Manchurian walnut (Juglans mandshurica Maxim.) is a tree with multiple industrial uses and medicinal properties in the Juglandaceae family (walnuts and hickories). J. mandshurica produces juglone, which is a toxic allelopathic agent and has potential utilization value. Furthermore, the seed of J. mandshurica is rich in various unsaturated fatty acids and has high nutritive value. Here, we present a high-quality chromosome-scale reference genome assembly and annotation for J. mandshurica (n = 16) with a contig N50 of 21.4 Mb by combining PacBio high-fidelity reads with high-throughput chromosome conformation capture data. The assembled genome has an estimated sequence size of 548.7 Mb and consists of 657 contigs, 623 scaffolds, and 40,453 protein-coding genes. In total, 60.99% of the assembled genome consists of repetitive sequences. Sixteen super-scaffolds corresponding to the 16 chromosomes were assembled, with a scaffold N50 length of 33.7 Mb and a BUSCO complete gene percentage of 98.3%. J. mandshurica displays a close sequence relationship with Juglans cathayensis, with a divergence time of 13.8 million years ago. Combining the high-quality genome, transcriptome, and metabolomics data, we constructed a gene-to-metabolite network and identified 566 core and conserved differentially expressed genes, which may be involved in juglone biosynthesis. Five CYP450 genes were found that may contribute to juglone accumulation. NAC, bZip, NF-YA, and NF-YC are positively correlated with the juglone content. Some candidate regulators (e.g., FUS3, ABI3, LEC2, and WRI1 transcription factors) involved in the regulation of lipid biosynthesis were also identified. Our genomic data provide new insights into the evolution of the walnut genome and create a new platform for accelerating molecular breeding and improving the comprehensive utilization of these economically important tree species.

The C-Repeat Binding Factor (CBF) gene family has been identified and characterized in multiple plant species, and it plays a crucial role in responding to low temperatures. Presently, only a few studies on tree species demonstrate the mechanisms and potential functions of CBFs associated with cold resistance, while our study is a novel report on the multi-aspect differences of CBFs among three tree species, compared to previous studies. In this study, genome-wide identification and analysis of the CBF gene family in Acer truncatum, Acer pseudosieboldianum, and Acer yangbiense were performed. The results revealed that 16 CBF genes (five ApseCBFs, four AcyanCBFs, and seven AtruCBFs) were unevenly distributed across the chromosomes, and most CBF genes were mapped on chromosome 2 (Chr2) and chromosome 11 (Chr11). The analysis of phylogenetic relationships, gene structure, and conserved motif showed that 16 CBF genes could be clustered into three subgroups; they all contained Motif 1 and Motif 5, and most of them only spanned one exon. The cis-acting elements analysis showed that some CBF genes might be involved in hormone and abiotic stress responsiveness. In addition, CBF genes exhibited tissue expression specificity. High expressions of ApseCBF1, ApseCBF3, AtruCBF1, AtruCBF4, AtruCBF6, AtruCBF7, and ApseCBF3, ApseCBF4, ApseCBF5 were detected on exposure to low temperature for 3 h and 24 h. Low expressions of AtruCBF2, AtruCBF6, AtruCBF7 were detected under cold stress for 24 h, and AtruCBF3 and AtruCBF5 were always down-regulated under cold conditions. Taken together, comprehensive analysis will enhance our understanding of the potential functions of the CBF genes on cold resistance, thereby providing a reference for the introduction of Acer species in our country.

The NAC (NAM, ATAF and CUC) gene family plays a crucial role in the transcriptional regulation of various biological processes and has been identified and characterized in multiple plant species. However, genome-wide identification of this gene family has not been implemented in Juglans mandshurica, and specific functions of these genes in the development of fruits remain unknown. In this study, we performed genome-wide identification and functional analysis of the NAC gene family during fruit development and identified a total of 114 JmNAC genes in the J. mandshurica genome. Chromosomal location analysis revealed that JmNAC genes were unevenly distributed in 16 chromosomes; the highest numbers were found in chromosomes 2 and 4. Furthermore, according to the homologues of JmNAC genes in Arabidopsis thaliana, a phylogenetic tree was constructed, and the results demonstrated 114 JmNAC genes, which were divided into eight subgroups. Four JmNAC gene pairs were identified as the result of tandem duplicates. Tissue-specific analysis of JmNAC genes during different developmental stages revealed that 39 and 25 JmNAC genes exhibited upregulation during the mature stage in walnut exocarp and embryos, indicating that they may serve key functions in fruit development. Furthermore, 12 upregulated JmNAC genes were common in fruit ripening stage in walnut exocarp and embryos, which demonstrated that these genes were positively correlated with fruit development in J. mandshurica. This study provides new insights into the regulatory functions of JmNAC genes during fruit development in J. mandshurica, thereby improving the understanding of characteristics and evolution of the JmNAC gene family.

Light is one of the most important environmental cues that affects plant development and regulates its behavior. Light stress directly inhibits physiological responses and plant tissue development and even induces mortality in plants. Korean pine (Pinus koraiensis) is an evergreen conifer species widely planted in northeast China that has important economic and ecological value. However, the effects of light stress on the growth and development of Korean pine are still unclear. In this study, the effects of different shading conditions on physiological indices, molecular mechanisms and metabolites of Korean pine were explored. The results showed that auxin, gibberellin and abscisic acid were significantly increased under all shading conditions compared with the control. The contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid also increased as the shading degree increased. Moreover, a total of 8556, 3751 and 6990 differentially expressed genes (DEGs) were found between the control and HS (heavy shade), control and LS (light shade), LS vs. HS, respectively. Notably, most DEGs were assigned to pathways of phytohormone signaling, photosynthesis, carotenoid and flavonoid biosynthesis under light stress. The transcription factors MYB-related, AP2-ERF and bHLH specifically increased expression during light stress. A total of 911 metabolites were identified, and 243 differentially accumulated metabolites (DAMs) were detected, among which flavonoid biosynthesis (naringenin chalcone, dihydrokaempferol and kaempferol) metabolites were significantly different under light stress. These results will provide a theoretical basis for the response of P. koraiensis to different light stresses.

Flowers are imperative reproductive organs and play a key role in the propagation of offspring, along with the generation of several metabolic products in flowering plants. In Juglans mandshurica, the number and development of flowers directly affect the fruit yield and subsequently its commercial value. However, owing to the lack of genetic information, there are few studies on the reproductive biology of Juglans mandshurica, and the molecular regulatory mechanisms underlying the development of female and male inflorescence remain unclear. In this study, phytohormones and transcriptomic sequencing analyses at the three stages of female and male inflorescence growth were performed to understand the regulatory functions underlying flower development. Gibberellin is the most dominant phytohormone that regulates flower development. In total, 14,579 and 7188 differentially expressed genes were identified after analyzing the development of male and female flowers, respectively, wherein, 3241 were commonly expressed. Enrichment analysis for significantly enriched pathways suggested the roles of MAPK signaling, phytohormone signal transduction, and sugar metabolism. Genes involved in floral organ transition and flowering were obtained and analyzed; these mainly belonged to the M-type MADS-box gene family. Three flowering-related genes (SOC1/AGL20, ANT, and SVP) strongly interacted with transcription factors in the co-expression network. Two key CO genes (CO3 and CO1) were identified in the photoperiod pathway. We also identified two GA20xs genes, one SVP gene, and five AGL genes (AGL8, AGL9, AGL15, AGL19, and AGL42) that contributed to flower development. The findings are expected to provide a genetic basis for the studies on the regulatory networks and reproductive biology in inflorescence development for J. mandshurica.