Comparative gene expression following 2,4-D treatment in two Red Clover (Trifolium pratense L.) populations with differential tolerance to the herbicide

Authors: DE ARAUJO, LUCAS - University Of Kentucky; BARRETT, MICHAEL - University Of Kentucky; Dinkins, Randy

Submitted to: Agronomy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2024
Publication Date: 6/1/2024
Citation: de Araujo, L.P., Barrett, M., Dinkins, R.D. 2024. Comparative gene expression following 2,4-D treatment in two Red Clover (Trifolium pratense L.) populations with differential tolerance to the herbicide. Agronomy. 14(6). Article 1198. https://doi.org/10.3390/agronomy14061198.
DOI: https://doi.org/10.3390/agronomy14061198

Interpretive Summary: Incorporation of red clover into grass pastures can reduce the need for nitrogen fertilizer applications and increase the nutritional value of the forage. However, red clover cultivars currently available for producers are highly susceptible to the herbicides used for pasture broadleaf weed control, namely 2,4-D and similar chemistries. To overcome this problem, ‘UK2014’ red clover was selected for increased tolerance to 2,4-D for the U.S. transition zone. A transcriptome approach was employed to compare the gene expression response following 2,4-D treatment of ‘UK2014’ to that of ‘Kenland’, a 2,4-D sensitive red clover. The objectives were to determine if the increased 2,4-D tolerance in ‘UK2014’ is reflected in a change of transcription response and/or a quicker recovery of a transcriptional response following 2,4-D treatment; and to identify genes, whether constitutively expressed or induced by 2,4-D, that could be the basis for the increased 2,4-D tolerance. Leaf tissues from the two red clovers grown in the field were collected at 4, 24, and 72 hours after application of 2,4-D (1.12 kg 2,4-amine a.e. ha-1) and untreated control plants. Global gene expression was determined by RNA-seq reads mapped against the red clover draft genome. The increased tolerance of the ‘UK2014’ was observed in the change in overall gene expression whereby ‘UK2014’ appeared less affected by the herbicide application when compared to the 2,4-D susceptible red clover ‘Kenland’. These results corroborate previous findings that suggests that 2,4-D tolerance in red clover is conferred by multiple genes, and additional work is needed to identify the genome regions, and genes. Additionally, while the application of 2,4-D elicited a significant increase in expression in both tolerant and susceptible red clover genotypes of several enzymes known to be involved in herbicide metabolism, different isoforms of these proteins were identified between the tolerant and susceptible plants using the RNA-seq data. Additional work is needed characterize the different isoforms in regard to 2,4-D metabolism, but this would allow for selection of more tolerant germplasm using DNA markers in breeding programs.

Technical Abstract: Incorporation of red clover (Trifolium pratense L.) into grass pastures can reduce the need for nitrogen fertilizer applications and increase the nutritional value of the forage. However, red clover cultivars available for Kentucky producers are highly susceptible to the herbicides, such as 2,4-D (2,4-dichlorophenoxy acetic acid), used for pasture broadleaf weed control. To overcome this problem, ‘UK2014’ red clover was selected for increased tolerance to 2,4-D. We employed a transcriptome analysis approach to compare the gene expression response following 2,4-D treatment of ‘UK2014’ to that of ‘Kenland’, a 2,4-D sensitive red clover and one of the parents of ‘UK2014’. The objectives were: one, determine if the increased 2,4-D tolerance in ‘UK2014’ is reflected in a change of transcription response and/or a quicker recovery of a transcriptional response following 2,4-D treatment; and second, to identify genes, whether constitutively expressed or induced by 2,4-D, that could be the basis for the increased 2,4-D tolerance. Leaf tissue from the two red clovers grown in the field were collected at 4, 24, and 72 hours after 2,4-D (1.12 kg 2,4-amine a.e. ha-1) treatment from both untreated and treated plants. Global gene expression was determined with reads from Illumina Hiseq 2500 mapped against the red clover draft genome, Tpv2.1 (GenBank Accession GCA_900079335.1). Genes that displayed differential expression (DEGs) following 2,4-D treatment were selected for further analysis. The number of DEGs was higher for ‘Kenland’ than for ‘UK2014’, suggesting that a lower transcriptional response corresponds with the higher 2,4-D tolerance in the ‘UK2014’ line. Similarly, gene ontology enrichment analysis revealed that expression of photosynthesis related genes was less affected by 2,4-D in the ‘UK2014’ line than ‘Kenland’. Although we were not able to identify any specific genes that are the basis for the increased 2,4-D tolerance of ‘UK2014’, we concluded that the increased 2,4-D tolerance of ‘UK2014’ correlates with a decreased transcription response to 2,4-D. Additionally, expression of several cytochrome P450 genes that had different isoforms between ‘UK2014’ and ‘Kenland’ increased significantly in both following 2,4-D treatment, one or more of these P450s could be mediators of 2,4-D metabolism and tolerance in red clover.