ated with the following symbols representing fold changes: ++++ > 5; 5 > +++ > 3; 3 > ++ > 1; 1 > + > 0; 0 < − < −1; -1 < −−− < −3; -3 < −−− < −5; −−− < −5. The symbol denotes the overall changes of predicted isoenzymes. Some co-substrates or co-products of some reactions are omitted for clarity. The one “+” in the fatty acid synthesis in plastids denotes the overall transcriptional changes of the enzymes involved. Detailed fold changes are listed in Additional file 3 and Additional file 1. Enzymes were based on the genome annotation and prediction. Locus tags of some ambiguous isoenzymes are indicated. The localization of most of the enzymes are based on prediction and have not been determined experimentally.
The primary effect of N deprivation is the reduced availability of nitrogen, an essential nutrient for de novo synthesis of amino acids, nucleic acids and various other cellular constituents. Unsurprisingly, therefore, it strongly affected transcript levels of genes involved in nitrogen assimilation and metabolism (Additional file 3: Table S1b). Notably, marked increases were observed in transcripts encoding five of at least six ammonium transporters present in the diatom, which transport ammonium ions across the cell membrane and are known to be activated by N deprivation . The rise in the level of one of them was also confirmed in our qPCR test (Phatrdraft_27877, 45-fold increase). In addition, there was a 7-fold increase in transcripts of glutamine synthase (GS, Phatrdraft_22357, EC 22.214.171.124), a key enzyme in ammonia assimilation in both plants and Gram-negative microorganisms. GS plays an important role in the efficient use of nitrogen sources and nitrogen metabolism. Other critical nitrogen metabolism enzymes, such as nitrate reductase (Phatrdraft_54983, EC126.96.36.199), a molybdoenzyme that reduces nitrate to nitrite, and ferredoxin-nitrite reductase (Phatrdraft_12902, EC188.8.131.52), which catalyzes conversion of nitrite to ammonia, also showed significant increases (4-fold and 33-fold, respectively) in transcript abundance. These observations indicate that N deprivation induces homeostatic responses, including activation of the glutamine synthesis pathway and increases in the cells’ capacity to utilize trace amounts of nitrogen resources, and also the possible redistribution of intracellular nitrogen such as increased amino acid catabolism in accordance with the study in T. pseudonana, ultimately yielding acetyl-CoA or succinyl-CoA as the TCA cycle intermediates.
Transcripts of photosynthesis-related genes decreased
As shown in Additional file 3: Table S1c, levels of most transcripts encoding proteins associated with photosynthesis decreased under N deprivation, implicating that photosynthesis could be inhibited or at a standstill. Notably, only one encoding a light harvesting complex (LHC) component was up-regulated (Phatrdraft_16481, fucoxanthin chlorophyll a/c protein; up-regulated 10-fold), while most of the others were down-regulated more than 10-fold. As previously demonstrated in C. reinhardtii, reductions in the abundance of light-harvesting complexes may reduce photosynthetic rates , in accordance with theoretical expectations, unless reductions in transcript levels are balanced by changes at other levels. Transcript levels of ferredoxin-NADP+ reductase (Phatrdraft_43018), which catalyzes the last electron transfer, from photosystem I to NADPH during photosynthesis , also decreased (4.6-fold).
The reductions in mRNA levels of genes encoding photosynthetic proteins under N deprivation were consistent with expectations since various stresses can inhibit photosynthesis, and upregulated light harvesting can result in excess production of highly toxic reactive oxygen species (ROS) . To examine whether the reduced expression of photosynthetic genes was reflected in physiological changes, we also measured and calculated chlorophyll fluorescence parameters of the cultures, including Fo, Fm, Fv/Fm and Fv/Fo. As shown in Table 3, all of these parameters significantly decreased under N deprivation, in