Abstract
Key message
Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere.
Abstract
Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.
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Data availability
Raw RNA-seq reads are deposited in the Short Read Archive database at GenBank under BioProject PRJNA657870.
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Acknowledgements
The authors wish to thank Dr. David McNair in the Plant and Soil Science Department at the University of Kentucky for advice and support on the root nodulation count procedures. This project was funded by the USDA-ARS Project no. 5042-21000-004-00D.
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RDD and HZ conceived and designed the experiments. JH, BLC, JBM, JPG, WTB, JL, YF and QZ performed the experiments and BLC, WTB and JL supervised the work. Data analysis was done by RDD, JBM, JPG and WTB. Manuscript was written by RDD, BLC, WTB and HZ. All authors read and approved the final manuscript.
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Dinkins, R.D., Hancock, J., Coe, B.L. et al. Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover. Plant Cell Rep 40, 517–528 (2021). https://doi.org/10.1007/s00299-020-02647-4
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DOI: https://doi.org/10.1007/s00299-020-02647-4