Transcriptome sequencing revealed the influence of blue light on the expression levels of light-stress response genes in Centella asiatica.

Centella asiatica is rich in medical and cosmetic properties. While physiological responses of C. asiatica to light have been widely reported, the knowledge of the effects of light on its gene expression is sparse. In this study, we used RNA sequencing (RNA-seq) to investigate the expression of the C. asiatica genes in response to monochromatic red and blue light. Most of the differentially expressed genes (DEGs) under blue light were up-regulated but those under red light were down-regulated. The DEGs encoded for CRY-DASH and UVR3 were among up-regulated genes that play significant roles in responses under blue light. The DEGs involved in the response to photosystem II photodamages and in the biosynthesis of photoprotective xanthophylls were also up-regulated. The expression of flavonoid biosynthetic DEGs under blue light was up-regulated but that under red light was down-regulated. Correspondingly, total flavonoid content under blue light was higher than that under red light. The ABI5, MYB4, and HYH transcription factors appeared as hub nodes in the protein-protein interaction network of the DEGs under blue light while ERF38 was a hub node among the DEGs under red light. In summary, stress-responsive genes were predominantly up-regulated under blue light to respond to stresses that could be induced under high energy light. The information obtained from this study can be useful to better understand the responses of C. asiatica to different light qualities.

Concentration of hinokinin, phenolic acids and flavonols in leaves and stems of Hydrocotyle leucocephala is differently influenced by PAR and ecologically relevant UV-B level.

We examined the effects of ambient, non-stressing ultraviolet (UV)-B (280-315nm) level combined with different intensities of photosynthetic active radiation (PAR, 400-700nm) on the accumulation of the lignan (-)-hinokinin, in leaves and stems of Hydrocotyle leucocephala. Plants were exposed in sun simulators under almost natural irradiance and climatic conditions to one of four light regimes, i.e. two PAR intensities (906 and 516µmolm(-2)s(-1)) including or excluding UV-B radiation (0 and 0.4Wm(-2)). Besides hinokinin, we identified three chlorogenic acid isomers, one other phenolic acid, 12 quercetin, and five kaempferol derivatives in the H. leucocephala extracts. Hinokinin was most abundant in the stems, and its accumulation was slightly enhanced under UV-B exposure. We therefore assume that hinokinin contributes to cell wall stabilization and consequently to a higher resistance of the plant to environmental factors. Quercetin derivatives increasingly accumulated under UV-B and high PAR exposure at the expense of kaempferols and chlorogenic acids, which was apparently related to its ability to scavenge reactive oxygen species. In general, the concentration of the constituents depended on the plant organ, the leaf age, the light regimes, and the duration of exposure. The distribution pattern of the compounds within the examined organs was not influenced by the treatments. Based on the chemical composition of the extracts a principal component analysis (PCA) enabled a clear separation of the plant organs and harvesting dates. Younger leaves mostly contained higher phenylpropanoid concentrations than older leaves. Nevertheless, more pronounced effects of the light regimes were detected in older leaves. As assessed, in many cases the individual compounds responded differently to the PAR/UV-B combinations, even within the same phenylpropanoid class. Since this is the first report on the influence of light conditions on the accumulation of lignans in herbaceous plants, it opens many perspectives for a more precise elucidation of all involved biochemical and molecular processes.

Ecologically relevant UV-B dose combined with high PAR intensity distinctly affect plant growth and accumulation of secondary metabolites in leaves of Centella asiatica L. Urban.

We investigated the effects of environmentally relevant dose of ultraviolet (UV)-B and photosynthetic active radiation (PAR) on saponin accumulation in leaves on the example of Centella asiatica L. Urban. For this purpose, plants were exposed to one of four light regimes i.e., two PAR intensities with or without UV-B radiation. The experiment was conducted in technically complex sun simulators under almost natural irradiance and climatic conditions. As observed, UV-B radiation increased herb and leaf production as well as the content of epidermal flavonols, which was monitored by non-destructive fluorescence measurements. Specific fluorescence indices also indicate an increase in the content of anthocyanins under high PAR; this increase was likewise observed for the saponin concentrations. In contrast, UV-B radiation had no distinct effects on saponin and sapogenin concentrations. Our findings suggest that besides flavonoids, also saponins were accumulated under high PAR protecting the plant from oxidative damage. Furthermore, glycosylation of sapogenins seems to be important either for the protective function and/or for compartmentalization of the compounds. Moreover, our study revealed that younger leaves contain higher amounts of saponins, while in older leaves the sapogenins were the most abundant constituents. Concluding, our results proof that ambient dose of UV-B and high PAR intensity distinctly affect the accumulation of flavonoids and saponins, enabling the plant tissue to adapt to the light conditions.

Discrimination of three Pegaga (Centella) varieties and determination of growth-lighting effects on metabolites content based on the chemometry of 1H nuclear magnetic resonance spectroscopy.

The metabolites of three species of Apiaceae, also known as Pegaga, were analyzed utilizing (1)H NMR spectroscopy and multivariate data analysis. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) resolved the species, Centella asiatica, Hydrocotyle bonariensis, and Hydrocotyle sibthorpioides, into three clusters. The saponins, asiaticoside and madecassoside, along with chlorogenic acids were the metabolites that contributed most to the separation. Furthermore, the effects of growth-lighting condition to metabolite contents were also investigated. The extracts of C. asiatica grown in full-day light exposure exhibited a stronger radical scavenging activity and contained more triterpenes (asiaticoside and madecassoside), flavonoids, and chlorogenic acids as compared to plants grown in 50% shade. This study established the potential of using a combination of (1)H NMR spectroscopy and multivariate data analyses in differentiating three closely related species and the effects of growth lighting, based on their metabolite contents and identification of the markers contributing to their differences.

Flavonoid and leaf gas exchange responses of Centella asiatica to acute gamma irradiation and carbon dioxide enrichment under controlled environment conditions.

The study was couducted to investigate the effects of gamma irradiation and CO2 on flavonoid content and leaf gas exchange in C.asiatica. For flavonoid determination, the design was a split split plot based on Randomized Complete Block Design (RCBD). For other parameters, the designs were split plots. Statistical tests revealed significant differences in flavonoid contents of Centella asiatica leaves between different growth stages and various CO2 treatments. CO2 400, G20 (400 = ambient CO2; G20 = Plants exposed to 20 Gy) showed 82.90% higher total flavonoid content (TFC) in the 5th week than CO2 400 as control at its best harvest time (4th week). Increasing the concentration of CO2 from 400 to 800 µmol/mol had significant effects on TFC and harvesting time. In fact, 800 µmol/mol resulted in 171.1% and 66.62% increases in TFC for control and irradiated plants, respectively. Moreover, increasing CO2 concentration reduced the harvesting time to three and four weeks for control and irradiated plants, respectively. Enhancing CO2 to 800 µmol/mol resulted in a 193.30% (CO2 800) increase in leaf biomass compared to 400 µmol/mol and 226.34% enhancement in irradiated plants (CO2 800, G20) [800 = Ambient CO2; G20 = Plants exposed to 20 Gy] than CO2 400, G20. In addition, the CO2 800, G20 had the highest amount of flavonoid*biomass in the 4th week. The results of this study indicated that all elevated CO2 treatments had higher PN than the ambient ones. The findings showed that when CO2 level increased from 400 to 800 µmol/mol, stomatal conductance, leaf intercellular CO2 and transpiration rate had the tendency to decrease. However, water use efficiency increased in response to elevated CO2 concentration. Returning to the findings of this study, it is now possible to state that the proposed method (combined CO2 and gamma irradiation) has the potential to increase the product value by reducing the time to harvest, increasing the yield per unit area via boosting photosynthesis capacity, as well as increasing biochemicals (flavonoids) per gram DM.

Effects of acute gamma irradiation on physiological traits and flavonoid accumulation of Centella asiatica.

In the present study, two accessions of Centella asiatica (CA03 and CA23) were subjected to gamma radiation to examine the response of these accessions in terms of survival rate, flavonoid contents, leaf gas exchange and leaf mass. Radiation Sensitivity Tests revealed that based on the survival rate, the LD(50) (gamma doses that killed 50% of the plantlets) of the plantlets were achieved at 60 Gy for CA03 and 40 Gy for CA23. The nodal segments were irradiated with gamma rays at does of 30 and 40 Gy for Centella asiatica accession 'CA03' and 20 and 30 Gy for accession 'CA23. The nodal segment response to the radiation was evaluated by recording the flavonoid content, leaf gas exchange and leaf biomass. The experiment was designed as RCBD with five replications. Results demonstrated that the irradiated plantlets exhibited greater total flavonoid contents (in eight weeks) significantly than the control where the control also exhibited the highest total flavonoid contents in the sixth week of growth; 2.64 ± 0.02 mg/g DW in CA03 and 8.94 ± 0.04 mg/g DW in CA23. The total flavonoid content was found to be highest after eight weeks of growth, and this, accordingly, stands as the best time for leaf harvest. Biochemical differentiation based on total flavonoid content revealed that irradiated plantlets in CA23 at 20 and 30 Gy after eight weeks contained the highest total flavonoid concentrations (16.827 ± 0.02; 16.837 ± 0.008 mg/g DW, respectively) whereas in CA03 exposed to 30 and 40 Gy was found to have the lowest total flavonid content (5.83 ± 0.11; 5.75 ± 0.03 mg/g DW). Based on the results gathered in this study, significant differences were found between irradiated accessions and control ones in relation to the leaf gas. The highest PN and gs were detected in CA23 as control followed by CA23 irradiated to 20Gy (CA23G20) and CA23G30 and the lowest PN and gs were observed in CA03 irradiated to 40Gy (CA03G40). Moreover, there were no significant differences in terms of PN and gs among the irradiated plants in each accession. The WUE of both irradiated accessions of Centella asiatica were reduced as compared with the control plants (p < 0.01) while Ci and E were enhanced. There were no significant differences in the gas exchange parameters among radiated plants in each accession. Moreover, malondialdehyde (MDA) of accessions after gamma treatments were significantly higher than the control, however, flavonoids which were higher concentration in irradiated plants can scavenge surplus free radicals. Therefore, the findings of this study have proven an efficient method of in vitro mutagenesis through gamma radiation based on the pharmaceutical demand to create economically superior mutants of C. asiatica. In other words, the results of this study suggest that gamma irradiation on C. asiatica can produce mutants of agricultural and economical importance.