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The SbASR-1 Gene Cloned from an Extreme Halophyte Salicornia brachiata Enhances Salt Tolerance in Transgenic Tobacco

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Abstract

Salinity severely affects plant growth and development. Plants evolved various mechanisms to cope up stress both at molecular and cellular levels. Halophytes have developed better mechanism to alleviate the salt stress than glycophytes, and therefore, it is advantageous to study the role of different genes from halophytes. Salicornia brachiata is an extreme halophyte, which grows luxuriantly in the salty marshes in the coastal areas. Earlier, we have isolated SbASR-1 (abscisic acid stress ripening-1) gene from S. brachiata using cDNA subtractive hybridisation library. ASR-1 genes are abscisic acid (ABA) responsive, whose expression level increases under abiotic stresses, injury, during fruit ripening and in pollen grains. The SbASR-1 transcript showed up-regulation under salt stress conditions. The SbASR-1 protein contains 202 amino acids of 21.01-kDa molecular mass and has 79 amino acid long signatures of ABA/WDS gene family. It has a maximum identity (73 %) with Solanum chilense ASR-1 protein. The SbASR-1 has a large number of disorder-promoting amino acids, which make it an intrinsically disordered protein. The SbASR-1 gene was over-expressed under CaMV 35S promoter in tobacco plant to study its physiological functions under salt stress. T0 transgenic tobacco seeds showed better germination and seedling growth as compared to wild type (Wt) in a salt stress condition. In the leaf tissues of transgenic lines, Na+ and proline contents were significantly lower, as compared to Wt plant, under salt treatment, suggesting that transgenic plants are better adapted to salt stress.

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Acknowledgments

The authors are thankful to the Council of Scientific and Industrial Research, New Delhi (NWP-020), for the financial assistance. VT is thankful to CSIR for the Junior Research Fellowship.

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  1. Discipline of Marine Biotechnology and Ecology, CSIR- Central Salt and Marine Chemicals Research Institute, G.B. Road, Bhavnagar, 364 002, Gujarat, India

    Bhavanath Jha, Sanjay Lal, Vivekanand Tiwari, Sweta Kumari Yadav & Pradeep K. Agarwal

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  1. Bhavanath Jha
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  2. Sanjay Lal
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  3. Vivekanand Tiwari
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  4. Sweta Kumari Yadav
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  5. Pradeep K. Agarwal
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Correspondence to Bhavanath Jha or Pradeep K. Agarwal.

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Supplementary Fig. 1

The phylogenetic tress was prepared using protein accession number, viz. AAB96681 (Oryza sativa), ACI15208 (Salicornia brachiata), ACZ60128 (Musa acuminate), AAA82741 (Citrus maxima), BAI94530 (Prunus mume), AAL26889 (Prunus persica), AAB97140 (Prunus armeniaca), AAP46155 (Hevea brasiliensis), AAA34137 (Solanum lycopersicum), AAY97998 (Solanum lycopersicum), AAZ93634 (Vitis vinifera), AAA21866 (Zea mays), AAB64185 (Solanum lycopersicum), AAM51877 (Lilium longiflorum), AAD00254 (Solanum tuberosum), AAL27560 (Cucumis melo), XP_002524296 (Ricinus communis), AAY97997 (Solanum cheesmaniae), AAY98001 (S. chilense), AAY98002 (Solanum corneliomuelleri), AAY98000 (Lycopersicon peruvianum), AAY97999 (Solanum habrochaites), AAR23420 (Ginkgo biloba), ABC86744 (Vitis pseudoreticulata), ACL68147 (Musa acuminata), AAB02692 (Pinus taeda) and BT114797 (Picea glauca). AAB96796 (Solanum lycopersicum ER5) was used as an out-group to make the root of the tree. The evolutionary history of ASR-1 amino acid sequences was inferred by using the maximum likelihood method based on the JTT matrix-based model using MEGA version 5 software. The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the taxa analysed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 28 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 222 positions in the final data set (JPEG 70 kb)

High resolution image (TIFF 62 kb)

Supplementary Fig. 2

Multiple alignment of ASR-1 amino acid sequences was carried out using the sequences as in Supplementary Fig. 1 by online ClustalW program. Conserved WDS/ABA domain annotated by query against InterPro is underlined. Other conserved domains are shaded in different colours and shown by double-sided solid arrows. A N-terminal His-rich domain is highlighted with yellow colour but absent in SbASR-1 and underlined. B Zn2+-dependent DNA-binding domain of tomato ASR-1 (Rom et al. 2006). C Hydrophobic tomato ASR-1 sequence hindering DNA binding (Rom et al. 2006). D and E C-terminal-conserved NLS sequence found in two K-rich amino acid clusters separated by non-conserved 11 amino acid residues in lily LLA23 (Wang et al. 2005) while by 16 amino acid residues in SbASR-1 (DOC 43 kb)

Supplementary Fig. 3

Secondary structure of SbASR-1 predicted by ExPasy tools shows five coils and four helix loops (JPEG 111 kb)

High resolution image (TIFF 258 kb)

Supplementary Table 1

Comparative analysis of SbASR-1 and other glycophytic ASR-1 proteins showed higher percentage of disorder-promoting amino acid residues and N-myristoylation site (DOC 44.0 kb)

Supplementary Table 2

Prediction of N-glycosylation sites, protein kinase C phosphorylation sites, tyrosine kinase phosphorylation sites and N-myristoylation sites by PROSCAN.BASE server (JPEG 67 kb)

High resolution image (TIFF 59 kb)

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Jha, B., Lal, S., Tiwari, V. et al. The SbASR-1 Gene Cloned from an Extreme Halophyte Salicornia brachiata Enhances Salt Tolerance in Transgenic Tobacco. Mar Biotechnol 14, 782–792 (2012). https://doi.org/10.1007/s10126-012-9442-7

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