Roles of the middle domain-specific WUSCHEL-RELATED HOMEOBOX genes in early development of leaves in Arabidopsis

Abstract

During leaf development in flowering plants, adaxial (upper) and abaxial (lower) side-specific genes are responsible for blade outgrowth, which takes places predominantly in the lateral direction, and for margin development as well as differentiation of adaxial and abaxial tissues. However, the underlying mechanisms are poorly understood. Here, we show that two WUSCHEL-RELATED HOMEOBOX (WOX) genes, PRESSED FLOWER (PRS)/WOX3 and WOX1, encoding homeobox transcription factors, act in blade outgrowth and margin development downstream of adaxial/abaxial polarity establishment. The expression of PRS and WOX1 defines a hitherto undescribed middle domain, including two middle mesophyll layers and the margin, as a center that organizes the outgrowth of leaf blades. The expression of PRS and WOX1 is repressed in the abaxial leaf domain by the abaxial-specific transcription factor KANADI. Furthermore, PRS and WOX1 coordinate adaxial/abaxial patterning together with adaxial- and abaxial-specific genes. Our data suggest a model of blade outgrowth and adaxial/abaxial patterning via the middle domain-specific WOX genes in Arabidopsis thaliana leaves.

Figure 1.

Function of PRS and WOX1 in the Outgrowth of the Leaf Blade. (A) Images of the eighth leaf of the different genotypes. (B) The lengths and widths of the first two leaves and the eighth leaves. White, the wild type; yellow, prs-2; cyan, wox1-101; magenta, prs wox1. (C) The number of palisade cells per 1000-μm width in the first two leaves and the eighth leaves. White, the wild type; magenta, prs wox1. Data in (B) and (C) are represented as the means ± sd. ***P < 0.001 by Tukey’s honestly significant different (HSD) test. The numbers in the bars indicate the number of samples. (D) to (F) P3 leaf primordia (D), P5 leaf primordia (E), and P8 leaf primordia (F) in cross section. Right drawings show illustrations of cell alignment ([E] and [F]). Yellow, epidermal layer; orange, subepidermal layer; magenta, third layer from surface. (G) RNA ISH of P3 or P4 leaf primordia cross sections with H4 antisense probe. The number of H4-positive cells per 1000-μm² area in the whole leaf primordia is indicated below. (H) The number of H4-positive cells per 1000-μm² area in cross sections of leaf primordia divided into five parts in width. The photo at right shows an example of the divided primordium. M, the SAM. Data in (G) and (H) are represented as the means ± se. In total, 41 sections from six wild-type plants and 50 sections from seven prs-2 wox1-101 plants were assessed. ***P < 0.001 by Wilcoxon rank-sum test. (I) RNA ISH of wild-type shoot apex in cross section with WOX1 probe. (J) Comparison of the expression domain of WOX1 with that of PRS in cross sections of P3 and P5-P6 leaf primordia. The “M” in (H) and (I) indicates SAM; asterisks in (J), edges of leaf primordia; ladders mark the cell layers of the blade region. Bars = 5 mm in (A), 50 μm in (D), (E), (I), and (J), and 100 μm in (F).

Figure 2.

Phenotypes of the FIL_pro:WOX1 Plants. (A) Live image of rosette leaves. The dashed oval denotes a leaf with leaflet-like structures. (B) to (F) The phenotypes of the FIL_pro:WOX1 plants. (B) A bifurcated leaf (arrowhead) with abaxial protrusions (arrows). (C) An abaxial protrusion (arrow) and long, rectangular cells (asterisks) on the abaxial side of a mature leaf. (D) An abaxial ridge with a hydathode-like structure (arrow) and long, rectangular cells (arrowheads) on the abaxial side of a leaf. (E) Cross section of an abaxial ridge consisting of compactly packed mesophyll cells (asterisks). (F) Adventitious outgrowths on the abaxial sides (arrowheads) and around the margins (arrows) of leaf primordia. (G) The relative level of WOX1 expression. (H) The relative expression levels of the abaxial-specific genes. FpG, FIL_pro:GUS; FpW, FIL_pro:WOX1. Data are represented as the means ± sd in (G) and (H). Bars = 1 mm in (B) and 100 μm in (C) to (F).

Figure 3.

Functions of PRS and WOX1 in the Margin Development of Leaf Primordia. (A) to (D) Scanning electron micrographs of the leaf margins of the genotypes as indicated. Arrowheads indicate long, rectangular margin cells. (E) and (F) The leaf margin of mature leaves in cross section. Asterisks, margin-specific compactly packed mesophyll cells; cyan dots, adaxial air spaces; orange dots, abaxial palisade-like cells. (G) The number of hydathodes. Data are represented as the means ± sd. ***P < 0.001 by Tukey’s HSD test. Numbers in the bars indicate the number of samples. (H) Fluorescence of KLU_pro:vYFPer in cross sections of the leaf blades. Green, YFP fluorescence; magenta, chlorophyll autofluorescence; asterisks, leaf edge. Bars = 100 μm in (A) to (F) and 50 μm in (H).

Figure 4.

Adaxial/Abaxial Patterning of Tissue Differentiation in the Wild-Type and prs wox1 Leaves. (A) and (B) Scanning electron micrographs of the adaxial surface of prs wox1. Yellow dashed lines, the boundary between the adaxial-type epidermis and abaxial-type epidermis. (C) and (D) Scanning electron micrographs of the abaxial (C) and adaxial (D) surfaces of the wild-type leaves. (E) The number of abaxial trichomes. Data are represented as the means ± sd. ***P < 0.001 by Tukey’s HSD test. In total, 11 wild-type and prs-2 and 9 wox1-101 and prs-2 wox1-101 samples were assessed. (F) and (G) Scanning electron micrographs of the wild-type (F) and prs wox1 leaves (G) in cross section. Asterisks, leaf edges; arrowheads, abaxial trichomes. (H) and (I) The medial region of the eighth leaves in cross section in the wild type (H) and prs wox1 (I). (J) Schematic view of leaf margin phenotypes. Bars = 500 μm in (A), (F), and (G), 200 μm in (C) and (D), and 100 μm in (H) and (I).

Figure 5.

Expression Patterns of AS2, FIL, and 35S_pro:miYFP-W in the Wild-Type and prs wox1 Leaf Primordia. (A) RNA ISH in cross sections of leaf primordia with AS2 and FIL probes. Asterisks, edges of leaf primordia; ladders, cell layers along the adaxial-abaxial axis; arrowheads, edges of the region where genes are expressed. (B) Schematic view of expression patterns of AS2 and FIL. (C) YFP fluorescence of 35S_pro:miYFP-W in cross sections of shoot apices. M, SAM; green, YFP fluorescence; red, chlorophyll autofluorescence. Bars = 50 μm in (A) and 100 μm in (C).

Figure 6.

Control of PRS and WOX1 Expression by the FIL/YAB and KAN Genes. (A) The relative expression levels of PRS and WOX1. Data are represented as the means ± sd. ***P < 0.001 by paired Student’s t test. (B) and (C) RNA ISH of cross sections of shoot apices with PRS (B) and WOX1 (C) probes. M, SAM. (D) RNA ISH of cross sections of leaf primordia with KAN1 probe. (E) The relative levels of expression of PRS and WOX1 with or without DEX and with or without CHX in each line as indicated. Data are represented as the means ± sd. Asterisks indicate a significant difference between expression levels with and without DEX (***P < 0.001 by Student’s t test). Bars = 100 μm in (B) and (C) and 50 μm in (D).

Figure 7.

Genetic Interaction between two WOX Genes and KAN Genes. (A) to (F) Scanning electron micrographs of the abaxial surfaces of leaves ([A] to [D]) and leaf primordia ([E] and [F]) for indicated genotypes. Arrowheads in (A), (C), and (E), abaxial protrusions; asterisks in (C), long, rectangular cells similar to margin cells; asterisks in (E), stipules; arrows in (F), abaxial trichomes. (G) and (H) RNA ISH of cross sections of shoot apex with AS2 probe. Arrowheads in (G), edges of AS2 expression domain; M, position above the SAM. (I) to (K) Phenotype of 35S_pro:KAN1:GR plants without (I) and with ([J] and [K]) DEX. Asterisks indicate elongated cells located at the margin. Bars = 1 mm in (A), (B), (I), and (J) and 100 μm in (C) to (H) and (K).

Figure 8.

Phenotypes of prs wox1 as2 and prs wox1 fil yab3. (A) to (I) Phenotypes of prs-2 wox1-101 ([A] and [D]), as2-1 ([B] and [E]), and prs wox1 as2 ([C], [F], and [G]). (A) to (C) Live images of 8-d-old plants with genotypes as indicated. (D) to (G) Scanning electron micrographs of leaf primordia ([D] to [F]) and developed filamentous leaves (G). (H) and (I) FIL expression patterns in cross sections of shoot apex. (J) to (O) Phenotypes of fil-1 yab3-2 ([J] and [M]) and prs wox1 fil yab3 ([K], [L], [N], and [O]). (J) and (K) Live images of eight-d-old mutants. (L) to (O) Scanning electron micrographs of leaves ([L] and [O]) and leaf primordia ([M] and [N]). The first leaf (L) and the third or later leaves ([M] to [O]). Arrowheads in (N), abaxial trichomes. Bars = 2 mm in (A) to (C) and (J) to (L) and 100 μm in (D) to (I) and (M) to (O).

Figure 9.

Schematic View of the Gene Expression Patterns and a Model of Early Leaf Development. (A) Schematic view of gene expression patterns in cross sections of P3 and P4-P6 leaf primordia. (B) Schematic view of the adaxial/middle/abaxial patterns in cross sections of leaf primordia that were observed in this study. (C) A model of early development of the leaf. A network among adaxial, middle, and abaxial domain–specific genes (below) regulates the establishment and maintenance of the three-domain structure. Lp, leaf primordia.