Multiple Herbicide Resistance in Lolium multiflorum and Identification of Conserved Regulatory Elements of Herbicide Resistance Genes

Abstract

Herbicide resistance is a ubiquitous challenge to herbicide sustainability and a looming threat to control weeds in crops. Recently four genes were found constituently over-expressed in herbicide resistant individuals of Lolium rigidum, a close relative of Lolium multiflorum. These include two cytochrome P450s, one nitronate monooxygenase and one glycosyl-transferase. Higher expressions of these four herbicide metabolism related (HMR) genes were also observed after herbicides exposure in the gene expression databases, indicating them as reliable markers. In order to get an overview of herbicidal resistance status of L. multiflorum L, 19 field populations were collected. Among these populations, four populations were found to be resistant to acetolactate synthase (ALS) inhibitors while three exhibited resistance to acetyl-CoA carboxylase (ACCase) inhibitors in our initial screening and dose response study. The genotyping showed the presence of mutations Trp-574-Leu and Ile-2041-Asn in ALS and ACCase, respectively, and qPCR experiments revealed the enhanced expression of HMR genes in individuals of certain resistant populations. Moreover, co-expression networks and promoter analyses of HMR genes in O. sativa and A. thaliana resulted in the identification of a cis-regulatory motif and zinc finger transcription factors. The identified transcription factors were highly expressed similar to HMR genes in response to xenobiotics whereas the identified motif is known to play a vital role in coping with environmental stresses and maintaining genome stability. Overall, our findings provide an important step forward toward a better understanding of metabolism-based herbicide resistance that can be utilized to devise novel strategies of weed management.

Keywords: genotyping; herbicide resistance; metabolic based resistance; promoter analysis; regulation of expression; weed management.

Figure 1

Gene expression pattern of Lolium multiflorum orthologs of GT, NMO, CYPs along with control genes in A. thaliana. (A) Developmental stage-specific expression pattern. Left to right, “germinating seed,” “seedling,” “young rosette,” “developed rosette,” “bolting rosette,” “young flower,” “developed flower,” “flower and silique,” “mature silique,” and “senescence.” Our selected genes were tending to show higher expression in senescence. “HIGH,” “MEDIUM,” and “LOW” expression were based on microarray gene expression data found in GENEVESTIGATOR (http://www.genevestigator.com). (B) Heat map of expression of selected genes in response to various external conditions such as chemicals were analyzed using Genevestigator perturbation tool. Relative expression of the genes was represented in log₂ ratio and significant change in expression were filtered out based on NMO, p < 0.001 and fold change >3. Expression of HMR genes strongly induced in response to various chemicals including herbicides and herbicide safeners.

Figure 2

Venn diagrams represent the transcription factors associated to the promoters of HMR genes of A. thaliana (A) and O. sativa (B). Analysis is done through The Plant Promoter Analysis Navigator (PlantPAN 2.0) using the promoter analysis function. (A) TFs associated to the promoters of HMR genes in Arabidopsis thaliana. (B) TFs associated to the promoters of HMR genes in Oryza sativa. ^*TFs associated to all four HMR genes of A. thaliana and O. sativa. One hundred and thirty-six TFs commonly associated to promoters of all four MR genes in A. thaliana and 85 TFs linked to the promoters of HMR genes of O. sativa. AT represents Arabidopsis thaliana. Venn diagrams were plotted using online tool Venny 2.0 http://bioinfogp.cnb.csic.es/tools/venny.

Figure 3

Classification of TFs based on InterPRO analysis in A. thaliana and O. sativa. Percent of TFs associated to the promoters of all four HMR genes were presented.

Figure 4

Expression pattern of identified conserved transcription factors ZAT10 and ZAT6 genes along with of HMR. (A) Developmental stage-specific expression pattern. Left to right, “germinating seed,” “seedling,” “young rosette,” “developed rosette,” “bolting rosette,” “young flower,” “developed flower,” “flower and silique,” “mature silique,” and “senescence.” Our identified TFs were tending to show higher expression in senescence similar to genes. “HIGH,” “MEDIUM,” and “LOW” expression were based on microarray gene expression data found in GENEVESTIGATOR (http://www.genevestigator.com). (B) Heat map of the expression of identified TFs along with HMR genes under various chemical stress were analyzed using Genevestigator perturbation tool. Relative expression of the genes was represented in log₂ ratio and significant change in expression were filtered out based on NMO, p < 0.001 and fold change >3. Expression of these genes strongly induced in response to various chemicals including herbicides and herbicide safeners.