Espen Granum

C3 and C4 Pathways of Photosynthetic Carbon Assimilation in Marine Diatoms Are under Genetic, Not Environmental, Control

Marine diatoms are responsible for up to 20% of global CO2 fixation. Their photosynthetic efficiency is enhanced by concentrating CO2 around Rubisco, diminishing photorespiration, but the mechanism is yet to be resolved. Diatoms have been regarded as C3 photosynthesizers, but recent metabolic labeling and genome sequencing data suggest that they perform C4 photosynthesis. We studied the pathways of photosynthetic carbon assimilation in two diatoms by short-term metabolic 14C 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 C3-C4 intermediate photosynthesis in T. weissflogii, but exclusively C3 photosynthesis in T. pseudonana. The labeling patterns were the same for cells grown at near-ambient (380 μL L−1) and low (100 μL L−1) CO2 concentrations. The lack of environmental modulation of carbon assimilatory pathways was supported in T. pseudonana by measurements of gene transcript and protein abundances of C4-metabolic enzymes (phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase) and Rubisco. This study suggests that the photosynthetic pathways of diatoms are diverse, and may involve combined CO2-concentrating mechanisms. Furthermore, it emphasizes the requirement for metabolic and functional genetic and enzymic analyses before accepting the presence of C4-metabolic enzymes as evidence for C4 photosynthesis.

Carbon acquisition by diatoms.

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 CO2 affinity and CO2/O2 selectivity, the low diffusion coefficient for CO2 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 CO2 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 CO2 and HCO3− 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 C4-like biochemistry which could act as, or be part of, a CCM. Alternative mechanisms which have not been eliminated include the production of CO2 from HCO3− at low pH maintained by a H+ pump, in a compartment close to that containing Rubisco.

A simple combined method for determination of β-1,3-glucan and cell wall polysaccharides in diatoms

A new method is described for the combined determination of β-1,3-glucan and cell wall polysaccharides in diatoms, representing total cellular carbohydrate. The glucan is extracted by 0.05 mol l−1 H2SO4 at 60 °C for 10 min, and the cell wall polysaccharides are subsequently hydrolyzed by 80% H2SO4 at 0–4 °C for 20 h. Each carbohydrate fraction is determined by the phenol-sulphuric acid method. The method has been demonstrated for axenic cultures of the marine diatom Skeletonema costatum and natural marine phytoplankton populations dominated by diatoms. Cellular glucan and cell wall polysaccharides were determined with standard deviations of 1–3% and 2–5%, respectively.

PRIMARY CARBON AND NITROGEN METABOLIC GENE EXPRESSION IN THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE): DIEL PERIODICITY AND EFFECTS OF INORGANIC CARBON AND NITROGEN 1

Diel periodicity and effects of inorganic carbon (Ci) and NO3− on the expression of 11 key genes for primary carbon and nitrogen metabolism, including potential C4 photosynthesis, in the marine diatom Thalassiosira pseudonana Hasle et Heimdal were investigated. Target gene transcripts were measured by quantitative reverse transcriptase–PCR, and some of the gene‐encoded proteins were analyzed by Western blotting. The diatom was grown with a 12 h photoperiod at two different Ci concentrations maintained by air‐equilibration with either 380 μL · L−1 (near‐ambient) or 100 μL · L−1 (low) CO2. Transcripts of the principal Ci and NO3− assimilatory genes RUBISCO LSU (rbcL) and nitrate reductase displayed very strong diel oscillations with peaks at the end of the scotophase. Considerable diel periodicities were also exhibited by the β‐carboxylase genes phosphoenolpyruvate carboxylase (PEPC1 and PEPC2) and phosphoenolpyruvate carboxykinase (PEPCK), and the Benson–Calvin cycle gene sedoheptulose–bisphosphatase (SBPase), with peaks during mid‐ to late scotophase. In accordance with the transcripts, there were substantial diel periodicities in PEPC1, PEPC2, PEPCK, and especially rbcL proteins, although they peaked during early to mid‐photophase. Inorganic carbon had some transient effects on the β‐carboxylase transcripts, and glycine decarboxylase P subunit was highly up‐regulated by low Ci concentration, indicating increased capacity for photorespiration. Nitrogen‐starved cells had reduced amounts of carbon metabolic gene transcripts, but the PEPC1, PEPC2, PEPCK, and rbcL transcripts increased rapidly when NO3− was replenished. The results suggest that the β‐carboxylases in T. pseudonana play key anaplerotic roles but show no clear support for C4 photosynthesis.

MOBILIZATION OF β-1,3-GLUCAN AND BIOSYNTHESIS OF AMINO ACIDS INDUCED BY NH4+ ADDITION TO N-LIMITED CELLS OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

Mobilization of the reserve β‐1,3‐glucan (chrysolaminaran) in N‐limited cells of the marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) was investigated. The diatom was grown in pH‐regulated batch cultures with a 14:10‐h light:dark cycle until N depletion. In a pulse‐chase experiment, the cells were first incubated in high light (200 μmol photons·m−2·s−1) with 14C‐bicarbonate until dissolved inorganic carbon was exhausted. Unlabeled bicarbonate (1 mM) was then added, and the cells were incubated in the dark and subsequently in low light (20 μmol photons·m−2·s−1) with additions of 40 μM NH4+. In the 14C pulse phase with high light and N depletion, β‐1,3‐glucan accumulated and accounted for 85% of incorporated 14C. In the subsequent 14C chase phases, added NH4+ was assimilated at an N‐specific rate of 0.11 h−1 in both the dark and low light, and in both cases it caused a significant mobilization of β‐1,3‐glucan (dark, 26%; low light, 19%). Biochemical fractionation of organic 14C showed that free amino acids were most rapidly labeled in the early stage of NH4+ assimilation, whereas proteins and polysaccharides were labeled more rapidly after 1.2 h. Analysis of the cellular free amino acids strongly indicated that de novo biosynthesis was occurring, with a Gln:Glu ratio increasing from 0.4 to 10 within 1.2 h. After the NH4+ was exhausted, the cellular pools of glucan and amino acids became constant or slowly decreased. In another experiment, N‐limited cells were first incubated in high light until dissolved inorganic carbon was exhausted and were further incubated in high light with 150 μM NH4+ under inorganic carbon limitation. Added NH4+ was assimilated at an N‐specific rate of 0.023 h−1, and cellular β‐1,3‐glucan decreased by 15% within 6 h. Hence, β‐1,3‐glucan was mobilized during NH4+ assimilation, even though inorganic carbon was modifying the metabolic rates. The results provide new evidence of β‐1,3‐glucan supplying essential precursors for biosynthesis of amino acids and other components in S. costatum in both the dark and subsaturating light and even saturating light under inorganic carbon limitation.

Effects of NH4+ assimilation on dark carbon fixation and β-1,3-glucan metabolism in the marine diatom Skeletonema costatum (Bacillariophyceae)

The effects of NH4+ assimilation on dark carbon fixation and β‐1,3‐glucan metabolism in the N‐limited marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) were investigated by chemical analysis of cell components and incorporation of 14C‐bicarbonate. The diatom was grown in pH‐regulated batch cultures with a 14:10 h LD cycle until N depletion. The cells were then incubated in the dark with 14C‐bicarbonate, but without a source of N for 2 h, then in the dark with 63 μmol·L−1 NH4+ for 3 h. Without N, the cellular concentration of free amino acids was almost constant (∼4.5 fmol·cell−1). Added NH4+ was assimilated at a rate of 12 fmol·cell−1·h−1, and the cellular amino acid pool increased rapidly (doubled in <1 h, tripled in <3 h). The glutamine level increased steeply (45× within 3 h), and the Gln/ Glu ratio increased from 0.1 to 2.4 within 3 h. The rate of dark C fixation during N depletion was only 1.0 fmol·cell−1·h−1. The addition of NH4+ strongly stimulated dark C fixation, leading to an assimilation rate of 4.0 fmol·cell−1·h−1, corresponding to a molar C/N uptake ratio of 0.33. Biochemical fractionation of organic 14C showed no significant 14C fixation into amino acids during N depletion, but during the first 1–2 h of NH4+ assimilation, amino acids were rapidly radiolabeled, accounting for virtually all net 14C fixation. These results indicate that anaplerotic β‐carboxylation is activated during NH4+ assimilation to provide C4 intermediates for amino acid biosynthesis. The level of cellular β‐1,3‐d‐glucan was constant (16.5 pg·cell−1) during N depletion, but NH4+ assimilation activated a mobilization of 28% of the reserve glucan within 3 h. The results indicate that β‐1,3‐glucan in diatoms is the ultimate substrate for β‐carboxylation, providing precursors for amino acid biosynthesis in addition to energy from respiration.

Biology of (1,3)-β-Glucans and Related Glucans in Protozoans and Chromistans

Publisher Summary This chapter discusses the occurrence and functional roles of (1,3)-β-glucans and related glucans are treated in a taxonomic framework starting with the protozoan Euglenophyta (Euglenophyceae), followed by the chromistans Haptophyta (Haptophyceae) and Heterokontophyta. (1,3)-β-Glucans and related glucans are important both as storage polysaccharides (laminarins and paramylon) and structural cell wall polysaccharides (callose, cellulose, chitin, etc.) in protozoans and chromistans. The nature of such polysaccharides is somewhat correlated with taxonomy, but there is considerable variation between, and sometimes even within, classes in each division. Furthermore, the contents and detailed structures of the polysaccharides vary with the state of growth and development. The storage polysaccharides in the euglenids (paramylon), haptophytes, diatoms and chrysophytes (chrysolaminarin), oomycetes (mycolaminarin) and brown algae (laminarin) are (1,3)-or (1,3;1,6)-β-glucans. Furthermore, (1,3)-β-glucans and related glucans are essential as structural cell wall components in many protozoans and chromistans, including callose ((1,3)-β-glucan), cellulose ((1,4)-β-glucan), chitin ((1,4)-β-N-acetylglucosamine) and (1,3:1,4)-β-glucans. There is considerable variation in the chemical structures and properties of different (1,3)-β-glucans, from the fibrillar callose and paramylon to the water-soluble laminarins.