David H. Turpin

EFFECTS OF INORGANIC N AVAILABILITY ON ALGAL PHOTOSYNTHESIS AND CARBON METABOLISM

The purpose of this contribution is to assess the effects of N-limitation on algal photosynthesis and address the key physiological and biochemical interactions that occur between the assimilation of inorganic nitrogen and algal photosynthesis and carbon metabolism

Limiting nutrient patchiness and its rôle in phytoplankton ecology

Limiting nutrient patchiness is examined as a factor affecting the community structure and species succession of natural phytoplankton communities held in ammonia limited continuous culture at a dilution rate of 0.3 day−1. It was found that under a homogeneous distribution of the limiting nutrient members of genus Chaetoceros dominated and when ammonia was added daily (patchy distribution), Skeletonema dominated. Intermediate patchiness gave rise to an assemblage dominated by both Chaetoceros and Skeletonema. The nutrient uptake ability of each assemblage was determined three weeks after experiment initiation. Each assemblage was best able to optimize uptake of ammonia under its particular patchy nutrient regime. Optimization of a patchy environment took place by an increased maximal uptake rate (Vmax) while optimization of a homogeneous environment appeared to take place by increased substrate affinity (i.e., low Ks). This study demonstrates that limiting nutrient patchiness can alter the relative abundance of populations within a community based on each populations ability to exploit the limiting resource under a particular degree of patchiness. We also show that coexistence of two populations might be expected due to the patchiness of a single limiting nutrient. The importance of patchiness in relation to other factors which determine community structure is discussed.

STEADY-STATE LUXURY CONSUMPTION AND THE CONCEPT OF OPTIMUM NUTRIENT RATIOS: A STUDY WITH PHOSPHATE AND NITRATE LIMITED SELENASTRUM MINUTUM (CHLOROPHYTA)1

Selenastrum minutum (Naeg.) Collins was grown over a wide range of growth rates under phosphate or nitrate limitation with non‐limiting nutrients added to great excess. This resulted in saturated luxury consumption. The relationships between growth rate and cell quota for the limiting nutrients were well described by the Droop relationship. The observed variability in N cell quota under N limitation as reflected in kQ·Qmax−1*, was similar in magnitude to previously reported values but kQ·Qmax−1* for P under P limitation was greater than previously reported for other species. These results were evaluated in light of the optimum ratio hypothesis. Our findings support previous work suggesting that the use of a single optimum ratio (kQi·KQj−1) is inappropriate for dealing with a species growing under steady‐state nutrient limitation. Under these conditions the optimum ratio should be viewed as a growth rate dependent variable. Two approaches for testing the growth rate dependency of optimum ratios are proposed.

SEXUALITY AND CYST FORMATION IN PACIFIC STRAINS OF THE TOXIC D1NOFLAGELLATE GONYAULAX TAMARENSIS1,2

Sexuality has been established for a culture of Gonyaulax tarnarensis Lebour (strain NEPCC–71). The addition of a thick inoculum to a nitrogen–deprived medium results in the occurrence of anisogamous sexual fusion within the first three days in the new culture. Planozygotes, large “lumpy” cells recognizable by their four flagella, may persist up to 2 wk before forming a smooth–walled, oval hypnozygote. The latter resembles cysts released asexually by ecdysis but has a slightly thicker wall. Viable cysts resembling hypnozygotes (zygotic cysts), but with reduced photosynthetic pigmentation, have been isolated from natural murine sediments in Hidden Basin, British Columbia, and a culture (strain NEPCC–254) was initiated from excysted individuals. Zygotic cysts of NEPCC–71 remained encysted in the light at 17 C for 8 wk before excysting. The presence of a ventral pen with toxicity in the latter strain indicates that the taxonomy of G. tamarensis‐like organisms is still in a stale of flux and the criteria for recognition of G. excavata (Braarud) Balech as a separate species are not satisfactory as presently formulated.

CARBOXYSOME CONTENT OF SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYTA) IN RESPONSE TO INORGANIC CARBON1

The carboxysome content of chemostat grown Synechococcus leopoliensis (Racib.) Komarek increases under inorganic carbon limitation. At growth rates of ca. 85%μmax the carboxysome content (±SE) was 0.57 ± 0.09 carboxysomes·cell section−1. Under severe carbon limitation (ca. 13%μmax) this increased to 3.4 · 0.3 carboxysomes·cell section−1. Corresponding to this change is a three order of magnitude decrease in the half‐saturation constant of photosynthesis for dissolved inorganic carbon. Nitrogen and phosphorus limitation had no effect on carboxysome content or the kinetics of photosynthesis with respect to inorganic carbon. These results are discussed in light of the apparent lack of photorespiration in these organisms.

Interactions between Photosynthesis and Respiration in the Green Alga Chlamydomonas reinhardtii (Characterization of Light-Enhanced Dark Respiration).

The rate of respiratory O2 consumption by Chlamydomonas reinhardtii cell suspensions was greater after a period of photosynthesis than in the preceding dark period. This “light-enhanced dark respiration” (LEDR) was a function of both the duration of illumination and the photon fluence rate. Mass spectrometric measurements of gas exchange indicated that the rate of gross respiratory O2 consumption increased during photosynthesis, whereas gross respiratory CO2 production decreased in a photon fluence rate-dependent manner. The rate of postillumination O2 consumption provided a good measure of the O2 consumption rate in the light. LEDR was substantially decreased by the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or glycolaldehyde, suggesting that LEDR was photosynthesis-dependent. The onset of photosynthesis resulted in an increase in the cellular levels of phosphoglycerate, malate, and phosphoenolpyruvate, and a decrease in whole-cell ATP and citrate levels; all of these changes were rapidly reversed upon darkening. These results are consistent with a decrease in the rate of respiratory carbon flow during photosynthesis, whereas the increase in respiratory O2 consumption during photosynthesis may be mediated by the export of photogenerated reductant from the chloroplast. We suggest that photosynthesis interacts with respiration at more than one level, simultaneously decreasing the rate of respiratory carbon flow while increasing the rate of respiratory O2 consumption.

Regulation of photosynthetic light harvesting by nitrogen assimilation in the green alga Selenastrum minutum

The interaction of whole cell metabolism with the distribution of excitation energy between photosystem 2 (PS2) and photosystem 1 (PS1), the light state transition, was investigated in vivo in the green alga Selenastrum minutum. Nitrogen limited cells of S. minutum were presented with a pulse of either NH4 + or NO3 − in the light. As shown previously, CO2 fixation is inhibited and high rates of N assimilation ensue [(1986) Plant Physiol. 81, 273–279]. NH4 + assimilation has a much higher requirement ratio for ATP/NADPH than either CO2 or NO3 − assimilation and thus drastically increases the demand for ATP relative to reducing power. Room temperature chlorophyll a fluorescence kinetic measurements showed that a reversible non‐photochemical quenching of PS2 fluorescence accompanied the assimilation of NH4 + but not the assimilation of NO3 − or CO2. 77K fluorescence emission spectra taken from samples removed at regular intervals during NH+ 4assimilation showed that the non‐photochemical quenching of PS2 was accompanied by a complementary increase in the fluorescence yield of PS1, characteristic of a transition to state 2. Our data suggests that S. minutum responds to the increased demand for ATP/NADPH during NH4 assimilation by inducing the light state transition to direct more excitation energy to PS1 at the expense of PS2 to increase the production of ATP by cyclic electron transport.

Pyruvate kinase isozymes from the green alga, Selenastrum minutum: II. Kinetic and regulatory properties☆

The kinetic and regulatory properties of two pyruvate kinase isozymes, PKp and PKc (apparent chloroplastic and cytosolic isozymes, respectively) from the green alga Selenastrum minutum were studied. The two isozymes differed greatly in several kinetic properties. Although both isozymes showed hyperbolic substrate saturation kinetics, the apparent Michaelis constants for PEP and ADP were about twofold and fourfold lower, respectively, for PKc as compared with PKp. ADP was the preferred nucleotide substrate for both isozymes. However, PKc utilized alternate nucleotides far more effectively than did PKp. PKc and PKp also differed strongly in the effect of activators and inhibitors on the enzymes. Although both isozymes were activated by dihydroxyacetone phosphate (DHAP) with a similar activation constant of about 30 microM, this activator (0.5 mM) caused an approximate 30% increase in the Vmax of PKc, but had no effect on the Vmax of PKp. PKp, but not PKc, was inhibited by ribose 5-phosphate, ribulose 1,5-bisphosphate, 2-phosphoglycerate, phosphoglycolate, and malate. Both isozymes were inhibited by MgATP, Mg2citrate, Mg2oxalate, and Pi. PKc was far more sensitive to inhibition by Pi, as compared with PKp. Pi was a competitive inhibitor of PKc with respect to phosphoenolpyruvate (PEP) (Ki = 1.3 mM). Glutamate was a potent inhibitor of PKc, but had no effect on PKp. In contrast with Pi, glutamate was a mixed-type inhibitor of PKc with respect to PEP (Ki = 0.7 mM). DHAP facilitated the binding of PEP by both isozymes and reversed or relieved the inhibition of PKc by Pi and/or glutamate. The regulatory properties of PKp indicate that it is likely less active in the light and more active in the dark. The in vivo activity of PKc is probably regulated by the relative cytosolic levels of DHAP, Pi, and glutamate; this provides a rationale for the activation of algal cytosolic pyruvate kinase which occurs during periods of enhanced ammonia assimilation.

Pyruvate kinase isozymes from the green alga, Selenastrum minutum. I. Purification and physical and immunological characterization.

Pyruvate kinase from the green alga Selenastrum minutum consists of two isoforms (PK1 and PK2) separable by Q-Sepharose chromatography. The two isoforms have been highly purified to respective final specific activities of 42 and 23 (mumol pyruvate produced/min)/mg protein. Purification steps included salt fractionation, anion-exchange, hydrophobic interaction, and gel filtration chromatography. The final enzyme preparations differ significantly in physical and immunological properties. PK1 is heat labile and is completely inactivated following reaction with N-ethylmaleimide. In contrast, PK2 is heat-stable and is only partially inactivated following N-ethylmaleimide treatment. PK1 appears to be homotetrameric with a native molecular mass of about 240 kDa, whereas PK2 appears to be homodecameric with a native molecular mass of approximately 590 kDa. The antigenic reaction of both final PK preparations to rabbit antiserum prepared against homogeneous germinating castor bean endosperm cytosolic pyruvate kinase was tested by immunoprecipitation and Western blotting. The two algal pyruvate kinases are immunologically unrelated as only PK2 cross-reacts with the cytosolic pyruvate kinase antibodies. These data indicate that the S. minutum pyruvate kinase isoforms, PK1 and PK2, are not interconvertible forms of the same protein, but probably represent chloroplastic and cytosolic isozymes, respectively.

Influence of changes in CO2 concentration and temperature on marine phytoplankton 13C/12C ratios: an analysis of possible mechanisms

Abstract The 13C/12C fractionation associated with net transport fluxes and chemical conversions, and with equilibria, associated with inorganic C assimilation processes in marine phytoplankton are quite well understood, though some gaps remain. These values are used in models of overall 13C/12C fractionation in inorganic C assimilation involving the two major mechanisms involved in inorganic C entry, i.e. diffusion of CO2 and active transport of CO2 and/or HCO3−. The CO2 diffusion model predicts the observed decrease in the 13C/12C of plankton organic C relative to source CO2 when CO2 concentration increases and/or temperature decreases. The inorganic C active transport model is complicated by repression of the active transport mechanism at high inorganic C levels, but this model also predicts the observed effect on cell 13C/12C of changes in CO2 partial pressure or temperature for cell growth. More refined modelling and more input data are needed for both transport processes. Operation of either of the alternative mechanisms for inorganic C entry can be consistent with growth rate not being limited by inorganic C supply even when the photosynthetic rate is inorganic C-limited.