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Comparative Study
. 2005 Jan;17(1):77-91.
doi: 10.1105/tpc.104.026898. Epub 2004 Dec 17.

Asymmetric Auxin Response Precedes Asymmetric Growth and Differentiation of Asymmetric leaf1 and Asymmetric leaf2 Arabidopsis Leaves

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Free PMC article
Comparative Study

Asymmetric Auxin Response Precedes Asymmetric Growth and Differentiation of Asymmetric leaf1 and Asymmetric leaf2 Arabidopsis Leaves

Jessie M Zgurski et al. Plant Cell. .
Free PMC article

Abstract

We have analyzed the development of leaf shape and vascular pattern in leaves mutant for ASYMMETRIC LEAVES1 (AS1) or AS2 and compared the timing of developmental landmarks to cellular response to auxin, as measured by expression of the DR5:beta-glucuronidase (GUS) transgene and to cell division, as measured by expression of the cycB1:GUS transgene. We found that the earliest visible defect in both as1 and as2 first leaves is the asymmetric placement of auxin response at the distal leaf tip. This precedes visible changes in leaf morphology, asymmetric placement of the distal margin gap, formation of margin gaps along the leaf border, asymmetric distribution of marginal auxin, and asymmetry in cell division patterns. Moreover, treatment of developing leaves with either exogenous auxin or an auxin transport inhibitor eliminates asymmetric auxin response and subsequent asymmetric leaf development. We propose that the initial asymmetric placement of auxin at the leaf tip gives rise to later asymmetries in the internal auxin sources, which subsequently result in asymmetrical cell differentiation and division patterns.

Figures

Figure 1.
Figure 1.
Development of Wild-Type, as1-16, and as2-1 First Leaves. (A) to (C) The wild type. (D) to (F) as1-16. (G) to (I) as2-1. At 5 DAG, elongated margin cells differentiate basipetally, beginning adjacent to a group of nonelongated cells that remains at the distal leaf tip (the distal margin gap) in all genotypes ([A] and [B], [D] and [E], and [G] and [H]). In both as1 and as2 leaves, the distal margin gap may be asymmetrically placed (arrows in [D]). At 10 DAG, files of margin cells are continuous in the wild type (C) but show gaps in as1-16 and as2-1 (indicated by arrows in [F] and [I]). Scale bars = 50 μm.
Figure 2.
Figure 2.
Development of Wild-Type, as1-16, and as2-1 Fifth Leaves. (A) to (D) The wild type. (E) to (H) as1-16. (I) to (L) as2-1. At 11 DAG, elongated margin cells differentiate in all genotypes ([A], [B], [D], [E], [I], and [J]), beginning adjacent to the distal margin gap. In as1 and as2 leaves, the distal margin gap may be asymmetrically placed (arrows in [F] and [I]). At maturity, margin cells usually form continuous files in the wild type (C), although rarely a margin gap is seen (arrows in [D]). Margin gaps are common in as1-16 and as2-1 leaves (arrows in [G], [H], [K], and [L]). Scale bars = 50 μm.
Figure 3.
Figure 3.
CycB1:GUS Expression in Developing First Leaves of the Wild Type and as1-16 and as2-1 Viewed with Phase Contrast or Bright-Field Optics. (A) to (C) The wild type. (D) to (F) as1-16. (G) to (I) as2-1. (A), (D), and (G) Phase contrast images. (B), (C), (E), (F), (H), and (I) Bright-field images. CycB1:GUS expression is similar in the wild type and as1-16 at 4 DAG ([A] and [D]) but is expressed in fewer cells in as2-1 (G). At 5 DAG, cycB1:GUS expression is lower in the distal half compared with the proximal half of all leaves ([B], [E], and [H]), with fewer cells expressing cycB1:GUS in as2-1 leaves. At 7 DAG, very few cells express cycB1:GUS in leaf distal halves, and in the proximal half, asymmetric expression is seen on either side of the midvein in as1-16 and as2-1 ([F] and [I]). Scale bars = 0.1 mm.
Figure 4.
Figure 4.
Vascular Pattern Development in the Wild Type, as1-16, and as2-1 Viewed with Phase Contrast or Dark-Field Optics. (A), (F), and (G) The wild type (B), (E), and (H) as1-16. (C), (F), and (I) as2-1. (A), (D), and (G) Phase contrast images. (B), (C), (E), (F), (H), and (I) Dark-field images. At 5 DAG, the xylem of the midvein is differentiating in the wild type (A) but not in the mutants ([D] and [G]). Distal secondary veins are initiated from the wild type 5 DAG ([A], [D], and [G]) but may be asymmetrically placed in the mutant (asterisks in [G] and [E]). Distal secondary maturation is evident by 6 DAG ([B], [E], and [H]), with the proximal point of secondary joining to the midvein lower in mutants (arrows in [E] and [H]) than in the wild type (arrows in [B]). Proximal secondaries are differentiating by day 7 and are often initiated at a more distal position in mutant leaves (asterisks in [F] and [I]) than in the wild type (asterisk in [C]).
Figure 5.
Figure 5.
DR5:GUS Expression in Developing First Leaves of the Wild Type, as1-16, and as2-1 Viewed with Phase Contrast Optics. (A), (D), (G), and (J) The wild type. (B), (E), (H), and (K) as1-16. (C), (F), (I), and (L) as2-1. At 3 DAG, DR5:GUS expression is visible in the distal tip of wild-type leaves (A), but this expression is often asymmetrically placed in as1-16 (B) and as2-1 (C) leaves. By 4 DAG, expression of DR5:GUS within the mutant leaf blades ([E] and [F]) is usually indistinguishable from the wild type (D), although asymmetries may exist within the blade or more frequently at the distal tip (E). At 6 DAG, DR5:GUS is expressed strongly in hydathodes (arrows), usually at opposite points along the leaf blade in the wild type (G), but often asymmetrically placed in as1-16 (H) and as2-1 (I). At high magnification, hydathodes show a larger number of small cells expressing GUS in as1-16 (K) and as2-1 (L) than in the wild type (J). Scale bars = 0.1 mm in (A) to (C) and (J) to (L), 0.2 mm in (D) to (F), and 0.25 mm in (G) to (I).
Figure 6.
Figure 6.
DR5:GUS Expression in Developing Leaves of the Wild Type and as1-16 Treated with 10−7 M 2,4-D, 10−6 2,4-D, and 10 μM NPA Viewed with Phase Contrast Optics or Differential Interference Contrast Optics. (A) to (D), (I) to (L), and (Q) to (T) The wild type. (E) to (H), (M) to (P), (U) to (X) as1-16. (A) to (H) Treated with 10−7 M 2,4-D. (I) to (P) Treated with 10−6 2,4-D. (Q) to (X) Treated with 10 μM NPA. (A), (B), (E), (F), (I), (K), (M), (O), (Q), (S), (U), and (W) Phase contrast images. (C), (D), (G), (H), (L), (P), (T), and (X) Differential interference contrast images. At 3 DAG, leaves treated with 10−7 M 2,4-D show DR5:GUS expression in the distal tip, which is somewhat more intense than in untreated leaves, and symmetrically placed in wild-type leaves (A), but is often asymmetrically placed in as1-16 (E) leaves. By 5 DAG, expression of DR5:GUS within the treated mutant leaf blades (F) is usually indistinguishable from the wild type (B). At 6 DAG, DR5:GUS is expressed strongly in hydathodes (arrows), usually at opposite points along the leaf blade in the wild type (C) but often asymmetrically placed in as1-16 (G). At 10 DAG, 10−7 M 2,4-D–treated wild-type leaves show symmetrical shape and vascular pattern (D), whereas in as1-16, shape and vascular pattern are often asymmetric (H). At 4 DAG, leaves treated with 10−6 M 2,4-D of both the wild type (I) and as1-16 (M) express DR5:GUS in a distal maximum containing more cells than that in untreated leaves. At 5 and 6 DAG in the wild type ([J] and [K]) and as1-16 ([O] and [P]), distal maximum expression increased both in intensity and number of cells. At 10 DAG, both wild-type (L) and as1-16 (P) leaves are small with little vascular development. At 4 DAG, leaves treated with 10 μM NPA of both the wild type (Q) and as1-16 (U) express DR5:GUS at a low level in a group of often disconnected cells across the distal region of the primordium. At 5 and 6 DAG in the wild type ([R] and [S]) and as1-16 ([V] and [W]), DR5:GUS is expressed in two loops of cells; the inner loop at 6 DAG is coincident with developing vascular tissue. At 10 DAG, both wild-type (T) and as1-16 (X) leaves have a loop of vascular tissue adjacent to the leaf margin with veins extending from the margin into the leaf interior. Scale bars = 0.05 mm in (A), (B), (E), (F), (I), (K), (M), (O), (Q), (R), (U), and (V), 0.1 mm in (C), (G), (P), (S), and (W), and 0.5 mm in (D), (H), (T), and (X).

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