Genetic Diversity,Genotype Discrimination,and Population Structure of Mexican <Emphasis Type="Italic">Opuntia</Emphasis> sp., Determined by SSR Markers

The Opuntia (prickly pear) genus, an important horticultural crop in Mexico, is essentially a fruit crop with two variants: sweet (“tunas”) or acid (“xoconostles”) fruits; it is also a source of vegetables “nopalitos” or fodder for livestock, among other uses. Its taxonomical classification has been reported as complex, although few studies on the genetic structure of Mexican Opuntia are available, and genetic differences between the two types of fruits are unknown. Opuntia genotype identification and classification are still mainly based on morphological characters. In this study, the genetic diversity of Mexican Opuntia germplasm with agronomic and economic importance was revealed, using 88 accessions and 13 SSR markers, in an attempt to explore the genetic relationships among them. A total of 159 alleles were detected ranging from 7 to 23 per locus with an average of 12.2. The SSR markers generated unique fingerprints for each Opuntia accession confirming their usefulness for genetic analysis. The accessions’ grouping was defined by several complementary clustering methods, and the moderate incongruences between the different methods did not influence the overall clustering. DAPC and STRUCTURE analyses grouped the accessions into five groups, thus confirming the incorrect delimitation of species in this genus. The following species had no clear boundaries: Opuntia ficus-indica, Opuntia albicarpa, Opuntia megacantha, Opuntia streptacantha, Opuntia lasiacantha, and Opuntia hyptiacantha. However, Opuntia robusta was separated from the rest of the species. Opuntia joconostle and Opuntia matudae, which produce acid fruits, tended to differ from the others. Median-joining simulation classified all genotypes into a complex network, and both linear and reticular ties between Mexican Opuntia genotypes were revealed. The genetic distance revealed in the present study shows the importance of Mexican accessions for conservation and use in breeding programs.     

Genetic diversity of <Emphasis Type="Italic">Opuntia</Emphasis> spp. varieties assessed by classical marker tools (RAPD and ISSR)

Opuntia, commonly named “nopal” in Mexico, is an important crop for its agronomical, economical, ecological and cultural value. Furthermore, it is known for its taxonomic complexity. In this paper, we report the genetic variability of 52 Opuntia cultivars with agronomic and economic importance, classified into 12 different species using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. Ten primers, five for each marker type, were selected to assess their ability to detect polymorphisms in this plant accesions/varieties. Both marker systems generated a total of 307 bands, of which 50.8 % were polymorphic with an average of 15.6 polymorphic bands per primer. Thus, we assume that Mexican Opuntia varieties present broad genetic variation. Based on percentage of polymorphic bands; resolving power; polymorphic information content; and Marker Index, the K-12 (RAPD) and IS-06 (ISSR) primers were the most informative ones. Clusters obtained from RAPD, ISSR and a combination of both data sets did not match the actual taxonomic classification. On the other hand, the putative varieties currently classified in the same species were not located in the same cluster. Besides, the varieties included in O. ficus-indica, O. albicarpa and O. megacantha showed broad variation but were not well defined into separate clades; these cultivars possibly have common ancestry. The results presented here support our hypothesis about the existence of a smaller number of Opuntia species in accordance with those currently described, but with high intraspecific genetic variation.     

MeJA is more effective than JA in inducing defense responses in <Emphasis Type="Italic">Larix olgensis</Emphasis>

The roles of jasmonic acid (JA) and methyl jasmonate (MeJA) in improving the inducible resistance of plants to biotic and abiotic stimuli/stresses have been well investigated. However, the differences in inducing effects between exogenous applications of JA and MeJA are poorly understood. In this study, we compared the inducing effects of exogenous spray applications of 0.1 mmol/L JA and MeJA onto four un-bagged lateral branches on defense response of Larix olgensis seedlings against the gypsy moth (Lymantria dispar). The bio-activities of three major defense enzymes (SOD, PAL, and PPO) plus two protease inhibitors (TI and CI) of the unsprayed larch seedling needles, and the growth, development and reproductive capacity of the gypsy moth were examined. The results show that partial spray of JA or MeJA on L. olgensis seedlings significantly increased the bio-activities of SOD, PAL, PPO, TI, and CI (P < 0.05), and strongly decreased the larval/pupal weights and survivals, as well as the fecundity of L. dispar that fed on the seedlings relative to the control. However, the MeJA treatment showed quicker inductive effects on SOD and PAL activities; longer and more significant effects on PPO, TI, and CI activities; better inhibitory effects on the larval/pupal weights and survivals, as well as the fecundity of L. dispar than did the JA treatment. Comparatively, MeJA in the current study showed stronger effects on inducing systemic resistance to the defoliator (L. dispar) in L. olgensis than did JA.     

Content of Osmolytes and Flavonoids under Salt Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Plants Defective in Jasmonate Signaling

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 μM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range in leaves. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.     

Protection from uv radiation in the economic crop, <Emphasis Type="Italic">Opuntia</Emphasis> SPP.

Cacti of the genus puntia are an economically important crop. Understanding the mechanisms they possess to protect against UV radiation is important for assessing their possible response to climatic change. Measurements of the concentrations of UV-screening compounds and epidermal transmittance for two species of platyopuntia, Opuntia engelmannii Salm-Dyck. and O. phaeacantha Engelm. during 1998 and 1999 were used to investigate the UV-protection afforded by the cactus epidermis. A UV-radiative transfer model was used to investigate the interception of UV radiation on differently oriented surfaces. We show that vertical morphology itself confers significant protection against UV radiation compared to a horizontal surface. Concentrations of UV-screening flavonoids were found to vary depending on the UV exposure of different surfaces. West-facing surfaces had lower concentrations than east-facing surfaces, although theoretically they should be identical. This might be explained by the higher mean temperatures on west-facing surfaces. Although UV-absorbing soluble flavonoids in the epidermis block both UV-B and UV-A, the structure of the epidermis alone may be sufficient to remove up to 94% of the UV-B portion of the spectrum. These data yield insights into possible mechanisms of recent declines in cacti populations.     

Evaluation of flavonoids from <Emphasis Type="Italic">Zostera asiatica</Emphasis> as antioxidants and nitric oxide inhibitors

Zostera asiatica is one of the five members of the genus Zostera that can be found in Korea. Studies have reported the phytochemical properties and bioactivities of Zostera species. Current study focused on the antioxidant effects of Z. asiatica as a part of ongoing research for bioactive substances from marine resources. Results indicated that a crude extract of Z. asiatica not only scavenged on peroxynitrite in vitro and on intracellular reactive oxygen species (ROS) but also inhibited production of nitric oxide (NO). The crude extract was subjected to solvent fractionation for bioactivity-based separation using aforementioned three bioassay systems. From the active n-butanol fraction, two flavonoids were isolated and characterized as luteolin (1) and luteolin-3’-sulfate (2). Both flavonoids showed significant antioxidant effects. In conclusion, Z. asiatica was demonstrated to possess antioxidant effect partly attributed to isolated flavonoids, the first such effect reported from Z. asiatica, to the best of our knowledge.     

Identification and comparative analysis of <Emphasis Type="Italic">Brassica juncea</Emphasis> pathogenesis-related genes in response to hormonal,biotic and abiotic stresses

Pathogenesis-related proteins (PRs) are the antimicrobial proteins which are commonly used as signatures of defense signaling pathways and systemic acquired resistance. However, in Brassica juncea most of the PR proteins have not been fully characterized and remains largely enigmatic. In this study, full-length cDNA sequences of SA (PR1, PR2, PR5) and JA (PR3, PR12 and PR13) marker genes were isolated from B. juncea and were named as BjPR proteins. BjPR proteins showed maximum identity with known PR proteins of Brassica species. Further, expression profiling of BjPR genes were investigated after hormonal, biotic and abiotic stresses. Pre-treatment with SA and JA stimulators downregulates each other signature genes suggesting an antagonistic relationship between SA and JA in B. juncea. After abscisic acid (ABA) treatment, SA signatures were downregulated while as JA signature genes were upregulated. During Erysiphe cruciferarum infection, SA- and JA-dependent BjPR genes showed distinct expression pattern both locally and systemically, thus suggesting the activation of SA- and JA-dependent signaling pathways. Further, expression of SA marker genes decreases while as JA-responsive genes increases during drought stress. Interestingly, both SA and JA signature genes were induced after salt stress. We also found that BjPR genes displayed ABA-independent gene expression pattern during abiotic stresses thus providing the evidence of SA/JA cross talk. Further, in silico analysis of the upstream regions (1.5 kb) of both SA and JA marker genes showed important cis-regulatory elements related to biotic, abiotic and hormonal stresses.     

Production of phenolic compounds,antioxidant and antimicrobial activities in hairy root and shoot cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.

Three hairy root clones of Hypericum perforatum (HR 2, HR 15 and HR 27) transformed with Agrobacterium rhizogenes A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-p-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-O-methylkaempferol-O-hexoside and quercetin 6-C-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-C-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa and Escherichia coli. Altogether, H. perforatum HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties.     

Exogenous 5-aminolevulinic acid alleviates the detrimental effects of UV-B stress on lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L) seedlings

One of the abiotic stress factors affecting plant metabolism is ultraviolet-B (UV-B) radiation. 5-Aminolevulinic acid (ALA), a key precursor of porphyrin biosynthesis, promotes plant growth and crop yields. To investigate the alleviating effects of exogenous ALA on the damages caused by UV-B exposure, two different concentrations [10 ppm (ALA1) and 25 ppm (ALA2)] of ALA were applied to lettuce seedlings for 24 h and then they were exposed to 3.3 W m^?2 UV-B. Results showed that UV-B treatment significantly decreased chlorophyll a and b (Chl a and b) concentration, enhanced the activity of antioxidant enzymes, total phenolic concentration, soluble sugar contents, expression of phenylalanine ammonia lyase (PAL) and γ-tocopherol methyltransferase (γ-TMT) genes, the concentration of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and the rate of superoxide radical (\({\text{O}}_{2}^{ - }\)) generation in the lettuce seedlings when compared to the control. Pre-treatment with exogenous ALA significantly enhanced UV-B stress tolerance in lettuce seedlings by decreasing the reactive oxygen species. On the other hand, ALA application caused more increases in the PAL and γ-TMT gene expression, antioxidant enzymes activities, Chl a and b concentration, total phenolic content, antioxidant capacity and the concentrations of soluble sugars. Obtained results indicated that UV-B radiation exerts an adverse effect on lettuce seedlings, and some of the negative effects of UV-B radiation can be alleviated by exogenous ALA.     

Phytochemistry and bioactivity of <Emphasis Type="Italic">Citrus</Emphasis> flavonoids: a focus on antioxidant,anti-inflammatory,anticancer and cardiovascular protection activities

Epidemiological studies have suggested an inverse relationship between increased consumption of fruits and reduced risk of chronic diseases, such as cardiovascular diseases, cancer, and diabetes. Citrus fruit is one of the mostly consumed fruits worldwide, and numerous studies have revealed its remarkable health-promoting activities, such as antioxidant, anticancer, anti-inflammatory, and cardiovascular protection activities. These activities largely depend upon the diverse chemical constituents of Citrus fruits, including vitamins, minerals, terpenoids, and flavonoids. Notably, dietary flavonoids occurring in Citrus fruits have attracted growing interest due to their distinct beneficial effects on human health. In this review, we outlined the main health-related properties of Citrus flavonoids, with a focus on antioxidant, anticancer, anti-inflammation, and cardiovascular protection activities. Also the bioavailability, a critical factor that influences the biological efficacy, of Citrus flavonoids was discussed. It was believed that insights about these advances may encourage researchers to discover new phytochemical components and further study specific bioactivities from Citrus fruits.     

Changes in phytochemical content and pharmacological activities of three <Emphasis Type="Italic">Chlorella</Emphasis> strains grown in different nitrogen conditions

The phytochemical content and biological activity of three Chlorella strains cultured in low (35 mg L^?1) or high (700 mg L^?1) nitrogen (N) and harvested on days 5 and 10 were evaluated. High N resulted in a higher biomass in Chlorella MACC 438 and MACC 452 while MACC 555 produced a higher biomass in low N. MACC 555 (low N/day 5) had the highest phenolic content, and MACC 438 in low N/day 5 and high N/day 5 accumulated the highest flavonoids and condensed tannins, respectively. Iridoids were most abundant in MACC 452 on low N/day 10. Harvest time had the greatest effect on the phytochemical content with phenolics, flavonoids, and condensed tannins decreasing over time and iridoids increasing in low N and decreasing in high N conditions. Extracts were more active in β-carotene-linoleic acid model compared to 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. Most extracts had good antimicrobial activity. Extracts became less potent over time in the antioxidant, acetylcholinesterase inhibitory (AChE), and antimicrobial assays when growing in low N and more potent in the antioxidant and AChE assays when grown in high N. Thus, phytochemical content and biological activities of the three Chlorella strains were affected by N levels, harvest time, and strain.     

Overexpression of the receptor-like cytoplasmic kinase gene <Emphasis Type="Italic">XCRK</Emphasis> enhances Xoc and oxidative stress tolerance in rice

In this paper, we characterized a differentially expressed receptor-like cytoplasmic kinase XCRK, which confers resistance to bacterial leaf streak (BLS). We analyzed the tissue expression of XCRK and showed that XCRK was widely expressed in multiple rice (Oryza sativa) organs, including internodes, roots, leaves and flowers. In addition, the expression of XCRK was significantly induced by ABA, salt and H₂O₂ treatments, suggesting its function in these pathways. The XCRK-overexpressing transgenic seedlings exhibited higher tolerance to Xanthomonas oryzae pv.oryzicola (Xoc) compared with the wild-type seedlings. Furthermore, XCRK-overexpressing seedlings showed stronger antioxidant capacity with reduced MDA and H₂O₂ content and higher antioxidant enzyme activities. It has been hypothesized that the enhanced Xoc tolerance was attributed to the improved expression of resistance-responsive factors positively regulated by XCRK. In accordance with this, the expression of resistance and oxidation-related genes Wrky77, Wrky13, PAL1, PR5, Fe-SOD and SodCc2 were up-regulated by the overexpression of XCRK, which might contribute collectively to the increased Xoc tolerance. Overall, overexpression of XCRK could enhance the antioxidant capacity and Xoc tolerance in rice.     

A novel co-culture system of adventitious roots of <Emphasis Type="Italic">Echinacea</Emphasis> species in bioreactors for high production of bioactive compounds

To efficiently produce bioactive compounds of Echinacea via adventitious root (AR) culture, we established an AR co-culture system of Echinacea species. ARs of different combination of Echinacea species [E. pallida (Epa), E. purpurea (Epu), and E. angustifolia (Ean)] were inoculated into 5 L balloon-type airlift bioreactors to select a suitable combination group. The biomass of ARs increased in the Epa+Epu group but decreased in co-culture groups of Epa+Ean and Epa+Epu+Ean. In the Epa+Epu group, the content and productivity of total phenolics, flavonoids, and caffeic acid derivatives increased, a monomer of caffeic acid derivatives (caffeic acid) that is absent in single-species cultures (Epa or Epu) were synthesized; moreover, Epa+Epu also showed the highest antioxidant activity. The inoculum proportions in Epa+Epu significantly influenced the co-culture effect; among the proportion groups (Epa:Epu?=?1:6, 2:5, 3:4, 1:1, 4:4, 5:2, and 6:1), the Epa:Epu proportion of 4:3 was the most favorable for AR biomass and bioactive compound accumulation, and the antioxidant activity also peaked at 4:3 proportion. In addition, the co-culture system is suitable for large bioreactors (10 and 20 L), wherein the AR biomass increased without a decrease in the amount of bioactive compounds. A co-culture system was thus successfully established in this study, and AR cultures are expected to be used as an alternative raw material for the production of Echinacea-derived products.     

Hydrogen sulfide is involved in the regulation of ascorbate and glutathione metabolism by jasmonic acid in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

This study investigated the role of hydrogen sulfide (H₂S) in the regulation of the ascorbate (AsA) and glutathione (GSH) metabolism by jasmonic acid (JA) in the leaves of Arabidopsis thaliana by using H₂S scavenger hypotaurine (HT) and H₂S synthetic mutant (SALK_041918, designated Atl-cdes). The results showed that JA significantly increased the H₂S content, the activities of L-cysteine desulfhydrase (L-CDes), D-cysteine desulfhydrase (D-CDes), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), L-galactono-1,4-lactone dehydrogenase (GalLDH) and γ-glutamylcysteine synthetase (γ-ECS), the ratio of AsA to dehydroascorbate (DHA), and decreased the content of malondialdehyde (MDA) and H₂O₂ in the wild type of A. thaliana, compared to control. The above effects of JA except the increased activities of L-CDes and D-CDes were suppressed by addition of HT. However, JA and HT+JA had no significant effects on the ratio of reduced GSH to oxidized GSH (GSSG) in the wild type of A. thaliana. Application of HT to the control decreased H₂S content, AsA/DHA ratio, and activities of APX, GR, DHAR, MDHAR, γ-ECS, and GalLDH, but had no effects on MDA content, activities of L-CDes and D-CDes, and GSH/GSSG ratio. In the H₂S synthetic mutant, JA had no obvious effects on above mentioned parameters except the D-CDes activity compared with the control. Our results suggest that JA-induced H₂S, which is a signal that leads to the up-regulation of the AsA and GSH metabolism.     

Changes in Formation and Localization of Phenolic Compounds in the Tissues of European and Canadian Yew during Dedifferentiation <Emphasis Type="Italic">In Vitro</Emphasis>

Changes in formation and localizations of phenolic compounds, including flavans, were investigated in the tissues of European and Canadian yew (Taxus baccata L. and T. canadensis Marsh.) during dedifferentiation in vitro. Annual shoots of European yew had the highest capacity for synthesizing these compounds. During the summer growth period, the content of total soluble phenolic compounds and flavans in these shoots was 30–40% higher than in the winter. Cell dedifferentiation and growth in vitro was accompanied by enhanced synthesis of phenolic compounds, including flavans, the change in tissue localization of these compounds, and an increase in the number of cells containing phenolics. Significant accumulation of phenolic compounds in callus cells resulted in necroses following two subcultures in the European and Canadian yew cultures initiated from summer explants, and following seven subcultures of the European yew calli initiated from winter explants. These data allow us to suggest that a high level of phenolic compounds in yew calli could be the reason for their necrosis.     

Phylogeny,antimicrobial, antioxidant and enzyme-producing potential of fungal endophytes found in <Emphasis Type="Italic">Viola odorata</Emphasis>

Viola odorata, a medicinal plant, is traditionally used to treat common cold, congestion and cough. Given its medicinal properties and occurrence in the northwestern Himalayas, we isolated and characterized endophytic fungi from this plant morphologically, microscopically and by internal transcribed spacer-based rDNA sequencing. In total, we isolated 27 morphotypes of endophytes belonging to phyla Ascomycota and Basidiomycota. The roots showed the highest diversity of endophyte as well as fungal dominance, followed by leaves and leaf nodes. The fungal extract of VOR16 (Fusarium oxysporum) displayed potent antimicrobial activity against Salmonella typhimurium, Klebsiella pneumoniae and Escherichia coli, with a minimum inhibitory concentration of 0.78, 0.78 and 1.56 μg/mL, respectively, while fungal extract VOLF4 (Aspergillus sp.) exhibited promising antioxidant activity (IC₅₀ of 17.4 μg/mL). To identify the components responsible for various bioactivities, we analyzed the content of penicillin G in the extract of bioactive endophytes. The results suggested that the expression of penicillin G under the fermentation conditions applied was too low to display antimicrobial effects. Thus, the activity may be contributed by a different, novel secondary metabolite. The antioxidant activity of VOLF4 may be attributed to its high content of flavonoids. Of the endophytic fungi assessed, 27% were found to be enzyme producers. The highest zone of clearance was observed in VOLN5 (Colletotrichum siamense) for protease production. Only VOR5 (Fusarium nematophilum) was found to be a producer of cellulase, glutenase, amylase and protease. In summary, this is the first report of the isolation of endophytes, namely Fusarium nematophilum, Colletotrichum trifolii, C. destructivum, C. siamense and Peniophora sp., from V. odorata and their bioactive and enzyme-producing potential.