Epidermal peel of wild-type adaxial blade and impressions of wild-type and mutant blade epidermises. (A) Wild-type epidermal tissue is characterized by a very regular pattern of specialized cells: macrohairs (mh), pricklehairs (ph), microhairs (mih), bulliform cells (bc), long cells (lc), silica cells (sc), and guard cells with associated subsidiary cells, which together make up stomatal complexes (st). (B) Macrohairs (white arrowhead) and bulliform cell rows (black arrowhead) are seen in the wild-type adaxial epidermis. In contrast, no macrohairs are seen in the wild-type abaxial epidermis (D). (C) Adaxial epidermis of Rolled leaf1-PB heterozygous mutant. (E) Abaxial epidermis of Rolled leaf1-PB heterozygous mutant. Arrow indicates macrohair.

Schiff staining of ‘clearing’ at the blade sheath boundary. (A) Rolled leaf1-PB mutant with region of pale tissue indicated by an arrowhead. (B) Abaxial view of veins at the blade-sheath boundary of a wild-type leaf, as observed with Schiff staining. (C) Abaxial view of blade-sheath boundary in a Rld1 leaf at region of pale tissue (arrowhead in A), located in same longitudinal region as the ligule flap (lf). Schiff-stained vasculature in Rld1-PB leaf shows lack of intermediate and transverse veins (black arrowhead) in this ‘clearing.’ (D) Enlarged view of boxed area in B, showing intermediate veins (iv) and transverse veins (tv) between the lateral veins (lv). (E) Enlarged view of boxed area in C showing lack of intermediate and transverse veins in the region of the ‘clearing’ (black arrowhead).

Subepidermal architecture. Hypodermal schlerenchyma (hs) in wild-type blade tissue is more frequently associated with the abaxial epidermis (A). In regions of Rolled leaf1 mutants where the epidermal characters have been reversed, hypodermal schlerenchyma is frequently seen on the other side near the adaxial epidermis (B).

Leaf phenotypes resulting from dosage series. Aneuploidy experiments showing three sibling leaves with one copy of Rolled leaf1-O, but differing in dosage of the wild-type allele, rld1⁺: (left) Rld1-O/rld1⁺rld1⁺(hyperploid), (middle) Rld1-O/rld1⁺ (euploid), (right) Rld1-O/– (hypoploid). Rld1-O/rld1⁺rld1⁺ exhibits the least severe mutant phenotype. The abaxial ligule (black arrowhead) is present in only a narrow portion (as shown by size of black bar above ligule) of the blade width and the vascular disturbance is mild resulting in negligible ‘clearings’ (red arrowheads). Clearings result from a disturbed pattern of venation in the mutant (Fig. 3E). The width of the blade lamina is very similar to that observed in wild-type siblings for the same dosage series (data not shown). As rld1⁺ alleles change in dose from 2 to 0, all components of Rld1 phenes become more severe as can be seen for leaf-width and clearings.

Construction and phenotypes of genetic mosaics. (A) Seeds were X-irradiated to generate mosaic plants. (B) Cartoon of a cell showing a pair of chromosome 9 and relative positions of alleles of rld1 and wlu4. The arrow indicates an X-ray breakage event. The somatic loss of the 9L arm will result in a lineage of cells mutant for wlu4 (white) and no longer carrying the mutant Rld1-O allele. (C) Mature leaf from plant heterozygous for Rld1-O and wlu4 (phenotype is Rolled leaf1 and green) with a white sector (hemizygous for rld1⁺wlu4). (D) Schematic showing cross section of a leaf (DV axis) with the five layers (TL1-TL5), and tissue types indicated. (E) Cartoons representing the different classes (i to xi) of leaf sectors found in X-irradiated Rolled leaf1-O plants, white indicates the rld1⁺wlu4 genotype and green indicates the Rld1-OWlu4⁺ genotype. The resulting phenotype (wild-type (wt) or Rolled leaf1(Rld1)) of each of the sectors and the total numbers for each sector class are shown. Sectors within each class came from different chromosome breakage events.

Leaf phenotypes of sectors from mosaic analysis. Transverse sections of Rolled leaf1 leaves containing non mutant white sectors. Plants are genotypically rld1⁺wlu4/ Rld1-OWlu4⁺ (Rolled leaf1 phenotype, green tissue). White tissue indicates removal of dominant mutant allele Rld1-O from particular tissue layers of sector. (A) Leaf with no white sectors as shown by inset cartoon. Macrohairs on the abaxial epidermis show the characteristic Rolled leaf1 phenotype. (B) White sector, indicated between arrowheads, marks the loss of Rld1-O. Close examination showed epidermal guard cells were still green, as shown by inset. This sector had the typical Rld1-O polarity as the presence of macrohairs on the abaxial epidermis in white tissue sector indicate. (C) Micrograph of abaxial epidermis with a sector running through it. To the left of the sector there are macrohairs (mh). No macrohairs are observed on this epidermis in the sector. (D) Transverse section of a sector where Rld1-O has been lost in all tissue layers except the abaxial epidermis (TL5) as shown by inset. Prickle hairs (ph) and bulliform cells (bc) are present on the abaxial epidermis. (E) Transverse section of a region of the sector seen in C. Prickle hairs and bulliform cells are present on the adaxial epidermis. (F) Adaxial epidermis of Rld1 sector seen in D, where Rld1-O was removed in all but the abaxial epidermis. Stomatal complexes (sc) contain guard cells that are white. (G) Abaxial surface of the region of sector in E marked by arrowheads. Rld1-O has been lost on this epidermis as observed in the stomatal complexes that are not fluorescing red. Inset cartoons show genotypes of each half of this sector. Tissue is phenotypically wild type. (H) Abaxial epidermis of same sector as F. Chloroplasts of guard cells (gc) are fluorescent red indicating presence of Rld1-O. The presence of macrohairs and prickle hairs on the abaxial surface indicate the sector is phenotypically Rolled leaf1.