Genome-wide analysis of the GRAS gene family in white clover (Trifolium repens L.) provides insight into its critical role in response to cold stress

Abstract

The GRAS transcription factor (TF) is a plant-specific regulator that plays a critical role in plant growth, development and response to various abiotic stresses. While the functions of many GRAS TFs have been extensively studied in numerous model plants with fully sequenced genomes, the GRAS TFs in white clover (Trifolium repens L.) remain elusive despite the sequencing of its genome. To bridge this knowledge gap, homologs of GRAS TFs from Arabidopsis were employed and subjected to a genome-wide blast against white clover proteins. This analysis identified and characterized 102 putative GRAS genes, designated as TrGRAS, based on the presence of GRAS domains in positive blast hits. Concurrently, an exploration of their gene structures, classification, evolutionary relationships and conservative motifs provided insights into the distinctive features of TrGRAS in white clover. Furthermore, gene duplication analysis revealed an expansion of TrGRAS genes in the PAT1 and LISCL subfamilies, indicating functional diversity within the white clover genome. Lastly, RNA-seq and quantitative reverse transcription-PCR (qRT-PCR) experiments confirmed their higher expression during the early or intermittent responses to cold stress, suggesting their potential and crucial roles in determining the cold tolerances of white clover. The analysis of TrGRAS genes, coupled with expression studies, contributes to the functional characterization of TrGRAS genes in white clover. This knowledge is pivotal for informing genetic improvement strategies in future breeding efforts aimed at enhancing cold tolerance in white clover.

Keywords:

• White clover
• GRAS
• genome duplication
• cold stress
• RNA-seq

Authors’ contributions

YS and MH conceived and the study; MH, ML and GZ conducted the experiments; MH, ML, HG and CL analyzed the data; YS and MH wrote the manuscript; MH, ML and YS acquired funding; all authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings reported in this study are available from the corresponding author [YS] upon reasonable request.

Additional information

Funding

This research was funded by Zhaoqing Science and Technology Innovation Project Fund (202004031116), the Innovative Project for Postgraduate Students of Harbin Normal University (grant number HSDSSCX2023-42) and the Natural Science Foundation of Heilongjiang Province (grant number LH2022C050).