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Discovery of E3 Ubiquitin Ligases That Alter Responses to Nitrogen Deficiency Using Rice Full-Length cDNA OvereXpressor (FOX)-Hunting System

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Abstract

Nitrogen is the most critical nutrient for plant growth. To find potential strategies for enhancing both nitrogen use and tolerance to nitrogen deficiency in rice plants, we used the rice Full-length-cDNA OvereXpressor (FOX)-hunting system, a high-throughput phenotyping screen. After screening 3229 rice FOX lines, we identified 82 FOX-hunting lines that responded differently to nitrogen starvation. Among them, 11 FOX-hunting lines overexpressed putative E3 ligases, of which 6 were RING-type and 5 were F-box type E3 ligases. Of these, two lines overexpressed the same F-box type E3 ligase, OsFBL15. In vitro ubiquitination assay confirmed the auto-ubiquitination activity of OsFBL15. The overexpression of these E3 ligases altered the rice response to nitrogen deficiency and suggests a way to develop rice that is tolerant to nitrogen-deficient field conditions.

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Abbreviations

AMT:

AMmonium Transporter

FOX:

Full-length cDNA OvereXpressor

NRT:

NitRate Transporter

RING:

Really Interesting New Gene

−N:

Nitrogen deficiency

+N:

Nitrogen sufficiency

N:

Nitrogen

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Acknowledgements

We thank Mr. Tsuzumi Mito for taking care of rice plants, and Ms. Keiko Iida-Okada and Ms. Etsuko Sugai for their subdivision work of FOX-rice seeds. This work was supported by a grant from the Japan Society for Promotion of Science bilateral joint program to R.S. and under the framework of the International Cooperation Program (2015K2A2A4000129) managed by the National Research Foundation of Korea to C.-J.P. The production of FOX-rice lines was supported by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project EF-1004 and Genomics for Agricultural Innovation, AMR-0001) and a funding for the biological resources from National Institute of Agrobiological Sciences (Tsukuba, Japan) to H.I.

Author information

Author notes

  1. Makoto Hakata

    Present address: Lowland Farming Research Division, NARO Kyushu Okinawa Agricultural Research Center, Izumi 496, Chikugo, Fukuoka, 833-0041, Japan

  2. Hidemitsu Nakamura

    Present address: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

  3. Hiroaki Ichikawa

    Present address: Division of Applied Genetics, NARO Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602, Japan

  4. H.T. and J.-P.H. contributed equally to this work.

Authors and Affiliations

  1. RIKEN Center for Sustainable Resource Science, 1-7-22 Shuehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan

    Hajime Takiguchi, Jong-Pil Hong, Hidetoshi Nishiyama & Ryoung Shin

  2. Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8602, Japan

    Makoto Hakata, Hidemitsu Nakamura & Hiroaki Ichikawa

  3. Department of Bioresources Engineering and PERI, Sejong University, Seoul, 05006, Republic of Korea

    Chang-Jin Park

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  1. Hajime Takiguchi
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Correspondence to Ryoung Shin.

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Electronic supplementary material

Fig. S1

Representative FOX-hunting lines selected during the primary screening in nitrogen deficient condition (-N). The left side of each photo is Nipponbare control plants (WT) grown in -N. The right side of each photo is a tested FOX line. AD157 (A), AR013 (B), BI246 (C) and BM107 (D) show a tolerance phenotype to nitrogen deficiency, and AY004 (E) and BM137 (F) show a more susceptible phenotype to nitrogen deficiency condition compared to the control. The selected lines were transferred to soil for phenotypic analysis and seed production (DOCX 685 kb)

Fig. S2

The deduced amino acid sequence of OsFBL15 protein. The grey highlight indicates a predicted F-box motif. The lines indicate five leucine-rich repeats, which are numbered in superscript numbers (DOCX 226 kb)

Fig. S3

In vitro auto-ubiquitination assay for OsFBL15. Recombinant GST-OsFBL15 and GST proteins were incubated with ubiquitin and rice total protein extracts for 0 or 2 hours at 37 ºC. The total protein was prepared from rice plants grown in -N conditions for 7 days. Reaction mixtures were separated on 8% SDS-PAGE gel and the blotted proteins were detected with anti-Ub antibodies. The red line indicates the ubiquitinated protein (line) (DOCX 144 kb)

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Takiguchi, H., Hong, JP., Nishiyama, H. et al. Discovery of E3 Ubiquitin Ligases That Alter Responses to Nitrogen Deficiency Using Rice Full-Length cDNA OvereXpressor (FOX)-Hunting System. Plant Mol Biol Rep 35, 343–354 (2017). https://doi.org/10.1007/s11105-017-1027-1

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