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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - FEAR-SAP (Function and Evolution of Attack and Response Strategies during Allelopathy in Plants)

Teaser

Summary

Plants grow as communities; these can be natural ecosystems with a diverse mixture of species or an agricultural monoculture of many genetically similar individuals. Every plant strives to secure optimal access to resources by outcompeting others in direct proximity; 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 â€˜donorâ€™ plant with a competitive advantage.
This process of chemical interference between organisms is called â€œallelopathyâ€ and has been known to farmers and gardeners for centuries. Many species that use allelopathy have been identified, ranging from trees (e.g. walnut) to shrubs and grasses, and include many of todayâ€™s major crops, e.g. wheat, rye, and maize. Although many of the â€œallelochemicalsâ€ involved have been identified, it remains unclear how most 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). Once these degradation products enter plant cells they 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. For this, we temporally analyse global changes at the protein and transcript level, coupled with biochemical assays using purified proteins. To determine the potency of allelochemicals, we use the model plant Arabidopsis and the weed Thlaspi arvense (pennycress) as a readout. For example, aminophenoxazinones inhibit root growth of these species in a dose-dependent manner (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, accessible in a large collection of more than 1,100 plants collected from across the Northern Hemisphere (1001genomes.org) and whose genomes have been sequenced. We have screened approximately half of this collection and 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 metabolize 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 neighboring 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.

Work performed

In the initial 18 months of the project, we have established the necessary material resources and protocols, and have carried out the first experiments in the four different areas of the project.
1. In order to identify the molecular target(s) of the allelochemical aminophenoxazinone (APO), we aim to purify plant histone deacetylases (HDACs) and test inhibition by APO in vitro. So far, we have succeeded in purifying HDACs from the different enzyme families that exist in the model plant Arabidopsis thaliana and have observed that APO is able to inhibit some but not all HDACs in vitro. 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 double and triple knockout combinations that we are going to characterize in detail in the coming project period.
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.
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.
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.

Final results

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.