Physiological and transcriptomic evidence for a close coupling between chloroplast ontogeny and cell cycle progression in the pennate diatom Seminavis robusta

Abstract

Despite the growing interest in diatom genomics, detailed time series of gene expression in relation to key cellular processes are still lacking. Here, we investigated the relationships between the cell cycle and chloroplast development in the pennate diatom Seminavis robusta. This diatom possesses two chloroplasts with a well-orchestrated developmental cycle, common to many pennate diatoms. By assessing the effects of induced cell cycle arrest with microscopy and flow cytometry, we found that division and reorganization of the chloroplasts are initiated only after S-phase progression. Next, we quantified the expression of the S. robusta FtsZ homolog to address the division status of chloroplasts during synchronized growth and monitored microscopically their dynamics in relation to nuclear division and silicon deposition. We show that chloroplasts divide and relocate during the S/G2 phase, after which a girdle band is deposited to accommodate cell growth. Synchronized cultures of two genotypes were subsequently used for a cDNA-amplified fragment length polymorphism-based genome-wide transcript profiling, in which 917 reproducibly modulated transcripts were identified. We observed that genes involved in pigment biosynthesis and coding for light-harvesting proteins were up-regulated during G2/M phase and cell separation. Light and cell cycle progression were both found to affect fucoxanthin-chlorophyll a/c-binding protein expression and accumulation of fucoxanthin cell content. Because chloroplasts elongate at the stage of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigments in concert with cell division might balance chloroplast growth, which confirms that chloroplast biogenesis in S. robusta is tightly regulated.

Figure 1.

Response of S. robusta to dark treatments. A, Flow cytometric DNA content histograms of an exponentially growing culture before transfer to darkness (top), after 24 h of dark incubation (middle), and after 24 h of light (bottom). B to D and F, Light microscopic photographs of the 24-h light-treated culture (strain F₁-8B; B), the 24-h dark-treated culture (strain F₁-8B; C), a 24-h dark-arrested cell in girdle view with focus on the ventral chloroplast (strain F₂-31B; D), and a 24-h dark-arrested cell in valve view (strain F₂-31B; F). The chloroplasts appear brownish, and the cell wall is visible due to its high contrast. E and G, Confocal fluorescence microscopic photographs of cells from dark-arrested cultures (strain F₂-31B) in which the chloroplastidic signal is red (autofluorescence), the nuclear signal is green (SYBR Safe), and the cell wall signal is blue (PDMPO; Lysosensor). Chloroplasts of dark-arrested cultures are undivided and pressed against the girdle. The ventral chloroplast has four subcentral lobes that extend across the valve toward the dorsal side of the cell. Bars = 10 μm.

Figure 2.

S. robusta cells in cell cycle inhibitor-treated cultures. A and B, Light-microscopic photographs. C and D, Fluorescence microscopic photographs. A, HU-treated cell (strain F₂-31B) arrested during S phase. B to D, Aphidicolin-treated cell (strain F₁-8B) in girdle view (B) with focus on the ventral chloroplast (C) and on the dorsal chloroplast (D). In each case, both chloroplasts are undivided and located against the valves. The difference in cell morphology between cells in A and B to D is due to the different average cell sizes of the strains used. Bars = 10 μm.

Figure 3.

Evaluation of the synchrony of cell and chloroplast division in S. robusta synchronized strains F₁-8B and F₁-9A. The proportion of dividing cells is presented as a function of time, during reillumination of the 24-h dark-arrested cultures. Data are means ± se of six microscopic fields (0.09 mm²). The predominant morphological stages in each cell cycle phase are represented from light to dark gray in this order: undivided chloroplasts, dividing chloroplasts, dividing cells, and separating cells. G1 corresponds to the phase in which cells with undivided girdle-located chloroplasts are present. The asterisk denotes the first observed cells with divided chloroplasts and also approximates the moment of S-phase initiation, according to the transcriptional induction of the MCM5 prereplication factor (Supplemental Fig. S8). G2/M corresponds to the phase in which dividing cells were increasingly represented with a maximum in the amount of dividing cells at 9 h. M/G1 corresponds to the stage of cell separation.

Figure 4.

Cytological events during synchronized growth of S. robusta observed by confocal fluorescence microscopy. The chloroplastidic signal is red (autofluorescence), the nuclear signal is green (SYBR Safe), and the silica tracer signal is blue (PDMPO; Lysosensor). In photographs A, C, D, and H, the transmission light channel was permitted in order to visualize the outline of the cell. B to D, Cells shown in valve view. A, E to J, and L, Cells shown in girdle view. A, Cell with focus on a dividing dorsal chloroplast, displaying the central constriction (arrow). B, Cell with the dorsal chloroplast divided. C, Cell with both chloroplasts divided. D and E, Cell with fully rotated, valve-located chloroplasts and containing an undivided nucleus. F, Cell with valve-located chloroplasts and showing a blue band in the middle of the cell, extending from pole to pole. This band probably represents a girdle band (Supplemental Fig. S4). G, Cell just after karyokinesis. Each daughter nucleus is situated on a different side of the future division plane. H, Cell during cytokinesis. The division of the protoplast is apparent by the cleavage furrow, visible as a line of higher contrast running from pole to pole and in between both daughter nuclei. The PDMPO signal is confined to two “vacuole-like” compartments; as such, it appears that deposition of silica at the site of the cleavage furrow has not yet begun. I, Dividing cell during frustule formation. Deposition of silica into the frustule is visible as a line running from pole to pole and situated between the chloroplast pairs and daughter nuclei. J, Dividing cell with chloroplasts moving back from the valves toward the girdle. K, Separating daughter cells in slightly tilt valve view with focus on the dorsal chloroplasts, which are completely covering the girdle area. From the ventral chloroplasts, only the subcentral lobes are visible. L, A newly divided cell with the newly formed valve stained with PDPMO. The other valve is inherited from the mother cell. Bar = 10 μm.

Figure 5.

Expression of the S. robusta FtsZ ortholog as a function of time after reillumination during two replicate synchronizations of strain F₁-8B. The proportion of dividing cells is presented as a function of time during reillumination together with the relative expression values of FtsZ. RT-PCR data were normalized against three cDNA-AFLP-acquired reference genes. [See online article for color version of this figure.]

Figure 6.

Hierarchical clustered expression profiles of 917 TDFs reproducibly modulated during the cell cycle of synchronized cultures of strain F₁-8B and F₁-9A. Clustering was performed using TMEV software (Saeed et al., 2003) and the hierarchical clustering algorithm (Eisen et al., 1998). Each row represents a tag with the relative transcript accumulation patterns shown over 12 consecutive time points (columns) after reillumination of dark-arrested cultures. Yellow and blue color intensities reflect up- and down-regulation of gene expression relative to a range of +3 to −3, respectively; gray represents missing data. Cluster names (in accordance with adaptive quality-based clustering) and cell cycle phases are indicated at right and left, respectively. The arrow corresponds with the expression profile of the DNA replication licensing factor MCM5.

Figure 7.

Comparison of fucoxanthin pigment turnover and FCP expression between light/dark-synchronized cultures and light/dark-synchronized cultures to which the cell cycle inhibitor aphidicolin was added shortly before reillumination. A difference in fucoxanthin pigment (μg mg⁻¹ dry weight) accumulation is apparent after 5 h of reillumination. In the S-phase-arrested cultures, the buildup is less pronounced. In parallel, FCP expression is up-regulated between 1 and 5 h after reillumination in both dividing and arrested cultures, but its expression is down-regulated after the initial increase in arrested cultures while it is maintained in the dividing cultures. [See online article for color version of this figure.]