Milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits on individual caseins.

In recent years, many attempts have been made to find new plant proteases to make artisan cheeses. The global increase in cheese consumption, together with a lower supply and increasing cost of calf rennet, religious factors (Islam and Judaism) and food choices (vegetarianism) have led to the search for suitable rennet substitutes for milk clotting. This study describes the milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits (SoAP) on individual caseins to explore its potential use as an alternative to animal rennet. The milk-clotting index obtained for SoAP was 8.4 times lower than that obtained for chymosin. SoAP showed a higher degree of hydrolysis on α-casein than on the other fractions under the proposed conditions. RP-HPLC, mass spectrometry analyses and sequencing of the hydrolysates allowed identifying five peptides from α-casein, one peptide from ß-casein, and three peptides from k-casein. In silico analysis showed that the peptides identified may display a wide variety of potential biological activities. These results demonstrate the possibility of using SoAP for the manufacture of new types or artisan cheeses, with the simultaneous added value of the potential health-promoting benefits of the bioactive peptides generated during the hydrolysis.

Genome-wide identification of small G protein ROPs and their potential roles in Solanaceous family.

Small GTPases function as molecular switches to active or inactive signaling cascades via binding or hydrolyzing GTP. A type of plant specific small GTPases, the ROPs are known to be involved in plant growth, development and immunity. We determined whether ROPs are conserved in Solanaceous species and whether they are involved in plant growth, development and resistance against Phytophthora capsisi. In genome-wide screening, a total of 66 ROPs in six Solanaceous species (SolROPs) were identified, including 16 ROPs in Solanum tuberosum L. (potato), 9 in Solanum lycopersicum L. (tomato), 5 in Solanum melongena L. (eggplant), 9 in Capsicum annuum L. (pepper), 13 in Nicotiana benthamiana Domin and 14 in Nicotiana tabacum L. (tobacco). Phylogenetic analysis revealed that 11 AtROPs and 66 SolROPs fall into five distinct clades (I-V) and hence a novel and systematic gene nomenclature was proposed. In addition, a comprehensive expression analysis was performed by making use of an online database. This revealed that ROP genes are differentially expressed during plant growth and development. Moreover, gene expression of SlROP-II.1 in S. lycopersicum could be significantly induced by P. capsici. Subsequently, SlROP-II.1 and its homologues in N. benthamiana and C. annuum (NbROP-II.1 and CaROP-II.1) were selected for functional analysis using virus-induced gene silencing. Infection assays with P. capsici on silenced plants revealed that SlROP-II.1, NbROP-II.1 and CaROP-II.1 play a role in P. capsici resistance, suggesting conserved function of ROP-II clade across different Solanaceous species. In addition, NbROP-II.1 is also involved in regulating plant growth and development. This study signified the diversity of Solanaceous ROPs and their potential roles in plant growth, development and immunity.

Bioinformatics study of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) genes in Solanaceae.

Isoprenoids, the largest and most diverse class of secondary metabolites in plants, play an important role in plant growth and development. Isoprenoids can be synthesized by two distinct pathways: the methylerythritol-4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is the first step and a key regulatory enzyme of the MEP pathway in plants. The DXS gene has been reported to play a key role in seedling development, flowering, and fruit quality in plants of the Solanaceae, such as tomato, potato and tobacco. However, to improve our understanding and utilization of DXS genes, a thorough bioinformatics study is needed. In this study, 48 DXS genes were aligned and analyzed by computational tools to predict their protein properties, including molecular mass, theoretical isoelectric point (pI), signal peptides, transmembrane and conserved domains, and expression patterns. Sequence comparison analysis revealed strong conservation among the 48 DXS genes. Phylogenetic analysis indicated that all DXS genes were derived from one ancestor and could be classified into three groups with different expression patterns. Moreover, the functional divergence of DXS was restricted after gene duplication. The results suggested that the function and evolution of the DXS gene family were highly conserved and that the DXS genes of Group I may play a more important role than those of other groups.

De Novo Production of the Plant-Derived Tropine and Pseudotropine in Yeast.

Tropine and pseudotropine with opposite stereospecific configurations as platform compounds are central building blocks in both biosynthesis and chemical synthesis of pharmacologically important tropane and nortropane alkaloids. The supply of plant-derived tropine and pseudotropine still heavily depends on either plant extraction or chemical synthesis. Advances in synthetic biology prompt the microbial synthesis of various valuable chemicals. With the biosynthetic pathway elucidation of tropine and pseudotropine in several Solanaceae plants, the key genes were sequentially identified. Here, the enzymes responsible for converting N-methylpyrrolinium into tropine and pseudotropine from Anisodus acutangulus were characterized. Reconstruction of the six-step biosynthetic pathways into Saccharomyces cerevisiae provides cell chassis producing tropine and pseudotropine with 0.13 and 0.08 mg/L titers from simple feedstocks in a shake flask, respectively. The strains described not only offer alternative sources of these central intermediates and their derived alkaloids but also provide platforms for pathway enzyme discovery.

Biochemical characterization reveals the functional divergence of two tropinone reductases from Przewalskia tangutica.

Przewalskia tangutica is a traditional medicinal plant from Tibet used for the analgesic effect from the tropane alkaloids (TAs) produced by the plant. Its roots have the highest yield of hyoscyamine in all plant species and so have been overharvested becoming an endangered medicinal plant species. Metabolic engineering is a good way to improve the yield of TAs in plants. In our study, two functionally distinct tropinone reductases genes, PtTRI and PtTRII, were cloned from P. tangutica and the functional divergence were characterized. The enzyme kinetics of PtTRI and PtTRII were investigated. The phylogenetic analysis classified them into different clades: PtTRI and PtTRII were in the clade of tropine-forming reductase and pseudotropine-forming reductase, respectively. We found PtTRI to be expressed in the roots but less in leaves, whereas PtTRII was expressed in the roots at higher levels than in the leaves. The kinetic parameters (Km , Vmax , and Kcat ) were analyzed using purified recombinant enzymes at their optimum pH. Enzymatic analysis results showed that tropinone is a better substrate for PtTRII compared with PtTRI, suggesting that PtTRII might be a potential gene target for TA biosynthesis engineering. Compared with the reported TRIs, PtTRI exhibited a higher affinity for tropinone.

BcXyl, a ß-xylosidase Isolated from Brunfelsia Calycina Flowers with Anthocyanin-ß-glycosidase Activity.

Brunfelsia calycina flowers lose anthocyanins rapidly and are therefore well suited for the study of anthocyanin degradation mechanisms, which are unclear in planta. Here, we isolated an anthocyanin-ß-glycosidase from B. calycina petals. The MS/MS (Mass Spectrometry) peptide sequencing showed that the enzyme (72 kDa) was a ß-xylosidase (BcXyl). The enzyme showed high activity to p-Nitrophenyl-ß-d-galactopyranoside (pNPGa) and p-Nitrophenyl-ß-d-xylopyranoside (pNPX), while no activity to p-Nitrophenyl-ß-d-glucopyranoside (pNPG) or p-Nitrophenyl-ß-D-mannopyranoside (pNPM) was seen. The optimum temperature of BcXyl was 40 °C and the optimum pH was 5.0. The enzyme was strongly inhibited by 1 mM D-gluconate and Ag⁺. HPLC (High Performance Liquid Chromatography) analysis showed that BcXyl catalyzed the degradation of an anthocyanin component of B. calycina, and the release of xylose and galactose due to hydrolysis of glycosidic bonds by BcXyl was detected by GC (Gas Chromatography) /MS. A full-length mRNA sequence (2358 bp) of BcXyl (NCBI No. MK411219) was obtained and the deduced protein sequence shared conserved domains with two anthocyanin-ß-glycosidases (Bgln and BadGluc, characterized in fungi). BcXyl, Bgln and BadGluc belong to AB subfamily of Glycoside hydrolase family 3. Similar to BcPrx01, an anthocyanin-degradation-related Peroxidase (POD), BcXyl was dramatically activated at the stage at which the rapid anthocyanin degradation occurred. Taken together, we suggest that BcXyl may be the first anthocyanin-ß-glycosidase identified in higher plants.

Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway.

The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome-localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non-model plant species that remain underexplored.

Solanesol Biosynthesis in Plants.

Solanesol is a non-cyclic terpene alcohol composed of nine isoprene units that mainly accumulates in solanaceous plants. Solanesol plays an important role in the interactions between plants and environmental factors such as pathogen infections and moderate-to-high temperatures. Additionally, it is a key intermediate for the pharmaceutical synthesis of ubiquinone-based drugs such as coenzyme Q10 and vitamin K2, and anti-cancer agent synergizers such as N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl) ethylenediamine (SDB). In plants, solanesol is formed by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway within plastids. Solanesol's biosynthetic pathway involves the generation of C5 precursors, followed by the generation of direct precursors, and then the biosynthesis and modification of terpenoids; the first two stages of this pathway are well understood. Based on the current understanding of solanesol biosynthesis, we here review the key enzymes involved, including 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), isopentenyl diphosphate isomerase (IPI), geranyl geranyl diphosphate synthase (GGPPS), and solanesyl diphosphate synthase (SPS), as well as their biological functions. Notably, studies on microbial heterologous expression and overexpression of key enzymatic genes in tobacco solanesol biosynthesis are of significant importance for medical uses of tobacco.

Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants.

In plants, indole-3-acetic acid (IAA) amido hydrolases (AHs) participate in auxin homeostasis by releasing free IAA from IAA-amino acid conjugates. We investigated the role of IAR3, a member of the IAA amido hydrolase family, in the response of Solanaceous plants challenged by biotrophic and hemi-biotrophic pathogens. By means of genome inspection and phylogenic analysis we firstly identified IAA-AH sequences and putative IAR3 orthologs in Nicotiana benthamiana, tomato and potato. We evaluated the involvement of IAR3 genes in defense responses by using virus-induced gene silencing. We observed that N. benthamiana and tomato plants with knocked-down expression of IAR3 genes contained lower levels of free IAA and presented altered responses to pathogen attack, including enhanced basal defenses and higher tolerance to infection in susceptible plants. We showed that IAR3 genes are consistently up-regulated in N. benthamiana and tomato upon inoculation with Phytophthora infestans and Cladosporium fulvum respectively. However, IAR3 expression decreased significantly when hypersensitive response was triggered in transgenic tomato plants coexpressing the Cf-4 resistance gene and the avirulence factor Avr4. Altogether, our results indicate that changes in IAR3 expression lead to alteration in auxin homeostasis that ultimately affects plant defense responses.

Comparative Biochemistry and In Vitro Pathway Reconstruction as Powerful Partners in Studies of Metabolic Diversity.

There are estimated to be >300,000 plant species, producing >200,000 metabolites. Many of these metabolites are restricted to specific plant lineages and are referred to as "specialized" metabolites. These serve varied functions in plants including defense against biotic and abiotic stresses, plant-plant and plant-microbe communication, and pollinator attraction. These compounds also have important applications in agriculture, medicine, skin care, and in diverse aspects of human culture. The specialized metabolic repertoire of plants can vary even within and between closely related species, in terms of the number and classes of specialized metabolites as well as their structural variants. This phenotypic variation can be exploited to discover the underlying variation in the metabolic enzymes. We describe approaches for using the diversity of specialized metabolites and variation in enzyme structure and function to identify novel enzymatic activities and understand the structural basis for these differences. The knowledge obtained from these studies will provide new modules for the synthetic biology toolbox.

Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing a S14P/K97A mutant of hyoscyamine 6ß-hydroxylase AaH6H.

Hyoscyamine 6ß-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6ß-hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus (AaH6H). A double mutant, AaH6HM1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34h cultivation of Escherichia coli cells harboring Aah6hm1 in a 5-L bioreactor with a working volume of 3L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500mgL(-1) (-)-hyoscyamine in 97% conversion, and 1.068g of the product were isolated, corresponding to a space-time yield of 251mgL(-1)d(-1). This study shows that the protein engineering of some key enzymes is a promising and effective way for improving the production of rare natural products such as scopolamine.

In silico prediction of proteins related to xyloglucan fucosyltransferases in Solanaceae genomes.

Two independent studies have shown that the cell wall of pollen tubes from tobacco and tomato species contained fucosylated xyloglucan (XyG). These findings are intriguing as many reports have shown that XyG of somatic cells of these species is not fucosylated but instead is arabinosylated. In order to produce fucosylated XyG, plants must express a functional galactoside α-2-fucosyltransferase. Here, using a bioinformatics approach, we show that several candidate genes coding for XyG fucosyltransferases are present in the genome of coffee and several Solanaceae species including tomato, tobacco, potato, eggplant and pepper. BLAST and protein alignments with the 2 well-characterized XyG fucosyltransferases from Arabidopsis thaliana and Pisum sativum revealed that at least 6 proteins from different Solanaceae species and from coffee displayed the 3 conserved motifs required for XyG fucosyltransferase activity.

Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside.

Genetic basis for a rare floral mutant in an Andean species of Solanaceae.

PREMISE OF THE STUDY: White forms of typically pigmented flowers are one of the most common polymorphisms in flowering plants. Although the range of genetic changes that give rise to white phenotypes is well known from model systems, few studies have identified causative mutations in natural populations. METHODS: Here we combine genetic studies, in vitro enzyme assays, and biochemical analyses to identify the mechanism underlying the loss of anthocyanin pigment production in the naturally occurring white-flowered morph of Iochroma calycinum (Solanaceae). KEY RESULTS: Comparison of anthocyanin gene sequences revealed a putative loss-of-function mutation, an 11 amino-acid deletion in dihydroflavonol 4-reductase (DFR), in the white morph. Functional assays of Dfr alleles from blue and white morphs demonstrated that this deletion results in a loss of enzymatic activity, indicating that the deletion could be solely responsible for the lack of pigment production. Consistent with this hypothesis, quantitative PCR showed no significant differences in expression of anthocyanin genes between the morphs. Also, thin layer chromatography confirmed that the white morph continues to accumulate compounds upstream of the DFR enzyme. CONCLUSIONS: Collectively, these experiments indicate that the structural mutation at Dfr underlies the rare white flower morph of I. calycinum. This study is one of only a few examples where a flower color polymorphism is due to a loss-of-function mutation in the coding region of an anthocyanin enzyme. The rarity of such mutations in nature suggests that negative consequences prevent fixation across populations.

In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.

The tomato kinome and the tomato kinase library ORFeome: novel resources for the study of kinases and signal transduction in tomato and solanaceae species.

Protein kinase-driven phosphorylation constitutes the core of cellular signaling. Kinase components of signal transduction pathways are often targeted for inactivation by pathogens. The study of kinases and immune signal transduction in the model crop tomato (Solanum lycopersicum) would benefit from the availability of community-wide resources for large scale and systems-level experimentation. Here, we defined the tomato kinome and performed a comprehensive comparative analysis of the tomato kinome and 15 other plant species. We constructed a tomato kinase library (TOKN 1.0) of over 300 full-length open reading frames (ORF) cloned into a recombination-based vector. We developed a high-throughput pipeline to isolate and transform tomato protoplasts. A subset of the TOKN 1.0 library kinases were expressed in planta, were purified, and were used to generate a functional tomato protein microarray. All resources created were utilized to test known and novel associations between tomato kinases and Pseudomonas syringae DC3000 effectors in a large-scale format. Bsk7 was identified as a component of the plant immune response and a candidate effector target. These resources will enable comprehensive investigations of signaling pathways and host-pathogen interactions in tomato and other Solanaceae spp.

[Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene].

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.

Functional evolution of an anthocyanin pathway enzyme during a flower color transition.

Dissecting the genetic basis for the evolution of species differences requires a combination of phylogenetic and molecular genetic perspectives. By mapping the genetic changes and their phenotypic effects onto the phylogeny, it is possible to distinguish changes that may have been directly responsible for a new character state from those that fine tune the transition. Here, we use phylogenetic and functional methods to trace the evolution of substrate specificity in dihydroflavonol-4-reductase (Dfr), an anthocyanin pathway gene known to be involved in the transition from blue to red flowers in Iochroma. Ancestral state reconstruction indicates that three substitutions occurred during the flower color transition, whereas several additional substitutions followed the transition. Comparisons of enzymatic function between ancestral proteins in blue- and red-flowered lineages and proteins from present-day taxa demonstrate that evolution of specificity for red pigment precursors was caused by the first three substitutions, which were fixed by positive selection and which differ from previously documented mutations affecting specificity. Two inferred substitutions subsequent to the initial flower color transition were also adaptive and resulted in an additional increase in specificity for red precursors. Epistatic interactions among both sets of substitutions may have limited the order of substitutions along branches of the phylogeny leading from blue-pigmented ancestors to the present-day red-flowered taxa. These results suggest that the species differences in DFR specificity may arise by a combination of selection on flower color and selection for improved pathway efficiency but that the exact series of genetic changes resulting in the evolution of specificity is likely to be highly contingent on the starting state.

Oxaloacetate and malate production in engineered Escherichia coli by expression of codon-optimized phosphoenolpyruvate carboxylase2 gene from Dunaliella salina.

A new phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina is identified using homology analysis was conducted using PEPC gene of Chlamydomonas reinhardtii and Arabidopsis thaliana. Recombinant E. coli SGJS115 with increased production of malate and oxaloacetate was developed by introducing codon-optimized phosphoenolpyruvate carboxylase2 (OPDSPEPC2) gene of Dunaliella salina. E. coli SGJS115 yielded a 9.9 % increase in malate production. In addition, E. coli SGJS115 exhibited two times increase in the yield of oxaloacetate over the E. coli SGJS114 having identified PEPC2 gene obtained from Dunaliella salina.

Enhancing the production of tropane alkaloids in transgenic Anisodus acutangulus hairy root cultures by over-expressing tropinone reductase I and hyoscyamine-6ß-hydroxylase.

Tropane alkaloids (TA) including hyoscyamine, anisodamine, scopolamine and anisodine, are used medicinally as anticholinergic agents with increasing market demand, so it is very important to improve TA production by metabolic engineering strategy. Here, we report the simultaneous introduction of genes encoding the branch-controlling enzyme tropinone reductase I (TRI, EU424321) and the downstream rate-limiting enzyme hyoscyamine-6ß-hydroxylase (H6H, EF187826) involved in TA biosynthesis into Anisodus acutangulus hairy roots by Agrobacterium-mediated gene transfer technology. Transgenic hairy root lines expressing both TRI and H6H (TH lines) produced significantly higher (P < 0.05) levels of TA compared with the control and single gene transformed lines (T or H lines). The best double gene transformed line (TH53) produced 4.293 mg g(-1) TA, which was about 4.49-fold higher than that of the control lines (0.96 mg g(-1)). As far as it is known, this is the first report on simultaneous introduction of TRI and H6H genes into TA-producing plant by biotechnological approaches. Besides, the content of anisodine was also greatly improved in A. acutangulus by over-expression of AaTRI and AaH6H genes. The average content of anisodine in TH lines was 0.984 mg g(-1) dw, about 18.57-fold of BC lines (0.053 mg g(-1) dw). This is the first time that this phenomenon has been found in TA-producing plants.