Functional characterization of PpGS2 promoter in response to hormonal and environmental signals in Kentucky bluegrass

Kentucky bluegrass (Poa pratensis) is an important cool-season turfgrass that requires high nitrogen (N) fertilizer inputs for optimal density, and turf quality. PpGS2 is a key gene involved in N uptake and assimilation; however, the core regulatory region of its promoter remains unclear. To characterize the transcriptional regulation of the PpGS2 promoter, a series of 5'-terminal deletion fragments were generated, each fused to the β-glucuronidase (GUS) reporter gene and introduced into Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). The transcriptional activity of all fragments decreased as fragment length shortened. The full-length promoter drove the strongest GUS expression in the leaves of transgenic Arabidopsis. Furthermore, exogenous hormone treatment, low-N stress, and high-light stress all significantly enhanced promoter activity. Among these treatments, methyl jasmonate (MeJA) elicited the strongest induction of promoter activity. Deletion analysis revealed distinct distributions of responsive elements of the PpGS2 promoter: gibberellin (GA) and abscisic acid (ABA) responsive elements were primarily located between - 2000 bp and - 1600 bp and - 1200 bp to -800 bp, respectively; auxin (IAA) and MeJA responses relied on the synergy of multiple regions. The low-N response was mediated by both positive and negative cis-acting elements. Specifically, the - 2000 bp to -1200 bp region likely contained repressive elements, whereas the - 1200 bp to -500 bp region harbored enhancing elements. Light-responsive elements were mainly concentrated within the - 1600 bp to -800 bp regions. In summary, this study provides a preliminary characterization of the regulatory pattern of the PpGS2 promoter, providing insight into the synergistic regulatory mechanisms of PpGS2 in response to hormonal and environmental signals.