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Plant Cell Reports

, Volume 35, Issue 8, pp 1687–1698 | Cite as

Wax crystal-sparse leaf 3 encoding a β-ketoacyl-CoA reductase is involved in cuticular wax biosynthesis in rice

  • Lu Gan
  • Xiaole Wang
  • Zhijun Cheng
  • Linglong Liu
  • Jiulin Wang
  • Zhe Zhang
  • Yulong Ren
  • Cailin Lei
  • Zhichao Zhao
  • Shanshan Zhu
  • Qibing Lin
  • Fuqing Wu
  • Xiuping Guo
  • Jie Wang
  • Xin Zhang
  • Jianmin WanEmail author
Original Article

Abstract

Key message

WSL3 encodes β-ketoacyl-CoA reductase (KCR) in rice, in a similar way to YBR159w in yeast, and is essential for VLCFA biosynthesis and leaf wax accumulation.

Abstract

Cuticular waxes on plant surfaces limit non-stomatal water loss, protect plants against deposits of dust and impose a physical barrier to pathogen infection. We identified a wax-deficient mutant of rice, wax crystal-sparse leaf 3 (wsl3), which exhibits a pleiotropic phenotype that includes reduced epicuticular wax crystals on the leaf surface and altered wax composition. Map-based cloning demonstrated that defects in the mutant were caused by two adjacent single-nucleotide changes in a gene encoding β-ketoacyl-CoA reductase (KCR) that catalyzes the second step of the fatty acid elongation reaction. The identity of WSL3 was further confirmed by genetic complementation. Transient assays of fluorescent protein-tagged WSL3 in tobacco protoplasts showed that WSL3 localizes to the endoplasmic reticulum, the compartment of fatty acid elongation in cells. Quantitative PCR and histochemical staining indicated that WSL3 is universally expressed in tissues. RNA interference of WSL3 caused a phenotype that mimicked the wsl3 mutant. Very long-chain fatty acids (VLCFAs) 20:0 and 22:0, or 20:1Δ11 and 22:1Δ13, were detected when WSL3 and Arabidopsis fatty acid elongation 1 (FAE1) were co-expressed in a yeast ybr159wΔ mutant strain. Our results indicated that WSL3 affects rice cuticular wax production by participating in VLCFA elongation.

Keywords

Wax crystal-sparse leaf 3 β-Ketoacyl-CoA reductase Cuticular wax Fatty acid elongase Rice (Oryza sativa

Abbreviations

BSA

Bulked segregant analysis

BSTFA

Bis-N, N-(trimethylsilyl) trifluoroacetamide

CER

Eceriferum

CoA

Coenzyme A

ECR

Enoyl-CoA reductase

ER

Endoplasmic reticulum

FAE

Fatty acid elongase

FAME

Fatty acid methyl ester

GC–MS

Gas chromatography–mass spectrometry

GFP

Green fluorescent protein

GUS

β-Glucuronidase

HCD

β-Hydroxyacyl–CoA dehydratase

InDel

Insertion and deletion

KCR

β-Ketoacyl-CoA reductase

KCS

β-Ketoacyl-CoA synthase

RNAi

RNA interference

SEM

Scanning electron microscope

SDR

Short-chain alcohol dehydrogenase reductase

TEM

Transmission electron microscopy

ORF

Open reading frame

PAS2

PASTICCINO2

qRT-PCR

Quantitative RT-PCR

UBQ

Ubiquitin

VLCFAs

Very long-chain fatty acids

WSL

Wax crystal-sparse leaf

Notes

Acknowledgments

The authors thank Dr. F. Beaudoin, Department of Biological Chemistry, Rothamsted Research, UK, for providing the yeast ybr159wΔ mutant strain. This work was supported by the National Special Project (2014ZX08009-003-003) and the National Natural Science Foundation of China (91535302, 31571629). We also acknowledge the support of the Jiangsu Collaborative Innovation Center for Modern Crop Production.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Supplementary material

299_2016_1983_MOESM1_ESM.tif (2.5 mb)
Supplementary material 1 Fig. 1. Amino acid sequence alignment of WSL3 with homologs from other species. WSL3, β-ketoacyl reductases from O. sativa (GenBank accession number NP_001053125.1); AtKCR1 from A. thaliana (GenBank accession number NP_564905.1); GL8a and GL8b from Z. mays (GenBank accession numbers, NP_001105406.1 and AAQ08990.1, respectively); GhKCR1 and GhKCR2 from G. hirsutum (GenBank accession numbers, AAY23354.1 and AAY23355.1, respectively); BnKCR1 and BnKCR2 from B. napus (GenBank accession numbers, AAO43448.1 and AAO43449.1, respectively); Ybr159w from S. cerevisiae (GenBank accession number NP_009717.1); HsKCR from human (GenBank accession number AAP36605.1); and MmKCR from mouse (GenBank accession number NP_062631.1). Putative NADH binding motif (GxxxGxGxxxAxxxAxxG), essential catalytic motif (SxxxxxxxxxxxxxxYxxxK), and dilysine ER retention motif are indicated (*). The mutant set of WSL3 is marked by an arrow. (TIFF 2544 kb)
299_2016_1983_MOESM2_ESM.docx (16 kb)
Supplementary material 2 (DOCX 16 kb)
299_2016_1983_MOESM3_ESM.xlsx (12 kb)
Supplementary material 3 (XLSX 12 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Lu Gan
    • 1
  • Xiaole Wang
    • 1
  • Zhijun Cheng
    • 1
  • Linglong Liu
    • 2
  • Jiulin Wang
    • 1
  • Zhe Zhang
    • 1
  • Yulong Ren
    • 1
  • Cailin Lei
    • 1
  • Zhichao Zhao
    • 1
  • Shanshan Zhu
    • 1
  • Qibing Lin
    • 1
  • Fuqing Wu
    • 1
  • Xiuping Guo
    • 1
  • Jie Wang
    • 1
  • Xin Zhang
    • 1
  • Jianmin Wan
    • 1
    • 2
    Email author
  1. 1.National Key Facility for Crop Gene Resources and Genetic ImprovementInstitute of Crop Science, Chinese Academy of Agricultural SciencesBeijingChina
  2. 2.National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research CenterNanjing Agricultural UniversityNanjingChina

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