Periodic Reporting for period 4 - FEAR-SAP (Function and Evolution of Attack and Response Strategies during Allelopathy in Plants)
Periodo di rendicontazione: 2022-01-01 al 2023-06-30
Plants grow as communities, on both natural ecosystems with a diverse mixture of species and agricultural monocultures. Every plant strives to secure optimal access to resources by outcompeting others; some engage in chemical warfare by releasing chemicals into the soil. These compounds enter nearby plants and interfere with molecular and cellular processes to prevent growth or development, leaving the ‘donor’ plant with a competitive advantage.
This process of chemical interference between organisms is called “allelopathy”, known to farmers and gardeners for centuries. Species using allelopathy range from trees (e.g. walnut) to shrubs and grasses, and include many major crops, e.g. wheat, rye, and maize. Although many allelochemicals have been identified, it remains unclear for most how they act in the plant and why they are toxic to some plants but not others.
We study allelochemicals produced by horticultural and agricultural crops. Upon release, some of them are only mildly toxic but are quickly converted to more toxic compounds in soil (Fig. 1). In the case of benzoxazinoids (BX), a compound class produced in, among others, wheat and maize, these degradation products enter plant cells and inhibit the activity of histone deacetylases (HDA). HDAs remove acetyl groups from proteins, particularly histones. Histones help organize DNA in the nucleus, and addition or removal of acetyl groups regulates the compaction of this DNA-protein complex. HDAs thus ultimately help regulate the accessibility of genes (Fig. 1). We showed that by inhibiting HDA activity, allelochemicals change the overall organization of the chromatin and thereby interfere with basic cellular functions.
Our group is working on solving the enzymatic specificity of these allelochemicals. To determine the potency of allelochemicals, we use the model plant Arabidopsis and the weed Thlaspi arvense (pennycress) as a readout (Fig. 2).
We also use Arabidopsis for another approach to identify genes that allow some plants to tolerate allelochemicals. We make use of the vast genetic diversity that exists in this species (1001genomes.org) and whose genomes have been sequenced. By screening appr. half of this collection, we identified a dozen genotypes that are resistant to aminophenoxazinones (Fig. 3). Using statistical analysis, we are searching for associations between specific genetic variants and increased resistance to identify the genes responsible.
Our analyses extend beyond plants: because the soil surrounding roots is populated by thousands of bacterial and fungal species, we ask if and to what extent the presence of allelochemicals affects the microbial community, and how microbes contribute to the chemical dynamics in soil (Fig. 4). Using high-throughput, automated culture handling, we are screening approximately 200 bacterial strains, individually and in different combinations, for resistance to different allelochemicals (Fig. 5). Our goal is to identify bacteria that metabolise or convert the compounds, and that might play a role in detoxifying them in soil.
Altogether, our research aims to resolve the intricate relationship between neighbouring plants. Our work will contribute to a better understanding of the dynamics of natural ecosystems and agricultural plant communities and could lead to the development of sustainable plant protection strategies.
This process of chemical interference between organisms is called “allelopathy”, known to farmers and gardeners for centuries. Species using allelopathy range from trees (e.g. walnut) to shrubs and grasses, and include many major crops, e.g. wheat, rye, and maize. Although many allelochemicals have been identified, it remains unclear for most how they act in the plant and why they are toxic to some plants but not others.
We study allelochemicals produced by horticultural and agricultural crops. Upon release, some of them are only mildly toxic but are quickly converted to more toxic compounds in soil (Fig. 1). In the case of benzoxazinoids (BX), a compound class produced in, among others, wheat and maize, these degradation products enter plant cells and inhibit the activity of histone deacetylases (HDA). HDAs remove acetyl groups from proteins, particularly histones. Histones help organize DNA in the nucleus, and addition or removal of acetyl groups regulates the compaction of this DNA-protein complex. HDAs thus ultimately help regulate the accessibility of genes (Fig. 1). We showed that by inhibiting HDA activity, allelochemicals change the overall organization of the chromatin and thereby interfere with basic cellular functions.
Our group is working on solving the enzymatic specificity of these allelochemicals. To determine the potency of allelochemicals, we use the model plant Arabidopsis and the weed Thlaspi arvense (pennycress) as a readout (Fig. 2).
We also use Arabidopsis for another approach to identify genes that allow some plants to tolerate allelochemicals. We make use of the vast genetic diversity that exists in this species (1001genomes.org) and whose genomes have been sequenced. By screening appr. half of this collection, we identified a dozen genotypes that are resistant to aminophenoxazinones (Fig. 3). Using statistical analysis, we are searching for associations between specific genetic variants and increased resistance to identify the genes responsible.
Our analyses extend beyond plants: because the soil surrounding roots is populated by thousands of bacterial and fungal species, we ask if and to what extent the presence of allelochemicals affects the microbial community, and how microbes contribute to the chemical dynamics in soil (Fig. 4). Using high-throughput, automated culture handling, we are screening approximately 200 bacterial strains, individually and in different combinations, for resistance to different allelochemicals (Fig. 5). Our goal is to identify bacteria that metabolise or convert the compounds, and that might play a role in detoxifying them in soil.
Altogether, our research aims to resolve the intricate relationship between neighbouring plants. Our work will contribute to a better understanding of the dynamics of natural ecosystems and agricultural plant communities and could lead to the development of sustainable plant protection strategies.
In the initial 30 months of the project, we have established the necessary material resources and protocols, and have carried out experiments in the four different areas of the project.
1. In order to identify the molecular target(s) of the allelochemical aminophenoxazinone (APO), we have studied allelochemical effects on purified plant histone deacetylases (HDAs). In a complementary approach, we have generated plants that lack the function of one or several of the HDT-class HDACs. There are four HDT gene copies in A. thaliana and we have succeeded in generating knockout combinations that we are going to characterize in detail in the coming project period. To understand the wider molecular effect of these allelochemicals, we have generated whole-genome transcriptomic as well as targeted and untargeted proteomic data.
2. To identify genes conferring tolerance towards APO, we have measured root growth under control and APO conditions in >500 natural genotypes of A. thaliana. We indeed found resistant accessions and were able to map several genomic loci associated with resistance that mapped to the sulfur metabolism of the plant. Follow-up experiments showed that additional sulfur supplementation can mitigate the allelochemicals' effect. Also related to aim 2 of the project, we have completed the genomic profiling of a European collection of the weed T. arvense and have contributed to building a new reference genome for that species.
3. As we are interested in the impact of allelochemicals on root-associated microbiota, we tested growth of 180 bacterial strains isolated from soil upon exposure to different compounds. Closely related strains showed a very diverse reaction to the compounds, and we used that information in combination with the available genome sequences to map bacterial genes associated with resistance or susceptibility. These genes are going to be characterized in the next phase of the project. Moreover, we monitored the effect of allelochemicals on bacterial community dynamics. Studying the feedback effects on plant growth, we observed intricate interplay between plant genotype and an allelochemical-primed microbiome.
4. The long-term goal of our project is to develop strategies by which crops could become more tolerant to allelochemicals. In the initial phase of the project, we have used the information gained from testing individual bacterial strains to build synthetic bacterial communities. We inoculated A. thaliana plants in a sterile environment with these mixed cultures and exposed the whole system to allelochemicals. Some of those mixed cultures indeed influenced plant growth in dependence of the presence of allelochemicals, indicating that bacteria can confer resistance or susceptibility to the chemical compound in the host plant.
1. In order to identify the molecular target(s) of the allelochemical aminophenoxazinone (APO), we have studied allelochemical effects on purified plant histone deacetylases (HDAs). In a complementary approach, we have generated plants that lack the function of one or several of the HDT-class HDACs. There are four HDT gene copies in A. thaliana and we have succeeded in generating knockout combinations that we are going to characterize in detail in the coming project period. To understand the wider molecular effect of these allelochemicals, we have generated whole-genome transcriptomic as well as targeted and untargeted proteomic data.
2. To identify genes conferring tolerance towards APO, we have measured root growth under control and APO conditions in >500 natural genotypes of A. thaliana. We indeed found resistant accessions and were able to map several genomic loci associated with resistance that mapped to the sulfur metabolism of the plant. Follow-up experiments showed that additional sulfur supplementation can mitigate the allelochemicals' effect. Also related to aim 2 of the project, we have completed the genomic profiling of a European collection of the weed T. arvense and have contributed to building a new reference genome for that species.
3. As we are interested in the impact of allelochemicals on root-associated microbiota, we tested growth of 180 bacterial strains isolated from soil upon exposure to different compounds. Closely related strains showed a very diverse reaction to the compounds, and we used that information in combination with the available genome sequences to map bacterial genes associated with resistance or susceptibility. These genes are going to be characterized in the next phase of the project. Moreover, we monitored the effect of allelochemicals on bacterial community dynamics. Studying the feedback effects on plant growth, we observed intricate interplay between plant genotype and an allelochemical-primed microbiome.
4. The long-term goal of our project is to develop strategies by which crops could become more tolerant to allelochemicals. In the initial phase of the project, we have used the information gained from testing individual bacterial strains to build synthetic bacterial communities. We inoculated A. thaliana plants in a sterile environment with these mixed cultures and exposed the whole system to allelochemicals. Some of those mixed cultures indeed influenced plant growth in dependence of the presence of allelochemicals, indicating that bacteria can confer resistance or susceptibility to the chemical compound in the host plant.
Our finding that HDT-type histone deacetylases have an HDAC-like activity is the first finding of this kind. The plant-specific clade of HDACs is enigmatic; HDTs do not have a classical HDAC domain arrangement and there are no known homologs known in other organisms. It has been speculated that HDTs are not histone deacetylases at all but rather act on other protein substrates. We hope to be able to narrow down those substrates in the future course of the project.
The bacterial work has progressed considerably faster than anticipated, as we did not expect to obtain positive results on plant-microbe interactions in dependence of allelochemicals this early into the project. If these results can be confirmed, they would constitute one of the first proven examples of specific feedback reactions between soil microbes and host plants.
With our work on the weed T. arvense, we have established the first large-scale resource of an agricultural weed in Europe that also comprises single-nucleotide-resolution genomic data. As T. arvense is currently being explored by our collaboration partners at the University of Minnesota, USA, as a sustainable biofuel crop, we hope to be able to contribute valuable information on genetic diversity to their breeding program.
The bacterial work has progressed considerably faster than anticipated, as we did not expect to obtain positive results on plant-microbe interactions in dependence of allelochemicals this early into the project. If these results can be confirmed, they would constitute one of the first proven examples of specific feedback reactions between soil microbes and host plants.
With our work on the weed T. arvense, we have established the first large-scale resource of an agricultural weed in Europe that also comprises single-nucleotide-resolution genomic data. As T. arvense is currently being explored by our collaboration partners at the University of Minnesota, USA, as a sustainable biofuel crop, we hope to be able to contribute valuable information on genetic diversity to their breeding program.