The PsnWRKY70 transcription factor (TF) was reported to play an important role in the salt stress response mechanism of Populus simonii × Populus nigra in our previous research, and we also produced several PsnWRKY70 overexpression (OEXs) and RNAi suppression (REXs) P. simonii × P. nigra lines. In order to further compare the photosynthetic and physiological characteristics of NT (non-transgenic line) and transgenic lines under salt stress, the dynamic phenotypic change, Na⁺ and K⁺ content in leaf and root tissues, superoxide dismutase (SOD) and peroxidase (POD) activity, malondialdehyde (MDA) content, chlorophyll content (Chl), photosynthesis parameters (net photosynthetic rate, P_n; stomatal conductance, Gs; intercellular CO₂ concentration, C_i; transpiration rate, T_r), chlorophyll fluorescence parameters (electron transport rate, ETR; maximum photochemical efficiency of photosystem II (PSII), F_v/F_m; actual efficiency of PSII, Φ_PSII; photochemical quenching coefficient, q_P; non-photochemical quenching, NPQ; the photosynthetic light-response curves of Φ_PSII and ETR) and RNA-seq of NT, OEX and REX lines were detected and analyzed. The phenotypic observation, MDA content and Chl detection results indicate that the stress damage of REXs was less severe than that of NT and OEX lines under salt stress. Photosynthesis parameter (P_n, Gs, T_r and C_i) and chlorophyll fluorescence parameter (ETR, F_v/F_m, Φ_PSII q_P and NPQ) detection results indicate that the REX lines exhibited much better photosynthetic adaptability than NT and OEX lines during salt stress. The photosynthetic light-response curves of Φ_PSII and ETR of NT, OEX and REX lines indicate that REXs exhibited better ability to activate the photosynthetic protection mechanism and adapt to a certain degree of strong light than NT and OEX lines under salt stress. RNA-seq analysis indicates that the DEGs between OEX1 vs. NT and REX1 vs. NT in different tissues (apical bud and fifth functional leaf) were all different in category and change trend. The expression of PsnWRKY70 was significantly up-regulated in both the apical bud and fifth functional leaf of OEX1, and showed no significant change (namely maintained low expression level) in both the apical bud and fifth functional leaf of REX1, thus indicating the negative regulation role of PsnWRKY70 in P. simonii × P. nigra under salt stress. Additionally, there were a lot of stress response-related TF genes (such as bHLH, WRKY, MYB, NAM and AP2/EREBP) and photosynthesis-related genes among all the DEGs. In REX1, the expression of three Photosystem I P700 chlorophyll a apoprotein A1 genes (Potri.003G065200, Potri.013G141800 and Potri.019G028100) and a Photosystem II protein D1 gene (Potri.013G138300) were significantly up-regulated after 6 days of salt stress. In OEX1, the Heterodimeric geranylgeranyl pyrophosphate synthase small subunit gene (Potri.015G043400) and Phospho-2-dehydro-3-deoxyheptonate aldolase 1 gene (Potri.007G095700) were significantly down-regulated after 6 days of salt stress. These photosynthesis-related genes are probably regulated by PsnWRKY70 TF in response to salt stress. In conclusion, the REX lines suffered less severe salt damage and exhibited better photosynthetic adaptability than NT and OEXs under salt stress. The differences among the DEGs between OEX1 vs. NT and REX1 vs. NT in apical bud and fifth functional leaf, and the significantly differentially expressed photosynthesis-related genes are probably the key clues for discovering the photosynthesis adaptability mechanism of PsnWRKY70 transgenic P. simonii × P. nigra under salt stress.

Pathogenesis-related protein 1 (PR-1) genes are involved in plant defense responses and abiotic stress responses. In this study, a total of 14 PfPR-1 family members were identified from the genome of Paulownia fortunei, and their physicochemical properties, phylogenetic relationships, conserved motifs, promoter cis-acting elements, chromosomal localization, and syntenic relationships were systematically analyzed. The results showed that PfPR-1 family members exhibited considerable variation in protein physicochemical characteristics and structural composition, suggesting evolutionary and functional divergence among different members. Promoter analysis revealed the presence of numerous hormone-responsive, stress-responsive, and light-responsive cis-acting elements, indicating that PfPR-1 genes may be involved in multiple biological processes and signaling pathways. Transcriptome sequencing analysis demonstrated that most PfPR-1 genes significantly responded to Paulownia witches’ broom (PaWB) disease, salt stress, and drought stress. Under phytoplasma infection, five PfPR-1 genes were markedly upregulated (PR-1-1, PR-1-3, PR-1-4, PR-1-8, and PR-1-14), whereas four PfPR-1 genes (PfPR-1-6, PfPR-1-7, PfPR-1-12, and PfPR-1-13) were downregulated, suggesting their differential responses to phytoplasma infection. Under salt- and drought-stress conditions, different PfPR-1 genes exhibited diverse expression patterns, indicating their potential functional specialization in stress-responsive regulation. Subsequent qRT-PCR analysis further confirmed the reliability of the transcriptome data. Notably, the expression of PR-1-3 was significantly upregulated under both biotic and abiotic stresses. Yeast one-hybrid and dual-luciferase reporter assays demonstrated that an ERF transcription factor (TF), PfERF1, could significantly activate the transcription of PfPR-1-3, suggesting that PfERF1 may participate in stress-responsive regulation in Paulownia fortunei through modulating PfPR-1 expression. Collectively, these findings provide a theoretical basis for further investigation of the biological functions and molecular regulatory mechanisms of the PfPR-1 gene family during stress responses in Paulownia fortunei.