Evaluation of reference genes for normalizing RT-qPCR in leaves and suspension cells of Cephalotaxus hainanensis under various stimuli.

BACKGROUND: Reverse transcription quantitative real-time PCR (RT-qPCR) is a widely used approach for investigating gene expression levels in plants because of its high reproducibility, sensitivity, accuracy and rapidness. Evaluation of reference genes for normalizing RT-qPCR data is a necessary step, especially in new plant varieties. Cephalotaxus hainanensis is a precious medicinal plant belonging to the family of Cephalotaxaceae and no RT-qPCR studies have been reported on it. RESULTS: In this study, 9 candidate reference genes were selected from the transcriptome data of C. hainanensis; 3 statistical algorithms (geNorm, NormFinder, BestKeeper) were applied to evaluate their expression stabilities through 180 samples under 6 stimuli treatments in leaves and leaf-derived suspension cultured cells; a comprehensive stabilities ranking was also performed by RefFinder. The results showed that suitable reference genes in C. hainanensis should be selected for normalization relative to different experimental sets. 18S showed a higher stability than other candidate reference genes which ranked at the top two suitable genes under all experimental setups in this study. CONCLUSION: This study is the first to evaluate the stability of reference genes in C. hainanensis and supply an important foundation to use the RT-qPCR for an accurate and far-reaching gene expression analysis in C. hainanensis.

Cell shape can be uncoupled from formononetin induction in a novel cell line from Callerya speciosa.

KEY MESSAGE: It is the first time that formononetin produced by cell culture and its accumulation was shown to be triggered by specific stress signalling linked jasmonate pathway. Callerya speciosa, an endangered traditional Chinese medicine plant, is intensively used in traditional folk medicine. To develop sustainable alternatives for the overexploitation of natural resources, a suspension cell line was created from C. speciosa. Ingredients of C. speciosa, for instance the isoflavone formononetin, are formed during a peculiar swelling response of the root, which is considered as a quality trait for commercial application. A cell strain with elongated cells was obtained by using synthetic cytokinin 6-benzylaminopurine (6-BA) and synthetic auxin picloram. Both, picloram and 6-BA, promote cell division, whereas picloram was shown to be crucial for the maintenance of axial cell expansion. We addressed the question, whether the loss of axiality observed in the maturating root is necessary and sufficient for the accumulation of formononetin. While we were able to mimic a loss of axiality for cell expansion, either by specific combinations of 6-BA and picloram, or by treatment with the anti-microtubular compound oryzalin, formononetin was not detectable. However, formononetin could be induced by the stress hormone methyl jasmonate (MeJA), as well as by the bacterial elicitor flagellin peptide (flg22), but not by a necrosis inducing protein. Combined the fact that none of these treatments induced the loss of axiality, we conclude that formononetin accumulates in response to basal defence and unrelated with cell swelling.

De novo leaf and root transcriptome analysis to identify putative genes involved in triterpenoid saponins biosynthesis in Hedera helix L.

Hedera helix L. is an important traditional medicinal plant in Europe. The main active components are triterpenoid saponins, but none of the potential enzymes involved in triterpenoid saponins biosynthesis have been discovered and annotated. Here is reported the first study of global transcriptome analyses using the Illumina HiSeq™ 2500 platform for H. helix. In total, over 24 million clean reads were produced and 96,333 unigenes were assembled, with an average length of 1385 nt; more than 79,085 unigenes had at least one significant match to an existing gene model. Differentially Expressed Gene analysis identified 6,222 and 7,012 unigenes which were expressed either higher or lower in leaf samples when compared with roots. After functional annotation and classification, two pathways and 410 unigenes related to triterpenoid saponins biosynthesis were discovered. The accuracy of these de novo sequences was validated by RT-qPCR analysis and a RACE clone. These data will enrich our knowledge of triterpenoid saponin biosynthesis and provide a theoretical foundation for molecular research on H. helix.