pmc.ncbi.nlm.nih.gov

OsDIS1-mediated stress response pathway in rice

Abstract

Ubiquitin-mediated protein degradation has been well demonstrated as a key regulatory mechanism in response to drought stress in Arabidopsis. However, the biological function of most E3 ligase genes in drought response is still unknown in rice. We recently showed that OsDIS1 (Oryza sativa drought-induced SINA protein 1), a SINA type E3 ligase, is involved in the drought-stress signal transduction in rice. OsDIS1 plays a negative role in drought stress tolerance through the transcriptional regulation of diverse stress-related genes and also possibly through the posttranslational regulation of its interacting protein OsNek6 in rice. Here we also show that OsDIS1 interacts with OsSKIPa, a drought and salt stress positive regulator in rice. Based on these results, we propose a working model for the function of OsDIS1 in regulating the stress signaling pathway in rice.

Keywords: E3 ligase, OsDIS1, rice, SINA, stress, ubiquitin

The SINA E3 Ligase OsDIS1 Negatively Regulates Drought Response in Rice

Drought is the most critical stress to crop yield and quality in many countries.1-3 With the global water shortage, it is also becoming a main constrain for rice production in major rice-producing areas.4 Although the transcriptional changes in drought-treated in rice are well studied,5-8 evidence for the involvement of E3 ligase-mediated protein degradation pathway in rice drought response is still lacking.9-13

Recently, we characterized a SINA-type E3 ligase, named OsDIS1 (Oryza sativa drought-induced SINA protein 1), which negatively regulates drought stress in rice.14 Microarray analysis revealed that a large number of drought-responsive genes are induced or suppressed in the OsDIS1 overexpression plants under normal and drought conditions. Importantly, we identified the OsDIS1 interacting protein OsNek6, a tubulin complex-related serine/threonine protein kinase in the yeast two-hybrid screening. OsDIS1 can promote OsNek6 degradation in vivo, and this ability is abolished by its mutant OsDIS1 (H71Y), which is essential for its E3 ligase activity. The function of OsNek6 in drought response is being investigated with the overexpression and RNAi silencing transgenic lines. The Nek6 gene in Arabidopsis positively regulates salt and osmotic stress response.15 It remains to be investigated whether OsNek6 also has similar functions in rice.

OsDIS1 interacts with OsSKIPa, a drought and salt stress positive regulator in rice

We found that OsSKIPa, a drought and salt positive regulator, interacts with SINA members in rice in the yeast two-hybrid screening.16 Previous research in animals showed that the C-terminal sequence of the SINA proteins regulates oligomerization and binding to target proteins,17 and the SINA members in rice contain a conserved C-terminal sequence.14 Therefore, we performed a yeast two-hybrid screen to determine whether OsSKIPa interacts with OsDIS1 in yeast. The assay showed that OsSKIPa interacted with both the wild type OsDIS1 and its mutant protein OsDIS1 (H71Y) in yeast (Fig. 1A). We also found that OsSKIPa degradation was inhibited by MG132, a 26S proteasome inhibitor, because OsSKIPa had more accumulation after the MG132 treatment than that in the control (DMSO) treatment (Fig. 1B), indicating that OsSKIPa degradation is via the 26S proteasome-dependent pathway. Whether OsDIS1 directly ubiquitinates OsSKIPa that leads its degradation remains to be resolved.

Figure 1.

Figure 1.

Interaction between OsDIS1 and OsSKIPa in yeast and MG132 inhibition of OsSKIPa degradation. (A) The full-length cDNAs of OsDIS1, OsDIS1(H71Y) were cloned into pGBKT7 while the full-length cDNA of OsSKIPa was cloned pGADT7. The derived constructs were transformed into the yeast strain HF7c. The transformed yeast cells were plated onto the SD/-Leu-Trp medium (left), SD/-Leu-Trp-His+4 mM AT medium (middle) and processed for X-gal assay (right). (B) MG132 inhibition of OsSKIPa degradation. The Agrobacterium strain carrying the GFP-OsSKIPa plasmid was infiltrated into Nicotiana benthamiana leaves, 50 μM MG132 was infiltrated into N. benthamiana 24 h before sampling with DMSO as a control. The extracted protein and RNA were used for protein gel blot and RT-PCR analysis, respectively.

Working model of the OsDIS1-mediated abiotic stress pathway in rice

Based on the results we have obtained, we proposed a working model for the function of OsDIS1 in the stress signaling pathway in rice (Fig. 2). In the rice cell, OsDIS1, a functional E3 ligase, interacts with an E2 (OsUBCx) and few substrates or partners to negatively regulates drought stress tolerance. Specifically, OsDIS1 interacts with a serine/threonine protein kinase OsNek6 and promotes its degradation via the 26S proteasome dependent pathway. In additional, OsDIS1 interacts with the drought and salt positive regulator OsSKIPa in rice. Future studies will be focused on the identification of the relationship between OsDIS1 and OsNek6 and OsSKIPa, and how this protein complex controls the drought stress tolerance in rice.

Figure 2.

Figure 2.

Working model of the OsDIS1-mediated stress pathway in rice

Acknowledgments

This project is supported by the grants from Chinese Academy of Sciences (KSCX3-EW-N-07–1), the 973 Program (2011CB915402) and NSF of China (30530400/90717006) to Q.X. and from the State Laboratory for Biology of Plant Diseases and Insect Pest to G.L.W.

Footnotes

Reference

  • 1.Boyer JS. Plant productivity and environment. Science. 1982;218:443–8. doi: 10.1126/science.218.4571.443. [DOI] [PubMed] [Google Scholar]
  • 2.Cushman JC, Bohnert HJ. Genomic approaches to plant stress tolerance. Curr Opin Plant Biol. 2000;3:117–24. doi: 10.1016/S1369-5266(99)00052-7. [DOI] [PubMed] [Google Scholar]
  • 3.Luo LJ. Breeding for water-saving and drought-resistance rice (WDR) in China. J Exp Bot. 2010;61:3509–17. doi: 10.1093/jxb/erq185. [DOI] [PubMed] [Google Scholar]
  • 4.Zhang Q. Strategies for developing Green Super Rice. Proc Natl Acad Sci USA. 2007;104:16402–9. doi: 10.1073/pnas.0708013104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Hadiarto T, Tran LS. Progress studies of drought-responsive genes in rice. Plant Cell Rep. 2011;30:297–310. doi: 10.1007/s00299-010-0956-z. [DOI] [PubMed] [Google Scholar]
  • 6.Kim YS, Kim JK. Rice transcription factor AP37 involved in grain yield increase under drought stress. Plant Signal Behav. 2009;4:735–6. doi: 10.4161/psb.4.8.9079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Nakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, Todaka D, et al. Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J. 2007;51:617–30. doi: 10.1111/j.1365-313X.2007.03168.x. [DOI] [PubMed] [Google Scholar]
  • 8.Xiang Y, Tang N, Du H, Ye H, Xiong L. Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice. Plant Physiol. 2008;148:1938–52. doi: 10.1104/pp.108.128199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Cho SK, Ryu MY, Song C, Kwak JM, Kim WT. Arabidopsis PUB22 and PUB23 are homologous U-Box E3 ubiquitin ligases that play combinatory roles in response to drought stress. Plant Cell. 2008;20:1899–914. doi: 10.1105/tpc.108.060699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Qin F, Sakuma Y, Tran LS, Maruyama K, Kidokoro S, Fujita Y, et al. Arabidopsis DREB2A-interacting proteins function as RING E3 ligases and negatively regulate plant drought stress-responsive gene expression. Plant Cell. 2008;20:1693–07. doi: 10.1105/tpc.107.057380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lee HK, Cho SK, Son O, Xu Z, Hwang I, Kim WT. Drought stress-induced Rma1H1, a RING membrane-anchor E3 ubiquitin ligase homolog, regulates aquaporin levels via ubiquitination in transgenic Arabidopsis plants. Plant Cell. 2009;21:622–41. doi: 10.1105/tpc.108.061994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ryu MY, Cho SK, Kim WT. The Arabidopsis C3H2C3-Type RING E3 Ubiquitin Ligase AtAIRP1 Is a Positive Regulator of an ABA-Dependent Response to Drought Stress. Plant Physiol. 2010;154:1983–97. doi: 10.1104/pp.110.164749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhang Y, Yang C, Li Y, Zheng N, Chen H, Zhao Q, et al. SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis. Plant Cell. 2007;19:1912–29. doi: 10.1105/tpc.106.048488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ning Y, Jantasuriyarat C, Zhao Q, Zhang H, Chen S, Liu J, et al. The SINA E3 ligase OsDIS1 Negatively Regulates Drought Response in Rice. Plant Physiol. 2011 doi: 10.1104/pp.111.180893. In press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhang B, Chen HW, Mu RL, Zhang WK, Zhao MY, Wei W, et al. NIMA-related kinase NEK6 affects plant growth and stress response in Arabidopsis. Plant J. 2011 doi: 10.1111/j.1365-313X.2011.04733.x. In press. [DOI] [PubMed] [Google Scholar]
  • 16.Hou X, Xie K, Yao J, Qi Z, Xiong L. A homolog of human ski-interacting protein in rice positively regulates cell viability and stress tolerance. Proc Natl Acad Sci USA. 2009;106:6410–5. doi: 10.1073/pnas.0901940106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hu G, Fearon ER. Siah-1 N-terminal RING domain is required for proteolysis function, and C-terminal sequences regulate oligomerization and binding to target proteins. Mol Cell Biol. 1999;19:724–32. doi: 10.1128/mcb.19.1.724. [DOI] [PMC free article] [PubMed] [Google Scholar]