Molecular cloning and characterization of genistein 4'-O-glucoside specific glycosyltransferase from Bacopa monniera.

Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 4'-O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 4'-O-glucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49% identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (+) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC-MS and HPLC, which confirmed its specificity for genistein 4'-O-glucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatio-temporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism.

Investigation of biochemical responses of Bacopa monnieri L. upon exposure to arsenate.

Widespread contamination of arsenic (As) is recognized as a global problem due to its well-known accumulation by edible and medicinal plants and associated health risks for the humans. In this study, phytotoxicity imposed upon exposure to arsenate [As(V); 0-250 µM for 1-7 days] and ensuing biochemical responses were investigated in a medicinal herb Bacopa monnieri L. vis-à-vis As accumulation. Plants accumulated substantial amount of As (total 768 µg g(-1) dw at 250 µM As(V) after 7 days) with the maximum As retention being in roots (60%) followed by stem (23%) and leaves (17%). The level of cysteine and total nonprotein thiols (NP-SH) increased significantly at all exposure concentrations and durations. Besides, the level of metalloid binding ligands viz., glutathione (GSH) and phytochelatins (PCs) increased significantly at the studied concentrations [50 and 250 µM As(V)] in both roots and leaves. The activities of various enzymes viz., arsenate reductase (AR), glutathione reductase (GR), superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and catalase (CAT) showed differential but coordinated stimulation in leaves and roots to help plants combat As toxicity up to moderate exposure concentrations (50 µM). However, beyond 50 µM, biomass production was found to decrease along with photosynthetic pigments and total soluble proteins, whereas lipid peroxidation increased. In conclusion, As accumulation potential of Bacopa may warrant its use as a phytoremediator but if Bacopa growing in contaminated areas is consumed by humans, it may prove to be toxic for health.

Use of the cryptogein gene to stimulate the accumulation of Bacopa saponins in transgenic Bacopa monnieri plants.

Genetic transformation of the Indian medicinal plant, Bacopa monnieri, using a gene encoding cryptogein, a proteinaceous elicitor, via Ri and Ti plasmids, were established and induced bioproduction of bacopa saponins in crypt-transgenic plants were obtained. Transformed roots obtained with A. rhizogenes strain LBA 9402 crypt on selection medium containing kanamycin (100 mg l(-1)) dedifferentiated forming callus and redifferentiated to roots which, spontaneously showed shoot bud induction. Ri crypt-transformed plants thus obtained showed integration and expression of rol genes as well as crypt gene. Ti crypt-transformed B. monnieri plants were established following transformation with disarmed A. tumefaciens strain harboring crypt. Transgenic plants showed significant enhancement in growth and bacopa saponin content. Bacopasaponin D (1.4-1.69 %) was maximally enhanced in transgenic plants containing crypt. In comparison to Ri-transformed plants, Ri crypt-transformed plants showed significantly (p ≤ 0.05) enhanced accumulation of bacoside A(3), bacopasaponin D, bacopaside II, bacopaside III and bacopaside V. Produced transgenic lines can be used for further research on elicitation in crypt-transgenic plants as well as for large scale production of saponins. Key message The cryptogein gene, which encodes a proteinaceous elicitor is associated with increase in secondary metabolite accumulation-either alone or in addition to the increases associated with transformation by A. rhizogenes.

Iron-induced oxidative stress in a macrophyte: a chemometric approach.

Iron-induced oxidative stress in plants of Bacopa monnieri L., a macrophyte with medicinal value, was investigated using the chemometric approach. Cluster analysis (CA) rendered two distinct clusters of roots and shoots. Discriminant analysis (DA) identified discriminating variables (NP-SH and APX) between the root and shoot tissues. Principal component analysis (PCA) results suggested that protein, superoxide dismutase (SOD), ascorbic acid, proline, and Fe uptake are dominant in root tissues, whereas malondialdehyde (MDA), guaiacol peroxidase (POD), cysteine, and non-protein thiol (NP-SH) in shoot of the stress plant. Discriminant partial-least squares (DPLS) results further confirmed that SOD and ascorbic acid contents dominated in root tissues, while NP-SH, cysteine, POD, ascorbate peroxidase (APX), and MDA in shoot. MDA and NP-SH were identified as most pronounced variables in plant during the highest exposure time. The chemometric approach allowed for the interpretation of the induced biochemical changes in plant tissues exposed to iron.

Effect of bavistin on in vitro plant conversion from encapsulated uninodal microcuttings of micropropagated Bacopa monnieri (L.)--an Ayurvedic herb.

A reliable and reproducible protocol for contamination free plant recovery system from alginated encapsulated uninodal microcuttings of micropropagated Bacopa monnieri L. have been developed after storage at 18 degrees C for 45 days. Node segments excised from freshly micropropagated plants were encapsulated as single explant beads with 3.0% sodium alginate and 80 mM CaCl2 2 H2O. To find out the optimal concentration of fungicide bavistin for efficient plant recovery different concentrations of bavistin (1.0 - 15 mg l(-1)) were incorporated in to the encapsulation medium. 3.0mg l(-1) bavistin showed no reduction in plant conversion and generated maximum number of shoots (45.6 +/- 1.69) at high frequency with out any contamination after storage up to 45 days at 18 degrees C. At high concentrations (13 and 15 mg l(-1)), rupturing of calcium alginate coats after 8 - 9 days and gradual decline in the number of shoots indicates the toxic effect of bavistin on plant conversion. Encapsulated node cuttings stored up to 45 days regenerated shoots (5.2) and multiple shoots (45.6) in MS basal and hormone medium respectively. Maximum shoot length (8.2 +/- 0.37 cm) was observed from encapsulsted node cuttings incorporated with 3.0 mg l(-1) bavistin on MS basal medium. 90% of the recovered plantlets were hardened off and successfully established in soil.

Improvement of pseudojujubogenin glycosides production from regenerated Bacopa monnieri (L.) Wettst. and enhanced yield by elicitors.

Bacopa monnieri (L.) Wettst. was studied for shoot induction and regeneration on Murashige and Skoog (MS) medium supplemented with different plant growth regulators. Stem explants cultured on medium containing 0.1 mg/l thidiazuron (TDZ) resulted in the highest number of shoots (117 shoots/explant). Regenerated plants from medium with 0.5 mg/l TDZ contained the highest level of pseudojujubogenin glycosides [(30.62 +/- 1.29) mg/g dry wt] which was 2-fold higher than that of in vitro grown plants of the same age [(16.96 +/- 1.49) mg/g dry wt]. Plantlets regenerated from 0.1 mg/l TDZ also showed a high level of pseudojujubogenin glycosides [(27.94 +/- 1.19) mg/g dry wt]. The effect of elicitor on pseudojujubogenin glycosides accumulation in B. monnieri whole plant cultures was investigated. Chitosan at 150 mg/l and yeast extract at 2 mg/ml increased the pseudojujubogenin glycosides production [(40.83 +/- 2.24) mg/g dry wt and (40.05 +/- 2.37) mg/g dry wt, respectively] after 7 days, which was 6-fold higher than in the control cultures.

Shoot bud regeneration from different explants of Bacopa monniera (L.) Wettst. by trimethoprim and bavistin.

A mass in vitro propagation system devoid of growth regulators for Bacopa monniera (L.) Wettst., a traditional Indian medicinal plant, has been developed. Direct shoot bud regeneration was induced by culturing internode and leaf explants on Murashige and Skoog's (MS) medium supplemented with an antibiotic (trimethoprim) or a fungicide (bavistin). Bavistin showed a marked cytokinin-like activity, as evident from high number of shoot buds induced in node, internode and leaf explants. Optimum adventitious shoot buds induction occurred at 300 mg/l bavistin from internode explants. In vitro regenerated shoots were elongated and rooted before transferred to field with 85% survival. The regeneration protocol developed in this study illustrates the usefulness of additives for mass propagation and germplasm conservation of B. monniera.

Effect of iron on lipid peroxidation, and enzymatic and non-enzymatic antioxidants and bacoside-A content in medicinal plant Bacopa monnieri L.

The effect of Fe was investigated in medicinally important plant, Bacopa monnieri L. and the response on malondialdehyde (MDA) content, superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) was found different in roots and leaves of the metal treated plants. Iron induced stress was observed as indicated by high level of lipid peroxidation, being more steep increase in leaves than roots. In roots, SOD activity was found to increase in metal treated plants except 80 and 160 microM at 72 h, whereas, it decreased in leaves except 10 and 40 microM after 48 h as compared to their respective controls. Among H2O2 eliminating enzymes, POD activity increased in roots, however, it decreased in leaves except at 10 and 40 microM Fe after 48 h as compared to control. At 24 and 48 h, APX activity and ascorbic acid content followed the similar trend and were found to increase in both parts of the metal treated plants as compared to their respective controls. The level of cysteine content in the roots increased at initial period of exposure; however, no marked change in its content was noticed in leaves. In both roots and leaves, non-protein thiol content was found to increase except at higher metal concentrations at 72 h. The data of proline content have shown significant (p<0.01) increase at 40 microM onwards in both part of the plants after 48 and 72 h. Correlation coefficient was evaluated between metal accumulations with various parameters and also between different antioxidant parameters with MDA. Since the level of bacoside-A (active constituent) content in metal treated plants increases, therefore, it is advisable to assess the biological activity of the plants before using for medicinal purposes, particularly in developing countries.