The Na+/H+ antiporter (NHX) gene subfamily is essential for plant adaptation to salt stress, contributing significantly to ion homeostasis. These antiporters play crucial function in various biological processes by regulating Na+ and H+ balance within plant cells. Yet their genomic features and roles of salicylic acid (SA) in regulating them, activating antioxidant defense mechanisms as well as nutrients acquisition under salt stress conditions remain largely unknown in many crop species. In this study, we identified the NHX gene family members of three Brassicaceae species including Brassica napus, Brassica rapa and Brassica oleracea and analyzed the expression patterns of NHX genes in response to salt stress and phytohormonal application (SA). Comprehensive computational analyses, encompassing domain and motif prediction, cellular localization, cis-regulatory elements (CREs), and post-translational modification (PTM) sites revealed distinct group-specific characteristics within the NHX gene family. These characteristics included chromosomal distribution, motif composition, exon count and protein length (amino acids). qRT-PCR analysis depicted significantly induced expression of NHXs, specifically, BnNHX7, BraNHX6 and BoNHX6 (4 ~ 6 folds) using different concentrations of NaCl (100 mM and 200 mM) and SA (10 mg/L and 20 mg/L) at different periods of time. SA induced antioxidant enzymatic activity (SOD 3 ~ 4 folds, POD 2 ~ 3 folds, CAT 1 ~ 2 folds and APX 11 ~ 12 folds), upregulated antioxidant genes (SOD, POD, APX, CAT) by 3 ~ 5 folds under salt stress, and improved nutrient acquisition in all respective Brassicaceae species. We have conducted the first molecular characterization of NHX genes in three Brassica species, revealing their dual role in salt stress adaptation through ion homeostasis and salicylic acid induced antioxidant regulation, thus identifying promising targets for crop improvement. Not applicable.

The plant hormone abscisic acid (ABA) plays a crucial role throughout the plant life cycle and in adaptive responses to environmental stresses. The pyrabactin resistance 1-like (PYR/PYL/RCAR) proteins act as key regulators in the ABA signal transduction pathway by functioning as direct receptors for ABA. Although PYL genes have been identified in a variety of plant species, their evolutionary and structural characteristics in tomatoes (Solanum lycopersicum) remain elusive. To address this gap, we identified nine SlPYL genes, which were classified into three subfamilies: I (two genes), II (three genes), and III (four genes), and their encoded proteins were predicted to be primarily localized in the cytosol and chloroplast. Structural analysis revealed diverse exon–intron organizations along with five conserved motifs. All identified SlPYLs contained the START domain (PF10604), validating their identity as actual PYL proteins. Prediction of cis-acting regulatory elements in SlPYL’s promoter regions was found to be associated with light responsiveness, hormone signaling, stress responses, and plant growth and development. Prediction of post-translational modification sites indicated that SlPYLs are predominantly phosphorylated and acetylated at serine and lysine residues, respectively. Tertiary structure modeling demonstrated conserved three-dimensional architectures among SlPYL proteins, supporting their functional conservation. Expression profiling revealed that specific SlPYL genes exhibit distinct expression patterns across different tissues (root, leaf, and bud) following ABA treatment, indicating functional diversification. Considering the well-established negative correlation between ABA accumulation and bud outgrowth, the ABA-induced differential expression (3~5-fold) of some SlPYL genes (SlPYL3, SlPYL4, SlPYL7, and SlPYL8), particularly in bud tissues after 24 hpt, suggests a potential role in ABA-mediated suppression of bud outgrowth. However, these functional inferences are primarily based on genome-wide computational analyses and expression profiling and therefore require further experimental validation.

Small heat shock proteins (sHSPs) are essential components of the plant proteostasis network under environmental stress; however, the cis-regulatory mechanisms underlying stress-responsive expression of individual sHSP genes remains insufficiently depicted. Here, we characterized the promoter of ClHSP22.9 from wild watermelon (Citrullus lanatus subsp. cordophanus) and evaluated its transcriptional responsiveness under different abiotic stresses. A semi-quantitative RT-PCR analysis revealed that ClHSP22.9 transcripts were strongly induced by heat, dehydration, and salinity treatments, whereas cold treatment caused comparatively limited transcriptional alterations. A 1482 bp promoter region upstream of the transcription start site was isolated using thermal asymmetric interlaced PCR (TAIL-PCR) and partially overlapping primer-based PCR (POP-PCR). An in silico promoter analysis identified multiple predicted stress and hormonal-associated cis-regulatory elements, including two heat shock elements (HSEs). Three serial 5′ promoter deletion fragments (−1482, −1012, and −522 bp) were fused to the GUS reporter gene and functionally analyzed in transgenic Brassica napus. Under heat stress, the −1482 and −1012 bp promoter fragments exhibited strong reporter activation, whereas deletion to −522 bp significantly reduced promoter activity, indicating that the −1012 to −522 bp region contributes substantially to maximal heat-responsive transcription. Furthermore, the −1012 bp proHSPII fragment retained an inducible GUS expression under heat, drought, and combined heat and drought stress conditions. These findings identify an important region within the ClHSP22.9 promoter and establish the −1012 bp promoter fragment as a promising candidate stress-responsive regulatory sequence for further mechanistic characterization and potential application in plant biotechnology.