Selection and validation of reference genes for RT-qPCR normalization in different tissues of milk thistle (Silybum marianum, Gaert.).

Quantitative reverse transcription PCR is a sensitive technique for evaluating transcriptional profiles in different experimental datasets. To obtain a reliable quantification of the transcripts level, data normalization with stable reference genes is required. Stable reference genes are identified after analysis of their transcripts profile in every new experiment and species of interest. In Silybum marianum, a widely cultivated officinal plant, only few gene expression studies exist, and reference genes for RT-qPCR studies in the diverse plant tissues have never been investigated before. In this work, the expression stability of 10 candidate reference genes was evaluated in leaves, roots, stems and fruits of S. marianum grown under physiological environmental condition. The stability values for each candidate reference gene were calculated by four canonical statistical algorithms GeNorm, NormFinder, Bestkeeper and ΔCt method in different subsets of samples, then they were ranked with RefFinder from the most to the least suitable for normalization. Best combinations of reference genes are finally proposed for different experimental data sets, including all tissues, vegetative, and reproductive tissues separately. Three target genes putatively involved in important biosynthetic pathway leading to key metabolites in the fruits of milk thistle, such as silymarin and fatty acids, were analyzed with the chosen panels of reference genes, in comparison to the ones used in previous papers. To the best of our knowledge, this is the first report on a reliable and systematic identification and validation of the reference genes for RT-qPCR normalization to study gene expression in S. marianum.

Gene Expression Analysis and Metabolite Profiling of Silymarin Biosynthesis during Milk Thistle (Silybum marianum (L.) Gaertn.) Fruit Ripening.

Mature fruits (i.e., achenes) of milk thistle (Silybum marianum (L.) Gaertn., Asteraceae) accumulate high amounts of silymarin (SILM), a complex mixture of bioactive flavonolignans deriving from taxifolin. Their biological activities in relation with human health promotion and disease prevention are well described. However, the conditions of their biosynthesis in planta are still obscure. To fill this gap, fruit development stages were first precisely defined to study the accumulation kinetics of SILM constituents during fruit ripening. The accumulation profiles of the SILM components during fruit maturation were determined using the LC-MS analysis of these defined developmental phases. The kinetics of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and peroxidase (POX) activities suggest in situ biosynthesis of SILM from l-Phenylalanine during fruit maturation rather than a transport of precursors to the achene. In particular, in contrast to laccase activity, POX activity was associated with the accumulation of silymarin, thus indicating a possible preferential involvement of peroxidase(s) in the oxidative coupling step leading to flavonolignans. Reference genes have been identified, selected and validated to allow accurate gene expression profiling of candidate biosynthetic genes (PAL, CAD, CHS, F3H, F3'H and POX) related to SILM accumulation. Gene expression profiles were correlated with SILM accumulation kinetic and preferential location in pericarp during S. marianum fruit maturation, reaching maximum biosynthesis when desiccation occurs, thus reinforcing the hypothesis of an in situ biosynthesis. This observation led us to consider the involvement of abscisic acid (ABA), a key phytohormone in the control of fruit ripening process. ABA accumulation timing and location during milk thistle fruit ripening appeared in line with a potential regulation of the SLIM accumulation. A possible transcriptional regulation of SILM biosynthesis by ABA was supported by the presence of ABA-responsive cis-acting elements in the promoter regions of the SILM biosynthetic genes studied. These results pave the way for a better understanding of the biosynthetic regulation of SILM during the maturation of S. marianum fruit and offer important insights to better control the production of these medicinally important compounds.

Silybin content and overexpression of chalcone synthase genes in Silybum marianum L. plants under abiotic elicitation.

Silymarin, a Silybum marianum seed extract containing a mixture of flavonolignans including silybin, is being used as an antihepatotoxic therapy for liver diseases. In this study, the enhancing effect of gamma irradiation on plant growth parameters of S. marianum under salt stress was investigated. The effect of gamma irradiation, either as a single elicitor or coupled with salinity, on chalcone synthase (CHS) gene expression and silybin A + B yield was also evaluated. The silybin A + B content in S. marianum fruits was estimated by liquid chromatography-mass spectrometry (LC-MS/MS). An increase in silybin content was accompanied by up-regulation of the CHS1, CHS2 and CHS3 genes, which are involved in the silybin biosynthetic pathway. The highest silybin A + B production (0.77 g/100 g plant DW) and transcript levels of the three studied genes (100.2-, 91.9-, and 24.3-fold increase, respectively) were obtained with 100GY gamma irradiation and 4000 ppm salty water. The CHS2 and CHS3 genes were partially sequenced and submitted to the NCBI database under the accession numbers KT252908.1 and KT252909.1, respectively. Developing new approaches to stimulate silybin biosynthetic pathways could be a useful tool to potentiate the use of plants as renewable resources of medicinal compounds.