Anath Bandhu Das

Defense potentials to NaCl in a mangrove, Bruguiera parviflora: differential changes of isoforms of some antioxidative enzymes.

In order to assess the role of the antioxidative defense system against salt treatment, the activities of some antioxidative enzymes and levels of antioxidants were monitored in a true mangrove, Bruguiera parviflora, subjected to varying levels of NaCl under hydroponic culture. In the leaves of B. parviflora, salt treatment preferentially enhanced the content of H2O2 as well as the activity of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), and superoxide dismutase (SOD), whereas it induced the decrease of total ascorbate and glutathione (GSH+GSSG) content as well as catalase (CAT) activity. Analysis of isoforms of antioxidative enzymes by native PAGE and activity staining revealed that leaves of B. parviflora had one isoform each of Mn-SOD and Cu/Zn-SOD and three isoforms of Fe-SOD. Expression of Mn-SOD and Fe-SOD-2 was preferentially elevated by NaCl. Similarly, out of the six isoforms of GPX, the GPX-1, 2, 3 and 6 were enhanced by salt treatment but the levels of GPX-4 and -5 changed minimally as compared to those of a control. Activity staining gel revealed only one prominent isoform of APX and two isoforms of GR (GR-1 and GR-2), all of these isoforms increased upon salt exposure. Four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced, suggesting differential down regulation of CAT isoforms by NaCl. The concentrations of malondialdehyde (MDA), a product of lipid peroxidation, remained unchanged in leaves of the plant treated with different concentrations of NaCl. This suggests that plants are protected against activated oxygen species by the elevated levels of certain antioxidative enzymes, thus avoiding lipid peroxidation during salt exposure. The differential changes in the levels of the isoforms due to NaCl treatment may be useful as markers for recognizing salt tolerance in mangroves.

NaCl stress causes changes in photosynthetic pigments, proteins, and other metabolic components in the leaves of a true mangrove,Bruguiera parviflora, in hydroponic cultures

We studied salt stress-induced biochemical changes in young, hydroponically grown plants of mangrove,Bruguiera parviflora (Rhizophoraceae). Our focus was on the effect of NaCI (applied at 100, 200, 400, or 500 mM) on leaf pigments, total soluble proteins, total free amino acids, carbohydrates, polyphenols, and proline. The total Chi content increased for 14 d after treatment with 100 mM NaCI, then gradually stabilized. At 400 mM, the total Chi content slowly decreased over the 45-d test period. However, the Chia:b ratio remained unchanged in isolated chloroplasts and in leaf tissue. Percent changes in the carotenoids content followed the same trend as for Chi, except for a 1.5-fold decrease during the 400-mM NaCI treatment, compared with the control. The total sugar content increased by 2.5-fold by Day 45 after treatment with 400 mM NaCI, whereas the starch content measured in the same treatment decreased by 40 to 45%. Leaf protein content decreased as salinity increased, which suggests either a possible disruption in the protein synthesis mechanism or, more likely, an increase in proteolytic activity. The total amino-acid pool increased steadily, by four-fold, in the 45-d, 400-mM treatment Both proline and polyphenols accumulated with increasing levels of salinity, which confirms the role of proline as a stress-induced protective metabolite in the adaptive process of this species. Our results showed that a true mangrove such as 8.parviflora can easily be sustained and propagated under low-salinity conditions. At high levels of salinity (~400 mM, beyond which they could not survive), the plants became adapted to salt stress after two to three weeks. During this adaptive period, changes in pigment and protein levels also occurred. The accumulation of proline and polyphenols played a key role in the plant’s stressinduced adjustment to NaCI under hydroponic culture conditions.

Investigations on the antioxidative defence responses to NaCl stress in a mangrove, Bruguiera parviflora: Differential regulations of isoforms of some antioxidative enzymes

Two-month-old healthy seedlings of a true mangrove, Bruguiera parviflora, raised from propagules in normal nursery conditions were subjected to varying concentrations of NaCl for 45 d under hydroponic culture conditions to investigate the defence potentials of antioxidative enzymes against NaCl stress imposed oxidative stress. Changes in the activities of the antioxidative enzymes catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), glutathione reductase (GR) and superoxide dismutase (SOD) were assayed in leaves to monitor the temporal regulation. Among the oxidative stress triggered chemicals, the level of H2O2 was significantly increased while total ascorbate and total glutathione content decreased. The ratio of reduced to oxidized glutathiones, however, increased due to decreased levels of oxidized glutathione in the leaf tissue. Among the five antioxidative enzymes monitored, the APX, POX, GR and SOD specific activities were significantly enhanced at high concentration (400 mM NaCl), while the catalase activities declined, suggesting both up and downregulations of antioxidative enzymes occurred due to NaCl imposed osmotic and ionic stress. Analysis of the stress induced alterations in the isoforms of CAT, APX, POX, GR and SOD revealed differential regulations of the isoforms of these enzymes. In B. parviflora one isoform of each of Mn-SOD and Cu/Zn-SOD while three isoforms of Fe-SOD were observed by activity staining gel. Of these, only Mn-SOD and Fe-SOD2 content was preferentially elevated by NaCl treatment, whereas isoforms of Cu/Zn-SOD, Fe-SOD1 and Fe-SOD3 remained unchanged. Similarly, out of the six isoforms of POX, the POX-1,-2,-3 and -6 were enhanced due to salt stress but the levels of POX-4 and -5 remained same as in control plants suggesting preferential upregulation of selective POX isoforms. Activity staining gel revealed only one prominent band of APX and this band increased with increased salt concentration. Similarly, two isoforms of GR (GR1 and GR2) were visualized on activity staining gel and both these isoforms increased upon salt stress. In this mangrove four CAT-isoforms were identified, among which the prominent CAT-2 isoform level was maximally reduced again suggesting differential downregulation of CAT isoforms by NaCl stress. The results presented in this communication are the first report on the resolutions of isoforms APX, POX and GR out of five antioxidative enzymes studied in the leaf tissue of a true mangrove. The differential changes in the levels of the isoforms due to NaCl stress may be useful as markers for recognizing salt tolerance in mangroves. Further, detailed analysis of the isoforms of these antioxidative enzymes is required for using the various isoforms as salt stress markers. Our results indicate that the overproduction of H2O2 by NaCl treatment functions as a signal of salt stress and causes upregulation of APX, POX, GR and deactivations of CAT in B. parviflora. The concentrations of malondialdehyde, a product of lipid peroxidation and lipoxygenase activity remained unchanged in leaves treated with different concentrations of NaCl, which again suggests that the elevated levels of the antioxidant enzymes protect the plants against the activated oxygen species thus avoiding lipid peroxidation during salt stress.

Salt-stress induced alterations in protein profile and protease activity in the mangrove Bruguiera parviflora

Two-month-old seedlings of Bruguiera parvifora were treated with varying levels of NaCl (100, 200 and 400 mᴍ) under hydroponic culture. Total proteins were extracted from leaves of control and NaCl treated plants after 7, 14, 30 and 45 d of treatment and analysed by SDS-PAGE. As visualized from SDS-PAGE, the intensity of several protein bands of molecular weight 17, 23, 32, 33 and 34 kDa decreased as a result of NaCl treatment. The degree of decrease of these protein bands seemed to be roughly proportional to the external NaCl concentration. The most obvious change concerned a 23 kDa-polypeptide (SSP-23), which disappeared after 45 d treatment in 400 mm NaCl. Moreover, the SSP-23 protein, which disappeared in B. parviflora under salinity stress, reappeared when these salinized seedlings were desalinized. These observations suggest the possible involvement of these polypeptides for osmotic adjustment under salt stress. NaCl stress also caused an increase in the activity of both acid and alkaline protease. The increasing activity of proteases functions as a signal of salt stress in B. parviflora, which induces the reduction of protein level.

Molecular stress physiology of plants

1.Title: Abiotic and biotic stress tolerance in plants. Author: Susana Redondo-Gomez.- 2.Title: Molecular Mechanism of stress-resistance of photosynthetic machinery.Authors: Vladimir D. Kreslavski, Ann Zorina, Dmitry A. Los, Irina R. Fomina, Suleyman I. Allakhverdiev.- 3.Title: Salinity induced genes and molecular basis of salt tolerance strategies in mangroves.Authors: Anath Bandhu Das, Reto J Strasser.- 4.Title: PSII fluorescence techniques for measurement of drought and high temperature stress signal in crop plants: protocols and applications. Authors: Marian Brestic, Marek Zivcak.- 5.Title: Salt tolerance in cereals: Molecular mechanism and applications.Authors: Allah Ditta.- 6.Title: Salt stress: A biochemical and physiological adaptation of some Indian halophytes of Sundarbans.Authors: Nirjhar Dasgupta, Paramita Nandy, Sauren Das.- 7.Title: Molecular physiology of osmotic stress in plants. Authors: H. Upadhyaya, L. Sahoo, S. K. Panda.- 8. Title: The physiology of reproductive-stage abiotic stress tolerance in cereals.Authors: Rudy Dolferus, Nicola Powell, Xuemei JI, Rudabe Ravash, Jane Edlington, Sandra Oliver, Joost Van Dongen, Behrouz Shiran.- 9.Title: Salicylic acid: role in plant physiology and stress tolerance.Author: Gopal K. Sahu.- 10. Title: Role of calcium-mediated CBl-CIPK network in plant mineral nutrition and abiotic stress.Authors: Indu Tokas, Amita Pandey, Girdhar K. Pandey.- 11. Title: Isothermal calorimetry and Raman spectroscopy to study response of plants to abiotic and biotic stress.Authors: Andrzej Skoczowski, Magdalena Troc.- 12. Title: Mechanism of plant tolerance in response to heavy metals.Authors: Jot Sharma, Nivedita Chakravarty.- 13. Title: Brassinosteroids - Biosynthesis and role in growth development and thermo tolerance responses.Author: Geetika Sirhindi.- 14. Title: Submergence stress tolerance in crop plants.Authors: Chinmay Pradhan, Monalisha Mohanty.- 15. Title: Stress tolerance in plants: a proteomics approach.Authors: Gyana Ranjan Rout, Sunil Kumar Senapati.- 16. Title: Marker-Assisted Breeding (MBA for stress resistance in crop plants. Authors: J. Panigrahi, R. R. Mishra, A. R. Sahu, S. C. Rath, C. R. Kole.- 17. Title: DNA Methylation Associated Epigenetic Changes in Stress Tolerance of Plants.Author: Mahmoud W. Yaish.

Metabolic changes during rooting in stem cuttings of five mangrove species

Vegetative propagation through rooting in stem cuttings in five tree mangroves namely Bruguiera parviflora, Cynometra iripa, Excoecaria agallocha, Heritiera fomes, and Thespesia populnea using IAA, IBA and NAA was reported. Spectacular increase in the root number was noted in the cuttings of H. fomes and C. iripa treated together with IBA (5000 ppm) and NAA (2500 ppm). The highest number of roots was obtained with IBA (2500 ppm) and NAA (500 ppm) in E. agallocha. B. parviflora and T. populnea responded better to IAA and IBA treatment. The species specific variation in the rooting response to exogenous application of auxins was reflected in the metabolic changes during initiation and development of roots in cuttings. Biochemical analysis showed increase of reducing sugar in the above-girdled tissues at initiation as well as subsequent development of roots which was further enhanced by the use of auxins. Decreases in the total sugar, total carbohydrate and polyphenols and increase in total nitrogen were recorded in the girdled tissues and the high C/N ratio at the initial stage helped in initiation of roots in all the species. Interaction of IBA and NAA promoted starch hydrolysis better than IAA and IBA during root development and subsequently reduced the C/N ratio and increased the protein-nitrogen activity during root development which suggest the auxin influenced mobilization of nitrogen to the rooting zone.

Antimicrobial effect of silver zinc oxide (Ag-ZnO) nanocomposite particles

The antimicrobial effects of silver nanocomposite particles (Ag-ZnO NC) on microorganisms, the antimicrobial mechanism and applications in medical devices are not yet clear. Stable Ag-ZnO NC were prepared and their morphological sizes and shapes were characterized by scanning electron microscopy. The effect of Ag-ZnO NC was tested on Bacillus thuringiensis, Escherichia coli and Pseudomonas aeruginosa in antibacterial tests including growth kinetics, antimicrobial susceptibility (disc diffusion) and minimal inhibitory concentration (MIC). Different concentrations of nanocomposites (i.e. 10, 20, 50,100, and 200 μg) showed concentration-dependant efficacy on all three tested microorganisms. E. coli was fairly sensitive in 200 μg of NC, forming a ∼15 mm inhibition zone; followed by B. thuringiensis, having ∼9 mm of inhibition zone, while P. aeruginosa, a pathogenic bacterium, showed negligible inhibition zone with Ag-ZnO NC. Growth of E. coli under Ag-ZnO NC treatment was significantly delayed with an extended lag phase of 2 hrs and 30 mins. Scanning electron microscopy confirmed the bacteriostatic effect of Ag-ZnO NC, which was manifested in cell division arrest with significant cell elongations compared to the control. The free radical generation effect of Ag-ZnO NC was tested against all these organisms. The results suggest that Ag-ZnO NC can be used effectively against microbial growth. This may be of use in diverse medical devices for antimicrobial control and can be a proper substitute for antibiotics in curing human health.

A novel cadmium induced protein in wheat: characterization and localization in root tissue

A 51-kDa soluble protein was over-expressed in wheat (Triticum aestivum) seedlings by the treatment of seeds before germination with 50 µM CdCl2 for 48 h and subsequently washed off Cd2+. This protein designated as Cd stress associated protein (CSAP), was purified. Polyclonal antibody was raised against CSAP for localizing the protein in root tissue of treated and control seedlings. It was observed that CSAP was located below the plasma membrane and outer periphery of the tonoplast. This unique type of organized localization of CSAP is suggestive of defensive role against metal phytotoxicity. N-terminal analysis of CSAP and expressed sequence tags (EST) database search of wheat sequences suggests that this protein has not been reported earlier in higher plants.

Karyotype diversity and genomic variability in some Indian tree mangroves

SUMMARYInvestigation on karyotype and genomic characteristics of 11 Indian tree mangroves belonging to the family Bignoniaceae, Malvaceae, Myrsinaceae, Rhizophoraceae and Sterculiaceae revealed wide genetic variation. Numerical and structural alteration of chromosomes at intergeneric and interspecific level were manifested in the species specific genome length, volume and form percentage of the karyotype. Somatic chromosome number varied from 2n = 26 in Thespesia populnea of Malvaceae to 2n = 44 in Aegiceras comiculatum of Myrsinaceae. The members of Rhizophoraceae and Sterculiaceae showed 2n = 34, 36, 38 chromosomes in the root tip cells. Correlation coefficient studies among the cytological parameters showed significant progressive interdependence within the species. Critical differences in the average genome length and genome volume showed significant variation.

Bioprospecting and Genetic Engineering of Mangrove Genes to Enhance Salinity Tolerance in Crop Plants

Salinity in agricultural land is a major problem world wide, placing a severe constraint on crop growth and productivity in many regions and increased salinization of arable land is expected to have devastating global effects. Though plants vary in their sensitivity to salt stress, high salinity causes water deficit and ion toxicity in many plant species. Considerable efforts have therefore been made to investigate how genes respond to salt stress in various plants by using several approaches, including proteomics. Proteomic approaches for identifying proteins that are regulated in response to salt stress are becoming common in the post-genomics era of crop research. In this chapter, a detailed description of physiological, biochemical and antioxidative genes of salt stress in mangroves is given. Recent developments on salt stress genes of mangrove origin are also discussed and their identification applying bioinformatics approach as well as their validation in lower unicellular organisms. In addition, genomic biological changes in the proteomes of mangroves under salt stress condition are discussed. This chapter will provide a viewpoint into how proteomics and genomic based research is likely to develop in this field. Accumulation of compatible solutes and induction of antioxidative enzymes are other mechanisms of salt tolerance in mangroves. Mangroves also provide a reservoir for some of the best known, and at times, novel genes and proteins, involved in tolerance to salinity stress, that are likely at work in other plants. The salt tolerance genes listed in this review most likely represent only the tip of the iceberg, and continuous efforts to isolate and identify novel useful genes and promoters from mangroves are necessary; DNA microarray technology in particular is likely to become a powerful tool for this purpose. Eventually, the largest challenge will be to combine these genes and promoters in a systematic and logical way in order to maximize plant salinity tolerance. When realized, genetic engineering of crop and industrial plant for salinity tolerance using genes isolated from mangroves will be a vitally important tool in the quest to alleviate the earth’s future problems concerning food, energy, and the environment.