Periodic Reporting for period 5 - FEAR-SAP (Function and Evolution of Attack and Response Strategies during Allelopathy in Plants)
Período documentado: 2023-07-01 hasta 2024-06-30
Plants usually grow in dense communities, in natural ecosystems with a diverse mixture of species, as well as in 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 often referred to as ‘allelopathy’ and has been known to farmers and gardeners for centuries, but we are still at the beginning of understanding the underlying molecular mechanisms. 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 from several chemical groups. In this project, we focused on benzoxazinoids, which are produced in may grasses, including major crops such as wheat and maize. Upon release from the plant roots, these compounds are at first only mildly toxic to other plants, but they are quickly converted to more toxic compounds in soil, upon which they are able to interfere with basic molecular functions once they enter the cells of neighbouring plants.
Since allelochemicals act against some plant species but are generally less toxic to the plants producing them, they have the potential to serve as the basis for novel herbicides. Given the detrimental impact of many conventional herbicides on human health and the environment, there is an urgent need to develop more sustainable and eco-friendly alternatives, and allelochemical-based products might be one option. However, to do so, we must first understand the chemical ecology and molecular mode of action of these compounds, as well as the tolerance or resistance mechanism that are at play in the producing plant species.
In FEARSAP, we followed four major objectives: (i) elucidating the molecular mode of action of benzoxazinoid-derived products; (ii) investigating the effect of benzoxazinoids on the plant-associated microbial communities; (iii) understanding the bacteria-mediated conversion of benzoxazinoids; (iv) uncovering the genetic basis of the plant’s response to benzoxazinoids.
This process of chemical interference between organisms is often referred to as ‘allelopathy’ and has been known to farmers and gardeners for centuries, but we are still at the beginning of understanding the underlying molecular mechanisms. 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 from several chemical groups. In this project, we focused on benzoxazinoids, which are produced in may grasses, including major crops such as wheat and maize. Upon release from the plant roots, these compounds are at first only mildly toxic to other plants, but they are quickly converted to more toxic compounds in soil, upon which they are able to interfere with basic molecular functions once they enter the cells of neighbouring plants.
Since allelochemicals act against some plant species but are generally less toxic to the plants producing them, they have the potential to serve as the basis for novel herbicides. Given the detrimental impact of many conventional herbicides on human health and the environment, there is an urgent need to develop more sustainable and eco-friendly alternatives, and allelochemical-based products might be one option. However, to do so, we must first understand the chemical ecology and molecular mode of action of these compounds, as well as the tolerance or resistance mechanism that are at play in the producing plant species.
In FEARSAP, we followed four major objectives: (i) elucidating the molecular mode of action of benzoxazinoid-derived products; (ii) investigating the effect of benzoxazinoids on the plant-associated microbial communities; (iii) understanding the bacteria-mediated conversion of benzoxazinoids; (iv) uncovering the genetic basis of the plant’s response to benzoxazinoids.
Objective 1: We sought to resolve the molecular mode of action and specificity of allelochemicals, notably of benzoxazinones (BX) in soil, later expanded to diterpenes.
Our studies uncovered that these two types of allelochemicals lead to surprisingly similar molecular effects: both compounds (APO, a BX conversion product, and momilactone, an allelochemical from rice) triggered very similar detoxification and transport processes, pointing at a general response pathway to these phytotoxins (Knoch et al. 2022).
Objective 2: To test the response of root-associated bacteria to the compound, we screened the A. thaliana RSphere collection and identified susceptible as well as resistant strains. Using machine learning, we identified bacterial orthologs associated with tolerance towards APO and continued with studying effects on synthetic communities (SynComs) rather than individual strains. Our results indicate that community development is strongly influenced by the presence of allelochemicals, and that slight modifications in community composition can have large effects on the community development under allelochemical treatment (Schandry et al. 2021; Schandry, Knoch et al., unpublished).
Objective 3: Conversely to Objective 2, here, we tested the effects of root-associated microbes on the chemical fate of allelochemicals, particularly benzoxazinoids (BX). We developed a protocol for the preparation of 13-carbon labeled BX from rye seedlings, by supplementing the growth medium with 13C-sugar and by removing the endosperm from the embryo tissue (Knoch et al., unpublished). We then fed 13C-labelled DIBOA to synthetic communities and carried out metabolomic and meta-transcriptomic analyses. To our surprise, besides expected and known compounds, we identified >30 unknown conversion products, which we currently try to structurally determine (Schandry and Knoch et al., unpublished).
In collaboration with the Schläppi lab, we were able to identify the genetic locus in bacterial strains isolated from maize roots that is responsible for the conversion of DIBOA to the phytotoxic compounds APO (Thönen et al. 2024).
In parallel, we conducted mutant screens in bacterial strains that we had identified to be tolerant towards APO to identify the genetic basis of tolerance. We identified an AcrB transporter mutation that caused decreased growth under APO conditions (Rouyer et al., in preparation).
Objective 4: Genome-wide association mapping in in >500 A. thaliana accessions revealed sulfur metabolism to be involved in promoting tolerance to APO. Our hypothesis is that naturally occurring alleles allow some genotypes to be tolerant to APO by more efficiently directing their sulfur resources into detoxification pathways. In line with this, we have been able to validate this hypothesis by rescuing the APO effect via supplementation of external sulfur donors (Hüther et al., in preparation).
To study allelochemical tolerance in another species, we contributed to a collection effort of >400 specimens of the weed Thlaspi arvense across Europe. We performed whole-(epi)genome sequencing on this collection using short-read sequencing (Galanti et al., 2022; Contreras-Garrido et al., 2024). To analyse natural variation in T. arvense, we first had to generate an improved version of the T. arvense reference genome (Nunn et al., 2022). However, carrying out APO-response screens in T. arvense proved to be impracticable. Instead, we studied response to allelochemical-treated soil in A. thaliana, of which we grew a genotype panel in soils that had been conditioned by growing benzoxazinoid-producing and non-producing maize. Through GWA mapping, we have identified a genomic locus that is strongly associated with the response of A. thaliana to the conditioning of the soil. This work was carried out in collaboration with the Schläppi lab (van Renseburg, Schandry et al., in preparation).
Our studies uncovered that these two types of allelochemicals lead to surprisingly similar molecular effects: both compounds (APO, a BX conversion product, and momilactone, an allelochemical from rice) triggered very similar detoxification and transport processes, pointing at a general response pathway to these phytotoxins (Knoch et al. 2022).
Objective 2: To test the response of root-associated bacteria to the compound, we screened the A. thaliana RSphere collection and identified susceptible as well as resistant strains. Using machine learning, we identified bacterial orthologs associated with tolerance towards APO and continued with studying effects on synthetic communities (SynComs) rather than individual strains. Our results indicate that community development is strongly influenced by the presence of allelochemicals, and that slight modifications in community composition can have large effects on the community development under allelochemical treatment (Schandry et al. 2021; Schandry, Knoch et al., unpublished).
Objective 3: Conversely to Objective 2, here, we tested the effects of root-associated microbes on the chemical fate of allelochemicals, particularly benzoxazinoids (BX). We developed a protocol for the preparation of 13-carbon labeled BX from rye seedlings, by supplementing the growth medium with 13C-sugar and by removing the endosperm from the embryo tissue (Knoch et al., unpublished). We then fed 13C-labelled DIBOA to synthetic communities and carried out metabolomic and meta-transcriptomic analyses. To our surprise, besides expected and known compounds, we identified >30 unknown conversion products, which we currently try to structurally determine (Schandry and Knoch et al., unpublished).
In collaboration with the Schläppi lab, we were able to identify the genetic locus in bacterial strains isolated from maize roots that is responsible for the conversion of DIBOA to the phytotoxic compounds APO (Thönen et al. 2024).
In parallel, we conducted mutant screens in bacterial strains that we had identified to be tolerant towards APO to identify the genetic basis of tolerance. We identified an AcrB transporter mutation that caused decreased growth under APO conditions (Rouyer et al., in preparation).
Objective 4: Genome-wide association mapping in in >500 A. thaliana accessions revealed sulfur metabolism to be involved in promoting tolerance to APO. Our hypothesis is that naturally occurring alleles allow some genotypes to be tolerant to APO by more efficiently directing their sulfur resources into detoxification pathways. In line with this, we have been able to validate this hypothesis by rescuing the APO effect via supplementation of external sulfur donors (Hüther et al., in preparation).
To study allelochemical tolerance in another species, we contributed to a collection effort of >400 specimens of the weed Thlaspi arvense across Europe. We performed whole-(epi)genome sequencing on this collection using short-read sequencing (Galanti et al., 2022; Contreras-Garrido et al., 2024). To analyse natural variation in T. arvense, we first had to generate an improved version of the T. arvense reference genome (Nunn et al., 2022). However, carrying out APO-response screens in T. arvense proved to be impracticable. Instead, we studied response to allelochemical-treated soil in A. thaliana, of which we grew a genotype panel in soils that had been conditioned by growing benzoxazinoid-producing and non-producing maize. Through GWA mapping, we have identified a genomic locus that is strongly associated with the response of A. thaliana to the conditioning of the soil. This work was carried out in collaboration with the Schläppi lab (van Renseburg, Schandry et al., in preparation).
Besides the above, we also developed open-source bioinformatic tools:
1. ARADEEPOPSIS is a transfer-learning-based plant phenotyping pipeline (Hüther et al. 2020); it is built as a Nextflow pipeline, which allows biologists without a bioinformatics background to use it on standard computer systems. For advanced needs, ARADEEPOPSIS can also be used on high-performance-computing platforms. The tool accurately segments plant rosettes and puts out a range of morphological parameters. Moreover, ARADEEPOPSIS is able to segment and quantify anthocyanine-rich and senescent leaf areas, enabling it to score effects of stress on plant growth and morphology.
2. MethylScore is a machine-learning-based computational pipeline that we developed to call differences in DNA methylation in large plant populations, such as the one we used for T. arvense (Hüther et al. 2022).
1. ARADEEPOPSIS is a transfer-learning-based plant phenotyping pipeline (Hüther et al. 2020); it is built as a Nextflow pipeline, which allows biologists without a bioinformatics background to use it on standard computer systems. For advanced needs, ARADEEPOPSIS can also be used on high-performance-computing platforms. The tool accurately segments plant rosettes and puts out a range of morphological parameters. Moreover, ARADEEPOPSIS is able to segment and quantify anthocyanine-rich and senescent leaf areas, enabling it to score effects of stress on plant growth and morphology.
2. MethylScore is a machine-learning-based computational pipeline that we developed to call differences in DNA methylation in large plant populations, such as the one we used for T. arvense (Hüther et al. 2022).