Oxidative stress enzymes in Ficus religiosa L.: biochemical, histochemical and anatomical evidences.

In order to unveil the reasons behind the successful survival of Ficus religiosa L. grown under normal and adverse habitats (AH), i.e., on concrete roof tops were subjected to biochemical, histochemical and physiological studies with a focus on reactive oxygen species (ROS) and oxidative stress enzymes (OSE). The specific objectives were: to localize the OSE, peroxidase (POX) and catalase (CAT); to localize and quantify the main ROS, hydrogen peroxide (H(2)O(2)); to estimate the activities of POX, CAT and glycolate oxidase (GO); and to study the diurnal variations in stomatal activity by scanning electron microscopy (SEM). From the results, plants grown in AH showed 55% higher H(2)O(2) production with about 30% increase in POX activity. Of the three substrates tested for POX activity (guaiacol, ascorbate and o-dianisidine), o-dianisidine was found as the preferred substrate of F. religiosa POX with about 7-fold more activity over its counterparts. Cytosolic POX activity showed 11-fold increase over cell wall bound POX. Similarly, CAT activity in specimens from AH showed about 2-fold increase during day time. The physiological interaction between CAT and its substrate H(2)O(2) in the plant was determined by quantifying H(2)O(2) and assaying the CAT, in which CAT showed 4-fold increases in activity, especially during night. F. religiosa has higher amount of H(2)O(2) deposition during night than day time, which was in correlation high CAT activity during night, coupled with scotoactive opening of stomata as shown by the SEM images. Moreover, GO did not show much habitat-dependent variation. In toto, F. religiosa grown in AH showed elevated production of ROS and their scavenging OSE, which is the direct evidence for drought stress and also giving an insight into its evolution and ecological niche.

Rapid detection and characterization of Chikungunya virus by RT-PCR in febrile patients from Kerala, India.

There has been a resurgence and prevalence of fever with symptoms of Chikungunya (CHIK) and increased death toll in Kerala, the southern-most state of India. The objective of this study was to develop a rapid detection method to determine the presence of CHIK- virus in the serum samples collected from febrile patients in Kerala, India. Serum specimens were analyzed for CHIK viral RNA by RT-PCR using primers specific for nsP1 and E1 genes. Five out of twenty clinical samples were positive for CHIK virus. The partial sequences of the E1 and nsP1 genes of the strain, IndKL01 were highly similar to the Reunion strains and the recently isolated Indian strains. A novel substitution, A148V, was detected in the E1 gene of the isolate, IndKL02. The detection procedure used in this study was simple, sensitive and rapid (less than 4 hr). This result suggests that CHIK viruses similar to the Reunion strains, which had resulted in high morbidity and mortality rates, may have caused the recent Chikungunya outbreak in India. The effect of the variant, E1-A148V, in the virulence and the rate of transmission of the virus deserves further investigation.

Purification and characterization of cinnamyl alcohol-NADPH-dehydrogenase from the leaf tissues of a basin mangrove Lumnitzera racemosa Willd.

Cinnamyl alcohol-NADPH-dehydrogenase (CAD), the marker enzyme of lignin biosynthesis was purified from the leaf tissues of a basin mangrove Lumnitzera racemosa by ammonium sulphate precipitation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the CAD enzyme was determined as 89 kDa, by size elution chromatography. SDS-PAGE of CAD revealed two closely associated bands of 45 kDa and 42 kDa as heterogenous subunits. The optimum pH of CAD was found to be 4.0. Km for the substrates cinnamaldehyde, coniferaldehyde and sinapaldehyde was determined. Cinnamaldehyde showed higher Km value than sinapaldehyde and coniferaldehyde. The correlation of activity of CAD with the amount of lignin was found less significant in L. racemosa, compared to plant species of other habitats viz., mesophytes, xerophytes and hydrophytes, suggesting that CAD possibly exhibits physiological suppression due to the saline habitat of the plant.

H+-ATPase as a biochemical marker for early detection of root (wilt) disease in coconut palms (Cocos nucifera L).

H+-ATPase activity in leaves and roots of coconut palms growing in 'root wilt disease-prevalent areas' was compared with that of coconut palms growing in 'disease-free areas'. The activity was found to be significantly less in the leaves and roots of palms in the disease-prevalent zone as compared to that in disease-free zone. Histochemical examination of the leaves showed results that corroborated the biochemical findings. The possible application of H+-ATPase activity as a marker for the early detection of wilt disease in coconut palms is suggested.

Association of lignifying enzymes in shell synthesis of oil palm fruit (Elaeis guineensis--dura variety).

Scanning electron microscopic (SEM) observation demonstrates the differentiation of mesocarp and endocarp tissues and their lignified nature in dura fruits at 8 weeks after pollination (WAP). During shell formation, the endocarp cells become lignified to a hard shell while the mesocarp tissue remains cellular and fibrous. A transition zone made up of fibrous units was also visible beneath the shell. The soluble phenols of mesocarp and endocarp tissues at their developmental stage was analyzed using Reverse phase high performance liquid chromatography (RP-HPLC). The appearance of ferulic acid at 4 WAP and its absence at 8 WAP indicates the role of ferulic acid in lignin synthesis. The HPLC data was supported by the lignin concentration. To ascertain the biochemical relationship of lignin pathway enzymes, phenylalanine ammonia lyase (PAL), cinnamyl alcohol-NADPH-dehydrogenase (CAD) and peroxidase (POD) with shell synthesis, the activities of these enzymes and lignin content were assessed during development of the shell between 4 and 8 WAP. The three enzymes, PAL, CAD and POD expressed high level of activity in the mesocarp and endocarp at 4 WAP. At 8 WAP a sharp decline in activity was observed in the endocarp whereas the mesocarp showed a moderate reduction. This variation is an indication of the role of these enzymes in shell formation.