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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01328

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Specification of adaxial cell fate during maize leaf development

Michelle T. Juarez^1,2, Richard W. Twigg^1,* and Marja C. P. Timmermans^1,

¹ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
² Graduate Program in Genetics, Stony Brook University, Stony Brook, New York 11794, USA

View larger version (58K): [in a new window]   Fig. 1. lbl1 and Rld1-O mutually suppress each other. Relative to wild-type B73 (A), weak lbl1-ref (B) and severe lbl1-rgd1 (C) mutants have a reduced stature and develop a variety of leaf phenotypes with the most severe leaves being thread-like and abaxialized. Rld1-O/+ plants (D) have a normal stature but their leaves are curled upwards because of a partial inversion of adaxial/abaxial polarity. lbl1-ref Rld1-O double mutant leaves (E,F) appear normal or display very mild lbl1- or Rld1-O-like phenotypes, but their subtending internodes elongate normally. The lbl1-rgd1 Rld1-O phenotypes (G) are also less severe than either single mutant and frequently resemble the weaker phenotypes of lbl1-ref. Detached leaves (H) illustrate the range of leaf phenotypes observed. (H1) wild type; (H2) lbl1-ref; (H3) Rld1-O; (H4) lbl1-ref Rld1-O, note the reduced rolling of the proximal leaf blade near the ligule/auricle when compared with the Rld1-O leaf; (H5) lbl1-rgd1 Rld1-O, note the morphological similarity to the lbl1-ref leaf. Scale bar: 7.5 inches (190.5 mm).

View larger version (106K): [in a new window]   Fig. 2. Rld1-O alters adaxial/abaxial polarity in the leaf. The maize leaf (A,B) comprises sheath (s) and blade (b) tissues separated by the auricle (a) and adaxial ligule (l). Rld1-O leaves (C) have narrow blades that roll upwards, and frequently develop an abaxial ligule (arrow) and clear sectors with fewer minor veins and no photosynthetic tissue (arrowhead). The ectopic abaxial ligule (D) is narrower, shorter and arises at a slightly different position along the proximodistal axis than the normal adaxial ligule. Rld1-O causes a partial inversion in adaxial/abaxial polarity and development of ectopic outgrowths on the abaxial leaf surface (E). Bulliform cells (arrows) are displaced to the abaxial epidermis and schlerenchyma cells (asterisk) develop on the adaxial rather than the abaxial side of intermediate veins. ad, adaxial; ab, abaxial.

View larger version (76K): [in a new window]   Fig. 3. lbl1 suppresses the epidermal patterning defects in Rld1-O. Scanning electron micrographs of the adaxial (A1-E1) and abaxial (A2-E2) epidermal surfaces of adjacent mature adult leaves samples. (A3-E3) High magnification of selected images to illustrate specific epidermal patterning defects. Compared with wild-type leaves (A), Rld1-O leaves (B) display normal polarity near the margins, but, in the center of the lamina, bulliform cells and macrohairs develop on the abaxial surface. Note the presence of isolated macrohairs (arrow in B3) and the overlap between macrohairs on the adaxial and abaxial epidermis. The regular spacing between bulliform cell files and between macrohairs is disrupted in weakly phenotypic lbl1-ref leaves (C), and bulliform cell files are frequently disrupted (arrow in C3). lbl1-ref Rld1-O leaves with a mild rolled phenotype (D) develop bulliform cells on both the adaxial and abaxial epidermis, and macrohairs on the abaxial epidermis in some regions of the blade. As in lbl1-ref, bulliform cell files are discontinuous and irregularly spaced (arrow in D3). Epidermal patterning in lbl1-ref Rld1-O leaves with a flattened morphology (E) is indistinguishable from that of wild type. ad, adaxial; ab, abaxial; mh, macrohair; bc, bulliform cell. All samples are oriented with the margin towards the right. Scale bars: in A1-E2, 1 mm; in A3-E3, 0.25 mm.

View larger version (101K): [in a new window]   Fig. 4. lbl1 acts upstream of rld1. In situ hybridization shows that rld1 expression in wild type (A,B) occurs in the SAM and vasculature, and on the adaxial side of leaf primordia. In lbl1 apices (C,D), rld1 expression persists in the vasculature but is variably reduced in the SAM and on the adaxial side of developing primordia. Arrows in A and B mark selected regions expressing rld1; arrows in C and D mark the absence of rld1 transcripts in the corresponding positions. (A,C) Longitudinal sections; (B,D) transverse sections. RT-PCR analysis (E) on vegetative apices and young leaf primordia from wild type, lbl1-rgd1, Rld1-O and the lbl1 Rld1-O double mutant indicates that rld1 transcript levels are reduced in lbl1-rgd1 but increased in Rld1-O. In the double mutant, rld1 transcripts accumulate to a level intermediate between that of either single mutant. Ubiquitin (ubi) transcripts were amplified as a control. A, apices comprising the meristem and approximately four young leaf primordia; LP, P5-P8 leaf primordia.

View larger version (43K): [in a new window]   Fig. 5. The maize yabby genes, zyb9, 10, 14 and 15, are homologs of FIL and YAB3. (A) Phylogenetic analysis of the Arabidopsis and maize YABBY proteins demonstrates that the maize yabby genes form a separate clade that is most closely related to FIL and YAB3. Bootstrap values based on 1000 repetitions are indicated. Amino acid sequence alignments of the Zn-finger domains (B), and of the YABBY and C-terminal domains (C), used in the phylogenetic analysis of the maize and Arabidopsis YABBY proteins highlight the high level of sequence conservation in these domains of all YABBY proteins. Identical amino acids are shaded dark gray; similar amino acids are shaded light gray. GenBank Accession numbers are: zyb9, AY313903; zyb10, AY313904; zyb14, AY313901; zyb15, AY313902.

View larger version (73K): [in a new window]   Fig. 6. Maize yabby genes are expressed on the adaxial side of incipient leaf primordia. (A) In situ hybridization with a knotted1 (kn1) specific probe shows expression in the meristematic cells of the SAM. (B-E) Hybridization of adjacent longitudinal sections with kn1 (B) and zyb14 (C), or kn1 (D) and zyb9 (E), shows that incipient leaf primordia defined by lack of kn1 expression comprise around six tiers of cells (indicated by arrowheads), and that zyb9 and zyb14 expression is limited to the adaxial side.

View larger version (188K): [in a new window]   Fig. 7. Maize yabby genes are expressed on the adaxial side of leaf primordia. (A-D) In situ hybridization of zyb14 (A,C) and zyb9 (B,D) shows that both genes are expressed on the adaxial side of incipient (arrows in A,B) and young leaf primordia. During primordium development expression of zyb9 and zyb14 becomes restricted to the margins and to the central layer of the ground tissue. Expression of zyb9 also persists in the vasculature (D). The black lines outline the margins of some primordia. (A,B) Longitudinal sections; (C,D) transverse sections.

View larger version (90K): [in a new window]   Fig. 8. The yabby genes act downstream of lbl1 and rld1. Relative to wild type (A), zyb14 expression is reduced in lbl1-rgd1 (B,C). In the narrow lbl1 leaf primordia, zyb14 is absent from mutant margins (arrows in C), but remains localized to the adaxial side of normal margins. Expression of zyb14 is induced uniformly in ectopic lamina on the adaxial surface of lbl1 leaf primordia (arrow in D). zyb14 is expressed more abundantly in Rld1-O (E), but the pattern of expression in young Rld1-O primordia resembles that observed in wild type. Ectopic outgrowths arise on the abaxial surface of Rld1-O leaf primordia at the boundary of sectors expressing zyb14 (F) or zyb9 (G), and sectors that lack yabby expression (e.g. asterisk in F). These abaxial ectopic outgrowths also induce both zyb14 and zyb9 expression (arrows in F and G, respectively). RT-PCR analysis on vegetative apices and young leaf primordia from wild type, lbl1-rgd1, and Rld1-O in the A158 inbred indicates that the relative expression levels of zyb14 (H) and zyb9 (I) are reduced in lbl1-rgd1 apices, and slightly increased in Rld1-O. ubi transcripts were amplified as a control. A, apices comprising the meristem and approximately four young leaf primordia; LP, P5-P8 leaf primordia.

View larger version (9K): [in a new window]   Fig. 9. Genetic pathway leading to adaxial cell fate and mediolateral growth of the maize leaf. lbl1 and miRNA166 have an opposing affect on the accumulation of rld1 transcripts, and together lead to the adaxial specific expression of rld1. RLD1 specifies adaxial cell fate, possibly upon activation by the proposed meristem-derived signal. RLD1 also induces yabby gene expression in the adaxial domain, and the juxtaposition of yabby-expressing and non-expressing cells mediates mediolateral outgrowth.

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