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 k_Q·Q_max^?1*, was similar in magnitude to previously reported values but k_Q·Q_max^?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 (k_Qi·K_Qj^?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. The capacity for luxury consumption differed between nutrients and was growth rate dependent. At low growth rates, the coefficient of luxury consumption (R_sat) for P was ca. four times that for N. The set of all possible relationships between N and P cell quota under these conditions was reported and these values were then used to establish the cellular N:P niche boundaries for S. minutum. Cell quotas of non-limiting nutrients were not described by the Droop equation. Analysis showed that as the cellular N:P ratio deviates from the optimum ratio, the ability of the Droop equation to describe the relationship between growth rate and non-limiting cell quotas decreases. When non-limiting nutrient cell quotas are saturated, the Droop equation appears to be invalid. Previously reported patterns of non-limiting nutrient utilization are summarized in support of this conclusion. The physiological and ecological consequences of luxury consumption and growth rate dependent optimum ratios are considered.     

PREDICTING THE KINETICS OF DISSOLVED INORGANIC CARBON LIMITED GROWTH FROM THE SHORT-TERM KINETICS OF PHOTOSYNTHESIS IN SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYTA)1

The blue-green alga (Cyanobacterium) Synechococcus leopoliensis (Racib.) Komarek was grown in dissolved inorganic carbon [DIC]-limited chemostats over the entire range of growth rates. At each growth rate, the kinetics of photosynthesis with respect to [DIC] and the maximal rate of photosynthesis (P_max) were determined. The half-saturation constant for [DIC]-limited photosynthesis (K_1/2^DIC) for cells growing below 1.7 d^?1 was constant (4.7 μM) whereas for growth rates between 1.7 d^?1 and 2.1 d^?1 (μ_max) the kinetics of photosynthesis were multiphasic with an apparent K_1/2^DIC between 1.5–2.0 mM. P_max increased in a linear fashion with growth rate for growth rates below 1.7 d^?1. No trend in P_max was apparent for growth rates greater than 1.7 d^?1. These kinetic parameters were used to predict a growth rate versus [DIC] relationship. Results show that the Monod relationship is a physiologically valid expression of growth as a function of [DIC] provided (K_1/2^DIC) remains constant. The major change in (K_1/2^DIC) as μ approaches μ_max results in the conclusion that two separate and distinct Monod equations must be used to describe growth as a function of DIC over the entire growth range. These results point to a major discontinuity in the μ vs. [DIC] curve at 1.7 d^?1 which corresponds to the change from high to low affinity photosynthetic kinetics. We believe these results account for the previously described deficiencies of the Monod equation in describing [DIC]-limited algal growth.     

The molecular force field of guanine and its deuterated species as determined from neutron inelastic scattering and resonance Raman measurements

Neutron inelastic scattering (NIS) spectra from polycrystalline samples and ultraviolet resonance Raman scattering (RRS) spectra from aqueous solutions of guanine and CS-deuterated and (N9, NI, C2-amino)-deuterated guanine are reported. These measurements allowed theoretical simulations of the vibrational wavenumbers and intensities of the NIS and RRS bands to be performed. Å valence force field enabled the normal mode wavenumbers, as well as the atomic displacements, to be calculated. The NIS intensities were simulated by considering multi-phonon interactions arising from the lattice mode couplings with the internal molecular vibrational modes. The RRS intensities were simulated within the framework of the so-called small shift approximation, by using the molecular bond-order changes induced by the electronic transition from the ground to the first electronic excited state. It is shown that NIS spectroscopy mainly provides information on the guanine out-of-plane modes of vibration, while RRS allows the in-plane stretching vibrational motions to be analyzed.     

Appearance of Novel Glucose-6-Phosphate Dehydrogenase Isoforms in Chlamydomonas reinhardtii during Growth on Nitrate

Extractable glucose-6-phosphate dehydrogenase activity is higher from N-limited Chlamydomonas reinhardtii cells than from N-sufficient cells. Native gels reveal that the isoform complexity varies depending on the form of N supplied. The isoforms associated with NO3- growth appear within 2 h of switching cells from NH4+ to NO3-.     

Effects of Phosphorus Limitation on Respiratory Metabolism in the Green Alga Selenastrum minutum

The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi-limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to “bypass” the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.     

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: III. Alanine Is the Product of Anaerobic Ammonium Assimilation

We have determined the flow of ¹⁵N into free amino acids of the N-limited green alga Selenastrum minutum (Naeg.) Collins after addition of ¹⁵NH₄⁺ to aerobic or anaerobic cells. Under aerobic conditions, only a small proportion of the N assimilated was retained in the free amino acid pool. However, under anaerobic conditions almost all assimilated NH₄⁺ accumulates in alanine. This is a unique feature of anaerobic NH₄⁺ assimilation. The pathway of carbon flow to alanine results in the production of ATP and reductant which matches exactly the requirements of NH₄⁺ assimilation. Alanine synthesis is therefore an excellent strategy to maintain energy and redox balance during anaerobic NH₄⁺ assimilation.     

Significance of Phosphoenolpyruvate Carboxylase during Ammonium Assimilation: Carbon Isotope Discrimination in Photosynthesis and Respiration by the N-Limited Green Alga Selenastrum minutum

The effect of N-assimilation on the partitioning of carbon fixation between phosphoenolpyruvate carboxylase (PEPcase) and ribulose bisphosphate carboxylase/oxygenase (Rubisco) was determined by measuring stable carbon isotope discrimination during photosynthesis by an N-limited green alga, Selenastrum minutum (Naeg.) Collins. This was facilitated by a two process model accounting for simultaneous CO₂ fixation and respiratory CO₂ release. Discrimination by control cells was consistent with the majority of carbon being fixed by Rubisco. During nitrogen assimilation however, discrimination was greatly reduced indicating an enhanced flux through PEPcase which accounted for upward of 70% of total carbon fixation. This shift toward anaplerotic metabolism supports a large increase in tricarboxylic acid cycle activity primarily between oxaloacetate and α-ketoglutarate thereby facilitating the provision of carbon skeletons for amino acid synthesis. This provides an example of a unique set of conditions under which anaplerotic carbon fixation by PEPcase exceeds photosynthetic carbon fixation by Rubisco in a C₃ organism.     

Short-Term Metabolite Changes during Transient Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum

In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH₄⁺ addition we found that: (a) N accumulated in glutamine while glutamate and α-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH₄⁺ addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru-1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH₄⁺ addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme.