Cryptomeria fortunei has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in C. fortunei have not been reported. Here, we tested the expression stability for 12 RGs in C. fortunei under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated CYP, actin, UBC, and 18S as good choices for studying C. fortunei gene expression. qRT-PCR analysis of the expression patterns of three target genes (CAT and MAPK1/6) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for C. fortunei gene expression standardization and quantification.

Cryptomeria fortunei (Chinese cedar) has outstanding medicinal value due to its abundant flavonoid and terpenoid contents. The metabolite contents of C. fortunei needles differ across different seasons. However, the biosynthetic mechanism of these differentially synthesized metabolites (DSMs) is poorly understood. To improve our understanding of this process, we performed integrated non-targeted metabolomic liquid chromatography and gas chromatography mass spectrometry (LC-MS and GC-MS), and transcriptomic analyses of summer and winter needles. In winter, the C. fortunei needle ultrastructure was damaged, and the chlorophyll content and F_v/F_m were significantly (p < 0.05) reduced. Based on GC-MS and LC-MS, we obtained 106 and 413 DSMs, respectively; based on transcriptome analysis, we obtained a total of 41.17 Gb of clean data and assembled 33,063 unigenes, including 14,057 differentially expressed unigenes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these DSMs/DEGs were significantly (p < 0.05) enriched in many biosynthesis pathways, such as terpenoids, photosynthates, and flavonoids. Integrated transcriptomic and metabonomic analyses showed that seasonal changes have the greatest impact on photosynthesis pathways, followed by terpenoid and flavonoid biosynthesis pathways. In summer Chinese cedar (SCC) needles, DXS, DXR, and ispH in the 2-methyl-pentaerythritol 4-phosphate (MEP) pathway and GGPS were highly expressed and promoted the accumulation of terpenoids, especially diterpenoids. In winter Chinese cedar (WCC) needles, 9 genes (HCT, CHS, CHI, F3H, F3'H, F3'5'H, FLS, DFR, and LAR) involved in flavonoid biosynthesis were highly expressed and promoted flavonoid accumulation. This study broadens our understanding of the metabolic and transcriptomic changes in C. fortunei needles caused by seasonal changes and provides a reference regarding the adaptive mechanisms of C. fortunei and the extraction of its metabolites.

Cryptomeria fortunei growth and development are usually affected by low temperatures. Despite the evergreen nature of this species, most needles turn yellowish-brown in cold winters. The underlying discoloration mechanisms that cause this phenomenon in response to cold acclimation remain poorly understood. Here, we measured the pigment content and ultrastructure of normal wild-type (Wt) and evergreen mutant (GM) C. fortunei needles and performed integrated transcriptomic and metabolomic analyses to explore potential discoloration mechanisms. The results showed that the needle chlorophyll content of these two genotypes decreased in winter. Wt needles showed greater decrease in the chlorophyll content and local destruction of chloroplast ultrastructure and contained larger amounts of flavonoids than GM needles, as shown by metabolomics analysis. We subsequently identified key differentially expressed genes in the flavonoid biosynthesis pathway and observed significantly upregulated flavonol synthase expression in Wt needles compared with GM needles that significantly increased the anthoxanthin (flavones and flavonols) content, which is likely a key factor underlying the difference in needle color between these two genotypes. Therefore, flavonoid metabolism may play important roles in the cold resistance and needle discoloration of C. fortunei, and our results provide an excellent foundation for the molecular mechanism of C. fortunei in response to cold stress.

Pinus massoniana and P. hwangshanensis are two conifer species located in southern China, which are of both economic and ornamental value. Around the middle and lower reaches of the Yangtze River, P. massoniana occurs mainly at altitudes below 700 m, while P. hwangshanensis can be found above 900 m. At altitudes where the distribution of both pines overlaps, a natural introgression hybrid exists, which we will further refer to as the Z pine. This pine has a morphological character that shares attributes of both P. massoniana and P. hwangshanensis. However, compared to the other two pines, its reproductive structure, the pinecone, has an ultra-low ripening rate with seeds that germinate poorly. In this study, we aimed to find the reason for the impaired cone maturation by comparing transcriptome libraries of P. massoniana and Z pine cones at seven successive growth stages. After sequencing and assembly, we obtained unigenes and then annotated them against NCBI’s non-redundant nucleotide and protein sequences, Swiss-Prot, Clusters of Orthologous Groups, Gene Ontology and KEGG Orthology databases. Gene expression levels were estimated and differentially expressed genes (DEGs) of the two pines were mined and analyzed. We found that several of them indeed relate to reproductive process. At every growth stage, these genes are expressed at a higher level in P. massoniana than in the Z pine. These data provide insight into understanding which molecular mechanisms are altered between P. massoniana and the Z pine that might cause changes in the reproductive process.

Shoot branching substantially impacts vegetative and reproductive growth as well as wood characteristics in perennial woody species by shaping the shoot system architecture. Although plant hormones have been shown to play a fundamental role in shoot branching in annual species, their corresponding actions in perennial woody plants are largely unknown, in part due to the lack of branching mutants. Here, we demonstrated the role of plant hormones in bud dormancy transition toward activation and outgrowth in woody plants by comparing the physiological and molecular changes in the apical shoot stems of ‘Yangkou’ 020 fir and ‘Dugan’ fir, two Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) clones with normal and completely abolished branching phenotypes, respectively. Our studies showed that the defect in bud outgrowth was the cause of failed shoot branching in ‘Dugan’ fir whereas apically derived signals acted as triggers of this ectopic bud activity. Further studies indicated that auxin played a key role in inhibiting bud outgrowth in ‘Dugan’ fir. During bud dormancy release, the differential auxin resistant 1/Like AUX1 (AUX1/LAX) and PIN-formed (PIN) activity resulted in an ectopic auxin/indole-3-acetic acid (IAA) accumulation in the apical shoot stem of ‘Dugan’ fir, which could inhibit the cell cycle in the axillary meristem by decreasing cytokinin (CK) biosynthesis but increasing abscisic acid (ABA) production and response through the signaling pathway. In contrast, during bud activation and outgrowth, the striking increase in auxin biosynthesis and PIN activity in the shoot tip of ‘Dugan’ fir may trigger the correlative inhibition of axillary buds by modulating the polar auxin transport stream (PATS) and connective auxin transport (CAT) in shoots, and by influencing the biosynthesis of secondary messengers, including CK, gibberellin (GA) and ABA, thereby inducing the paradormancy of axillary buds in ‘Dugan’ fir by apical dominance under favorable conditions. The findings of this study provide important insights into the roles of plant hormones in bud outgrowth control in perennial woody plants.

Cryptomeria fortunei, also known as the Chinese cedar, is an important timber species in southern China. The primary component of its woody tissues is lignin, mainly present in secondary cell walls. Therefore, continuous lignin synthesis is crucial for wood formation. In this study, we aimed to discover key genes involved in lignin synthesis expressed in the vascular cambium of C. fortunei. Through transcriptome sequencing, we detected expression of two genes, 4CL and CCoAOMT, known to be homologous to enzymes involved in the lignin synthesis pathway. We studied the function of these genes through bioinformatics analysis, cloning, vascular cambium expression analysis, and transgenic cross-species functional validation studies. Our results show that Cf4CL and CfCCoAOMT do indeed function in the pathway of lignin synthesis and likely perform this function in C. fortunei. They are prime candidates for future (gene-editing) studies aimed at optimizing C. fortunei wood production.