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

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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.

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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

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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.

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Correspondence to Jianmin Wan.

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The authors declare that they have no conflict of interests.

Additional information

Communicated by K. Chong.

L. Gan, X. Wang, and Z. Cheng contribute equal to this work.

Electronic supplementary material

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299_2016_1983_MOESM1_ESM.tif

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)

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Gan, L., Wang, X., Cheng, Z. et al. Wax crystal-sparse leaf 3 encoding a β-ketoacyl-CoA reductase is involved in cuticular wax biosynthesis in rice. Plant Cell Rep 35, 1687–1698 (2016). https://doi.org/10.1007/s00299-016-1983-1

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