Figure 1.
Cell density as a function of culture time.
A) Growth of A. anophagefferens grown on urea, nitrate, and a mixture of urea and nitrate. B) Growth of A. anophagefferens under nitrogen-replete and limited conditions. Cell density under nitrogen-limited condition was compared with that under nitrogen-replete condition. Significance values were expressed as follows: * P <0.05, ** P <0.001. C) Growth rates of A. anophagefferens grown on urea, nitrate, and a mixture of urea and nitrate. Error bars represent standard deviation of the mean for the three biological replicates.
Figure 2.
Photosystem II efficiency (Fv/Fm).
A) A. anophagefferens cultures grown on urea, nitrate, and a mixture of urea and nitrate at the 6-, 9- and 10-day sampling points; B) A. anophagefferens cultures grown under nitrogen-replete (Nr), limited (Nli) and recovery conditions. Nir and Nur represent nitrate and urea addition, respectively. Nli was compared with Nr while Nir and Nur were compared with Nli. Significance values were expressed as follows: * P <0.05, ** P <0.001. Error bars represent standard deviation of the mean for the three biological replicates.
Figure 3.
Relative electron transport rate (ETR) as a function of PAR.
A. anophagefferens was grown on urea, nitrate, and a mixture of urea and nitrate, and samples were meaused at the 6-, 9- and 10-day. Error bars represent standard deviation of the mean for the three biological replicates.
Figure 4.
Concentrations of urea and nitrate as a function of culture time in media.
Solid triangle represents nitrate concentration in medium with mixture N while solid and open circles represent urea concentrations in media with urea and mixture N, respectively. The difference between time points with asterisk was significant. Significance values were expressed as follows: * P <0.05, ** P <0.001. Error bars represent standard deviation of the mean for the three biological replicates.
Table 1.
Summary of RNA-seq sequencing data (mapping to the reference genome).
Table 2.
Summary of RNA-seq sequencing data (mapping to the reference transcript).
Figure 5.
The number of differentially expressed genes among cells that were grown on the three N sources.
Nitr, urea and mix represent cells grown on nitrate, urea and mixture N, respectively.
Figure 6.
Proposed model showing the urea cycle, nitrate transport and assimilation, and the glutamine-glutamate cycle.
Enzymes that are involved in these pathways are labeled with colors that indicate the fold change (log2) in their transcript levels in urea-grown cells relative to nitrate-grown cells (color code is provided in the figure). NRT, nitrate transporter; NR nitrate reductase; NAR1, nitrite transporter; NasB, NADPH nitrite reductase; GS, glutamine synthetase; Fd-GOGAT, ferredoxin-dependent glutamate synthase; CPS, carbamoyl phosphate synthase; OTC, ornithine transcarboxylase; AsuS, argininosuccinate synthase; ASL, argininosuccinate lyase; ARG, arginase; Ure, urease; DUR, urea transporter.
Figure 7.
Pathways for starch synthesis, glycolysis, aromatic amino acid synthesis and the TCA cycle.
Enzymes that are involved in these pathways are labeled with colors that indicate the fold change (log2) in their transcript levels in urea-grown cells relative to nitrate-grown cells (color code is provided in the figure). Dashed lines mean that no differentially expressed genes were detected in these pathways. AL, aldolase; PGM, phosphoglucomutase; UGPase, UDP-glucose-pyrophosphorylase; PFK, phosphofructokinase; TPI, triose-phosphate isomerase; PHM, phosphoglycerate mutase; PK, pyruvate kinase, PC, pyruvate carboxylase; OXH, oxoglutarate dehydrogenase; SCS, succinyl-CoA ligase; MDH, malate dehydrogenase; EPSPS, 3-phosphoshikimate 1-carboxyvinyltransferase; CHM, chorismate mutase; DES, dehydroquinate synthase; ASPA, aspartate aminotransferase; ACCase, Acetyl-CoA carboxylase; GLPI, glucose-6-phosphate isomerase.
Figure 8.
Specific genes transcriptionally regulated by urea.
A) Number of common genes differentially regulated between the mixture N and urea groups compared with the reference nitrate group; B) the pattern of regulation of these common genes.
Table 3.
GO function class of the genes differentially regulated that are common between the urea and mixture N groups compared with the reference nitrate group.
Table 4.
List of the genes differentially regulated among the nitrogen-replete (Nrep), limited (Ndep) and 24 h recovery (urea recovery, Urecov; nitrate recovery, Nrecov) libraries.
Figure 9.
Absolute abundances of transcripts related to N assimilation pathway and the urea cycle.
The transcript abundance that was obtained from RNA-seq data is indicated as RPKM (see Methods). The abundance of NasB transcript is shown on the right Y axis, while that of other transcripts is shown on the left Y axis. NAR1, nitrite transporter NAR1; NRT, nitrate high affinity transporter; AMT, ammonium transporter; NasB, NADPH nitrite reductase; NR, nitrate reductase; CPS, CPSase; AsuS, argininosuccinate synthase; ASL, argininosuccinate lyase.