Methyl jasmonate promote protostane triterpenes accumulation by up-regulating the expression of squalene epoxidases in Alisma orientale.

Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The natural distribution of protostane triterpenes is limited mainly to members of the botanical family Alismataceae. Squalene epoxidase (SE) is the key rate-limiting enzyme in triterpene biosynthesis. In this study, we report the characterization of two SEs from A. orientale. AoSE1 and AoSE2 were expressed as fusion proteins in E. coli, and the purified proteins were used in functional research. In vitro enzyme assays showed that AoSE1 and AoSE2 catalyze the formation of oxidosqualene from squalene. Immunoassays revealed that the tubers contain the highest levels of AoSE1 and AoSE2. After MeJA induction, which is the main elicitor of triterpene biosynthesis, the contents of 2,3-oxidosqualene and alisol B 23-acetate increased by 1.96- and 2.53-fold, respectively. In addition, the expression of both AoSE proteins was significantly increased at four days after MeJA treatment. The contents of 2,3-oxidosqualene and alisol B 23-acetate were also positively correlated with AoSEs expression at different times after MeJA treatment. These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2.

[Quadratic Orthogonal Rotation Combination Design on Alisma orientalis of Fertilization].

OBJECTIVE: To study the effects of combined N, P, K and micronutrient fertilizers on the yield of Alisma orientalis tuber, and to optimize the fertilizer application rate. METHODS: Four factors five levels quadratic orthogonal rotation combination design was used. A function was established on nitrogen, phosphor, potassium and microelement fertilizer application rate with the yield of Alisma orientalis tuber. RESULTS: The established mathematical model was of high reliability for prediction with quadratic regression equation of R2 = 0. 8980. The order of increasing Alisma orientalis tuber yield was nitrogen > micronutrient fertilizer > potassium > phosphor. The results of the frequency analysis showed that for the target yield over 8 250 kg/hm2 and the confidence interval of 95%, the optimal fertilizer application rates were as follows :nitrogen of 241. 45 - 283. 55 kg/hm2, phosphor of 81. 14 - 208. 44 kg/hm2, potassium of 95. 57 - 239. 42 kg/hm2, and zinc fertilizer of 14. 32 - 16. 18 kg/hm2, boron fertilizer of 18. 84 - 19. 86 kg/hm2, and molybdenum fertilizer of 0. 151 -0. 159 kg/hm2 in micronutrient fertilizer. CONCLUSION: Nitrogen is related to the growth of Alisma orientalis, potassium promotes tuber bulking, micronutrient fertilizer consisted of zinc, boron and molybdenum fertilizer promotes Alisma orientalis growth and the absorption of nitrogen, phosphor and potassium. Moderate application of nitrogen, phosphorus, potassium, zinc, boron and molybdenum fertilizer can promote Alisma orientalis tuber yield. The nitrogen has the best effect.

Characterization and function of the 3-hydroxy-3-methylglutaryl-CoA reductase gene in Alisma orientale (Sam.) Juz. and its relationship with protostane triterpene production.

Protostane triterpenes from Alisma orientale (Sam.) Juz. have exhibited distinct pharmacological properties that are currently in high demand. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered the first rate-limiting enzyme in isoprenoid biosynthesis via the mevalonic acid (MVA) pathway. In this study, we cloned a full-length cDNA of A. orientale (Sam.) Juz. HMGR (AoHMGR; 2252 bp; GenBank accession no. KP342318) with an open reading frame (ORF) of 1809 bp. The deduced protein sequence contained four conserved motifs and exhibited homology with HMGR proteins from other plants. We next expressed the cloned gene in Escherichia coli BL21 (Rosetta) cells, collected the expressed products, and incubated those with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to determine enzymatic activity. GC/MS analysis revealed that the products were able to catalyze HMG-CoA and NADPH to form MVA. The purified protein was used to immunize New Zealand rabbits and prepare an antibody against AoHMGR. Western blot results demonstrated that the antibodies specifically recognized AoHMGR protein in A. orientale (Sam.) Juz. We then established a rapid test to detect AoHMGR protein in the plant, and found the tuber to be the most AoHMGR protein-abundant organ in A. orientale (Sam.) Juz. Furthermore, we detected the expression level of AoHMGR and contents of the main active component, Alisol B 23-acetate, at different growth phases of A. orientale (Sam.) Juz. A significant positive correlation was identified, indicating that AoHMGR represents a key enzyme in the synthetic pathway of protostane triterpenes.

[Effect of climate change on genuine medicinal materials producing areas during last 2 000 years--Alisma orientale and Citrus aurtantium as examples].

Climate in China has fluctuated greatly for last two thousand years. Also, the temperate-subtropical transition zone, as well as the distribution boundaries of subtropical biology and growth of suitable areas appear north-south lapse. In historical period, significant climate change will also be bound to the changes of the medicinal organism distribution suitable areas. The past dynasties herbal herbs have documented origin in detail, especially genuine medicinal materials producing areas. In this paper, Alisma orientale and Citrus aurtantium as examples, were used to elaborate the impact of climate change fluctuations on genuine producing area by evolution and change of genuine producing areas. The results showed that medicinal species were more sensitive to climate change such as A. orientale and C. aurtantium, its main producing areas or genuine producing area from north to south shifted in the Ming and Qing dynasties, consistent with the characteristics of climate change in China in last two thousand years. Factors prompted producing areas southward are not only low temperature and cold damage, and temperature and humidity have often synergistic effect. The human activities are more likely to exacerbate the impact.

[Distribution characteristics and correlation analysis of inorganic elements in Alismatis rhizoma and rhizospheric soils in different habitats].

OBJECTIVE: To investigate the distribution characteristics and correlation of inorganic element contents in Alismatis Rhizoma and rhizospheric soils from three habitats (Jianou, Fujian; Guangchang, Jiangxi; Pengshan, Sichuan). METHODS: The inorganic element contents were detected with inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: The results showed that inorganic element contents in Alismatis Rhizoma from different habitats presented regular distribution characteristics. In all samples, the contents of S, P, K, Mg and Ca were relatively high. The contents of S, P, K, Fe, Mg, Ca, Al and Zn in Alismatis Rhizoma from Fujian with low-content harmful heavy metal were more than those of other two places. Alismatis Rhizoma from different habitats showed remarkable ability to enrich P, S, Zn, Mg and Cu, especially to P and S. The contents of inorganic element in Alismatis Rhizoma and rhizosperic soil showed certain correlation. CONCLUSION: These results provide scientific evidence for artificial modification of effective components biosynthesis in Alisma orientalis, and it could also provide a reference for forming mechanism of genuine characteristics and quality of Alismatis Rhizoma.

[Studies on shapes and germination characters of Alisma plantago-aquatica seeds].

OBJECTIVE: To explore the differences of the shapes of seeds and germination characters of Alisma plantago-aquatica. METHOD: The seeds were collected from four regions in Sichuan province, characters of the seeds' appearance were observed. The purification, weights per thousand seeds, content of moisture, seed vigor and different germination rates of A. plantago-aquatica were measured. The germination rates of the seeds were determined under different temperature treatments. RESULT: It was markedly varied in shapes of the seeds, rates of germination and all quality characters of the four local species. The seeds from Pengshan showed the best quality, the weights per thousand, the seed vigor and germination rate were 0.4308 g, 6% and 82%, respectively, and the size of the seed, its width and length were 1.882, 1.455 mm, respectively. The length was positively correlated with the width, and so did the weights per thousand seeds with the seed vigor and germination rates. The rate of seed vigor was positively correlated with the rate of germination. Results indicated that temperature had affected the germination of the seed. CONCLUSION: The seed from Pengshan was the best. The result of this study provides scientific evidences for identification, growth and cultivation of A. plantago-aquatica.

[Comparative studies on genetic resource of characteristics of Alisma].

Based on the introduction and cultivation of Alisma germplasm which were from Fujian, Jiangxi and Sichuan provinces, the biological characteristics, morphological characteristics and quality were observed and studied. After three-year continuous experiment and monographic study, there were remarkable difference in the biological characteristics, morphological characteristics and product quality of Fujian Alisma, Sichuan Alisma and Jiangxi Alisma. Fujian Alisma and Jiangxi Alisma were the same plant species of A. orientalis, whereas Sichuan Alisma and Fujian Alisma were the different plant species of A. plantago-aquatica. The study results will provide the theoretical and practical basis for the genuine medicinal materials research and good agricultural practice (GAP) of Alisma.

[Variation in yield and quality of Alisma orientalis grown under different ecological climatic regions].

UNLABELLED: To compare yield, alisol content of Alisma orientalis planted at different ecological climatic regions, and explore further the impact of environmental factors on the yield and quality. METHOD: Different local varieties were planted at varing ecological climatic conditions. Diameter, yield was measured after harvest, the contents of 23-acetyl alisol B and 24-acetyl alisol A were quantitatively analyzed by HPLC. RESULT: The result revealed that ecological condition had significant impacts on yield and alisol content. Yield of MeiShan was the highest which was up to 1 200.72 kg x hm(-2). The contents of 23-acetyl alisol B and 24-acetyl alisol A of A. orientalis cultivated in Dujiangyan were significantly higher than those of other regions, the values were up to 4.222, 2.727 g x kg(-1), respectively. 23-acetyl alisol B content was positively correlated with 24-acetyl alisol A content (P < 0.01). The diameter was positively correlated with yield (P < 0.01). CONCLUSION: Considering yield and medicinal ingredients, Dujiangyan may be the most suitable region to plant A. orientalis.

[Effects of S-3307 on the yield and main ingredients of Alisma plantago-aquatica].

UNLABELLED: To study the effect of S-3307 on the yield and main ingredients of Alisma plantago-aquatica. METHOD: The contents of 24-acetyl alisol A and the 23-acetyl alisol B in tuber were determined by HPLC. RESULTS: The contents of 24-acetyl alisol A and the 23-acetyl alisol B as well as yield were significantly increased in all groups applied with different concentrations of S-3307 comparing with control group. The optimal concentration of S-3307 was 80 mg x kg(-1). The residues of S-3307 was detected under 0.316 8 mg x kg(-1) (detecting limit). CONCLUSION: The optimal concentration of S-3307 is 80 mg x kg(-1), it reached the best result when applied 36 d after seedling.

[Quality evaluation of Alisma orientalis from different habitats].

OBJECTIVE: To study the seed quality of Alisma orientalis (Sam.) Juzep. METHOD: Collecting seeds from different habitats and studying the thousand-grain weight, moisture content, purity and germination percentage. RESULT: The seeds reaped in summer friom Pengshan, Sichuan province are better than those from other habitats. Its thousand-grain weight is 0.4682 g, moisture content is 12.48%, purity of seeds is 89.18% and germination percentage is above 95%. CONCLUSION: The cause of qualitative differences in seeds of Alisma orientalis from different habitats were analysed in this paper.

Biotypes resistant to herbicides in paddy fields in the Iberian Peninsula.

A consequence of modern agriculture and its dependence on herbicides, is the appearance of resistant weed populations. The first case of resistance on the Iberian Peninsula was found in Portugal and it was in a population of Alisma plantago-aquatica, resistant to bensulfuronmethyl, an ALS-inhibitor. For the same herbicide, control problems with Cyperus difformis were found in some Spanish rice fields. In the same rice field area, tolerance was observed in some Echinochloa spp. when auxinic herbicides (quinclorac) were used and some resistant Echinochloa spp. populations were found due to the long use of PS II inhibitors herbicides (propanil). The association of active ingredients, rotation of crops and adoption of mechanical control methods are the main strategies to combat resistance to herbicides.