A detailed study was made of cellular and extracellular production of carbohydrates and amino acids by the marine diatom Skeletonema costatum (Grev.) Cleve during different growth phases. Batch cultures were run with a 14:10 h light:dark cycle in N-limited media at 2 different nutrient strengths. The exponential growth rate was 2.0 div. d -1, and balanced growth took place except for significant diel variation in chemical composition. Inorganic C and N were primarily assimilated during the photophase, and the elemental cell quotas increased accordingly. The level of storage polysaccharide, β-1, 3-glucan, oscillated between 17% (end of scotophase) and 42% (end of photophase) of cellular organic C, and the corresponding protein:glucan ratio alternated between 2.3 and 0.7. Cell wall polysaccharides constituted 6 to 10% of cellular organic C. Concurrently, the cellular free amino acid pool oscillated between 8% (end of scotophase) and 22% (end of photophase) of cellular organic N. Glutamine emerged as the principal amino acid during photosynthesis, increasing from 0.2 to 12 fmol cell -1 , and the corresponding glutamine:glutamate ratio increased from 0.05 to 2. Upon NO 3 -exhaustion, the glucan level increased rapidly for 3 to 4 d, and then stabilized at 75 to 80% of cellular organic C with little diel variation. In contrast, the cellular N quota decreased by 80%, and the cell wall polysaccharide quota decreased by 35%. Consequently, the protein:glucan ratio decreased to < 0.1. The cellular free amino acid pool decreased by 90% within 24 h of N depletion, and continued to decrease slowly throughout the stationary phase. Glutamine decreased most rapidly, and constituted <1% of the free amino acids in the stationary phase. Extracellular production accounted for 4% of total photosynthetic production during both exponential and stationary growth phase, but the absolute excretion rate (per cell) was markedly higher in the exponential phase. A transient high release occurred in the transition phase in 1 case, which was probably caused by cell leakage. Extracellular production by 'healthy' cells contained 33% polysaccharides, 15% monosaccharides and 5% free amino acids (as C). The composition of the extracellular amino acids differed from the intracellular ones, and changed considerably from exponential to stationary growth phase. This study illustrates the rapid response of carbohydrate and amino acid dynamics to ambient N and light conditions at the cellular level.

Marine diatoms are responsible for up to 20% of global CO 2 fixation. Their photosynthetic efficiency is enhanced by concentrating CO 2 around Rubisco, diminishing photorespiration, but the mechanism is yet to be resolved. Diatoms have been regarded as C 3 photosynthesizers, but recent metabolic labeling and genome sequencing data suggest that they perform C 4 photosynthesis. We studied the pathways of photosynthetic carbon assimilation in two diatoms by short-term metabolic 14 C labeling. In Thalassiosira weissflogii, both C3 (glycerate-P and triose-P) and C4 (mainly malate) compounds were major initial (2-5 s) products, whereas Thalassiosira pseudonana produced mainly C3 and C6 (hexose-P) compounds. The data provide evidence of C 3 -C 4 intermediate photosynthesis in T. weissflogii, but exclusively C 3 photosynthesis in T. pseudonana. The labeling patterns were the same for cells grown at near-ambient (380 mL L 21 ) and low (100 mL L 21 ) CO 2 concentrations. The lack of environmental modulation of carbon assimilatory pathways was supported in T. pseudonana by measurements of gene transcript and protein abundances of C 4 -metabolic enzymes (phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase) and Rubisco. This study suggests that the photosynthetic pathways of diatoms are diverse, and may involve combined CO 2 -concentrating mechanisms. Furthermore, it emphasizes the requirement for metabolic and functional genetic and enzymic analyses before accepting the presence of C 4 -metabolic enzymes as evidence for C 4 photosynthesis.

Diatoms are responsible for up to 40% of primary productivity in the ocean, and complete genome sequences are available for two species. However, there are very significant gaps in our understanding of how diatoms take up and assimilate inorganic C. Diatom plastids originate from secondary endosymbiosis with a red alga and their Form ID Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase) from horizontal gene transfer, which means that embryophyte paradigms can only give general guidance as to their C acquisition mechanisms. Although diatom Rubiscos have relatively high CO(2) affinity and CO(2)/O(2) selectivity, the low diffusion coefficient for CO(2) in water has the potential to restrict the rate of photosynthesis. Diatoms growing in their natural aquatic habitats operate inorganic C concentrating mechanisms (CCMs), which provide a steady-state CO(2) concentration around Rubisco higher than that in the medium. How these CCMs work is still a matter of debate. However, it is known that both CO(2) and HCO (3) (-) are taken up, and an obvious but as yet unproven possibility is that active transport of these species across the plasmalemma and/or the four-membrane plastid envelope is the basis of the CCM. In one marine diatom there is evidence of C(4)-like biochemistry which could act as, or be part of, a CCM. Alternative mechanisms which have not been eliminated include the production of CO(2) from HCO (3) (-) at low pH maintained by a H(+) pump, in a compartment close to that containing Rubisco.

Diatoms are responsible for at least a quarter of inorganic carbon fixed each year in the ocean. Despite very considerable research over the last 30 years, there are still a number of fundamental unresolved aspects of inorganic carbon assimilation by marine diatoms. It is not clear how the carbon-concentrating mechanism functions and whether it is based on the direct acquisition of inorganic carbon or on a C4 pathway, or a combination of both. Although evidence for the operation of a C4 pathway is accumulating, the role(s) of the enzyme(s) responsible for "C3 + C1" inorganic carbon assimilation in the light and dark are still matters of controversy. In this review, we discuss whether diatoms possess the enzymic and structural components necessary for a C4-type CO2-concentrating mechanism. These are compared and contrasted with other C4 systems, both single-celled and those in terrestrial plants, which are based on Kranz anatomy. New data are presented on expression of genes that might be involved in C4 photosynthesis, including phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase.Key words: CO2-concentrating mechanism, C4 photosynthesis, marine diatoms, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase.