ECERIFERUM 10 Encoding an Enoyl-CoA Reductase Plays a Crucial Role in Osmotolerance and Cuticular Wax Loading in Arabidopsis

Abstract

Acquired osmotolerance induced after salt stress is widespread across Arabidopsis thaliana (Arabidopsis) accessions (e.g., Bu-5). However, it remains unclear how this osmotolerance is established. Here, we isolated a mutant showing an acquired osmotolerance-defective phenotype (aod2) from an ion-beam-mutagenized M2 population of Bu-5. aod2 was impaired not only in acquired osmotolerance but also in osmo-shock, salt-shock, and long-term heat tolerances compared with Bu-5, and it displayed abnormal morphology, including small, wrinkled leaves, and zigzag-shaped stems. Genetic analyses of aod2 revealed that a 439-kbp region of chromosome 4 was translocated to chromosome 3 at the causal locus for the osmosensitive phenotype. The causal gene of the aod2 phenotype was identical to ECERIFERUM 10 (CER10), which encodes an enoyl-coenzyme A reductase that is involved in the elongation reactions of very-long-chain fatty acids (VLCFAs) for subsequent derivatization into cuticular waxes, storage lipids, and sphingolipids. The major components of the cuticular wax were accumulated in response to osmotic stress in both Bu-5 WT and aod2. However, less fatty acids, primary alcohols, and aldehydes with chain length ≥ C30 were accumulated in aod2. In addition, aod2 exhibited a dramatic reduction in the number of epicuticular wax crystals on its stems. Endoplasmic reticulum stress mediated by bZIP60 was increased in aod2 under osmotic stress. The only cer10 showed the most pronounced loss of epidermal cuticular wax and most osmosensitive phenotype among four Col-0-background cuticular wax-related mutants. Together, the present findings suggest that CER10/AOD2 plays a crucial role in Arabidopsis osmotolerance through VLCFA metabolism involved in cuticular wax formation and endocytic membrane trafficking.

Keywords: Arabidopsis thaliana accession; ER stress; cuticular wax; enoyl-CoA reductase; osmotolerance; very-long-chain fatty acid.

Figure 1

Identification of acquired osmotolerance-defective (aod2) mutant. (A) Flow chart of the acquired osmotolerance assay. A total of 30,000 ion-beam-mutagenized, salt-acclimated, 2-week-old seedlings of accession Bu-5 were transferred to Murashige and Skoog agar plates containing 750 mM sorbitol for 15 days. Seedlings showing osmo-hypersensitivity (red circle) were selected as mutants showing an acquired osmotolerance-defective phenotype. The mutant aod2 was identified by using this approach. (B) Chlorophyll content as an index of acquired osmotolerance in Bu-5 (WT) and aod2. FW, fresh weight. Differences between WT (white bar) and aod2 (black bar) were analyzed by Student’s t-test (mean ± SE, n = 3, **p < 0.01).

Figure 2

Morphogenesis of aod2 mutant. Representative images of 4-week-old WT and aod2 plants grown in soil under normal growth conditions; the images show leaves (A), stems and mechanically opened flowers (B), and leaf trichomes (C).

Figure 3

Characterization of acquired osmotolerance-defective (aod2) mutant. (A) Top: flow chart of the salt- and osmo-shock tolerance assay. Two-week-old seedlings were transferred to Murashige and Skoog agar plates containing 200 mM NaCl for 8 days or 600 mM sorbitol for 21 days. Middle and bottom: Chlorophyll content as an index of the salt- and osmo-shock tolerances of aod2 and Bu-5 (WT). FW, fresh weight. (B) Expression of osmostress-responsive marker genes in WT and aod2 under normal (control) and acquired osmotic stress (100 mM NaCl for 7 days and subsequent 750 mM sorbitol for 8 h) conditions; expression levels were determined by quantitative real-time polymerase chain reaction relative to those of Actin2 (mean ± SE, n = 3). (C) Long-term heat tolerance of aod2. Ten-day-old WT and aod2 seedlings were grown initially at 22°C, then at 37°C for 4 days, and then at 22°C for 5 days. Then, chlorophyll content was determined as an index of heat tolerance. Differences between WT and aod2 were analyzed by Student’s t-test (mean ± SE, n = 3, **p < 0.01).

Figure 4

Identification of the causal gene of the acquired osmotolerance-defective phenotype. (A) High-resolution mapping of the causal locus in aod2 by using F₂ progeny from a cross between aod2 and Pog-0. The scores indicate recombination frequencies (%). Arabidopsis Genome Initiative numbers are shown above the genes. The red bar shows the 439-kbp insertion from Chr. 4. (B) Complementation test performed by transforming aod2 with AOD2/At3g55360. T₂ plants transformed with the native promoter At3g55360 (aod2_At3g55360) derived from Bu-5 (WT) were used. Top panel: Representative images showing acquired osmotolerance in the complementation lines. Lower panel: Chlorophyll content of WT, aod2, and aod2_At3g55360. Differences between WT and aod2 or between aod2 and aod2_At3g55360 were analyzed by Student’s t-test (mean ± SE, n = 3, **p < 0.01). (C) Expression profiles of CER10 in WT under normal and acquired osmotic stress conditions; expression levels were determined by quantitative real-time PCR relative to those of Actin2 (mean ± SE, n = 3). Differences between normal and acquired osmotic stress conditions were analyzed by Student’s t-test. **p < 0.01.

Figure 5

Cuticular wax content and composition in acquired osmotolerance-defective (aod2) mutant. (A) Total wax content (including fatty acids, primary alcohols, aldehydes, and alkanes) in Bu-5, aod2, and Col-0 seedlings under normal (cont) and osmotic stress (osmo) conditions. (B) Waxes identified in Bu-5, aod2, and Col-0 seedlings under normal (cont) and osmotic stress (osmo) conditions. Data are presented as mean ± SE, n = 6. Letters at the top of columns are grouped with each chain length based on one-way ANOVA and Tukey’s test, p < 0.05.

Figure 6

Wax layer of acquired osmotolerance-defective (aod2) mutant. (A) Left: representative images from the toluidine blue (TB) test. Plants with a normal cuticle repel TB, but a deficient cuticle allows TB to permeate the epidermal surface. Two-week-old Bu-5 (WT) and aod2 plants were used for the experiment. Right: TB uptake was examined spectrophotometrically by measuring absorbance at 630 nm (A630). The major peak of absorbance due to plant material (A435) was used for normalization. Relative levels of TB were calculated as the ratio of A630 to A435. (B) Scanning electron microscopy images of the surface of the stems of WT and aod2. Bars = 100 μm.

Figure 7

Water loss from detached acquired osmotolerance-defective (aod2) mutant leaves. (A) Leaves of 4-week-old plants grown in soil under normal growth conditions were detached (normal condition) and then left in ambient conditions for 9 h (drought). (B) Percentage decreases of fresh weight are expressed as percentage water loss. Differences between Bu-5 (WT) and aod2 were analyzed by Student’s t-test (mean ± se, n = 3, **p < 0.01, ***p < 0.001).

Figure 8

Effect of the sloh4 mutation on expression of endoplasmic reticulum stress-related genes under osmotic stress. (A) Transcript levels of bZIP17, bZIP28, and bZIP60 in Bu-5 (WT) and aod2 plants under normal and acquired osmotic stress (100 mM NaCl for 7 days and subsequent 750 mM sorbitol for 10 days) conditions. (B) Expression of the target genes for the transcription factor bZIP60 in WT and aod2 under normal and osmotic stress conditions; expression levels were determined by quantitative real-time polymerase chain reaction relative to those of Actin2 (mean ± SE, n = 3). Differences between WT and aod2 were analyzed by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 9

Osmotolerance of mutants defective in wax biosynthesis. (A) Top: representative images showing osmo-shock tolerance in Col-0 and Col-0-background cer10, cer2, cer5, and lcr mutants. Two-week-old seedlings were mesh-transferred to MS agar plates containing 600 mM sorbitol for 11 days. Bottom: Chlorophyll contents of the mutant plants shown in the top panel. (B) Toluidine blue (TB) test using Col-0 and the Col-0-background mutants. TB uptake was examined spectrophotometrically by measuring absorbance at 630 nm (A630). The major peak of absorbance due to plant material (A435) was used for normalization. Relative levels of TB were calculated as the ratio of A630 to A435. (C) Scanning electron microscopy images of the surfaces of the stems of Col-0 and the Col-0-background mutants. Bars = 100 μm. Data are presented as mean ± SE, n = 3. Letters at the top of columns are based on one-way ANOVA and Tukey’s test, p < 0.05.