TO-GCN analysis reveals the salt response mechanism in Carya illinoinensis and a Preliminary Exploration of the ‘CiPLATZ23-PIP2;8’ module

Carya illinoinensis was a crop with high value and was widely cultivated in China. Particularly under the increasingly severe soil salinization background, the promotion of salt-tolerant C. illinoinensis varieties provided substantial economic benefits to local regions. However, the molecular mechanisms underlying C. illinoinensis's salt response remained unclear. Two-year-old C. illinoinensis saplings exhibited significant damage after treatment with 600 mM NaCl solution. Transcriptome data at nine salt treatment time points were obtained from two-year-old C. illinoinensis saplings using RNA-seq, and 7840 differentially expressed genes (DEGs) were identified to construct a time-ordered gene co-expression network (TO-GCN). DEGs in the TO-GCN were classified into 10 levels corresponding to salt treatment time points. Genes in Level 1 (L1) and Level 2 (L2) were enriched in Gene Ontology (GO) terms associated with photosynthesis and transport channel proteins. Cluster analysis identified three aquaporin-coding genes among highly expressed genes. Based on promoter cis-acting elements, A/T-rich sequence features, and antagonistic gene expression patterns in transcriptomic data, it was speculated that CiPIP2;8 might be transcriptionally repressed by the upstream transcription factor CiPLATZ23. This hypothesis was validated through a Dual-luciferase reporter (DLR) assay. The presence of ABA-responsive elements (ABREs) in ProCiPLATZ23 suggested its involvement in the abscisic acid (ABA) pathway. Transient expression of ProCiPLATZ23::GUS in Nicotiana benthamiana exhibited enhanced histochemical staining following ABA treatment. Within 24 h after spraying walnuts with ABA, the transcription level of CiPLATZ23 rapidly increased. Furthermore, 35S::CiPLATZ23 overexpression lines in Arabidopsis thaliana displayed reduced ABA sensitivity compared to Wild-type (WT) A. thaliana. Reverse transcription quantitative PCR (RT-qPCR), yeast one-hybrid, and DLR assays confirmed that CiPLATZ23 participated in regulating ABA sensitivity by binding to the promoter of AtPIP2;8, the homolog of CiPIP2;8 in A. thaliana. Characterization of canonical ABA pathway families confirmed that C. illinoinensis ABA responses were implicated under salt stress. This study employed TO-GCN analysis of RNA-seq data to elucidate salt stress response mechanisms in C. illinoinensis and identified a key module 'CiPLATZ23-PIP2; 8' that may be associated with ABA.