Deliverables

Identify the overall biological (e.g. age, size) and environmental (e.g. temperature, tides,day length, dehydration, tearing, wounding) parameters triggering the shift to the reproductive phase

Identify the chemical compounds and signalling molecules (morphogens) mediating the differentiation of the reproductive cells (gametes or spores)

Identify the mode of action of the signalling molecules within the whole seaweed tissue:localisation of biosynthesis, transport and receptors

Characterize the cell differentiation steps leading to the development of reproductive organs, at both the cellular (microscopy) and the transcriptional level (microdissection followed by transcriptomics)

Identify cell markers (transcripts and proteins) allowing early identification of reproductive cells prior to morphological differentiation. This will generate tools capable of speeding up the reproductive shift (to be developed in Task 4)

Identify the parameters triggering the release of reproductive cells into seawater.

Characterize the physiological parameters of germ cells’ motility within the seawater: velocity, survival, phototaxis.

Characterize the chemo-attraction of female and male germ cells and identify the molecular factors enabling physical recognition and contact between the female and male germ cells. Describe their fusion mechanisms at the cellular and molecular levels.

Identify the processes of cytoplasmic heredity in the first steps of zygotic cell division (contributing to improved seaweed selection for algoculture).

Characterize the establishment of the cell polarity axis at the sub-cellular level prior to and after the first cell division (cytoskeleton, cell nucleus position and orientation of the division plan). This will impact on the further developmental pattern of the seaweed.

Identify the molecular factors involved in determining cell fate at the embryo stage and during sporulation events by e.g. cell wall determinants and external factors.

Characterize the expression pattern (overall transcript composition) of the zygote prior to its entry into mitosis or meiosis; Seaweeds have complex and diverse life cycles and some skip the diploid stage as the zygote immediately enters into meiosis: a better knowledge of this process might allow generation of artificial life cycle stages useful in aquaculture (e.g. haploid / diploid stages).

Characterize whether cell-cell communication occurs within the embryo (apoplastic or symplastic communication). This will allow practice of excision of seaweed parts, which might be necessary in aquaculture processes.

Assess the impact of external and internal forces (mainly mechanical) on embryo fate. Most seaweed embryos develop directly in the sea, exposed to strong mechanical forces (such as sea current, waves and tides). In addition, growth and cell multiplication generate compression and shearing forces, which cells need to account for and respond to.

Identify bacterial chemicals used as external seaweed morphogens. This will lead to possible control of seaweed developmental steps by external application of morphogens present in natural compounds (in hatchery).

Characterize seaweed tissues or cellular structures enabling long-range transport through the seaweed body (differentiation of sieve elements and/or plasmodesmata, transport velocity). The intention would be to eventually manipulate the size of seaweed whole-body or specific organ to optimise yield should account for these knowledge.

Determine the existence of processes such as apical dominance, leading to the possibility of modifying overall seaweed morphology by promoting branching, thereby increasing seaweed biomass or production of reproductive organs, similarly to crop plants on land (e.g. “Green Revolution” breeds of wheat and rice).

Establishment of cultivation protocols enabling the whole seaweed life-cycle to occur in laboratories conditions. This will be developed on specific seaweeds with aquaculture interests.

Establishment of cryopreservation protocols. Different stages of the life cycles (zygote, embryo, germlings, specific tissues/organs of the adult body, such as reproductive organs, gametes or spores), will be tested for cryopreservation (-120°C, -80°C, with different cooling processes). Alternatively, shorter-term storage systems (cold room for only a few months) will be developed.

Identification of detectable markers (RNA, proteins, chemicals) used to identify the reproductive cells prior to morphological (visible) differentiation. That will allow definition of the vegetative-reproductive shift, and hence anticipation of further steps in the aquaculture process.

Implementation of high-throughput “-OMICS” tools, already established in some seaweeds by academic partners, which will be transferred to and developed in other seaweeds more specifically dedicated to aquaculture (upon requirement to achieve other Deliverables).

Development of reverse genetic and transformation protocols in seaweeds. First, a selection of genetically tractable seaweeds and cell types (e.g. germ cells) will be performed, relying on COST partners skilled in green or brown seaweed transgenics.

Develop cell biology (state-of-the-art imaging, in situ localisation, reporter transgenics) in relevant seaweeds. This will complement “-omics” and genetics to define spatio-temporal molecular expression patterns during the different steps of seaweed development.