbonate use for photosynthesis, morphological features to reduce desiccation and morphological and biochemical modifications to enhance photosynthetic carbon fixation. In many marine macroalgae, fixation occurs solely by ribulose bisphosphate carboxylase/oxygenase (RuBPCO) and the photosynthetic carbon reduction cycle, and in some, considerable photorespiration is evident. But, in others photorespiration is suppressed, and studies are only now beginning to elucidate the methods that accomplish this effect. A C 4 -like system based on phosphoenolpyruvate carboxykinase appears to operate in the green alga Udotea flabellum (Ellis & Solander) Lamouroux, while in the red alga, Gracilaria corticata J. Agardh, phosphoenolpyruvate carboxylase is used. Among brown algae CAM-type systems or bicarbonate uptake may reduce photorespiration. Bicarbonate uptake in association with carbonic anhydrase activity may be responsible for low photorespiration in some red algae. Regardless of the mechanism, reduced photorespiration is proposed to be achieved by concentrating CO 2 at the RuBPCO fixation site. However, elevated internal CO 2 levels have yet to be demonstrated in any marine macroalga. Although photorespiration seems suppressed in many marine macroalgae, further studies are needed to comprehend fully the mechanisms involved.

Photosynthetic and Other Phosphoenolpyruvate Carboxylase Isoforms in the Single-Cell, Facultative C4 System of Hydrilla verticillata

The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C4 plant. It typically exhibits C3 photosynthetic characteristics, but exposure to low [CO2] induces a C4 system in which the C4 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 C4 induction. This and enzyme kinetic data were consistent with it being the C4 photosynthesis isoform. However, the C4 signature serine of terrestrial plant C4isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C3 sequences, was present. Western analyses of C3 and C4 leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C3, non-graminaceous C4, and Crassulacean acid metabolism PEPCs but not with the graminaceous C4, and formed a clade with a gymnosperm, which is consistent with H. verticillataPEPC predating that of other C4 angiosperms.

Immunogold localization of primary carboxylases in leaves of aquatic and A C3-C4 intermediate species

Abstract Using protein A-gold labeled antibodies, attempts were made to determine the cellular location of ribulose bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) in two aquatic macrophytes and a terrestrial C 3 -C 4 intermediate plant. Hydrilla verticillata and Myriophyllum spicatum are submersed angiosperms which exhibit variable photorespiratory states. Leaf sections indicated that neither possessed Kranz anatomy. In Myriophyllum , the chloroplasts were confined to a single epidermal layer. The Hydrilla leaf was composed of two morphologically distinct chloroplastic cell layers. Immunocytochemical studies showed that Rubisco was distributed in the chloroplasts of all the cells of both layers, and that no difference in Rubisco labeling between cells was apparent, even though low-photorespiration Hydrilla has many biochemical and physiological characteristics of C 4 -photosynthesis. In Hydrilla the location of PEPC was largely cytosolic and was in all leaf cells. These data support the postulate that an intracellular separation of C 4 and C 3 fixation events may account for the low photorespiration state in Hydrilla . PEPC labeling was undectectable in Myriophyllum , which correlates with its lack of C 4 -like photosynthetic biochemistry. The terrestrial C 3 -C 4 intermediate species, Moricandia arvensis , has leaves with photosynthetic cells differentiated into a chloroplast-containing bundle sheath layer surrounded by mesophyll tissue. Rubisco labeling was found in the chloroplasts of both cell types. Thus, the reduced apparent photorespiration in Moricandia is not achieved through a separation of CO 2 fixation events into different cells, as occurs in C 4 plants with Kranz anatomy, but is more likely due to efficient refixation of photorespiratory CO 2 by Rubisco.

Hormonal Control of Sexual Morphogenesis in Achlya: Dependence on Protein and Ribonucleic Acid Syntheses

The induction of the male sexual organ primordia (antheridial hyphae) by the steroid hormone antheridiol in the water mold Achlya ambisexualis requires both transcription and translation. Inhibition of either of these processes eliminates the expected increase in the production and release of the enzyme cellulase, which accompanies the formation of the antheridial hyphae.

Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C4 NADP-malic enzyme species in which the C4 and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C4 system. Using differential display, mRNA populations from C3 and C4H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C4 leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C4 upregulated genes were identified. Among these one encoded a previously recognized C4 phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C4 system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C4 system in a C3 crop species.

A CO2-flux mechanism operating via pH-polarity in Hydrilla verticillata leaves with C-3 and C-4 photosynthesis

The aquatic angiosperm Hydrilla verticillata lacks Kranz anatomy, but has an inducible, C4-based, CO2 concentrating mechanism (CCM) that concentrates CO2 in the chloroplasts. Both C3 and C4Hydrilla leaves showed light-dependent pH polarity that was suppressed by high dissolved inorganic carbon (DIC). At low DIC (0.25 mol m−3), pH values in the unstirred water layer on the abaxial and adaxial sides of the leaf were 4.2 and10.3, respectively. Abaxial apoplastic acidification served as a CO2 flux mechanism (CFM), making HCO3− available for photosynthesis by conversion to CO2. DIC at 10 mol m−3 completely suppressed acidification and alkalization. The data, along with previous results, indicated that inhibition was specific to DIC, and not a buffer effect. Acidification and alkalization did not necessarily show 1:1 stoichiometry; their kinetics for the apolar induction phase differed, and alkalization was less inhibited by 2.5 mol m−3 DIC. At low irradiance (50 μmol photons m−2 s−1), where CCM activity in C4 leaves is minimized, both leaf types had similar DIC inhibition of pH polarity. However, as irradiance increased, DIC inhibition of C3 leaves decreased. In C4 leaves the CFM and CCM seemed to compete for photosynthetic ATP and/or reducing power. The CFM may require less, as at low irradiance it still operated maximally, if [DIC] was low. Iodoacetamide (IA), which inhibits CO2 fixation in Hydrilla, also suppressed acidification and alkalization, especially in C4 leaves. IA does not inhibit the C4 CCM, which suggests that the CFM and CCM can operate independently. It has been hypothesized that irradiance and DIC regulate pH polarity by altering the chloroplastic [DIC], which effects the chloroplast redox state and subsequently redox regulation of a plasma-membrane H+-ATPase. The results lend partial support to a down-regulatory role for high chloroplastic [DIC], but do not exclude other sites of DIC action. IA inhibition of pH polarity seems inconsistent with the chloroplast NADPH/NADP+ ratio being the redox transducer. The possibility that malate and oxaloacetate shuttling plays a role in CFM regulation requires further investigation.

Dissolved inorganic carbon influences the photosynthetic responses of Hydrilla to photoinhibitory conditions.

Abstract Hydrilla verticillata (L.f.) Royle, like other submersed angiosperms, exhibits shade plant characteristics. However, Hydrilla not only grows well at low irradiance in benthic habitats, but also at the water surface where its canopy is exposed to solar irradiance of 1800 μmol photons m−2 s−1. Leaves from plants grown at a moderate irradiance of 300 μmol m−2 s−1 had low light compensation (Ic), onset of light saturation (Ik), and light saturation (LSP) points of 10 μmol photons m−2 s−1, 47 μmol photons m−2 s−1, and 280 μmol photons m−2 s−1, respectively. Plants exposed to 1800 μmol photons m−2 s−1 for 15 min at only 0.6 mM dissolved inorganic carbon (DIC) exhibited about 50% and 30% photoinhibition of the photosynthetic rate, and apparent quantum yield, respectively. However, the presence of 2 mM DIC protected the leaves from photoinhibition, and allowed them to acclimate, with a higher LSP and photosynthetic rate. Superoxide dismutase, an enzyme which scavenges ·O−2 radicals produced under light and O2 stress, increased over three-fold in activity within only a 15 min exposure to high irradiance, irrespective of the DIC. Ascorbate peroxidase, which detoxifies H2O2, did not increase in activity. Two other enzymes of the ascorbate/glutathione cycle changed activity under high light and low DIC: monodehydroascorbate reductase which increased, and dehydroascorbate reductase which decreased. These changes presumably enhanced the scavenging of toxic ·O−2 radicals, and the recycling of NADP+ to photosystem I under a high light/low DIC regime which reduced the capacity of the Calvin cycle to utilize NADPH.

Inorganic Carbon Concentrating Systems from an Environmental Perspective

Submersed aquatic organisms exist in an environment where light may be selectively attenuated, and diel temperature fluctuations are buffered; where they are continuously bathed in an exogenous H+ ion concentration, that may show substantial diel variations [up to pH 11]; where, in addition to free CO2, they are exposed to HCO3 - and CO3 2- as potential dissolved inorganic carbon [DIC] sources; and where the diffusion of O2 and CO2 is greatly retarded in comparison with movement in air. This diffusion resistance can result in large deviations from air-equilibrium values for dissolved O2 and CO2, especially in freshwater. The dissolved O2 can range from near zero to over 200% air-saturation [0.48 mol m-3], and DIC from zero to 100 mol m-3, though in freshwater the DIC is usually closer to 1 mol m-3. In marine waters these deviations are less pronounced, with O2 being 0.24 and DIC about 2 mol m-3, and the pH 8.2; though in lagoons or rock pools, due to rain, evaporation, and dense vegetation, considerable variation can occur. Furthermore, in terrestrial plants, plastid placement within micrometers of an air-water interface [i.e., the leaf mesophyll cell walls] that is many-fold greater than the ground area subtended by the leaf, facilitates the O2 and CO2 exchange process (1). By comparison, the air-water interface may be meters away from the plastids of submersed plants, and only mechanical factors, such as wind and wave action, facilitate exchange across the interface.

Photosynthetic responses and anatomical features of two marine macroalgae with different CO2 compensation points

Abstract Codium decorticatum (Woodward) Howe and Udotea flabellum (Ellis and Solander) Lamouroux are marine, siphonaceous macroalgae in the Chlorophyta. The C. decorticatum thallus consisted of chloroplastic and achloroplastic filaments of 132 μm diameter, while the filaments of the U. flabellum thallus were small (24 μm diameter) and chloroplastic. At 2 mM dissolved inorganic carbon (DIC), photosynthesis in C. decorticatum , but not in U. flabellum , was inhibited by O 2 ; the CO 2 compensation points in these algae were high and low (54 and 7 μl CO 2 l −1 ), respectively. Growth under different temperature, photoperiod and CO 2 regimes failed to alter the CO 2 compensation points, indicating stability in the high and low photorespiratory states. Both macroalgae were shade species, with light compensation and saturation points for photosynthesis of 5–10 and 250 μmol quanta m −2 s −1 , respectively. Although U. flabellum had a greater photosynthetic affinity for CO 2 and HCO 3 − than C. decorticatum , neither was saturating at seawater DIC levels (2.0 mM at pH 8). Apparent K m values indicated free CO 2 was preferred, but the high HCO 3 − in seawater was probably the major photosynthetic DIC source. The photosynthetic Q 10 of U. flabellum was almost twice that of C. decorticatum ; the former was able to photosynthesize and grow at temperatures above 30°C, whereas the latter could not. For U. flabellum , a C 4 -like mechanism is probably a factor minimizing photorespiration and influencing its tropical distribution, whereas C 3 -like photosynthesis in C. decorticatum may account for its high photorespiration and more temperate range.