Fig 1.
Phenotype of the nonglaucous mutant.
(A) The mutant was found in a small F2 population derived from a cross between two RNAi transgenic plants in BW background due to premature drying out. (B) Adult plants of BW (left) and the nonglaucous mutant (right) at anthesis. (C) Adult plants of BW (left) and the nonglaucous mutant (right) at grain-filling stage.
Fig 2.
Genetic analysis of w3 mutant.
(A) The F1 hybrid between BW and the nonglaucous mutant is intermediate between its parents in glaucousness intensity. (B) The F1 hybrid between the mutant and w1w2 double recessive line was glaucous. The scale bars indicate 1 cm.
Table 1.
Phenotypes and segregations of glaucousness in hybrids and their progenies.
Fig 3.
Chromosomal localization of the W3 locus.
(A) Molecular mapping of the W3 locus on the chromosome arm 2BS. The markers are listed at the right side of the map, and genetic distances (cM) between the marker loci are indicated at the left side of the map. The W3 locus is indicated in bold. The top of the map is towards the telomere and the bottom is towards the centromere. (B) The peduncles of CS N2B-T2D (1), F1 hybrids of NG2 with N2B-T2D (2), with deletion line 2BS-1 (3), with 2BS-2 (4), with 2BS-3 (5), with 2BS-5 (6), with 2BS-10 (7) and with 2BS-14 (8). All the flag-leaf sheaths are nonglaucous. The scale bar indicate 1 cm.
Fig 4.
SEM micrographs of cuticle surfaces of flag leaf sheaths.
(A) BW and (B) w3 mutant. The scale bars indicated 1 μm.
Fig 5.
Analysis of cuticle permeability of BW and w3 mutant.
Cuticle permeability was evaluated by air drying at room temperature (A) and by chlorophyll leaching in 80% ethanol (B). The numbers on the x-axes represent hours of treatment. Water loss or chlorophyll leaching at each time point is represented on the y-axes as percentages of the total water content or total chlorophyll content in the tissue. Measurements taken from six individuals were averaged.
Fig 6.
Spikes responses to dehydration.
Spike of BW (W1W1W2W2W3W3), w3 mutant (W1W1W2W2w3w3), w1w2 double recessive line (w1w1w2w2W3W3), and the F1 hybrid between w3 and w1w2 (W1w1W2w2W3w3) at 0 (A), 6 (B) and 12 h of dehydration (C). The designations are indicated on the top. The scale bars indicate 1 cm.
Fig 7.
Wax composition of BW and w3 mutant.
Total wax load and content of alkanes, β-diketones, primary alcohols (alkan-1-ols), aldehydes, wax esters, and fatty acids of the flag leaf sheaths were measured by GC-MS. The numbers on the y-axes indicate average content expressed as μg per g dried tissue (dry weight, DW). The error bars indicate standard deviation of the mean estimated from five biological replicates.
Fig 8.
Variation of wax homologues between BW and w3 mutant.
Carbon atom numbers of alkanes, β-diketones, primary alcohols (alkan-1-ols), fatty acids, wax esters, and aldehydes are indicated on the x-axes. Their contents are indicated on y-axes as μg per g dried tissue (dry weight, DW). The error bars indicate standard deviation of the mean calculated from five biological replicates. β-D, β-diketone; and OH-β, hydroxy-β-diketones.
Fig 9.
Transcriptional changes of wax genes in w3 mutant against BW.
Genes with significant fold changes are depicted. The error bars represent standard deviation of the mean fold-change of mRNA levels calculated from four biological replicates. Asterisks indicate that the difference is significant at P< 0.05 (*) or at P < 0.01 (**). Expression data for all genes analyzed are listed in S2 Table.