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. 2012 Aug;159(4):1385-95.
doi: 10.1104/pp.112.199646. Epub 2012 Jun 11.

RDR1 and SGS3, components of RNA-mediated gene silencing, are required for the regulation of cuticular wax biosynthesis in developing inflorescence stems of Arabidopsis

Patricia Lam  1 , Lifang Zhao, Heather E McFarlane, Mytyl Aiga, Vivian Lam, Tanya S Hooker, Ljerka Kunst
Affiliations

RDR1 and SGS3, components of RNA-mediated gene silencing, are required for the regulation of cuticular wax biosynthesis in developing inflorescence stems of Arabidopsis

Patricia Lam et al. Plant Physiol. 2012 Aug.

Abstract

The cuticle is a protective layer that coats the primary aerial surfaces of land plants and mediates plant interactions with the environment. It is synthesized by epidermal cells and is composed of a cutin polyester matrix that is embedded and covered with cuticular waxes. Recently, we have discovered a novel regulatory mechanism of cuticular wax biosynthesis that involves the ECERIFERUM7 (CER7) ribonuclease, a core subunit of the exosome. We hypothesized that at the onset of wax production, the CER7 ribonuclease degrades an mRNA specifying a repressor of CER3, a wax biosynthetic gene whose protein product is required for wax formation via the decarbonylation pathway. In the absence of this repressor, CER3 is expressed, leading to wax production. To identify the putative repressor of CER3 and to unravel the mechanism of CER7-mediated regulation of wax production, we performed a screen for suppressors of the cer7 mutant. Our screen resulted in the isolation of components of the RNA-silencing machinery, RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, implicating RNA silencing in the control of cuticular wax deposition during inflorescence stem development in Arabidopsis (Arabidopsis thaliana).

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Figures

Figure 1.
Figure 1.
CER3, under the control of the CER6 promoter, can complement cer7-3. A, Stems of 5-week-old wild type (WT; Columbia-0), cer7-3, and cer7-3 transformed with the ProCER6:CER3 transgene showing restored wax in the transgenic plant. B, Quantitative RT-PCR showing that CER3 expression levels are restored to wild-type levels in plants carrying the ProCER6:CER3 transgene. ACTIN2 was used as an internal control, and control samples were normalized to 1. Values represent means ± sd (n = 4). Statistically significant differences from cer7-3 (P < 0.05) are indicated by asterisks.
Figure 2.
Figure 2.
Summary of the suppressor screen.
Figure 3.
Figure 3.
Analysis of war mutants. A, Stems of 6-week-old wild type (WT; Ler), cer7-1, and four war mutant plants showing the suppression of the cer7-1 wax-deficient phenotype in the war mutants as indicated by glaucous stems. B, Stem wax loads of war1 to war4 compared with the wild type and cer7-1. Values represent means ± sd (n = 3). Statistically significant differences between samples (P < 0.05) are indicated by asterisks. C, Stem wax composition of war1 to war4 compared with the wild type and cer7-1. Wax compositions for all war mutants are restored to near wild-type-like ratios of major wax components. D, Quantitative RT-PCR showing that CER3 transcript levels are restored to wild-type levels in the war mutants. ACTIN2 was used as an internal control, and control samples were normalized to 1. Values represent means ± sd (n = 4). Statistically significant differences between samples (P < 0.05) are indicated by asterisks.
Figure 4.
Figure 4.
Positional cloning of war3 and war4, and RDR1 and SGS3 gene structures. A, Schematic representation of the chromosomal location of war3 as determined by fine-mapping. The markers used for mapping and the number of recombinants are indicated. B, Schematic representation of the RDR1 gene structure. The 5′ and 3′ untranslated regions are indicated as gray boxes, exons as white boxes, and introns as black lines. The translational start site is represented by the bent arrow. The positions and types of the mutations in rdr1 mutant alleles are also shown. C, Schematic representation of the chromosomal location of war4 as determined by fine-mapping. The markers used for mapping and the number of recombinants are indicated. D, Schematic representation of the SGS3 gene structure and the positions and types of mutations in sgs3 alleles. The 5′ and 3′ untranslated regions are indicated as gray boxes, exons as white boxes, and introns as black lines. The translational start site is represented by the bent arrow.
Figure 5.
Figure 5.
Expression analysis of RDR1 and SGS3 in different organs and tissues of wild-type Arabidopsis (Columbia-0) as determined by quantitative RT-PCR. ACTIN2 was used as an internal control, and control samples were normalized to 1. Values represent means ± sd (n = 4).
Figure 6.
Figure 6.
Expression of RDR1 and SGS3. A and B, Tissue-specific expression of ProRDR1:GUS and ProSGS3:GUS in Arabidopsis stems. Stems of 4-week-old transgenic plants expressing ProRDR1:GUS (A) and ProSGS3:GUS (B) were stained for GUS activity. Cross-sections from the top 3 cm of the stem are shown. Bar = 0.1 mm. C and D, Localization of SGS3 by confocal microscopy. In stems, SGS3:YFP is localized to the ER (C). In leaves, SGS3:YFP is localized to the cytoplasm and to punctae (D). Images are Z-projections of confocal stacks. Bars = 10 µm.
Figure 7.
Figure 7.
CER3 expression levels in the top 3 cm and the bottom 3 cm of a 10-cm stem as measured by quantitative RT-PCR. ACTIN2 was used as an internal control, and control samples were normalized to 1. Values represent means ± sd (n = 4), and statistically significant differences (P < 0.05) are indicated by asterisks. WT, Wild type.
Figure 8.
Figure 8.
Model illustrating the roles of RDR1 and SGS3, components of RNA silencing, in regulating cuticular wax biosynthesis at the top of the stem. A, In the wild type (WT), the precursor of the small RNA (smRNA) that regulates the expression of CER3 is degraded by CER7; therefore, CER3 is expressed and cuticular wax production ensues. B, In the cer7 mutant, the smRNA precursor is not degraded and is used for the production of a smRNA species by a pathway that involves RDR1 and SGS3. smRNA functions to silence CER3, leading to decreased cuticular wax biosynthesis. C, In either rdr1 or sgs3, suppressors of cer7, the smRNA species responsible for CER3 silencing will not be synthesized, resulting in CER3 expression and wax production in the absence of CER7 activity. DCL, DICER-LIKE; HEN1, HUA ENHANCER1; AGO, ARGONAUTE.
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