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3.21 Verification of the diurnally-entrained model system for chloroplast growth and division: T-zone localized PSII biogenesis was shown to occur during y-1 chloroplast differentiation (Chapter 3.1) and within light-stimulated dark-adapted mature cells [2]. The previous mature cell model system had two critical issues: PSI biogenesis was not stimulated and the percentage of cells displaying T-zone localization were limited due to asynchronous algal growth. Therefore, it

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was advantageous to use another mature algal model system which is undergoing coordinated thylakoid biogenesis and thus can still stimulate both photosystems in a temporal manner.

The use of diurnally-entrained cultures provided both a synchronized cell cycle and the coordination of thylakoid membrane biogenesis for mature chloroplast growth. Synchronization of algal cultures was achieved using a home-made system in substitute of a bioreactor. Through the control of environmental parameters, this system provided us with reproducible 24-hour diurnally-entrained cells which were verified for markers of synchrony and then subsequently used for experimentation. An acceptable level of synchrony was achieved if the following markers were observed: a visible cell size increase throughout the light cycle, coordinated division during mid- dark phase, and a 10 fold increase in cell density between 24-hour cycles [3], [52], [67]. Light microscope images qualitatively assessed synchrony throughout the 24-hour cycle (Figure 8).

Diurnally-entrained cells displayed the expected qualitative changes in cellular morphology and cell-cycle events (Figure 8). Zeitgeber Time 0 cells (ZT0) the first time point of the 24-hour cycle, displayed morphology typical of newly released progeny after division: pale green color, actively swimming, a small size, and oblong in shape. Cells at the end of that same 12-hour light phase (ZT12) exhibited the expected morphology of grown chloroplasts ready to

Figure 8. Qualitative verification of diurnally-entrained cells

Light microscope images of synchronized cells entrained under a 24-hour light/dark cycle. Scale bar is 10 µm, indicating comparative change in cell size.

*All work conducted by Melissa V.P.

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begin division: darker green color, relatively immobile, a larger size, and a rounded shape. This change in cell shape and size is one diurnally-entrained marker of synchrony. Another hallmark of synchrony is denoted by the characteristics of cells preparing for division, where the noticeable qualitative cellular changes were; the re-absorption of the two flagella resulting in limited cellular movement, and the less-clear internal chloroplast morphology (Figure 8) [3]. These light phase changes in chloroplast morphology are accompanied by the intra-chloroplastic accumulation of starch for use as an energy source during the dark phase [20]. Upon entering the dark phase these diurnally-entrained cells begin dividing by multiple fission which must be completed before the beginning of the next 24-hour cycle [3], [55].

Another marker of synchrony is the timing of division, where the expected mid-dark phase divisional timing was confirmed within our diurnally-entrained cultures (Figure 8). The presence of dividing cells and newly released progeny at ZT18 denotes that cells began this divisional process during the early-to-mid dark phase (Figure 8). Moreover, by the end of this dark phase (ZT24) a large majority of cells had completed division and thus were newly released progeny (Figure 8). Therefore, our diurnally-entrained cells displayed the expected dark phase coordination of division and the required completion of this process before the subsequent light phase. Lastly, the final marker of synchrony was the rapid and large increase in culture density.

The increase in culture density was qualitatively suggested by the light microscope images and was quantitatively confirmed through counting (Figure 8 and 9A). Comparison of the ZT0 and the ZT24 light microscope images revealed a visible augmentation in cell number and hence cell density during the 24-hour light/dark cycle (Figure 8). This cell density increase began after ZT16 which supports the commencement of division during early-to-mid dark phase (Figure 9A). Moreover, the surge in diurnal cell density between ZT16 and ZT24 temporally coincided with the previously observed cell division within the ZT18 cell population image (Figure 8 and 9A). Therefore, this rapid 7-fold dark-phase increase in cell density confirms the final marker of synchrony within our diurnally-entrained cells. Having numerically supported this hallmark of synchrony we sought to do the same for the other qualitative observations covered previously.

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Imaged cells were electronically measured thus their cell size increase throughout the 12- hour light phase was numerically quantified (Figure 9B, 9C and Supplemental Figure 5). Previous qualitative analysis during the 12-hour light phase reveled a change in cell size and shape, where cells became larger and rounder (Figure 8). Comparison between ZT0 cells and ZT12 cells displayed an increase of average cell length from 7 µm to 10 µm while the average cell width increased from 4 µm to 9 µm (Supplemental Figure 5A). This numerical cell size increase supports the growth of cells from the beginning to the end of the light phase and indicates a change in cell shape as suggested by the 1.4-fold increase in cell length as compared to the 2.25-fold increase in cell width. This change in cell shape was supported by the calculated decline in length-to-width ratio and by the increase of an arbitrary Fiji parameter called ‘roundness’ (Figure 9B and

Figure 9. Quantitative verification of diurnally-entrained cells

(A) Cell density (cells/mL) counted over the 24-hour cycle. (B) Calculated length to width ratio based on manual measurements of light microscope images.

Sample sizes of cells: ZT0 (n=58), ZT2 (n=52), ZT4 (n=50), ZT6 (n=68), ZT8 (n=69), ZT10 (n=59), ZT12 (n=63).

(C) Cell area, calculated through use of a Fiji macro and light microscope images. Sample sizes of

cells: ZT0 (n=60), ZT2 (n=51), ZT4 (n=50), ZT6 (n=67), ZT8 (n=68), ZT10 (n=58), ZT12 (n=65).

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Supplemental Figure 5B). Lastly, cell size increase was further confirmed using calculated cell area. The comparison of the cell area between ZT0 and ZT12 cells denoted an increase from 7 µm2 to 18 µm2 _{(Figure 9C). These quantitative analyses confirm that our diurnally-entrained cells are} changing in cell size, cell shape, and cell area satisfying more markers of culture synchrony.

In summary, our synchronized cell system reproducibly generates diurnally-entrained algal cultures which demonstrate all expected markers of synchrony. Qualitative visual analyses supported these known hallmarks of diurnally-entrained cells and quantitative measurements confirmed these observed changes in cell size, cell shape, and divisional timing. Most importantly, these results have shown that our diurnally-entrained cultures are equivalent to those grown within more sophisticated apparatuses and thus they are suitable for subsequent experimental use.

Supplemental Figure 5. Additional quantitative verification of diurnally-entrained cells (A) Average length and width of cells per 2-hour ZT time point, derived through manual

measurements of light microscope images. Sample sizes of cells: ZT0 (n=58), ZT2 (n=52), ZT4

(n=50), ZT6 (n=68), ZT8 (n=69), ZT10 (n=59), ZT12 (n=63).

(B) Fiji calculated roundness of cells per 2-hour ZT time point, derived through light microscope

images. Sample sizes of cells: ZT0 (n=60), ZT2 (n=51), ZT4 (n=50), ZT6 (n=67), ZT8 (n=68), ZT10 (n=58), ZT12 (n=65).

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3.22 Characterization of the diurnally-entrained model system for chloroplast growth and division: