Lipid Droplets of Bacteria, Algae and Fungi and a Relationship between their Contents and Genome Sizes as Revealed by BODIPY and DAPI Staining

Abstract

SummaryWe have developed a convenient method to visualize triacylglycerol-filled lipid droplets (LDs) in some species of bacteria, algae and fungi by staining with borondipyrromethene difluoride (BODIPY). When BODIPY was excited by blue light, LDs emitted green fluorescence, which was distinguished easily from the red autofluorescence of chloroplasts. This makes BODIPY staining suitable for the identification of small amounts of LDs, especially in plants. We first ensured that in Chlamydomonas reinhardtii cells growing in nitrogen-replete (+N) and -deficient (-N) media, the spots of BODIPY-stained LDs coincided with those of Nile Red-stained LDs. In addition, it was shown that the LD content per cell in N-starved cells was 200-fold higher than those of the control (+N) using a video-intensified microscope photoncounting system (VIMPCS). BODIPY staining was applied to visualize LD in bacteria, algae and fungi, and included those algae regarded as nonoleaginous. We identified LD spots in unicellular and multicellular bacteria and eukaryotes, namely Cyanidioschyzon merolae, Cyanidium caldarium delta, Chlamydomonas reinhardtii, Klebsormidium nitens and Penicillium sp., but not in Anabaena flos-aquae. We also examined the relationship between the contents of LDs and the genome size in the algae and fungi using VIMPCS but were unable to find a strong relationship between genome size and production of LDs. Finally, the location of LDs was considered in relation to organelles including the endoplasmic reticulum and chloroplasts, which are related to the formation of LDs.Key words Lipid droplets, Genome size, BODIPY, Cyanidioschyzon merolae, Algae, Fungi.There is considerable interest in the development of technologies that harvest lipids from microalgae and fungi and convert them into diesel fuel (Hu et al. 2008, Radakovits et al. 2010, Scott et al. 2010, Wijffels and Barbosa 2010. A widely adopted approach is to collect non-food algal

Lipid Droplet Formation in Cells of the Filamentous Green Alga Klebsormidium nitens as Revealed by BODIOY-DiOC6 and BODIPY-Nile Red Double-Staining Microscopy

“…Microscopic analyses of the stained cells showed that LDs form at the edge of the ER, but never at sites between the ER and the chloroplast periphery. The results suggest that in this green alga, LDs form at the ER, like in red algae (Kuroiwa et al 2012). …”

“…Goodson et al (2011) proposed that LDs in C. reinhardtii are synthesized from both the ER and chloroplasts. In the primitive red alga Cyanidioschyzon merolae, LDs are associated with only the ER (Kuroiwa et al 2012). These results suggest that the formation of LDs is related to both chloroplast growth (in young cells) and chloroplast degradation (in starved cells).…”

“…In cells of Chlamydomonas reinhardtii, LDs are associated with the chloroplast and the ER (Goodson et al 2011). In our previous study, we observed that LDs were closely associated with the chloroplast periphery in nitrogen-starved C. reinhardtii cells (Kuroiwa et al 2012). When the chloroplasts began to decompose, large LDs formed (Kuroiwa et al 2014), indicating that in C. reinhardtii cells, chloroplast components are converted to LDs.…”

“…Indeed, K. nitens and Klebsormidium flaccidum have large nuclear genomes of 310.8 Mbp (Kuroiwa et al 2012) and 117.1 Mbp (Hori et al 2014), respectively, and K. nitens cells form large LDs. Thus, it is easy to observe LD formation in K. nitens (Kuroiwa et al 2012).…”

Primitive red alga <i>Cyanidioschyzon merolae</i> accumulates storage glucan and triacylglycerol under nitrogen depletion

“…LBs can be visualized by microscopy after preferential staining with fluorescent dyes, such as Nile Red or BODIPY, but the latter is thought to be more specific to LBs (Gocze and Freeman, 1994). Some BODIPY dyes also have an advantage over Nile Red in that they can be observed more easily, especially in algae and plants, because their fluorescence color is not masked by red chlorophyll autofluorescence (Kuroiwa et al, 2012). Therefore, we used BODIPY493/503 to visualize LBs in cells during nitrogen depletion (Fig.…”