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Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes

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Abstract

Bax inhibitor-1 (BI-1) is a widely conserved cell death suppressor localized in the endoplasmic reticulum membrane. Our previous results revealed that Arabidopsis BI-1 (AtBI-1) interacts with not only Arabidopsis cytochrome b 5 (Cb5), an electron transfer protein, but also a Cb5-like domain (Cb5LD)-containing protein, Saccharomyces cerevisiae fatty acid 2-hydroxylase 1, which 2-hydroxylates sphingolipid fatty acids. We have now found that AtBI-1 binds Arabidopsis sphingolipid Δ8 long-chain base (LCB) desaturases AtSLD1 and AtSLD2, which are Cb5LD-containing proteins. The expression of both AtBI-1 and AtSLD1 was increased by cold exposure. However, different phenotypes were observed in response to cold treatment between an atbi-1 mutant and a sld1sld2 double mutant. To elucidate the reasons behind the difference, we analyzed sphingolipids and found that unsaturated LCBs in atbi-1 were not altered compared to wild type, whereas almost all LCBs in sld1sld2 were saturated, suggesting that AtBI-1 may not be necessary for the desaturation of LCBs. On the other hand, the sphingolipid content in wild type increased in response to low temperature, whereas total sphingolipid levels in atbi-1 were unaltered. In addition, the ceramide-modifying enzymes AtFAH1, sphingolipid base hydroxylase 2 (AtSBH2), acyl lipid desaturase 2 (AtADS2) and AtSLD1 were highly expressed under cold stress, and all are likely to be related to AtBI-1 function. These findings suggest that AtBI-1 contributes to synthesis of sphingolipids during cold stress by interacting with AtSLD1, AtFAH1, AtSBH2 and AtADS2.

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Abbreviations

ADS:

Acyl lipid desaturase

BI-1:

Bax inhibitor-1

BiFC:

Bimolecular fluorescence complementation

BLAST:

Basic local alignment search tool

CaMV:

Cauliflower mosaic virus

Cb5:

Cytochrome b 5

Cb5LD:

Cytochrome b 5 -like domain

Cer:

Ceramide containing non-hydroxy fatty acid

COR:

Cold-responsive

CYP83A1:

Cytochrome P450 83A1

ER:

Endoplasmic reticulum

FAD:

Flavin adenine dinucleotide

FAH:

Fatty acid 2-hydroxylase

FOA:

Fluoroorotic acid

GCS:

Glucosylceramide synthase

GIPC:

Glycosylinositolphosphoceramide

GlcCer:

Glucosylceramide

hCer:

Ceramide containing 2-hydroxy fatty acid

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

Moco:

Molybdenum-pterin cofactor

MRM:

Multiple-reaction monitoring

MS:

Murashige and Skoog

NIA:

Nitrate reductase

PCD:

Programmed cell death

RLF:

Reduced lateral root formation

RT-PCR:

Reverse transcription polymerase chain reaction

SBH:

Sphingolipid base hydroxylase

SLD:

Sphingolipid Δ8 desaturase

suY2H:

Split-ubiquitin yeast two-hybrid

VLCFA:

Very long chain fatty acid

WT:

Wild type

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Acknowledgments

Plasmids and strains used for the suY2H system were generously provided by Dr. Ralph Panstruga (Max-Planck Institute, Saabruecken, Germany) and Dr. Imre E. Somssich (Max-Planck Institute). Plasmids used for the BiFC assay were kindly provided by Dr. Tsuyoshi Nakagawa (Shimane University, Japan). We appreciate Dr. Noriko Inada (Nara Institute of Science and Technology, Japan) for technical advice of confocal laser microscopy and FRET analysis. We are grateful to Dr. Ikuo Nishida (Saitama University, Japan) for cold stress analysis and to Dr. Hiroyuki Imai (Konan University, Japan) for lipid analysis. This work was supported by a Grant from the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for Next Generation World-Leading Researchers (NEXT program, to M.K.-Y.),” initiated by the Council for Science and Technology Policy (CSTP), a Grant-in-Aid for JSPS Fellows (to M.N. and T.I.), and a Grant from the Ministry of Agriculture, Forestry and Fishery, Japan (Genomics for Agricultural Innovation, IPG-0014). Minoru Nagano and Toshiki Ishikawa contributed equally to this work.

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Correspondence to Maki Kawai-Yamada.

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Dedicated to K. Shimamoto who passed away on September 28, 2013.

M. Nagano and T. Ishikawa contributed equally to this work.

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Nagano, M., Ishikawa, T., Ogawa, Y. et al. Arabidopsis Bax inhibitor-1 promotes sphingolipid synthesis during cold stress by interacting with ceramide-modifying enzymes. Planta 240, 77–89 (2014). https://doi.org/10.1007/s00425-014-2065-7

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