Table 1.
Primers used in this study.
Figure 1.
P. donghaiense form II RuBisCO gene (Pdrbc) structure and copy number in the genome.
A) Gene structure (horizontal bars) inferred from isolated cDNA sequences using various primer sets (arrows with labels below the bars). 5-UTR, 5′-untranslated region; HSP, hydrophobic signal peptide; cTP, chloroplast transit peptide; HPD, hydrophilic domain; CU, coding unit; S, spacer sequence; 3-UTR, 3′-untranslated region; The primer sequences are shown in Table 1. B) Estimation of Pdrbc copy number in P. donghaiense genome using qPCR. Standard curve (solid triangle and line) was constructed using decadal dilution series of purified Pdrbc cDNA, from 107 to 102 copies, each in triplicate. Three different amounts of P. donghaiense genomic DNA (1 ng, 5 ng and 10 ng), each in triplicate, were used as unknown samples, which gave estimate of 117±40 copies of Pdrbc in one cell.
Figure 2.
Phylogenetic tree of dinoflagellate form II RuBisCO based on full CU sequences.
Tree topology shown is from Neighbor-Joining (NJ) analysis; Bayesian analysis (BE) gave similar tree topology. Support of nodes >70% in NJ bootstrap values (left) and >0.70 in Bayesian posterior probability (right) are both shown.
Figure 3.
Cell cycle profiles over a 24-h sampling period in the 14∶10 light∶dark cycle (A), continuous light (B) and continuous dark (C) cultures.
Grey shading indicates dark period. The progression of the cell cycle can be observed from the time-sequential succession of the peaks of the G1, S, and G2M phase fractions (e.g. A and B), the lack of which is indication of no cell cycle progression (C).
Figure 4.
Gene transcription dynamics of reference genes (A–B) and Pdrbc normalized to total RNA (C1, D1, E1) and to the reference genes (C2, C3, D2, E2).
LD: under light/dark cycle. LL: under continuous light. DD: under continuous darkness. Sampling of the LL and DD cultures started after the cultures had been placed under LL and DD conditions for 22 h, respectively. Hatched bars denote dark cycle. Dotted lines in vertical denote the peak expression time point. Error bars indicate ± standard deviation.
Figure 5.
Correlation between Pdrbc mRNA abundance and percentage of G2M-phase cells as well as time distance from onset of the light period.
A1–A2: correlation between calm-normalized Pdrbc mRNA abundance and percentage of G2M-phase cells (A1) and time distance (A2) under light/dark cycle. B1–B2: correlation between gapdh-normalized Pdrbc mRNA abundance and percentage of G2M-phase cells (B1) and time distance (B2) under light/dark cycle. C1–C2: correlation between QTR- or calm-normalized Pdrbc mRNA abundance and percentage of G2M-phase cells under continuous light. Time distance, regardless before or after onset of the light period, is taken as positive values. As the culture was grown under a 14 h∶10 h light∶dark regime, time points over 10 hours backward from the onset of the light period would be in the light period again, we limited the correlation analysis to data within 10 hours from the onset of the light period. LD: under light/dark cycle. LL: under continuous light. DD: under continuous dark.
Figure 6.
Schematic of PdRBC import pathway into the chloroplast in P. donghaiense.
The precursor polypeptide consists of a signal peptide (orange) followed by a chloroplast transit peptide (deep green) and a hydrophobic signal peptide (blue). The signal peptide leads the polyprotein through the outermost membrane. The chloroplast transit peptide guides the polyprotein into the two inner membrane and then is cleaved off, unmasking the hydrophobic region peptide to stop transfer. The dashed arrows depict unknown cleave mechanism.