Yaguang Zhan

Fungal endophytes from Acer ginnala Maxim: isolation, identification and their yield of gallic acid

Aims:  The aim of the study was to isolate the endophytic fungi from Acer ginnala and screen isolates rich in gallic acid.

Genome-Scale Transcriptome Analysis in Response to Nitric Oxide in Birch Cells: Implications of the Triterpene Biosynthetic Pathway

Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10−5) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis.

Drought physiology and gene expression characteristics of Fraxinus interspecific hybrids

In this study, we investigated the physiological and gene expression responses of Fraxinus interspecific hybrids seedlings to short-term artificially-applied drought stress. Experiment results showed that there were differences in physiological and gene expression responses between hybrids and their parents. Hybrid D110 kept higher levels of photosynthetic parameters and antioxidant enzyme activities than its female parents, as well as ABA contents and expressions of ABA-related genes. These results showed that hybrid D110 improved the suitability which could be due to the change of ABA signal transduction under drought conditions. The expression characteristics of circadian clock gene TOC1 and LHY in Fraxinus hybrid plants and their parents under drought conditions showed that altered amplitude of circadian gene expression might be a possible molecular mechanism for drought-advantage in hybrids. Combined with the physiological response of each hybrid under drought stress, these more sensitive responses of NCED, PYR1 and SnRK2.6 expressions in hybrid D110 compared to its female parent and other hybrids, along with the higher levels of PYR1 and SnRK2.6 expressions might coursed the drought-advantage in hybrid D110.

Crosstalk among nitric oxide, calcium and reactive oxygen species during triterpenoid biosynthesis in Betula platyphylla

We analysed NO, reactive oxygen species (ROS) and Ca2+ crosstalk during triterpenoid biosynthesis in white birch (Betula platyphylla Suk.) cells. Cells were pretreated with diphenyleneiodonium, sodium diethyldithiocarbamate (DDTC) or catalase (CAT), or a Ca2+ channel blocker or chelator before sodium nitroprusside treatment. Changes in triterpenoid, malondialdehyde and proline levels, cell viability, and CAT, ascorbate peroxidase and peroxidase activity were recorded. Furthermore, enzyme gene expression levels related to triterpene biosynthesis, endogenous signalling and antioxidase activity, and cell apoptosis and death rates were measured. Sodium nitroprusside elevated ROS and Ca2+ levels. Oleanolic acid levels in cells pretreated with diphenyleneiodonium and CAT reduced significantly, but it increased with DDTC pretreatment. ROS inhibition downregulated BpDXR, BpCALM and BpNIA expression. Oleanolic acid, BpMnSOD expression, and CAT, ascorbate peroxidase and peroxidase activities reduced when the Ca2+ signalling pathway was blocked. The apoptosis rates of cells pretreated with DDTC and CAT decreased significantly; cell death rates also reduced in groups Ca2+ pretreated with channel blocker and chelator . Thus ROS and Ca2+ participate in triterpenoid biosynthesis, cell apoptosis and death induced by exogenous NO application. Further, NO causes oxidative stress and restricts the level of intracellular ROS through the Ca2+ signalling pathway.

Cross-talk of polyamines and nitric oxide in endophytic fungus-induced betulin production in Betula platyphylla plantlets

AbstractKey messageThis paper showed that NO, PAs, PA-induced NO, and NO-induced PAs mediate fungus-induced betulin accumulation in birch plantlets.Abstract The aim of this study was to investigate the relationship between nitric oxide (NO) and polyamines (PAs) and to determine their roles in betulin accumulation induced by the endophytic fungus Phomopsis in Betula platyphylla. Treatment of birch plantlets with the endophytic fungus Phomopsis promoted an NO burst and accumulation of PAs and betulin. Birch plantlets were treated with the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) and the PA synthesis inhibitor d-arginine (d-arg). cPTIO and d-arg inhibited the fungus-induced NO burst and accumulation of PAs and betulin. The exogenous NO donor sodium nitroprusside promoted PA production and betulin accumulation, whereas an exogenous PA, putrescine, promoted an NO burst and betulin accumulation. In addition, d-arg inhibited NO production and cPTIO decreased PA production during fungus-induced betulin accumulation. Our results indicate that NO, PAs, PA-induced NO, and NO-induced PAs mediate fungus-induced betulin accumulation in birch plantlets.

Exogenous Feeding of Fructose and Phenylalanine Further Improves Betulin Production in Suspended Betula platyphylla Cells under Nitric Oxide Treatment

The aim of this study was to assay by NMR the metabolites which contribute to betulin production. 8-day-old suspended birch (Betula platyphylla) cells were treated by sodium nitroprusside (SNP) treatment, an NO donor, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), an NO-specific scavenger. The results showed that betulin production was increased by five times after SNP treatment, similar with that of the control under cPTIO treatment. Forty one metabolites were detected after SNP treatment or cPTIO treatment. Among them, 10 were found to significantly contribute to the differences observed between controls and treated cell culture samples. To validate the contribution of the above 10 metabolites to betulin production, myo-inositol, fructose and phenylalanine based on correlation analysis between the content of 12 metabolites and betulin were used to feed birch suspension cell cultures under SNP treatment. Exogenous feeding of fructose or phenylalanine further enhanced the betulin production under SNP treatment, but myo-inositol had the opposite result.

Effects of High Temperature Stress on Transgene Expression and DNA Methylation Pattern

Abstract-Studies about the variation of DNA methylation pattern under high temperature stress (37˚C) contribute to a comprehensive understanding of the apparent regulation of biological functions. Based on the in vitro cultivation of genetically modified birch (Betula platyphylla Suk.) clones for high-temperature stress, the paper detected exogenous gene transcription level differences with RT-PCR. Methylation-sensitive amplified polymorphism (MSAP) analysis the variation of DNA methylation pattern in transgenic birch. In this paper we used ten pairs of primers MSAP to deal with genetically modified DNA screening birch. The study revealed that high temperature stress can lead the level of transcriptional expression of transgene decrease, while endogenous levels of DNA methylation, including the overall methylation, the lateral semi-cytosine methylation as well as the inside of cytosine methylation level of the whole is almost twice than the control.

Characteristics of Seasonal and Spatial Variations of Proanthocyanidins Content in Elaeagnus angustifolia and Effect of Salicylic Acid on Its Accumulation

In this paper, we took Elaeagnus angustifolia as material. The paper studied characteristics of seasonal and spatial variations of proanthocyanidins content. There was an apparent difference in proanthocyanidins content of the leave from May to October, the maximim content of proanthocyanidins in October was 34.36 mgldrg -1 DW, while proanthocyanidins content in July was only 17.16 mgldrg-1 DW. Content of proanthocyanidins in different organs of Elaeagnus angustifolia from the high to low was bark > root > leave > annual branch. We also studied the effects of salicylic acid on its accumulation among the different organs of Elaeagnus angustifolia were studied. The result showed that spraying 400 mgldrL -1 salicylic acid chould enhance remarkablely proanthocyanidins content in the leave, annual branch and bark of Elaeagnus angustifolia, the maximal increment of proanthocyanidins content was 76.48%, 53.27% and 28.06% respectively on the third day after treatment.

Preliminary Study on the Expression of BGT Gene in Transgenic Birch

In this paper, Southern blot analysis of the transgenic birch (Betula platyphylla Suk.) plants indicated that the the copy number of bgt varies from one to four. Of which 18.2 percent were single copy. Transcriptional expression of bgt gene in transgenic birch plants was analyzed by Northern blot. TGS has been found in four transgenic lines with one, three and four copies of bgt. Bgt gene was inserted into the prokaryotic vector pET 28a and transferred into E.coli BL21 (DE3) for expression of BGT fusion protein. After overnight cleavage with Thrombin and purification by immobilized metal ion affinity chromatography, the final BGT proteins were obtained with purity of 90%. The ELISA result showed that the BGT protein expression level in the transgenic birch plants ranged from 0.000% to 0.283% of total soluble protein. No significant correlation was found between copy number and expression level of bgt gene.

Establishment of the Callus Culture of Elaeagnus angustifolia to Produce Proanthocyanidins and Effect of UV-B Radiation on Its Accumulation

Callus culture of Elaeagnus angustifolia was established with the objective to produce proanthocyanidins. The effects of different conditions on the callus growth and the production of proanthocyanidins were researched. The leaf tissue part of Elaeagnus angustifolia was optimum explant of callus induction. The best callus growth and the highest proanthocyanidins production was obtained under the culture on MS basal medium containing 4.50 μmol·L 2, 4-D and 2.20 μmol·L BA. The optimum time of subculture was 20-25 d. The content of proanthocyanidins in callus was up to 56.25 mg·g DW. Callus bud differentiation exhibited a negative correlation with proanthocyanins accumulation, UV-B radiation had remarkable promoting effects on the accumulation of proanthocyanidins in the callus of Elaeagnus angustifolia. KeywordsElaeagnus angustifolia L, callus, proanthocyanidins Abbreviations PGR, plant growth regulator; DW, dry weight; FW, fresh weight; 2,4-D, 2,4-dichlorophenoxyacetic acid; BA, 6bezyladenine; NAA, α-naphthaleneacetic acid; IAA, Indole-3-acetic acid