Anil Kush

Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Hevea brasiliensis

Hevea brasiliensis is the major producer of natural rubber which is cis-1,4-polyisoprene. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is involved in the biosynthesis of rubber and other plant products. We have used a hamster HMGR cDNA clone as a heterologous hybridization probe to isolate and characterize cDNA and genomic clones of HMGR from H. brasiliensis. Sequence analysis revealed that these clones fall into two different classes, HMGR1 and HMGR2. Comparison of the two classes shows 86% nucleotide sequence homology and 95% amino acid homology. The carboxy-termini of Hevea HMGRs are highly homologous to those of hamster, yeast and Arabidopsis HMGR. The amino-terminus of Hevea HMGR contains two potential membrane-spanning domains as in Arabidopsis HMGR while seven such domains are found in the HMGRs of other organisms. The apparent molecular mass of Hevea HMGR was estimated in western blot analysis to be 59 kDa. Northern blot analysis indicated that the HMGR1 transcript of 2.4 kb is more highly-expressed in laticifer than in leaf. Genomic Southern analysis using 3′-end cDNA probes indicates the presence of at least two HMGR genes in Hevea.

Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis).

Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level.

cDNA cloning and characterisation of a maize homologue of the MCM proteins required for the initiation of DNA replication.

A central question in cell cycle regulation is how DNA replication is initiated and executed only once in each cell cycle. The cell cycle-regulated assembly of specific initiation protein complexes at chromosomal origins appears to specify the initial sites and timing of DNA replication, and to restrict this process to only one round in the somatic cell cycle. Among the enzymes involved in origin activation, the MCM proteins play a conserved key role. In particular, MCM3 homologues have been shown to be components of the DNA replication licensing activity in yeast and vertebrates. In spite of our detailed knowledge of the regulation of the initiation of DNA synthesis in yeast, there is virtually no information available on the molecules involved in origin activation in higher plants. We have isolated a cDNA from maize root apices, termedROA (Replication Origin Activator), encoding a protein which shares a high degree of homology with the MCM3 subfamily of MCM proteins. Analysis of gene organisation by Southern blotting shows 2–4 copies per haploid genome of closely relatedROA sequences and the presence of further less related sequences in a multigene family. The steady-state levels ofROA mRNA are under developmental control, being relatively high in proliferative tissues such as the root apex, the developing cob and the coleoptile, and are strongly correlated with that of the histone H4 transcript.In situ hybridisation analysis in the root apex reveals thatROA mRNA expression is limited to specific subpopulations of cycling cells, which is typical of cell cycle-regulated expression. The isolation of nearly identical sequences from barley andArabidopsis by the polymerase chain reaction indicates that MCM-related proteins are conserved in higher plants.

Distribution patterns of 104 kDa stress-associated protein in rice

A 104 kDa protein (SAP 104) accumulates in rice seedlings in response to several abiotic stress conditions and immunological homologues of rice SAP 104 have been detected in several monocot and dicot species, as also Neurospora crassa, a fungus. In this report, we show that the amino acid sequence of a tryptic peptide generated from purified SAP 104 bears significant homology with an ATP-binding domain of the HSP 100 family proteins of Arabidopsis thaliana and Glycine max. It is further shown that differential uninduced and induced (by high-temperature stress) levels of this protein are accumulated in various organs of the mature rice plant grown under field conditions. Significant uninduced levels of this protein were in particular found in developing and mature rice grains. Seeds/grains of several other plant genera (i.e. Triticum aestivum, Zea mays, Brassica juncea) were also found to contain high uninduced levels of SAP 104. Importantly, the levels of uninduced SAP 104 in rice grains were found to decline during the seed germination phase: after two days of germination, this protein was undetectable in tissues representing pooled sample of seeds and just-emerged seedlings. Tissue print-immunoblotting analysis has indicated that in seeds high levels of this protein are specifically present in the embryo portion.

Spatial and temporal volatile organic compound response of select tomato cultivars to herbivory and mechanical injury.

Plants resist an insect or pathogen attack through a range of direct and indirect defense signals. Vegetable crop plants constitutively synthesize and store a battery of volatiles implicated in defense against herbivores. We examined the effect of herbivory (Spodoptera litura Fab.) and mechanical injury on the release of volatile organic compounds (VOCs) in five tomato (Solanum lycopersicum Mill.) cultivars followed by behavioral assays on Trichogramma chilonis Ishii. T. chilonis is an egg parasitoid widely used as biocontrol agent in integrated pest management practices. Our goal was to assess the VOC variability across cultivars under different induction regimes. The findings reveal variability among cultivars in the absolute quantity of VOCs emitted as well as qualitative composition of the blend. Herbivore and mechanical wounding caused a substantial quantitative shift in the emission profile of select cultivars. The uninduced and induced volatiles of the tomato cultivars predominantly constitute monoterpenes (65%) followed by sesquiterpenes (15%) and aldehydes (10%). These ubiquitous and induced signals triggered a distinct cultivar specific olfactory response in T. chilonis. The spatial and temporal variations in induced VOCs across tomato cultivars suggest that these unique metabolite profiles are largely intrinsic and genetically determined. This study highlights the genotype based volatile profile and indicates their potential role in host, pest and natural enemy interactions.

Distribution patterns of HSP 90 protein in rice

Rice seedlings exposed to high and low temperatures, salinity and water stress accumulate 87 and 85 kDa stress-associated-proteins (SAP), collectively referred to as SAP 90. In the present study, we provide evidence that SAP 87 of rice is a member of the eukaryotic stress 90/HSP 90 family. Further, high uninduced levels of rice SAP/HSP 90 exist in lemma, palea and culm tissues. Dry seeds of rice, wheat, maize and sorghum also contain high uninduced amounts of this protein. Seed-derived callus tissue show high constitutive levels of this protein as well. When seed and callus tissues were subjected to heat shock, the levels of this protein declined. Tissue print-immunoblot analysis using anti SAP 90 antibodies revealed higher accumulation of SAP/HSP 90 in the vascular bundles/procambial cells and in the outermost cell layers of various leaves as well as in the meristematic cells of the stem apex. In seeds, this protein was most abundant in the seed coat and whole embryo.

Molecular regulation of santalol biosynthesis in Santalum album L.

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 - a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 - an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis.

Lactobacillus plantarum mediated fermentation of Psidium guajava L. fruit extract.

Sixteen hour fermentation of the white flesh raw guava Lucknow 49 cultivar using Lactobacillus plantarum NCIM 2912 was taken up for enhancing the antioxidant potential. The fermented guava product with high antioxidant potential, total phenolic content and short and medium chain fatty acids can be used as functional food.

Synthetic racemates of abyssinone I and II induces apoptosis through mitochondrial pathway in human cervix carcinoma cells

Abyssinones I and II are prenylated flavanones existing in plant Erythrina abyssinica showing diverse biological activities including anticancer activities. We synthesized racemic mixtures of these flavanones from corresponding chalcones and herein we report for the first time the molecular mechanisms of cell death, anti-proliferative effect and ability to induce apoptosis in human cervical carcinoma (HeLa) cells. Cytotoxicity was assessed by MTT assay to determine LD50 for prenylated chalcones and their corresponding flavones. Abyssinones promoted apoptosis by up regulation of p53 and Bax, along with down regulation of Bcl-2. Apoptosis induction was mediated through mitochondrial pathway releasing cytochrome c and Apaf-1 into cytosol; associated with activation of caspase-3. Further they were able to decrease the expression of cell proliferation markers PCNA and cyclin D1 indicating anti proliferative activity. These observations demonstrate that abyssinones trigger apoptosis via mitochondrial pathway by activation of caspase-3 and disrupts cell cycle.

NeeMDB: Convenient Database for Neem Secondary Metabolites

Indian Neem tree is known for its pesticidal and medicinal properties for centuries. Structure elucidation of large number of secondary metabolites responsible for its diverse properties has been achieved. However, this data is spread over various books, scientific reports and publications and difficult to access. We have compiled and stored structural details of neem metabolites in NeeMDB, a database which can be easily accessed, queried and downloaded. NeeMDB would be central in dissipating structural information of neem secondary metabolites world over.