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 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.

MicroRNA (miRNA) expression analysis is very important for investigating its functions. To date, no research on reference genes (RGs) for miRNAs in gymnosperms, including Cryptomeria fortunei, has been reported. Here, ten miRNAs (i.e., pab-miR159a, cln-miR162, cas-miR166d, pab-miR395b, ppt-miR894, cln-miR6725, novel1, novel6, novel14 and novel16) and three common RGs (U6, 5S and 18S) were selected as candidate RGs. qRT-PCR was used to analyse their expressions in C. fortunei under various experimental conditions, including multiple stresses (cold, heat, drought, salt, abscisic acid and gibberellin) and in various tissues (roots, stems, tender needles, cones and seeds). Four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) were employed to assess the stability of candidate RG expression; the geometric mean and RefFinder program were used to comprehensively evaluate RG stability. According to the results, novel16, cln-miR6725, novel1 and U6 were the most stable RGs for studying C. fortunei miRNA expression. In addition, the expression of three target miRNAs (aly-miR164c-5p, aly-miR168a-5p and smo-miR396) was examined to verify that the selected RGs are suitable for miRNA expression normalisation. This study may aid further investigations of miRNA expression/function in the response of C. fortunei to abiotic stress and provides an important basis for the standardisation of miRNA expression in other gymnosperm species.

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.