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.
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
References
Aggarwal C, Banas AK, Kasprowicz-Maluski A, Borghetti C, Labuz J, Dobrucki J, Gabrys H (2014) Blue-light-activated phototropin2 trafficking from the cytoplasm to Golgi/post-Golgi vesicles. J Exp Bot 65:3263–3276
Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16:2463–2480
Bach L, Michaelson LV, Haslam R, Bellec Y, Gissot L, Marion J, Da CM, Boutin JP, Miquel M, Tellier F, Domergue F, Markham JE, Beaudoin F, Napier JA, Faure JD (2008) The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development. Proc Natl Acad Sci USA 105:14727–14731
Bach L, Gissot L, Marion J, Tellier F, Moreau P, Satiat-Jeunemaitre B, Palauqui JC, Napier JA, Faure JD (2011) Very-long-chain fatty acids are required for cell plate formation during cytokinesis in Arabidopsis thaliana. J Cell Sci 124:3223–3234
Batoko H, Zheng HQ, Hawes C, Moore I (2000) A rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for normal golgi movement in plants. Plant Cell 12:2201–2218
Beaudoin F, Michaelson LV, Hey SJ, Lewis MJ, Shewry PR, Sayanova O, Napier JA (2000) Heterologous reconstitution in yeast of the polyunsaturated fatty acid biosynthetic pathway. Proc Natl Acad Sci USA 97:6421–6426
Beaudoin F, Gable K, Sayanova O, Dunn T, Napier JA (2002) A Saccharomyces cerevisiae gene required for heterologous fatty acid elongase activity encodes a microsomal β-keto-reductase. J Biol Chem 277:11481–11488
Beaudoin F, Wu X, Li F, Haslam RP, Markham JE, Zheng H, Napier JA, Kunst L (2009) Functional characterization of the Arabidopsis β-ketoacyl-coenzyme A reductase candidates of the fatty acid elongase. Plant Physiol 150:1174–1191
Bottanelli F, Gershlick DC, Denecke J (2012) Evidence for sequential action of Rab5 and Rab7 GTPases in prevacuolar organelle partitioning. Traffic 13:338–354
Broun P, Poindexter P, Osborne E, Jiang CZ, Riechmann JL (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci USA 101:4706–4711
Dietrich CR, Perera MA, Yandeau-Nelson D, Meeley RB, Nikolau BJ, Schnable PS (2005) Characterization of two GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development. Plant J 42:844–861
Espana L, Heredia-Guerrero JA, Reina-Pinto JJ, Fernandez-Munoz R, Heredia A, Dominguez E (2014) Transient silencing of CHALCONE SYNTHASE during fruit ripening modifies tomato epidermal cells and cuticle properties. Plant Physiol 166:1371–1386
Gallavotti A, Whipple CJ (2015) Positional cloning in maize (Zea mays subsp. mays, Poaceae). Appl. Plant Sci 3:1400092
Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34
Hiei Y, Komari T (2008) Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3:824–834
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405
Jetter R, Schaffer S (2001) Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development. Plant Physiol 126:1725–1737
Jung JH, Kim H, Go YS, Lee SB, Hur CG, Kim HU, Suh MC (2011) Identification of functional BrFAD2-1 gene encoding microsomal delta-12 fatty acid desaturase from Brassica rapa and development of Brassica napus containing high oleic acid contents. Plant Cell Rep 30:1881–1892
Kim J, Jung JH, Lee SB, Go YS, Kim HJ, Cahoon R, Markham JE, Cahoon EB, Suh MC (2013) Arabidopsis 3-ketoacyl-coenzyme a synthase9 is involved in the synthesis of tetracosanoic acids as precursors of cuticular waxes, suberins, sphingolipids, and phospholipids. Plant Physiol 162:567–580
Kunst L, Samuels L (2009) Plant cuticles shine: advances in wax biosynthesis and export. Curr Opin Plant Biol 12:721–727
Lee SB, Suh MC (2013) Recent advances in cuticular wax biosynthesis and its regulation in Arabidopsis. Mol Plant 6:246–249
Lee SB, Jung SJ, Go YS, Kim HU, Kim JK, Cho HJ, Park OK, Suh MC (2009) Two Arabidopsis 3-ketoacyl CoA synthase genes, KCS20 and KCS2/DAISY, are functionally redundant in cuticular wax and root suberin biosynthesis, but differentially controlled by osmotic stress. Plant J 60:462–475
Li H, Jiang L, Youn JH, Sun W, Cheng Z, Jin T, Ma X, Guo X, Wang J, Zhang X, Wu F, Wu C, Kim SK, Wan J (2013) A comprehensive genetic study reveals a crucial role of CYP90D2/D2 in regulating plant architecture in rice (Oryza sativa). New Phytol 200:1076–1088
Mao B, Cheng Z, Lei C, Xu F, Gao S, Ren Y, Wang J, Zhang X, Wang J, Wu F, Guo X, Liu X, Wu C, Wang H, Wan J (2012) Wax crystal-sparse leaf2, a rice homologue of WAX2/GL1, is involved in synthesis of leaf cuticular wax. Planta 235:39–52
Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L (1999) CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11:825–838
Panikashvili D, Savaldi-Goldstein S, Mandel T, Yifhar T, Franke RB, Hofer R, Schreiber L, Chory J, Aharoni A (2007) The Arabidopsis DESPERADO/AtWBC11 transporter is required for cutin and wax secretion. Plant Physiol 145:1345–1360
Qin YM, Pujol FM, Shi YH, Feng JX, Liu YM, Kastaniotis AJ, Hiltunen JK, Zhu YX (2005) Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers. Cell Res 15:465–473
Qin BX, Tang D, Huang J, Li M, Wu XR, Lu LL, Wang KJ, Yu HX, Chen JM, Gu MH, Cheng ZK (2011) Rice OsGL1-1 is involved in leaf cuticular wax and cuticle membrane. Mol Plant 4:985–995
Raffaele S, Leger A, Roby D (2009) Very long chain fatty acid and lipid signaling in the response of plants to pathogens. Plant Signal Behav 4:94–99
Rao X, Huang X, Zhou Z, Lin X (2013) An improvement of the 2^(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath 3:71–85
Sieber P, Schorderet M, Ryser U, Buchala A, Kolattukudy P, Metraux JP, Nawrath C (2000) Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions. Plant Cell 12:721–738
Sturaro M, Hartings H, Schmelzer E, Velasco R, Salamini F, Motto M (2005) Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production. Plant Physiol 138:478–489
Tel-Zur N, Abbo S, Myslabodski D, Mizrahi Y (1999) Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Mol Biol Rep 17:249–254
Todd J, Post-Beittenmiller D, Jaworski JG (1999) KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. Plant J 17:119–130
Weidenbach D, Jansen M, Franke RB, Hensel G, Weissgerber W, Ulferts S, Jansen I, Schreiber L, Korzun V, Pontzen R, Kumlehn J, Pillen K, Schaffrath U (2014) Evolutionary conserved function of barley and Arabidopsis 3-KETOACYL-CoA SYNTHASES in providing wax signals for germination of powdery mildew fungi. Plant Physiol 166:1621–1633
Yu D, Ranathunge K, Huang H, Pei Z, Franke R, Schreiber L, He C (2008) Wax Crystal-Sparse Leaf1 encodes a β–ketoacyl CoA synthase involved in biosynthesis of cuticular waxes on rice leaf. Planta 228:675–685
Zheng H, Rowland O, Kunst L (2005) Disruptions of the Arabidopsis Enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell 17:1467–1481
Zhou L, Ni E, Yang J, Zhou H, Liang H, Li J, Jiang D, Wang Z, Liu Z, Zhuang C (2013) Rice OsGL1-6 is involved in leaf cuticular wax accumulation and drought resistance. PLoS One 8:e65139
Zhou X, Li L, Xiang J, Gao G, Xu F, Liu A, Zhang X, Peng Y, Chen X, Wan X (2015) OsGL1-3 is involved in cuticular wax biosynthesis and tolerance to water deficit in rice. PLoS One 10:e116676
Zhu X, Xiong L (2013) Putative megaenzyme DWA1 plays essential roles in drought resistance by regulating stress-induced wax deposition in rice. Proc Natl Acad Sci USA 110:17790–177905
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
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
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-016-1983-1