Multi-omics investigation across the life-history stages of the red seaweed, Asparagopsis taxiformis
Creators: name: Patwary, Zubaida Parveen
Publisher: name: University of the Sunshine Coast, Queensland
Year: 2023
Subjects: Asparagopsis
seaweed
multi-omics
transcriptomics
proteomics
gametophyte
sporophyte
reproduction
Fisheries sciences

description: The red seaweed Asparagopsis taxiformis is recognised for its ability as a feed additive to mitigate methane production during fermentation of ruminant livestock. This has driven interest in the development of A. taxiformis aquaculture, however, difficulties associated with stimulating life-history transitions has impacted commercial production. A better knowledge of its molecular biology, including insights into the mechanisms that regulate processes such as signalling, metabolite biosynthesis, growth and reproduction could inform future developments towards scaling the aquaculture of both the sporophyte and gametophyte life-history stages. Towards achieving this, the Thesis provides a multi-omics investigation across the life-history stages.
description: In Chapter 2, to establish a better understanding of the most up-to-date literature relevant to seaweed -omics, I conducted a systematic review of published manuscripts between 1994 to 2021. The majority of the 312 studies on seaweed-omics research had been performed on the red seaweeds (45% of total studies), with more than half of these studies based upon two commercially relevant genera, Pyropia (nori, previously Porphyra) and Gracilaria (agar production). Overall, the combined research aligned most closely with the field of evolution (46% of total studies), followed by investigations into their ecology, natural products and biosynthesis, omics methodologies and seaweed-microbe interactions. A single study had explored A. taxiformis genomics, however, the focus was specifically on the vanadium and bromoform-related gene cluster and subsequent chemical analysis. Also, the exact lineage of A. taxiformis was not reported, amongst which we now know the phylogeny differs markedly across the globe.
description: In Chapter 3, a high-quality draft nuclear genome of domesticated diploid A. taxiformis sporophyte from Queensland (Lineage 6), was used as a reference to perform a comparative transcriptomic and proteomic analysis. First, a differential gene expression analysis identified that cultured (domesticated and non-reproductive) sporophytes demonstrate remarkable variation with wild (non-reproductive) sporophytes. Cultured sporophytes demonstrated an enrichment of regulatory factors (kinases, transcription factors), whereas wild sporophytes were enriched with defence and stress-related genes, including those involved in protein folding (heat shock proteins) and halogenated metabolite production. Despite this, cultured sporophytes contained 4-times more bromoform (the key anti-methanogenic natural product in Asparagopsis) compared to wild sporophytes. Wild sporophytes also expressed a relatively high level of novel secreted proteins, with similarity to collagen-alpha proteins (termed rhodophyte collagen-alpha-like proteins, RCAPs). The A. taxiformis genome enabled the development of a protein database, which was used for a proteomic investigation of cultured sporophytes, resulting in the identification of over 400 proteins, including RCAPs, numerous enzymes and phycobiliproteins, which can now be used for functional characterisations.
description: In Chapter 4, the A. taxiformis (Lineage 6) proteomics research was extended through the interrogation and comparison of proteins in male and female haploid gametophytes, using different extraction procedures to partition soluble and insoluble proteins derived from the nuclear genome. The outcome was the identification of 741 unique non-redundant proteins using a genome-derived database, highlighting a relatively large proportion of ion-binding proteins (i.e., iron, zinc, manganese, potassium and copper) which may play a role in seaweed heavy metal tolerance. Additionally, 58 stress-related (e.g., heat shock and vanadium-dependent haloperoxidases) and 44 photosynthesis-related (e.g., phycobilisomes, photosystem I, photosystem II and ATPase) proteins were identified which were generally more abundant in the female gametophyte. Forty proteins were predicted to be secreted, including 10 rhodophyte collagen-alpha-like proteins (RCAPs), which displayed overall high gene expression levels.
description: In Chapter 5, a comparative transcriptome analysis of male, female and immature A. taxiformis (Lineage 6) was performed with the aim of identifying sex-biased genes, which are likely associated with sex determination and reproduction. Initially, gametophyte ‘tips’ (branchlets where both male and female reproductive structures are found) were found to contain a relatively higher abundance of differentially expressed genes, compared with the main axis and the rhizome. Subsequently, 65 reproductive sex-biased genes were confirmed through comparison of reproductive mature and immature gametophyte tips, of which 92% were exclusive or significantly more highly expressed in males. Functional annotation of these reproductive sex-biased genes revealed that around half (46%) could be categorised as either novel, hypothetical or unknown. Also, transcription factors and enzymes associated with signalling or cell regulation were enriched. Female gametophytes contained a higher concentration of bromoform compared to males (8.5±1.0 mg/g dry weight versus 6.5±1.0 mg/g dry weight), although no significant difference was observed in the gene expression of marine bromoform biosynthesis locus genes (relevant to bromoform biosynthesis). Upon further confirmation, the identified sex-biased genes could be used as sex markers and enable approaches to manipulate sex in A. taxiformis.
description: In Chapter 6, a comparative investigation was performed on cultured non-reproductive and wild non-reproductive sporophytes, as well as with wild reproductive sporophytes (sporangia present) of A. taxiformis (Lineage 6). The outcome validated the previous findings (Chapter 3), that transcriptional profiles of cultured sporophytes are remarkably different to wild sporophytes (both non-reproductive and reproductive). In addition, 44 candidate sporophyte reproduction-associated genes were identified, which show significant up- or down-regulation in reproductive sporophytes compared to non-reproductive sporophytes. Candidate sporophyte reproduction-associated genes encode for enzymes and proteins relevant to cell structure, transport and the cell cycle. A preliminary experiment exploring the effects of altering culture temperature (higher and lower) on non-reproductive sporophytes found that prolonged exposure (1 week) to an elevated temperature (28°C) promoted reproduction-associated gene expression changes similar to that observed in reproductive sporophytes (i.e., 87% similar upregulation, 50% similar downregulation). This supported a role for temperature increase in reproductive maturation of sporophytes. No clear effects were observed with reduced temperature (16°C).
description: In summary, this Thesis provides a significant improvement on the molecular knowledge of A. taxiformis (Lineage 6) by creating multi-omics datasets across life-history stages (i.e., sporophytes and gametophytes). The outcomes highlight the potential of omics investigation to deliver targeted gene and protein analysis towards applications in aquaculture. For example, this research should be the foundation for deciphering the molecular mechanism of reproduction transitions of gametophytes with potential benefits for identification of candidate sex-specific markers, as well as the transition of sporophytes towards sporogenesis, which in turn could overcome bottlenecks in aquaculture.