Methyl jasmonate enhanced production of rosmarinic acid in cell cultures of Satureja khuzistanica in a bioreactor

The growing interest in rosmarinic acid (RA), an ester of caffeic acid and 3,4‐dihydroxyphenyl lactic acid, is due to its biological activities, which include cognitive‐enhancing effects, slowing the development of Alzheimer's disease, cancer chemoprotection, and anti‐inflammatory activity. Inspired by the challenge of meeting the growing demand for this plant secondary metabolite, we developed a biotechnological platform based on cell suspension cultures of Satureja khuzistanica. The high amounts of RA produced by this system accumulated mainly inside the cells. To further improve production, two elicitors, 100 μM methyl jasmonate (MeJA) and 40 mM cyclodextrin (CD), were tested, separately and together. MeJA increased RA productivity more than 3‐fold, the elicited cultures achieving an RA production of 3.9 g L^?1 without affecting biomass productivity. CD did not have a clear effect on RA production, and under the combined treatment of MeJA + CD only a small amount of RA was released to the medium. When the cell culture was transferred from a shake flask to a wave‐mixed bioreactor, a maximum RA production of 3.1 g L^?1 and biomass productivity of 18.7 g L^?1 d^?1 was achieved under MeJA elicitation, demonstrating the suitability of S. khuzistanica cell suspensions for the biotechnological production of this bioactive plant secondary metabolite.     

Fe2O3 nanoparticles induced biochemical responses and expression of genes involved in rosmarinic acid biosynthesis pathway in Moldavian balm under salinity stress

The effect of iron oxide nanoparticle (NP) at four concentrations (0, 30, 60 and 90 ppm) and salinity at three levels (0, 50 and 100 mM) were investigated on rosmarinic acid (RA) production in 5-week-old Moldavian balm (Dracocephalum moldavica L.) plants. Salinity and spraying iron oxide NPs significantly affected tyrosine (Tyr), phenylalanine (Phe) and proline (Pro) amino acids content, Phenylalanine Ammonia-Lyase (PAL), Tyrosine Aminotransferase (TAT) and Rosmarinic Acid Synthase (RAS) genes expression levels, RA content, Polyphenol Oxidase (PPO), PAL and Superoxide Dismutase (SOD) activities, malondialdehyde (MDA) content and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity. PAL, TAT and RAS genes expression rate and content of RA were enhanced in Moldavian balm plants exposed by NaCl + NPs. The results of high performance liquid chromatography (HPLC) revealed that simultaneous application of 50 mM NaCl and 90 ppm NPs increases the RA content in leaf by 81.15% as compared to control plants. The Tyr and Phe contents decreased in Moldavian balm plants exposed to salt stress. Application of NPs had a positive effect on the content of these amino acids. Proline content increased under salinity stress and application of iron NPs induced a significant increase in the Pro content of leaf. The results revealed that PAL, PPO and SOD enzymes activities increased under salinity conditions. The highest activity of PPO and SOD was observed in 100 mM NaCl + 60 ppm NPs treatment. Simultaneous application of 100 mM NaCl + 90 ppm NPs increased the MDA content and DPPH radical scavenging activity compared to control plants. It can be concluded that the application of appropriate levels of NPs moderates the effect of salinity stress in D. moldavica L. and results in an increased amount of RA compared to control plants.     

Alleviation of drought stress and the physiological mechanisms in Citrus cultivar (Huangguogan) treated with methyl jasmonate

ABSTRACT

The role of exogenous methyl jasmonate (MeJA) in alleviating drought stress was investigated on Huangguogan. Except for intercellular CO₂ concentration, MeJA had little effect on net photosynthetic rate, stomatal conductance, and transpiration rate under drought stress. Compared with drought stress, MeJA significantly alleviated the decrease of chlorophyll content. However, chlorophyll a/b ratio was significantly increased. MeJA significantly increased proline and soluble sugar contents, significantly decreased the O₂ ^?· and H₂O₂ levels, and increased SOD and POD activities. In addition, the MDA content of drought stress was the highest of all treatments. MeJA significantly reduced MDA content in drought-stressed Huangguogan leaves. Although the Ascorbic acid (AsA) contents of 500 and 1000 mg L^?1 MeJA treatments were lower than that of 250 mg L^?1 MeJA, but all concentration of MeJA treatments delayed the decline of AsA content. Therefore, MeJA could induce drought stress tolerance by increasing the osmotic adjustment substances and antioxidant activities.     

In vitro mature embryo culture protocol of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum L.) wheat under boron stress

Mature embryos of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum L.) wheat were used for callus induction on media containing four different doses (0, 1, 2 and 4 mg L^?1) of 2,4-D and dicamba supplemented with five different boron concentrations (0, 6.2, 12.4, 24.8, and 37.2 mg L^?1). The obtained callus was transferred to culture media with three (0, 0.5, and 2 mg L^?1) different BAP doses with five boron concentrations for further regeneration. The maximum callus weight in einkorn wheat was in culture media with 1 mg L^?1 dicamba and 6.2 mg L^?1 (3.71?±?0.13 g). Bread wheat had the maximum callus weight on culture media with 4 mg L^?1 dicamba and 12.4 mg L^?1 (3.46?±?0.40 g). The highest plantlet numbers were in only 2 mg L^?1 BAP (2.92?±?0.88) for einkorn wheat and 0.5 mg L^?1 BAP supplemented with 6.2 mg L^?1 boron (3.71?±?1.12) for bread wheat. This indirect regeneration protocol using mature embryos of einkorn and bread wheat under boron stresses expected to be useful for future wheat breeding studies.

     

Expression of Flowering Repressor Gene CsSVP,Carbohydrates, and Antioxidants Affected by Plant Growth Regulators in Saffron

Improving flower yield through lengthening flowering duration is a primary breeding objective in saffron (Crocus sativus L.). Asexual reproduction in saffron limits biodiversity and conventional breeding. Hence, eliciting flowering-related gene expression by plant growth regulators is one way to achieve this aim. The phytohormones methyl jasmonate (MeJA) and 6-benzyl amino purine (BAP) signals are received by the MADs-box gene family. In this study, to elucidate the role of phytohormones on flower development, plant were treated with BAP (0 and 5 mg L^?1), and methyl jasmonate (MeJA) (0, 20, and 100 mM) at three developmental stages of the saffron life cycle. Then, the expression of the SHORT VEGETATIVE PHASE (CsSVP) gene as a MADS-box gene family was assessed in the saffron corm. The activities of antioxidant enzymes, soluble sugar, starch content, and soluble protein content were also measured in corm, leaf, and root tissues. The application of MeJA and BAP treatments resulted in down-regulation of CsSVP expression in the corm during dormancy. At the dormancy stage, catalase, peroxidase activity decreased, and ascorbate peroxidase activity increased following MeJA treatment. In contrast, an increment in catalase and peroxidase activity and reduction of ascorbate peroxidase activity were observed after treatment with MeJA during the flowering stage. This change in enzyme activity is most likely due to flowering, which demands the re-allocation of resources. As flowering is a process heavily influenced by the environment, plants treated with MeJA, which may mimic environmental stress, showed changes in antioxidant enzyme activity. Overall, these results suggested that MeJA and BAP treatments play a significant role in the vegetative-to-reproductive phase change in saffron.

     

The effect of explant origin and collection season on stilbene biosynthesis in cell cultures of Vitis amurensis Rupr.

Plant cell and tissue cultures are considered as a source of valuable secondary metabolites but usually produce insufficient level of the compounds, which is the limiting factor for their application in biotechnology. We obtained 18 callus cell cultures from different organs of wild grape Vitis amurensis Rupr. collected at different seasons and analyzed stilbene accumulation in combination with calli growth parameters. This analysis showed that temporal and tissue origin of the calli affected the rate of stilbene biosynthesis. Stem-derived calli accumulated higher stilbene levels and exhibited a higher expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes than calli derived from the leaves and petioles. The highest content of stilbenes was detected in the calli initiated from grapevine stems collected in the autumn. In general, all “autumn” cell cultures contained more than 2 mg g^??1 dry wt (up to 11 mg g^??1 dry wt) and exhibited high PAL and STS genes expression in comparison with the calli initiated in the summer. The content of stilbenes in the “autumn” cell cultures were comparable to the highest stilbene contents detected in other plant sources described in the literature. Thus, selecting the most optimal explant source for cell culture establishment could be an effective approach towards developing plant cell cultures producing high stilbene levels.

     

Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in <Emphasis Type="Italic">Zoysia japonica</Emphasis>

In this study, we have demonstrated that Zoysia japonica callus induced from mature seeds can produce high frequencies of plant regeneration and somatic embryogenesis, even following a prolonged period of subculturing. Initial callus cultures were induced from mature seeds of Japanese lawngrass (Z. japonica Steud.) incubated on a medium containing major N₆ medium salts, minor Murashige and Skoog (MS) medium salts, and modified MS medium organic elements supplemented with 3 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01–0.02 mg L⁻¹ 6-benzyladenine. Compact callus were selected and subcultured monthly on a medium containing 2 mg L⁻¹ 2,4-D, 0.5 mg L⁻¹ kinetin, 500 mg L⁻¹ casein hydrolysate, 500 mg L⁻¹ proline, and 500 mg L⁻¹ myoinositol. Callus maintained in vitro for 18 mo could be induced to regenerate plantlets with a frequency of >90%. By contrast, 36-mo-old callus cultures failed to produce normal shoot regeneration. However, the addition of CuSO₄ to the subculture media maintained >90% regeneration frequencies in such long-term callus cultures. Histological observations revealed that plant regeneration occurred both through somatic embryogenesis and organogenesis pathways. The ability to sustainable regeneration in long-term callus cultures will be valuable to the program of genetic transformation and somaclonal variant selection.     

Efficient plant regeneration from in vitro leaves and petioles via shoot organogenesis in Sapium sebiferum Roxb.

Sapium sebiferum Roxb. is a widespread and economically important multipurpose tree due to its high value in ornamental, and biodiesel production as well as medicine. A highly efficient in vitro plant regeneration system through direct shoot organogenesis was established for the first time from leaves and petioles of S. sebiferum. The results showed that plant growth regulators (PGRs), mechanical damage, explant orientation, explant source, and developmental stage had a strong influence on the in vitro morphogenesis of S. sebiferum. For shoot organogenesis from leaves, the highest adventitious shoot induction rate (96.67%) with 25.67 shoots per explant was obtained when mechanically damaged leaves (the first three leaf explants at the top, leaf #1–3) were cultured with the abaxial surface placed down on Murashige and Skoog (MS) medium containing 0.5 mg L^?1 thidiazuron (TDZ). For in vitro morphogenesis of petioles, the combination of 1-naphthylacetic acid (NAA) and 6-benzylainopurine (6-BA) played a key role in cell fate determination. All of the in vitro petioles produced adventitious shoots on MS medium containing 1.0 mg L^?1 6-BA and 0.1 mg L^?1 NAA, while they produced green calli on medium fortified with 0.5 mg L^?1 6-BA and 1.0 mg L^?1 NAA. The shoots were subcultured in medium fortified with 0.5 mg L^?1 6-BA and 0.1 mg L^?1 NAA for multiplication and elongation. The elongated shoots successfully rooted on half-strength MS (1/2 MS) medium fortified with 0.5 mg L^?1 indole-butyric acid (IBA) and 0.25 mg L^?1 indole-3-acetic acid (IAA), and the regenerated plantlets successfully acclimatized with a survival rate of 92.56% in the greenhouse. The genetic fidelity of in vitro regenerated plants was evaluated using inter simple sequence repeat molecular markers. The in vitro regenerated plants were found to be the true to their mother plant. This study will be beneficial for the large-scale propagation as well as the genetic improvement of S. sebiferum.

     

Influence of light spectra and elicitors on growth and ascaridole content using in vitro cultures of Dysphania ambrosioides L.

Dysphania ambrosioides L. is a medicinal plant with anti-helmintic potential. The aim of this study was to evaluate separately the effect of light spectra and elicitors on Dysphania ambrosioides growth and volatile constituents in vitro. Thus, plantlets were first cultured under blue (B), red (R), white, combinations of B:R (1:1, 2:1, 1:2) from LEDs and fluorescent lamps. Secondly, nodal segments were inoculated in the medium supplemented with chitosan (0, 50, 100, 150, and 200 mg L^??1) and salicylic acid (0, 3, 6, 9, and 12 mg L^??1). After 40 days of cultivation, the growth parameters and chemical composition of volatile constituents were evaluated. The light spectra significantly influenced in vitro growth of D. ambrosioides. The best growth occured using white LED or a blue:red combination of 2:1. It was also observed that the blue LEDs inhibited the synthesis of Z-ascaridole, while fluorescent light promoted a greater conversion of α-terpinene into ascaridole. The elicitors, chitosan and salicylic acid had a negative effect on the growth of nodal segments. However, the highest Z-ascaridole content was obtained at 50 to 100 mg L^??1 of chitosan and with 6 to 9 mg L^??1 of salicylic acid. The present study demonstrates that shoots regenerated from nodal segments exposed to different light spectra or on MS medium containing chitosan and salicylic acid can exhibit an altered growth and increased volatile constituents of interest.

     

Salicylic acid improves podophyllotoxin production in cell cultures of Linum album by increasing the expression of genes related with its biosynthesis

Treatment of Linum album cell cultures with 10 μM salicylic acid (SA) for 3 days improved podophyllotoxin (PTOX) production up to 333 μg/g dry weight (DW): over three times that of the control cultures. qPCR analyses showed that in SA-treated cells, the expression of the genes coding for phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD), all involved in the first steps of PTOX biosynthesis, also increased reaching a peak 8–12 h after the treatment. Expression of the pinoresinol-lariciresinol reductase gene (PLR), which is involved in one of the last biosynthetic steps, was not affected by SA. The selective action of SA on these genes can be applied to control the biotechnological production of this anticancer agent.     

Producing Biochemicals in Yarrowia lipolytica from Xylose through a Strain Mating Approach

Enabling xylose catabolism is challenging, especially for unconventional yeasts and previously engineered background strains. In this study, the efficacy of a yeast mating approach with Yarrowia lipolytica that can combine a previously engineering and evolved xylose phenotype with a metabolite overproduction phenotype is demonstrated. Specifically, several engineered Y. lipolytica strains that produce α‐linolenic acid (ALA), riboflavin, and triacetic acid lactone (TAL) with an engineered and adapted xylose‐utilizing strain to obtain three diploid strains that rapidly produce these molecules directly from xylose are mated. Titers of 0.52 g L^?1 ALA, 96.6 mg L^?1 riboflavin, and 2.9 g L^?1 TAL, are obtained from xylose in flask cultures and 1.42 g L^?1 production of ALA is obtained using bioreactor condition. This total production level is generally on par or higher than the parental strain cultivated on glucose, although specific productivities decreased as a result of improved overall cell growth by the diploid strains. In the case of ALA, this lipid content reached similar levels to that of flaxseed oil. This result showcases the first study using strain mating in Y. lipolytica for producing biomolecules from xylose, and thus demonstrates the utility of this approach as a routine tool for metabolic engineering.     

Constructing E. coli Co‐Cultures for De Novo Biosynthesis of Natural Product Acacetin

Modular co‐culture engineering is an emerging approach for biosynthesis of complex natural products. In this study, microbial co‐cultures composed of two and three Escherichia coli strains, respectively, are constructed for de novo biosynthesis of flavonoid acacetin, a value‐added natural compound possessing numerous demonstrated biological activities, from simple carbon substrate glucose. To this end, the heterologous biosynthetic pathway is divided into different modules, each of which is accommodated in a dedicated E. coli strain for functional expression. After the optimization of the inoculation ratio between the constituent strains, the engineered co‐cultures show a 4.83‐fold improvement in production comparing to the mono‐culture controls. Importantly, cultivation of the three‐strain co‐culture in shake flasks result in the production of 20.3 mg L^?1 acacetin after 48 h. To the authors' knowledge, this is the first report on acacetin de novo biosynthesis in a heterologous microbial host. The results of this work confirm the effectiveness of modular co‐culture engineering for complex flavonoid biosynthesis.     

RNAi-mediated suppression of the phenylalanine ammonia-lyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis

Medicinal Salvia miltiorrhiza contains two main groups of active pharmaceutical ingredients: lipid-soluble tanshinones and water-soluble phenolic acids, including rosmarinic acid and salvianolic acid B. Phenylalanine ammonia-lyase (PAL) catalyzes the first step in the phenylpropanoid pathway and is assumed to be closely related to the accumulation of rosmarinic acid and its derivatives. We selected a 217-bp fragment, located at the 3′ end of the coding region of PAL1, to establish an RNA interference construct that was introduced into S. miltiorrhiza via Agrobacterium tumefaciens-mediated transformation. PAL-suppressed plants exhibited several unusual phenotypes such as stunted growth, delayed root formation, altered leaves, and reduced lignin deposition. The total phenolic content was decreased by 20–70% in PAL-suppressed lines, and was accompanied by lower PAL activity. Down-regulation of PAL also affected the expression of C4H, 4CL2, and TAT, which are related genes in the rosmarinic acid pathway. Moreover, rosmarinic acid and salvianolic acid B were markedly reduced in PAL-suppressed lines, as detected by HPLC analysis. Our results indicate that PAL is very important for the synthesis of major water-soluble pharmaceutical ingredients within S. miltiorrhiza.     

Morpho-anatomical and physiological changes of Indian sandalwood (Santalum album L.) plantlets in ex vitro conditions to support successful acclimatization for plant mass production

Santalum album L. (Indian sandalwood) is an economically important but vulnerable tropical tree species. Cultures were established via direct shoot regeneration from axillary buds on Murashige and Skoog (MS) medium supplemented with 2.5 mg L^?1 6-benzylaminopurine (BAP). The shoots were multiplied using MS medium containing 1.0 mg L^?1 BAP and 0.5 mg L^?1 indole-3 acetic acid and rooted on half strength MS medium containing 1.0 mg L^?1 indole-3 butyric acid. The rooted plantlets were hardened and acclimatized in greenhouse using soilrite® and cocopeat (1:1) mixture. The concentrations of photosynthetic pigments were analyzed and detected less under in vitro conditions (6.05 μg g^?1 FW) as compared to the 4 weeks old hardened (6.91 μg g^?1 FW) and 12 weeks old acclimatized plantlets (7.8 μg g^?1 FW) under greenhouse (ex vitro) environment. The anatomical evaluation of plantlets at subsequent stages of propagation suggested that the in vitro raised plantlets possessed structural abnormalities such as underdeveloped cuticle, unorganized tissue systems, reduced mesophyll tissues, fewer vascular elements and mechanical tissues, and loosely arranged thin walled paranchymatous ground tissues, which were slowly repaired during ex vitro hardening and acclimatization process to validate the developmental adaptation of micropropagated plantlets for maximum survival in the field (98.0% survival rate). The findings could help in the optimization of high-frequency commercial micropropagation of S. album for year-round production, and supply of this economically prominent vulnerable plant species to the farmers and the industries that rely on it.

     

Experimental evaluation of using calcein and alizarin red S for immersion marking of bighead carp Aristichthys nobilis (Richardson, 1845) to assess growth and identification of marks in otoliths,scales and fin rays

In order to evaluate the effects of immersion marking with calcein (CAL) and alizarin red S (ARS) on growth and mortality of juvenile bighead carp Aristichthys nobilis, and assess mark quality in otoliths, scales, and fin rays, CAL from 50 to 200 mg L^?1 and ARS from 150 to 300 mg L^?1 concentrations were used. With the exception of non‐lateral line scales from 50 mg L^?1 CAL treatments, immersion for 24 h produced detectable marks in sagittae, lateral line and non‐lateral line scales, and fin rays (dorsal, pectoral, ventral, anal, and caudal) at 100 days post‐marking. Detectable fluorescent marks in sagittae were readily observed at concentrations of 150–200 mg L^?1 CAL or 150–300 mg L^?1 ARS. Marks were poorly visible in all non‐lateral line scales from both CAL‐ and ARS‐treated groups. Fluorescent marks were readily detected in lateral line scales at 100–200 mg L^?1 CAL or 150–300 mg L^?1 ARS, and in fin rays at 150–200 mg L^?1 CAL or 150–300 mg L^?1 ARS. In particular, optimal marks were observed at the highest concentrations investigated in sagittae (300 mg L^?1 ARS), lateral line scales (150–200 mg L^?1 CAL or 250–300 mg L^?1 ARS), and fin rays (200 mg L^?1 CAL or 250–300 mg L^?1 ARS). However, fluorescent marks visible to the naked eye were not produced by any of the CAL or ARS treatments in sagittae, scales, or fin rays during this experiment. In addition, there was no significant difference on survival and growth of marked fish compared to controls throughout the experiment (P > 0.05).     

Photoperiod and elicitors increase steviol glycosides,phenolics, and flavonoid contents in root cultures of Stevia rebaudiana

The establishment of green root cultures of Stevia rebaudiana Bertoni, and the effect of elicitors such as hydrogen peroxide (H₂O₂) and methyl jasmonate (MeJA), is shown in the present study. Stevioside, rebaudioside A, and the isomers steviol/isosteviol were identified through DFI-ESI-IT-MSⁿ and UPLC-TOFMS spectrometric systems, in combination with solid-phase extraction. The accumulation of steviol glycosides increased by 2.4 times (compared to the control value of 22.35 μgSG per gDW), with the addition of 250 μM H₂O₂. The non-enzymatic antioxidant response, which resulted from production of phenolic and flavonoid compounds, was modified based on the elicitor and the dose used. The maximum accumulation of flavonoids was induced on the third day with the addition of H₂O₂ (250 or 500 μM), and with MeJA (250 or 500 μM); the increase was observed on the fifth day. The enzymatic antioxidant response of the catalase and peroxidase from the roots under elicitation confirmed the stress conditions.

     

Elicitation of anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> and the involvement of methyl jasmonate and salicylic acid

The effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the anthocyanin accumulation, endogenous titres of polyamines and ethylene production in callus cultures of Daucus carota were studied. The interaction of these signaling molecules with elicitors from Aspergillus niger was investigated and the involvement of MeJA was elucidated through the use of the jasmonic acid (JA) biosynthetic inhibitor ibuprofen. MeJA and SA were both found to stimulate the anthocyanin production in the callus cultures. The highest levels of anthocyanin was observed in the cultures treated with 200 μM SA 0.36 % and 0.01 μM MeJA 0.37 %. The MeJA and SA treatments were also found to result in higher activity of Ca²⁺ ATPase suggesting that the enhancement of anthocyanin by SA and MeJA could be mediated through the involvement of the calcium channel. The treatment of the callus cultures with SA was found to result in marginally higher titres of endogenous polyamines (PAs) whereas MeJA resulted in lower levels of PAs as compared to the control. The SA treatment was found to result in lower ethylene production and the treatment with MeJA stimulated the ethylene production. These results suggest that the stimulation of anthocyanin production by MeJA and SA in callus cultures of D. carota is not related to the ethylene production.