Guanine, a high-capacity and rapid-turnover nitrogen reserve in microalgal cells

Abstract

Nitrogen (N) is an essential macronutrient for microalgae, influencing their productivity, composition, and growth dynamics. Despite the dramatic consequences of N starvation, many free-living and endosymbiotic microalgae thrive in N-poor and N-fluctuating environments, giving rise to questions about the existence and nature of their long-term N reserves. Our understanding of these processes requires a unequivocal identification of the N reserves in microalgal cells as well as their turnover kinetics and subcellular localization. Herein, we identified crystalline guanine as the enigmatic large-capacity and rapid-turnover N reserve of microalgae. The identification was unambiguously supported by confocal Raman, fluorescence, and analytical transmission electron microscopies as well as stable isotope labeling. We discovered that the storing capacity for crystalline guanine by the marine dinoflagellateAmphidiniumcarteraewas sufficient to support N requirements for several new generations. We determined that N reserves were rapidly accumulated from guanine available in the environment as well as biosynthesized from various N-containing nutrients. Storage of exogenic N in the form of crystalline guanine was found broadly distributed across taxonomically distant groups of microalgae from diverse habitats, from freshwater and marine free-living forms to endosymbiotic microalgae of reef-building corals (Acropora millepora,Euphyllia paraancora). We propose that crystalline guanine is the elusive N depot that mitigates the negative consequences of episodic N shortage. Guanine (C5H5N5O) may act similarly to cyanophycin (C10H19N5O5) granules in cyanobacteria. Considering the phytoplankton nitrogen pool size and dynamics, guanine is proposed to be an important storage form participating in the global N cycle.

Paradigm shift in eukaryotic biocrystallization

“…Particular triggers for purine inclusion formation are unknown, but they are often produced after transfer to fresh growth media, media containing surplus sources of nitrogen, and under stress conditions 4,6 . As previously shown, purine crystals may act as nitrogen storage for microalgae in which they are formed in a type of luxury uptake resulting in net removal of nitrogen from the medium 8 .…”

“…Measurements and data-processing were performed as described elsewhere 6,8,24 . The advantages and possible drawbacks of this method have recently been discussed 25 .…”

“…To the best of our knowledge, this is the first report confirming the occurrence of crystalline guanine monohydrate in any microorganism. To date, only a β-polymorph of crystalline guanine anhydride was detected in various organisms, including some microalgae and protists 4,5,8 . In Flectonema sp., guanine monohydrate was detected in the form of pure crystals (Fig.…”

“…To date, the investigation of crystalline inclusions and their biological importance in unicellular eukaryotes has been impeded by a lack of methods for their non-destructive in situ and in vivo characterization. Although Raman microscopy 8 provides just such capabilities (as detailed in Supplementary Materials and Methods), this methodology has not yet been fully exploited in the study of intracellular crystalline inclusions. Compared to other tools for microscopic elemental analysis (nanoSIMS, EELS, EDX TEM), or chemical analytical approaches (GC and LC MS, NMR, X-ray crystallography), Raman microscopy offers direct visualization of molecular profiles for various cellular compartments in single cells with the resolution of confocal microscopy and without the need for fixation, time-consuming and laborious sample preparation, or the need for large amounts of biological material necessary for chemical extractions.…”

“…purine inclusions of guanine, xanthine, hypoxanthine, or uric acid are often overlooked [4][5][6][7][8] . The recent resurgence of research on purine inclusions has shown rapid uptake kinetics of different nitrogen compounds that are converted into massive guanine nitrogen stores with sufficient capacity, in some cases, to support three consecutive cell cycles 8 . As a fundamental biogenic element, nitrogen represents a great share of biotic elemental stoichiometry, from cellular to global scales, impacting Earth's climate 9 .…”