Profiling safener-responsive GST expression in grain

sorghum lines that differ in s-metolachlor tolerance

Loren Goodrich, Rong Ma, Christopher Kaiser, Patrick Brown, and Dean E. Riechers

Department of Crop Sciences, University of Illinois, Urbana, IL 61801

Outline

o Background on Safeners

o Objectives and Hypothesis

o Materials and Methods

o Results

o Conclusions

o Future Research

Herbicide Detoxification

Herbicide safeners are non-toxic compounds that confer protection to cereal crops by inducing detoxification and defense systems.

These include massive increases in the expression and activity of glutathione S-transferases (GSTs) and cytochrome P450s.

The precise molecular mechanisms for induction of expression of GSTs and P450s remain unknown.

Herbicide Detoxification

Control PRE Herbicide Herbicide + Safener

14 DAT

Preliminary Data

A genome-wide association study (GWAS) was conducted in the greenhouse with preemergence s-metolachlor

The safener fluxofenim was used to determine phenotypes for natural herbicide tolerance and herbicide-safener responses in 400 diverse sorghum inbred lines

– Identify phenotypic variability

Phenotypic Variability among Sorghum Lines

Through an association panel screening of these 400 sorghum lines, two phenotypically diverse sorghum inbreds were identified:

1. high natural tolerance to s-metolachlor (with or without safener)

2. high sensitivity to s-metolachlor (with or without safener)

Phenotypic Variability among Sorghum Lines

Safener-induced

tolerance

(N = normal)

Natural tolerance (T)

Sensitive (S)

+ safener- safener

Herbicide treatment : 2.25 lbs s-metolachlor / Acre

Identify Genes of Interest

Associate phenotypic variability (traits) with genotypic variability (SNPs):

The top GWAS hits:

- Safener-induced GST gene expression located on chromosome 9

- Basal GST gene expression located on chromosome 3

Both hits in close proximity to GST gene clusters containing GST genes identified in the preliminary RNAseq studies.

Each specific GST is located within distinct gene clusters identified as hits through GWAS.

(1kb from GST cluster on chrom 9)

Manhattan plot of marker-trait associations of safener response across the

Sorghum genome (BTx623). Significance thresholds were determined using a

false discovery rate of 0.1 (black horizontal line).

Safener Response

Objectives

Design gene-specific primers (GSPs) from each GST gene identified through GWAS

Quantify transcript levels of the two candidate SbGSTs in coleoptile tissue at 8 and 12 hours after treatment

Hypothesis

Expression of either or both SbGST genes will correlate with natural or safener-enhanced herbicide responses to s-metolachlor in the three diverse sorghum genotypes.

Materials and Methods

• Three sorghum lines were examined: 2 lines identified through GWAS and inbred BTx623

• Seeds grown in vermiculite for 72 h then treated with either fluxofenim (20µM) or DMSO via a soil drench

• Sorghum shoots were harvested 8 and 12 hours after treatment (HAT)

• Total RNA was extracted with Trizol reagent

• GSPs for two GSTs of interest designed using NCBI Primer-BLAST

• Basal GST expression and safener-induced GST expression levels were compared via RT-PCR

RNA Extraction

3 Sorghum Lines:

1) Safener Induced Tolerance

- BTx623

2) Natural Tolerance

3) Sensitive

Harvested at 8 and 12 HAT with control or safener

High quality RNA

Ethidium Bromide Stained Agarose Gel

5µg total RNA / lane

Results

RT-PCR• Strong correlation

between basal GST expression and phenotype

• Need to confirm correlation between safener-induced GST expression and phenotype using quantitative RT-PCR

GST on chromosome 9

8 HAT

Results

RT-PCR

• Induction by safener is evident

• Cannot infer correlation between basal GST expression nor induced GST expression and phenotype

GST on chromosome 3

8 HAT

Results

Conclusions

Strong correlation between phenotype and basal expression of the SbGST on chromosome 9 at 8 HAT

– Indicates that this GST is a key gene involved in s-metolachlor detoxification

Correlation between basal expression of the SbGSTon chromosome 3 at 8 HAT and phenotype was not observed in spite of safener induction

– Lack of correlation indicates that this GST is not key gene associated with s-metolachlor tolerance

Interpretation of Data

• Identify additional key GST genes

– Multiple GST genes in each GST cluster on the chromosome identified in the RNAseq study

• Examine early GST responses in the pathway

– Measure at earlier time points: 2-8 HAT

• Shoot tissue used for RNA extraction contains coleoptile and leaf tissue

Future Research

• Quantitative RT-PCR

– Measurements of basal and safener-induced GST gene expression from each sorghum line at multiple time points

• Expand time course

– 2, 4, 8, and 12 HAT

• Isolate coleoptile tissue – main target for safener activity

• Identify GSTs and other genes to determine additional steps of the safener-induced detoxification pathway

– Markers to rapidly screen sorghum lines for increased herbicide tolerance

Acknowledgements

• Riechers and Brown Labs

• This project was supported by the Agriculture and Food Research Initiative, Competitive grant # 2015-67013-22818 of the USDA National Institute of Food and Agriculture.

Questions?