Allelopathy is widespread and significantly influences plant growth and development. Here, we aimed to explore the effects of poplar root aqueous extracts on tall fescue (Festuca arundinacea Schreb.) plants. Gas chromatography–mass spectrometry (GC-MS) analysis, using area–normalization, revealed distinct species–specific relative chemical abundance profiles: eight compounds were shared, while 18 and 13 unique compounds were identified in P. tomentosa and P. euphratica, respectively. Notably, 1,2-cyclohexanediol (26.51

Zelkova schneideriana Hand.-Mazz. is an ecologically and economically valuable tree species in China, but its sensitivity to low temperature restricts its use in colder regions. To identify genes and pathways associated with chilling responses, we combined physiological screening, full-length transcriptome construction, Illumina RNA-seq, and time-ordered gene co-expression network analysis (TO-GCN). Fifty seedling lines were evaluated after 7 d at 4 °C, and the line with the lowest relative electrolyte leakage (REL) and malondialdehyde (MDA) content was selected for transcriptome profiling at 0, 3, and 6 d of chilling treatment. RNA-seq detected 5875 differentially expressed genes. Most expression changes occurred by day 3 and were largely maintained on day 6. Pathway reconstruction showed contrasting dynamics between antioxidant-related systems: Flavonoid and anthocyanin biosynthesis were progressively activated, whereas glutathione-related genes, particularly glutathione S-transferase family members, were broadly repressed. TO-GCN highlighted ZeF3H (Ze_transcript_14112) as a late-stage hub associated with anthocyanin biosynthesis and ZeDRT102 (Ze_transcript_44427) as an early-stage hub linked to DNA damage repair and glutathione-associated genes. These results indicate that the chilling response in Z. schneideriana involves activation of flavonoid-based protection together with weakened glutathione-mediated redox regulation, providing candidate genes for functional validation and cold-resistance breeding. These insights provide a critical foundation for molecular breeding strategies that will ultimately facilitate the successful introduction and stable cultivation of this valuable species in northern temperate climates.

Expansins are crucial components in plant cell walls and are classified into four subfamilies based on their sequences. To investigate their function differentiation in plant growth and stress resistance, four expansin subfamily genes—PtoEXPA8, PtoEXPB3, PtoEXLA2, and PtoEXLB1—were cloned from Populus tomentosa Carr. TC1521. Their sequence and structure exhibited typical subfamily characteristics. They were individually introduced in tobacco plants and made different contributions to the plants’ performances. Compared to wild-type plants, the PtoEXPA8 overexpressing lines increased the leaf area, and PtoEXPB3 facilitated floral organ development and earlier flowering and increased flower diameter. PtoEXLA2 increased plant height but reduced flower diameter and 1000-seeds weight. Finally, PtoEXLB1 increased plant height and decreased flower diameter. Under heat stress conditions, compared to the wild-type plants, the PtoEXPA8 overexpressing plants performed the best, while the other three genes barely contributed to heat resistance. The results indicate that the expansin subfamily genes underwent significant function differentiation, playing distinct roles in plant growth and stress resistance in poplar.

Expansins are cell wall proteins that loosen cellulose networks by breaking noncovalent bonds between polysaccharides, thus regulating plant growth, development, and stress responses. The family is divided into four subfamilies based on gene structure and phylogeny, with EXPA usually being the largest. To explore the functional differentiation within the EXPA subfamily, we selected four representative EXPA genes from Populus tomentosa Carr. for functional comparison: PtoEXPA1, PtoEXPA3, PtoEXPA8, and PtoEXPA12. These genes share amino acid sequence identities ranging from 43.12

Bursaphelenchus xylophilus is a pathogen that causes devastating mortality in pine forests, characterized by high virulence, rapid transmission, and difficulty for control. Exploring resistance genes and resistance mechanisms are effective strategies for addressing pine wilt disease. In this study, a pathogen induced gene, PtCRK2, was cloned from the pathogen-resistant line of Pinus tabuliformis, which is 1,188 bp in length and encodes a receptor kinase. The gene was introduced into P. tabuliformis callus. Triphenyltetrazolium chloride (TTC) staining assay revealed that the PtCRK2-overexpression callus and control performed better under regular conditions without significant difference. When subjected to the treatment of B. xylophilus, the pinus callus displayed the color fading, indicating the cell activity decreased. The PtCRK2-overexpression calli performed better than the control with the callus color redder. Measurement of triphenyltetrazolium formazan (TTF) content showed 1.84-fold higher in the transgenic calli than the control. The physiological index tests showed that the relative electrical leakage and MDA content of the control were 1.33-fold and 1.46-fold higher than the PtCRK2-overexpression callus, respectively. All these findings indicated that PtCRK2 gene significantly enhanced the resistance of P. tabuliformis against B. xylophilus and can serve as an effective gene resource for molecular breeding.

Plasma membrane intrinsic proteins (PIPs) are involved in plant growth and stress adaptation through their dynamic gating mechanism. Nevertheless, pinpointing the specific roles of individual isoforms remains challenging because of their functional pleiotropy and integrated responses to diverse cues. In this study, we characterized a salt-responsive aquaporin gene, designated SmPIP1;3, isolated from a salt-resistant Salix matsudana variety. Bioinformatics analysis confirmed that it encodes a protein that possesses canonical PIP features with six transmembrane domains, five interhelical loops, and seven serine phosphorylation sites involved in phosphorylation-mediated regulation. The SmPIP1;3 gene was introduced in tobacco plants, and its heterologous expression conferred significant morphological improvements, including taller plant height, larger leaves, longer roots, and increased biomass. Under salt, drought, cold, and heat stresses, transgenic plants showed substantially alleviated membrane damage, as evidenced by weakened Evans blue staining. Consistently, their malondialdehyde contents were 1.48-, 1.47-, 1.57-, and 1.62-fold lower, while relative electrolyte leakage values were 1.56-, 1.35-, 1.53-, and 1.61-fold lower than those of wild-type plants, respectively. SmPIP1;3 orchestrates multi-stress tolerance by sustaining physiological homeostasis and limiting membrane damage. Its performance positions it as a valuable genetic asset for molecular breeding programs.

Bursaphelenchus xylophilus is a pine wood nematode capable of destroying pine forests. Exploring the genes providing resistance to this pathogen and understanding their resistance mechanisms is thus necessary and constitutes an effective way to tackle this problem. We used Pinus tabuliformis Carriere to dissect its response to B. xylophilus strain BxFC. The 30 d inoculation results showed that the P. tabuliformis germplasms exhibited a wide resistance spectrum. Some lines were sensitive with the needles fully wilted and the MDA content and the relative conductivity of needles greatly increased, while some lines demonstrated strong resistance with good needle vigor and better physiological conditions. Moreover, the transcriptome analysis revealed 7928 differentially expressed genes (DEGs) between the resistant and sensitive germplasm pools, including 3754 upregulated and 4174 downregulated genes in the resistant lines. These DEGs were specially enriched in the pathways of plant–pathogen interaction (318 genes), phenylpropanoid biosynthesis (108 genes), ubiquitin-mediated proteolysis (47 genes), carotenoid biosynthesis (18 genes), and monoterpenoid biosynthesis (9 genes). Accordingly, P. tabuliformis utilized multiple ways to control the proliferation and activity of B. xylophilus, such as immune response, ubiquitination, thickening plant cell walls, and increasing its terpenoid and antioxidant contents. Our results could thus help in better understanding the resistance process of P. tabuliformis against B. xylophilus and offer some new strategies and gene resources for a molecular breeding program of resistant P. tabuliformis.

The flavonoid 3′-hydroxylase gene (F3H), in relation to flavonoid biosynthesis, is widely involved in stress tolerance. To understand its contribution to chilling stress, we cloned a ZeF3H gene—1092 bp long and encoding 363 amino acids—from the chilling-tolerant line of Zelkova schneideriana. Under a cold treatment, ZeF3H’s expression level in the Zelkova genotypes was found to be significantly related to its morphological performance, with a correlation coefficient of −0.8735. The ZeF3H gene was introduced into tobacco plants. When subjected to 4 °C for 10 h, the ZeF3H-transgenic tobacco plants performed better and had relatively low electrical leakage and malondialdehyde contents—0.76-fold and 0.70-fold lower than the wild-type plant—and had a high proline content and soluble sugar content—1.40- and 1.20-fold higher than that of the WT plants, respectively. In conclusion, ZeF3H can significantly improve plants’ tolerance to chilling stress and can be a candidate gene for molecular breeding programs.

Chilling is a type of abiotic stress that limits plant distribution and production. To improve plant chilling resistance, the chilling response gene ZsDRT102 was cloned from Zelkova schneideriana Hand.-Mazz. This gene is 942 bp long and encodes a protein related to DNA damage repair (DDR). Expression analysis of ZsDRT102 showed a 3.6-fold higher level in the chilling-resistant lines compared to the chilling-sensitive lines. Tobacco plants with ZsDRT102 overexpression significantly improved the morphological performance under chilling stress compared with wild-type (WT) plants. Under chilling stress, physiological index assays showed that the malondialdehyde (MDA) content was 10.3–26.2

The expansin genes are commonly expressed in plant cells, and the encoded proteins influence plant growth and stress resistance by loosening the structure and increasing the flexibility of the cell wall. The objective of this study was to characterize expansin gene promoters in Populus trichocarpa to clarify the regulatory mechanisms underlying gene expression and evolution. Sequence alignments revealed that the similarity among 36 poplar expansin genes was greater for the coding sequences than for the promoter sequences, which suggested these promoter sequences evolved asynchronously. The bases flanking the start codon exhibited a usage bias, with sites +3, +4, and +5 biased toward GC, whereas the other sites were biased toward AT. The flanking sites were significantly correlated with gene expression, especially sites −10 and −17, in which C and G are the bases positively associated with gene expression. A total of 435 regulatory elements (61 types) were identified on the promoters of the poplar expansin genes; Skn-1 was the most common element in 23 promoters. Some expansin genes had more regulatory elements on their promoters (e.g., PtrEXPA4, PtrEXPA3, PtrEXPB3, and PtrEXPB1), whereas some others had less (e.g., PtrEXLA2, PtrEXLB1, and PtrEXPA23). Furthermore, 26 types of elements were involved in expansin gene expression, 25 of which positively affected expression in all analyzed samples. The exception was the endosperm expression-related element Skn-1, which negatively regulated expression in four tissues or treatments. Expression analysis showed that the expansin genes in Populus trichocarpa performed much differently under regular and abiotic stress conditions, which well matched the diversity of their promoter sequences. The results show that expansin genes play an important role in plant growth and development and stress resistance through expression adjustment.

Expansins, cell wall proteins, play a significant role in plant stress resistance. Our previous study confirmed the expression of the expansin gene SmEXPA13 from Salix matsudana Koidz. enhanced salt tolerance of plants. This report presented an assay that the expression of SmEXPA13 was higher in the salt-resistant willow variety 9901 than in the salt -sensitive variety Yanjiang. In order to understand the possible reasons, a study of the regulation process was conducted. Despite being cloned from both varieties, SmEXPA13 and its promotor showed no significant differences in the structure and sequence. A transcription factor (TF), SmMYB1R1-L, identified through screening the yeast library of willow cDNA, was found to regulate SmEXPA13 . Yeast one -hybrid (Y1H) assay confirmed that SmMYB1R1-L could bind to the MYB element at the -520 bp site on the SmEXPA13 promotor. A dual-luciferase reporter assay also demonstrated that SmMYB1R1-L could greatly activate SmEXPA13 expression. The willow calli with over -expression of SmMYB1R1-L exhibited better physiological performance than the wild type under salt stress. Further testing the expression of SmMYB1R1-L displayed it significantly higher in 9901 willow than that in Yanjiang under salt stress. In conclusion, the high accumulation of SmMYB1R1-L in 9901 willow under salt stress led to the high expression of SmEXPA13 , resulting in variations in salt stress resistance among willow varieties. The SmMYB1R1-L/ SmEXPA13 cascade module in willow offers a new perspective on plant resistance mechanisms.

Transgenic technology is a potent tool for verifying gene functions, and poplar serves as a model system for genetically transforming perennial woody plants. However, the current poplar genetic transformation system is limited to a few genotypes. In this study, we developed an efficient transformation system based on the Agrobacterium-mediated transformation of Populus wulianensis, a rare and endangered tree species endemic to Shandong Province. Aseptic seedlings of P. wulianensis were used as experimental materials, and the optimal medium for inducing adventitious buds was explored as 1/2(NH4NO3) MS + 0.05 mg/L naphthalene acetic acid (NAA) + 0.5 mg/L 6-benzylaminopurine (6-BA), resulting in up to 35 adventitious buds. The selection resistance critical pressure of 300 mg/L for timentin can effectively inhibit the growth of Agrobacterium while promoting the induction of adventitious buds in leaves. The critical screening pressure for kanamycin for producing resistant adventitious buds and inducing resistant rooting seedlings was 100 mg/L. We optimized several independent factors, which significantly enhanced the efficiency of genetic transformation. The leaves were infected with Agrobacterium suspension diluted twice by adding 100 μmol/L acetylsyringone (β-AS) (OD600 = 0.6) for 15 min, followed by co-culture in the dark for 3 d. Using this improved transformation system, we obtained transgenic P. wulianensis clones overexpressing the enhanced green fluorescent protein (EGFP) gene through direct organogenesis. Among the 112 resistant buds obtained, 17 developed resistant rooting in seedlings. Eight positive plants were identified through DNA, RNA, and protein level analyses, with a positivity rate of 47.06%. This study provides a foundation for developing and utilizing P. wulianensis germplasm resources and lays the groundwork for resource improvement.

Zelkova schneideriana is a landscaping flowering and deciduous plant whose leaf color changes in the fall season. In the discoloration period, the anthocyanin content in the red lines of Z. schneideriana is 3.52 times greater than that in the green lines, whereas the chlorophyll content in the red lines is 20.10 times lower. To understand the molecular mechanism of the leaf discoloration, transcriptome sequencing was performed. A total of 3965 differentially expressed genes (DEGs) were identified between the red and green lines in the discoloration period. These DEGs were mainly involved in plant–pathogen interactions, the MAPK signaling pathway, plant hormone signal transduction, flavonoid biosynthesis, and anthocyanin biosynthesis. Among them, three downregulated genes were involved in chlorophyll synthesis; these genes exhibited a maximum decrease in the red lines that was 11.13 times greater than their expression in the green lines. In contrast, 33 upregulated genes were involved in anthocyanin biosynthesis; they exhibited a maximum increase in the red lines 4645.33 times greater than their expression in the green lines. The results of an association analysis revealed that four transcription factors, including ZeBHLH42, ZeMYB123, ZeMYB113, and ZeWRKY44, positively regulated the anthocyanin synthesis genes. These results explained the mechanism underlying the discoloration of Zelkova leaves and provided a basis for molecular breeding programs of colorful plants.

The cytokinin signaling pathway is important for plant growth and development. To understand the regulatory process, a type A response regulator, SmRR5, in Salix matsudana Koidz., was characterized and functionally analyzed. Gene expression tests showed that SmRR5 was distinctly higher in the leaves and roots of the fast-growing S. matsudana variety 9901 than in those of the slow-growing variety Yanjing (YJ). The transcript abundance was highest in the meristem zone (MEZ), followed by the elongation zone (EZ) and maturation zone (MAZ) in 9901 roots, but it was identically low in YJ roots. Overexpression of SmRR5 in tobacco plants significantly improved plant height, maximum root length (MRL), lateral root number (LRN), fresh weight (FW), dry weight (DW), and flowering time compared with wild-type plants. Transcript profiling revealed that multiple genes associated with flowering (SWEET1, FPF1, and COL12), plant growth (YUCCA8, PIN5, and ARF9a), and adventitious root (AR) formation (Hox3, MYC2, and AGL46) were highly expressed in the overexpression of leaves and roots. Thus, SmRR5 effectively facilitated plant growth and development.

WRKY transcription factors play key roles in plant responses to abiotic stress. In this study, we cloned and characterized the drought-induced WRKY gene SmWRKY12 from Salix matsudana Koidz. Following drought treatment, SmWRKY12 was significantly upregulated in the roots of the drought-tolerant willow variety 9901. Overexpressing SmWRKY12 in willow calli significantly increased drought tolerance. The results of yeast onehybrid and dual-luciferase reporter assays showed that SmWRKY12 can bind to the promoter of the expansin gene SmEXPA13 and activate its expression. The results of yeast two-hybrid and split luciferase complementation assays showed that SmWRKY12 can interact with SmRAP2-7. The results of dual-luciferase and transgenic experiments showed that the combination of SmWRKY12 and SmRAP2-7 significantly increased the transcriptional regulation of SmWRKY12 on SmEXPA13. SmEXPA13 was introduced into willow calli and tobacco plants. Overexpressing SmEXPA13 significantly improved their performance under drought conditions. The results revealed a novel mechanism to tolerate drought stress through the SmWRKY12-SmRAP2-7-SmEXPA13 module in willow. This study also provided a new strategy for the molecular design and breeding of drought-tolerant plants.

Alternative splicing (AS) is an important post-transcriptional regulatory model that can change the normal transcript expression level and possibly result in protein diversity. In this study, we conducted the full-length transcript sequencing of Salix matsudana Koidz 9901 leaves under salt treatment using the PromethION platform. A total of 4786 AS genes (9307 AS events) were determined, accounting for 7.45% of all the transcribed genes. Of them, intron retention (IR) events accounted for the most AS events (46.05%), followed by alternative 3′ splice sites (A3SS). During salt stress, the percentage of IR events decreased, and the percentage of the others increased. Statistical results showed that 5′GG was the most common motif at the 5′ end of the intron in the AS events, and GG3′ was the most common motif at the 3′ end. 5′GG-AG3′ was the most common splice mode in the AS events. The occurrence of AS events was significantly related to the exon number, exon length, intron length, GC content, and expression abundance of the genes. During salt stress, the number of AS genes gradually increased, and they mainly participated in purine and chlorophyll metabolism, RNA transport, and autophagy. Meanwhile, the AS sites of the gene increased during salt treatment, indicating the complexity of the AS events by salt stress. A comparison of differentially expressed genes (DEGs) and differentially alternative splicing (DAS) genes during salt stress revealed that they had a different mechanism of gene expression regulation when subjected to salt stress. These results expand our knowledge of AS events and shed light on and improve our understanding of plant resistance to salt tolerance in willow.

Populus wulianensis mainly grows in hilly and sloped areas and has strong resistance to adversity. Previous transcriptome studies have shown that a WRKY gene PwuWRKY48 is expression-induced under drought stress. In this study, we aim to characterize the gene’s structure and investigate its role in plant drought resistance. The results show that PwuWRKY48 (1113 bp) belongs to a class IIc WRKY subfamily and it was determined as a nuclear localization protein. The gene promoter region contains a variety of cis-elements in relation to stress resistance. Under drought stress, PwuWRKY48 was expression-induced in leaves and stems, 29.7 and 16.6 times those before treatment, respectively. Overexpressing PwuWRKY48 lines were associated with increased activities of peroxidase (POD) and superoxide dismutase (SOD), 2.5 and 1.6 times higher than those of the wild type. While malondialdehyde content (MDA), superoxide anion radical (O2·−), and relative conductivity were decreased by 20%, 30%, and 21.3%, proline and chlorophyll contents increased by 37.5% and 11.2%, respectively. This indicates that PwuWRKY48 efficiently improved the drought tolerance of transgenic plants. PwuWRKY48 can be used as a gene resource for molecular breeding of plant drought resistance.

Expansin plays a crucial role in plant growth and stress resistance as a cell wall relaxation protein. The expansin family consists of four subfamilies: EXPA, EXPB, EXLA, and EXLB. However, a few reports have been previously published investigating EXLA genes. The research here aimed to characterize the PtEXLA1 gene from a popular species ( P. alba × P. glandulosa CV.84K) and evaluate its role through genetic transformation to understand its contribution to plant growth and stress resistance. The results showed that the PtEXLA1 gene was 780 bp in length, encoded 259 amino acids, and had typical characteristics of EXLA. The PtEXLA1 transgenic tobacco plants had a larger corolla in comparison to wild-type plants, and exhibited higher resistance to drought, high temperature, and salt stress based on the evaluation of chlorophyll content, relative conductivity, and malondialdehyde content. PtEXLA1 can be an efficient gene resource for stress resistance breeding of plants.

Expansin is a vital plant cell wall protein that loosens cell wall components and increases cell flexibility. This study aimed to evaluate its association with salt stress defense. The results showed that an expansin gene SmEXPA13 was express induced in salt conditions; the expression was 9.8