Molecular diversity of tuliposide B-converting enzyme in tulip (Tulipa gesneriana): identification of the root-specific isozyme.

6-Tuliposide B (PosB) is a glucose ester accumulated in tulip (Tulipa gesneriana) as a major secondary metabolite. PosB serves as the precursor of the antimicrobial lactone tulipalin B (PaB), which is formed by PosB-converting enzyme (TCEB). The gene TgTCEB1, encoding a TCEB, is transcribed in tulip pollen but scarcely transcribed in other tissues (e.g. roots) even though those tissues show high TCEB activity. This led to the prediction of the presence of a TCEB isozyme with distinct tissue specificity. Herein, we describe the identification of the TgTCEB-R gene from roots via native enzyme purification; this gene is a paralog of TgTCEB1. Recombinant enzyme characterization verified that TgTCEB-R encodes a TCEB. Moreover, TgTCEB-R was localized in tulip plastids, as found for pollen TgTCEB1. TgTCEB-R is transcribed almost exclusively in roots, indicating a tissue preference for the transcription of TCEB isozyme genes.

A protocol for axenic liquid cell cultures of a woody leguminous mangrove, Caesalpinia crista, and their amino acids profiling.

Callus induction, maintenance and protoplast cultures were achieved from immature seeds of a woody leguminous mangrove, Caesalpinia crista. Axenic cultures were possible during 1.5 months of pod storage in 0.1% benzalkonium chloride solution. Callus induction was achieved using 1 mL liquid medium in a 10 mL flat-bottomed culture tube. Protoplasts were isolated using Cellulase R10, Hemicellulase, and Driselase 20 in 0.6 M mannitol solution and sub-culturable calluses were obtained in 50 µL liquid medium using a 96-microplate method. The optimal hormonal concentration was 10 µM each of 2,4-dichlorophenoxyacetic acid and benzyladenine in liquid Murashige and Skoog's basal medium for both callus induction and maintenance, and protoplast cultures. Similarities and differences in amino acid profiles and culture conditions are discussed among woody mangrove species and non-mangrove leguminous species. Caesalpinia crista cultures were unique as they secreted a large amount of amino acids, including proline, into the liquid culture medium.

The biosynthetic activities of primary and secondary metabolites in suspension cultures of Aquilaria microcarpa.

Two types of suspension-cultured Aquilaria microcarpa cells, friable and aggregated, were selectively generated. The biosynthetic activities of primary and secondary metabolites in target cells were detected using laser scanning microscopy (LSM) imaging with diphenylboric acid 2-amino ethyl ester (DPBA) and 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) staining. Scanned friable cells produced weakly fluorescent images revealing low productivity of metabolites. On the other hand, scanning of aggregated cells produced clear fluorescent images depicting the accumulations of flavonoids and lipids. Furthermore, abundant deposition of an unknown resinous compound in extracellular portion of aggregated cells could be visualized. The resinous compound was white to whitish-gray in color and highly sedimented in the medium. Based on these observations, we focused our investigation of metabolite productivity on aggregated suspension cells. Some prominent extracellular compounds were detected in the used liquid medium, as well as in the resinous residue within the medium. The characteristics of these metabolites were investigated in detail via gas chromatography-mass spectrometry (GC-MS) analysis.

A stepwise protocol for induction and selection of prominent coniferous cell cultures for the production of ß-thujaplicin.

In order to demonstrate the potential of plant cell culture systems to produce a target natural bioactive compound, we proposed a stepwise protocol for ß-thujaplicin production as follows. 1. Induction phase: Characteristics of callus cultures originating from newly flushed shoots of 10 conifer species were evaluated on different basal media such as Murashige and Skoog (MS), Schenk and Hildebrandt (SH), and Lloyd and McCown's Woody Plant medium (WP) containing 10 µM 2,4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 1 µM of N6-benzyladenine (BA). The conifer species used were as follows: Chamaecyparis (C. obtusa Sieb. et Zucc. and C. pisifera Sieb. et Zucc.), Juniperus (J. chinensis L. 'Kaizuka', J. chinensis L. var. sargentii, and J. conferta Parlatore), Thuja (T. occidentalis L. and T. standishii (Gord.) Carr.), Thujopsis (T. dolabrata Sieb. et Zucc. and T. dolabrata Sieb. et Zucc. var. hondae), and Cryptomeria (C. japonica D. Don). We observed the phenotypes of each callus to determine the optimal conditions for callus induction and to infer biosynthetic activity of the calli over 4-8 weeks. 2. Habituation phase: Each of the cell cultures obtained was transferred to a modified MS medium containing 680 mg L(-1) KH2PO4 and 10 µM Picloram to select the habituated cells with synchronous growth pattern. The growth of each cell culture was highly improved in the habituation medium, except that of J. chinensis 'Kaizuka'. 3. Metabolite-production phase: The concentration of ß-thujaplicin (known as hinokitiol in Japan) in the shoots of donor trees and the habituated cell cultures was analyzed via high-performance liquid chromatography (HPLC). Histochemical characteristics of the cells were also observed using laser scanning microscopy (LSM) imaging. After the third step, we tested the biosynthetic activity of two habituated calli (C. obtusa and J. conferta) on a 0.3%, w/v, yeast extract (YE)-containing medium. We found significant improvement in ß-thujaplicin production in J. conferta callus (4600 µg g DW-1), which was up to 20-fold higher than in the habituation phase.

Plant cell, tissue and organ culture: the most flexible foundations for plant metabolic engineering applications.

Significant advances in plant cell, tissue and organ culture (PCTOC) have been made in the last five decades. PCTOC is now thought to be the underlying technique for understanding general or specific biological functions of the plant kingdom, and it is one of the most flexible foundations for morphological, physiological and molecular biological applications of plants. Furthermore, the recent advances in the field of information technology (IT) have enabled access to a large amount of information regarding all aspects of plant biology. For example, sequencing information is stored in mega repositories such as the National Center for Biotechnology Information (NCBI), which can be easily accessed by researchers worldwide. To date, the PCTOC and IT combination strategy for regulation of target plant metabolism and the utilization of bioactive plant metabolites for commercial purposes is essential. In this review, the advantages and the limitations of these methodologies, especially regarding the production of bioactive plant secondary metabolites and metabolic engineering in target plants are discussed mainly from the phenotypic view point.

Molecular identification of tuliposide B-converting enzyme: a lactone-forming carboxylesterase from the pollen of tulip.

6-Tuliposides A (PosA) and B (PosB), which are the major secondary metabolites in tulip (Tulipa gesneriana), are enzymatically converted to the antimicrobial lactonized aglycons, tulipalins A (PaA) and B (PaB), respectively. We recently identified a PosA-converting enzyme (TCEA) as the first reported member of the lactone-forming carboxylesterases. Herein, we describe the identification of another lactone-forming carboxylesterase, PosB-converting enzyme (TCEB), which preferentially reacts with PosB to give PaB. This enzyme was isolated from tulip pollen, which showed high PosB-converting activity. Purified TCEB exhibited greater activity towards PosB than PosA, which was contrary to that of the TCEA. Novel cDNA (TgTCEB1) encoding the TCEB was isolated from tulip pollen. TgTCEB1 belonged to the carboxylesterase family and was approximately 50% identical to the TgTCEA polypeptides. Functional characterization of the recombinant enzyme verified that TgTCEB1 catalyzed the conversion of PosB to PaB with an activity comparable with the native TCEB. RT-qPCR analysis of each part of plant revealed that TgTCEB1 transcripts were limited almost exclusively to the pollen. Furthermore, the immunostaining of the anther cross-section using anti-TgTCEB1 polyclonal antibody verified that TgTCEB1 was specifically expressed in the pollen grains, but not in the anther cells. N-terminal transit peptide of TgTCEB1 was shown to function as plastid-targeted signal. Taken together, these results indicate that mature TgTCEB1 is specifically localized in plastids of pollen grains. Interestingly, PosB, the substrate of TgTCEB1, accumulated on the pollen surface, but not in the intracellular spaces of pollen grains.

Environmentally benign process for the preparation of antimicrobial α-methylene-ß-hydroxy-γ-butyrolactone (tulipalin B) from tulip biomass.

Tulipalin B (α-methylene-ß-hydroxy-γ-butyrolactone, PaB) is an antimicrobial natural product occurring in tulip (Tulipa gesneriana). PaB is directly formed from the precursor glucose ester 6-tuliposide B (PosB) by endogenous Pos-converting enzyme (TCE). Despite the potential usefulness of antibacterial PaB in various industrial applications, lack of facile synthetic schemes hampers its practical use. Herein, we describe an environmentally benign and facile process for the preparation of PaB using tulip biomass materials based on one-step enzyme reaction catalyzed by TCE without the use of petroleum-derived solvents. By screening 115 tulip cultivars, we found three elite cultivars, which accumulated PosB almost exclusively in flower tissues. The flower extracts with aqueous ethanol were partially purified with activated charcoal and subjected to the enzyme reaction with reusable immobilized TCE prepared from bulb crude extracts. The reaction was completed in a few hours at room temperature, and PaB was purified with activated charcoal and ethanol in a batch-wise manner.

Methyl jasmonate-induced enhancement of expression activity of Am-FaPS-1, a putative farnesyl diphosphate synthase gene from Aquilaria microcarpa.

A cDNA clone, designated Am-FaPS-1 (1310 bp), was isolated from callus culture derived from the leaf tissues of Aquilaria microcarpa. This gene contains an open reading frame encoding the protein of 342 amino acid residues with high homology to farnesyl diphosphate synthase from various plant sources. An appreciable increase in the transcriptional level of Am-FaPS-1 was reproducibly observed by the exposure of the cell culture to methyl jasmonate. The expression activity of the gene was also elevated when the cells were treated with yeast extract and Ca(2+)-ionophore A23187. These results suggest that Am-FaPS-1 and its translate play roles in methyl jasmonate- and yeast extract-induced responses of A. microcarpa, and Ca(2+) functions as an important messenger molecule in these processes. This set of the results would support our hypothesis that the activation of Ca(2+)-cascade evoked by the elevation of cytoplasmic Ca(2+) concentration is an essential early event in methyl jasmonate-induced responses of higher plant cells.

Transcriptional activation of putative calmodulin genes am-cam-1 and am-cam-2 from Aquilaria microcarpa, in response to external stimuli.

A homology-based cloning strategy yielded two cDNA clones designated Am-cam-1 and Am-cam-2, presumably encoding calmodulin protein from a callus culture derived from the leaf tissues of Aquilaria microcarpa. An appreciable increase in the transcriptional activity of Am-cam-1 was reproducibly observed by exposure of the cell culture to methyl jasmonate, as analyzed by a reverse-transcription polymerase chain reaction. The expression level of the gene also increased when the cells were treated with yeast extract. The transcription of Am-cam-2 was similarly stimulated by the treatment with methyl jasmonate and yeast extract, however, the intensities of the enhanced expression appeared to be lower as compared with that of Am-cam-1. In contrast, Ca(2+)-ionophore A23187 did not show inducing activity for the expression of these two calmodulin genes. These results suggest that Am-cam-1 and Am-cam-2 and their products play important roles in signal transduction processes in methyl jasmonate- and yeast extract-treated cells of A. microcarpa, accompanying the change in the transcriptional activities.

Distribution and biosynthesis of theanine in Theaceae plants.

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (<0.2 mumol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [(14)C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.

Ethylamine content and theanine biosynthesis in different organs of Camellia sinensis seedlings.

We examined the distribution of ethylamine, glutamic acid and alanine, which are utilized in theanine biosynthesis, and other major amino acids in leaves, stems, cotyledons and roots of 6-week-old tea seedlings. Ethylamine and glutamic acid, which are substrates of theanine synthetase, were distributed almost uniformly in all parts of the seedlings; the contents in micromol/g fresh wt varied from 0.44-0.88 (ethylamine) and 1.6-2.4 (glutamic acid). The content of alanine, a possible precursor of ethylamine synthesis, was significantly higher in roots (3.1 micromol/g fresh wt) than in other parts. Incorporation of radioactivity from [U-14C]-alanine into theanine was also higher in roots than in other organs. In 10-week-old seedlings, [1-14C]ethylamine was converted to theanine in young and developed leaves, stems, main and lateral roots; the highest rates of conversion were detected in the main and lateral roots. These results suggest that the theanine synthesis preferentially takes place in roots but is not restricted to them; substrates and the enzymatic machinery for theanine synthesis are available in all parts of tea seedlings.

Possibility for selective accumulation of polyphenolics in tissue cultures of senno (Lychnis senno Siebold et Zucc.).

Senno (Lychnis senno Siebold et Zucc.), a traditional ornamental plant in Japan had been used as a crude drug acting as natural blood thinners. Since tissue culture protocols have been established, we analyzed polyphenol accumulation profiles in shoot culture, multiple shoot culture, and callus culture using the technique of HPLC with a Photodiode Array Detector. By comparing the HPLC profiles at 220-400 nm from extracts of different cultures, 14 putative flavonoids were confirmed as major metabolites in the cultures of senno. Among the 14 compounds detected, 6 were tissue specific metabolites. It appears that the biosynthetic pathway of polyphenolics in Senno is regulated or strongly influenced by how tissues are regenerated and maintained in the in vitro environment. Hence, it may be possible to selectively produce novel secondary metabolites including flavonoids by engineering a target tissue culture procedure developed in the present study.

A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress.

A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 muM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1-2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 muM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.

Expression of caffeine biosynthesis genes in tea (Camellia sinensis).

Using semi-quantitative reverse transcription-PCR, we studied the expression of genes encoding caffeine synthase (TCS1), inosine-5'-monophosphate dehydrogenase (TIDH), S-adenosyl-L-methionine synthase (sAMS), phenylalanine ammonia-lyase (PAL) and alpha-tubulin (Tua1) in young and mature leaves, stems and roots of 4-month-old tea seedlings and young and old tea tissue cultures. The amounts of transcripts of TCS1 were much higher in young leaves than in other parts of the plant. Expression of TIDH was greater in leaves than in other parts. Little difference in the amounts of transcripts of PAL, sAMS and Tua1 was found between various organs of tea seedlings. Larger amounts of transcripts of TCS1 and PAL were found in young callus tissues than in old tissues. These results support our conclusion deriving from previous enzymatic and metabolic studies that caffeine is synthesized mainly in young leaf tissues. We propose that marked caffeine biosynthesis in young leaves is dependent on a greater expression of the TCS1 gene in the organ.