Plant Cell Reports (2021) 40:517–528

https://doi.org/10.1007/s00299-020-02647-4

ORIGINAL ARTICLE

Isoflavone levels, nodulation and gene expression profiles of a CRISPR/

Cas9 deletion mutant in the isoflavone synthase gene of red clover
Randy D. Dinkins1   · Julie Hancock2 · Brenda L. Coe1 · John B. May3 · Jack P. Goodman3 · William T. Bass1 · Jinge Liu3 ·
Yinglun Fan3,4 · Qiaolin Zheng3,5 · Hongyan Zhu3

Received: 23 September 2020 / Accepted: 1 December 2020 / Published online: 2 January 2021
© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2021

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 includ-
ing 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.

Keywords  Red clover · Isoflavones · Isoflavone synthase · Nodulation · Gene expression

Communicated by Baochun Li.

Introduction
Supplementary Information  The online version contains
supplementary material available at https​://doi.org/10.1007/s0029​ Clovers and other forage legumes are commonly deployed
9-020-02647​-4.
in pastures due to their symbiotic relationship with rhizobia.
* Randy D. Dinkins This particular symbiosis induces the formation of root nod-
randy.dinkins@usda.gov ules. These microbes convert atmospheric nitrogen to plant-
available nitrogen forms. Red clover (Trifolium pratense)
1
USDA-ARS, Forage-Animal Production Research Unit, is an ideal choice for pastures as it is well suited to grazing
Lexington, KY, USA
and hay production when interseeded with common forage
2
College of Agriculture, Food and Environment, University grasses (Ball et al. 2007; Taylor 2008), and can contribute
of Kentucky, Lexington, KY, USA
more than 200 kg/ha of nitrogen per year to the cropping
3
Department of Plant and Soil Sciences, University system (Carlsson and Huss-Danell 2003). This reduces the
of Kentucky, Lexington, KY, USA
monetary costs of nitrogen fertilizer application, thus aids
4
Present Address: College of Agriculture, Liaocheng in mitigating potential environmental impacts and improv-
University, Liaocheng, China
ing overall agricultural system sustainability (Evers 2011).
5
Present Address: Department of Plant Pathology, University
of Florida, IFAS, Fort Pierce, FL, USA

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