Gamma irradiation of medicinally important plants and the enhancement of secondary metabolite production.

PURPOSE: The profitable production of some important plant-based secondary metabolites (ginsenosides, saponins, camptothecin, shikonins etc.) in vitro by gamma irradiation is a current area of interest. We reviewed different types of secondary metabolites, their mode of synthesis and effect of γ-radiation on their yield for different plants, organs and in vitro cultures (callus, suspension, hairy root). Special effort has been made to review the biochemical mechanisms underlying the increase in secondary metabolites. A comparison of yield improvement with biotic and abiotic stresses was made. RESULTS: Phenolic compounds increase with γ-irradiation in whole plants/plant parts; psoralen content in the common herb babchi (Psoralea corylifolia) was increased as high as 32-fold with γ-irradiation of seeds at 20 kGy. The capsaicinoids, a phenolic compound increased about 10% with 10 kGy in paprika (Capsicum annum L.). The in vitro studies show all the three types of secondary metabolites are reported to increase with γ-irradiation. Stevioside, total phenolic and flavonoids content were slightly increased in 15 Gy-treated callus cultures of stevia (Stevia rebaudiana Bert.). In terpenoids, total saponin and ginsenosides content were increased 1.4- and 1.8-fold, respectively, with 100 Gy for wild ginseng (Panax ginseng Meyer) hairy root cultures. In alkaloids, camptothecin yield increased as high as 20-fold with 20 Gy in callus cultures of ghanera (Nothapodytes foetida). Shikonins increased up to 4-fold with 16 Gy in suspension cultures of purple gromwell (Lithospermum erythrorhizon S.). The enzymes associated with secondary metabolite production were increased with γ-irradiation of 20 Gy; namely, phenylalanine ammonia-lyase (PAL) for phenolics, chalcone synthase (CHS) for flavonoids, squalene synthase (SS), squalene epoxidase (SE) and oxidosqualene cyclases (OSC) for ginsenosides and PHB (p-hydroxylbenzoic acid) geranyl transferase for shikonins. CONCLUSIONS: An increase in secondary metabolites in response to various biotic and abiotic stresses is compared with ionizing radiation. A ∼5- to 20-fold increase is noted with ∼20 Gy irradiation dose. It increases the yield of secondary metabolites by enhancing the activity of certain key biosynthetic enzymes. Identification of the optimum dose is the important step in the large-scale production of secondary metabolites at industrial level.

UV-A radiation effects on higher plants: Exploring the known unknown.

Ultraviolet-A radiation (UV-A: 315-400nm) is a component of solar radiation that exerts a wide range of physiological responses in plants. Currently, field attenuation experiments are the most reliable source of information on the effects of UV-A. Common plant responses to UV-A include both inhibitory and stimulatory effects on biomass accumulation and morphology. UV-A effects on biomass accumulation can differ from those on root: shoot ratio, and distinct responses are described for different leaf tissues. Inhibitory and enhancing effects of UV-A on photosynthesis are also analysed, as well as activation of photoprotective responses, including UV-absorbing pigments. UV-A-induced leaf flavonoids are highly compound-specific and species-dependent. Many of the effects on growth and development exerted by UV-A are distinct to those triggered by UV-B and vary considerably in terms of the direction the response takes. Such differences may reflect diverse UV-perception mechanisms with multiple photoreceptors operating in the UV-A range and/or variations in the experimental approaches used. This review highlights a role that various photoreceptors (UVR8, phototropins, phytochromes and cryptochromes) may play in plant responses to UV-A when dose, wavelength and other conditions are taken into account.

Comparison of phenolic metabolism and primary metabolism between green 'Anjou' pear and its bud mutation, red 'Anjou'.

Green 'Anjou' pear and its bud mutation, red 'Anjou' were compared to understand their differences in phenolic metabolism and its effect on primary metabolism. In the flesh of the two cultivars, no difference was detected in the concentration of any phenolic compound, the transcript level of MYB10 or the transcript levels or activities of key enzymes involved in anthocyanin synthesis. Compared with green 'Anjou', the shaded peel of red 'Anjou' had higher anthocyanin concentrations, higher transcript levels of MYB10 and higher activity of UDP-glucose:flavonoid 3-O-glycosyltransferase (UFGT), suggesting that MYB10 regulates UFGT to control anthocyanin synthesis in red 'Anjou' peel. In the sun-exposed peel, activities of phenylalanine ammonia lyase, dihydroflavonol reductase, flavonol synthase and anthocyanidin synthase as well as UFGT were higher in red 'Anjou' than in green 'Anjou'. The peel of red 'Anjou' had higher activities of sorbitol dehydrogenase, raffinose synthase and sucrose synthase and higher levels of raffinose, myo-inositol and starch, indicating that sorbitol metabolism, raffinose synthesis and starch synthesis were upregulated in red 'Anjou'. The flesh of red 'Anjou' had higher concentrations of glucose, but lower activities of ATP-dependent phosphofructokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase and lower dark respiration. The peel of red 'Anjou' had higher activities of glutaminase, asparagine synthetase and asparaginase, and higher concentrations of asparagine, aspartate, alanine, valine, threonine and isoleucine. The effects of anthocyanin synthesis on primary metabolism in fruit peel are discussed.

Aroma characterisation and UV elicitation of purple basil from different plant tissue cultures.

Exposure to stressful environmental conditions can induce severe metabolic variations in basil (Ocimum basilicum) aroma. The aromatic profiles of Dark Opal and Red Rubim varieties (in vivo plants, in vitro shoots, callus, and suspension cultures) were investigated for the first time. The established calli represented the most interesting miniaturised aromatic plant systems, as they were able to emit many typical basil volatiles with very low amounts of phenylpropanoids (1-2%). The hydrocarbon monoterpenes and oxygenated volatiles emitted from calli of both varieties were greatly and conversely affected by UV-C and UV-B, in comparison with the non-irradiated samples. As calli of both varieties still maintained very low levels of phenylpropanoids even after UV elicitation, they might be regarded not only as efficient in vitro plant models to study volatile compounds under UV stress conditions, but also as safe aromatic biomass in comparison with in vivo basil plants.

Light-induced degradation of starch granules in turions of Spirodela polyrhiza studied by electron microscopy.

Spirodela polyrhiza forms turions, starch-storing perennial organs. The light-induced process of starch degradation starts with an erosion of the surface of starch grains. The grain size decreases over a period of red irradiation and the surface becomes rougher. The existence of funnel-shaped erosion structures demonstrates that starch degradation is also possible inside the grains. Neither etioplasts nor clues as to their transition into chloroplasts were found in the storage tissue by transmission electron microscopy. Juvenile chloroplasts always contained the starch grains which remained from amyloplasts. No chloroplasts were found which developed independently of starch grains. Amyloplasts are therefore the only source of chloroplasts in the cells of irradiated turions. The intactness of amyloplast envelope membranes could not be directly proved by electron microscopy. However, the light-induced transition of amyloplasts into chloroplasts provides indirect evidence for the integrity of the envelope membranes throughout the whole process. The starch grains are sequestered from the cytosolic enzymes, and only plastid-localized enzymes, which have access to the starch grains, can carry out starch degradation. In this respect the turion system resembles transitory starch degradation as known from Arabidopsis leaves. On the other hand, with α-amylase playing the dominant role, it resembles the mechanism operating in the endosperm of cereals. Thus, turions appear to possess a unique system of starch degradation in plants combining elements from both known starch-storing systems.

Development of radiation indicators to distinguish between irradiated and non-irradiated herbal medicines using HPLC and GC-MS.

The effects of high dose γ-irradiation on six herbal medicines were investigated using gas chromatography-mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC). Herbal medicines were irradiated at 0-50 kGy with (60)Co irradiator. HPLC was used to quantify changes of major components including glycyrrhizin, cinnamic acid, poncirin, hesperidin, berberine, and amygdalin in licorice, cinnamon bark, poncirin immature fruit, citrus unshiu peel, coptis rhizome, and apricot kernel. No significant differences were found between gamma-irradiated and non-irradiated samples with regard to the amounts of glycyrrhizin, berberine, and amygdalin. However, the contents of cinnamic acid, poncirin, and hesperidin were increased after irradiation. Volatile compounds were analyzed by GC/MS. The relative proportion of ketone in licorice was diminished after irradiation. The relative amount of hydrocarbons in irradiated cinnamon bark and apricot kernel was higher than that in non-irradiated samples. Therefore, ketone in licorice and hydrocarbons in cinnamon bark and apricot kernel can be considered radiolytic markers. Three unsaturated hydrocarbons, i.e., 1,7,10-hexadecatriene, 6,9-heptadecadiene, and 8-heptadecene, were detected only in apricot kernels irradiated at 25 and 50 kGy. These three hydrocarbons could be used as radiolytic markers to distinguish between irradiated (>25 kGy) and non-irradiated apricot kernels.

Distribution of resveratrol and stilbene synthase in young grape plants (Vitis vinifera L. cv. Cabernet Sauvignon) and the effect of UV-C on its accumulation.

Current research indicated that the resveratrol was mainly accumulated in the skin of grape berry, however, little is yet known about the distribution of resveratrol, as well as the regulation mechanism at protein level and the localization of stilbene synthase (malonyl-CoA:4-coumaroyl-CoA malonyltransferase; EC 2.3.1.95; STS), a key enzyme of resveratrol biosynthesis, in young grape plants (Vitis vinifera L. cv. Cabernet Sauvignon). Resveratrol, whose constitutive level ranged from 0.2 mg kg(-1) FW to 16.5 mg kg(-1) FW, could be detected in stem, axillary bud, shoot tip, petiole, root and leaf of grape plants. Among them, stem phloems presented the most abundant of resveratrol, and the leaves presented the lowest. Interestingly, the level of STS mRNA and protein were highest in grape leaves. And the analysis of immunohistochemical showed the tissue-specific distribution of STS in different organs, presenting the similar results compared with the amount of protein. And the subcellular localization revealed that the cell wall in different tissues processed the most golden particles representing STS. Subjecting to UV-C irradiation, resveratrol and STS were both intensely stimulated in grape leaves, with the similar response pattern. Results above indicated that distribution of resveratrol and STS in grape was organ-specific and tissue-specific. And the accumulation of resveratrol induced by UV-C was regulated by transcriptional and translational level of STS.

Continuous UV-B irradiation induces endoreduplication and peroxidase activity in epidermal cells surrounding trichomes on cucumber cotyledons.

Most trichomes on the surface of cucumber (Cucumis sativus L.) cotyledons consist of three cells. We previously showed that continuous UV-B (290-320 nm) irradiation induces rapid cellular expansion and the accumulation of polyphenolic compounds, possibly stress lignin, in epidermal cells around these trichomes.(1)) To examine the mechanism of the UV-B-induced cellular expansion and to determine which step is stimulated by UV-B irradiation in the lignin synthesis pathway, we investigated relative DNA contents in epidermal cells, including trichomes, and enzyme activity and gene expression in the phenylpropanoid pathway. UV-B irradiation increased the ploidy level over 15 days, specifically in the epidermal cells surrounding trichomes, but not in the other epidermal cells or trichomes. In epidermal cells surrounding trichomes, UV-B irradiation induced peroxidase (POX) activity from days 7 to 15. In cotyledons, UV-B exposure induced CS-POX1 and CS-POX3 gene expression within 2 days, and it also induced two other enzymes in the phenylpropanoid pathway, sinapyl alcohol dehydrogenase and coniferyl alcohol dehydrogenase, from days 9 to 11. Thus, exposure to UV-B induces expansion, endoreduplication, POX activity, and the accumulation of polyphenolic compounds in epidermal cells surrounding the trichomes of cucumber cotyledons. Because polyphenolic compounds such as lignin absorb UV-B, our data indicate a physiological protective mechanism against UV-B irradiation in cucumber.

Phytochrome Interacting Factors: central players in phytochrome-mediated light signaling networks.

To adapt to the surrounding environment, plants constantly monitor and respond to changes in the red and far-red regions of the light spectrum through the phytochrome family of photoreceptors. Extensive efforts using genetic, molecular and photobiological techniques have led to the identification of a group of basic helix-loop-helix transcription factors called the Phytochrome Interacting Factors, PIFs, which directly bind to the photoactivated phytochromes. Members of the PIF family have been shown to control light-regulated gene expression directly and indirectly. PIF1, PIF3, PIF4 and PIF5 are degraded in response to light signals, and physical interaction of PIF3 with phytochromes is necessary for the light-induced phosphorylation and degradation of PIF3. PIFs constitute an excellent model for the investigation of the biochemical mechanisms of signal transfer from photoactivated phytochromes and the light-regulation of gene expression that controls photomorphogenesis in plants.

The effect of ultraviolet-depleted light on the flavonol contents of the cactus species Opuntia wilcoxii and Opuntia violacea.

An early investigation at the Biosphere-2 Laboratory, an artificial ecosystem in the Arizona desert, had shown that the flavonoid content of cacti grown in glass-filtered solar light was lower than of cacti grown in normal solar light. This was attributed to the absence of ultraviolet (UV) radiation, which is required for flavonoid biosynthesis. In this study, two species of Opuntia cacti were grown in solar and UV-depleted light, and their flavonol contents of different tissues were determined by HPLC. O. wilcoxii, previously raised in the absence of UV light, was exposed to normal solar light. The flavonol content of young O. wilcoxii pads was 28-fold higher when grown in solar light as compared to UV-depleted light. The flavonol contents of mature outer tissues were only slightly higher. O. violacea, previously raised in solar light, was also maintained in the same UV-depleted artificial ecosystem. The flavonol content after hydrolysis of outer tissues was similar, whether grown in solar light or UV-depleted light. We attribute these responses to different biosynthetic and metabolic rates of young vs. mature plant tissues; slow-growing mature tissues neither produce nor metabolize compounds as quickly as immature tissues. These findings indicate that artificial ecosystems can influence the production of natural products in cultivated plants.

Evaluation of toxic and genotoxic effects of low-level 137Cs ionising radiation on plants.

The aim of this study was to evaluate the impact of low internal exposure to 137Cs on L. sativum meristem cells and Tradescantia stamen hair cells. It also compared the impact of 137Cs internal and external irradiation of similar level on the plant seed germination and root growth. Compared to control, the tested internal (0.0007 mGy to 0.7 mGy) and external (0.04 mGy to 5.5 mGy) 137Cs ionising radiation doses stimulated the elongation of L. sativum roots by 11% to 12% and 24% to 33%, respectively. Internal 137Cs exposure (0.0003 mGy to 0.5 mGy) for 14 days caused 1.2% to 1.6% of somatic mutations and 19% to 87% of non-viable stamen hair in Tradescantia.

(137)Cs and (90)Sr uptake by sunflower cultivated under hydroponic conditions.

The (90)Sr and (137)Cs uptake by the plant Helianthus annuus L. was studied during cultivation in a hydroponic medium. The accumulation of radioactivity in plants was measured after 2, 4, 8, 16 and 32 days of cultivation. About 12% of (137)Cs and 20% of (90)Sr accumulated during the experiments. We did not find any differences between the uptake of radioactive and stable caesium and strontium isotopes. Radioactivity distribution within the plant was determined by autoradiography. (137)Cs was present mainly in nodal segments, leaf veins and young leaves. High activity of (90)Sr was localized in leaf veins, stem, central root and stomata. The influence of stable elements or analogues on the transfer behaviour was investigated. The percentage of non-active caesium and strontium concentration in plants decreased with the increasing initial concentration of Cs or Sr in the medium. The percentage of (90)Sr activity in plants decreased with increasing initial activity of the nuclide in the medium, but the activity of (137)Cs in plants increased. The influence of K(+) and NH(4)(+) on the uptake of (137)Cs and the influence of Ca(2+) on the uptake of (90)Sr was tested. The highest accumulation of (137)Cs (24-27% of the initial activity of (137)Cs) was found in the presence of 10 mM potassium and 12 mM ammonium ions. Accumulation of about 22% of initial activity of (90)Sr was determined in plants grown on the medium with 8 mM calcium ions.

Phospholipase C-dependent phosphoinositide breakdown induced by ELF-EMF in Peganum harmala calli.

With the aim of examining the response of plant cells to extremely low frequency (ELF) electromagnetic fields (EMF), we investigated the behaviour of the phosphatidylinositol 4,5 bisphosphate (PtdIns 4,5-P(2)) molecule (the precursor of the phosphoinositide signal transduction cascade) by exposing callus cells from Peganum harmala to 50 Hz, 1 gauss EMF for 10 min and by examining the level and the fatty acid composition of PtdIns 4,5-P(2) after the exposure. Our results evidenced a statistically significant decrease in PtdIns 4,5-P(2) concentrations and a different involvement of the constituting fatty acids in the induced breakdown. The manipulation of the lipid-based signalling pathway by phosphoinositide-phospholipase C (PI-PLC) inhibitors (i.e., neomycin, U-73122 and ET-18-OCH(3)) seems to support the hypothesis that, as in animals, also in plants, the cell membrane is the primary impact site of ELF electromagnetic stimulus and that this interaction could probably involve the activation of PI signal transduction pathway including a heterotrimeric G protein.

Light induces phosphorylation of glucan water dikinase, which precedes starch degradation in turions of the duckweed Spirodela polyrhiza.

Degradation of storage starch in turions, survival organs of Spirodela polyrhiza, is induced by light. Starch granules isolated from irradiated (24 h red light) or dark-stored turions were used as an in vitro test system to study initial events of starch degradation. The starch-associated pool of glucan water dikinase (GWD) was investigated by two-dimensional gel electrophoresis and by western blotting using antibodies raised against GWD. Application of this technique allowed us to detect spots of GWD, which are light induced and absent on immunoblots prepared from dark-adapted plants. These spots, showing increased signal intensity following incubation of the starch granules with ATP, became labeled by randomized [betagamma-33P]ATP but not by [gamma-33P]ATP and were removed by acid phosphatase treatment. This strongly suggests that they represent a phosphorylated form(s) of GWD. The same light signal that induces starch degradation was thus demonstrated for the first time to induce autophosphorylation of starch-associated GWD. The in vitro assay system has been used to study further effects of the light signal that induces autophosphorylation of GWD and starch degradation. In comparison with starch granules from dark-adapted plants, those from irradiated plants showed increase in (1) binding capacity of GWD by ATP treatment decreased after phosphatase treatment; (2) incorporation of the beta-phosphate group of ATP into starch granules; and (3) rate of degradation of isolated granules by starch-associated proteins, further enhanced by phosphorylation of starch. The presented results provide evidence that autophosphorylation of GWD precedes the initiation of starch degradation under physiological conditions.

N,beta-D-Glucopyranosyl vincosamide, a light regulated indole alkaloid from the shoots of Psychotria leiocarpa.

From leaves of Psychotria leiocarpa, an indole alkaloid was isolated to which the structure N,beta-D-glucopyranosyl vincosamide (1) was assigned. This represents the first report of an N-glycosylated monoterpenoid indole alkaloid. In field-grown plants highest amounts of 1 were found in the leaves (2.5% of dry wt) and fruit pulp (1.5% dry wt). Lower amounts were found in the stems (0.2% dry wt) and the seeds (0.1% of dry wt), whereas the alkaloid was not detected in the roots. The accumulation of 1 in aseptic seedlings was also restricted to the shoots and increased with plant age and light exposure, independent of the supply of sucrose in the culture medium.

[Mechanism of induction of cytogenetic damage in plant meristematic cells caused by combined effect of heavy natural radionuclides and heavy and alkaline metals]. / Zakonomernosti induktsii tsitogeneticheskikh narushenii v kletkakh meristem rastenii pri sochetannom deistvii tiazhelykh estestvennykh radionuklidov, tiazhelykh i shchelochnykh metallov.

The analysis of experimental data on the combined effect of 232Th and water from melted snow containing metal ions from industrally loaded territories on Tradescantia (clone 02) and on Allium shoenoprasum meristematic root tip cells treated with heavy natural radionuclides from natural water sources in the area of radioactive wastes storehouse is presented. It was demonstrated that plant meristematic cells response to heavy metals and heavy natural radionuclides, both from two-component model system and from natural water sources, have commom features.

Direct current electrical field effects on intact plant organs.

Intact plant tissues (of hypocotyls, radicles, cotyledons and leaves) were contracted by applying a low DC electrical field through them. Stomatal opening as a result of the electrical treatment of leaves was observed, presumably due to the differential influence of the electrical treatment on guard cell turgor pressure versus turgor pressure of the surrounding epidermal cells. In addition, leakage of minerals from the treated leaves was detected (higher contents of potassium, sodium, calcium and sulfur), as was leakage of betanin from electrically treated red beet roots (higher OD value of the immersion solution with increasing time of applied electrical field). Application of such a treatment can be used for initial drying or as part of another more drastic drying method. The advantages of this method lie in its nonthermal character and its potential to increase the quality of processed foods by maintaining their "like-fresh" quality, e.g., fruits and vegetables that are less damaged by browning. An understanding of the mechanism involved in this nonthermal application can help in controlling the process and predicting its outcome.

[Changes in vectors of endogenously generated ion currents in light-induced germination of turions of Spirodela polyrrhiza]. / Izmenenie vektorov éndogenno-generiruemykh ionnykh tokov pri svetoindutsirovannom prorastanii turionov Spirodela polyrrhiza.

Nondormant turions of Spirodela polyrhiza (L.) Schleiden were utilized to investigate endogenous ion currents in light-induced germination and early growing processes of higher plants. A small outward current was detected at the ventral side of the turions near the pocket containing the most developed sprout primordium. After a light pulse, the direction of the endogenous current changed from outward to inward. These ion currents are most likely conditioned by unspecific diffusion of cations (probably H+) inside the cell. Three-day-old sprouts of Spirodela showed the highest inward current near the sprout base which decreases toward its edge. Newly formed sprouts demonstrated a strong gravity effect (bending), which was preceded by a lowering of the Z-component of vectors close to the sprout base after a change of the turion fixation.

Early development in fern gametophytes: interpreting the transition to prothallial architecture in terms of coordinated photosynthate production and osmotic ion uptake.

Gametophytes of Onoclea sensiblis L. were grown under various light and media-ion conditions to gain a better understanding of the source/sink relationships between photosynthetic and ion-absorbing cells. There was a clear interdependency between green cell and rhizoid functions, such that the growth and development of the rhizoids was completely dependent on the internal delivery of photosynthates from green cells, and conversion of the one-dimensional filament into the two-dimensional prothallus required monovalent cations that could only be provided by rhizoid uptake. The need for monovalent cations was related to osmotic demands of dividing and expanding cells; prothallial development was blocked by monovalent cation deficiency, and the system resorted to Na+ uptake to support cell expansion when K+ was absent. Surgical excisions of filament cells further demonstrated the high degree of coordinated growth between the light-absorbing and ion-absorbing regions. It was also learned that excised sub-apical cells of the protonemata, like the intensively studied apical cell, were capable of remodelling remnants of the filament into a normal prothallus.

Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch.

The phototropins are flavoprotein kinases that control phototropic bending, light-induced chloroplast movement, and stomatal opening in plants. Two flavin mononucleotide binding light, oxygen, or voltage (LOV) domains are the sites for initial photochemistry in these blue light photoreceptors. We have determined the steady state, photoexcited crystal structure of a flavin-bound LOV domain. The structure reveals a unique photochemical switch in the flavin binding pocket in which the absorption of light drives the formation of a reversible covalent bond between a highly conserved Cys residue and the flavin cofactor. This provides a molecular picture of a cysteinyl-flavin covalent adduct, the presumed signaling species that leads to phototropin kinase activation and subsequent signal transduction. We identify closely related LOV domains in two eubacterial proteins that suggests the light-induced conformational change evident in this structure is an ancient biomolecular response to light, arising before the appearance of plants.