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The homeodomain leucine zipper (HD-Zip) transcription factors are involved in plant responses to abiotic stresses. In this study, we have identified a total of 52 HD-Zip genes from the released genome of perennial ryegrass (Lolium perenne), a cool-season turfgrass and forage species, which is relatively sensitive to heat and drought stresses. These HD-Zip genes are classified into classes I-IV, of which, 49 are evenly distributed on all seven chromosomes of perennial ryegrass whereas other 3 are on scaffolds. Cis -elements analysis showed that phytohormone, light signaling, and stress responsive elements are abundant in the promoter region of the HD-Zip genes. Expression analysis indicated that LpHOX24 and its paralog LpHOX22 were significantly induced by heat stress treatment. Ectopic overexpressing (OE) of LpHOX22 and LpHOX24 in Arabidopsis compromised plant osmotic stress tolerance as evidenced by declined survival rate with elevated electrolyte leakage (EL) and malondialdehyde (MDA) accumulation. Expression levels of several osmotic responsive genes were downregulated in OE lines. Additionally, heat stress tolerance was impaired in LpHOX22 and LpHOX24 overexpression transformants, along with increased EL and MDA content. Further analysis revealed that overexpression of LpHOX22 and LpHOX24 repressed the expressions of AtHSFAs and AtDREB2A when compared to wild type after heat stress treatment. In summary, LpHOX22 and LpHOX24 potentially functioned as negative regulators in perennial ryegrass in response to osmotic and heat stresses. • Fifty-two HD-Zip genes were identified from the perennial ryegrass genome. • LpHOX22 and LpHOX24 were significantly induced by heat stress. • LpHOX22 and LpHOX24 transgenes impaired osmotic and heat stress tolerances. • LpHOX22 and LpHOX24 repressed expression of stress responsive genes. [ABSTRACT FROM AUTHOR]

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 auto-transactivation 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. • Overexpressing LpARR10 resulted in a reduction in Na+ content compared to WT plants. • The LpARR10 -OE lines exhibited significantly higher expression levels of SOS1 and SOS3. • LpARR10 acts as a transcriptional activator and positively regulates salt tolerance by up-regulating the expressions of SOS1 and SOS3. [ABSTRACT FROM AUTHOR]