Article Figures & Data
Figures
- Figure 1.
Map-based cloning of CER9. A, The mutated gene was mapped between SSLP markers Fo12.3MB and Fo21.2MB using 96 F2 plants. B, Fine mapping narrowed cer9-1 down to a 63-kb region between markers Fo16.296MB and Fo16.359MB using 2,000 F2 plants. Numbers above the horizontal lines in A and B are the number of recombinants between the indicated marker and cer9-1. C, Sequenced region of the cer9-1 mutant and the T-DNA insertion allele cer9-2. The white box represents the sequenced region between markers Fo16.296MB and Fo16.359MB. Gray arrows represent genes in the sequenced region. The dashed line shows the mutated site of cer9-1. Start, Start codon; Stop, stop codon. The black bar represents the untranslated region, and the gray bar represents the coding region. The black triangle indicates the T-DNA insertion site of the cer9-2 allele. D, RT-PCR analysis of CER9 transcript levels in leaves of cer9 mutants compared with the corresponding wild type using primer pairs as shown in C.
- Figure 2.
Gene expression pattern of CER9 using the promoter::GUS transgenic line. Stained tissues expressing the CER9pro::GUS reporter gene fusion are shown as follows: 2-d-old seedling after germination (A); 5-d-old seedling (B); 10-d-old seedling (C); inflorescence stem of a 6-week-old plant showing high expression in the upper stem (D); cross-section from the top portion of a stem showing expression throughout the epidermis and mesophyll of a 5-week-old plant (E); GUS staining on leaf (F), silique (G), and flowers (H) of a 6-week-old plant.
- Figure 3.
Cuticular wax composition of inflorescence stems and leaves of wild-type Ler-0 and the cer9-1 mutant. Wax coverage is expressed as µg dm−2 leaf (A) and stem (B) surface area. Each wax constituent is designated by carbon chain length and is labeled by chemical class along the x axis. Values shown are means ± sd (n = 4).
- Figure 4.
SEM results showing stem epicuticular wax crystals in wild-type Ler-0 (A), cer9-1 (B), wild-type Col-0 (C), and cer9-2 (D). Bars = 5 µm.
- Figure 5.
Cutin monomer composition of inflorescence stems and rosette leaves of wild-type Ler-0 and the cer9-1 mutant. The C16 and C18 labels on the x axis represent the 16- and 18-carbon acid chains, respectively, whereas the number preceding “OH” indicates chain insertion point(s). Dioic represents dioic acid. The number of double bonds is indicated after the colon. Monomer amounts are expressed as µg dm−2 leaf (A) and stem (B). Values shown are means ± sd (n = 4). ** P ≤ 0.01.
- Figure 6.
Transmission electron micrographs of the cuticle layer of epidermal cells. A, Ler-0 leaf adaxial layer. B, cer9-1 leaf adaxial layer. C, Ler-0 leaf abaxial layer. D, cer9-1 leaf abaxial layer. E, Ler-0 stem. F, cer9-1 stem. Bars = 0.2 μm. The cuticular layer is indicated by the black arrows. G, Ler-0 leaf stomata. H, cer9-1 leaf stomata. I, Ler-0 stem stomata. J, cer9-1 stem stomata. Bars = 1 μm. The cuticular ridges are indicated by the black arrows.
- Figure 7.
Both cer9-1 and cer9-2 are more resistant to drought stress compared with the corresponding wild-type Ler-0 and Col-0, respectively. The percentage relative water content (RWC%) shown for each representative plant is the average of 10 plants.
- Figure 8.
cer9 mutants have reduced transpiration rate and improved WUE. A and B, Transpiration rates of 5-week-old wild-type and mutant plants. Five-week-old plants of Ler-0, cer9-1, Col-0, cer9-2, lacs2-3, and the lacs2-3 cer9-2 double mutant grown under 12-h/12-h day/night were used for the transpiration experiment. Water loss from each plant was measured as weight change at 5-min intervals over 36 h. At the end of the experiment, leaf area was measured and transpiration rate was calculated. Data represent means ± se of five replicate plants for each genotype. Black horizontal bars represent the nighttime period, and white horizontal bars represent the daytime period. C to E, Stomatal density (number of stomata per area; C), pavement cell density (D), and stomatal index (number of stomata per total epidermal cells; E) were analyzed in the leaf abaxial epidermal layers from wild-type, cer9-2, lacs2-3, and lacs2-3 cer9-2 double mutant plants. Data are means ± se of seven individual plants. F, Carbon isotope analysis of cutin mutants of att1, cer9, lacs1, lacs2, and wax2 compared with the wild type. Values are carbon isotope discrimination relative to the Pee Dee Belemnite standard. More negative values correspond to less water-use-efficient plants. Error bars indicate se. * P ≤ 0.05.
- Figure 9.
Suberin monomer composition in roots of the wild type (Col-0 and Ler-0) and cer9 mutants. The cer9 mutants exhibit higher aliphatic suberin monomer amounts than the wild type. Values shown are means ± sd (n = 4). * P ≤ 0.05.
- Figure 10.
Wilting test of grafted wild-type Col-0 and mutant plants. After grafting, plants were grown for 4 weeks in soil with regular watering, after which time watering was stopped, and the plants’ wilting status was recorded at 11 d after water withdrawal. The percentage relative water content (RWC%) shown for each representative plant is the average of 10 plants. A, Wild-type Col-0 without water withdrawal. B, cer9-2 without water withdrawal. C, Wild-type Col-0 with water withdrawal. D, cer9-2 with water withdrawal. E, Self-grafted wild-type Col-0 with water withdrawal. F, Self-grafted cer9-2 with water withdrawal. G, Wild-type Col-0 shoot/cer9-2 root grafted with water withdrawal. H, cer9-2 shoot/wild-type Col-0 root grafted.
- Figure 11.
DNA microarray analysis reveals altered gene expression in cer9-2 leaves relative to the wild type. Functional categorization is shown for mRNAs that differentially accumulate in cer9-2 and wild-type leaf tissues. mRNAs identified as having at least 2-fold change relative to the wild type are placed in separate categories based on predicted function (numbers in parentheses). A, Genes that are up-regulated in cer9-2 leaves relative to the wild type. B, Genes that are down-regulated in cer9-2 relative to the wild type. For additional information about these genes, see Supplemental Tables S2 and S3.
Tables
- Table I. Cuticular wax composition of inflorescence stems of Arabidopsis Ler-0 and cer9-1 and of leaves of Ler-0 and cer9-1
Values shown are means ± sd (µg dm−2) total wax amounts and coverage of individual compound classes (n = 3–4). –, Undetectable.
Sample Total Load Fatty Acids Aldehydes 1-Alcohols Alkanes 2-Alcohols Ketones Esters Inflorescence stems Ler-0 2,175.0 ± 159.1 29.9 ± 3.2 105.4 ± 15.1 137.4 ± 9.2 1,196.0 ± 90.4 85.5 ± 9.9 589.1 ± 42.4 31.7 ± 8.2 cer9-1 947.6 ± 27.7 65.9 ± 2.7 53.3 ± 4.6 194.9 ± 7.3 310.1 ± 8.9 31.3 ± 4.4 222.3 ± 8.5 69.7 ± 6.2 Rosette leaves Ler-0 128.9 ± 11.1 11.2 ± 1.6 4.6 ± 0.2 23.4 ± 3.7 89.1 ± 6.3 – 0.65 ± 0.3 – cer9-1 164.3 ± 16.7 150.7 ± 15.2 1.4 ± 0.2 3.9 ± 0.4 7.8 ± 1.5 – 0.16 ± 0.06 –
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-5
- Supplemental Data - Supplemental Table I
- Supplemental Data - Supplemental Table II
- Supplemental Data - Supplemental Table III
