Defining the role of a caffeic acid 3-O-methyltransferase from Azadirachta indica fruits in the biosynthesis of ferulic acid through heterologous over-expression in Ocimum species and Withania somnifera.

MAIN CONCLUSION: The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis. Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem (NCOMT) fruits has been isolated and heterologously expressed in E. coli. The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta. Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.

Identification of key enzymes responsible for protolimonoid biosynthesis in plants: Opening the door to azadirachtin production.

Limonoids are natural products made by plants belonging to the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, contribution to bitterness in citrus fruits, and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranortriterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of 4 carbons with associated furan ring formation, by an as yet unknown mechanism. Here we have mined genome and transcriptome sequence resources for 3 diverse limonoid-producing species (A. indica, Melia azedarach, and Citrus sinensis) to elucidate the early steps in limonoid biosynthesis. We identify an oxidosqualene cyclase able to produce the potential 30-carbon triterpene scaffold precursor tirucalla-7,24-dien-3ß-ol from each of the 3 species. We further identify coexpressed cytochrome P450 enzymes from M. azedarach (MaCYP71CD2 and MaCYP71BQ5) and C. sinensis (CsCYP71CD1 and CsCYP71BQ4) that are capable of 3 oxidations of tirucalla-7,24-dien-3ß-ol, resulting in spontaneous hemiacetal ring formation and the production of the protolimonoid melianol. Our work reports the characterization of protolimonoid biosynthetic enzymes from different plant species and supports the notion of pathway conservation between both plant families. It further paves the way for engineering crop plants with enhanced insect resistance and producing high-value limonoids for pharmaceutical and other applications by expression in heterologous hosts.

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.

Insights on germinability and desiccation tolerance in developing neem seeds (Azadirachta indica): Role of AOS, antioxidative enzymes and dehydrin-like protein.

The germinability and desiccation tolerance (DT) in developing seed are regulated by cellular metabolism involving active oxygen species (AOS) and protective proteins during maturation drying. The aim of the present investigation was to unravel the functions of AOS (superoxide, H2O2 and OH-radical), antioxidative enzymes (SOD, CAT and APX) and dehydrin-like proteins in regulating the germinability and DT in undried and artificially desiccated developing neem seeds. Germination was first observed in seeds of 8 weeks after anthesis (waa) whereas DT was noticed from 9 waa. High levels of superoxide in undried and artificially desiccated seeds of 9 waa were rapidly declined up to 15 waa with simultaneous increase in levels of SOD (quantitative and isoenzymes) that dismutates superoxide with corresponding formation and accumulation of H2O2. Activities and isoenzymes of APX and CAT were promoted in seeds from 9 to 12 waa. Intensity of dehydrin-like proteins increased as development progressed in seeds with higher intensities in slow dried (SD) seeds. Desiccation modulated the metabolism for the acquisition of germinability and DT in the developing neem seeds from 8 to 15 waa by altering the levels of superoxide, H2O2 and OH-radical those possibly act as signalling molecules for reprogramming protective proteins. Desiccation mediated the expression of new bands of SOD and APX in undried as well as SD seeds during 9-12 waa but the bands were more intense in SD seeds. The superoxide and H2O2-regulated intensity of dehydrin-like protein in SD seeds further validated our conclusion.

Antioxidant enzyme changes in neem, pigeonpea and mulberry leaves in two stages of maturity.

Differential expression of antioxidant enzymes in various growth and differentiation stages has been documented in several plant species. We studied here, the difference in the levels of protein content and antioxidant enzymes activity at two stages of maturity, named young and mature in neem (Azadirachta indica A. Juss), pigeonpea (Cajanus cajan (L.) mill sp) and mulberry (Morus Alba L.) leaves. The results showed that detached neem and pigeonpea mature leaves possessed higher activities of catalase (CAT) and peroxidase (POD) and lower activities of polyphenol oxidase (PPO) and ascorbate peroxidase (APX) as compared with young leaves. However, glutathione reductase (GR) showed higher activity in mature leaves of neem, whereas no change in its activity was observed in pigeonpea. On the other hand, antioxidant enzymes in mulberry showed either positive (PPO) or negative (POD, GR, APX) correlation with the progression of leaf maturity. Apparently the trend of changes in antioxidant enzymes activity during leaf development is species-specific: their activity higher at mature stage in some plants and lower in others.

Role of chloroplastidial proteases in leaf senescence.

In this report the effect of hydrogen peroxide (H2O2) on peroxidase (POD) activity during leaf senescence was studied with and without phenylmethylsulfonyl fluoride (PMSF) pre-treatment in detached neem (Azadirachta indica A. juss) leaf chloroplasts. Increased POD activity was detected in natural and H2O2-promoted senescent leaf chloroplasts compared to untreated control mature green leaf chloroplasts. However, under H2O2 POD activity markedly increased at 1 day, and then significantly decreased until 4 days. In the presence of H2O2, PMSF, the induction of POD activity was alleviated at 1 day, whereas reduced after 4 days. In contrast, in the presence of H2O2, cycloheximide (CX), the induction of POD activity was reduced at 1 day, whereas alleviated after 4 days. The was a partial reduction in H2O2-induced POD activity with PMSF and CX, indicating the presence of pre-existing inactive PODs in chloroplasts. We also propose a new role for chloroplastidial proteases as activators of pre-existing inactive PODs during leaf senescence.

Oxidative metabolism-related changes in cryogenically stored neem (Azadirachta indica A. Juss) seeds.

The seeds of Azadirachta indica were successfully cryopreserved for 12 months with 45% survival following drying to 0.16 g H(2)O g(-1) dry mass (DM). Highest survival (94-96%) was recorded during the first month of cryostorage. Subsequent cryopreservation up to 12 months resulted in decreasing germination. Post-thawing pre-heat treatment enhanced the recovery marginally in seeds cryopreserved from 3 to 12 months. Viability of cryostored seeds was negatively correlated with leachate conductivity and accumulation of thiobarbituric acid reactive substances (TBRS) estimated in cotyledons and axes. Leachate conductivity of imbibed seeds was low during the first month of cryostorage but increased gradually with the duration of cryostorage to a maximum after 12 months. TBRS accumulation was gradual throughout cryostorage. Relatively low amounts of active oxygen species (AOS) detected during the first month of cryostorage were closely associated with very high activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in seeds (cotyledons and axes). Marked accumulation of AOS from 3 to 12 months was associated with decrease in antioxidant enzyme activity.