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.

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.

Low temperature (LT) is an important abiotic factor affecting plant survival, growth and distribution. The response of Cryptomeria fortunei (Chinese cedar) to LT is not well known, limiting its application in production and ornamental value. In this study, we first screened ten clones (#3, #25, #32, #42, #54, #57, #68, #66, #74, #X1), originating from five different locations in China, for their degrees of cold resistance. We then selected the two showing the highest (#32) and lowest (#42) cold resistance to see the physiological and morphological response of different cold-resistant C. fortunei clones to LT. We found that the electrolyte leakage of all ten clones increased strongly between 0 and −8 °C, while below −8 or between 4 and 0 °C did not yield additional increases. Under cold stress, clones #32 and #42 showed different degrees of needle browning. From 25 to −20 °C, maximum and effective quantum yields of photosystem II (F_v/F_m and YII) and photochemical and non-photochemical quenching (qP and NPQ) decreased continuously in two clones with decreasing temperature, where #42 was more strongly affected compared with #32. The chlorophyll content first decreased significantly to the lowest from 25 to −12 °C, then increased significantly at −16/−20 °C compared with −12 °C. We observed changes in needle cellular ultrastructure at −8 °C, with chloroplasts of #32 swelling, while those of #42 were destroyed. Correlation analysis indicated that needle browning and chlorophyll fluorescence were closely related to temperature, and cellular ultrastructure changed notably around semi-lethal temperature (LT₅₀), which can be used as physiological indicators for the identification of cold resistance. We found a clear difference in cold tolerance between clones of #32 and #42, with #32 being more tolerant, which can be exploited in breeding programs. We conclude that strongly cold-resistant clones have more stable physiological states and a wider adaptability to LT compared with weak ones.

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.

Cryptomeria fortunei Hooibrenk (Chinese cedar) is a coniferous tree from southern China that has an important function in landscaping and timber production. Lignin is one of the key components of secondary cell walls, which have a crucial role in conducting water and providing mechanical support for the upward growth of plants. It is mainly biosynthesized via the phenylpropanoid metabolic pathway, of which the molecular mechanism remains so far unresolved in C. fortunei. In order to obtain further insight into this pathway, we performed transcriptome sequencing of the C. fortunei cambial zone at 5 successive growth stages. We generated 78,673 unigenes from transcriptome data, of which 45,214 (57.47%) were successfully annotated in the non-redundant protein database (NR). A total of 8975 unigenes were identified to be significantly differentially expressed between Sample_B and Sample_A after analyzing their expression profiles. Of the differentially expressed genes (DEGs), 6817 (75.96%) and 2158 (24.04%) were up- and down-regulated, respectively. 83 DEGs were involved in phenylpropanoid metabolism, 37 DEGs that encoded v-Myb avian myeloblastosis viral oncogene homolog (MYB) transcription factor (TF), and many candidates that encoded lignin synthesizing enzymes. These findings contribute to understanding the expression pattern of C. fortunei cambial zone transcriptome. Furthermore, our results provide additional insight towards understanding the molecular mechanisms of wood formation in C. fortunei.