High NaCl (200 mM) increases the transcription of phospholipase Dδ (PLDδ) in roots and leaves of the salt-resistant woody species Populus euphratica. We isolated a 1138 bp promoter fragment upstream of the translation initiation codon of PePLDδ. A promoter–reporter construct, PePLDδ-pro::GUS, was introduced into Arabidopsis plants (Arabidopsis thaliana) to demonstrate the NaCl-induced PePLDδ promoter activity in root and leaf tissues. Mass spectrometry analysis of DNA pull-down-enriched proteins in P. euphratica revealed that PeGLABRA3, a basic helix–loop–helix transcription factor, was the target transcription factor for binding the promoter region of PePLDδ. The PeGLABRA3 binding to PePLDδ-pro was further verified by virus-induced gene silencing, luciferase reporter assay (LRA), yeast one-hybrid assay, and electrophoretic mobility shift assay (EMSA). In addition, the PeGLABRA3 gene was cloned and overexpressed in Arabidopsis to determine the function of PeGLABRA3 in salt tolerance. PeGLABRA3-overexpressed Arabidopsis lines (OE1 and OE2) had a greater capacity to scavenge reactive oxygen species (ROS) and to extrude Na⁺ under salinity stress. Furthermore, the EMSA and LRA results confirmed that PeGLABRA3 interacted with the promoter of AtPLDδ in transgenic plants. The upregulated AtPLDδ in PeGLABRA3-transgenic lines resulted in an increase in phosphatidic acid species under no-salt and saline conditions. We conclude that PeGLABRA3 activated AtPLDδ transcription under salt stress by binding to the AtPLDδ promoter region, conferring Na⁺ and ROS homeostasis control via signaling pathways mediated by PLDδ and phosphatidic acid.

The transcription of glycine-rich RNA-binding protein 2 (PeGRP2) transiently increased in the roots and shoots of Populus euphratica (a salt-resistant poplar) upon initial salt exposure and tended to decrease after long-term NaCl stress (100 mM, 12 days). PeGRP2 overexpression in the hybrid Populus tremula × P. alba ‘717-1B4’ (P. × canescens) increased its salt sensitivity, which was reflected in the plant’s growth and photosynthesis. PeGRP2 contains a conserved RNA recognition motif domain at the N-terminus, and RNA affinity purification (RAP) sequencing was developed to enrich the target mRNAs that physically interacted with PeGRP2 in P. × canescens. RAP sequencing combined with RT-qPCR revealed that NaCl decreased the transcripts of PeGRP2-interacting mRNAs encoding photosynthetic proteins, antioxidative enzymes, ATPases, and Na⁺/H⁺ antiporters in this transgenic poplar. Specifically, PeGRP2 negatively affected the stability of the target mRNAs encoding the photosynthetic proteins PETC and RBCMT; antioxidant enzymes SOD[Mn], CDSP32, and CYB1-2; ATPases AHA11, ACA8, and ACA9; and the Na⁺/H⁺ antiporter NHA1. This resulted in (i) a greater reduction in Fv/Fm, YII, ETR, and Pn; (ii) less pronounced activation of antioxidative enzymes; and (iii) a reduced ability to maintain Na⁺ homeostasis in the transgenic poplars during long-term salt stress, leading to their lowered ability to tolerate salinity stress.

Expansins are cell wall (CW) proteins that mediate the CW loosening and regulate salt tolerance in a positive or negative way. However, the role of Populus trichocarpa expansin A6 (PtEXPA6) in salt tolerance and the relevance to cell wall loosening is still unclear in poplars. PtEXPA6 gene was transferred into the hybrid species, Populus alba × P. tremula var. glandulosa (84K) and Populus tremula × P. alba INRA ‘717-1B4’ (717-1B4). Under salt stress, the stem growth, gas exchange, chlorophyll fluorescence, activity and transcription of antioxidant enzymes, Na⁺ content, and Na⁺ flux of root xylem and petiole vascular bundle were investigated in wild-type and transgenic poplars. The correlation analysis and principal component analysis (PCA) were used to analyze the correlations among the characteristics and principal components. Our results show that the transcription of PtEXPA6 was downregulated upon a prolonged duration of salt stress (48 h) after a transient increase induced by NaCl (100 mM). The PtEXPA6-transgenic poplars of 84K and 717-1B4 showed a greater reduction (42–65%) in stem height and diameter growth after 15 days of NaCl treatment compared with wild-type (WT) poplars (11–41%). The Na⁺ accumulation in roots, stems, and leaves was 14–83% higher in the transgenic lines than in the WT. The Na⁺ buildup in the transgenic poplars affects photosynthesis; the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT); and the transcription of PODa2, SOD [Cu-Zn], and CAT1. Transient flux kinetics showed that the Na⁺ efflux of root xylem and leaf petiole vascular bundle were 1.9–3.5-fold greater in the PtEXPA6-transgenic poplars than in the WT poplars. PtEXPA6 overexpression increased root contractility and extensibility by 33% and 32%, indicating that PtEXPA6 increased the CW loosening in the transgenic poplars of 84K and 717-1B4. Noteworthily, the PtEXPA6-promoted CW loosening was shown to facilitate Na⁺ efflux of root xylem and petiole vascular bundle in the transgenic poplars. We conclude that the overexpression of PtEXPA6 leads to CW loosening that facilitates the radial translocation of Na⁺ into the root xylem and the subsequent Na⁺ translocation from roots to leaves, resulting in an excessive Na⁺ accumulation and consequently, reducing salt tolerance in transgenic poplars. Therefore, the downregulation of PtEXPA6 in NaCl-treated Populus trichocarpa favors the maintenance of ionic and reactive oxygen species (ROS) homeostasis under long-term salt stress.

The toxic metal cadmium (Cd) poses a serious threat to plant growth and human health. Populus euphratica calcium-dependent protein kinase 21 (CPK21) has previously been shown to attenuate Cd toxicity by reducing Cd accumulation, enhancing antioxidant defense and improving water balance in transgenic Arabidopsis. Here, we confirmed a protein–protein interaction between PeCPK21 and Arabidopsis nuclear transcription factor YC3 (AtNF-YC3) by yeast two-hybrid and bimolecular fluorescence complementation assays. AtNF-YC3 was induced by Cd and strongly expressed in PeCPK21-overexpressed plants. Overexpression of AtNF-YC3 in Arabidopsis reduced the Cd inhibition of root length, fresh weight and membrane stability under Cd stress conditions (100 µM, 7 d), suggesting that AtNF-YC3 appears to contribute to the improvement of Cd stress tolerance. AtNF-YC3 improved Cd tolerance by limiting Cd uptake and accumulation, activating antioxidant enzymes and reducing hydrogen peroxide (H₂O₂) production under Cd stress. We conclude that PeCPK21 interacts with AtNF-YC3 to limit Cd accumulation and enhance the reactive oxygen species (ROS) scavenging system and thereby positively regulate plant adaptation to Cd environments. This study highlights the interaction between PeCPK21 and AtNF-YC3 under Cd stress conditions, which can be utilized to improve Cd tolerance in higher plants.

The toxic cadmium (Cd) poses a serious threat to plant growth and human health. Populus euphratica CPK21 has previously been shown to attenuate Cd toxicity by reducing Cd accumulation, enhancing antioxidant defense and improving water balance in transgenic Arabidopsis. Here, we confirmed a protein-protein interaction between PeCPK21 and AtNF-YC3 by yeast two-hybrid, HaloTag pull-down and bimolecular fluorescence complementation assays. AtNF-YC3 was induced by Cd and strongly expressed in PeCPK21-overexpressed plants. Overexpression of AtNF-YC3 in Arabidopsis reduced salt inhibition of root length, fresh weight and membrane stability under Cd stress conditions (100 µM, 7 d), suggesting that AtNF-YC3 appears to contribute to the improvement of Cd stress tolerance. AtNF-YC3 improved Cd tolerance by limiting Cd uptake and accumulation, activating antioxidant enzymes, and reducing H2O2 production under Cd stress. We conclude that PeCPK21 interacts with AtNF-YC3 to limit Cd accumulation, enhance the ROS scavenging system, and thereby positively regulate plant adaptation to Cd environments. This study highlights the interaction between PeCPK21 and AtNF-YC3 under Cd stress conditions, which can be utilized to improve Cd tolerance in higher plants.

Expansins are cell wall (CW) proteins that mediate the CW loosening and regulate salt tolerance in a positive or negative way. The transcription of Populus trichocarpa expansin A6 (PtEXPA6) was downregulated upon prolonged duration of salt stress (48 h) after a transient increase induced by NaCl (100 mM). The role of PtEXPA6 in salt tolerance was determined by transferring the PtEXPA6 gene into the hybrid species Populus alba × P. tremula var. glandulosa clone 84K (84K) and Populus tremula × P. alba INRA '717-1B4' (717-1B4). The PtEXPA6-transgenic poplars of 84K and 717-1B4 showed a greater reduction in photosynthesis, stem height and diameter growth under salt stress and a reduced capacity for ROS scavenging by antioxidant enzymes, which is associated with excessive Na+ accumulation in roots and shoots. NMT flux recordings showed that the radial translocation of Na+ salt into the root xylem and real-time Na+ translocation from roots to leaves were greater in PtEXPA6-transgenic poplars than in wild-type poplars. Analysis of comparative contractility and comparative extensibility of intact root tips indicated that PtEXPA6 increased the CW loosening in the transgenic poplars of 84K and 717-1B4. Noteworthy, the PtEXPA6-promoted CW loosening was shown to facilitate radial and longitudinal transport of Na+ in transgenic poplars. We conclude that overexpression of PtEXPA6 leads to CW loosening that facilitates radial translocation of Na+ into the root xylem and subsequent Na+ translocation from roots to leaves, resulting in excessive Na+ accumulation and consequently reduced salt tolerance in transgenic poplars.