Salicylic acid (SA) is a natural signaling molecule involved in plant defense response against pathogen infection. This article covers the recent key works contributing to our understanding of SA signaling and biosynthesis leading to a controlled SA level in plants.
Camptothecin derivatives are clinically used as anti-tumor alkaloids that are currently obtained by extraction from intact plants. Seeking for the alternative sources for commercial production and for fundamental study, cell and tissue cultures have been investigated. In the present study, we developed a method for regeneration of Ophiorrhiza pumila plant from hairy roots transformed with Agrobacterium rhizogenes. The regeneration frequency was over 83%. Integration of a rol B gene from T-DNA of A. rhizogenes was confirmed by polymerase chain reaction in both of the hairy roots and the regenerated plants. The transformed plants accumulated camptothecin in amounts of 66–111% compared with that in the wild-type plants.
The Eucalyptus perriniana cultured cells are widely used to biotransform a variety of compounds. The glucosyltransferase activity of a crude protein extract of E. perriniana cultured cells was maximized when cell growth was in the pre-logarithmic to logarithmic phase. We cloned a cDNA encoding glucosyltransferase (EPGT) from E. perriniana cultured cells by RT-PCR using a degenerated primer and RACE-PCR. The cDNA contained an open reading frame encoding 467 amino acids with a calculated molecular mass of 51.6 kDa. The consensus sequence of the plant glucosyltransferases was included in the deduced amino acid sequence. The amino acid sequence of EPGT showed a high identity to glucosyltransferases from tobacco and petunia. The recombinant EPGT was expressed in Escherichia coli and its substrate specificity was examined using UDP-[U-14C] glucose. Cinnamic acid was the best sugar acceptor in the compounds tested.
We report generation of 9,301 expressed sequence tags (ESTs) derived from callus cells of Euphorbia tirucalli in search of candidate genes involved in the triterpenoid and sterol biosyntheses. After assembling 4,342 redundant ESTs into 1,252 clusters, a total of 6,211 non-redundant sequences were obtained. Database search revealed that 4,449 out of the 6,211 sequences shared significant similarities to known nucleotide or amino acid sequences, while the remaining 1,762 showed no significant matches and appear to represent novel genes in E. tirucalli. The annotations assigned to the hit database entries suggest that 48 of the unique sequences are involved in triterpenoid and sterol biosyntheses. Although functions of genes tagged by the 48 sequences are yet to be determined, the EST resource described here should contribute to identification of genes participating in the triterpenoid and sterol biosyntheses in E. tirucalli.
Cultured cells of Sophora flavescens produce (2S)-naringenin-derived prenylated flavanone sophoraflavanone G and liquiritigenin-derived trifolirhizin 6′-O-malonate. The regulation of flavonoid biosynthesis was examined by analyzing the metabolites produced in the cultured cells fed (2RS)-naringenin. The amount of sophoraflavanone G in cells fed 0.1 or 0.3 mM (2RS)-naringenin was two-fold that in control cells, although the conversion ratio was only 5 to 10% of the administered (2S)-naringenin. On the other hand, (2R)-naringenin, which does not occur naturally, was efficiently converted into its 4′,7-di-O-β-D-glucoside. (2S)-Naringenin prenylation activity was higher at the logarithmic growth stage. The cells fed (2RS)-naringenin at a lower concentration (below 0.1 mM), accumulated sophoraflavanone G as the main prenylated flavanone. In contrast, cells fed 0.3 mM (2RS)-naringenin accumulated 8-prenylnaringenin and leachianone G, intermediates of sophoraflavanone G in large amounts. Accumulation of trifolirhizin 6′-O-malonate was suppressed by the addition of naringenin.
Somatic embryos of Corydalis ambigua (Papaveraceae), which were cultured in liquid Linsmaier and Skoog medium supplemented with 0.1 µM IAA and 3% sucrose, produced two tetrahydroprotoberberine alkaloids, corydaline (0.03% of dry cell weight) and cavidine (1.09%). Among various plant growth regulators tested, two phenylurea derivatives, thidiazuron and N-(2-chloro-4-pyridyl)-N′-phenylurea enhanced the alkaloid production. Addition of 1 µM thidiazuron to the medium gave the maximum alkaloid content, 0.12% corydaline and 1.91% cavidine, after 21 days incubation. These results were compared with those from callus cultures and various intact organs. Corydaline and cavidine were accumulated in leaf, tuber and somatic embryos, whereas corybulbine was detected only in tubers. Callus cultures and immature seeds, which lack embryos, contained only trace amounts of these alkaloids, suggesting the necessity for organ differentiation for alkaloid production in C. ambigua.
Yellow petals of carnations contain chalcone 2′-O-glucoside. The glucosylation occurs after the p-coumaroyl CoA and malonyl-CoA condensation reaction by chalcone synthase (CHS), but the enzyme(s) transferring glucose from UDP-glucose to the 2′-OH position of chalcone has not been identified. The full-length cDNA clones for 18 glucosyltransferase (GT) genes were isolated from petal tissue of carnation (Dianthus caryophyllus) bearing various flower colors. The 18 GTs encoded in the cDNAs were enzymatically characterized in an E. coli expression system using chalcone, flavanone, flavone, flavonol and anthocyanidin as substrates. Three of the 18 were characterized as 3-GT possessing different substrate specificities for flavonoids and anthocyanidin and another two GTs catalyzed the transfer of glucose to the 2′-hydroxyl group of chalcone. In addition, these two enzymes glucosylated flavonol (3-OH and 7-OH), flavanone (7-OH), flavone (7-OH) and anthocyanidin (3-OH and 7-OH).
Petunia flower colors are mainly due to flavonoids. The flower color of commercial varieties of Petunia hybrida was successfully modified by the suppression of endogenous flavonoid biosynthetic genes, the expression of a hetelorogous flavonoid biosynthetic gene, and the combination of both. Flower color changed from purple to almost white or from purple to red by the suppression of the endogenous gene expression, from red to orange by the down-regulation of the flavonoid 3′-hydroxylase gene and the expression of the rose dihydroflavonol 4-reductase gene, and from violet to pale violet by the expression of the flavonol synthase or flavone synthase gene. These results clearly indicate the usefulness of metabolic engineering of the flavonoid biosynthetic pathway to modify flower color. Only a few of the transgenic petunia exhibited phenotypic stability. For commercialisation, it is necessary to generate many independent transgenic lines, select elite lines with stable phenotypes and maintain them in tissue culture.
The adventitious roots of Raphanus sativus L. cv. Peking Koushin were established by culturing root segments of in vitro seedlings in 1/2 Murashige and Skoog (MS) liquid medium supplemented with 0.5 mg/l IBA. The adventitious roots cultured in 1/2 MS liquid medium supplemented with 0.5 mg/l IBA produced anthocyanin in the dark. When cultured under the 14 h/day light condition, the adventitious roots cultured with 0.5 mg/l IBA produced more anthocyanin than those cultured with 0.1 or 0.5 mg/l NAA. The acid hydrolysis of the extract prepared from the adventitious roots revealed that the main anthocyanidin was pelargonidin. In addition, the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the adventitious root extract was comparable to that of roots of the intact plant grown in the field.
A drastic increase in rosmarinic acid (RA) accumulation is induced in cultured cells of Lithospermum erythrorhizon after their exposure to yeast extract or methyl jasmonate. Addition of the elicitors to the cell cultures only slightly enhanced the activity of rosmarinic acid synthase, which catalyzes formation of 4-coumaroyl-4′-hydroxyphenyllactic acid (CHPL), the first reaction specific to RA biosynthesis. In contrast, the two-step cytochrome P450-catalyzed hydroxylations of CHPL to form RA were dramatically up-regulated by the elicitor treatments, indicating that these hydroxylation activities are likely to play a key regulatory role in elicitation of RA biosynthesis.
Euphorbia tirucalli is a potential source of commercially important chemicals such as sterols. Here we report the first successful plant regeneration from internode explants of E. tirucalli. Adventitious buds were efficiently induced on LS medium supplemented with 0.02 mg l-1 thidiazuron. On average of four experiments, 17.3 adventitious buds were induced from 12 explants on this medium. The adventitious buds grew into shoots during subsequent cultures on a hormone-free LS medium. For rooting treatment, we cultured these shoots on the LS medium containing 0.02 mg l-1 naphthalenacetic acid, followed by on the half-strength LS medium without vitamins, and were successful to obtain whole plantlets.
Variations in swollen hypocotyl of red and white radish (Raphanus sativus) varieties are attributed to the level of anthocyanin accumulation. However, it is not known which genes in the biosynthetic pathway are involved in this variation. Upon treatment with exogenous sucrose, hypocotyl of red radish produced a large amount of anthocyanins, while that of white radish produced little. Six genes involved in anthocyanin production and one gene in lignin biosynthesis in red and white varieties were tested to demonstrate which genes were expressed on addition of sucrose. In red radish, transcripts of all six anthocyanin-related genes were remarkably induced by sucrose, while none of them were induced in white radish. Transcripts for cinnamoyl-CoA reductase involved in lignin biosynthesis were not influenced by sucrose in either variety. These results suggest that the low level of anthocyanin accumulation in white radish is due to the weak response of anthocyanin-related genes to sucrose.
To examine the biotransformation of (+)-catechin by the plant cultured cells of Eucalyptus perriniana, three new biotransformation products, (+)-catechin-3′-O-β-D-glucoside, (+)-catechin-5-O-β-D-glucoside and (+)-catechin-7-O-β-D-glucoside were isolated after 3 days incubation. The cultured cells of E. perriniana were found to be capable of glycosylation at the 5, 7-positions on the A ring and the 3′-position on the B ring of (+)-catechin.
We obtained hairy roots of Ipomoea batatas cv. Ayamurasaki by infection of Agrobacterium rhizogenes A13. Accumulation of anthocyanins in hairy roots was observed when they were cultured under continuous light irradiation, whereas it was not observed when they were cultured in the dark. The amount of accumulated anthocyanins in hairy roots increased as a result of choosing the medium and increasing the sucrose concentration in the medium. Accumulation of anthocyanins was also observed in the hairy roots cultured in liquid medium in the dark by choosing medium.