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Main conclusion Representational difference analysis of cDNA was performed and differential products were sequenced and annotated. Candidate genes involved in biosynthesis of diosgenin in fenugreek were identified. Detailed mechanism of diosgenin synthesis was proposed.Fenugreek (Trigonella foenum-graecum L.) is a valuable medicinal and crop plant. It belongs to Fabaceae family and has a unique potential to synthesize valuable steroidal saponins, e.g., diosgenin. Elicitation (methyl jasmonate) and precursor feeding (cholesterol and squalene) were used to enhance the content of sterols and steroidal sapogenins in in vitro grown plants for representational difference analysis of cDNA (cDNA-RDA). To identify candidate genes involved in diosgenin biosynthesis, differential, factor-specific libraries were subject to the nextgeneration sequencing. Approximately 9.9 million reads were obtained, trimmed, and assembled into 31,491 unigenes with an average length of 291 bp. Then, functional annotation and gene ontogeny enrichment analysis was performed by aligning all-unigenes with public databases.

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen ( O ) formed during high light stress in higher plants. Although quenching of O by prenylquinols has been previously studied, direct evidence for chemical quenching of O by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol-9 (PQH -9) in chemical quenching of O was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH -9 and plastochromanol-8 biosynthesis. In this work, direct evidence for chemical quenching of O by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of O was associated with consumption of PQH -9 and formation of its various oxidized forms. Oxidation of PQH -9 by O leads to plastoquinone-9 (PQ-9), which is subsequently oxidized to hydroxyplastoquinone-9 [PQ(OH)-9]. We provide here evidence that oxidation of PQ(OH)-9 by O results in the formation of trihydroxyplastoquinone-9 [PQ(OH) -9]. It is concluded here that PQH -9 serves as an efficient O chemical quencher in Arabidopsis, and PQ(OH) -9 can be considered as a natural product of O reaction with PQ(OH)-9. The understanding of the mechanisms underlying O chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.

Changes in fenugreek transcriptome related to enhanced production of steroids were induced by methyl jasmonate, cholesterol and squalene, and recorded using RNA-seq. A total of 112,850 unigenes were obtained after de novo assembling of next generation sequencing data, and used for functional annotations. In steroidal saponins pathway, transcripts involved in mevalonate, terpenoid backbone and plant sterol synthesis were annotated. Overexpression of several transcripts from phytosterol biosynthesis pathway was confirmed by quantitative RT-PCR. In diosgenin biosynthesis pathway, fatty acid ω-hydroxylase (CYP86A2) and steroid 22-alpha-hydroxylase (CYP90B1) genes were annotated in all induced transcriptomes. Moreover, direct sequencing confirmed increased levels of CYP90B1, unspecific monooxygenase and 26-hydroxylase genes in plants with elevated level of diosgenin. New unigenes corresponding to enzymes involved in biosynthesis of diosgenin from cycloartenol via cholesterol were obtained and the role of CYP72A family in steroidal saponin biosynthesis was proposed. Additional support for biosynthetic pathway from cycloartenol to diosgenin was provided.

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Veratrum-type steroidal alkaloids (VSA) are the major bioactive ingredients that strongly determine the pharmacological activities of Veratrum nigrum. Biosynthesis of VSA at the molecular and genetic levels is not well understood. Next-generation sequencing of representational difference analysis (RDA) products after elicitation and precursor feeding was applied to identify candidate genes involved in VSA biosynthesis. A total of 12,048 contigs with a median length of 280 bases were received in three RDA libraries obtained after application of methyl jasmonate, squalene and cholesterol. The comparative analysis of annotated sequences was effective in identifying candidate genes. GABAT2 transaminase and hydroxylases active at C-22, C-26, C-11, and C-16 positions in late stages of jervine biosynthesis were selected. Moreover, genes coding pyrroline-5-carboxylate reductase and enzymes from the short-chain dehydrogenases/reductases family (SDR) associated with the reduction reactions of the VSA biosynthesis process were proposed. The data collected contribute to better understanding of jervine biosynthesis and may accelerate implementation of biotechnological methods of VSA biosynthesis.

Main conclusion Changes in proteome level as a result of methyl jasmonate and cholesterol treatment were investigated. The identified proteins were often involved in response to stress caused by various treatments. Furthermore, 18 proteins were expressed in treatmentspecific manner. In this study, the fenugreek plants were treated with methyl jasmonate (as an elicitor) and cholesterol (as a precursor of steroids and steroidal saponins) to check reaction at the level of the proteome to stress and to investigate steroidal saponin (diosgenin) biosynthesis. Proteins were separated by two-dimensional electrophoresis (2-DE) and identified by MALDI-ToF/ToF followed by database searches using Mascot search engine. Totally, 63 and 41 protein spots were differentially expressed after methyl jasmonate and cholesterol treatment, respectively. These proteins were classified into seven groups: photosynthesis, energy, metabolism, protein metabolism, secondary metabolism, stress and defense, and other. We found that 9 proteins were responsive to all treatments, and 18 proteins expressed in treatment-specific manner. Higher level of photosynthetic proteins sensitive to both biotic and abiotic stimuli was detected. In addition, proteins related to the stress (especially oxidative) and defense, protein, and secondary metabolism were overexpressed. The results indicate that methyl jasmonate and cholesterol elicited a defense reaction at the proteome level as a response to stress. The usefulness of 2-DE method for identification of proteins related with species-specific metabolic pathways is restricted. Integration of transcriptome data with proteomic analysis improved annotation process.