An inducible potato (E,E)-farnesol synthase confers tolerance against bacterial pathogens in potato and tobacco.

Terpene synthases (TPSs) have diverse biological functions in plants. Though the roles of TPSs in herbivore defense are well established in many plant species, their role in bacterial defense has been scarce and is emerging. Through functional genomics, here we report the in planta role of potato (Solanum tuberosum) terpene synthase (StTPS18) in bacterial defense. Expression of StTPS18 was highest in leaves and was induced in response to Pseudomonas syringae and methyl jasmonate treatments. The recombinant StTPS18 exhibited bona fide (E,E)-farnesol synthase activity forming a sesquiterpenoid, (E,E)-farnesol as the sole product, utilising (E,E)-farnesyl diphosphate (FPP). Subcellular localization of GFP fusion protein revealed that StTPS18 is localized to the cytosol. Silencing and overexpression of StTPS18 in potato resulted in reduced and enhanced tolerance, respectively, to bacterial pathogens P. syringae and Ralstonia solanacearum. Bacterial growth assay using medium containing (E,E)-farnesol significantly inhibited P. syringae growth. Moreover, StTPS18 overexpressing transgenic potato and Nicotiana tabacum leaves, and (E,E)-farnesol and P. syringae infiltrated potato leaves exhibited elevated expression of sterol pathway and members of pathogenesis-related genes with enhanced phytosterol accumulation. Interestingly, enhanced phytosterols in 13 C3 -(E,E)-farnesol infiltrated potato leaves were devoid of any noticeable 13 C labeling, indicating no direct utilization of (E,E)-farnesol in phytosterols formation. Furthermore, leaves of StTPS18 overexpressing transgenic lines had no detectable (E,E)-farnesol similar to the control plant, and emitted lower levels of sesquiterpenes than the control. These findings point towards an indirect involvement of StTPS18 and its product (E,E)-farnesol in bacterial defense through upregulation of phytosterol biosynthesis and defense genes.

lncRNADetector: a bioinformatics pipeline for long non-coding RNA identification and MAPslnc: a repository of medicinal and aromatic plant lncRNAs.

Long non-coding RNAs (lncRNAs) are an emerging class of non-coding RNAs and potent regulatory elements in the living cells. High throughput RNA sequencing analyses have generated a tremendous amount of transcript sequence data. A large proportion of these transcript sequences does not code for proteins and are known as non-coding RNAs. Among them, lncRNAs are a unique class of transcripts longer than 200 nucleotides with diverse biological functions and regulatory mechanisms. Recent emerging studies and next-generation sequencing technologies show a substantial amount of lncRNAs within the plant genome, which are yet to be identified. The computational identification of lncRNAs from these transcripts is a challenging task due to the involvement of a series of filtering steps. We have developed lncRNADetector, a bioinformatics pipeline for the identification of novel lncRNAs, especially from medicinal and aromatic plant (MAP) species. The lncRNADetector has been utilized to analyse and identify more than 88,459 lncRNAs from 21 species of MAPs. To provide a knowledge resource for the plant research community towards elucidating the diversity of biological roles of lncRNAs, the information generated about MAP lncRNAs (post-filtering steps) through lncRNADetector has been stored and organized in MAPslnc database (MAPslnc, https://lncrnapipe.cimap.res.in). The lncRNADetector web server and MAPslnc database have been developed in order to facilitate researchers for accurate identification of lncRNAs from the next-generation sequencing data of different organisms for downstream studies. To the best of our knowledge no such MAPslnc database is available till date.

Characterization of a class III peroxidase from Artemisia annua: relevance to artemisinin metabolism and beyond.

KEY MESSAGE: A class III peroxidase from Artemisia annua has been shown to indicate the possibility of cellular localization-based role diversity, which may have implications in artemisinin catabolism as well as lignification. Artemisia annua derives its importance from the antimalarial artemisinin. The -O-O- linkage in artemisinin makes peroxidases relevant to its metabolism. Earlier, we identified three peroxidase-coding genes from A. annua, whereby Aa547 showed higher expression in the low-artemisinin plant stage whereas Aa528 and Aa540 showed higher expression in the artemisinin-rich plant stage. Here we carried out tertiary structure homology modelling of the peroxidases for docking studies. Maximum binding affinity for artemisinin was shown by Aa547. Further, Aa547 showed greater binding affinity for post-artemisinin metabolite, deoxyartemisinin, as compared to pre-artemisinin metabolites (dihydroartemisinic hydroperoxide, artemisinic acid, dihydroartemisinic acid). It also showed significant binding affinity for the monolignol, coniferyl alcohol. Moreover, Aa547 expression was related inversely to artemisinin content and directly to total lignin content as indicated by its transient silencing and overexpression in A. annua. Artemisinin reduction assay also indicated inverse relationship between Aa547 expression and artemisinin content. Subcellular localization using GFP fusion suggested that Aa547 is peroxisomal. Nevertheless, dual localization (intracellular/extracellular) of Aa547 could not be ruled out due to its effect on both, artemisinin and lignin. Taken together, this indicates possibility of localization-based role diversity for Aa547, which may have implications in artemisinin catabolism as well as lignification in A. annua.

PlantAFP: a curated database of plant-origin antifungal peptides.

Emerging infectious diseases (EIDs) are a severe problem caused by fungi in human and plant species across the world. They pose a worldwide threat to food security as well as human health. Fungal infections are increasing now day by day worldwide, and the current antimycotic drugs are not effective due to the emergence of resistant strains. Therefore, it is an urgent need for the finding of new plant-origin antifungal peptides (PhytoAFPs). Huge numbers of peptides were extracted from different plant species which play a protective role against fungal infection. Hundreds of plant-origin peptides with antifungal activity have already been reported. So there is a requirement of a dedicated platform which systematically catalogs plant-origin peptides along with their antifungal properties. PlantAFP database is a resource of experimentally verified plant-origin antifungal peptides, collected from research articles, patents, and public databases. The current release of PlantAFP database contains 2585 peptide entries among which 510 are unique peptides. Each entry provides comprehensive information of a peptide that includes its peptide sequence, peptide name, peptide class, length of the peptide, molecular mass, antifungal activity, and origin of peptides. Besides this primary information, PlantAFP stores peptide sequences in SMILES format. In order to facilitate the user, many tools have been integrated into this database that includes BLAST search, peptide search, SMILES search, and peptide-mapping is also included in the database. PlantAFP database is accessible at http://bioinformatics.cimap.res.in/sharma/PlantAFP/.

ISSR-Derived Species-Specific SCAR Marker for Rapid and Accurate Authentication of Ocimum tenuiflorum L.

Ocimum tenuiflorum has been widely used in traditional medicine and has high medicinal value. High volume trade of this potential medicinal plant species led to unscrupulous adulteration of both crude drugs as well as formulations. Morphology-based authentication is difficult in cases of incomplete or damaged samples and in dried herbal materials. In such cases, PCR-based molecular methods may aid in accurate identification. The present study aimed at developing species-specific DNA marker(s) for the authentication of O. tenuiflorum. A species-specific amplicon (279 bp) generated through an inter-simple sequence repeat marker (UBC 835) in all individuals of O. tenuiflorum was cloned, sequenced, and a primer pair was developed (designated as CIM-OT-835F/CIM-OT-835R). The newly developed sequence characterized amplified region marker was validated through PCR amplification in all available seven species of Ocimum, and its specificity for O. tenuiflorum was confirmed with the consistent generation of an amplicon of 177 bp. The developed marker can be used for accurate and rapid identification of the species for certification purposes and will be useful in quality control of medicinal preparations containing this important medicinal species.

A WRKY transcription factor from Withania somnifera regulates triterpenoid withanolide accumulation and biotic stress tolerance through modulation of phytosterol and defense pathways.

Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated. A jasmonate- and salicin-inducible WRKY transcription factor from W. somnifera (WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay. WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W. somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W. somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, WsWRKY1 binds to W-box sequences in promoters of W. somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while WsWRKY1 silencing in W. somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance. Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense.

Latex-less opium poppy: cause for less latex and reduced peduncle strength.

A genotype 'Sujata' developed earlier at CSIR-CIMAP from its parent 'Sampada' is considered to be the latex-less variety of Papaver somniferum. These two genotypes are contrasting in terms of latex and stem strength. Earlier we have carried out microarray analysis to identify differentially expressing genes from the capsules of the two genotypes. In this study, the peduncles of the two genotypes were compared for the anatomy revealing less number of laticifers in the cortex and vascular bundles. One of the important cell wall-related genes (for laccase) from the microarray analysis showing significantly higher expression in 'Sampada' capsule was taken up for further characterization in the peduncle here. It was functionally characterized through transient overexpression and RNAi suppression in 'Sujata' and 'Sampada'. The increase in acid insoluble lignin and total lignin in overexpressed tissue of 'Sujata', and comparable decrease in suppressed tissue of 'Sampada', along with corresponding increase and decrease in the transcript abundance of laccase confirm the involvement of laccase in lignin biosynthesis. Negligible transcript in phloem compared to the xylem tissue localized its expression in xylem tissue. This demonstrates the involvement of P. somniferum laccase in lignin biosynthesis of xylem, providing strength to the peduncle/stem and preventing lodging.

4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway.

RNAi suppression of Arogenate Dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals.

l-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development. ADT1 showed strict substrate specificity toward arogenate, although with the lowest catalytic efficiency among the three ADTs. ADT1 suppression via RNA interference in petunia petals significantly reduced ADT activity, levels of Phe, and downstream phenylpropanoid/benzenoid volatiles. Unexpectedly, arogenate levels were unaltered, while shikimate and Trp levels were decreased in transgenic petals. Stable isotope labeling experiments showed that ADT1 suppression led to downregulation of carbon flux toward shikimic acid. However, an exogenous supply of shikimate bypassed this negative regulation and resulted in elevated arogenate accumulation. Feeding with shikimate also led to prephenate and phenylpyruvate accumulation and a partial recovery of the reduced Phe level in transgenic petals, suggesting that the phenylpyruvate route can also operate in planta. These results provide genetic evidence that Phe is synthesized predominantly via arogenate in petunia petals and uncover a novel posttranscriptional regulation of the shikimate pathway.

A transcriptomic approach for exploring the molecular basis for dosha-balancing property-based classification of plants in Ayurveda.

In Ayurveda, a healthy body is defined by a balance among the three doshas (Vata, Pitta, Kapha) and ailments result due to imbalances among them. It prescribes specific plant parts/tissues collected in a season-specific manner for curing dosha-related imbalances but the plants prescribed for treating a particular dosha imbalance belong to taxonomically diverse families and often contain similar classes of phytomolecules, making it difficult to provide a phytochemical validation for any similarity that might exist among them. This exploratory study hypothesised that plants of the same dosha-curing group may have similarity at the transcript level. For proving/disproving the hypothesis, cDNA-AFLP and specific expression subset analysis (SESA) were carried out on the Ayurveda-defined active tissues of four representative plants each of the three dosha-balancing groups. cDNA-AFLP analyses indicated that even though the plants belonging to a particular dosha-group may widely differ at the transcript level, there is a small fraction of transcripts that is monomorphic among their active tissues. SESA (Tester-active tissue cDNA; Driver-cDNA from other major tissue[s]) generated 803 subtractive ESTs from the twelve plants that yielded 150 unigenes upon assembly (of ESTs from each plant separately). Cross-plant EST assembly for plants in the same dosha group also corroborated the results. Although a distinct pattern of transcripts was not observed across all the plants in a particular dosha group, some commonalities were obtained that need further characterization towards searching for the hitherto elusive similarity among plants of the same group.

Expression of CspE by a psychrotrophic bacterium Enterobacter ludwigii PAS1, isolated from Indian Himalayan soil and in silico protein modelling, prediction of conserved residues and active sites.

Proteome analysis of Enterobacter ludwigii PAS1 provide a powerful set of tool to study the cold shock proteins along with that combination of bioinformatics is useful for interpretation of comparative results from many species. There is a considerable interest in the use of psychrotrophic bacteria for nitrogen fixation, especially at hilly regions, thus better understanding of cold adaptation mechanisms too. The psychrotrophic E. ludwigii PAS1 grown at 30 and 4 °C, isolated from Himalaya soil was undertaken for proteomic responses during optimal and cold shock conditions. Comparative proteomic analyses using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS revealed the presence of Cold shock protein E (CspE). Three-dimensional structure of CspE of E. ludwigii PAS1 divulge the presence of five antiparallel ß-sheets forming a ß-barrel structure with surface exposed aromatic and basic residues that were responsible for nucleic acid binding and also reveals the presence of highly conserved nucleic acid-binding motifs RNP1 and RNP2 in Csp family.

Molecular characterization of three CYP450 genes reveals their role in withanolides formation and defense in Withania somnifera, the Indian Ginseng.

The medicinal properties of Ashwagandha (Withania somnifera) are attributed to triterpenoid steroidal lactones, withanolides, which are proposed to be derived from phytosterol pathway, through the action of cytochrome P450 (CYP450) enzymes. Here, we report the characterization of three transcriptome-mined CYP450 genes (WsCYP749B1, WsCYP76 and WsCYP71B10), which exhibited induced expression in response to methyl jasmonate treatment indicating their role in secondary metabolism. All three WsCYP450s had the highest expression in leaf compared to other tissues. In planta characterization of WsCYP450s through virus induced gene silencing (VIGS) and transient overexpression approaches and subsequent metabolite analysis indicated differential modulation in the accumulation of certain withanolides in W. somnifera leaves. While WsCYP749B1-vigs significantly enhanced withaferin A (~ 450%) and reduced withanolide A (~ 50%), its overexpression drastically led to enhanced withanolide A (> 250%) and withanolide B (> 200%) levels and reduced 12-deoxywithastramonolide (~ 60%). Whereas WsCYP76-vigs led to reduced withanolide A (~ 60%) and its overexpression increased withanolide A (~ 150%) and reduced 12-deoxywithastramonolide (~ 60%). Silencing and overexpression of WsCYP71B10 resulted in significant reduction of withanolide B (~ 50%) and withanolide A (~ 60%), respectively. Further, while VIGS of WsCYP450s negatively affected the expression of pathogenesis-related (PR) genes and compromised tolerance to bacteria P. syringae DC3000, their overexpression in W. somnifera and transgenic tobacco led to improved tolerance to the bacteria. Overall, these results showed that the identified WsCYP450s have a role in one or several steps of withanolides biosynthetic pathway and are involved in conferring tolerance to biotic stress.

Proteomics indicates modulation of tubulin polymerization by L-menthol inhibiting human epithelial colorectal adenocarcinoma cell proliferation.

Menthol is a naturally occurring cyclic monoterpene used in oral hygiene products, confectionary, pharmaceuticals, cosmetics, pesticides, and as a flavoring agent. In the present study, we analyzed the differentially expressing proteome in L-menthol-treated Caco-2 cell line as it was found to inhibit cell proliferation. Interestingly, free tubulin proteins were observed to be limited after menthol treatment. Semiquantitative RT-PCR with α-tubulin primers showed no change in the level of RNA expression in menthol-treated cell line. However, tubulin polymerization assay with menthol indicated a trend similar to taxol in promoting microtubule assembly. Further, physical counting of apoptotic nuclei and active caspase-3 assays confirmed onset of apoptosis though the rate was slower as compared with that of taxol treatment. This study is the first report of a monoterpene L-menthol modulating tubulin polymerization and apoptosis to inhibit cancer cell proliferation.

PhytoAFP: In Silico Approaches for Designing Plant-Derived Antifungal Peptides.

Emerging infectious diseases (EID) are serious problems caused by fungi in humans and plant species. They are a severe threat to food security worldwide. In our current work, we have developed a support vector machine (SVM)-based model that attempts to design and predict therapeutic plant-derived antifungal peptides (PhytoAFP). The residue composition analysis shows the preference of C, G, K, R, and S amino acids. Position preference analysis shows that residues G, K, R, and A dominate the N-terminal. Similarly, residues N, S, C, and G prefer the C-terminal. Motif analysis reveals the presence of motifs like NYVF, NYVFP, YVFP, NYVFPA, and VFPA. We have developed two models using various input functions such as mono-, di-, and tripeptide composition, as well as binary, hybrid, and physiochemical properties, based on methods that are applied to the main data set. The TPC-based monopeptide composition model achieved more accuracy, 94.4%, with a Matthews correlation coefficient (MCC) of 0.89. Correspondingly, the second-best model based on dipeptides achieved an accuracy of 94.28% under the MCC 0.89 of the training dataset.

Recombinant Expression and Characterization of Lemon (Citrus limon) Peroxidase.

BACKGROUND: Class III plant peroxidases play important role in a number of physiological processes in plants such as lignin biosynthesis, suberization, cell wall biosynthesis, reactive oxygen species metabolism and plant defense against pathogens. Peroxidases are also of significance in several industrial applications. In view of this, the production and identification of novel peroxidases having resistance towards temperature, pH, salts is desirable. OBJECTIVE: The objective of the present work was to clone and characterize a novel plant peroxidase suitable for industrial application. METHODS: A full length cDNA clone of lemon peroxidase was isolated using PCR and RACE approaches, characterized and heterologously expressed in Escherichia coli using standard protocols. The expressed peroxidase was purified using Ni-NTA agarose column and biochemically characterized using standard protocols. The peroxidase was also in-silico characterized at nucleotide as well as protein levels using standard protocols. RESULTS: A full length cDNA clone of lemon peroxidase was isolated and expressed heterologously in E. coli. The expressed recombinant lemon peroxidase (LPRX) was activated by in-vitro refolding and purified. The purified LPRX exhibited pH and temperature optima of pH 7.0 and 50°C, respectively. The LPRX was found to be activated by metal ions (Na+, Ca2+, Mg2+ and Mn2+) at lower concentration. The expressional analysis of the transcripts suggested involvement of lemon peroxidase in plant defense. The lemon peroxidase was in silico modelled and docked with the substrates guaiacol, and pyrogallol and shown the favourability of pyrogallol over guaiacol, which is in agreement with the in-vitro findings. The protein function annotation analyses suggested the involvement of lemon peroxidase in the phenylpropanoid biosynthesis pathway and plant defense mechanisms. CONCLUSION: Based on the biochemical characterization, the purified peroxidase was found to be resistant towards the salts and thus, might be a good candidate for industrial exploitation. The in-silico protein function annotation and transcript analyses highlighted the possible involvement of the lemon peroxidase in plant defense response.

Genetic variation revealed in the chloroplast-encoded RNA polymerase beta' subunit of downy mildew-resistant genotype of opium poppy.

Two accessions of opium poppy, Pps-1 (dark green leaves, highly resistant to downy mildew [DM]) and H-9 (yellowish green leaves, susceptible to DM), which originated from common progenitor SPS49 were selected, and their F(1) and F(2) progenies showed that leaf color trait was governed by single recessive nuclear gene, whereas DM resistance appeared to be the interaction between cytoplasmic and nuclear genes. Chloroplast DNA (cpDNA) analysis of these 2 accessions through arbitrarily-primed polymerase chain reaction generated a unique fragment in Pps-1. Subsequent sequence analysis upon cloning of this cpDNA fragment revealed its similarity with the plastid-encoded RNA polymerase beta' subunit (rpoCI). Full-length rpoCI DNA was therefore isolated from both the genotypes that was 2707 bp long with a 658-bp intron (436-1093) and a 2049-bp open reading frame encoding 682 amino acid long polypeptide. Comparative sequence analysis of the rpoC1 gene from both the genotypes, revealed 4 single-nucleotide substitutions at 4 positions that caused 3 amino acid changes in the protein sequence--1) A to C transversion at position 825 (Glu275Asp), 2) A to G transition at position 1203 (Ile401Met), and 3) T to C transition at position 1422 and G to A transition at position 1423 both in same codon of the reading frame (Ala475Thr). This investigation is the first report indicating base substitution changes in the plastid-encoded rpoCI gene in DM-resistant genotypes of opium poppy. This finding may lead to implication of possible role of RNA polymerase beta' subunit in resistance to DM caused by Peronospora arborescens.

An alpha-1 antitrypsin genetic variant from India.

The highly polymorphic human alpha-1 antitrypsin (AAT) gene codes for the most abundant circulating plasma serine protease inhibitor. Previously, genetic variants of the AAT gene were reported from different regions of the world. In the present study, the AAT gene was characterized in an Indian sample. The AAT gene was isolated and cloned from a liver biopsy sample through RT-PCR and the full-length gene was sequenced. Nucleotide sequence comparison with the human genome and the AAT sequences available in the GenBank (NCBI) demonstrated four unique variations--(i) an A to G variation at position 286 (Thr96Ala), (ii) an A to G variation at position 839 (Asp280Gly), (iii) a T to C variation at position 1182 that did not result in any change in the protein sequence (TTT to TTC both code for Phe) and (iv) an A to C variation at position 1200 (Glu400Asp) that resulted in replacement by an amino acid of similar nature. Other variations found were T to C at position 710 (Val273Ala) and T to C position 863 (Val288Glu), which were also reported earlier. In conclusion, this study reports the entire 1257 bp nucleotide sequence of protein coding region of the human AAT gene from an Indian sample. This preliminary finding is significant, as it reports for the first time the AAT gene sequence in the Indian sample.

Scent from Jasminum grandiflorum flowers: Investigation of the change in linalool enantiomers at various developmental stages using chemical and molecular methods.

Jasminum species are among the most preferred fresh cut flowers in India since ancient times. The plant produces small and fragrant flowers, which are of great demand in the preparation of fragrant garlands and also in perfume industries. Floral volatile of Jasminum grandiflorum L. (Family: Oleaceae) was extracted using solid-phase microextraction and analyzed in enantioselective gas chromatography. Chemical classes of identified volatiles revealed the presence of terpenoids, phenylpropanoids, and fatty acid derivatives. Marker constituent of flower volatiles, linalool was selected for analytical characterization on ethyl- and acetyl-ß-cyclodextrin stationary phase. (R)-(-)-Linalool was found as major enantiomer in volatiles of floral buds whereas (S)-(+)-linalool predominated in the volatiles of matured flowers. Simultaneously, a quantitative real-time PCR was performed to find the gene expression of linalool synthase to investigate the mechanism of enantiomeric inversion. The emission pattern of (R)-(-)-linalool at different flower developmental stages was well correlated (P = 0.01) with the gene expression of the cloned linalool synthase from J. grandiflorum. We observed that the successive change in (R)- to (S)-linalool ratio from bud to mature flower was mainly due to the enantio- specific transformation and temporal decline of (R)-linalool producing gene in J. grandiflorum. This enantiomeric change also leads to the difference in flower aroma. Furthermore, this is probably the reason behind consumer's acceptance for jasmine buds rather than bloomed flowers in cut flower segments.

Chitosan immobilized novel peroxidase from Azadirachta indica: Characterization and application.

In the present paper, a peroxidase was purified from the leaves of a medicinal tree, namely Azadirachta indica, to 45.2 folds with overall recovery of 61%. Based on the subunit size, the purified peroxidase was suggested to be a monomeric structure of size 50kDa and exhibited good thermostability as it was fully stable at 65°C for 1hr and also retained about 73% activity at 70°C till 30min. The substrate affinity was found to be in order of guaiacol>pyrogallol>o-dianisidine. The purified peroxidase was found to be insensitive towards high concentrations of Na+, Ca2+, Mg2+ and Mn2+. Heavy metals, namely Cs2+, Co2+ and Cd2+ activated the peroxidase while that of Hg2+ deactivated the peroxidase in concentration dependent manner. The purified peroxidase exhibited tolerance towards organic solvents in order of ethanol>butanol>isopropanol>acetone. Immobilization of purified peroxidase by entrapment into chitosan beads led to shift in its optimum pH from pH 5 to 7 and considerable enhancement in dye decolorization ability as compared to that of free enzyme. Thus, based on all the above properties, it may be suggested that the purified A. indica peroxidase is a promising candidate for industrial applications.

Identification of miRNAs and their targets involved in the secondary metabolic pathways of Mentha spp.

The endogenous, small and non-coding functional microRNAs govern the regulatory system of gene expression and control the growth and development of the plants. Mentha spp. are well known herbs for its flavor, fragrance and medicinal properties. In the present study, we used a computational approach to identify miRNAs and their targets involved in different secondary metabolic pathways of Mentha spp. Additionally, phylogenetic and conservation analysis were also done for the predicted miRNAs. Eleven miRNAs families were identified from Mentha spp., out of which five miRNA families were reported for the first time from Lamiaceae. Overall, 130 distinct target transcripts were predicted for eight miRNAs families. All the predicted targets regulated by predicted miRNAs control the reproduction, signaling, stimulus response, developmental and different metabolic process. miRNA mediated gene regulatory network was also constructed on the basis of hybridized minimum free energy of identified miRNAs and their targets. The study revealed that the gene regulatory system of essential oil biosynthesis may be governed by miR156, miR414 and miR5021 in mint family. Furthermore, three miRNA candidates (miR156, miR5021, and miR5015b) were observed to be involved in trichome development also. This is the first in-silico study describing miRNAs and their role in the regulation of secondary metabolic pathways in Mentha spp.