WRKY is an important complex family of transcription factors involved in plant immune responses. Among them, WRKY70 plays an important role in the process of the plant defense response to the invasion of pathogens. However, the defense mechanism of PsnWRKY70 is not clear in Populus nigra. In this study, we showed that PsnWRKY70-overexpression lines (OE) had fewer leaf blight symptoms than PsnWRKY70-repressing lines (RE). PsnWRKY70 activated MAP kinase cascade genes (PsnM2K4, PsnMPK3, PsnM3K18), calcium channel proteins-related genes (PsnCNG3, PsnCNGC1, PsnCNG4), and calcium-dependent protein kinases genes (PsnCDPKL, PsnCDPKW, PsnCDPKS, PsnCDPKQ). Furthermore, 129 genes of PsnWRKY70 putative genome-wide direct targets (DTGs) were identified by using transcriptome (RNA-seq) and DNA affinity purification sequencing (DAP-seq). PsnWRKY70 directly binds to the promoters of homologous genes and LRR domain proteins to promote the expression of WRKY6, WRKY18, WRKY22, and WRKY22–1, LRR domain proteins LRR8, LRR-RLK, ADR1-like 2, NB-ARC, etc. Our study suggests that PsnWRKY70 enhances the resistance of A. alternata in poplar by activating genes in both pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).

Poplar is an important afforestation and ornamental tree species in Northeast China. The distribution area of saline-alkali land is approximately 765 hm² in Northeast China. The breeding of saline-alkali-resistant transgenic trees could be an effective method of afforestation in saline-alkali land. WRKY transcription factors play a crucial role in abiotic stress. In this study, we analyzed the genetic stability of the two-year-old PsnWRKY70 transgenic poplars. The results showed that PsnWRKY70 of transgenic poplars had been expressed stably and normally at the mRNA level. The gene interference expression (RE) lines had no significant effect on the growth of PsnWRKY70 under NaHCO₃ stress, and the alkali damage index of RE lines was significantly lower than that of WT and overexpression (OE) lines at day 15 under NaHCO₃ stress. POD activity was significantly higher in RE lines than in WT. The MDA content of the RE line was lower than that of the WT line. Transcriptome analysis showed that RE lines up-regulated genes enriched in cell wall organization or biogenesis pathway-related genes such as EXPA8, EXPA4, EXPA3, EXPA1, EXPB3, EXP10, PME53, PME34, PME36, XTH9, XTH6, XTH23, CESA1, CESA3, CES9; FLA11, FLA16 and FLA7 genes. These genes play an important role in NaHCO₃ stress. Our study showed that the interference expression of the PsnWRKY70 gene can enhance the tolerance of NaHCO₃ in poplar.

The CBL-interacting protein kinases’ (CIPKs) gene family plays an important role in plants under salt stress. In this study, a total of 31 PtrCIPK genes were identified in poplar. CIPKs’ gene family was divided into two categories, few intron classes and multi-intron classes. They all have the core components of the kinase domain and regulatory domain unique to the CIPK gene family and share most of the same motifs. PtrCIPKs have 17 fragment repeat events and have high homology with Arabidopsis thaliana and Betula platyphylla, and partial homology with Zea mays. Prediction of cis-acting elements found that the PtrCIPK gene family has the most elements in terms of stress. Under NaCl stress, all members of poplar CIPKs’ gene family were significantly expressed. There were fourteen up-regulated genes and four down-regulated genes. Candidate gene expression was significantly higher in the phloem than in other tissues. In this study, characterization of CBL-interacting protein kinases’ gene family and expression pattern reveal their important roles in response to salt stress in poplar.

The new variety Betula pendula ‘Dalecarlica’ selected from Betula pendula, shows high ornamental values owing to its lobed leaf shape. In this study, to identify the genetic components of leaf shape formation, we performed bulked-segregant analysis (BSA) and molecular marker-based fine mapping to identify causal gene responsible for lobed leaves in B. pendula ‘Dalecarlica’. The most significant variations associated with leaf shape were identified within the gene BpPIN1 encoding a member of PIN-FORMED family, responsible for the auxin efflux carrier. We further confirmed the hypomethylation at the promoter region promoting the expression level of BpPIN1, which cause stronger and longer veins and lobed leaf shape in B. pendula ‘Dalecarlica’. These results indicated that DNA methylation at the BpPIN1 promoter region is associated with leaf shapes in Betula pendula. Our findings revealed an epigenetic mechanism of BpPIN1 in the regulation of leaf shape in birch, which could help in molecular breeding of ornamental traits.

Hybrid larch is an excellent afforestation species in northern China. The instability of seed yield is an urgent problem to be solved. The biological characteristics related to seed setting in larch are different from those in angiosperms and other gymnosperms. Studying the developmental mechanism of the larch sporophyll can deepen our understanding of conifer reproductive development and help to ensure an adequate supply of seeds in the seed orchard. The results showed that the formation of microstrobilus primordia in hybrid larch could be observed in anatomical sections collected in the middle of July. The contents of endogenous gibberellin 3 (GA3) and abscisic acid (ABA) were higher and the contents of GA4, GA7, jasmonic acid and salicylic acid were lower in multiseeded larch. Transcriptome analysis showed that transcription factors were significantly enriched in the AP2 family. There were 23 differentially expressed genes in the buds of the multiseeded and less-seeded types, and the expression of most of these genes was higher in the buds than in the needles. We conclude that mid-July is the early stage of reproductive organ development in hybrid larch and is suitable for the study of reproductive development. GA3 and ABA may be helpful for improving seed setting in larch, and 23 AP2/EREBP family genes are involved in the regulation of reproductive development in larch.

Betula pendula ‘Purple Rain’ is characterized by its purple leaves and has ornamental applications. A green mutant line NL, which was mutated by line NZ of B. pendula ‘Purple Rain’ during tissue culture, shows green leaves instead of the typical purple color of B. pendula ‘Purple Rain’. This study quantified the leaf color traits of NL and a normal B. pendula ‘Purple Rain’ line NZ, and uncovered differentially expressed genes involved in flavonoid biosynthesis pathway genes in NL through RNA-Seq analysis. Compared to NZ, reduced levels of six anthocyanins contained in NL were revealed via flavonoids-targeted metabolomics. Sequence mutations in transcription factors that could explain NL’s phenotype failed to be screened via whole-genome resequencing, suggesting an epigenetic basis for this variant. Therefore, a key gene, BpMYB113, was identified in NL via the combined analysis of small RNA sequencing, whole-genome methylation sequencing, and transcriptomics. In NL, this gene features a hyper CHH context methylation site and a lower transcription level compared to NZ, disrupting the expression of downstream genes in the phenylalanine metabolism pathway, and thereby reducing flavonoid biosynthesis. Our study elucidates an epigenetic mechanism underlying color variation in variegated trees, providing pivotal insights for the breeding and propagation of colored-leaf tree species.