Mitochondrial metabolism is central for response and resistance of Saccharomyces cerevisiae to exposure to a glyphosate-based herbicide

Abstract

Glyphosate-based herbicides, the most extensively used herbicides in the world, are available in an enormous number of commercial formulations with varying additives and adjuvants. Here, we study the effects of one such formulation, Credit41, in two genetically diverse yeast strains. A quantitative trait loci (QTL) analysis between a sensitive laboratory strain and a resistant strain linked mitochondrial function to Credit41 resistance. Two genes encoding mitochondrial proteins identified through the QTL analysis were HFA1, a gene that encodes a mitochondrial acetyl CoA carboxylase, and AAC3, which encodes a mitochondrial inner membrane ATP/ADP translocator. Further analysis of previously studied whole-genome sequencing data showed that, although each strain uses varying routes to attain glyphosate resistance, most strains have duplications of mitochondrial genes. One of the most well-studied functions of the mitochondria is the assembly of Fe–S clusters. In the current study, the expression of iron transporters in the transcriptome increased in cells resistant to Credit41. The levels of iron within the cell also increased in cells exposed to Credit41 but not pure glyphosate. Hence, the additives in glyphosate-based herbicides have a significant contribution to the negative effects of these commercial formulations on biological systems.

Introduction

The ubiquitous use of glyphosate-based herbicides around the world has led to extensive research of the effects of glyphosate on plants, bacteria, and higher eukaryotes. Decades of research has been carried out on herbicide resistance, mainly focusing on glyphosate. However, it is only in the past few years that the potential negative impacts of glyphosate-based herbicides (GBHs) and their additives have been acknowledged. The aromatic amino acid pathway is the canonical target of glyphosate (Amrhein et al. 1980; Steinrücken and Amrhein, 1980). Herbicidal additives, although claimed to be neutral, have interactive effects when combined with glyphosate (Ravishankar et al., 2019). Evaluating genetic variation in glyphosate-based herbicide resistance is one approach to address the pathways of toxicity of these additives. Variation in response to GBHs lies not only in the genetic make-up of organisms but also in the variation in components and the concentrations of the adjuvants and surfactants added to the herbicides with the same active ingredient (Ravishankar et al. 2019). Recent studies have shown that the toxicity of glyphosate-based herbicides is not only due to the principal ingredient, but more so the additives in the various formulations (Mesnage et al. 2015; Jacques et al. 2019).

Saccharomyces cerevisiae is used extensively in the toxicogenomics study of various chemicals. Its benefits include a unique combination of highly conserved mechanisms of adaptation, tolerance, and resistance related to higher eukaryotes (Parsons et al. 2003; dos Santos et al. 2012), along with the broad spectrum of variation that lies within the Saccharomyces species at the genome level. The environment from which the strains are isolated has a significant influence on this genetic variation between the strains (Landry et al. 2006; Smith and Kruglyak, 2008; Lee et al. 2019). The two strains used throughout this study were S288c and RM11. RM11 is a Californian vineyard strain that was isolated from a vineyard in 1993 (Mortimer et al. 1994). As glyphosate-based herbicides were introduced in the 1970s, it is likely that this strain was exposed to this stressor and has developed resistance mechanisms, as it is resistant to most GBH. S288c is a laboratory strain that is sensitive to GBH.

A QTL study carried out using petite frequency phenotype to identify polymorphisms in multiple genes contributing to mitochondrial instability also used S288c and RM11 (Dimitrov et al. 2009), in turn pointing out the diversity within their mitochondrial genomes and making them good candidates for this QTL analysis. Among other differences, there are multiple polymorphisms in the RM11 allele of PDR5 (Wei et al., 2007) which is a pleiotropic drug marker involved in glyphosate export for the cell (Rong-Mullins et al. 2017). S288c and RM11 have been shown to be suitable strains for comparative genomics studies catering to understanding the effects of stressors, due to their extensive differences in sensitivity to different stressors, variation in gene expression, Ty copy number variation, and even mitochondrial content (Kvitek et al. 2008).

Commercially available herbicides are comprised of an active ingredient and proprietary additives, of which only the active ingredients have been studied extensively. Glyphosate, the active ingredient in GBHs is known to target the shikimate pathway. Here, we hypothesized that GBHs affect numerous important biological processes that lie outside the shikimate pathway due to the additives in the commercial formulations, that are yet to be deciphered. To test our hypothesis, we used the genetic variation in yeast to identify the differences between strains contributing to their ability to tolerate the stress induced by GBHs. In this study, the variation in response between RM11 and S288c upon Credit41 (Cr41) exposure was used to perform quantitative trait loci (QTL) analysis. We found resistance was associated with loci containing genes encoding mitochondrial proteins involved in mitochondrial fatty acid biosynthesis. Transcriptomic data and whole-genome sequencing data of cells that evolved resistance to Cr41 implied that iron homeostasis within the mitochondria contributes to the resistant phenotype of cells exposed to this environmental stressor, potentially involving cytochromes and iron-sulfur (Fe–S) clusters (Mühlenhoff and Lill, 2000; Foury and Talibi, 2001). A significant increase in iron concentrations in resistant cells (RM11) was found with Credit41 exposure, which may contribute to it’s resistant phenotype. The increased levels of iron transport were specific to the commercial formulation, Cr41, and not pure glyphosate (PG), indicating that the effects observed were not a result of the principal component but the additives and their collaborative effect.