Lipidomics of Icelandic bryophytes: Discovering novel sources of polysaturated fatty acids

Bryophytes (mosses, liverworts, and hornworts) are the second largest group in the plant kingdom. From an evolutionary point of view, bryophytes were the first lineage of the terrestrial plants about 450 million years ago. They comprise the intermediate phylogenic position between green algae and tracheophytes.

From a chemical perspective, bryophytes, albeit morphologically small and structurally simple, possess a complex chemical diversity. Bryophytes produce high contents of very long-chain polyunsaturated fatty acids (vl-PUFAs), including arachidonic acid (20:4, AA) and eicosapentaenoic acid (20:5, EPA).

Fatty acids with longer than 20 carbons are seldom found in higher plants, but are commonly produced by algae and bryophytes. These vl-PUFAs are beneficial for human health and are consumed by humans as a food supplement. The current source of vl-PUFAs are mostly from algae and marine fish (which accumulates vl-PUFAs by consuming algae).

However, concerns have been raised since the marine fish population is reduced, and the heavy metal accumulation is an unignorable issue. Purifying vl-PUFA from algae is a more sustainable option but it is currently still costly to harvest the algal biomass and extract the oil. Thus, finding an alternative green and sustainable source of vl-PUFAs is urgent.

Here, we propose a novel potential vl-PUFA source from bryophytes. Due to several obstacles (e.g., morphologically insignificant, slow growth speed in nature, difficult to obtain bulk materials), research conducted on bryophytes are limited. The lipid composition, lipid metabolisms of bryophytes, and the differences between bryophytes and other vascular plants, are largely unexplored.

Advanced analytical methods, such as mass spectrometry (MS)-based untargeted lipidomics, have seldom applied on bryophyte studies. This study aims to find valuable lipids produced by Icelandic bryophytes by using ultra-performance liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLCESI-QTOF-MS) and to investigate the possibility of using bryophytes for vl-PUFA production in an industrial scale.

My research can be divided into three main objectives. 1). Establish a lipid extraction protocol and set up an untargeted lipid profiling method by using UPLCESI-QTOF-MS ( Paper 3 ). We evaluated the effects of different cell disruption methods (no disruption, shake, ultrasound, and bead beating), and storage conditions (air-dried, freeze-dried, and fresh frozen) of five moss species (including Racomitrium lanuginosum B and D, Philonotis fontana, Sphagnum teres, and Hylocomium splendens).

The results were analyzed by multivariate data analysis including unsupervised principal component analysis (PCA) and supervised orthogonal projections to latent structures discriminant analysis (OPLS-DA). We concluded that lipid extraction using fresh frozen samples with ultrasound assistance provide the most original lipid composition and gave a relatively high lipid content. 2).

By using the method developed from the first objective, we screened 39 Icelandic bryophyte species (including 32 mosses and 7 liverworts) by using UPLC-ESI-QTOF-MS ( Paper 4 ). The fatty acid distribution and relative concentrations were investigated with an aim of finding high AA and EPA producers.

We found six bryophyte species that contained high contents of AA and EPA in the form of phospholipids and glycerolipids. Two of the six species also accumulated large amounts of PUFA-enrichedtriacylglycerides (TG). 3). We have chosen five moss species for in vitro cultivation; the selection was based on the conclusion from the second objective and the availability of axenic in vitro cultures from our collaborator Lund University.

Bryophytes are generally cold- or even freeze-tolerant, low temperature usually stimulates the bryophytes to accumulate lipids with longer carbon chains and higher unsaturation levels. To have a better understanding of the lipid metabolism of bryophytes under cold stress condition, we investigated the lipid compositional changes of two moss species (Bryum pseudotriquetrum and the model moss species Physcomitrium patens) at room temperature and under cold stress by using untargeted lipidomics ( Paper 5 ).

The potential biomarkers of cold stress for B.pseudotriquetrum and P.patens were discovered, respectively. In addition, two review articles have been published to provide a more comprehensive understanding of the lipids and other chemicals isolated from bryophytes. The first review article ( Paper 1 ) summarized the valuable lipids that have been found in bryophytes and the biotic and abiotic approaches of enhancing the productions of AA and EPA.

The second review article ( Paper 2 ) focus on all kinds of natural products produced by bryophytes. This is a collaboration with other students from the joint project MossTech, and my main contribution is the chapter “Chemical diversity of bryophytes”.