Periodic Reporting for period 1 - SUBTOL (Understanding seaweed submergence tolerance mechanisms and translating them into land plants)
Reporting period: 2018-09-05 to 2020-09-04
Loss of agricultural productivity due to flooding is a major global issue that is worsening due to ongoing climate change, including increases in annual rainfall in many areas of the world. This challenged the global food production needs to increase at least 50% by 2050 to feed the growing world population if current dietary habits do not change.
Waterlogging and submergence cause cells to experience low oxygen (hypoxia), affecting the metabolism, cell injury and cell death. Flooding also affects the availability of light for photosynthesis. When floods subside, cells experience oxidative stress due to rapidly increasing oxygen levels and plants can also experience stress from changing salinity and desiccation.
Thus, it is important to develop crops that are tolerant to flooding stresses. This has been done successfully with some cereal crops (rice, barley) using relatively species-specific mechanisms. The SUBTOL project takes a more general approach applicable to multiple plants by stepping further back in evolutionary time and examining marine green seaweeds, a naturally submergence-tolerant group of organisms that diverged from the land plant lineage about 1.2 billion years ago. The objectives of SUBTOL are to:
1) Identify the large-scale gene expression changes that occur during the green seaweed Ulva submergence and exposure.
2) Compare Ulva submergence gene networks with those in land plants.
3) Use Ulva submergence/exposure gene networks to improve land plant flood tolerance.
The project was able to accomplish all of these objectives. SUBTOL-developed the proof-of-concept that plant stress tolerance can be enhanced by a naturally submergence-tolerant group of organisms that diverged from the land plant lineage about 1.2 billion years ago.
Waterlogging and submergence cause cells to experience low oxygen (hypoxia), affecting the metabolism, cell injury and cell death. Flooding also affects the availability of light for photosynthesis. When floods subside, cells experience oxidative stress due to rapidly increasing oxygen levels and plants can also experience stress from changing salinity and desiccation.
Thus, it is important to develop crops that are tolerant to flooding stresses. This has been done successfully with some cereal crops (rice, barley) using relatively species-specific mechanisms. The SUBTOL project takes a more general approach applicable to multiple plants by stepping further back in evolutionary time and examining marine green seaweeds, a naturally submergence-tolerant group of organisms that diverged from the land plant lineage about 1.2 billion years ago. The objectives of SUBTOL are to:
1) Identify the large-scale gene expression changes that occur during the green seaweed Ulva submergence and exposure.
2) Compare Ulva submergence gene networks with those in land plants.
3) Use Ulva submergence/exposure gene networks to improve land plant flood tolerance.
The project was able to accomplish all of these objectives. SUBTOL-developed the proof-of-concept that plant stress tolerance can be enhanced by a naturally submergence-tolerant group of organisms that diverged from the land plant lineage about 1.2 billion years ago.
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.
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.
The previous state of the art was restricted to understand the submergence tolerance mechanisms in flood-tolerant, semi-aquatic key crop plant rice and the land plant model (but flood-sensitive) Arabidopsis. It defined two submergence tolerance strategies (i) low-oxygen quiescence, (ii) low-oxygen escape. A “core” set of 49 genes involved in land plant submergence tolerance mechanisms has been gathered from experiments in Arabidopsis and rice. Moreover, some submergence tolerance mechanisms (e.g. those involving group VII ethylene response factors (ERFs) that have been well characterised in Arabidopsis are not fully conserved in key crop species such as rice.
By identifying Ulva genes involved in seaweed submergence responses, we have uncovered both (i) new candidate land plant genes that play a role in hypoxia and also (ii) some unique Ulva genes that have sufficient similarity to those in land plants that they are able to promote submergence tolerance in cross-species transfer experiments, which have not been performed across this large an evolutionary distance before. This moves the field significantly beyond the previous state of the art and provides new avenues for future strategies to improve the flooding tolerance of land plants, including crops. The project has also demonstrated that the genes involved in seaweed submergence responses have the potential to also promote tolerance to additional abiotic stresses, in particular salinity, in land plants. As salinity of agricultural land is a problem that is getting worse due to climate change, this is an added beneficial outcome of the project. Improving crop production is a major global challenge so this work will in the longer term benefit agriculturalists, farmers and the general public.
By identifying Ulva genes involved in seaweed submergence responses, we have uncovered both (i) new candidate land plant genes that play a role in hypoxia and also (ii) some unique Ulva genes that have sufficient similarity to those in land plants that they are able to promote submergence tolerance in cross-species transfer experiments, which have not been performed across this large an evolutionary distance before. This moves the field significantly beyond the previous state of the art and provides new avenues for future strategies to improve the flooding tolerance of land plants, including crops. The project has also demonstrated that the genes involved in seaweed submergence responses have the potential to also promote tolerance to additional abiotic stresses, in particular salinity, in land plants. As salinity of agricultural land is a problem that is getting worse due to climate change, this is an added beneficial outcome of the project. Improving crop production is a major global challenge so this work will in the longer term benefit agriculturalists, farmers and the general public.