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

Abstract

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.

Keywords: C4 photosynthesis; CO2-concentrating mechanism; Thalassiosira pseudonana; anaplerosis; carbon fixation; diatom; gene expression; nitrogen assimilation; photorespiration.