1. 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
2. Outline
o Background on Safeners
o Objectives and Hypothesis
o Materials and Methods
o Results
o Conclusions
o Future Research
3. 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.
5. 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
6. 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)
8. 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.
9. 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
10. 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
11. 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.
12. 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
13. 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
14. 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
15. 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
16. 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 SbGST
on 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
17. 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
18. 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
19. 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.
