Ajay Parida

Antioxidative response mechanisms in halophytes: their role in stress defence.

Normal growth and development of plants is greatly dependent on the capacity to overcome environmental stresses. Environmental stress conditions like high salinity, drought, high incident light and low or high temperature cause major crop losses worldwide. A common denominator in all these adverse conditions is the production of reactive oxygen species (ROS) within different cellular compartments of the plant cell. Plants have developed robust mechanisms including enzymatic or nonenzymatic scavenging pathways to counter the deleterious effects of ROS production. There are a number of general reviews on oxidative stress in plants and few on the role of ROS scavengers during stress conditions. Here we review the regulation of antioxidant enzymes during salt stress in halophytes, especially mangroves. We conclude that (i) antioxidant enzymes protect halophytes from deleterious ROS production during salt stress, and (ii) genetic information from mangroves and other halophytes would be helpful in defining the roles of individual isoforms. This information would be critical in using the appropriate genes for oxidative stress defence for genetic engineering of enhanced stress tolerance in crop systems.

Generation and analysis of expressed sequence tags from the salt-tolerant mangrove species Avicennia marina (Forsk) Vierh.

Salinization poses an increasingly serious problem in coastal and agricultural areas with negative effects on plant productivity and yield. Avicennia marina is a pantropical mangrove species that can survive in highly saline conditions. As a first step towards the characterization of genes that contribute to combating salinity stress, the construction of a cDNA library of A. marina genes is reported here. Random expressed sequence tag (EST) sequencing of 1,841 clones produced 1,602 quality reads. These clones were classified into functional categories, and blast comparisons revealed that 113 clones were homologous to genes earlier implicated in stress responses, of which the dehydrins are the most predominant in this category. Of the ESTs analyzed, 30% showed homology to previously uncharacterized genes in the public plant databases. Of these 30%, 52 clones were selected for reverse Northern analysis: 26 were shown to be up-regulated and five shown to be down-regulated. The results obtained by reverse Northern analysis were confirmed by Northern analysis for three clones.

Monitoring expression profiles of antioxidant genes to salinity, iron, oxidative, light and hyperosmotic stresses in the highly salt tolerant grey mangrove, Avicennia marina (Forsk.) Vierh. by mRNA analysis

Plant photosynthesis results in the production of molecular oxygen. An inevitable consequence of this normal process is the production of reactive oxygen species (ROS) by the transfer of electrons to molecular oxygen. Plants are adequately protected by the presence of multiple antioxidative enzymes in different organelles of the plant such as chloroplasts, cytosol, mitochondria and peroxisomes. Under high light and CO2 limiting conditions caused by environmental stress like salinity, these antioxidative enzymes play an important role in scavenging toxic radicals. To investigate the functions of antioxidative enzymes in a mangrove plant, we isolated three cDNAs encoding cytosolic Cu–Zn SOD (Sod1), catalase (Cat1) and ferritin (Fer1) from Avicennia marina cDNA library. Sod1, Cat1 and Fer1 cDNA encoded full-length proteins with 152, 492 and 261 amino acids respectively. We studied the expression of these antioxidant genes in response to salt, iron, hydrogen peroxide, mannitol and light stress by mRNA expression analysis. Cat1, Fer1 showed short-term induction while Sod1 transcript was found to be unaltered in response to NaCl stress. A decrease in mRNA levels was observed for Sod1, Cat1 while Fer1 mRNA levels remained unaltered with osmotic stress treatment. Sod1, Cat1 and Fer1 mRNA levels were induced by iron, light stress and by direct H2O2 stress treatment, thus confirming their role in oxidative stress response.

MOLECULAR PHYLOGENY OF MANGROVES IX: Molecular marker assisted intra-specific variation and species relationships in the Indian mangrove tribe Rhizophoreae

Molecular phylogeny and genetic diversity in all the nine species (Rhizophora mucronata, R. apiculata, R. stylosa, Bruguira cylindrical, B. parviflora, B. gymnorriza, Ceriops tagal and C. decandra) and a natural Rhizophora hybrid, of the mangrove Rhizophoraceae, occurring in the Indian sub-continent were analysed using random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and RFLP of polymerase chain reaction amplified chloroplast genes (PCR-RFLP) as markers. Intra-specific variability as revealed by RAPDs were low in all the analysed species. Inter-specific RFLP analysis revealed species-specific profiles in some probe-enzyme combinations. The rDNA repeat units, as flanked by the Hind III restriction sites was found to be very conserved within each genus and three different rDNA repeat units were observed among the four genera. Generic differences in PCR-RFLP were observed only in rbcL and trnS-psbC gene regions. Species differences were observed in Rhizophora in the PCR-RFLP of trnS-psbC and trnL-UAA with Hae III and Taq I, respectively. Rhizophora mucronata was found to be the chloroplast donor for the natural inter-specific hybrid. A dendrogram based on the data sets from all the three marker systems revealed that the four genera segregated into three species groups.

A chloroplast-localized and auxin-induced glutathione S-transferase from phreatophyte Prosopis juliflora confer drought tolerance on tobacco

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Prosopis juliflora, a drought-tolerant tree species of Fabaceae, as a model plant system for mining genes functioning in abiotic stress tolerance. Large-scale random EST sequencing from a cDNA library obtained from drought-stressed leaves of 2-month-old P. juliflora plants resulted in identification of three different auxin-inducible glutathione S-transferases. In this paper, we report the cellular localization and the ability to confer drought tolerance in transgenic tobacco of one of these GSTs (PjGSTU1). PjGSTU1 was overexpressed in Escherichia coli and GST and GPX activities in total protein samples were assayed and compared with controls. The results indicated that PjGSTU1 protein forms a functional homo-dimer in recombinant bacteria with glutathione transferase as well as glutathione peroxidase activities. PjGSTU1 transgenic tobacco lines survived better under conditions of 15% PEG stress compared with control un-transformed plants. In vivo localization studies for PjGSTU1 using GFP fusion revealed protein localization in chloroplasts of transgenic plants. The peroxidase activity of PjGSTU1 and its localization in the chloroplast indicates a possible role for PjGSTU1 in ROS removal.

Molecular phylogeny of mangroves I. Use of molecular markers in assessing the intraspecific genetic variability in the mangrove species Acanthus ilicifolius Linn. (Acanthaceae)

Abstract Mangroves, the intertidal ecosystems occurring primarily in the tropical regions of the world, are valuable natural resources with high productivity and unique habitat value. However, the genetic structure of plant species within the mangrove ecosystem is poorly understood. The present communication is the first report on the use of molecular markers in assessing intra-site and intra-specific polymorphism in one of the mangrove species, Acanthus ilicifolius, for identifying/ detecting distinct genotypes for long-term conservation. Random amplified polymorphic DNAs (RAPDs) and restriction fragment length polymorphisms (RFLPs) were used to elucidate the intra- and inter-population variability in this widely distributed mangrove species. In all, 48 genotypes representing eight distinct populations were analysed. A low level of polymorphism was detected at the intra-population level through both RAPD (3.8–7.3%) and RFLP (3.2–9.1%) analyses. At the inter-population level, 25 of the 73 RAPD loci (34%) detected through the use of 13 random primers and 44 of the 96 RFLP loci (45.8%) revealed through 15 probe/enzyme combinations were polymorphic. RFLP analyses were carried out using genomic clones developed from the same species. The somatic cells of the species displayed 48 chromosomes, with no numerical changes at either intra- or inter- population levels.

A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants.

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate-glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.

Molecular phylogeny of mangroves VII. PCR-RFLP of trnS-psbC and rbcL gene regions in 24 mangrove and mangrove-associate species.

Abstract Chloroplast DNA (cpDNA) regions, trnS-psbC and rbcL, from 120 individuals of 24 mangrove and mangrove associate species belonging to 11 orders, 13 families and 17 genera of Angiospermae were amplified by the polymerase chain reaction (PCR) and restriction-digested with HaeIII. Analysis of polymorphism in the restriction fragments (PCR-RFLP) revealed 18 classes of restriction banding pattern in trnS-psbC region. This has provided molecular evidence for diversity in the mangrove floral component at the above-species level. Intra-generic variations were observed in three genera, viz. Rhizophora, Avicennia and Suaeda. Species-specific restriction patterns were found in the genera Rhizophora and Suaeda. A natural hybrid belonging to the genus Rhizophora was also analysed, and its restriction pattern was the same as that of a putative parental species.PCR-RFLP analysis of rbcL gene region was less differentiating. However, it showed 13 different classes of restriction patterns and revealed the usefulness of these investigations for genome analysis at a higher taxonomic level. Intra-specific variation was not observed in any of the species in either of the cpDNA regions analysed. This is the first report which describes variations in the chloroplast genome of mangrove species.

Molecular Phylogeny of mangroves III Parentage analysis of a Rhizophora hybrid using random amplified polymorphic DNA and restriction fragment length polymorphism markers

Abstract In this study, random amplified polymorphic DNA (RAPD) markers of genomic DNA and restriction fragment length polymorphism (RFLP) of mitochondrial DNA using a mitochondrial genome specific probe, atp 6 (from maize), were used to identify the parentage of a Rhizophora hybrid. The combined RAPD profiles of two species, Rhizophora apiculata and R. mucronata had 96.5% similarity with that of the hybrid, indicating that these two species are the probable parents. RFLPs for the mitochondrial genome were readily observed, and the RFLP profile of R. apiculata was exactly the same as that of the hybrid, establishing its maternal status for the hybrid. This is the first report on characterising the parentage of a Rhizophora hybrid using molecular markers.

Species identification in seven small millet species using polymerase chain reaction - Restriction fragment length polymorphism of trnS-psbC gene region

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCR-RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.