Trichomes can be found on the surfaces of the leaves, stems, and other organs of many angiosperm plants. Plant trichomes are commonly divided into two classes: glandular trichomes and non-glandular trichomes. Glandular trichomes produce large quantities of specialized natural compounds of diverse classes and are regarded as ‘chemical factories’ due to their impressively efficient biosynthetic capacities. This efficiency makes glandular trichomes an excellent experimental system for the elucidation of both the biosynthesis and the mechanisms of regulation of natural product pathways. The development of various -omics techniques has greatly accelerated experimental procedures that are typically used in combination with trichome studies. The purpose this review is to provide an introduction to the methods and technologies used for the investigation of glandular trichomes, to summarize current progress in the field, and to highlight the potential applications of trichome studies in metabolic engineering using the strategies of synthetic biology.
The family Myrtaceae is known for its high foliar terpene concentrations as well as significant qualitative and quantitative variation in foliar terpenes between taxa, populations and individuals. To date, few studies have investigated the genetic and biochemical processes, which underlie this variation, much of which is known to be under genetic control. Differences in yield are both ecologically and commercially important and a better understanding of its basis will allow a greater understanding of Australian ecosystems as well as improve commercial viability of essential oil industries. Over the past decade a good understanding of the genes involved in terpene biosynthesis has developed in other species and several important regulatory steps have been identified. Much of this work has been done in transgenic plants, so our understanding at a molecular level is strong. Nonetheless, it remains unclear if these processes are transferrable to wild populations, or indeed how ecologically important quantitative variation in terpenoids arise and are maintained in natural ecosystems. In this review we will summarize what is known about terpene biosynthesis and the control of flux through the terpene biosynthetic pathways. We will then argue that this platform of work provides a great resource for Myrtaceae, as well as other plants, to identify candidate genes that control flux through the biosynthetic pathways and how this will inform further studies into the ecological implications of quantitative variation of terpenes. Work into terpene biosynthesis would also provide a framework to improve the profitability of essential oil crops.
Plants have the ability to produce vast amounts of secondary metabolites, including terpenoids, flavonoids, alkaloids, and their derivatives. These metabolites sometimes affect the growth of other organisms, either positively or negatively. Production of defensive compounds, for example phytoalexins, is a well-known strategy used by plants to defend against and withstand invading pathogens. As phytoalexins serve an obvious function in plant defense, they are known as specialized metabolites, substances with a specific biological role that function during specific circumstances. In rice, 15 diterpenoid-type phytoalexins and one flavonoid-type phytoalexin have been identified. Extensive studies have explored the biochemistry, biosynthesis, and biological functions of phytoalexins in rice, specifically the mechanism by which they function in disease resistance. This review focuses on our current knowledge of the transcriptional and hormonal regulation of the production of phytoalexins in rice.
Japanese cultivated gentians (Gentiana triflora, G. scabra and their hybrids), some of the most important ornamental flowers in Japan, have vivid blue flowers that accumulate polyacylated anthocyanins such as gentiodelphin. To breed attractive flower colors in Japanese gentians, our research group has been studying the molecular mechanisms that control flower pigmentation. Flavonoids, including anthocyanins, are widely distributed in the plant kingdom and are found in almost all plant organs. Along with longstanding genetic and molecular biological analyses of flavonoid biosynthesis, recent studies have revealed that transcription activators and repressors are involved in sophisticated control of temporal and spatial flavonoid accumulation in various plant organs. In this review, we summarize recent research on the transcriptional regulation of flavonoid biosynthesis in flowers, with a special focus on our findings using Japanese gentians. We also introduce and discuss the potential application of these transcription factor genes as novel tools to engineer flower color intensity and patterns in floricultural plants.
Phenylpropenes such as eugenol, isoeugenol, chavicol, and anethole are C6–C3 volatile compounds derived from phenylalanine by modification of its benzene ring and reduction of its propyl side chain, with the final reduction step catalyzed by a product-specific phenylpropene synthase. Recent advances in the biochemical and molecular analysis of phenylpropene synthases have improved our understanding of their evolution, structural properties, and reaction mechanism, providing insights into how plants produce and regulate the many types of phenylpropene volatiles. Since phenylpropenes are important in determining the flavor of foods and the quality of essential oils in cosmetics, the identification of the genes and enzymes responsible for the biosynthesis of phenylpropene volatiles has also provided us with tools to meet the challenge of improving plant aromas through genetic engineering.
When exposed to herbivore-infested plant volatiles or volatiles from artificially damaged plants, intact plants enhance their defense against herbivores. This phenomenon is called plant-plant communication. Here, we outline studies on plant-plant communication from both ecological and plant physiological perspectives. Regarding the ecological perspective, we give an overview of studies showing that plant–plant communication affect direct and indirect defense levels of exposed plants, and herbivore performance on exposed plants. Cases of kin selection in plant–plant communications and intra-plant communication via airborne signals are also summarized. Regarding the plant physiological perspective, we give an overview of studies that showed specific responses of receiver plants to a volatile molecular species, to different configurations of a volatile molecular species and to blends of volatiles. Furthermore, we review the signaling pathways involved, priming, sensitivity, and how plants receive volatile compounds in plant–plant communications.
Multidrug and toxic compound extrusion (MATE) transporters are a family of cation antiporters occurring in most organisms from prokaryotes to eukaryotes. This family constitutes one of the largest transporter families in plants, with, for example, more than 50 MATE genes in the Arabidopsis genome. Moreover, MATE transporters are involved in a wide variety of physiological functions throughout plant development, transporting a broad range of substrates such as organic acids, plant hormones and secondary metabolites. This review categorizes plant MATE transporters according to their physiological roles and summarizes their tissue specificity, membrane localization, and transport substrates. We also review the molecular evolutionary development of plant MATE transporters.
Flavonoids, one of the most-described group of plant “specialized metabolites”, consist of more than 10,000 structurally diverse compounds. Most flavonoids accumulate in plant vacuoles as glycosides, with some released by the roots into rhizospheres. These flavonoids are involved in biological communications with rhizobia, arbuscular mycorrhizal fungi, plant growth promoting rhizobacteria, pathogens, nematodes, and other plant species. Both aglycones and glycosides of flavonoids are found in root exudates and in soils. This review describes researches on the mechanisms of flavonoid secretion and the fate of flavonoids released into rhizospheres. This review also discusses the direction of future research that may elucidate the specific roles of flavonoids in biological communications in rhizospheres, enabling the utilization of flavonoid activities and functions in agricultural practice.
Green leaf volatiles (GLVs) are six-carbon volatile compounds. They are formed from fatty acids by a dioxygenation reaction catalyzed by lipoxygenase and a subsequent cleavage reaction catalyzed by hydroperoxide lyase. GLVs are involved in direct and indirect plant defense against herbivores and pathogens. In intact plant tissues, GLVs are usually present at low concentrations, but upon wounding, GLVs are synthesized rapidly: within seconds to minutes. It has been hypothesized that this ‘GLV burst’ is supported by activation of pre-existing enzymes on endogenous substrates; however, the detailed mechanism of the GLV burst has not been elucidated. Recently, we found that a certain portion of GLVs is formed without liberation of free fatty acids from lipids. Accordingly, we hypothesized that lipoxygenase plays an essential role in the GLV burst. In particular, direct oxygenation by lipoxygenase on membrane lipids seems to be responsible. Lipoxygenase is also a target for controlling GLV levels in food derived from plants.
Plants produce a multitude of secondary metabolites, including alkaloids with biological activities, and many alkaloids have been used for medicinal purposes. The biosynthetic enzymes and genes involved in alkaloid metabolic pathways exhibit divergent localizations, implying that alkaloid metabolites, including pathway products and intermediates, travel from organelle to organelle, cell to cell, and organ to organ. Biochemical studies have indicated that specific transporters move these metabolites. Indeed, molecular and cellular approaches have identified alkaloid transporters of the ATP-binding cassette (ABC) protein, multidrug and toxic compound extrusion (MATE), and purine permease (PUP) families. Interestingly, some of these transporters were found to be required for the efficient biosynthesis of alkaloids in plants. Here, we provide an updated inventory of alkaloid transporters and discuss the possibility of genetically manipulating the expression of these transporters to increase the accumulation of valuable alkaloid compounds.
Plant specialized metabolites play important roles in human life. These metabolites, however, are often produced in small amounts in particular plant species. Moreover, some of these species are endangered in their natural habitats, thus further limiting the availability of some plant specialized metabolites. Microbial production of these compounds may circumvent this problem. Considerable progress has been made in the microbial production of various plant specialized metabolites over the past decade. Now, the microbial production of these compounds is becoming robust, fine-tuned, and commercially relevant systems using the methods of synthetic biology. This review describes the progress of microbial production of plant specialized metabolites, including phenylpropanoids, flavonoids, stilbenoids, diarylheptanoids, phenylbutanoids, terpenoids, and alkaloids, and discusses future challenges in this field.
Artemisinin is the most effective antimalarial compound isolated from Artemisia annua. Artemisinic aldehyde Δ11(13)-reductase (DBR2) catalyzes the reduction of artemisinic aldehyde into dihydroartemisinic aldehyde, switching the pathway towards artemisinin production. Although other Artemisia species cannot produce artemisinin, we found a putative DBR2 ortholog expressed in A. absinthium (abDBR2). We examined the catalytic activity of abDBR2 in vitro and found that it shows comparable activity to that of DBR2 based on the reduction of artemisinic aldehyde into dihydroartemisinic aldehyde. Furthermore, we found that dihydroartemisinic aldehyde was detected in the extract of A. absinthium leaves fed with artemisinic aldehyde, suggesting the presence of active abDBR2 in planta. Our results indicate that A. absinthium may be a potential host for the production of artemisinin through metabolic engineering.
(+)-Sesamin is a major furofran-class lignan in sesame seeds and harbors characteristic two methylenedioxy bridges (MDB) that are sequentially formed from (+)-pinoresinol via (+)-piperitol by a Sesamum indicum P450, CYP81Q1. However, the molecular basis for this unique catalytic activity of CYP81Q1 has been poorly understood. To elucidate MDB formation, we tested various natural and non-natural metabolites as substrates for CYP81Q1. A synthetic (+)-SC1mr and a naturally occurring (+)-kobusin showed inhibitory effect on the production of (+)-sesamin by CYP81Q1 unlike (+)-epipinoresinol and (−)-pinoresinol, indicating the strict diastereomer and enantiomer selectivity. Homology modeling followed by site-directed mutagenesis of CYP81Q1 showed that an amino acid residue crucial for MDB formation is a unique Ala residue (A308), located in I-helix proximal to the substrate pocket, responsible to the conserved distal-Thr residue. MDB by CYP81Q1 is produced possibly through the formation of a substrate-participated hydrogen-bonding network, since single replacement of the Ala by Thr severely and specifically lowered the MDB forming activity. This hypothesis is supported by a newly identified MDB-forming enzyme CYP81Q38 from Phryma leptostachya harboring an Ala responsible to Ala308 in CYP81Q1. An evolutional perspective of CYP81Q1 is discussed in relation to another MDB-forming CYP719As functionally conserved in Ranunculales.
Heartwood of Caesalpinia sappan L. has been traditionally used to many diseases such as homoptysis, syphilis, eye disease, dysentery, depurative and prevention of osteoporosis. Our previous in vitro screening of Indonesian plants revealed that an ethanolic extract of the heartwood of C. sappan exhibits a proliferation stimulating activity against primary osteoblastic cells. In our continued interest to this plant, we further fractionated the extract and isolated active constituents on the basis of the stimulating activity in the osteoblastic cells. The fractionation and isolation were carried out with various chromatography methods and the structure of isolated compounds was elucidated based on NMR, IR, UV and MS spectroscopic data. From an active fraction, a new biphenyl dimer, namely caesappanin C (1), along with two known compounds, protosappanin A (2) and sappanchalcone (3), were isolated. Among them, the new compound 1 exhibited the strongest activity and significantly increased the cell viability up to 276±5%. The other two compounds 2 and 3 also stimulated the cell proliferation and increased the cell viability up to 233±8% and 187±4%, respectively.
Anabasine is an alkaloid found in a small number of Nicotiana species. The components of the anabasine biosynthetic pathway have yet to be identified. Here, we report the reinvestigation of biosynthetic pathways of anabasine and related tobacco alkaloids in genetically engineered cells. Hairy roots of N. tabacum harboring a lysine/ornithine decarboxylase gene from Lupinus angustifolius (La-L/ODC) were fed with labeled [ε-15N]- or [α-15N]-L-lysine. Relative to the unfed control, feeding of labeled 15N-L-lysine greatly enhanced anabasine levels 13.5-fold in La-L/ODC-expressing line compared to 5.3-fold in the control line, suggesting that both LDC activity and substrate supplied are important factors for the efficient production of anabasine. GUS-expressing line showed preferential incorporation of [ε-15N]-L-lysine into anabasine, indicating the main biosynthetic pathway of Δ1-piperideine intermediate in tobacco is asymmetrically processes. In contrast, the expression of La-L/ODC showed the symmetric labeling of 15N atom into anabasine, implying the occurrence of free cadaverine, which is produced by La-L/ODC enzyme, during the biosynthesis of Δ1-piperideine intermediate. No considerable incorporation of 15N into other tobacco alkaloids such as, nicotine, anatabine, and anatalline, was detected. Detailed analysis using ultra-high resolution mass spectrometry indicated that two 15N atoms were incorporated into anabasine in La-L/ODC-expressing lines after feeding [ε-15N]- or [α-15N]-L-lysine. Our results not only provide information insight into the biosynthesis of anabasine but also suggest an alternative route for the production of anabasine by genetic engineering.
C-Glucosides are glucose-containing glycosides that have carbon–carbon bonds between the anomeric carbon of the sugar moieties and aglycon, rendering the molecules remarkably stable against hydrolysis by enzymes or acids. In this work, we showed the production of C-glucosides of flavonoids and related compounds (i.e., 2-hydroxyflavanone, dihydrochalcone, and trihydroxyacetophenone) by Escherichia coli expressing buckwheat C-glucosyltransferase. The substrates in their respective cultures were taken up by the cells and C-glucosylated, and the products were released into the culture media. The bioconversion process was completed in 1–2 h, but products were already observed immediately after addition of the substrates (200 µM). The conversion rates of these substrates reached 80–95%. Without addition of glucose to the conversion media, almost no C-glucosides were produced. Although the amounts of the substrates fed to their respective cultures were limited by their solubility in water, repeated addition of the substrate to the culture at regular time intervals effectively increased the total amount of product obtained.
Fresh ginger (Zingiber officinale) rhizome is characterized by a pleasant citrus aroma and pungent flavor. The majority of the aroma-contributing volatiles are monoterpenoids, especially geraniol derivatives such as geranial, geranyl acetate, geraniol and citronellol. In this study, we investigated the interconversion of geraniol derivatives by incorporation experiments using deuterium-labeled geraniol and geranyl acetate. GC-MS analysis revealed that the incorporated geraniol and geranyl acetate were transformed and detected as geranial, geraniol, geranyl acetate and citronellol; however, nerol and neral were hardly detected. Next, we isolated and characterized a cDNA encoding geraniol dehydrogenase (ZoGeDH) by expressed sequence tag database mining. Phylogenic analysis of ZoGeDH resulted in its categorization into the cinnamyl alcohol dehydrogenase (CAD) group, along with the previously reported GeDHs of sweet basil (Ocimum basilicum) and wild perilla (P. setoyensis, P. citriodora, and P. frutescens). The recombinant ZoGeDH catalyzed the NADP-dependent oxidation from geraniol to citral. Furthermore, its substrate specificity was the highest for geraniol and nerol, while that for cinnamyl alcohol was 32% of the activity observed for geraniol. The expression levels of ZoGeDH in various ginger plant tissues were in accordance with the accumulation of geranial, except in old rhizome.
Aldehydes and ketones produced from lipid peroxides (oxylipin carbonyls) exhibit a variety of biological activities ranging from the induction of defense genes to the irreversible damage to cells. Short oxylipin carbonyls such as acrolein and (E)-2-pentenal are responsible for tissue injury under environmental stress, but their production mechanism remains unclear. In this study, we elucidated the source fatty acids of short oxylipin carbonyls in leaves. We first established a comprehensive analysis of oxylipin carbonyls for quantitation and structural estimation. Carbonyls were extracted from rosette leaves of Arabidopsis thaliana, derivatized with 2,4-dinitrophenylhydrazine and separated with a reverse-phase HPLC equipped with a photodiode array detector and an Fourier transform ion cyclotron resonance mass spectrometer. Thirty-three distinct carbonyls were detected, in which 19 species were identified on the MS/MS spectrum. Using this analysis system, we compared the carbonyl composition in the leaves between two A. thaliana lines that have different fatty acid composition. The mutant fad7fad8, which lacks the biosynthesis of trienoic fatty acids in the plastid, contained significantly lower amounts of malondialdehyde, acrolein and (E)-2-pentenal and higher amounts of acetone, 3-pentanone, and n-hexanal than the wild type Col-0. This difference in the carbonyl composition agreed with the oxidative degradation of dienoic and trienoic fatty acids in vitro, showing that similar non-enzymatic reactions occur in the thylakoid membrane. Consideration of the formation mechanism of acrolein from trienoic fatty acid suggests that membrane lipids in chloroplasts are constitutively oxidized by singlet oxygen.
A novel type of O-methyltransferase (OMT) cDNA was isolated from maturing seeds of Carthamus tinctorius (safflower). The deduced sequence of the OMT protein showed moderate sequence identity (52%) with C. tinctorius 5-hydroxyconiferaldehyde O-methyltransferase 1 (CAldOMT1). Phylogenetic analysis showed that the novel OMT did not belong to the typical CAldOMT [=caffeic acid OMT (CAOMT)] cluster. The recombinant protein of the OMT catalyzed 3- (or 5-) O-methylation of hydroxycinnamaldehydes and hydroxycinnamyl alcohols, while it showed only weak or moderate activity toward hydroxycinnamates and hydroxycinnamoyl coenzyme A esters. Therefore, this OMT was designated as C. tinctorius 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol OMT (CtAAOMT). The time profile of CtAAOMT gene expression in C. tinctorius matched the patterns of lignin accumulation. Taken together, our data strongly suggest that along with CtCAldOMT1, CtAAOMT is involved in biosynthesis of syringyl lignin.
Canterbury bells (Campanula medium) have deep purple petals due to the accumulation of 7-polyacylated anthocyanin molecules. The first step in the production of 7-polyacylated anthocyanins is glucosylation at the C7 position of anthocyanidin mediated by an acyl-glucose dependent anthocyanin 7-O-glucosyltransferase (AA7GT). To date, two such enzymes have been identified: DgAA7GT from delphinium (Delphinium grandiflorum) and AaAA7GT from African lily (Agapanthus africanus). Here, we describe the isolation of AA7GT cDNA from C. medium and the characterization of the enzymatic properties of a recombinant protein. The CmAA7GT protein belongs to glycoside hydrolase family 1, similarly to other AA7GTs; a phylogenetic analysis revealed that CmAA7GT was in the same clade as other AA7GTs. The CmAA7GT gene showed expression only in flowers, with a peak level of expression at the middle stage of floral development. A recombinant CmAA7GT protein showed significant preference for interaction with anthocyanidin 3-O-rutinoside rather than anthocyanidin 3-O-monoglycoside, which is the preferred target of other AA7GTs. This difference in target preference may reflect a conformational difference in the acceptor pocket of the enzyme protein that recognizes the anthocyanidin glycoside.
Lignans are a class of phenylpropanoids that are widely distributed in the plant kingdom and some lignans are known to be present as glycosides. In the model plant Arabidopsis thaliana, pinoresinol and lariciresinol as well as their glucosides are found to be accumulated in the roots, but the enzymes involved in the glucosylation of lignans remain to be characterized. UGT71C1 showed activity towards several phenylpropanoids in previous studies, although its activity towards lignans has not been investigated. In the present study, the involvement of UGT71C1 in lignan glucosylation was examined. Quantification of lignans in a T-DNA knockout line of the UGT71C1 gene, ugt71c1, by an ultra performance liquid chromatography-tandem mass spectrometry showed that the content of pinoresinol glucoside decreased in parallel with an increase of pinoresinol at the corresponding degree. Two major peaks corresponding to lariciresinol glucosides were detected in the mass chromatogram of the extract from the wild type and one of the peaks decreased in the ugt71c1 line suggesting that the amount of lariciersinol glucoside also decreased in the mutant. UGT71C1 expressed in Escherichia coli showed glucosyltransferase activity towards pinoresinol and lariciresinol. The present results suggest that UGT71C1 is involved in lignan glucosylation in A. thaliana.
Flavonoids are phenolic secondary metabolites commonly occurring in plants. In particular, prenylated flavonoids exhibit a wide range of biological activities, including antitumor, antibacterial and antioxidant activities. In this study, we attempted to produce prenylated flavonoids in tomato as a host plant by means of metabolic engineering through the introduction of both the naringenin 8-dimethylallyltransferase (N8DT) gene, encoding a prenyltransferase from Sophora flavescens, and the chalcone isomerase (CHI) gene from Nicotiana tabacum cv. Samsun-NN. Liquid chromatography-tandem mass spectrometry analysis revealed the production of 8-dimethylallyl naringenin in the double transformants, while the production level was lower than N8DT single transformants. In addition, tomato fruits over-expressing both N8DT and CHI genes accumulated high levels of rutin compared with wild-type tomato. A possible endogenous regulation of the synthesis of flavonoid derivatives is discussed.
Tea plant (Camellia sinensis) biosynthesizes a wide variety of specialized metabolites, including phenolic compounds such as catechins. Flavonol, one of the major flavonoid subclasses, in C. sinensis is present in the O-glycoside form, such as quercetin 3-O-β-D-glucopyranoside, kaempferol 3-O-β-D-glucopyranoside, and rutin (quercetin 3-O-β-glucopyranosyl-6-O-α-rhamnoside). These flavonol glycosides are highly accumulated, constituting up to 2–3% (w/w dry weight) of tea leaves; however, their biosynthetic machinery in C. sinensis remains elusive. Using high-throughput RNA sequencing from the fresh leaves of a cultivar (C. sinensis var sinensis cv Yabukita) and rapid amplification of cDNA ends (RACE) cloning with degenerate oligonucleotide primers, we identified a full-length cDNA of UDP-glycosyltransferase, designated as UGT73A17, and characterized the biochemical and molecular functions of UGT73A17. Recombinant UGT73A17 protein catalyzed 3-O-glucosylation of quercetin, yielding quercetin 3-O-β-D-glucopyranoside in vitro. The preferential expression of UGT73A17 gene in the mature, relative to young leaves, stems and roots, is roughly consistent with the accumulation pattern of flavonol glycosides in C. sinensis, suggesting that UGT73A17, in part, participates in the biosynthesis of flavonol glycosides in planta.
Specialized metabolism in land plants produces the diverse array of compounds, which is important in interaction with the environments. Generally, specialized metabolism-related genes consist of large gene families (superfamily), including cytochrome P450 monooxygenases (CYPs), 2-oxoglutarate-dependent dioxygenases (DOXs), and family-1 UDP-sugar dependent glycosyltransferases (UGTs), especially in angiosperms and gymnosperms. We investigated the changes in the numbers of these superfamily genes during the evolution of angiosperms by inferring gain and loss events in ancestral lineages of 5 angiosperms and 1 lycophyte. We observed the clear difference in the changes in the gene number among ancestral lineages. Intriguingly, gene gain events were coordinately occurred among CYP, DOX and UGT in lineage-specific manner, and the gain events were in good accordance with ancient whole genome duplication (WGD) events. Thus, the WGD events in angiosperms would have an important role in the expansion and evolution of specialized metabolism by providing prerequisite genetic resources for subsequent lineage-specific local tandem duplication (LTD) of superfamily genes as well as functional differentiation of these superfamily genes.
The sesquiterpenoid rich essential oils of sandalwood (Santalum album L.) stems and roots are a highly sought commodity in the fragrance industry. Plantations of sandalwood are being established in northern Australia, however the valuable heartwood essential oils do not accumulate in substantial amounts before 10 years, while commercially viable harvests do not normally take place for at least 15 years. Inducing essential oil accumulation at an earlier stage, or increasing oil yield in mature trees, may have the potential to enhance the oil productivity of plantations. In this study, we investigated the effects of foliar application of methyl jasmonate on less than one-year-old sandalwood seedlings. Essential oil accumulation was unaffected in both stems and roots. However, at the gene transcript level, several key genes early in the biosynthesis of sandalwood oil components were induced in both leaves and stems. These results suggest that terpenoid biosynthesis in S. album does indeed respond to foliar application of methyl jasmonate, however the effects are small and the full biosynthesis of santalols is likely to be developmentally regulated.
Hevea brasiliensis is the key source of latex for commercial natural rubber production. Genetic improvement of H. brasiliensis is required to enhance natural rubber production, although the biosynthetic mechanism has not been fully elucidated. In this study, we established a cell suspension culture from petiole explants of H. brasiliensis clone RRIM 600 for basic research on the biosynthesis of natural rubber. Calli were induced from petiole explants on callus induction medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg l−1 6-benzylaminopurine (BA). Then, the calli were suspension cultured in MS basal medium supplemented with 2 mg l−1 2,4-D and 2 mg l−1 BA. Although transcripts of some laticifer-specific genes were detected in the cultured cells, their levels were much lower than those in the other laticifer-containing tissues. Additionally, there was no detectable activity of rubber transferase in this cell line. The laticifer-specific genes in the cell line showed transcriptional responses to phytohormone treatments. Among them, up-regulations of Rubber Elongation Factor by the ethophon treatment were concordant with those in laticifers, suggesting that these cells retained at least some of the cytochemical properties of laticifers. The cell line established in this study could be useful for biochemical and molecular studies on natural rubber biosynthesis.