Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover

Authors: Dinkins, Randy; HANCOCK, JULIE - University Of Kentucky; Coe, Brenda; MAY, JOHN - University Of Kentucky; GOODMAN, JACK - University Of Kentucky; Bass, William - Troy; LIU, JINGE - University Of Kentucky; FAN, YINGLUN - University Of Kentucky; ZHENG, QIAOLIN - University Of Kentucky; ZHU, HONGYAN - University Of Kentucky

Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2020
Publication Date: 1/2/2021
Citation: Dinkins, R.D., Hancock, J., Coe, B.L., May, J.B., Goodman, J.P., Bass, W.T., Liu, J., Fan, Y., Zheng, Q., Zhu, H. 2021. Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover. Plant Cell Reports. 40(3):517-528. https://doi.org/10.1007/s00299-020-02647-4.
DOI: https://doi.org/10.1007/s00299-020-02647-4

Interpretive Summary: Red clover is a high-quality forage legume, well suited for grazing and hay production, and like other legumes, forms a symbiosis with nitrogen fixing bacteria, thus aids in mitigating the environmental impact of nitrogen fertilizers. Red clover produces a number of isoflavonoid compounds that have come into the forefront of biomedical and agricultural research due to their potential for medicinal and antimicrobial applications. In addition, one of the postulated roles for isoflavones in soybeans is that they function as a signal molecule exuded from the roots in attracting nitrogen fixing rhizobia. In order to study the role, and effects of lack of, isoflavonoids in red clover, CRISPR/Cas9 technology was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase, and gene expression profiles were compared between the mutant and wild-type plants by RNA-seq technology. Mutant plants, carrying a deletion in the isoflavone synthase gene, had significantly reduced levels of the isoflavones formononetin, biochanin A and genistein. However, no differences in the number of nodules were observed on the wild-type and mutant roots when inoculated with rhizobium, suggesting that these isoflavones are probably not the signaling molecule in red clover. However, when gene expression profiles were compared between the mutant and wild-type plants, it was observed that genes involved in defense response were more highly expressed in the mutant plants. This result suggests that the exuded isoflavones may aid to function in the control of harmful bacteria in the root zone, and illustrates the putative role for the production of these compounds in legumes.

Technical 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, suggesting that the plants may have been responding to biotic stress possibly due to lower levels of isoflavones in the surrounding rhizosphere.