Glutathione S-transferases (GSTs) play a crucial role in responding to abiotic stress and are an important target for research on plant stress tolerance mechanisms. Populus euphratica is a promising candidate species for investigating the abiotic tolerance mechanisms in woody plants. In our previous study, PeGSTU58 was identified as being associated with seed salinity tolerance. In the present study, PeGSTU58 was cloned from P. euphratica and functionally characterized. PeGSTU58 encodes a Tau class GST and is located in both the cytoplasm and nucleus. Transgenic Arabidopsis overexpressing PeGSTU58 displayed enhanced tolerance to salt and drought stress. Under salt and drought stress, the transgenic plants exhibited significantly higher activities of antioxidant enzymes, including SOD, POD, CAT, and GST, compared to the wild-type (WT) plants. Additionally, the expression levels of several stress-responsive genes, including DREB2A, COR47, RD22, CYP8D11, and SOD1 were upregulated in PeGSTU58 overexpression lines compared to those in WT Arabidopsis under salt and drought stress conditions. Furthermore, yeast one-hybrid assays and luciferase analysis showed that PebHLH35 can directly bind to the promoter region of PeGSTU58 and activate its expression. These results indicated that PeGSTU58 was involved in salt and drought stress tolerances by maintaining ROS homeostasis, and its expression was positively regulated by PebHLH35.

The growth and production of poplars are usually affected by unfavorable environmental conditions such as soil salinization. Thus, enhancing salt tolerance of poplars will promote their better adaptation to environmental stresses and improve their biomass production. Stress-associated proteins (SAPs) are a novel class of A20/AN1 zinc finger proteins that have been shown to confer plants’ tolerance to multiple abiotic stresses. However, the precise functions of SAP genes in poplars are still largely unknown. Here, the expression profiles of Populus trichocarpa SAPs in response to salt stress revealed that PtSAP13 with two AN1 domains was up-regulated dramatically during salt treatment. The β-glucuronidase (GUS) staining showed that PtSAP13 was accumulated dominantly in leaf and root, and the GUS signal was increased under salt condition. The Arabidopsis transgenic plants overexpressing PtSAP13 exhibited higher seed germination and better growth than wild-type (WT) plants under salt stress, demonstrating that overexpression of PtSAP13 increased salt tolerance. Higher activities of antioxidant enzymes were found in PtSAP13-overexpressing plants than in WT plants under salt stress. Transcriptome analysis revealed that some stress-related genes, including Glutathione peroxidase 8, NADP-malic enzyme 2, Response to ABA and Salt 1, WRKYs, Glutathione S-Transferase, and MYBs, were induced by salt in transgenic plants. Moreover, the pathways of flavonoid biosynthesis and metabolic processes, regulation of response to stress, response to ethylene, dioxygenase activity, glucosyltransferase activity, monooxygenase activity, and oxidoreductase activity were specially enriched in transgenic plants under salt condition. Taken together, our results demonstrate that PtSAP13 enhances salt tolerance through up-regulating the expression of stress-related genes and mediating multiple biological pathways.

Understanding the genetic architecture of adventitious root and related shoot traits will facilitate the cultivation of superior genotypes. In this study, we measured 12 adventitious root and related shoot traits of 434 F₁ genotypes originating from Populus deltoides ‘Danhong’ × Populus simonii ‘Tongliao1’ and conducted an integrative analysis of quantitative trait locus (QTL) mapping and RNA-Seq data to dissect their genetic architecture and regulatory genes. Extensive segregation, high repeatability, and significant correlation relationship were detected for the investigated traits. A total of 150 QTLs were associated with adventitious root traits, explaining 3.1–6.1% of phenotypic variation (PVE); while 83 QTLs were associated with shoot traits, explaining 3.1–19.8% of PVE. Twenty-five QTL clusters and 40 QTL hotspots were identified for the investigated traits. Ten QTL clusters were overlapped in both adventitious root traits and related shoot traits. Transcriptome analysis identified 10,172 differentially expressed genes (DEGs) among two parents, three fine rooting and three poor-rooting genotypes, 143 of which were physically located within the QTL intervals. K-means cluster and weighted gene co-expression network analysis showed that PtAAAP19 (Potri.004G111400) encoding amino acid transport protein was tightly associated with adventitious roots and highly expressed in fine-rooting genotypes. Compare with ‘Danhong’, 153 bp deletion in the coding sequence of PtAAAP19 in ‘Tongliao1’ gave rise to lack one transmembrane domain, which might cause the variation of adventitious roots. Taken together, this study deciphered the genetic basis of adventitious root and related shoot traits and provided potential function genes for genetic improvement of poplar breeding.

Small heat shock proteins (sHsps) function mainly as molecular chaperones that play vital roles in response to diverse stresses, especially high temperature. However, little is known about the molecular characteristics and evolutionary history of the sHsp family in Salix suchowensis, an important bioenergy woody plant. In this study, 35 non-redundant sHsp genes were identified in S. suchowensis, and they were divided into four subfamilies (C, CP, PX, and MT) based on their phylogenetic relationships and predicted subcellular localization. Though the gene structure and conserved motif were relatively conserved, the sequences of the Hsp20 domain were diversified. Eight paralogous pairs were identified in the Ssu-sHsp family, in which five pairs were generated by tandem duplication events. Ka/Ks analysis indicated that Ssu-sHsps had undergone purifying selection. The expression profiles analysis showed Ssu-Hsps tissue-specific expression patterns, and they were induced by at least one abiotic stress. The expression correlation between two paralogous pairs (Ssu-sHsp22.2-CV/23.0-CV and 23.8-MT/25.6-MT) were less than 0.6, indicating that they were divergent during the evolution. Various cis-acting elements related to stress responses, hormone or development, were detected in the promoter of Ssu-sHsps. Furthermore, the co-expression network revealed the potential mechanism of Ssu-sHsps under stress tolerance and development. These results provide a foundation for further functional research on the Ssu-sHsp gene family in S. suchowensis.

WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors play critical roles in cell fate determination during plant development. As the founding member of the WOX family, WUSCHEL (WUS) is characterized for its role in maintaining stem cell in meristem. In this study, we investigated the function of Populus tomentosa WUSCHELa (PtoWUSa) in adventitious roots (ARs) in poplar. Expression profile analysis showed that PtoWUSa was not only expressed in shoot apical meristem and stem, but also expressed in ARs. Ectopic expression of PtoWUSa in Arabidopsis resulted in shortened primary root, as well as agravitropism and multiple branches. Overexpression of PtoWUSa in poplar increased the number of ARs but decreased their length. Moreover, the AR tip and lateral root tip became larger and swollen. In addition, the expression of auxin transporter genes PIN-FORMED were downregulated in ARs of transgenic plant. Taken together, these results suggest that PtoWUSa could be involved in AR development in poplar through regulating the polar auxin transport in ARs.

Drought is a major environmental constraint to plant growth, development and productivity. Compared with most willows that are generally susceptible to drought, the desert willow Salix psammophila has extraordinary adaptation to drought stress. However, its molecular basis of drought tolerance is still largely unknown. During polyethylene glycol 6000 (PEG 6000)-simulated drought stress, we found that the osmotic adjustment substances were accumulated and the antioxidant enzyme activities were enhanced in S. psammophila roots. A total of 8172 differentially expressed genes were identified in roots of S. psammophila through RNA-Sequencing. Based on K-means clustering, their expression patterns were classified into nine clusters, which were enriched in several stress-related processes including transcriptional regulation, response to various stresses, cell death, etc. Moreover, 672 transcription factors from 45 gene families were differentially expressed under drought stress. Furthermore, a weighted gene co-expression network was constructed, and eight genes were identified as hub genes. We demonstrated the function of two hub genes, magnesium-dependent phosphatase 1 (SpMDP1) and SpWRKY33, through overexpression in Arabidopsis thaliana. Overexpression of the two hub genes enhanced the drought tolerance in transgenic plants, suggesting that the identification of candidate drought tolerance genes in this study was highly efficient and credible. Our study analyzed the physiological and molecular responses to drought stress in S. psammophila, and these results contribute to dissect the mechanism of drought tolerance of S. psammophila and facilitate identification of critical genes involved in drought tolerance for willow breeding.