Search

Vinblastine is a chemotherapy agent produced by the plant

Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudo-aspidosperma alkaloids, pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates, and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors.

Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear. We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism. We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein-protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism. These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.

Understanding when and where metabolites accumulate provides important cues to the gene function. Mass spectrometry imaging (MSI) enables in situ temporal and spatial measurement of a large assortment of metabolites, providing mapping information regarding their cellular distribution. To describe the current state and technical advances using MSI in plant sciences, we employed MSI to demonstrate its significant contribution to the study of plant specialised metabolism. We show that coupling MSI with: (1) RNA interference (RNAi), (2) virus induced gene silencing (VIGS), (3) agroinfiltration or (4) samples derived from plant natural variation provides great opportunities to understand the accurate gene-metabolite relationship and discover novel gene-associated metabolites. This was exemplified in three plant species (i.e. tomato, tobacco and wheat) by mapping the distribution of metabolites possessing a range of polarities. In particular, we demonstrated that MSI is able to spatially map an entire metabolic pathway, including intermediates and final products, in the intricate biosynthetic route to tomato fruit steroidal glycoalkaloids. We therefore envisage MSI as a key component of the metabolome analysis arsenal employed in plant gene discovery strategies.

Steroidal glycoalkaloids (SGAs) are defense specialized metabolites produced by thousands of Solanum species. These metabolites are remarkable in structural diversity formed following modifications in their core scaffold. In recent years, it became clear that a large portion of this chemical repertoire was acquired through various molecular mechanisms involving 'hijacking' of core metabolism enzymes. This was typically accompanied by gene duplication and divergence and further neofunctionalization as well as modified subcellular localization and evolution of new substrate preferences. In this review, we highlight recent findings in the SGAs biosynthetic pathway and elaborate on similar occurrences in other chemical classes that enabled evolution of specialized metabolic pathways and its underlying structural diversity.

Wild tomato species represent a rich gene pool for numerous desirable traits lost during domestication. Here, we exploited an introgression population representing wild desert-adapted species and a domesticated cultivar to establish the genetic basis of gene expression and chemical variation accompanying the transfer of wild-species-associated fruit traits. Transcriptome and metabolome analysis of 580 lines coupled to pathogen sensitivity assays resulted in the identification of genomic loci associated with levels of hundreds of transcripts and metabolites. These associations occurred in hotspots representing coordinated perturbation of metabolic pathways and ripening-related processes. Here, we identify components of the Solanum alkaloid pathway, as well as genes and metabolites involved in pathogen defense and linking fungal resistance with changes in the fruit ripening regulatory network. Our results outline a framework for understanding metabolism and pathogen resistance during tomato fruit ripening and provide insights into key fruit quality traits., This project has received funding from the European Research Council (grant agreement 204575-SAMIT), EU Framework Program FP7/2007–2013 (grant agreement 613692-TriForC) and the Israel Science Foundation (grant number 1805/15). We thank the Adelis Foundation, Leona M. and Harry B. Helmsley Charitable Trust, Jeanne and Joseph Nissim Foundation for Life Sciences, Tom and Sondra Rykoff Family Foundation Research and Raymond Burton Plant Genome Research Fund for supporting A.A.’s laboratory activity. A.K. is thankful for a short-term EMBO fellowship (EMBO-ASTF-146-2014).

The development of genetic transformation methods is critical for enabling the thorough characterization of an organism and is a key step in exploiting any species as a platform for synthetic biology and metabolic engineering approaches. In this work we describe the development of an Agrobacterium rhizogenes-mediated hairy root transformation protocol for the crop and medicinal legume fenugreek (Trigonella foenum-graecum). Fenugreek has a rich and diverse content in bioactive specialised metabolites, notably diosgenin, which is a common precursor for synthetic human hormone production. This makes fenugreek a prime target for identification and engineering of specific biosynthetic pathways for the production of triterpene and steroidal saponins, phenolics, and galactomanans. Through this transformation protocol, we identified a suitable promoter for robust transgene expression in fenugreek. Finally, we establish the proof of principle for the utility of the fenugreek system for metabolic engineering programs, by heterologous expression of known triterpene saponin biosynthesis regulators from the related legume Medicago truncatula in fenugreek hairy roots.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

Climacteric ripening is related to a respiratory climax accompanied by an exponential increase in ethylene production. This association is not fully understood. Tomato fruit were exposed to the ethylene antagonist 1-methylcyclopropene (1-MCP) at different ripening stages and their transcriptome was monitored. ISOCITRATE DEHYDROGENASE 1 (ICDH, Solyc01g005560.2.1) was the only respiratory process related gene that was down regulated at all ripening stages including the mature green. Silencing SlICDH1using VIGS (virus indused gene silencing), to about 50 % of its native expression, in Micro-Tom tomatoes, reduced both respiration and ethylene production of the harvested fruit. Silenced SlICDH1 tomatoes also exhibited prolonged ripening and reduced susceptibility to the pathogen Botrytis cinerea. In addition, ethylene response factors binding sites were identified in the promotor and in the first intron of SlICDH1. These results suggest that regulation of SlICDH1 is a key step in the initiation of the climacteric rise of respiration via ethylene regulation.

With approximately 450 species, spiny Solanum species constitute the largest monophyletic group in the Solanaceae family, but a high-quality genome assembly from this group is presently missing. We obtained a chromosome-anchored genome assembly of eggplant (Solanum melongena), containing 34,916 genes, confirming that the diploid gene number in the Solanaceae is around 35,000. Comparative genomic studies with tomato (S. lycopersicum), potato (S. tuberosum) and pepper (Capsicum annuum) highlighted the rapid evolution of miRNA:mRNA regulatory pairs and R-type defense genes in the Solanaceae, and provided a genomic basis for the lack of steroidal glycoalkaloid compounds in the Capsicum genus. Using parsimony methods, we reconstructed the putative chromosomal complements of the key founders of the main Solanaceae clades and the rearrangements that led to the karyotypes of extant species and their ancestors. From 10% to 15% of the genes present in the four genomes were syntenic paralogs (ohnologs) generated by the pre-γ, γ and T paleopolyploidy events, and were enriched in transcription factors. Our data suggest that the basic gene network controlling fruit ripening is conserved in different Solanaceae clades, and that climacteric fruit ripening involves a differential regulation of relatively few components of this network, including CNR and ethylene biosynthetic genes.

The genus Solanum comprises three food crops (potato, tomato, and eggplant), which are consumed on daily basis worldwide and also producers of notorious anti-nutritional steroidal glycoalkaloids (SGAs). Hydroxylated SGAs (i.e. leptinines) serve as precursors for leptines that act as defenses against Colorado Potato Beetle (Leptinotarsa decemlineata Say), an important pest of potato worldwide. However, SGA hydroxylating enzymes remain unknown. Here, we discover that 2-OXOGLUTARATE-DEPENDENT-DIOXYGENASE (2-ODD) enzymes catalyze SGA-hydroxylation across various Solanum species. In contrast to cultivated potato, Solanum chacoense, a widespread wild potato species, has evolved a 2-ODD enzyme leading to the formation of leptinines. Furthermore, we find a related 2-ODD in tomato that catalyzes the hydroxylation of the bitter α-tomatine to hydroxytomatine, the first committed step in the chemical shift towards downstream ripening-associated non-bitter SGAs (e.g. esculeoside A). This 2-ODD enzyme prevents bitterness in ripe tomato fruit consumed today which otherwise would remain unpleasant in taste and more toxic., Steroidal glycoalkaloids (SGAs) accumulate in Solanum, but their hydroxylating enzymes are unknown. Here, the authors report 2-OXOGLUTARATE DEPENDENT DIOXYGENASE enzymes that catalyze the committed hydroxylation steps in the biosynthesis of leptinine insecticidal compounds in wild potato or non-bitter SGAs in cultivated tomato.

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein-ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2-3 fold and 6-7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.

Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpressing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.

The widespread pharmaceutically important triterpenoid saponins are synthesized via isoprenoid pathway. The formation of squalene is the key regulatory point in triterpene biosynthesis, catalyzed by squalene synthase (SQS). The present study deals with the detailed characterization of SQS by molecular, biochemical, and computational means from Bacopa monniera, an immensely important medicinal plant rich in triterpenoid saponin, bacosides. A full-length SQS gene was isolated from B. monniera, characterized as B. monniera squalene synthase (BmSQS) (1242 bp) encoding 414 amino acids. Deduced amino acid sequence of BmSQS showed highly conserved consensus aspartate-rich motifs (DXXXD) and catalytic site residues. Phylogenetic analysis showed that BmSQS belongs to dicot group having closest relationship with Salvia miltiorrhiza. Semiquantitative and real-time PCR studies showed that the BmSQS messenger RNA (mRNA) expression level was higher in vegetative parts (roots) as compared to floral parts. Methyl jasmonate induces the BmSQS mRNA expression in all tissues tested, while salicylic acid, cold, and salt induce much higher expression in roots. Homology modeling and docking simulations of BmSQS showed the pivotal roles of Asp77, Asp81, Asp213, Asp217, and Tyr168 in catalysis.

Nature Plants 3, 16205 (2016); published 22 December 2016; corrected 12 June 2017. Two of the Supplementary Information files originally published contained errors. In Supplementary Table 1a, the SolycIDs for SMO1 and SMO2 were interchanged. In Supplementary Data 1, the SolycIDs for SMO3 and SMO4 were interchanged.

Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K a = 3.72 × 105 M−1) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100 % of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f a = 1.0) was significantly higher than cesium ions (f a = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f a = 0.54), whereas quenching with iodide ion was decreased (f a = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions.

Flavonoids are a large class of phenylpropanoid-derived secondary metabolites, which are usually glycosylated by UDP-glycosyltransferases with one or more sugar groups. Here, we report the cloning and biochemical characterization of a flavonoid glycosyltransferase gene from Withania somnifera (WsGT), which is an important medicinal plant used in Ayurvedic formulations. Using PCR primers, designed for a highly conserved region of previously reported glycosyltransferases, we were able to isolate the corresponding fragment of the WsGT gene. Rapid amplification of cDNA ends (RACE) was then employed to isolate full-length cDNA, which had an open reading frame of 1,371 bp that encode for 456 amino acids. Phylogenetic analysis indicated that WsGT was similar to that of family 1 GT-B glycosyltransferase. Biochemical analysis revealed that WsGT interacts with UDP-glucose and was capable of regiospecifically glycosylating flavonoid-7-ols, such as apigenin, naringenin, luteolin, diadzein and genistein. Expression profiling studies showed that WsGT was highly expressed in young and mature leaves of W. somnifera. Furthermore, exposure to salicylic acid enhanced the expression of WsGT in the leaves and heat shock treatment resulted in decreased expression of WsGT after an initial increase. This may suggest the role of WsGT in response to abiotic/biotic stresses.

Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out. Homology modeling based modeled 3D structure of Ll-CCRH1 was used as template for in Silico mutant preparations. Docking simulations of Ll-CCRH1 mutants with CoA esters by AutoDock Vina tools showed altered substrate specificity as compared to wild type. The study evidences that conformational changes, and change in geometry or architecture of active site pocket occurred following mutations. The altered substrate specificity for active site mutants suggests the possible physiological role of CCR either in lignin formation or in defense system in plants. Abbreviations Ll-CCRH1 - Leucaena leucocephala cinnamoyl CoA reductase 1, OPLS - Optimized Potentials for Liquid Simulations, RMSD - Root Mean Square Deviation.

The amount of cholesterol made by many plants is not negligible. Whereas cholesterogenesis in animals was elucidated decades ago, the plant pathway has remained enigmatic. Among other roles, cholesterol is a key precursor for thousands of bioactive plant metabolites, including the well-known Solanum steroidal glycoalkaloids. Integrating tomato transcript and protein co-expression data revealed candidate genes putatively associated with cholesterol biosynthesis. A combination of functional assays including gene silencing, examination of recombinant enzyme activity and yeast mutant complementation suggests the cholesterol pathway comprises 12 enzymes acting in 10 steps. It appears that half of the cholesterogenesis-specific enzymes evolved through gene duplication and divergence from phytosterol biosynthetic enzymes, whereas others act reciprocally in both cholesterol and phytosterol metabolism. Our findings provide a unique example of nature's capacity to exploit existing protein folds and catalytic machineries from primary metabolism to assemble a new, multi-step metabolic pathway. Finally, the engineering of a 'high-cholesterol' model plant underscores the future value of our gene toolbox to produce high-value steroidal compounds via synthetic biology.

Farnesyl pyrophosphate synthase (FPS; EC 2.5.1.10) is a key enzyme in isoprenoid biosynthetic pathway and provides precursors for the biosynthesis of various pharmaceutically important metabolites. It catalyzes head to tail condensation of two isopentenyl pyrophosphate molecules with dimethylallyl pyrophosphate to form C15 compound farnesyl pyrophosphate. Recent studies have confirmed FPS as a molecular target of bisphosphonates for drug development against bone diseases as well as pathogens. Although large numbers of FPSs from different sources are known, very few protein structures have been reported till date. In the present study, FPS gene from medicinal plant Bacopa monniera (BmFPS) was characterized by comparative modeling and docking. Multiple sequence alignment showed two highly conserved aspartate rich motifs FARM and SARM (DDXXD). The 3-D model of BmFPS was generated based on structurally resolved FPS crystal information of Gallus gallus. The generated models were validated by various bioinformatics tools and the final model contained only α-helices and coils. Further, docking studies of modeled BmFPS with substrates and inhibitors were performed to understand the protein ligand interactions. The two Asp residues from FARM (Asp100 and Asp104) as well as Asp171, Lys197 and Lys262 were found to be important for catalytic activity. Interaction of nitrogen containing bisphosphonates (risedronate, alendronate, zoledronate and pamidronate) with modeled BmFPS showed competitive inhibition; where, apart from Asp (100, 104 and 171), Thr175 played an important role. The results presented here could be useful for designing of mutants for isoprenoid biosynthetic pathway engineering well as more effective drugs against osteoporosis and human pathogens. Abbreviations IPP - Isopentenyl Pyrophosphate, DMAPP - Dimethylallyl Pyrophosphate, GPP - Geranyl Pyrophosphate, FPP - FPPFarnesyl Pyrophosphate, DOPE - Discrete Optimized Protein Energy, BmFPS - Bacopa monniera Farnesyl Pyrophosphate Synthase, RMSD - Root Mean square Deviation, OPLS-AA - Optimized Potentials for Liquid Simulations- All Atom, FARM - First Aspartate Rich Motif, SARM - Second Aspartate Rich Motif.

Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 °C, respectively. The enzyme was most stable at pH 8 at 25 °C with deactivation rate constant (Kd*) 1.398 × 10(-4) and half life (t1/2) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg(2+), having Km and Vmax 331.9 μM and 719.1 pKat μg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2.

Triterpenoid saponins are the class of secondary metabolites, synthesized via isoprenoid pathway. Oxidosqualene cyclases (OSCs) catalyzes the cyclization of 2, 3-oxidosqualene to various triterpene skeletons, the first committed step in triterpenoid biosynthesis. A full-length oxidosqualene cyclase cDNA from Bacopa monniera (BmOSC) was isolated and characterized. The open reading frame (ORF) of BmOSC consists of 2,292 bp, encoding 764 amino acid residues with an apparent molecular mass of 87.62 kDa and theoretical pI 6.21. It contained four QxxxxxW motifs, one Asp-Cys-Thr-Ala-Glu (DCTAE) motif which is highly conserved among the triterpene synthases and another MWCYCR motif involved in the formation of triterpenoid skeletons. The deduced amino acid sequence of BmOSC shares 80.5 % & 71.8 % identity and 89.7 % & 83.5 % similarity with Olea europaea mixed amyrin synthase and Panax notoginseng dammarenediol synthase respectively. Phylogenetic analysis revealed that BmOSC is closely related with other plant OSCs. Quantitative real-time PCR (qRT-PCR) data showed that BmOSC is expressed in all tissues examined with higher expression in stem and leaves as compared to roots and floral parts.

Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (Ll-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 °C and starts forming aggregates at 55 °C. Ll-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of L1-CCRH1 at pH 2.0 was stable up to 90 °C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCl), it got transformed into different structure which was stable in the vicinity of 2 M GdnHCl (as compared to drastic loss of native structure in 2 M GdnHCl) as seen in far UV-CD spectra. The structural transition of Ll-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein.

Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25 °C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×106 M−1 s−1), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20–50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm3 with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme.

Glycosylation of flavonoids is mediated by family 1 uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). Until date, there are few reports on functionally characterized flavonoid glycosyltransferases from Withania somnifera. In this study, we cloned the glycosyltransferase gene from W. somnifera (UGT73A16) showing 85-92 % homology with UGTs from other plants. UGT73A16 was expressed as a His(6)-tagged fusion protein in Escherichia coli. Several compounds, including flavonoids, were screened as potential substrates for UGT73A16. HPLC analysis and hypsochromic shift indicated that UGT73A16 transfers a glucose molecule to several different flavonoids. Based on kinetic parameters, UGT73A16 shows more catalytic efficiency towards naringenin. Here, we explored UGT73A16 of W. somnifera as whole cell catalyst in E. coli. We used flavonoids (genistein, apigenin, kaempferol, naringenin, biochanin A, and daidzein) as substrates for this study. More than 95 % of the glucoside products were released into the medium, facilitating their isolation. Glycosylation of substrates occurred on the 7- and 3-hydroxyl group of the aglycone. UGT73A16 also displayed regiospecific glucosyl transfer activity towards 3-hydroxy flavone compound, which is the backbone of all flavonols and also for a chemically synthesized compound, not found naturally. The present study generates essential knowledge and molecular as well as biochemical tools that allow the verification of UGT73A16 in glycosylation.

The phenylpropanoid pathway is responsible for the biosynthesis of a variety of products that include lignin, flavonoids and hydroxycinnamic acid conjugates. Many intermediates and end products of this pathway play important role in plants as phytoalexins, antiherbivory compounds, antioxidants, ultra-violet (UV) protectants, pigments and aroma compounds. Lignin has far reaching impacts on agriculture, industry and the environment, making phenylpropanoid metabolism a globally important part of plant biochemistry. The mechanical support provided by lignin prevents lodging, a problem in many agronomically important plants, it also provides a hydrophobic surface, essential for longitudinal water transport, and provides a barrier against pathogens. Finally the many functions of lignin and related products in resistance to biotic and abiotic stresses make the phenylpropanoid pathway vital to the health and survival of plants. Besides its critical role in normal plant health and development, high lignin levels are problematic in the agro-industrial exploitation of various plant species. Lignin is considered an undesirable component in paper manufacture, and has a negative impact on forage crop digestibility. Leucaena leucocephala is one of the most versatile, fast growing commercially important trees for paper and pulp industry in India, contributing 1/4th of the total raw material. Lignin composition, quantity and distribution are known to affect the agro-industrial utilization of plant biomass. High quantity and low Syringyl (S) to Guaiacyl (G) lignin ratio plays a detrimental role in economy and ecology of paper production. Every unit increase in S/G ratio decreases the cost of paper production by two and half times. Moreover chemical processing of pulp for lignin removal releases chlorinated organic compounds in effluent, which are hazardous and a serious threat to the environment. Hence, there is currently intense interest in modifying the content and/or composition of the cell wall structural polymer (lignin) as a means of improving the efficiency of the paper pulping process for forest trees. To engineer plants with agronomically useful lignin related traits, we need to devise strategies that can flexibly and predictably yield reductions in lignin content and/or changes in lignin monomer composition. Our studies have concentrated on attempts to alter the levels of enzymes involved in early as well as late phenylpropanoid pathway, mainly by downregulation or upregulation of the phenylpropanoid pathway genes in transgenic L. leucocephala and tobacco plants. Besides, we are also working on some R2R3 type MYB transcription factors supposed to play important role in lignin biosynthesis and some other genes which are not directly involved in phenylpropanoid pathway, but are important for carrying out polymerization of monolignols (peroxidases) and defense mechanism of plants (β glucosidase: family 1 Glycosyl hydrolase). Major phenylpropanoid pathway genes (C4H, 4-CL, CCoAOMT, CCR, Cald5H and CAD) were isolated using PCR based approach. Their 5’ and 3’ UTR determined by rapid amplification of cDNA race (RACE). We could isolate multiple isoforms of most of the genes in this way well supported by Southern hybridization experiment. All the genes were expressed in E. coli and/or yeast with/without the signal sequence. The over-expressed proteins were purified using suitable methods and were used for raising polyclonal antibodies against them. The most un-conserved region of each gene was cloned in antisense orientation in suitable binary vector and L. leucocephala and tobacco explants were transformed using the antisense construct to down-regulate the targeted gene. In order to increase the S/G ratio of lignin monomers sense construct of the target gene was used. We have also done RNAi based downregulation of Cinnamate 4-Hydroxylase (C4H), a key enzyme of phenylpropanoid pathway and a member of cytochrome P450 family in tobacco. Spatio-temporal expression of each gene was studied in L. leucocephala in different tissues at different periods of their growth. Kinetics study of some of the enzymes has been carried out in our lab. We are also trying to establish the structure function correlation of some enzymes. We have also isolated promoters of some phenylpropanoid pathway genes and have identified R2-R3 type MYB binding domain(s) in them. Two MYB genes have been isolated from L. leucocephala and heterologously expressed in Escherichia coli. Their DNA binding efficiency and role in regulating phenylpropanoid pathway remain to be seen. All the isolated genes shared 70-80% homology with the already reported sequences from other species at nucleotide level and more than 80% identity at amino acid level. Different isoforms of different genes had varying degree of identity between them ranging from 80% to more than 95%. We could easily locate the ribosome binding site in the 5’ UTR and the polyadenylation signal in the 3’ UTR in all the genes. Proteins were expressed in both prokaryotic and eukaryotic system. Some of the genes were difficult to express in BL21 (DE3), primarily because of the translational incompatibility of some of the codons in E. coli and may be partly because of the signal sequence present in most of the proteins. All the genes were found to be actively expressing in lignifying tissues and roots in comparison to leaves albeit a time dependent regular expression pattern could not be drawn in case of every gene. We used three methods of genetic transformation for transferring our gene constructs to L. leucocephala embryo viz: Agrobacterium mediatd, gene gun mediated and gene gun followed by cocultivation with Agrobacterium. The transformation and regeneration efficiency varied with each protocol. The transgenic plants invariably showed stunted vigour and slow growth irrespective of the nature of the gene downregulated or upregulated. Initial screening of the transformants was done on MS medium containing appropriate antibiotic and later confirmation was done using PCR with hptII/nptII, gus specific and CaMV35S promoter specific primers. At least one tobacco plant downregulated for C4H showed rudimentary root system and curled leaves with brown tip. Transformed Leucaena and tobacco plants had reduced lignin content with varying degree in case of every downregulated gene. Histochemical staining of transverse root and stem tissue sections showed reduced lignification as evidenced by immunocytolocalization patterns of the candidate protein under study. Promoters of few lignin biosynthetic pathway genes having R2R3 type MYB-binding signal sequences (AC elements) have been cloned. Two R2R3 type MYB transcription factors from L. leucocephala that have been expressed and purified from E. coli will be used for gel retardation studies with the promoter sequences. Also in vitro synthesized oligonucleotides having the highly conserved MYB-binding motifs will be designed to determine the most probable binding sites of the two MYB proteins. Sense construct for the two MYB genes have been transformed in tobacco and antisense construct have been transformed in Leucaena plants to study the after effects of gene manipulation. In short, we have isolated and characterized several genes belonging to phenylpropanoid pathway and have expressed them in different systems. Transgenics for down-regulation or upregulation of those genes have shown very interesting results.