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.
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 (Fv/Fm 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.
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 (FLS) expression in Wt needles compared to 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.
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.
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