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.

Wood is an important raw material for global industries with rapidly increasing demand. To isolate the genes differentially expressed during xylogenesis of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.), we used a novel system. Forward and reverse subtracted cDNA libraries were constructed using the suppression subtractive hybridization method; for the forward library we used cDNA from the mutant Dugansha as the tester and cDNA from the wild-type clone Jurong 0 as the driver, and for the reverse library we used Jurong 0 cDNA as the tester and Dugansha cDNA as the driver. Transcriptional profiling was performed using a macroarray with 4 digoxigenin-labeled probes. We obtained 618 and 409 clones from the forward and the reverse subtracted library, respectively. A total of 405 unique expressed sequence tags (ESTs) were obtained. Forty percent of the ESTs exhibited homologies with proteins of known function and fell into 4 major classes: metabolism, cell wall biogenesis and remodeling, signal transduction, and stress. Real-time PCR was performed to confirm the results. The expression levels of 11 selected ESTs were consistent with both macroarray and real-time PCR results. The systematic analysis of genes involved in wood formation in Chinese fir provides valuable insights into the molecular mechanisms involved in xylem differentiation and is an important resource for forest research that can be directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

QRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate.