H. C. Kapoor

Antioxidants in tomato (Lycopersium esculentum) as a function of genotype

Consumption of tomato products has been associated with decreased risk of some cancer types. Epidemiological findings confirm the observed health effects due to the presence of varied antioxidants in tomato. The bio-antioxidant content and antioxidant activity of 12 tomato genotypes was therefore studied. Significant differences were found between lycopene, ascorbic acid and phenolic contents among various genotypes. Lycopene and ascorbic acid contents showed 1-4 fold and 1-2 fold variation on both fresh and dry weight basis, respectively. Antioxidant activity was found to vary significantly among genotypes. In both free radical quenching assay and FRAP assay, significant activity was found. Activity was higher in the hexane fraction containing lycopene than the methanol fraction containing phenolics. Tomato peels, in addition to lycopene, contain significantly high amounts of ascorbic acid and phenols. Cherry tomatoes, particularly variety 818 cherry, with the highest contents of antioxidants (lycopene, ascorbic acid and phenols) and highest antioxidant activity represents a valuable genotype not only for improving the status of dietary antioxidants in our diet but also for increasing nutritional value through germplasm enhancement programmes. The cherry varieties also merit considerable attention for processing because of their high total soluble solids and titrable acidity.

Purification and properties of growth stage-dependent antiviral proteins from the leaves of Celosia cristata.

Two antiviral glycoproteins, active against mechanical transmission of two tobamoviruses, tobacco mosaic virus and sunnhemp rosette virus, and citrus ring spot virus (ungrouped), were purified from the dried leaves of Celosia cristata. These proteins, called CCP-25 and CCP-27, have M(r) 25 and 27 kDa, respectively. Their concentration was found to vary between the pre-flowering and post-flowering stages of C. cristata90% lesion formation at a concentration of 20-30 µg ml(-1). They were resistant to proteases in the native state, but were readily digested when denatured. Both of them imparted actinomycin D sensitive resistance by inhibiting local lesions on Nicotiana tabacum cv. Samsun NN by tobacco mosaic virus. Their application, individually, also resulted in high resistance in systemic hosts to sunnhemp rosette virus, and citrus ring spot virus, respectively.

Antioxidant Activity of Antiviral Proteins from Celosia cristata

The antioxidant activity test of Celosia cristata antiviral proteins (CCP-25 and CCP-27) using ferric reducing antioxidant power (FRAP) assay in vitro indicated that these proteins are strong antioxidants. The increase in activities of redox-enzymes such as peroxidase, catalase and polyphenol oxidase on tobacco mosaic virus (TMV) inoculation of test plants, was inhibited when plants were treated with CCP-25 before TMV inoculation. The activity of phenylalanine ammonia lyase, involved in biosynthesis of antioxidative compounds was also inhibited. This is the first ever report where plant antiviral proteins have been shown to have strong antioxidative property. A possible correlation between antioxidant activity of CCPs and their antiviral activity is speculated.

Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).

Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus‐induced cell‐death phenomenon in which cysteine proteinases are active. To test this idea, a full‐length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cocks comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins. It was expressed in an Escherichia coli expression system as a fusion protein. The purified recombinant product, termed ‘celostatin’ (Celosia cystatin), inhibited the enzymatic activity of papain indicating its cystatin activity and prevented TMV (tobacco mosaic virus)‐induced hypersensitive‐response cell death in Nicotiana glutinosa (a wild species of tobacco) leaves by 65–70% at the concentration of approx. 50 ng/ml. It also offered resistance against TMV and caused normal growth of the test plant. Since the activity of cysteine proteinases is not involved in the TMV replication process, we speculated that inhibition of the hypersensitive response by celostatin may be due to the inactivation of proteolysis involved in the plant cell death programme, a phenomenon that has already been reported in animal systems.

Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP) from the leaves of Bougainvillea xbuttiana was isolated. The cDNA consisted of 1364 nucleotides with an open reading frame (ORF) of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids. The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species. The deduced protein has been designated BBAP1 (Bougainvillea xbuttiana antiviral protein1). The ORF was cloned into an expression vector and expressed in E. coli as a fusion protein of ∼78 kDa. The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity, and imparted a high level of resistance against the tobacco mosaic virus (TMV).

Single step isolation of plant RNA

A rapid method for preparation of plant RNA by single step extraction and instantaneous precipitation using sec-butanol is standardised.

An antiviral protein having deoxyribonuclease and ribonuclease activity from leaves of the post-flowering stage of Celosia cristata

An antiviral protein named CCP-27 was purified from the leaves of Celosia cristata at the post-flowering stage by anion-exchange, cation-exchange, and gel-filtration chromatography. It exhibited resistance against sunnhemp rosette virus in its test host Cyamopsis tetragonoloba. It also exhibited deoxyribonuclease activity against supercoiled p BlueScript SK+ plasmid DNA. It was found to nick supercoiled DNA into nicked circular form at lower prote in concentration followed by nicked to linear form conversion at higher protein concentration. CCP-27 also possesses strong ribonuclease activity against Torula yeast rRNA.

Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ∼57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome inactivating site. We nominate this short cDNA frag ment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.

Production, Partial Purification and Characterization of Extracelluar Xylanase from Chaetomium globosum

Culture conditions for efficient production of extracellular xylanase by fungus, Chaetomium globosum isolate Cg2, have been standardized. Further, xylanase has been partially purified and characterized. Xylanase activity was maximum after 9 days of incubation when amended in medium with 1.5 % xylan as carbon source and 0.6% NH4H2PO4 as nitrogen source. Partial purification of the xylanase was accomplished by ammonium sulphate precipitation, followed by further purification by anion exchange chromatography on DEAE-Sephadex A-50 column. The partially purified enzyme was electrophoresed on SDS-PAGE and a single band produced corresponded to molecular weight, 32 kD. The optimum temperature and pH for maximum activity of purified xylanase were 30°C and 5.5, respectively. Both the purified xylanase and culture filtrate have shown the antifungal activity against Bipolaris sorokiniana, a causal organism of spot blotch of wheat. Purified xylanase at 100 μg ml−1 concentration caused 100 per cent inhibition of conidia germination of B. sorokiniana, whereas the culture filtrate was able to inhibit germination up to 67.5 per cent.

Modifications in the purification protocol of Celosia cristata antiviral proteins lead to protein that can be N-terminally sequenced.

Plants antiviral proteins are being used as anticancer agents and inhibit other viral diseases in humans. We modified the purification protocol of the two N-terminally blocked antiviral glycoproteins, CCP-25 and CCP-27, purified from the leaves of Celosia cristata. This not only gave rise to single pure samples with few steps of purification but also resulted in N-terminally free proteins. The extra purity of the samples was analyzed by reverse phase HPLC. Deglycosylation studies of CCP-25 with PNGase F enzyme revealed that its asparagine or asparagine-linked glycon contents are negligible. Partial N-terminal sequence of the CCP-25 showed the sequence (ANDIS), which seems to be conserved among plant antiviral proteins.