Gtgen3A, a novel plant GH3 ß-glucosidase, modulates gentio-oligosaccharide metabolism in Gentiana.

Gentiobiose, a ß-1,6-linked glycosyl-disaccharide, accumulates abundantly in Gentianaceae and is involved in aspects of plant development, such as fruits ripening and release of bud dormancy. However, the mechanisms regulating the amount of gentio-oligosaccharide accumulation in plants remain obscure. The present study aimed to identify an enzyme that modulates gentio-oligosaccharide amount in gentian (Gentiana triflora). A protein responsible for gentiobiose hydrolysis, GtGen3A, was identified by partial purification and its peptide sequence analysis. The enzyme had a molecular mass of ∼67 kDa without a secretory signal peptide sequence. Sequence analysis revealed that GtGen3A could be a ß-glucosidase member belonging to glycoside hydrolase family 3 (GH3). GtGen3A showed a homology to GH3 ß-glucan exohydrolases, ExoI of Hordeum vulgare, and ExgI from Zea mays, which preferentially hydrolyzed ß-1,3- and ß-1,4-linked oligosaccharides. The purified recombinant GtGen3A (rGtGen3A) produced in Escherichia coli showed optimal reaction at pH 6.5 and 20°C. The rGtGen3A liberated glucose from ß-1,2-, ß-1,3-, ß-1,4-, and ß-1,6-linked oligosaccharides, and showed the highest activity toward gentiotriose among the substrates tested. Kinetic analysis also revealed that rGtGen3A preferentially hydrolyzed gentiotriose. Virus-induced gene silencing of Gtgen3A in gentian plantlets resulted in predominant accumulation of gentiotriose rather than gentiobiose. Furthermore, the expression level of Gtgen3A was almost similar to the amount of gentiobiose in field-grown gentians. These findings suggest that the main function of GtGen3A is the hydrolysis of gentiotriose to gentiobiose, and that GtGen3A plays a role in modulating gentiobiose amounts in gentian.

Variations in ramet performance and the dynamics of an alpine evergreen herb, Gentiana nipponica, in different snowmelt conditions.

PREMISE OF THE STUDY: Variation in demographic parameters reflects the life-history strategies of plants in response to specific environments. We aimed to investigate the intraspecific variation in life-history traits of a clonal alpine herb, Gentiana nipponica, in various snowmelt conditions. METHODS: Individual ramets within genets accumulate leaves for 7-9 yr without shedding, and die after reproduction. We tested the physiological function of accumulated leaves for reproduction and monitored the ramet demography in early, intermediate, and late snowmelt populations over 3 yr. Then, we simulated ramet dynamics using the demographic parameters. KEY RESULTS: Old leaves had a carbon storage function, and the initiation of reproduction depended on the amount of ramet leaves. Growth and reproductive performance were highest in the population with an intermediate snowmelt period. The early snowmelt population showed short persistence periods due to restricted growth and high mortality of the ramets. The late snowmelt populations showed slow growth, but high survival rate of the ramets, in which the ramet size at reproduction was smallest and fruit formation was often suppressed by the short growing period. CONCLUSIONS: Limiting factors dictating the distribution of G. nipponica differed between the early and late snowmelt habitats. High mortality and restricted growth, because of the harsh environment, determine the distribution limit toward earlier snowmelt locations. By contrast, late snowmelt strongly limited fecundity because of the short period for fruit maturation. The difference in snowmelt time provides a clear gradient of selective forces that may promote local adaptation among neighboring populations.

Rapid temporal changes in root colonization by arbuscular mycorrhizal fungi and fine root endophytes, not dark septate endophytes, track plant activity and environment in an alpine ecosystem.

Fungal root endophytes play an important role in plant nutrition, helping plants acquire nutrients in exchange for photosynthates. We sought to characterize the progression of root colonization by arbuscular mycorrhizal fungi (AMF), dark septate endophytes (DSE), and fine root endophytes (FRE) over an alpine growing season, and to understand the role of the host plant and environment in driving colonization levels. We sampled four forbs on a regular schedule from June 26th-September 11th from a moist meadow (3535 m a.s.l) on Niwot Ridge, Rocky Mountain Front Range, CO, USA. We quantified the degree of root colonization by storage structures, exchange structures, and hyphae of all three groups of fungi. AMF and FRE percent colonization fluctuated significantly over time, while DSE did not. All AMF structures changed over time, and the degree of change in vesicles differed by plant species. FRE hyphae, AMF arbuscules and AMF vesicles peaked late in the season as plants produced seeds. AMF hyphae levels started high, decreased, and then increased within 20 days, highlighting the dynamic nature of plant-fungal interactions. Overall, our results show that AMF and FRE, not DSE, root colonization rapidly changes over the course of a growing season and these changes are driven by plant phenology and seasonal changes in the environment.

Allelic variants of the esterase gene W14/15 differentially regulate overwinter survival in perennial gentian (Gentiana L.).

Overwinter survival has to be under critical regulation in the lifecycle of herbaceous perennial plants. Gentians (Gentiana L.) maintain their perennial life style through producing dormant and freezing-tolerant overwinter buds (OWBs) to overcome cold winter. However, the mechanism acting on such an overwinter survival and the genes/proteins contributing to it have been poorly understood. Previously, we identified an OWB-enriched protein W14/15, a member of a group of α/ß hydrolase fold superfamily that is implicated in regulation of hormonal action in plants. The W14/15 gene has more than ten variant types in Gentiana species. However, roles of the W14/15 gene in OWB survival and functional difference among those variants have been unclear. In the present study, we examined whether the W14/15 gene variants are involved in the mechanism acting on overwinter survival, by crossing experiments using cultivars carrying different W14/15 variant alleles and virus-induced gene silencing experiments. We found that particular types of the W14/15 variants (W15a types) contributed toward obtaining high ability of overwinter survival, while other types (W14b types) did not, or even interfered with the former type gene. This study demonstrates two findings; first, contribution of esterase genes to winter hardiness, and second, paired set or paired partner among the allelic variants determines the ability of overwinter survival.

Apple latent spherical virus vector-induced flowering for shortening the juvenile phase in Japanese gentian and lisianthus plants.

MAIN CONCLUSION: Infection by apple latent spherical virus (ALSV) vectors that promote the expression of Arabidopsis thaliana FLOWERING LOCUS T ( AtFT ) or Gentiana triflora GtFT s accelerates flowering in gentian and lisianthus plants. Apple latent spherical virus (ALSV) has isometric virus particles (25 nm in diameter) that contain two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp20, and Vp24). ALSV vectors are used for foreign gene expression and virus-induced gene silencing in a broad range of plant species. Here, we report the infection by ALSV vectors that express FLOWERING LOCUS T (AtFT) from Arabidopsis thaliana or its homolog GtFT1 from Gentiana triflora in three gentian cultivars ('Iwate Yume Aoi' [early flowering], 'Iwate' [medium flowering], and 'Alta' [late flowering]), and two lisianthus cultivars ('Newlination Pink ver. 2' and 'Torukogikyou daburu mikkusu') promotes flowering within 90 days post-inoculation using particle bombardment. Additionally, seedlings from the progeny of the early-flowering plants were tested by tissue blot hybridization, and the results showed that ALSV was not transmitted to the next generation. The promotion of flowering in the family Gentianaceae by ALSV vectors shortened the juvenile phase from 1-3 years to 3-5 months, and thus, it could be considered as a new plant breeding technique in ornamental gentian and lisianthus plants.

[Quality regionalization study on Gentianae Macrophyllae Radix].

The 79 samples of Gentianae Macrophyllae Radix were collected based on the distributed information by document literature. Based on sample information, and using the regression model of Gentianae Macrophyllae Radix index component and environmental factors, and combined with the prediction results of ecological suitability by MaxEnt and principal component analysis results of index component, the space distribution of Gentianae Macrophyllae Radix was estimated with the spatial analysis function of ArcGIS. The results showed that it had a higher comprehensive quality in south of Shaanxi, south of Gansu, middle of Sichuan and southeast of Xizang. The study results were coinciding with the producing regions of Gentianae Macrophyllae Radix. It can provide reference for Gentianae Macrophyllae Radix resource conservation, development and utilization.

[Ecology suitability study of Chinese materia medica Gentianae Macrophyllae Radix].

This paper is aimed to predict ecology suitability distribution of Gentianae Macrophyllae Radix and search the main ecological factors affecting the suitability distribution. The 313 distribution information about G. macrophylla, 186 distribution information about G. straminea, 343 distribution information about G. dauricaand 131 distribution information about G. crasicaulis were collected though investigation and network sharing platform data . The ecology suitable distribution factors for production Gentianae Macrophyllae Radix was analyzed respectively by the software of ArcGIS and MaxEnt with 55 environmental factors. The result of MaxEnt prediction was very well (AUC was above 0.9). The results of predominant factors analysis showed that precipitation and altitude were all the major factors impacting the ecology suitable of Getiana Macrophylla Radix production. G. macrophylla ecology suitable region was mainly concentrated in south of Gansu, Shanxi, central of Shaanxi and east of Qinghai provinces. G. straminea ecology suitable region was mainly concentrated in southwest of Gansu, east of Qinghai, north and northwest of Sichuan, east of Xizang province. G. daurica ecology suitable region was mainly concentrated in south and southwest of Gansu, east of Qinghai, Shanxi and north of Shaanxi province. G. crasicaulis ecology suitable region was mainly concentrated in Sichuan and north of Yunnan, east of Xizang, south of Gansu and east of Qinghai province. The ecological suitability distribution result of Gentianae Macrophyllae Radix was consistent with each species actual distribution. The study could provide reference for the collection and protection of wild resources, meanwhile, provide the basis for the selection of cultivation area of Gentiana Macrophylla Radix.

[Analysis of commercial specifications and grades of wild and cultivated Gentianae Macrophyllae Radix based on multi-indicative constituents].

The study is aimed to analyze the commercial specifications and grades of wild and cultivated Gentianae Macrophllae Radix based on multi-indicative constituents. The seven kinds of main chemical components containing in Gentianae Macrophyllae Radix were determined by UPLC, and then the quality levels of chemical component of Gentianae Macrophyllae Radix were clustered and classified by modern statistical methods (canonical correspondence analysis, Fisher discriminant analysis and so on). The quality indices were selected and their correlations were analyzed. Lastly, comprehensively quantitative grade division for quality under different commodity-specifications and different grades of same commodity-specifications of wild and planting were divided. The results provide a basis for a reasonable division of specification and grade of the commodity of Gentianae Macrophyllae Radix. The range of quality evaluation of main index components (gentiopicrin, loganin acid and swertiamarin) was proposed, and the Herbal Quality Index (HQI) was introduced. The rank discriminant function was established based on the quality by Fisher discriminant analysis. According to the analysis, the quality of wild and cultivated Luobojiao, one of the commercial specification of Gentianae Macrophyllae Radix was the best, Mahuajiao, the other commercial specification, was average , Xiaoqinjiao was inferior. Among grades, the quality of first-class cultivated Luobojiao was the worst, of second class secondary, and the third class the best; The quality of the first-class of wild Luobojiao was secondary, and the second-class the best; The quality of the second-class of Mahuajiao was secondary, and the first-class was the best; the quality of first-class Xiaoqinjiao was secondary, and the second-class was the better one between the two grades, but not obvious significantly. The method provides a new idea and method for evaluation of comprehensively quantitative on the quality of Gentianae Macrophyllae Radix.

[Study on Genetic Stablity of Cryopreservation Regeneration Plant of Gentiana straminea with ISSR-PCR].

OBJECTIVE: To establish ISSR-PCR system of cryopreservation regeneration plant of Gentiana straminea, and to select appropriate primers and analyze the genetic stability. METHODS: DNA was extracted by CTAB, the optimal ISSR-PCR system was established by orthogonal experiment,and genetic stability was analyzed. RESULTS: The optimal ISSR-PCR system (25 µL) was established: dNTPs 0.50 µL, Mg2+ 1.00 µL, 10 x PCR Buffer 2.00 µL, primer 0.60 µL, Taq DNA polymerase 1.25 µL, template DNA 1.30 µL, and ddH2O 18.35 µL. The amplification program was devised: 94 degrees C for 5 min, denaturing at 94 degrees C for 30 s, annealing of 1 min due to denaturing temperature of different primer,extension at 72 degrees C for 1.5 min, 35 cycles, last extension at 72 degrees C for 7 min, conservation at 4 degrees C . The DNA mutation rate of cryopreservation regeneration plant of Gentiana straminea was 1.05%. CONCLUSION: The cryopreservation regeneration plant of Gentiana straminea retains very good genetic stability, there is little variation between each plant, so the cryopreservation can be used as a feasible method for resource protection of Gentiana straminea.

[Fertilizer Effect on Yield and Quality of Gentiana crassicaulis].

OBJECTIVE: To find the best fertilizer formula of yield-quality-fertilizer effect, and to establish a new comprehensive evaluation-fertilizer effect method. METHODS: Grey related degree analysis and DTOPSIS method were combined to comprehensive evaluate the 11 indexes of 14 fertilizer formulas of nitrogen, phosphorus and potassium, in order to explore the best dosage and ratio. RESULTS: The fertilizer requirements of the factors which contribute to the yield and quality of Gentiana crassicaulis were different and the most appropriate rates of nitrogen, phosphorus and potassium fertilizer were 21.49~42.03 kg/667 m2, 49.53~65.75 kg/667 m2 and 26.34~ 42.97 kg/667 m2, respectively. CONCLUSION: The fertilizer formula in this study can be widely used in the standardized plantation of Gentiana crassicaulis.

[Influences of Base Fertilizer Dosage and Root Size on Quality and Yield of Gentiana crassicaulis Root].

OBJECTIVE: In order to optimize the base fertilizer dosage and root size of Gentiana crassicaulis in Ludian,Yulong, Yunnan,the differences in yield and quality of Gentiana crassicaulis caused by different base fertilizer dosage and root size were investigated. METHODS: The variation in fresh weight, dry weight, drying rate, length, diameter, branches as well as the contents of gentiopicroside, loganin acid, alcohol-soluble extract and total ash were determined. The ideal distance grey relational degree comprehensive evaluation method was applied to make a comprehensive evaluation on the quality and yield of Gentiana crassicaulis. RESULTS: There were big differences in yield and quality of Gentiana crassicaulis among different base fertilizer dosage and root size. 2 000 kg/667 m2 of manure, 20 kg/667 m2 of superphosphate, 100 kg/667 m2 of plant ash and mix size of transplanting roots showed the best comprehensive quality and the highest yield. CONCLUSION: 2 000 kg/667 m2 of manure, 20 kg/667 m2 of superphosphate,100 kg/667 m2 of plant ash, high quality transplanting roots and stagger certain depth to transplant are highly recommended to produce high quality and high yield of Gentiana crassicaulis in Ludian, Yulong, Yunnan.

[Study on Embryonic Development of Four Species of Gentiana (Gentianaceae)].

OBJECTIVE: To study the embryonic development of Gentiana straminea, G. robusta, G. crassicaulis and G. tibetica. METHODS: The seed germination rates, length and width of embryos, starch grains and chloroplasts were observed and analyzed by statistic software. RESULTS: The seed germination rates of the four species were all high. The increase of the length of embryo depended mainly on the increase of the length of hypocotyl. Starch grains were stored in cells and chloroplasts appeared in cotyledons. The process of germination was divided into eight periods. CONCLUSION: The embryo growth characteristics of the four species are recognized, and the results can be used to study the in situ conservation, genetic breeding and cultivation of Sect. Cruciata.

[Study on Dormant Culture and Plant Regeneration via Cluster Buds of Gentiana straminea].

OBJECTIVE: In order to provide scientific basis for micropropagation and cryopreservation of Gentiana straminea,plantlets were regenerated from dormant buds by cluster buds. METHODS: Based on MS medium, dormant buds were inoculated in mediums containing different type and concentration of cytokinin and auxin for inducing cluster buds. 1/2MS medium with different concentration of auxin were used for inducing root. RESULTS: The medium of MS with 2.0 mg/L 6-BA, 0.01 mg/L NAA,30 g/L sucrose and 7 g/L agar was suitable for cluster buds' primary culture and subculture. The cluster buds inducing rate was 93. 3%. Multiple shoot clumps multiplication factor was 5.6. The medium of 1/2MS with 2.0 mg/L IAA, 0.5 mg/L IBA, 15 g/L sucrose and 7 g/L agar was suitable for root induction, its inducing rate was 93.5% with plantlets growing well. CONCLUSION: Plantlets regenerated from dormant buds of Gentiana straminea via cluster buds are established in this study.

[Optimum harvest study of Gentiana crassicaulis in Ludian].

The paper is aimed to study the difference in yield and quality at different harvest time and determine the optimum harvest of planting Gentiana in Ludian traditional harvest period. The authors analyzed the variation in fresh weight, dry weight, dry discount rate, length, diameter, volume and the content of gentiopicroside, loganin acid, alcohol-soluble extract and total ash and made a comprehensive appraisal of yield, appearance quality and intrinsic quality by gray relational distance ideal Comprehensive Evaluation method. The results showed that there is a big difference in yield and quality both 2-year-old and 3-year-old Gentiana harvested in traditional harvest period and the comprehensive evaluation more better when harvested more later. It can be seen, Gentiana harvested the later had a better yield and quality in Ludian traditional harvest period. The harvest of Gentiana can be appropriate delayed depending on the particular circumstances of production.

[Determination of polysaccharides content of Gentiana farreri from different producing areas based on anthrone-sulfuric acid method].

Study a method for the detemination of the content of polysaccharides in Gentiana farreri, and analysis of the content of polysaccharides from different producing areas. The results showed that using the anthrone-sulfuric acid method, simple operation, accurate result. Sample was measured at 620 nm absorbance after anthrone-sulfuric acid color, at this wavelength, solution absorption and glucose showed a good linear relationship; The linearity was in the range of 0.01-0.07 g x L(-1) (r = 0.996 7). The recovery rate was 99.41%, with RSD of 2.0%. Considering the experimental conditions, to determine the solid-liquid ratio 1:60, extracting time 50 min, concentration of ethanol 80%. The mass fraction of polysaccharides was the highest to reached 0.743% in G. farreri from Gansu Xiahe. This experiment has laid a good foundation for further study on G. farreri.

[Varieties textual research and usage investigation of white flos gentianae, a Tibetan medicine].

OBJECTIVE: To clarity the original plants and the main application varieties of White Flos Gentianae. METHOD: Herbal textual research, wild specimen collection, investigation and collection of the samples from Tibetan hospital, Tibetan pharmaceutical factory and medical material market were carried out simultaneously to identify the original plants of White Flos Gentianae. RESULT: The results of varieties textual research and specimen identification showed that Gentiana szechenyii, G. purdomii and G. algida were in accord with the record of Tibetan herbal textual The three species above were the original plants of White Flos Gentianae. The identification of 20 batches samples showed that G. szechenyii was the main application variety. The other varieties were only used in Tibetan hospitals. All the samples above were flowering branches. CONCLUSION: It was necessary to strengthen the research on variety systematization of White Flos Gentianae make a further discussion on the taxonomy position of G. purdomii, G. algida and the white flos population. Its was also nessary to establish and improve the quality standard of different variety based on the principle of "one species, one name". The quality specification of White Flos Gentianae should be established and improved to standard clinical utilization and produce feeding. More study of resources investigation and cultivation of G. szechenyii should be carried on to meet the demand of produce and clinic.

[Genetic polymorphism of Gentiana lutea L. (Gentianaceae) populations from Chornohora Ridge of Ukrainian Carpathians].

The features of genetic structure and level of diversity were investigated for G. lutea populations from Chornohora Ridge of Ukrainian Carpathians using RAPD- and ISSR-PCR. We have shown a high level of genetic diversity for investigated populations. The differences between populations account for 59-72% of the total genetic variation, whereas intrapopulation polymorphism makes up 28-41%. The relationships among genetic variability level and ecological-geographical conditions as well as biological features of the species were assumed to be possible. The obtained results indicate the genetic isolation of G. lutea Chornohora populations from Ukrainian Carpathians. Pozhyzhevska agropopulation was characterized by a high level of polymorphism that means the possibility to use artificial plantings of the investigated species for its conservation.

An in vitro perspective to cholinesterase inhibitory and antioxidant activity of five Gentiana species and Gentianella caucasea.

The dichloromethane (DCM), ethyl acetate (EtOAc), and methanol extracts from the leaves, roots, and flowers of the five species of Gentiana (Gentiana asclepiadea, Gentiana cruciata, Gentiana olivieri, Gentiana septemfida, and Gentiana verna) and Gentianella caucasea were investigated for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and antioxidant effect using 2,2-diphenyl-1-picrylhydrazyl radical scavenging, metal-chelation capacity, and ferric-reducing antioxidant power assays. Total phenol and flavonoid contents of the extracts were determined spectrophotometrically. The presence of some characteristic compounds found in Gentiana species (gentiopicroside, swertiamarin, isoorientin, isovitexin and vitexin) was analyzed in the extracts by thin layer chromatography. The flower DCM extract of G. verna exerted the highest inhibition against AChE (53.65 ± 1.03%), whereas the root EtOAc extract of G. cruciata was the most effective in BChE inhibition assay (50.72 ± 0.75%) at 100 µg ml⁻¹. The extracts of G. verna were also found to be more active in the antioxidant tests.

[Macroscopic identification of cultivated and wild Gentiana macrophylla and preliminary screening for high quality germplasm].

OBJECTIVE: To investigate the difference of macroscopic characteristics between cultivated Gentiana macrophylla and wild one, analyze the correlation among its main quality traits and preliminary screen cultivated Gentiana macrophylla from different habitats based on quality traits. METHODS: Measured and obtained characteristic index, including main root length, lateral root number, taproot diameter, residue stem number and dry weight, and analyzed data by SPSS 12.0 statistical software. RESULTS: The main characteristics of cultivated Gentiana macrophylla were all better than those of the wild one. The characteristics was sequenced as follows: lateral root number > dry weight > main root lengthy > taproot diameter > residue stem number in order of difference. There were remarkable positive correlations among main quality traits. The optimal germplasm was located in Yulinpu of Liufengguan Village, Nanxing Town, Feng County in Shaanxi Province. CONCLUSION: Cultivated Gentiana macrophylla is relatively robust, having more branching roots with light colour of surface and section compared with the wild one. The paper provides suggestions for the revising of macroscopic characteristics of Gentiana macrophylla in the next edition of pharmacopoeia; The data offer scientific reference for screening excellent germplasm, introduction expansion and reasonable utilization of resources as well.

[Cryopreservation and plantlet regeneration of dormant buds of Gentiana straminea by vitrification].

OBJECTIVE: To investigate the detailed techniques for cryopreservation of Gentiana straminea dormant buds by vitrification. METHODS: Dormant buds as an experimental material,the influence of the different size of dormant buds,preculture and PVS etc. on cryopreservation of Gentiana straminea were studied. RESULTS: The optimal procedures were as follows: 10-11 mm long dormant buds which were cultured on MS medium supplemented with different sucrose concentration (0. 3,0. 5 or 0.7 mol/L) for 1 day respectively. The buds were immersed in loading solution for 20 min at 20 degrees C, and then treated in PVS2 solution for 40 min at 0 degrees C and finally plunged into liquid nitrogen quickly. After 24 hours, the buds were rapidly thawed in a water bath at 40 degrees C for 2 - 3 min and washed twice with MS medium supplemented with 1/2 MS liquid medium containing 1. 2 mol/L sucrose. Finally the buds were transferred to regeneration medium (MS + 0.5 mg/L 6-BA + 0.1 mg/L NAA + 3% sucrose + 0.7% agar), the survival rate was up to 83.3%. CONCLUSION: A high-efficiency cryopreservation protocol of Gentiana straminea is set up.