Leaf transcriptome analysis of a subtropical evergreen broadleaf plant, wild oil-tea camellia (Camellia oleifera), revealing candidate genes for cold acclimation.

BACKGROUND: Cold tolerance is a key determinant of the geographical distribution range of a plant species and crop production. Cold acclimation can enhance freezing-tolerance of plant species through a period of exposure to low nonfreezing temperatures. As a subtropical evergreen broadleaf plant, oil-tea camellia demonstrates a relatively strong tolerance to freezing temperatures. Moreover, wild oil-tea camellia is an essential genetic resource for the breeding of cultivated oil-tea camellia, one of the four major woody oil crops in the world. The aims of our study are to identify variations in transcriptomes of wild oil-tea camellia from different latitudes and elevations, and discover candidate genes for cold acclimation. RESULTS: Leaf transcriptomes were obtained of wild oil-tea camellia from different elevations in Lu and Jinggang Mountains, China. Huge amounts of simple sequence repeats (SSRs), single-nucleotide polymorphisms (SNPs) and insertion/deletions (InDels) were identified. Based on SNPs, phylogenetic analysis was performed to detect genetic structure. Wild oil-tea camellia samples were genetically differentiated mainly between latitudes (between Lu and Jinggang Mountains) and then among elevations (within Lu or Jinggang Mountain). Gene expression patterns of wild oil-tea camellia samples were compared among different air temperatures, and differentially expressed genes (DEGs) were discovered. When air temperatures were below 10 °C, gene expression patterns changed dramatically and majority of the DEGs were up-regulated at low temperatures. More DEGs concerned with cold acclimation were detected at 2 °C than at 5 °C, and a putative C-repeat binding factor (CBF) gene was significantly up-regulated only at 2 °C, suggesting a stronger cold stress at 2 °C. We developed a new method for identifying significant functional groups of DEGs. Among the DEGs, transmembrane transporter genes were found to be predominant and many of them encoded transmembrane sugar transporters. CONCLUSIONS: Our study provides one of the largest transcriptome dataset in the genus Camellia. Wild oil-tea camellia populations were genetically differentiated between latitudes. It may undergo cold acclimation when air temperatures are below 10 °C. Candidate genes for cold acclimation may be predominantly involved in transmembrane transporter activities.

Polymorphism and evolution of ribosomal DNA in tea (Camellia sinensis, Theaceae).

Ribosomal DNA (rDNA) repeats often exhibit a high level of within-species homogeneity, but intra-individual polymorphism of rDNA has been found in Camellia species. In order to reveal the evolutionary pattern of rDNA repeats in tea [Camellia sinensis (L.) O. Kuntze], we identified the 45S rDNA loci, estimated their copy number, and cloned partial regions of them from different PCR products and from digested genomic DNA. The results show that there are 3 loci (6 sites) containing 45S rDNA in the tea genome; they are located at the ends of the short arms of 6 chromosomes and consist of a maximum of ∼6500 repeat units. On one hand, 164 sequences of the 26S rDNA cloned from PCR products contained 147 haplotypes, consisting of 62% pseudogenes, 24% putative functional genes, and 14% PCR-mediated recombinants, suggesting that the 45S rDNA of tea maintains an extremely high level of polymorphism and divergence; on the other hand, rDNA fragments cloned directly from genomic DNA exhibited a very high level of homogeneity: only one of 33 rDNA fragments was from a pseudogene. These results demonstrate that although over 60% of the 26S rDNA sequences identified in PCR products belong to pseudogenes, most 45S rDNA repeats are functional genes and have undergone concerted evolution. In this study, strong PCR bias and PCR-mediated recombination greatly increased the apparent proportion of pseudogenes in PCR products. Phylogenetic analysis and genetic divergence values for 26S rDNA sequences obtained in this study show that many pseudogenes have originated independently from functional genes at different times, and despite thus escaping from concerted evolution, they have failed to be eliminated from the tea genome over a long period, some of them having even produced addition copies by rapid expansion. Importantly, our study suggests that in order to determine the true pattern of evolution of rDNA it is necessary to combine data from more than one method rather than relying only on sequences from PCR products.

Understanding plant-microbe interactions for phytoremediation of petroleum-polluted soil.

Plant-microbe interactions are considered to be important processes determining the efficiency of phytoremediation of petroleum pollution, however relatively little is known about how these interactions are influenced by petroleum pollution. In this experimental study using a microcosm approach, we examined how plant ecophysiological traits, soil nutrients and microbial activities were influenced by petroleum pollution in Phragmites australis, a phytoremediating species. Generally, petroleum pollution reduced plant performance, especially at early stages of plant growth. Petroleum had negative effects on the net accumulation of inorganic nitrogen from its organic forms (net nitrogen mineralization (NNM)) most likely by decreasing the inorganic nitrogen available to the plants in petroleum-polluted soils. However, abundant dissolved organic nitrogen (DON) was found in petroleum-polluted soil. In order to overcome initial deficiency of inorganic nitrogen, plants by dint of high colonization of arbuscular mycorrhizal fungi might absorb some DON for their growth in petroleum-polluted soils. In addition, through using a real-time polymerase chain reaction method, we quantified hydrocarbon-degrading bacterial traits based on their catabolic genes (i.e. alkB (alkane monooxygenase), nah (naphthalene dioxygenase) and tol (xylene monooxygenase) genes). This enumeration of target genes suggests that different hydrocarbon-degrading bacteria experienced different dynamic changes during phytoremediation and a greater abundance of alkB was detected during vegetative growth stages. Because phytoremediation of different components of petroleum is performed by different hydrocarbon-degrading bacteria, plants' ability of phytoremediating different components might therefore vary during the plant life cycle. Phytoremediation might be most effective during the vegetative growth stages as greater abundances of hydrocarbon-degrading bacteria containing alkB and tol genes were observed at these stages. The information provided by this study enhances our understanding of the effects of petroleum pollution on plant-microbe interactions and the roles of these interactions in the phytoremediation of petroleum-polluted soil.

Plants' use of different nitrogen forms in response to crude oil contamination.

In this study, we investigated Phragmites australis' use of different forms of nitrogen (N) and associated soil N transformations in response to petroleum contamination. 15N tracer studies indicated that the total amount of inorganic and organic N assimilated by P. australis was low in petroleum-contaminated soil, while the rates of inorganic and organic N uptake on a per-unit-biomass basis were higher in petroleum-contaminated soil than those in un-contaminated soil. The percentage of organic N in total plant-assimilated N increased with petroleum concentration. In addition, high gross N immobilization and nitrification rates relative to gross N mineralization rate might reduce inorganic-N availability to the plants. Therefore, the enhanced rate of N uptake and increased importance of organic N in plant N assimilation might be of great significance to plants growing in petroleum-contaminated soils. Our results suggest that plants might regulate N capture under petroleum contamination.

[Identification of four medicines of Panax L. revealed by mitochondrial nad 1 gene b/c intron].

OBJECTIVE: To study the identification method and phylogenetic relationships of four medicines of Panax L genus: Panax ginseng C. A. Meyer, P. quinquefolicum L. , P. notoginseng (Burk.) F. H. Chen and P. japonicus C. A. Meyer. METHODS: The mitochondrial nad 1 gene was amplified. Their sequence differences were analyzed after sequencing and alignment. RESULTS: The sequence lengths of P. ginseng (including Chinese transplanted ginseng and Korean ginseng), P. japonicus were 1 290 bp, and those of P. quinquefolicum and P. notoginseng were 1 269 bp and 1 522 bp respectively. The main difference among these sequences was in nad 1 gene b/c intron. The NJ phylogenetic tree showed that P. ginseng was most closely related to P. japonicus, next was closer to P. quinquefolicum, and P. notoginseng was comparatively distantly related to P. ginseng. CONCLUSION: P. quinquefolicum and P. notoginseng can be identified from the 4 medicines of Panax L. based on the sequence difference in mitochondrial nad 1 gene. The mitochondrial nad 1 gene b/c intron can provide some evolutionary information, therefore, it is useful to identify and phylogenetically analyse for the medicines of Panax L. genus.

Do plants modulate biomass allocation in response to petroleum pollution?

Biomass allocation is an important plant trait that responds plastically to environmental heterogeneities. However, the effects on this trait of pollutants owing to human activities remain largely unknown. In this study, we investigated the response of biomass allocation of Phragmites australis to petroleum pollution by a ¹³CO2 pulse-labelling technique. Our data show that plant biomass significantly decreased under petroleum pollution, but the root-shoot ratio for both plant biomass and ¹³C increased with increasing petroleum concentration, suggesting that plants could increase biomass allocation to roots in petroleum-polluted soil. Furthermore, assimilated ¹³C was found to be significantly higher in soil, microbial biomass and soil respiration after soils were polluted by petroleum. These results suggested that the carbon released from roots is rapidly turned over by soil microbes under petroleum pollution. This study found that plants can modulate biomass allocation in response to petroleum pollution.

Genetic characterization and phytochemical analysis of wild and cultivated populations of Scutellaria baicalensis.

Scutellaria baicalensis was collected from four wild and four cultivated populations from different locations in China. Forty-two samples were analyzed using random amplification of polymorphic DNA (RAPD) techniques for genetic profiling, and high performance liquid chromatography (HPLC) techniques to determine the flavonoid content. The selected 23 RAPD primers yielded a total of 838 clear and reproducible bands of which 237 were found to be polymorphic. The wild population exhibited higher polymorphism than that of the cultivated population. The dendrogram generated by the UPGMA method via Nei's genetic distance revealed three distinct genotypes from the cultivated populations and several branches from the wild populations. The contents of baicalin and wogonoside in dried roots of the samples ranged from (w/w) 8.63 to 17.84%, and from 1.99 to 4.21%, respectively, whereas their aglycones, baicalein and wogonin, were within the range of only 0.04-0.23%. The total content of the four flavonoids varied from 9.45 to 26.24%. Comparatively, the cultivated populations contained much higher levels of baicalin and total flavonoids than those in the wild populations. The results from genetic characterization and phytochemical analysis demonstrated that the quality variation of this drug was mainly determined by extrinsic environmental or agricultural factors, rather than by genetic differences. Our findings can be used for the commercial production and germplasm management of this medicinal plant.

Phenylalanine ammonia lyase functions as a switch directly controlling the accumulation of calycosin and calycosin-7-O-beta-D-glucoside in Astragalus membranaceus var. mongholicus plants.

Previously it had been shown that calycosin and calycosin-7-O-beta-D-glucoside (CGs) accumulate in whole plants, mainly in leaves, of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus) plants in response to low temperature. In this work, it was demonstrated that the influences of different conditions on CGs biosynthesis, by examining the changes in CGs content, as well as the expression of related genes, including phenylalanine ammonia lyase (PAL1), cinnamic acid 4-hydroxylase (C4H), chalcone synthase (CHS), chalcone reductase (CHR), chalcone isomerase (CHI), isoflavone synthase (IFS), and isoflavone 3'-hydroxylase (I3'H). The seven gene mRNAs accumulated in leaves of A. mongholicus upon exposure to low temperature in a light-dependent manner, though they exhibited different expression patterns. Transcriptions of CHS, CHR, CHI, IFS, and I3'H of the calycosin-7-O-beta-D-glucoside pathway were all up-regulated when plants were transferred from 16 degrees C to 2 degrees C or 25 degrees C or from 2 degrees C (kept for 24 h) to 25 degrees C. However, fluctuations in temperature influenced differently the transcriptions of PAL1 and C4H of the general phenylpropanoid pathway in leaves. Moreover, the amount of PAL1 expression changed sharply up and down, consistent with the variation of the content of CGs. PAL enzyme activity appears to be the limiting factor in determining the CGs levels. The inhibitor of PAL enzyme, L-alpha-aminooxy-beta-phenylpropionic acid, almost entirely shut down CGs accumulation at low temperature. All these results confirmed that PAL1, as a smart gene switch, directly controls the accumulation of CGs in A. mongholicus plants, in a light-dependent manner, during low temperature treatment.

A new phenolic glycoside from the aerial parts of Solidago canadensis.

A new phenolic glycoside, 2'-hydroxy-4',6'-di-O-beta-D-glucopyranosyl-butyrrophenone (1), was isolated from the aerial parts of Solidago canadensis. The structure was elucidated on the basis of spectroscopic methods.

[Determination of lignin content in tiny Panax ginseng by UV spectrophotometry].

OBJECTIVE: To establish the UV spectrophotometry for determining lignin content in tiny Panax ginseng powder. METHODS: Classical Klason method and UV spectrophotometry were used. RESULTS: The lignin contents measured by UV spectrophotometry were higher, more repeatable and accurate as comparison with the Klason method. The specific absorptance peak of ginseng lignin appeared at 260 nm. The acetyl bromide treatment of the UV spectrophotometry was processed for ginseng powder at 70 degrees C for 30 minutes. It is also concluded that the lignin contents were obvious different among various ginsengs. CONCLUSION: UV spectrophotometry is simple, accurate and just need a little materials. It is especially suitable to determine the lignin content for ginseng and other precious Chinese traditional medicines.

Antioxidative flavanone glycosides from the branches and leaves of Viscum coloratum.

Two new flavanone glucosides, (2S)-homoeriodictyol 7,4'-di-O-beta-D-glucopyranoside (4) and (2R)-eriodictyol 7,4'-di-O-beta-D-glucopyranoside (5) were isolated from the branches and leaves of Viscum coloratum (KOMAR) NAKAI (Loranthaceae), along with three known flavanone glucosides: (2S)-homoeriodictyol 7-O-beta-D-glucopyranoside (1), (2S)-eriodictyol 7-O-beta-D-glucopyranoside (2), and (2S)-naringenin 7-O-beta-D-glucopyranoside (3). The structures of these compounds were elucidated using spectroscopic methods. The antioxidant activities of these isolated compounds were evaluated by colorimetric methods based on their scavenging effects on hydroxyl radicals and superoxide anion radicals, respectively. All the compounds showed potent albeit varied degrees of antioxidative activities and the structure-activity relationship is discussed.

Rapid analysis of essential oil from Fructus Amomi by pressurized hot water extraction followed by solid-phase microextraction and gas chromatography-mass spectrometry.

In this paper, a simple, rapid, solvent-free and low-cost method of pressurized hot water extraction (PHWE) followed by headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) was developed for the analysis of essential oil in a traditional Chinese medicine (TCM) of the dried ripe fruit of Fructus Amomi (Sha Ren). The essential oil in the TCM (0.050 g) was extracted by water at 50 bar and 150 degrees C, followed by extraction and concentration by SPME fibers at 80 degrees C for 15min and analysis by GC-MS. The PHWE and HS-SPME parameters were optimized. Thirty-five compounds in the TCM were identified by PHWE-HS-SPME. Among them, camphor, an active compound, in the TCM samples was quantitatively analyzed. The proposed method required little time to prepare the sample. Moreover, little sample mass and no organic solvent was needed. The precision of the present method was found to be good (R.S.D. <10.0%). It is shown that PHWE-SPME-GC-MS is an alternative method for the determination of volatile components in TCMs and can be used as a powerful tool for TCM quality assessment.

Comparison of solid-phase microextraction, supercritical fluid extraction, steam distillation, and solvent extraction techniques for analysis of volatile consituents in Fructus Amomi.

Four sampling techniques, solid-phase microextraction (SPME), supercritical fluid extraction (SFE), steam distillation (SD), and solvent extraction (SE), were compared for the analysis of volatile constituents from a traditional Chinese medicine (TCM) of the dried ripe fruit of Fructus Amomi (Sha Ren). A total of 38 compounds were identified by gas chromatography/mass spectrometry. Different SFE and SPME parameters (modifier content, extraction pressure, and temperature for SFE and fibers, extraction temperature, and time for SPME) were studied. The results by SFE and SPME were compared with those obtained by conventional SD and SE methods. The results showed that SFE and SPME are better sample preparation techniques than SD and SE. Due to SFE's requirement for expensive specialized instrumentation, the simplicity, low cost, and speed of SPME make it a more appropriate technique for extraction of volatile constituents in TCMs.

Quality assessment of Flos Chrysanthemi Indici from different growing areas in China by solid-phase microextraction-gas chromatography-mass spectrometry.

Flos Chrysanthemi Indici is a common traditional Chinese medicine (TCM). In this paper, headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS) was developed for quality assessment of Flos Chrysanthemi Indici from different growing areas in China. SPME parameters such as extraction fibers, extraction temperature, extraction time and sample mass were investigated to achieve identical results to those obtained by the steam distillation (SD). The selected SPME conditions were as follows: SPME fiber coated with 65-microm PDMS/DVB, extraction temperature of 60 degrees C, extraction time of 30 min and sample mass of 1.0 g. Furthermore, four active compounds (eucalyptol, camphor, borneol and bornyl acetate) presented in the TCM were applied to evaluating the quality of Flos Chrysanthemi Indici from 20 various areas. The quality assessment was successfully performed to compare the similarity value (S) between different sample vector of Flos Chrysanthemi Indici and the standard profile vector (SPV). The results showed that the proposed HS-SPME-GC-MS was an alternative technique for quality assessment of Flos Chrysanthemi Indici samples.