Ashok Kumar Bhatnagar

Effects of arbuscular mycorrhiza and phosphorus application on artemisinin concentration in Artemisia annua L.

Annual wormwood (Artemisia annua L.) produces an array of complex terpenoids including artemisinin, a compound of current interest in the treatment of drug-resistant malaria. However, this promising antimalarial compound remains expensive and is hardly available on the global scale. Synthesis of artemisinin has not been proved to be feasible commercially. Therefore, increase in yield of naturally occurring artemisinin is an important area of investigation. The effects of inoculation by two arbuscular mycorrhizal (AM) fungi, Glomus macrocarpum and Glomus fasciculatum, either alone or supplemented with P-fertilizer, on artemisinin concentration in A. annua were studied. The concentration of artemisinin was determined by reverse-phase high-performance liquid chromatography with UV detection. The two fungi significantly increased concentration of artemisinin in the herb. Although there was significant increase in concentration of artemisinin in nonmycorrhizal P-fertilized plants as compared to control, the extent of the increase was less compared to mycorrhizal plants grown with or without P-fertilization. This suggests that the increase in artemisinin concentration may not be entirely attributed to enhanced P-nutrition and improved growth. A strong positive linear correlation was observed between glandular trichome density on leaves and artemisinin concentration. Mycorrhizal plants possessed higher foliar glandular trichome (site for artemisinin biosynthesis and sequestration) density compared to nonmycorrhizal plants. Glandular trichome density was not influenced by P-fertilizer application. The study suggests a potential role of AM fungi in improving the concentration of artemisinin in A. annua.

Variability in protein content of pollen of Castor bean (Ricinus communis) before and after exposure to the air pollutants SO {\rm _{2}} and NO {\rm _{2}}

The role of air pollution has been suspected to be an important factor in the recent increase in the incidence of respiratory diseases. The ambient air, which is the carrier of pollen grains, also incorporates pollutants from the atmosphere, leading to alteration of the pollen proteins. Several studies have reported higher risk of development of pollinosis in the areas of dense vehicular traffic as compared to the less polluted areas. The objective of the present investigation was to study the effect of chemical air pollutants (SO2 and NO) on the soluble total protein content of the pollen of Ricinus communis taken as a model system. Pollen grains of R. communis were collected and exposed to different (100, 200, 300, 400 and 500 μg/m) concentrations of SO and NO individually, as well as together, for 8, 16, 32 and 48 hours. Proteins were extracted from exposed pollen and also unexposed pollen, which acted as control. The variation found in the protein content was analysed statistically. In the exposed pollen, total protein content showed a significant decrease (p<0.05) in the extracts from the unexposed pollen to SO and NO. It varied from 1.84 to 11.01 mg/ml for the extracts from pollen exposed to SO only, whereas NO exposure recorded values for total protein from 0.82 to 3.44 mg/ml only. There was a gradual decrease in the soluble protein content with increase in the concentration and duration of exposure in the pollen exposed to SO and NO together (p<0.05).

Effect of elevated [CO2] on cell structure and function in seed plants

Unprecedented increase in atmospheric carbon dioxide concentration [CO2] is regarded as one of the key factors responsible for global climate change. Evidence from palaeo-records indicates a close relationship between variation in atmospheric [CO2] and corresponding structural adaptations in plants. Exposure to elevated [CO2] alters plant structure by inducing changes in rate of cell division, cell expansion and cell cycling. Alterations in plant structure under elevated [CO2] at organ level is possibly a result of metabolic changes induced at cellular level. Chloroplast, the light-harvesting organelle, has been shown to be most seriously affected by high [CO2]. Prolonged exposure to elevated [CO2] induces accumulation of starch grains within chloroplasts leading to distortion of thylakoids. Higher rate of photosynthesis in plants grown at high [CO2] is accompanied by increased number of mitochondria to meet high cellular energy demand. These ultrastructural observations are corroborated by studies on nuclear genome that have revealed up as well as down-regulation of several genes involved in photosynthetic and respiratory pathways. In some species, exposure to elevated [CO2] results in accumulation of tannin-like vacuolar deposition, suggesting activation of phenylpropanoid pathway. Ultrastructural alterations in peroxisomes, endoplasmic reticulum, Golgi apparatus and cytoskeleton due to CO2 enrichment have received less attention. It is imperative to understand cellular changes occurring with elevated [CO2]so as to correlate these with the physiological alterations at plant level. A thorough insight into ultrastructural changes in response to increased carbon dioxide will provide a better, generalized understanding of plant response to rising atmospheric [CO2] in future. Disruption of cell organelle structure and function may also indicate a wider impact of climate change across plant (and animal) groups.

Influence of phosphorus and pH on the fungicidal potential of Anabaena strains

The genus Anabaena is known to be a rich source of bioactive metabolites, but the biocontrol potential of this genus, mediated through hydrolytic enzymes is less investigated. In our investigation, five Anabaena strains – A. laxa RPAN8, A. iyengarii RPAN9, A. variabilis RPAN59 and A. oscillarioides RPAN69 (with A. variabilis RPAN16 serving as negative control) were evaluated in time course studies involving incubation under three levels of phosphorus and pH conditions. Total chlorophyll, proteins, chitosanase, endoglucanase and CMCase activity were measured and inhibition assayed against phytopathogenic fungi. The four weeks old RPAN69 culture showed significantly higher chlorophyll which was 41% higher than control. This was also linked with an enhancement of 18.26% and 9.18% in chitosanase and CMCase activity respectively over control in the treatment involving half dose of phosphorus. Chlorophyll and CMCase activity showed a high degree of correlation with highest values at pH 9.5. A pH of 5.5 was the most suitable condition for the maximum activity of chitosanase for all the strains except RPAN16. The strains RPAN8 and RPAN9 showed the highest activity of endoglucanase at pH 5.5 while the other strains exhibited maximum activity at pH 7.5. This study provides insight into the role of P and pH in modulating fungicidal activity in different Anabaena strains, which can be valuable for enhancing their efficiency as a biocontrol agent.

Analyses of diversity among fungicidal Anabaena strains

This study characterized 28 Anabaena strains exhibiting fungicidal activity using different molecular approaches. Based on 16S rDNA and internal transcribed spacer region sequences, all the Anabaena strains isolated from India were found genetically diverse from the other reference strains in the National Center for Biotechnology Information database. Out of these, six belonging to five different species were found to produce microcystin toxins as confirmed by polymerase chain reaction amplification of microcystin synthase gene and an enzyme-linked immunosorbent assay-based method. All the sequenced Anabaena strains with the fungicidal trait formed a separate group in both the trees, when compared to other reference Anabaena strains. Thus, it can be hypothesized that the antifungal trait has led to evolutionary divergence among these strains. This study illustrates the diversity among the Anabaena strains with fungicidal activity and their uniqueness vis a vis Anabaena strains available globally.