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Expression of a mutant maize gene in the ventral leaf epidermis is sufficient to signal a switch of the leaf’s dorsoventral axis

Jennifer M. Nelson, Barbara Lane and Michael Freeling*

Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA



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  		Fig. 1. Leaf phenotypes. (A) Wild-type blade (b), sheath (s), ligule/auricle region (a) indicated by arrows. (B) Field of Rolled leaf1-0/+ mutants. The spiky appearance of leaves is caused by leaf blades being inwardly rolled, as opposed to lying flat, as seen in wild-type leaves in A. The leaf blades of Rld1 mutants are often caught in the preceeding rolled-up blade and need to be unrolled in order to free the tassel (arrowhead). (C) Blade-sheath boundary of Rolled leaf1 leaf, with an abaxial ligule (l).

 


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  		Fig. 2. 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.

 


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  		Fig. 3. 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).

 


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  		Fig. 4. 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).

 


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  		Fig. 5. 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.

 


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  		Fig. 6. 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.

 


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  		Fig. 7. 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.

 


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  		Fig. 8. One model that explains how RLD1 normally functions in the abaxial epidermis to promote adaxial identity. In this model, dorsoventrality is initiated by a peripheral signal and RLD1 is involved in its maintenance. At P0, the leaf founder initials lack polarity. As these initials develop into the early leaf founder cells, they develop polarity in response to a peripheral/adaxialization signal emanating from the meristem. The surface closest to the meristem takes on adaxial identity and the surface further from the meristem might have abaxial identity as a default or abaxial identity might be specified on tissue with undefined cell fate (see inset) as a result of a concentration differential or via a signal cascade started by the peripheral signal. By the late leaf primordium, as the developing primordium is moving further away from the meristem, the peripheral signal is no longer a strong enough influence on the primordia and another mechanism for polarity maintenance is necessary. One possible model for polarity maintenance is a polarity maintenance signal (PMS) emanating from tissue with adaxial identity (light blue arrow). This reinforces its adaxial identity via adaxial factors (AdFs) such as LEAFBLADELESS1 (LBL1), and signals to the opposite half of the leaf to maintain abaxial identity. Abaxial factors (AbFs) such as ROLLED LEAF1 (RLD1) are responsible for maintenance of abaxial polarity. As the blade primordium develops, this ‘I’m AD, you be AB maintenance signal remains important to maintain correct polarity. In Rld1 mutants, the mutant RLD1 interferes with the function of wild-type RLD1. The mutant AbF is unable to maintain abaxial identity in the abaxial tissue and results in a blade phenotype of abaxial tissue being switched to adaxial identity as a mutant response to either the weaker peripheral/adaxialization signal or to the PMS from the adaxial tissue. The lower tissue having taken on adaxial identity, now sends out the PMS to the upper surface, and sometimes in Rld1 mutants, some part of this upper surface (adaxial tissue) responds to the signal, becoming abaxialized. This results in the dorsoventral axis being flipped over in some portion of a Rld1 mutant leaf. However, an alternative model where the abaxial epidermis generates an ‘I’m ab so you are ad’ trans-tissue signal is equally likely (not shown).

 

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© The Company of Biologists Ltd 2002