Regulatory protein genes and microRNAs in response to selenium stimuli in Pueraria lobata (Willd.) Ohwi.

Regulatory protein genes and microRNAs (miRNAs) play important roles in response to abiotic and biotic stress, and the biosynthesis of secondary metabolites in plants. However, their responses to selenium (Se) stimuli have not been comprehensively studied in Pueraria lobata (Willd.) Ohwi, a selenocompound-rich medicinal and edible plant. In this study, we identified a total of 436/556/1161/624 transcription factors, 134/157/308/172 transcriptional regulators, and 341/456/250/518 protein kinases, which were co-expressed with at least one selenocompound-related structural gene/sulfate transporter or phosphate transporter/reactive oxygen species (ROS) scavenging structural gene/isoflavone-related structural gene, respectively. Then, we identified a total of 87 expressed miRNAs by Se disposure, in which 11 miRNAs, including miR171f-3p, miR390b-3P, miR-N111b, miR-N118, miR-N30, miR-N38-3P, miR-N61a, miR-N61b, miR-N80-3p, miR-N84-3P, and miR-N90.2-3P, were significantly upregulated. We also identified a total of 1172 target genes for the 87 expressed miRNAs. Gene Ontology enrichment analysis of these target genes showed that regulation of transcription, DNA-templated, integral component of membrane, nucleus, ATP binding, and plasma membrane are the top five subclassifications. Finally, we revealed that 5 miRNAs targeted 10 regulatory protein genes, which are highly correlated with at least one selenocompound-related structural gene or transporter gene; 5 miRNAs targeted 10 regulatory protein genes, which are highly correlated with at least one ROS scavenging structural gene; and 5 miRNAs targeted 9 regulatory protein genes, which are potentially involved in the isoflavone biosynthesis. Overall, the study provides us the comprehensive insight into the roles of regulatory proteins and miRNAs in response to Se stimuli in P. lobata.

Molecular cloning and characterization of an isoflavone 7-O-glucosyltransferase from Pueraria lobata.

KEY MESSAGE: A novel isoflavone 7- O -glucosyltransferase PlUGT1 was isolated from Pueraria lobata . PlUGT1 could convert daidzein to daidzin, genistein to genistin as well as formononetin to ononin. Pueraria lobata roots are traditionally consumed as a rich source of isoflavone glycosides that have various human health benefits. However, to date, the genes encoding isoflavone UDP-glycosyltransferases (UGTs) have only been isolated from the roots of soybean seedlings (GmIF7GT), soybean seeds (UGT73F2) and Glycyrrhiza echinata cell suspension cultures (GeIF7GT). To investigate the isoflavone metabolism in P. lobata, 40 types of partial UGT cDNAs were isolated from P. lobata, and seven full-length UGT candidates with preferential expression in roots were identified. Functional assays in yeast (Saccharomyces cerevisiae) revealed that one of these UGT candidates, designated PlUGT1 (official UGT designation UGT88E12), efficiently glycosylated isoflavone aglycones at the 7-hydroxy group. Recombinant PlUGT1 purified from Escherichia coli cells was characterized and shown to be relatively specific for isoflavone aglycones, while flavonoid substrates were poorly accepted. The biochemical results suggested that PlUGT1 was an isoflavone 7-O-glucosyltransferase. The deduced amino acid sequence of PlUGT1 shared only 26 % identity with GeIF7GT, 27 % with UGT73F2 and 63 % with GmIF7GT. The PlUGT1 gene was highly expressed in P. lobata roots relative to other organs and strongly induced by methyl jasmonate signal in P. lobata cell suspension culture. The transcript abundance of PlUGT1 was correlated with the accumulation pattern of isoflavone glycosides such as daidzin in P. lobata plants or in cell suspension culture. The biochemical properties and gene expression profile supported the idea that PlUGT1 could play a role in isoflavone glycosylation in P. lobata.

[Effects of 6-benzylaminopurine and α-naphthaleneacetic acid on growth and isoflavone contents of Pueraria phaseoloides hairy roots].

In order to study the effect of phytohormone on growth and isoflavones contents of Pueraria phaseoloides hairy roots, we cultured the hairy roots with different concentrations of 6-benzylaminopurine (6-BA) alone or in combination with α-naphthaleneacetic acid (NAA). Then we determined the effects of 6-BA alone or in combination with NAA on the growth and the contents of isoflavones compounds and levels of antioxidase activities of hairy roots by spectrophotometry. The results show that 6-BA inhibited the growth, and decreased biomass and total isoflavones compounds of P. phaseoloides hairy roots. Furthermore, the inhibition was increased with the concentrations of 6-BA. Compared with the controls, different concentrations of 6-BA in combination with NAA 2.0 mg/L could inhibit the growth of hairy roots and decrease the content of total isoflavone compounds, and also significantly enhanced the contents of soluble protein and levels of peroxidase (POD) activities, but decreased the activities of superoxide dismutase (SOD). DNA ladders detected by agarose gel electrophoresis can be observed after hairy roots of P. phaseoloides were cultured with 6-BA alone for 30 days, but can appear on the 20th day after culture with 6-BA in combination with NAA 2.0 mg/L. This result indicates that 6-BA or 6-BA in combination with NAA can both stimulate appearance of programmed cell death (PCD), and NAA may play a synergistic role on PCD.

Involvement of abscisic acid in ozone-induced puerarin production of Pueraria thomsnii Benth. suspension cell cultures.

Exposure to ozone induced a rapid increase in the levels of the sesquiterpene phytohormone abscisic acid (ABA) and the isoflavone puerarin in suspension cell cultures of Pueraria thomsnii Benth. The observed increases in ABA and puerarin were dependent on the concentration of ozone applied to P. thomsnii cell cultures. In order to examine the role of ABA in ozone-induced puerarin production, cell suspensions were pretreated with the ABA biosynthetic inhibitor fluridone. Following ozone exposure, fluridone treatment suppressed ABA accumulation suggesting ABA was normally synthesized de novo through the carotenoid pathway. Fluridone also blocked ozone-induced puerarin production, which could be reversed through application of exogenous ABA. However, in the absence of ozone, ABA itself had no effect on puerarin accumulation in the suspension cells. Taken together, the data indicate that ozone is an efficient elicitor of puerarin production and may be particularly applicable for improving puerarin production in plant cell cultures. Furthermore, we demonstrate that ABA is one factor associated with ozone-induced puerarin production in P. thomsnii cell cultures.

[Study on cryopreservation of Pueraria lobata germplasm by vitrification].

The experiment of the cryopreservation technique on Pueraria lobata showed that: At first, the aseptic explants were treated at 4 degrees C for 5 days. Then the stems with buds were cut and precultured at 4 degrees C for 1 day in 5% DMSO + 5% sucrose media. They were dehydrated with 60% and 100% PVS2 (30% glycerol + 15% glycol + 15% dimethyl sulfoxide + 0.4 mol/L sucrose) at 0 degrees C for 30 minutes respectively. At last the stems were immersed immediately into liquid nitrogen directly and conserved for 24 hours. After rapidly thawing in a water bath at 40 degrees C for 90 seconds, the stems were washed two times with MS media supplemented with 1.2 mol/L sucrose and 10 minutes each time, then transferred on the MS media supplemented with BA 2 mg/L and NAA 1 mg/l, in dark for 7 days and then in light. The survival rate was up to 57-58% . The regenerated plant of Pueraria lobata were the same as the normal in morphology.

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate.

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g(-1) dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.

[Effect of sucrose concentration on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots].

Effects of sucrose concentrations on the growth and production of puerarin and isoflavones compounds in Pueraria phaseoloides hairy roots induced by Agrobacterium rhizogenes ATCC15834 were investigated. Changes of sucrose consumption in the medium during liquid culture were also determined. The results showed cultured for 16 days in MS medium with 5%, 4%, 3% and 2% sucrose, the proliferation times of dry weight of hairy roots were 11.7, 11.9, 10.1 and 5.9, respectively. 3% sucrose concentration in liquid medium was the best for accumulation of puerarin and isoflavones in the hairy roots. The highest content of puerarin, 5.147 mg/g DW, was obtained after 12 days of liquid culturing while the highest content of isoflavones, about 27.76 mg.g.DW, was gained after 16 days in culturing. Sucrose concentration decreased as hairy root growth proceeded. The growth rate and the content of soluble sugar in hairy roots of P. phaseoloides was directly proportional to the rate of sucrose utilization in the liquid medium during the whole culture. It was observed that the highest content of soluble sugar in hairy roots was at day 12 of liquid culture and sucrose in the liquid medium was used up at the end of 16 days of culture.

[Induction of hairy roots of Pueraria phaseoloides and its culture in liquid and solid medium].

An efficient transformation system for genetic transformation of medicinal plant, Pueraria phaseoloides, which contains puerarin and daidzein with hypothermic, spasmolytic, hypotensive and anti-arrhythmic activities, by using agropine-type Agrobacterium rhizogenes ATCC 15834 was developed. Hairy roots could be obtained directly from the cut edges of petioles of leaf explants of P. phaseoloides or via callus 20 days after inoculation with agrobacterium. The percentage of rooted leaf explants 35 days after infection was about 85%. Hairy roots could have a rapid growth on solid or liquid growth regulator-free MS medium. The transformation of hairy roots was confirmed by PCR amplification of rol B and rol C genes of Ri plasmid from A. rhizogenes. To investigate the physiological difference between solid and liquid culture, the biomass (fresh weight and dry weight), the reactive oxygen species (ROS) and the total content of soluble sugar in hairy roots cultured for 15 days in solid and liquid medium were detected, respectively, by the method of fluorescence labeling of 2',7'-dichlorofluorescein diacetate (2',7'-DCFH-DA) and by the anthrone colourimetry. Compared to hairy roots in solid medium, hairy roots grew more rapidly in liquid medium but formed no callus and appeared to become brown earlier during culture. The fresh weight, the dry weight, the total content of soluble sugar and the levels of reactive oxygen species of hairy roots cultured into liquid medium MS without plant growth regulators for 15 days were 1.59 times, 1.18 times, 5.25 times and 1.16 times, respectively as much as that of hairy roots cultured onto solid medium. Our results firstly indicate that P. phaseoloides hairy roots in solid medium can utilize or metabolize more soluble sugar but produce less reactive oxygen species than that in liquid medium. This may be related to the fact that hairy roots are easier to turn brown in liquid medium than that onto solid medium. Our results have laid a foundation for defining optimum culture manner for large-scale cultivation and large-scale production of secondary metabolites of P. phaseoloides hairy roots.