Producing Medicinal Phytochemicals from Phyllanthus acuminatus in Plant - Cell Suspension Cultures

Pharmaceutical use is not feasible for important medicinal compounds derived from certain plant materials, including Phyllanthus acuminatus roots, due to their low natural abundance. New technologies in non-traditional biomass generation are needed to produce these remarkable natural compounds. Therefore, this article describes a methodology for establishing Phyllanthus acuminatus plant-cell suspensions from callus cultures: An evaluation on inoculum concentration and agitation speed displayed significant changes in plant cell growth kinetics. It was determined that treatment with 2 g of inoculum in 25 mL of medium and 100 rpm agitation creates the best conditions for generating thick cell suspensions. Likewise, treatment with 2 g of inoculum and 120 rpm agitation produces the best conditions for establishing fine cell suspensions. Phytochemical comparison through high-resolution mass spectrometry of P. acuminatus roots and plant cell suspension extracts confirmed presence in the plant cell culture of multiple phyllantostatins of pharmaceutical interest. Here, we demonstrate that Phyllanthus acuminatus can be cultured in plant cell suspensions to produce secondary metabolites of medical interest – technology that could be scaled up for implementation in industrial bioprocesses.

LEDitSHAKE: a lighting system to optimize the secondary metabolite content of plant cell suspension cultures

Plant secondary metabolites are widely used in the food, cosmetic and pharmaceutical industries. They can be extracted from sterile grown plant cell suspension cultures, but yields and quality strongly depend on the cultivation environment, including optimal illumination. Current shaking incubators do not allow different light wavelengths, intensities and photoperiods to be tested in parallel. We therefore developed LEDitSHAKE, a system for multiplexed customized illumination within a single shaking incubator. We used 3D printing to integrate light-emitting diode assemblies into flask housings, allowing 12 different lighting conditions (spectrum, intensity and photoperiod) to be tested simultaneously. We did a proof of principle of LEDitSHAKE using the system to optimize anthocyanin production in grapevine cell suspension cultures. The effect of 24 different light compositions on the total anthocyanin content of grapevine cell suspension cultures was determined using a Design of Experiments approach. We predicted the optimal lighting conditions for the upregulation and downregulation of 30 anthocyanins and found that short-wavelength light (blue, UV) maximized the concentration of most anthocyanins, whereas long-wavelength light (red) had the opposite effect. Therefore our results demonstrate proof of principle that the LEDitSHAKE system is suitable for the optimization of processes based on plant cell suspension cultures.

LEDitSHAKE – A Lighting System to Optimize the Secondary Metabolite Content of Plant Cell Suspension Cultures

1.1 Background Plant secondary metabolites are widely used in the food, cosmetic and pharmaceutical industries. They can be extracted from naturally grown plants or plant cell suspension cultures grown under sterile conditions. In the latter case, yields and quality strongly depend on the cultivation environment, including optimal illumination. Current shaking incubators do not allow different light wavelengths, intensities and photoperiods to be tested in parallel. We therefore developed a system for multiplexed customized illumination within a single shaking incubator, and used it to optimize anthocyanin production in grapevine cell suspension cultures. 1.2 Results We used 3D printing to integrate light-emitting diode assemblies into flask housings to develop the LEDitSHAKE system, allowing 12 different lighting conditions (spectrum, intensity and photoperiod) to be tested simultaneously in a single shaking incubator. We used a Design of Experiments approach to determine the effect of 24 different light compositions on the total anthocyanin content of replicate grapevine cell suspension cultures. All tested conditions achieved higher yields than standard illumination or dark cultivation, and the optimal spectrum (8.3 µmol m− 2 s− 1 red, 8.3 µmol m− 2 s− 1 green, 33.3 µmol m− 2 s− 1 blue, and UV turned on) increased the total anthocyanin concentration by 2.42-fold after 4 weeks. Based on the resulting model, we predicted the optimal lighting conditions for the upregulation and downregulation of 30 anthocyanins, and found that short-wavelength light (blue, UV) maximized the concentration of most anthocyanins, whereas long-wavelength light (red) had the opposite effect. For example, the cyanidin glucoside concentration was predicted to increase 2.99-fold compared to the source culture with optimized illumination (12 µmol m− 2 s− 1 green, 38 µmol m− 2 s− 1 blue, and UV turned on for 1 h/day). 1.3 Conclusions The LEDitSHAKE system enables the screening of up to 12 different lighting conditions in terms of spectrum, intensity and photoperiod within a single shaking incubator using a Design of Experiments approach, as exemplified by the optimization of the anthocyanin content and composition in grapevine cell suspension cultures. Our results demonstrate proof of principle that the LEDitSHAKE system is suitable for the optimization of processes based on plant cell suspension cultures.

Root Cultures for Secondary Products

Plants are source of many high-value secondary compounds used as drugs, food additives, flavors, pigments and pesticides. The production of these compounds in nature faces to many difficulties because of the dependence on weather, soil … Furthermore, these compounds are usually limited by species, periods of growth or stress. The utilization of plant cells in vitro for the secondary compounds has gained increasing attention over past decades. However, the yield is still low, probably due to the degree of cell differentiation. Therefore, root culture is focused on research as an alternative to cell cultures to produce secondary compounds because of high rate proliferation, great potential in the production with high and stable yields. Hairy roots and adventitious roots have a high ability to biosynthesize secondary compounds in vitro with high and fairly stable in yield in comparison with plant cell suspension cultures. Nowadays, it is feasible to expand the scale of root cultures in bioreactors, which makes it possible to produce secondary compounds on an industrial scale.

Phospholipid Signaling Is a Component of the Salicylic Acid Response in Plant Cell Suspension Cultures

Salicylic acid (SA) is an important signaling molecule involved in plant defense. While many proteins play essential roles in SA signaling, increasing evidence shows that responses to SA appear to involve and require lipid signals. The phospholipid-generated signal transduction involves a family of enzymes that catalyze the hydrolysis or phosphorylation of phospholipids in membranes to generate signaling molecules, which are important in the plant cellular response. In this review, we focus first, the role of SA as a mitigator in biotic/abiotic stress. Later, we describe the experimental evidence supporting the phospholipid–SA connection in plant cells, emphasizing the roles of the secondary lipid messengers (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA)) and related enzymes (phospholipase D (PLD) and phospholipase C (PLC)). By placing these recent finding in context of phospholipids and SA in plant cells, we highlight the role of phospholipids as modulators in the early steps of SA triggered transduction in plant cells.

The Synergistic Effect of Co-Treatment of Methyl Jasmonate and Cyclodextrins on Pterocarpan Production in Sophora flavescens Cell Cultures

Pterocarpans are derivatives of isoflavonoids, found in many species of the family Fabaceae. Sophora flavescens Aiton is a promising traditional Asian medicinal plant. Plant cell suspension cultures represent an excellent source for the production of valuable secondary metabolites. Herein, we found that methyl jasmonate (MJ) elicited the activation of pterocarpan biosynthetic genes in cell suspension cultures of S. flavescens and enhanced the accumulation of pterocarpans, producing mainly trifolirhizin, trifolirhizin malonate, and maackiain. MJ application stimulated the expression of structural genes (PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I3’H, and IFR) of the pterocarpan biosynthetic pathway. In addition, the co-treatment of MJ and methyl-β-cyclodextrin (MeβCD) as a solubilizer exhibited a synergistic effect on the activation of the pterocarpan biosynthetic genes. The maximum level of total pterocarpan production (37.2 mg/g dry weight (DW)) was obtained on day 17 after the application of 50 μM MJ on cells. We also found that the combined treatment of cells for seven days with MJ and MeβCD synergistically induced the pterocarpan production (trifolirhizin, trifolirhizin malonate, and maackiain) in the cells (58 mg/g DW) and culture medium (222.7 mg/L). Noteworthy, the co-treatment only stimulated the elevated extracellular production of maackiain in the culture medium, indicating its extracellular secretion; however, its glycosides (trifolirhizin and trifolirhizin malonate) were not detected in any significant amounts in the culture medium. This work provides new strategies for the pterocarpan production in plant cell suspension cultures, and shows MeβCD to be an effective solubilizer for the extracellular production of maackiain in the cell cultures of S. flavescens.