Crystalline guanine packed within vacuoles serves as nitrogen store in Chromera velia

Nitrogen is an important element for all living organisms. Photoautotrophic organisms need to assimilate nitrogen from the environment, therefore changes in nitrogen availability have a strong influence on their growth and metabolism. Many microalgae have been known to contain crystalline inclusions, and recently, it has been shown that many of these consist of purines like guanine and thus must be linked to the cellular nitrogen metabolisms. The alveolate alga Chromera velia contains such guanine crystals, and during its lifecycle, the alga is thought to be subjected to strong changes in external nitrogen availability. Here, we investigated the formation or decline of crystalline guanine in dependence of the availability of inorganic nitrogen in the growth medium. Cells were examined using polarised light microscopy, Raman micro-spectroscopy, chromatography (HPLC), transmission and scanning electron microscopy. The cellular guanine crystal content decreased during nitrogen starvation and increased upon transfer of the cells back to standard growth medium containing nitrate. Raman micro-spectroscopy showed that the crystals were composed of anhydrous guanine in beta-polytype. They appear in unspecific positions throughout the cell, and staining with the green dye Lysotracker DND-26 suggests that they are within vacuoles. Stacks of crystals could be observed in cells via freeze fracture and freeze etching electron microscopy, which unambiguously showed a membrane around the crystal aggregates, in a similar arrangement as has been shown for guanine storage vacuoles (GSV) in Chlamydomonas reinhardtii. We developed a method to isolate the guanine crystals from whole cells, and were able to obtain crystals which retained their flat, plate-like structure, matching the electron microscopic observations from whole cells. The isolated crystals were shown to consist of nitrogen rich compounds via energy-dispersive X-ray (EDX) analysis, and Raman micro-spectroscopy confirmed that they consist of guanine.