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UV‐B activates a ‘group A’ mitogen activated protein kinase in Oryza sativa

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Abstract

Increased level of Ultra violet-B radiation at earth’s surface has several deleterious consequences for plants and ecosystems. Higher UV-B level affects crop plants in several ways and also gives penalty in terms of crop yield. With changing climatic conditions it is crucial to elucidate signal transduction pathway involved in UV-B stress. Here, involvement of mitogen activated protein kinases (MAPKs) in UV-B stress was investigated in rice (Oryza sativa). Transcripts profiling of all 15 rice MAPKs have shown UV-B induced expressions of a few MAPK genes including OsMPK3, OsMPK6, OsMPK4, OsMPK17-1, OsMPK17-2, OsMPK20-1, OsMPK20-4 and OsMPK20-5. In-gel kinase assay as well as immuno-kinase assay showed activation of single MAPK of size ~45 kDa in response to UV-B treatment. Further UV-B responsive activity of rice MAPKs, OsMPK3, OsMPK4 and OsMPK6 was checked using antibodies for the respective orthologs in Arabidopsis which indicated activation of OsMPK3 in UV-B stress. Additionally, GST: OsMPK3 protein showed UV responsive phosphorylation when incubated with crude protein extract from UV exposed plants in an in-vitro phosphorylation assay. These results indicate activation of OsMPK3 in UV-B stress. Further, possible involvement of UV-B responsive OsWRKY89 downstream of MAPK cascade was studied. It was observed that staurosporin and other specific chemical inhibitors of MAPK could attenuate UV-B induced expression of OsWRKY89. OsMPK3 was also found to interact with OsWRKY89 in yeast two-hybrid assay, although the interaction was weak in nature. The present work gives an account of MAPKs in UV-B stress in rice.

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Abbreviations

UV-B:

Ultra Violet B

MAPK:

Mitogen Activated Protein Kinase

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Acknowledgments

DPW, KPR thank University Grant Commission, India and MJ, SKR, PS thank Council of Scientific and Industrial Research, India for fellowships. This work is supported by core grant of National Institute of Plant Genome Research, New Delhi, India from Department of Biotechnology, India.

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Authors and Affiliations

  1. National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India

    Dhammaprakash Pandhari Wankhede, Pallavi Singh, Monika Jaggi, Kudupudi Prabhakara Rao, Susheel Kumar Raina & Alok Krishna Sinha

  2. ICAR-National Bureau of Plant Genetic Resources, New Delhi, India

    Dhammaprakash Pandhari Wankhede

  3. ICAR-Central Tobacco Research Institute, Bhaskarnagar, Rajahmundry, Andhra Pradesh, India

    Kudupudi Prabhakara Rao

  4. ICAR-National Institute of Abiotic Stress Management, Malegaon (Kh), Baramati, India

    Susheel Kumar Raina

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  1. Dhammaprakash Pandhari Wankhede
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  2. Pallavi Singh
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Correspondence to Dhammaprakash Pandhari Wankhede.

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Supplementary Fig. 1

Expression pattern of rice MAPKs in response to UV-B as shown by sqRT-PCR. Expression of rice Actin was used as a loading control. (PPT 395 kb)

Supplementary Fig. 2

Specificity of AtMPK3 antibody against rice OsMPK3. AtMPK3 antibody was used against crude protein extract of rice leaves, bacterially expressed and purified GST-OsMPK3 and GST alone. Left panel shows the immunoblot whereas right one shows ponceau stained blot of the same. (PPT 216 kb)

Supplementary Fig. 3

OsWRKY89 fused with GAL4 DNA-binding domain (pGBKT7 vector) showing auto-activation of reporter genes. (PPT 137 kb)

Supplementary Fig. 4

In-silico predictions of kinase specific protein phosphoylation sites in OsWRKY89. Amino acids sequence of OsWRKY89 was given as input following default parameters at NetPhosK 1.0 server (http://www.cbs.dtu.dk/services/NetPhosK/). Blue coloured, symbol ‘X’ denotes MAPK specific phosphorylation site. Kinase Landscapes (Graphics) methods was used to view output results. (PPT 331 kb)

Supplementary Fig. 5

In-vitro phosphorylation of GST-OsWRKY89 by GST-OsMPK3. GST-OsWRKY89 was incubated with (+) or without (−) GST-OsMPK3 alone or with GST-OsMKK6WT or GST-OsMKK6EE in the kinase reaction buffer and radiolabelled ATP. After stopping the reaction the proteins were separated on SDS-PAGE and autoradiographed. (PPT 510 kb)

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Wankhede, D.P., Singh, P., Jaggi, M. et al. UV‐B activates a ‘group A’ mitogen activated protein kinase in Oryza sativa . J. Plant Biochem. Biotechnol. 25, 392–399 (2016). https://doi.org/10.1007/s13562-016-0351-0

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