Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses

Abstract

Land plant aerial organs are covered by a hydrophobic layer called the cuticle that serves as a waterproof barrier protecting plants against desiccation, ultraviolet radiation, and pathogens. Cuticle consists of a cutin matrix as well as cuticular waxes in which very-long-chain (VLC) alkanes are the major components, representing up to 70% of the total wax content in Arabidopsis (Arabidopsis thaliana) leaves. However, despite its major involvement in cuticle formation, the alkane-forming pathway is still largely unknown. To address this deficiency, we report here the characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis. Analysis of CER1 expression showed that CER1 is specifically expressed in the epidermis of aerial organs and coexpressed with other genes of the alkane-forming pathway. Modification of CER1 expression in transgenic plants specifically affects VLC alkane biosynthesis: waxes of TDNA insertional mutant alleles are devoid of VLC alkanes and derivatives, whereas CER1 overexpression dramatically increases the production of the odd-carbon-numbered alkanes together with a substantial accumulation of iso-branched alkanes. We also showed that CER1 expression is induced by osmotic stresses and regulated by abscisic acid. Furthermore, CER1-overexpressing plants showed reduced cuticle permeability together with reduced soil water deficit susceptibility. However, CER1 overexpression increased susceptibility to bacterial and fungal pathogens. Taken together, these results demonstrate that CER1 controls alkane biosynthesis and is highly linked to responses to biotic and abiotic stresses.

Figure 1.

Expression analysis of the CER1 gene family in Arabidopsis. A, Differential expression analysis of Arabidopsis CER1-like, CER3, and MAH1 genes in various organs of Arabidopsis. The gene expression level was determined by real-time RT-PCR analysis. Results are presented as relative transcript abundance. The relative transcript abundance of ACT2, EF-1α, eIF-4A-1, UBQ10, and PP2A in each sample was determined and used to normalize for differences of total RNA amount. The data represent means ± sd of five replicates. Total RNA was isolated from 15-d-old seedlings, roots, stems, cauline leaves, rosette leaves, flowers, and siliques. B, Spatial expression patterns of the CER1 gene in transgenic Arabidopsis plants harboring the CER1 promoter fused to the GUS gene. Promoter activity was visualized through histochemical GUS staining on 10-d-old seedlings (a), young leaf of a 3-week-old plant (b), transverse section of the stem (S), secondary meristem (SM), and cauline leaf (CL; c), leaves of a 3-week-old plants (d), and flowers (e). C, Cortex; cb, cambium; ep, epidermis; pa, parenchyma; ph, phloem; pi, pith; vb, vascular bundle; xy, xylem.

Figure 2.

Molecular and phenotypic characterization of cer1 mutants and CER1-overexpressing lines. A, Schematic of CER1 gene structure indicating the positions of the T-DNA inserts in cer1 mutant alleles. Dark boxes indicate exons, black lines indicate introns, and gray boxes indicate 5′ and 3′ untranslated regions. The arrows underneath the gene structure are the positions of convergent primers used for PCR on genomic DNA. B, PCR on genomic DNA of wild-type Col-0 (WT) plants, cer1-1 and cer1-2 mutants, the cer1-1R rescued line, and CER1ox1 and CER1ox2 overexpressing lines. Amplification of the CER2 gene was used as a positive control. C, Semiquantitative RT-PCR analysis of steady-state CER1 transcripts in 4-week-old plants of the different lines compared with the wild-type plants as indicated above. The Actin2 gene was used as a constitutively expressed control. D, Real-time RT-PCR analysis of CER1 gene expression in 4-week-old plants of the different lines compared with wild-type plants as indicated above. Results are presented as relative transcript abundance. The data represent means ± sd of three replicates. E, Stems from 6-week-old cer1-1 and cer1-2 mutants showing glossy phenotypes compared with wild-type plants, the cer1-1R rescued line, and CER1ox1 and CER1ox2 overexpressing lines. F, Epicuticular wax crystal formation on Arabidopsis wild-type (Col-0), cer1-1, cer1-1R, and CER1ox1 stem surfaces detected by scanning electron microscopy at 5,000× magnification. Bars = 10 μm.

Figure 3.

Cuticular wax composition of cer1 mutants and CER1-overexpressing lines. A, Cuticular wax composition of inflorescence stems of wild-type Col-0 (WT), cer1-1, cer1-2, cer1-1R, CER1ox1, and CER1ox2 lines. B, Cuticular wax composition of rosette leaves of Col-0, cer1-1, cer1-2, cer1-1R, CER1ox1, and CER1ox2 lines. Amounts of major components are expressed as μg dm⁻² stem or leaf surface area. Each wax constituent is designated by carbon chain length and is labeled by chemical class along the x axis. The data represent means ± sd of four replicates.

Figure 4.

Phenotypes of cer1 mutants and CER1-overexpressing lines. A, Phenotypes of 4-week-old rosettes of wild-type Col-0 (WT), cer1-1, cer1-2, cer1-1R, CER1ox1, and CER1ox2 lines. B, The cer1-2 mutant is fertile in normal conditions, whereas the cer1-1 mutant shows conditional male sterility that can be restored in high-humidity conditions (cer1-1*) or by functional complementation (cer1-1R). Bars = 1 cm.

Figure 5.

Modulation of CER1 expression in Arabidopsis seedlings subjected to stress conditions. A, Modulation of CER1 expression in plants exposed to low-humidity conditions for 24 h. B, Modulation of CER1 expression in plants exposed to different ABA concentrations for 24 h. C, Time-course analysis of CER1 expression in plants exposed to 10 μm ABA for 24 h. RD29A gene expression was used as a control for treatment efficiency. The gene expression level was determined by real-time RT-PCR analysis. Results are presented as relative transcript abundance. The data represent means ± sd of three replicates. D, Quantitative assay and histochemical staining revealed induction of the GUS reporter activity in a 2-week-old ProCER1:GUS transgenic line exposed to different ABA concentrations for 24 h. The data represent means ± sd of four replicates. MU, Methylumbelliferone.

Figure 6.

CER1 deregulation affects cuticle properties and response to soil water deficit. A, Water-loss rates (expressed as a percentage of initial water-saturated weight) of isolated rosettes from wild-type Col-0 (WT), cer1-1, and CER1ox1 plants. Four-week-old plants were dark acclimated for 3 h, excised, and placed immediately in water in the dark for 1 h to equilibrate water contents. Rosette weights were determined gravimetrically using a microbalance. The data represent means ± sd of five replicates. B, Chlorophyll extraction rates (expressed as a percentage of total chlorophyll extracted after 24 h) of rosettes from Col-0, cer1-1, and CER1ox1 plants. Four-week-old plants were dark acclimated for 3 h, and rosettes were immersed in 80% ethanol for 24 h. The data represent means ± sd of five replicates. C, Water soil deprivation experiment. Three-week-old plants were exposed to 20 d of water deprivation (WD) alongside well-watered plants (WW). D, Well-watered and water-deprived plants were used to determine leaf RWC. The data represent means ± sd of five replicates (the experiments were repeated once with similar results). Significance was assessed by Student’s t test (* P < 0.01).

Figure 7.

CER1-overexpressing lines show enhanced susceptibility to Pst and Sclerotinia. A, Phenotype of wild-type Col-0, cer1-1, CER1ox1, and CER1ox2 lines 3 d post inoculation (dpi) with the Pst DC3000 virulent strain at 5 × 10⁵ colony-forming units (cfu) mL⁻¹. Growth of Pst DC3000 in the different lines was measured at 0 d (black bars) and 3 d (gray bars) after inoculation performed with a bacterial suspension at 5 × 10⁵ cfu mL⁻¹. B, Phenotypes of Col-0, cer1-1, CER1ox1, and CER1ox2 lines 2 d after inoculation with the Pst DC3000/avrRpt2 avirulent strain at 5 × 10⁶ cfu mL⁻¹. Growth of Pst DC3000/avrRpt2 in the different lines was measured at 0 d (black bars) and 3 d (gray bars) after inoculation performed with a bacterial suspension at 5 × 10⁵ cfu mL⁻¹. C, Macroscopic observation of symptoms 7 d after inoculation with the Sclerotinia mycelium of Col-0, cer1-1, CER1ox1, and CER1ox2 lines. Disease symptoms were scored 7 d after inoculation with the mycelium. The Arabidopsis ecotypes Shahdara (sha) and Rubezhnoe-1 (rub) were used as susceptible and resistant controls, respectively. The data represent average disease scores ± sd of three independent experiments. Significance was assessed by Student’s t test (** P < 0.05, *** P < 0.01).