4.7 Article

A novel class I Chitinase from Hippophae rhamnoides: Indications for participating in ICE-CBF cold stress signaling pathway

Journal

PLANT SCIENCE
Volume 259, Issue -, Pages 62-70

Publisher

ELSEVIER IRELAND LTD
DOI: 10.1016/j.plantsci.2017.03.004

Keywords

Chitinase; 3-D structure; Meta; SA; Cold stress; CBF dependent pathway

Funding

  1. Department of Biotechnology, Government of India [BT/PR10799/NDB/51/171/2008]
  2. University of Delhi

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Plant chitinases are the members of PR (Pathogenesis related) proteins family and protect plants from biotic and abiotic stress. A novel chitinase HrCHI1 (Accession number JQ289153) of 954 by ORF encoding 317 amino acids protein was cloned, expressed and characterized from seabuckthorn, a cold/freeze tolerant shrub. The 3D structure (predicted with I-TASSER server) showed highest homology with Oryza sativa class I chitinase (PDB 2dkvA). Putative promoter region (obtained by genome walking) showed GCC box, E-boxes, the binding site for bHLH proteins and DRE elements, the CBF (C-repeat binding factor) binding site besides TATA and CAAT boxes. The gel shift assay with the nuclear extract indicated that the HrCHI1 might be participating in CBF/ERF dependent cold stress signaling pathway. The quantitative transcript profiling supported this observation as cold induced expression of HrCBF peaked earlier (at 1 h) while HrCHI1 peaked latter (after 3 h) indicating HrCHI1 expression might be induced by HrCBF. Further, HrCHI1 expression was methyl jasmonate (Meta) dependent and salicylic acid(SA) independent. HrCHI1 was expressed in E. coil and purified using chitin affinity chromatography. It showed 512 U/mg chitinase hydrolytic activity and resolved as a 34 kDa spot with a slightly basic pI (8.5) on a 2-D gel. The E. coli cells containing recombinant chitinase showed higher rate of growth in cold in comparison with the cells containing the empty vector. In conclusion, we have isolated and characterized a cold responsive basic class I chitinase which is regulated by MeJa and seems to be functioning via CBF/ERF dependent cold stress signaling pathway. (C) 2017 Elsevier B.V. All rights reserved.

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