Diurnal changes in photosynthesis and antioxidants of Angelica sinensis as influenced by cropping systems

Diurnal dynamics of photosynthetic character of Angelica sinensis, as well as effect of continuous cropping on leaf photosynthetic character, antioxidant enzyme activity and growth of A. sinensis were investigated under field condition. The results showed that the diurnal net photosynthetic rate of A. sinensis in sunny day exhibited a double-peak pattern, and the peaks occurred at 9:45 and 16:45 h, respectively. There was a significant midday depression with A. sinensis, which was caused principally by stomatal factors such as stomatal conductance. The results also showed that net photosynthetic rate (P _N), transpiration rate (E), stomatal conductance (g _s), intercellular CO₂ concentration (C _i), and chlorophyll content (Chl) of A. sinensis plants under continuous cropping were significantly lower than those of the control. The activity of total superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and growth parameters of A. sinensis plants were significantly decreased under continuous cropping condition. This study provides evidence of continuous cropping obstacle effect on photosynthesis, antioxidant enzyme activity, and growth parameters of A. sinensis in a field experiment, which partly explained the yield reduction of A. sinensis in the field, when it was cultivated continuously on the same soil.     

Polyphenolic compounds in the fruits of Egyptian medicinal plants (Terminalia bellerica, Terminalia chebula and Terminalia horrida): Characterization, quantitation and determination of antioxidant capacities

Thirty-four polyphenolic substances in methanol extracts of the fruits of Terminalia bellerica, Terminalia chebula and Terminalia horrida, three plants used in Egyptian folk medicine, were initially identified by HPLC-ESI-MS and quantitated by analytical HPLC after column chromatography on Sephadex LH-20. After purification by semi-preparative HPLC the compounds were identified by their mass and fragmentation patterns using ESI-MS-MS. For several compounds detailed ¹H/¹³C NMR analysis at 600 MHz was performed. Two polyphenolics, namely 4-O-(4″-O-galloyl-α-l-rhamnopyranosyl)ellagic acid and 4-O-(3″,4″-di-O-galloyl-α-l-rhamnopyranosyl)ellagic acid were identified by NMR. Antioxidant capacities of the raw fruit extracts and the major isolated substances were determined using the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), oxygen radical absorbance capacity (ORAC) and ferric reducing ability of plasma (FRAP) in vitro assays and indicated that chebulic ellagitannins have high activity which may correlate with high potential as cancer chemopreventive agents. Therefore, further studies (metabolism, bioavailability and toxicity) of the polyphenolics in Terminalia species using preclinical models and in vivo human intervention trials are warranted.     

Novel Endophytic Nitrogen-Fixing Clostridia from the Grass Miscanthus sinensis as Revealed by Terminal Restriction Fragment Length Polymorphism Analysis

Anaerobic nitrogen-fixing consortia consisting of N₂-fixing clostridia and diverse nondiazotrophic bacteria were previously isolated from various gramineous plants (K. Minamisawa, K. Nishioka, T. Miyaki, B. Ye, T. Miyamoto, M. You, A. Saito, M. Saito, W. Barraquio, N. Teaumroong, T. Sein, and T. Tadashi, Appl. Environ. Microbiol. 70:3096-3102, 2004). For this work, clostridial populations and their phylogenetic structures in a stand of the grass Miscanthus sinensis in Japan were assessed by a 16S rRNA gene-targeted terminal restriction fragment length polymorphism (TRFLP) analysis combined with most-probable-number (MPN) counts. PCR primers and restriction enzymes were optimized for analyses of the plant clostridia. Clostridia were detected in strongly surface-sterilized leaves, stems, and roots of the plants at approximately 10⁴ to 10⁵ cells/g of fresh weight; they made up a large proportion of N₂-fixing bacterial populations, as determined by MPN counts associated with an acetylene reduction assay. Phylogenetic grouping by MPN-TRFLP analysis revealed that the clostridial populations belonged to group II of cluster XIVa and groups IV and V of cluster I; this result was supported by a culture-independent TRFLP analysis using direct DNA extraction from plants. When phylogenetic populations from M. sinensis and the soil around the plants were compared, group II clostridia were found to exist exclusively in M. sinensis.     

Chemical constituents from the petioles and leaves of Aquilaria sinensis

Nineteen reported compounds (1–19) were obtained from the dried petioles and leaves of Aquilaria sinensis (Thymelaeacea) by phytochemical methods. Their structures were determined on the basis of spectroscopic methods. Among them, compounds 6–14 and 16 were firstly obtained from the genus Aquilaria, 2 and 3 were obtained from A. sinensis for the first time. In addition, the chemotaxonomic relationships between A. sinensis and some other plants were also discussed.     

Diversity of Endophytic Bacterial Populations and Their Interaction with Xylella fastidiosa in Citrus Plants

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.     

Establishment of <Emphasis Type="Italic">Miscanthus sinensis</Emphasis> with decreased lignin biosynthesis by <Emphasis Type="Italic">Agrobacterium</Emphasis>–mediated transformation using antisense <Emphasis Type="Italic">COMT</Emphasis> gene

This study was to determine a transformation system for Miscanthus sinensis, and to optimize factors and conditions required for expression of an antisense caffeic acid O-methyltransferase gene in the M. sinensis (MsCOMT-AS). Transformation of callus derived from seeds and immature inflorescences of M. sinensis was established by using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pMBP1. In order to establish the stable transformation system, several transformation factors such as explant type, strain, co-culture periods, acetosyringone concentration, and selective markers were assessed. In this study, seven putative transgenic plants were obtained from callus transformation and plantlet regeneration. Various tests including PCR analysis and RT-PCR were used to detect the transgenic insert. The transgenic plants were also characterized for their agronomic and morphological characteristics, expression of MsCOMT-AS gene, and variation in lignocellulosic content. Biomass related traits such as plant height, number of leaves, length of leaf, stem diameter, fresh weight, dry weight, and cell size of the control plants were superior to transgenic plants. Total lignin content of transgenic plants was lower than that of the control plant due to reduced caffeic acid O-methyltransferase (COMT) gene expression related to lignin production. Cellulose and hemicellulose content in transgenic plants were not increased. Variation in cellulose and hemicellulose content had no correlation with variation in lignin content of transgenic plants. In conclusion, transgenic M. sinensis was obtained with down-regulated COMT gene. Lignin synthesis was decreased what offers possibility of crop modification for facilitated biofuel production.     

Biochemical characterization of sericin isolated from cocoons of tropical tasar silkworm Antheraea mylitta raised on three different host plants for its prospective utilization

Sericin were isolated and characterized from tasar cocoons raised in three different food plants i.e. Terminalia arjuna, T. tomentosa and Shorea robusta for its applications. Their molecular composition, structure and physical nature were determined by elemental analysis, amino acid analysis, Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC) analysis. These results show that tasar food plants influence the physical and chemical properties of sericin. Further, sericin isolated from cocoons of S. robusta food plant shows better antioxidant potential and inhibition of tyrosinase, elastase and glutathione-S-transferase activity than other food plants. This may be attributed to its amino acid variations and associated phenolic content. The present study appears to be useful in utilizing tasar sericin as a potential bio-molecule for its prospective utilization in pharmaceuticals and its associated fields.     

云南不同土壤铅背景值下大叶茶种群对铅的吸收积累特征及其遗传分化

在具有不同土壤铅(Pb)背景值的云南大叶茶主产区,分析了11个大叶茶种群（简写为P1, P2, …… P11）所在地的土壤Pb含量,相应各种群的老、嫩叶Pb含量和富集系数,并利用ISSR分子标记研究了这些种群的遗传特征,以期认识不同大叶茶种群在不同Pb背景值下对Pb的吸收积累特征及其遗传分化状况。结果表明：（1）在本研究区域内,大叶茶种群间土壤Pb含量、老叶和嫩叶Pb含量、富集系数差异显著,土壤有效Pb、嫩叶Pb含量分别在0.78—15.20mg/kg和2.03—7.02 mg/kg之间,嫩叶Pb富集系数变化范围为0.001—0.24;种群内差异小,例如P6种群内嫩叶Pb含量在2.82—2.84 mg/kg之间,嫩叶Pb富集系数变幅为0.09—0.10。（2）筛选的10个ISSR引物扩增出81条带,平均多态位点百分率（PPB）为75.25%;Shannon’s指数（I）估算出种群间的变异为34.28%,利用POPGENE软件计算出种群间遗传分化系数GST为0.3116,分子方差分析（AMOVA）也显示种群间变异占35.37%（P<0.001）,表明不同种群的大叶茶出现了遗传分化。（3）UPGMA聚类分析发现,11个种群可分为5个类群,对Pb吸收累积能力高的与能力低的种群在聚类分析中存在明显分异;相关性分析表明,土壤有效Pb含量与PPB、I、Nei’s基因多样性指数（H）的 Pearson相关系数r分别为-0.633,-0.786,-0.581（P<0.05）,土壤有效Pb含量与大叶茶种群遗传多样性水平程度不同呈负相关。讨论分析认为,在土壤Pb高背景值条件下,部分大叶茶种群遗传多样性水平降低,不同种群对Pb的吸收累积能力存在明显差异,种群间出现了显著的遗传分化。对低铅富集的遗传分化现象的深入研究将可能为遴选拒吸收污染物的洁净种质、在污染条件下进行无公害生产提供新途径。     

Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment

Isolation of intact and pure genomic DNA (gDNA) is essential for many molecular biology applications. It is difficult to isolate pure DNA from mature trees of hot and dry desert regions because of the accumulation of high level of polysaccharides, phenolic compounds, tannins etc. We hereby report the standardized protocol for the isolation and purification of gDNA from seven ecologically and medically important tree species of Combretaceae viz. Anogeissus (Anogeissus sericea var. nummularia, Anogeissus pendula, and Anogeissus latifolia) and Terminalia (Terminalia arjuna, Terminalia bellirica, Terminalia catappa and Terminalia chebula). This method involves (i) washing the sample twice with Triton buffer (2%) then (ii) isolation of gDNA by modified-CTAB (cetyl trimethyl ammonium bromide) method employing a high concentration (4%) of PVP (Polyvinylpyrrolidone) and 50 mM ascorbic acid, and (iii) purification of this CTAB-isolated gDNA by spin-column. gDNA isolated by modified CTAB or spin-column alone were not found suitable for PCR amplification. The Triton washing step is also critical. The quality of DNA was determined by the A₂₆₀/A₂₈₀ absorbance ratio. gDNA was also observed for its intactness by running on 0.8% agarose gel. The suitability of extracted DNA for PCR was tested by amplification with RAPD primers, which was successful. Further, rbcLa (barcoding gene) was amplified and sequenced to check the quality of extracted gDNA for its downstream applications.     

A real-time qPCR assay to quantify Ophiocordyceps sinensis biomass in Thitarodes larvae

Ophiocordyceps sinensis, an entomogenous fungus parasitic in the larvae of moths (Lepidoptera), is one of the most valuable medicinal fungi, and it only distributed naturally on the Tibetan Plateau. The parasitical amount of O. sinensis in various tissues of the host Thitarodes larvae has an important role in study the occurrence and developmental mechanisms of O. sinensis, but there no an effective method to detect the fungal anamorph. A real-time quantitative PCR (qPCR) system, including a pair of species-specific ITS primers and its related program, was developed for O. sinensis assay with high reliability and efficiency. A calibration curve was established and exhibited a very good linear correlation between the fungal biomass and the C _T values (R ²=0.999419) by the qPCR system. Based on this method, O. sinensis was detected rapidly in four tissues of its host caterpillars, and the results were shown as following: the maximum content of O. sinensis parasitized in the fat-body, and next came body-wall; both of them were much larger than that observed in the haemolymph and intestinal-wall. Taken together, these results show that qPCR assays may become useful tools for study on developmental mechanism of O. sinensis.     

Chemical composition,aphicidal and antiacetylcholinesterase activities of essential oils against Aphis nerii Boyer de Fonscolombe (Hemiptera: Aphididae)

Aphids are important sucking insects that attack many crops and cause huge economic loss. Essential oils have been proposed to be appropriate alternative to synthetic insecticides due to their low impact on environment and human health. In this work, the chemical compositions of eight essential oils extracted from plants growing in Egypt were determined by gas chromatography/mass spectrometry (GC–MS). The aphicidal activities of the isolated oils were examined against the adults of oleander aphid, Aphis nerii by a leaf-dipping assay. The inhibitory effect of essential oils on acetylcholinesterase (AChE) activity was also tested in vitro. Chemical analyses showed the essential oils consisted mainly of monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons and oxygenated sesquiterpenes except the oil of Artemisia monosperma which contained high content of benzenes. The results of toxicity assay showed that the oils of A. monosperma and Citrus sinensis were the most potent toxicants displaying LC₅₀ value of 0.06 mg/L. The oils of Callistemon viminals, Schinus terebinthifolius and Schinus molle were also highly effective against aphid as their LC₅₀ values were lower than 1 mg/L. In addition, the highest inhibitory effect on AChE was recorded by 0.5 mg/L C. sinensis with inhibition of 49.33%, while 0.1 mg/L Citrus lemon oil recorded the lowest inhibitory effect with inhibition of 0.4%. In general, six of tested essential oils showed high toxicity against A. nerii and they might be applied in the IPM programs for this insect.     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

Characterization of β-galactosidases from the germinating seeds of Vigna sinensis

Four forms of β-galactosidase from the germinating seeds of Vigna sinensis were separated and partially purified by ammonium sulphate precipitation, ion exchange chromatography (DE-52) and gel filtration to more than 50% purity as judged by PAGE. The pH and temperature optima, stability, M_r, kinetic parameters and energy of activation of each enzyme have been determined. The four forms differed in their M,_s and ionic charges.     

Distribution and biosynthesis of theanine in Theaceae plants

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (<0.2 μmol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [¹⁴C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.     

Regeneration and Agrobacterium-mediated genetic transformation of Terminalia bellerica Roxb.: a multipurpose tree species

An efficient in vitro transformation and plant regeneration protocol was developed for Terminalia bellerica using cotyledonary node cultures. High-frequency shoot bud proliferation was obtained on medium with 6-benzyladenine. Significant improvements in plant regeneration occurred using elevated levels of CuSO₄ and CoCl₂. Rooting occurred on a half-strength Murashige and Skoog medium containing indole-3-butyric acid. The rooted plants were acclimatized and transferred to field conditions. The genetic fidelity of the regenerated plants was confirmed using randomly amplified polymorphic DNA analysis. An Agrobacterium-mediated genetic transformation protocol was developed for Terminalia by varying several factors which influence T-DNA delivery. Southern blot analysis of regenerated plants confirmed selectable marker gene integration in transgenic plants. This transformation protocol can be utilized for further genetic manipulation of T. bellerica.     

Hydrogen peroxide induces vessel occlusions and stimulates sesquiterpenes accumulation in stems of Aquilaria sinensis

Agarwood is highly valuable resinous and fragrant heartwood, produced principally from tropical tree species in the genus Aquilaria, which is used widely in countries of the Middle East, Southeast Asia and Japan. Generally, healthy trees will not produce agarwood, but wounding of the tree initiates the production of agarwood. In this study, the pruning of actively growing saplings of Aquilaria sinensis resulted in hydrogen peroxide (H₂O₂) burst, which was followed by formation of vessel occlusions and sesquiterpene biosynthesis in the pruned stems. Treatment of the pruned stems with scavenger of H₂O₂ (ascorbate, AsA) greatly reduced the amount of H₂O₂ released, the number of vessel occlusions, and the amount of sesquiterpenes produced. In addition, exogenous H₂O₂ also induced A. sinensis plants to form vessel occlusions and produce sesquiterpenes as pruning treatment. The results indicated that H₂O₂ may be an important post-wounding signal in A. sinensis that leads to the induction of vessel occlusions formation and sesquiterpene biosynthesis, and thus H₂O₂ might play a vital role in agarwood formation in pruned stems of A. sinensis.     

In Vitro Screening for Anti-Cholinesterase and Antioxidant Activity of Methanolic Extracts of Ayurvedic Medicinal Plants Used for Cognitive Disorders

Inhibition of Acetylcholinesterase (AChE) is still considered as the main therapeutic strategy against Alzheimer’s disease (AD). Many plant derived phytochemicals have shown AChE inhibitory activity in addition to the currently approved drugs for AD. In the present study, methanolic extracts of 20 plants used in Indian Ayurvedic system of medicine for improving cognitive function were screened for acetylcholinesterase inhibitory activity by Ellman’s microplate colorimetric method. Out of 20 extracts, Emblica officinalis, Nardostachys jatamansi, Nelumbo nucifera, Punica granatum and Raulfia Serpentina showed IC₅₀ values <100 µg/ml for acetylcholinesterase inhibitory activity. Antioxidant activities of these plants were assessed by DPPH scavenging assay. Among the extracts used, antioxidant activity was highest for Terminalia chebula and Emblica officinalis with IC₅₀ values <10 µg/ml. Considering the complex multifactorial etiology of AD, these plant extracts will be safer and better candidates for the future disease modifying therapies against this devastating disease.     

Synergistic effect of kinetin and spermine on some physiological aspects of seawater stressed Vigna sinensis plants

The effect of kinetin, spermine and their combination on growth vigor, photosynthetic pigments, some metabolites, some enzymes, polyamines and productivity of salt-stressed Vigna sinensis plants was investigated. Salt stress reduced all evaluated growth criteria and yield components of used plants. Chlorophyll (CHL (a, b, carotenoids, carbohydrates, protein, spermidine and spermine level as well as and amylase activity were also decreased in response to salinity. On the other hand, proline, K⁺, Na⁺ and putrescine concentration, and peroxidase activity were increased in the salt-stressed plants. Exogenous application of kinetin and spermine mitigated the deleterious effects of salinity stress on growth and yield of the used plants. Conversely, the combined treatment of kinetin and spermine induced additional reduction in growth and yield of the stressed plants, and the effect appeared to be constitutive. The protective effect of kinetin and spermine on V. sinensis plants appeared mainly due to the enhancement effect of these growth regulators on chlorophylls and protein content and polyamines titer.     

Population structure and genetic variation in the genus Dipteronia Oliv. (Aceraceae) endemic to China as revealed by cpSSR analysis

The genus Dipteronia Oliv. endemic to central and southern China consists of two species, D. sinensis Oliv. and D. dyerana Henry, both of them are rare and endangered. In the present study, genetic variation in 17 populations of D. sinensis and four populations of D. dyerana was estimated using ten polymorphic chloroplast microsatellite loci (cpSSR). Forty-nine chloroplast haplotypes were identified from 204 individuals analyzed. Thirty-nine haplotypes were found in D. sinensis, while ten in D. dyerana. No haplotype was shared between the species. AMOVA analysis revealed that the majority of the genetic variation was partitioned among populations within D. sinensis (F _ST = 0.7980) and D. dyerana (F _ST = 0.654). Cluster analysis grouped the 21 populations into two groups according to their species delimitation. The populations of D. sinensis were further divided into two subgroups corresponding to their geographical distributions. Correlation analysis revealed significant correlation between geographical distance and genetic distance of these populations (r = 0.326, p < 0.01 for D. sinensis vs. r = 0.777, p < 0.05 for D. dyerana). Genetic structure of these populations and a calculated pollen-to-seed flow ratio of (3.2:1 vs. 0.6:1) within the species suggested that little gene flow has occurred among the populations over an extended period. Thus, it implies that the genus Dipteronia might have experienced a genetic bottleneck and limited expansion during its evolutionary history.