Objective 1 and 2: defining the molecular mechanisms underpinning submergence tolerance in green seaweeds and comparing them to those in land plants.
U. lactuca is a marine intertidal organism adapted to both drying and submergence. Since we were particularly interested in the molecular changes occurring during flooding/hypoxia, U. lactuca samples were subjected to an intense period of drying followed by gradual resubmergence. RNA was extracted and then transcriptome analysis was performed from sample time points. A summary of the key findings is as follows:
1) A suite of genes in Ulva lactuca is changed (up- or downregulated) in response to submergence.
2) Most of these genes are not related to the set of 49 “core hypoxia” genes, thus represent novel possibilities for engineering submergence tolerance in land plants. Indeed, the published Ulva genome lacks homologues about half of the “core hypoxia” genes and of the homologues are present, we showed that only about half are upregulated in response to submergence in Ulva. Thus, the mechanisms underpinning the submergence response in Ulva are largely different to those in land plants.
3) Some Ulva genes upregulated by submergence have clear homologues in Arabidopsis with no known submergence function. These Arabidopsis genes are good candidates to test directly for a submergence function.
4) Some Ulva genes upregulated by submergence are members of signalling pathways which have known roles in stress tolerance in land plants –namely transcription factors and kinases. These are good candidates to test for a submergence function using heterologous expression in Arabidopsis.
Objective 3: translating seaweed submergence-tolerance mechanisms into Arabidopsis.
The transcriptome data from Objective 1 was used to test gene function in two different ways.
1) Identified Ulva submergence-response genes that had clear homologues in Arabidopsis with unknown function were tested in Arabidopsis by analysis of loss-function mutant plants. After genotyping the mutants for homozygosity, the plants were subjected to seedling-based hypoxia assays. A number of mutants showed impaired growth under hypoxic conditions, compared to wild-type genes. Thus, SUBTOL has identified new land plant genes involved in the hypoxia response.
2) Identified Ulva submergence response genes that did not have close Arabidopsis homologues but were members of transcription factor or kinase groups (4 genes in total) were cloned from Ulva tissue into vectors that could drive ubiquitous expression in Arabidopsis. These constitutive-expression vectors were transformed into Arabidopsis plants and transgenic homozygous expression lines were generated that expressed the Ulva genes of interest. As above, these transgenic plants and corresponding wild-type controls were subjected to seedling-based hypoxia assays. Constitutive expression of three out of the four Ulva genes translated into Arabidopsis plants that showed elevated root growth under hypoxic conditions. Thus, SUBTOL has identified Ulva genes implicated in key stress signalling pathways that can improve the land plant submergence response.
The transgenic plants generated in 1) and 2) were also assayed for resistance to a post-flooding stress, salinity, and data from salt stress assays demonstrates that some transgenic lines show altered tolerance to salinity stress.
These results will be written up for open-access publication.
