Elucidating genes involved in sesquiterpenoid and flavonoid biosynthetic pathways in Saussurea lappa by de novo leaf transcriptome analysis.

Saussurea lappa (family Asteraceae) possesses immense pharmacological potential mainly due to the presence of sesquiterpene lactones. In spite of its medicinal importance, S. lappa has been poorly explored at the molecular level. We initiated leaf transcriptome sequencing of S. lappa using the illumina highseq 2000 platform and generated 62,039,614 raw reads. Trinity assembler generated 122,434 contigs with an N50 value of 1053 bp. The assembled transcripts were compared against the non-redundant protein database at NCBI. The Blast2GO analysis assigned gene ontology (GO) terms, categorized into molecular functions (3132), biological processes (4477) and cellular components (1.927). Using KEGG, around 476 contigs were assigned to 39 pathways. For secondary metabolic pathways, we identified transcripts encoding genes involved in sesquiterpenoid and flavonoid biosynthesis. Relatively low number of transcripts were also found encoding for genes involved in the alkaloid pathway. Our data will contribute to functional genomics and metabolic engineering studies in this plant.

[Population characteristics and ecological suitability of Saussureae hieracioides].

OBJECTIVE: To analyze the population characteristics and the appropriate producing area of Saussureae hieracioides in China. METHODS: Chuanxibei plateau, one of the main producing areas of Saussureae hieracioides, was selected as the analytical basal place. Ecological methods were used to investigate the density and biomass of Saussureae hieracioides. Traditional Chinese Medicine Geographic Information System (TCMGIS-II) was used to analyze the appropriate producing area of Saussureae hieracioides. RESULTS: Saussureae hieracioides could form the dominant species in its distribution area. The proper region (with similarity of 90% - 100%) of Saussureae hieracioides accounted for 338 776.89 km2, including 5 provinces/municipalities and 226 counties/cities. The largest area among them was Tibet Autonomous Region with area of 148 175.55 km2, followed by Sichuan Province (110 216.46 km2), Qinghai Province (62 947.61 km2), Gansu Province (16 233.09 km2) and Yunnan Province (1 177.18 km2). CONCLUSION: TCMGIS is much valuable to the recognition of formation of producing area, the division of adaptive area, introduction and acclimatization of medicinal materials, it also provides a scientific reference for the introduction and cultivation of Saussureae hieracioides.

[Regulation of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid biosynthesis in cell suspension cultures of Saussurea involucrata].

Syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid are three main bioactive ingredients in herbs of Saussurea involucrata with various pharmacological properties, while their contents are very low. In this study, the biosynthesis of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid in the cell suspension cultures of S. involucrata were regulated by feeding carbon sources and precursors, which resulted in a great increase of the contents and yields of the above three bioactive ingredients. After 16 days of fermentation, the yields of syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid reached 339.0, 225.3, 512.7 mg x L(-1), respectively. Meanwhile, their contents increased up to 67.9, 1.9, 10.6 times of wild medicinal material, respectively. The results provided a solid basis for further studies on application of cell suspension cultures of S. involucrata for large-scale production of bioactive compounds syringin, chlorogenic acid and 1,5-dicaffeoylquinic acid.

Composition and antioxidant activity of the polysaccharides from cultivated Saussurea involucrata.

Polysaccharides from cultivated Saussurea involucrata (CSIP) were purified, two major fractions (CSIP1-2 and CSIP2-3) were investigated for their molecular weights, monosaccharide compositions and in vitro antioxidant activities. The results suggested that the molecular weights of CSIP1-2 and CSIP2-3 were approximately 163.5 kDa and 88.6 kDa, respectively. CSIP1-2 was composed of glucose, galactose, xylose, rhamnose, arabinose and galacturonic acid with a molar ratio of 1.651:0.39:0.062:8.331:1.759:40.426. CSIP2-3 was composed of glucose, galactose, xylose, rhamnose, arabinose and galacturonic acid with a molar ratio of 0.762:0.657:0.112:5.587:0.318:44.655. Different scavenging activities on superoxide radical, DPPH radical and hydroxyl radical were observed in CSIP1-2 and CSIP2-3 at tested concentrations.

[Effects of phytohormones on plant regeneration and production of flavonoids in transgenic Saussurea involucrata hairy roots].

We investigated the plant regeneration and production of flavonoids in three high-yield flavonoids transgenic Saussurea involucrata hairy roots C17, C27 and C46 by quantification of two phytohormones GA3 and IAA. The results showed that GA3 concentration at more than 1.0 mg/L could induce adventitious shoots in the hairy root lines. The highest shoot regeneration rate, about 82%, was obtained when the hairy roots C17 were cultured with 2.0 mg/L GA3. The results on HPLC and UV spectrophotometry showed that exogenous application of both GA3 and IAA increased the content of flavonoids in the hairy roots. The contents of flavonoids and apigenin in the hormone-treated hairy roots and regenerates were higher comparing with those in the untreated hairy roots and the regenerates. However, the content of flavonoids was not related to tissue weight, and was negatively related to the regeneration efficiency.

Plant adaptation to frequent alterations between high and low temperatures: remodelling of membrane lipids and maintenance of unsaturation levels.

One major strategy by which plants adapt to temperature change is to decrease the degree of unsaturation of membrane lipids under high temperature and increase it under low temperature. We hypothesize that this strategy cannot be adopted by plants in ecosystems and environments with frequent alterations between high and low temperatures, because changes in lipid unsaturation are complex and require large energy inputs. To test this hypothesis, we used a lipidomics approach to profile changes in molecular species of membrane glycerolipids in two plant species sampled from alpine screes and in another two plant species grown in a growth chamber, with the temperature cycling daily between heat and freezing. We found that six classes of phospholipid and two classes of galactolipid showed significant changes, but the degree of unsaturation of total lipids and of three lysophospholipid classes remained unchanged. This pattern of changes in membrane lipids was distinct from that occurring during slow alterations in temperature. We propose two types of model for the adaptation of plants to temperature change: (1) remodelling of membrane lipids but maintenance of the degree of unsaturation are used to adapt to frequent temperature alterations; and (2) both remodelling and changes in the degree of unsaturation to adapt to infrequent temperature alterations.

[Comparison of rutin and syringin content between tissue culturing seedlings and botanical drug of Saussurea involucrata].

OBJECTIVE: To compare the rutin and syringin content in tissue culturing seedlings and in botanical drug of Saussurea involucrata. METHOD: The HPLC with Hydro-RP C18 (4.6 mm x 250 mm, 5 microm) column was used, a mixture of acetonitrile-water (5:95) was used as a mobile phase, with flow rate of 1 mL x min(-1), column temperature at 25 degrees C and detection wavelength at 220 nm. RESULT: The effective constituents of tissue culturing seedlings were almost similar to the botanical drug. And syringin in tissue culturing seedlings was increased 4.35 times. CONCLUSION: It has a good prospect to acquire high-quality S. involucrata by tissue culturing seedlings.

The promoting role of an isolate of dark-septate fungus on its host plant Saussurea involucrata Kar. et Kir.

A dark-septate endophytic (DSE) fungus EF-37 was isolated from the roots of Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant. The molecular identification of the fungus was based on internal transcribed spacer regions and the result showed that EF-37 was congeneric to Mycocentrospora. This study was conducted to clarify the influence of the root endophyte EF-37 on the host plant S. involucrata using material grown in a sterile culture bottle. After cultivation for 40 days, fungal hyphae were found to be branching repeatedly and forming "hyphae nets" in the epidermal layers. Significant differences were detected between the study groups in plant dry weight, plant height, root dry weight, shoot dry weight, and the number of hair root tips. There was a positive effect of endophyte EF-37 on plant root development, with results showing that cortical cells dissolved and formed aerate structures. There was a positive effect of endophyte EF-37 on plant growth, but chlorophyll fluorescence analysis showed that there were no significant differences between the study groups. In addition, analysis of the chemical composition of seedlings showed that the level of rutin was higher in plants cultivated with the EF-37 fungus compared to the controls. This study helps to establish a basis for germplasm conservation and for further investigation of the interaction between dark-septate fungi and this alpine plant.

Interaction between an isolate of dark-septate fungi and its host plant Saussurea involucrata.

A dark-septate endophytic (DSE) fungus EF-M was isolated from the roots of an alpine plant Saussurea involucrata Kar. et Kir. ex Maxim. The fungus was identified by sequencing the PCR-amplified rDNA 5.8S gene and ITS regions. The sequence was compared with similar sequences in the GenBank, and results showed that EF-M was congeneric to Leptodontidium. Resynthesis study was conducted to clarify the relationship between the root endophyte EF-M and the host plant S. involucrata using the material grown in sterile culture bottle. In roots recovered 6 weeks after inoculation, epidermal cells were colonized by intercellular and intracellular hyphae and "microsclerotia" formed within individual cells in the epidermis layers. However, hyphae did not invade the cortex and the stele. There were no profound effects of endophyte EF-M on plant root development, but significant differences were detected in plant height and shoot dry weight between the treatments. The present study is the first report hitherto on DSE fungi in S. involucrata.

In vitro propagation of an endangered medicinal plant Saussurea involucrata Kar. et Kir.

An efficient micropropagation system for Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant, has been developed. Shoot organogenesis occurred from S. involucrata leaf explants inoculated on medium with appropriate supplements of plant growth regulators. 66.0% of shoot regeneration frequency and 5.2 shoots per leaf explant were achieved when cultured on a medium containing 10 microM 6-benzylaminopurine (BAP) and 2.5 microM 1-naphthaleneacetic acid (NAA). Shoot organogenesis was improved further when the leaf explants were pre-incubated at low temperature, and 80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant at 4 degrees C by 5-day pretreatment period. Up to 87.0% of the regenerated shoots formed complete plantlets on a medium containing 2.5 microM indole-3-acetic acid (IAA) within 28 days, and 85.2% of the regenerated plantlets survived and grew vigorously in greenhouse condition. The phytochemical profile of the micropropagated plants was similar to that of wild plants. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite medicinal plant.

[Cell suspension culture and flavonoids production in Saussurea involucrate].

OBJECTIVE: To establish a system of cell suspension culture for Saussurea involucrate. METHOD: The effects of different factors on cell growth and flavonoids production of S. involucrate were systematically studied including the media, initial pH values of the medium, carbon sources, inoculum quantity, and plant growth substance. RESULT: The optimum medium was N6, initial pH values of the medium was 5.8, sucrose concentration was 50 g x L(-1), inoculum quantity was 60-80 g x L(-1) FW. Medium supplemented with BA (0.5 mg x L(-1)) and NAA (3 mg x L(-1)) was suitable for cell growth, but medium containing BA (0.2 mg x L(-1)) and NAA (2 mg x L(-1)) was suitable for flavonoids production. CONCLUSION: Cell growth and flavonoids production in the suspention of S. involucrate culture cell should be optimized.

[Tissue culture and plantlet regeneration from embryo of Saussurea involucrate].

OBJECTIVE: To establish a protocol of rapid clonal propagation of Saussurea involucrate by using embryo as explants. METHOD: MS medium was used as basal medium and BA and NAA were supplemented to find out the optimal hormone combinations for adventitious buds initiation, adventitious bud multiplication and rooting. RESULT: All embryo explants started to grow adventitious buds within 45 days when they were cultured on the media supplemented with 0.02-0.1 mg x L(-1) NAA and 2 mg x L(-1) BA; The adventitious buds multiplicated within 30 days when they were transferred to the media containing 0.1-0.2 mg x L(-1) NAA and 2-3 mg x L(-1) BA; 92% of the adventitious buds rooted well after they were planted on the MS medium containing macroelements at half strength and 0.4 mg x L(-1) NAA for 25 days. The regenerated plantlets grew well after they were transplanted and the survival rate was up to 70%. CONCLUSION: Plantlets of S. invducrate regenerated high frequence through adventitious bud.

Human-induced dwarfing of Himalayan snow lotus, Saussurea laniceps (Asteraceae).

Because evolutionary processes such as genetic drift and natural selection play a crucial role in determining the response that species will have to human-induced disturbances, there is increasing interest in the evolutionary aspects of conservation biology. Harvesting select individuals in natural plant populations can bring about unforeseen impacts that may negatively affect fitness. We analyzed how human harvesting affects two congeners known as snow lotus. Over a period of 100 years, there was a negative trend in plant height (r2= 0.4361, P < 0.001) for the intensely collected and rare species, Saussurea laniceps, but not in the less intensely collected species, Saussurea medusa. Additionally, S. laniceps were significantly smaller in areas of high harvest than in areas with low harvest (Z = 4.91, P < 0.0001), but this was not so for S. medusa. Humans can unconsciously drive evolution and must be considered when managing threatened species.

[Effects of physical and chemical factors on hairy root growth and flavonoids biosynthesis in the cultures of Saussurea medusa Maxim hairy root].

The effects of different physical and chemical factors on hairy root growth and flavonoids production were studied in suspension culture of Saussurea medusa hairy root in 1/2 MS medium. The results showed that the following culture conditions, nitrogen concentratiaon (involved NH4+ and NO3-), 30 mmol/L; the ratio of ammonium to nitrate, 5:25; the combination of 2% sucrose and 3% glucose; 0.5 mg/L GA3; 0.5 mg/L IBA; initial pH 5.8; light cycle, 18 h/d (3500lx); temperature, 24 degrees C; shaker revolutions per minute, 100 r/min, were favourable to hairy root growth and flavonoids production. Under the above culture conditions, up to 12.8 g/L (DW) of hairy root and 1922 mg/L of flavonoids were obtained after 21 days of culture. The content of total flavonoids in hairy root was 15%, which was about 25 times as that in the wild plantlet.

Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators.

A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and alpha-naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 microM BA and 1.0 microM NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 microM BA and 1.0 microM NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 microM indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.

Transformation of Saussurea medusa for hairy roots and jaceosidin production.

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.

[Establishment of Saussurea involucrata hairy roots culture and plantlet regeneration].

Hairy root clones of Saussurea involucrata transformed with Agrobacterium rhizogenes strains R1601, R1000, and LBA9402 were established to investigate the flavonoid production. Opine synthesis and PCR analysis confirmed the integration of the T-DNA fragment of Ri plasmid from A. rhizogenes strain R1601 into the transformed root genome. The frequency of hairy root formation from root segments, which were pre-cultured 2 days in N6 solid medium without plant growth regulators, amounted to 100% following infection with R1601 strain of A. rhizogenes. The transformed roots were kept in hormone-free N6 liquid medium in the dark at 25 degrees C, 110r/min and routinely subcultured every 20 - 24 days. One hairy root clone, which grew vigorously with lateral branches, was periodically examined for the ability to produce flavonoid. The maximum of biomass and flavonoid yield achieved 66.7 g/L (fresh weight) and 102.3mg/g dry weight after incubation 20 days. The calli were induced from the hairy root culture in the presence of 0.5mg/L IBA and intact plantlets were regenerated from these calli. The regeneration plantlets from hairy roots, in which the flavonoid content were 53% in that of untransformed plants, weren't different in growth and morphology of the untransformed plantlets. Therefore plant regeneration from hairy roots may be also a means for producing transformed S. involucrata plants. Hairy root cultures of S. involucrata clearly showed higher flavonoid contents compared to the wild plant or the regeneration seedlings. As the wild S. involucrata grows only in special regions with peculiar climate, and cultivation of this species in a normal climate has been unsuccessful so far. The success in obtaining a method for high production of flavonoid might very well be one of the solutions for this problem in the future.

In vitro propagation of Saussurea obvallata (DC.) Edgew.--an endangered ethnoreligious medicinal herb of Himalaya.

This is the first report of a micropropagation protocol for Saussurea obvallata (DC.) Edgew. (Asteraceae), a rare, threatened and near-endemic medicinal herb of the Indian Himalayan region. Multiple shoots were formed from epicotyle explants on Murashige and Skoog (MS) medium supplemented with 1.0 microM kinetin and 0.25 microM alpha-naphthaleneacetic acid. A maximum of five shoots were obtained from one explant in a 75-day culture period. The effect of subsequent subcultures on shoot formation was also studied. After 100% in vitro rooting was obtained in half-strength MS supplemented with 2.5 microM indole-3-butyric acid, the plantlets were transferred to ex vitro conditions. Following a 15-day in vitro rooting period and 12 days of ex vitro acclimatization, 66.7% of the plantlets had established in the field. Application of this protocol has the potential to substantially reduce the pressure on natural populations.