The green seaweed Ulva is an ecologically-important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed Ulva mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed.
The molecular mechanism underlying sexual reproduction in land plants is well understood in model plants and is a target for crop improvement. However, unlike land plants, the genetic basis involved in triggering reproduction and gamete formation remains elusive in most seaweeds, which are increasingly viewed as an alternative source of functional food and feedstock for energy applications. Here, gametogenesis of Ulva mutabilis, a model organism for green seaweeds, is studied. We analyze transcriptome dynamics at different time points during gametogenesis following induction of reproduction by fragmentation and removal of sporulation inhibitors. Analyses demonstrate that 45% of the genes in the genome are differentially expressed during gametogenesis. We identified several transcription factors that potentially play a key role in the early gametogenesis of Ulva given the function of their homologs in higher plants and microalgae. In particular, the detailed expression pattern of an evolutionary conserved transcription factor containing an RWP-RK domain suggests a key role during Ulva gametogenesis. The identification of putative master regulators of gametogenesis provides a starting point for further functional characterization.
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