Showing papers by "Narendra Tuteja published in 2005"

Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield

Abstract: Salt tolerance is an important trait that is required to overcome salinity-induced reduction in plant productivity. We have reported previously the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with the eukaryotic translation initiation factor eIF-4A. Here, we report that PDH45 mRNA is induced in pea seedlings in response to high salt, and its overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants confers salinity tolerance, thus suggesting a previously undescribed pathway for manipulating stress tolerance in crop plants. The T0 transgenic plants showed high levels of PDH45 protein in normal and stress conditions, as compared with WT plants. The T0 transgenics also showed tolerance to high salinity as tested by a leaf disk senescence assay. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress without any reduction in plant yield in terms of seed weight. Measurement of Na+ ions in different parts of the plant showed higher accumulation in the old leaves and negligible accumulation in seeds of T1 transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance is discussed. Overexpression of PDH45 provides a possible example of the exploitation of DNA/RNA unwinding pathways for engineering salinity tolerance without affecting yield in crop plants.

Cold- and salinity stress-induced bipolar pea DNA helicase 47 is involved in protein synthesis and stimulated by phosphorylation with protein kinase C.

Abstract: Helicases are involved in the metabolism of nucleic acid; this is very sensitive to the abiotic stresses that reduce plant growth and productivity. However, the molecular targets responsible for this sensitivity have not been well studied. Here we report on the isolation and characterization of cold- and salinity stress-induced pea DNA helicase 47 (PDH47). The transcript of PDH47 was induced in both shoots and roots under cold (4 degrees C) and salinity (300 mm NaCl) stress, but there was no change in response to drought stress. Tissue-specific differential regulation was observed under heat (37 degrees C) stress. ABA treatment did not alter expression of PDH47 in shoots but induced its mRNA in roots, indicating a role for PDH47 in both the ABA-independent and ABA-dependent pathways in abiotic stress. The purified recombinant protein (47 kDa) contains ATP-dependent DNA and RNA helicase and DNA-dependent ATPase activities. With the help of photoaffinity labeling, PDH47 was labeled by [alpha-32P]-ATP. PDH47 is a unique bipolar helicase that contains both 3' to 5' and 5' to 3' directional helicase activities. Anti-PDH47 antibodies immunodeplete the activities of PDH47 and inhibit in vitro translation of protein. Furthermore, the PDH47 protein showed upregulation of protein synthesis. The activities of PDH47 are stimulated after phosphorylation by protein kinase C at Ser and Thr residues. Western blot analysis and in vivo immunostaining, followed by confocal microscopy, showed PDH47 to be localized in both the nucleus and cytosol. The discovery of cold- and salinity stress-induced DNA helicase should make an important contribution to a better understanding of DNA metabolism and stress signaling in plants. Its bipolar helicase activities may also be involved in distinct cellular processes in stressed conditions.

Structural and mechanistic aspect of DEAD-box helicases

Abstract: DFAD-box helicases are ubiquitous enzymes that catalyze the unwinding of energetically stable duplex DNA (DNA helicases) or duplex RNA secondary structures (RNA helicases). DNA helicases are involved in replication, repair, recombination, and transcription, while the DEAD-box RNA helicases are involved in modulation of RNA structure and thereby influence RNA synthesis, splicing, replication, translation initiation, editing, rRNA processing, ribosome assembly, nuclear mRNA export, mRNA stabilization and degradation. They are associated with intrinsic ssDNA-dependent or RNA-dependent ATPase activities, which provide the energy for the helicase action. Mechanistically, there are two classes of helicases, those that can translocate 3'- to 5'-, or 5' to 3' directions with respect to the strand on which they initially bind. A hallmark of most of the helicases (but not for all) is the existence of a set of nine highly conserved amino acid sequences called 'helicase motifs', which are clustered together for helicase function. One of the important helicase motifs is DEAD (Asp-Glu-Ala-Asp) that is why the family of these proteins is called DEAD-box helicases. PcrA helicase from Bacillius stearothermophilus was the first helicase whose crystal structure was solved in 1996 by an Indian scientist H. S. Subramanya in Dale B. Wigley's group at University of Oxford, UK. After this several DEAD-box helicases have been crystallized and suggested a common structural fold for their function. Furthermore, the information on the function of conserved helicase motifs and mechanism of DNA or RNA unwinding is also emerging. In this article we have covered possibly all the helicases whose structure have been solved and the possible mechanisms of unwinding.