Molecular characterization of the cytokinin responsive type-B response regulators in perennial ryegrass highlights LpARR10 conferring salt tolerance via trans-activating SOS1 and SOS3

The type-B response regulator (ARR) family members represent the elements of two-component regulatory systems and play crucial roles in the cytokinin signaling, plant growth, and stress responses. The objective of this study was to characterize type-B ARR genes from perennial ryegrass, and to investigate their roles in regulating to salt tolerance. Twelve type-B ARRs, namely LpARR1-12, were identified in perennial ryegrass. They were clustered into three subgroups and were unevenly distributed on chromosomes. The stress responsive elements (SREs) and hormone responsive elements (HREs) were identified in LpARRs promoters and the transcription of LpARRs genes were responsive to various abiotic stress (heat, salt, and osmotic) and hormonal (cytokinin, ABA, and ethylene) treatments. LpARR10 was strong induced by salt stress, therefore it was selected for function analysis. Overexpressing LpARR10 in Arabidopsis led to a significant improvement in salt tolerance. The LpARR10-overexpressing (LpARR10-OE) lines exhibited lower levels of Na+ content compared to WT plants, and the expression levels of SOS1 and SOS3 were significantly higher in the LpARR10-OE lines. Results of yeast autotransactivation and plant cell transcriptional activity assays shows LpARR10 is a nucleus localized transcriptional activator and directly bind and transactivated the promoter of SOS1 and SOS3. Together, LpARR10 enhances salt tolerance may via trans-activating SOS1/SOS3 in Arabidopsis. Knowledge gained in this study will facilitate further functional analysis of LpARRs for molecular breeding of ryegrass and other related grass species.