Contrasting growth, physiological and gene expression responses of Clematis crassifolia and Clematis cadmia to different irradiance conditions.

Clematis crassifolia and Clematis cadmia Buch.-Ham. ex Hook.f. & Thomson are herbaceous vine plants native to China. C. crassifolia is distributed in shaded areas, while C. cadmia mostly grows in bright, sunny conditions in mountainous and hilly landscapes. To understand the potential mechanisms involved in the irradiance responses of C. crassifolia and C. cadmia, we conducted a pot experiment under three irradiance treatments with natural irradiation and two different levels of shading. Various growth, photosynthetic, oxidative and antioxidative parameters and the relative expression of irradiance-related genes were examined. In total, 15 unigenes were selected for the analysis of gene expression. The exposure of C. crassifolia to high irradiance resulted in growth inhibition coupled with increased levels of chlorophyll, increased catalase, peroxidase, and superoxide dismutase activity and increased expression of c144262_g2, c138393_g1 and c131300_g2. In contrast, under high irradiance conditions, C. cadmia showed an increase in growth and soluble protein content accompanied by a decrease in the expression of c144262_g2, c133872_g1, and c142530_g1, suggesting their role in the acclimation of C. cadmia to a high-irradiance environment. The 15 unigenes were differentially expressed in C. crassifolia and C. cadmia under different irradiance conditions. Thus, our study revealed that there are essential differences in the irradiance adaptations of C. crassifolia and C. cadmia due to the differential physiological and molecular mechanisms underlying their irradiance responses, which result from their long-term evolution in contrasting habitats.

A metabolomic strategy revealed the role of JA and SA balance in Clematis terniflora DC. Response to UVB radiation and dark.

Clematis terniflora DC. is a valuable resource with potential high pharmaceutical value. Proteomic, transcriptomic and metabolomic analyses of C. terniflora that has been exposed to high levels of UVB irradiation and dark conditions (HUVB + D) have revealed the mechanisms underlying its medicinal potential. However, the signal transduction pathways and the mechanisms of regulation for the accumulation of secondary metabolites remain unclear. In this study, we show that the jasmonic acid (JA) and salicylic acid (SA) signals were activated in C. terniflora in response to HUVB + D. Metabolomic analysis demonstrated that the perturbation in JA and SA balance led to additional reallocation of carbon and nitrogen resources. Evaluating the fold change ratios of differentially changed metabolites proved that JA signal enhanced the transformation of nitrogen to carbon through the 4-aminobutyric acid (GABA) shunt pathway, which increased the carbon reserve to be utilized in the production of secondary metabolites. However, SA signal induced the synthesis of proline, while avoiding the accumulation of secondary metabolites. Over all, the results indicate that the co-increase of JA and SA reconstructed the dynamic stability of transformation from nitrogen to carbon, which effectively enhanced the oxidative defense to HUVB + D in C. terniflora by increasing the secondary metabolites.

Transcriptomic and proteomic analyses of leaves from Clematis terniflora DC. under high level of ultraviolet-B irradiation followed by dark treatment.

High level of UV-B irradiation followed by dark treatment (HUV-B+D) causes accumulation of secondary metabolites in Clematis terniflora DC. To investigate the response mechanism under HUV-B+D, transcriptomic and proteomic analyses were performed in leaves of C. terniflora. The number of genes related to tetrapyrrole synthesis, amino acid metabolism, tricarboxylic acid cycle, and mitochondrial electron transport chains was hierarchically changed in leaves of C. terniflora under HUV-B+D. Data from RNA-sequencing transcriptomics and gel-free/label-free proteomics were integrated. The genes related to biosynthesis of lignins and flavonoids/isoflavonoids were significantly upregulated. Luteolin 7-O-ß-D-glucosiduronic acid, rutin, and kaempferol 3-O-rutinose were accumulated. The number of differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) related to protein metabolism were largely changed in posttranslational modification, ubiquitin proteasome, and ribosomal protein. The expression of NADP-dependent malic enzyme and the abundance of NADP-malate dehydrogenase were upregulated and increased, respectively. The activities of these two enzymes were also enhanced. These results suggest that the secondary metabolism pathway and tricarboxylic acid cycle might be activated in leaves of C. terniflora in response to HUV-B+D. BIOLOGICAL SIGNIFICANCE: This study reported response mechanism in leaves of Clematis terniflora DC. under high level of UV-B irradiation followed by dark treatment using transcriptomic and proteomic techniques. Transcriptomic analysis revealed that the number of genes related to secondary metabolism was increased by >2 times. Phenylalanine ammonia-lyase, 4-Coumarate: CoA ligase, chalcone synthase, isoflavone reductase homolog, and cinnamoyl-CoA reductase were significantly upregulated, which suggests that the secondary metabolism pathway related to lignins and flavonoids/isoflavonoids might be activated. Genes and proteins related to protein metabolism were largely changed in postranslational modification, ubiquitin proteasome, and ribosomal protein. NADP-dependent malic enzyme and NADP-malate dehydrogenase were activated in tricarboxylic acid cycle, which suggests that tricarboxylic acid cycle might be enhanced in leaf of C. terniflora in response to HUV-B+D.

Enhanced metabolic process to indole alkaloids in Clematis terniflora DC. after exposure to high level of UV-B irradiation followed by the dark.

BACKGROUND: Indole alkaloids, which characteristically contain an indole nucleus, have pharmaceutical potential in a diverse range of applications. UV-B can elicit the accumulation of indole alkaloids. The indole alkaloid (6-hydroxyl-1H-indol-3-yl) carboxylic acid methyl ester with cytotoxic activity was found to accumulate in Clematis terniflora DC. leaves after exposure to high level of UV-B irradiation and the dark. However, a more in-depth analysis of the process behind this response has not yet been performed. Therefore, an integrated approach involving metabolomic, proteomic, and transcriptomic analyses is essential to detail the biosynthetic mechanisms of the regulation of indole alkaloid under binary stress. RESULTS: Indole alkaloid (6-hydroxyl-1H-indol-3-yl) carboxylic acid methyl ester was found to increase 7-fold in C. terniflora leaves post-treatment with high level of UV-B irradiation followed by an incubation in the dark compared with pre-treatment. Analysis by proteomics and metabolomics indicates a decrease in photosynthesis and carbohydrate metabolism, respectively. By contrast, amino acid metabolism was activated by this binary stress, and, specifically, the genes involved in the metabolic pathway converting shikimate to L-tryptophan were concurrently upregulated. Metabolites involved in indole biosynthesis (shikimate metabolic) pathway were anthranilate, indole, and L-tryptophan, which increased 2-, 441-, and 1-fold, respectively. In addition, there was an increase of 2- and 9-fold in L-serine deaminase (L-SD) and L-tryptophan synthase activity in C. terniflora leaves after exposure to high level of UV-B irradiation and the dark. CONCLUSIONS: (6-hydroxyl-1H-indol-3-yl) carboxylic acid methyl ester was found to increase in response to high level of UV-B irradiation followed by an incubation in the dark, implying that indole alkaloid biosynthesis was activated in C. terniflora leaves. Analysis of perturbations in metabolism in these leaves demonstrated that amino acid metabolism was specifically activated by this binary stress. In addition, an enhancement in serine level and L-SD activity was noted, which likely leads to an accumulation of pyruvate that, in turn, supplies shikimate metabolic pathway. The genes, metabolites, and L-tryptophan synthase activity that are involved in the metabolic pathway leading from shikimate to L-tryptophan all increased under the experimental binary stress, resulting in an enhancement of indole biosynthesis (shikimate metabolic) pathway. Therefore, the metabolic process to indole alkaloids in C. terniflora was enhanced after exposure to high level of UV-B irradiation followed by the dark.

Proteomic and Metabolomic Analyses of Leaf from Clematis terniflora DC. Exposed to High-Level Ultraviolet-B Irradiation with Dark Treatment.

Clematis terniflora DC. has potential pharmaceutical value; on the contrary, high-level UV-B irradiation with dark treatment led to the accumulation of secondary metabolites. Metabolomic and proteomic analyses of leaf of C. terniflora were performed to investigate the systematic response mechanisms to high-level UV-B irradiation with dark treatment. Metabolites related to carbohydrates, fatty acids, and amino acids and/or proteins related to stress, cell wall, and amino acid metabolism were gradually increased in response to high-level UV-B irradiation with dark treatment. On the basis of cluster analysis and mapping of proteins related to amino acid metabolism, the abundances of S-adenosylmethionine synthetase and cysteine synthase as well as 1,1-diphenyl-2-picrylhydrazyl scavenging activity were gradually increased in response to high-level UV-B irradiation with dark treatment. Furthermore, the abundance of dihydrolipoyl dehydrogenase/glutamate dehydrogenase and the content of γ-aminobutyric acid were also increased following high-level UV-B irradiation with dark treatment. Taken together, these results suggest that high-level UV-B irradiation with dark treatment induces the activation of reactive oxygen species scavenging system and γ-aminobutyric acid shunt pathway in leaf of C. terniflora.