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First published online May 28, 2004
doi: 10.1242/10.1242/dev.01186

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Asymmetric leaf development and blade expansion in Arabidopsis are mediated by KANADI and YABBY activities

Yuval Eshed^1,2,*,, Anat Izhaki^1,*, Stuart F. Baum^1,, Sandra K. Floyd¹ and John L. Bowman^1,

¹ Section of Plant Biology, Division of Biological Sciences, University of California, One Shields Avenue, Davis, CA 95616, USA
² Department of Plant Sciences, The Weizmann Institute of Science, Rehovot, 76100 Israel

View larger version (102K): [in a new window]   Fig. 1. KANADI loss-of-function morphological phenotypes. (A-C) All three KANADI genes, KAN1 (A), KAN2 (B), and KAN3 (C) are expressed in the abaxial regions of developing embryos. Only one presumptive cotyledon is visible in the section showing KAN3 expression. Unlike the mild or lack of phenotypic alterations of the single mutants kan1 and kan2, respectively, kan1-2 kan2-1 plants exhibit gross morphological aberrations in all lateral organs. (D) A kan1-2 kan2-1 plant with narrow leaves which have outgrowths formed on their abaxial side (D). (G,H) The abaxial outgrowths (arrows) are visible shortly after leaf primordia have expanded, appearing first as a row along the bottom third of the leaf, and later in a less organized pattern as the leaf elongates. (E) A wild-type plant. (F) In wild-type leaves, two stipules (*) are formed on the flanks of each leaf, obscuring the apical meristem from view. (I) Leaves of kan1-2 kan2-1 kan3-1 plants exhibit much less lamina expansion than do those of the double mutant, being radial except at their distal tips. (L) Leaves of the triple mutant are radial at inception, but they still retain some polar characteristics, such as a lack of abaxial trichomes on the first two leaves. (J-L) As in phb-1d homozygotes, axillary meristems (arrows) may form on the abaxial sides of kan1 kan2 kan3 leaves (J,K) and similar to kan1-2 kan2-1 and phb-1d/+ leaves, stipules (arrows) develop all around the base of the leaves (L). (M) Disorganized blade tissue often forms at the distal tips of kan1 kan2 kan3 leaves. ab, abaxial; ad, adaxial.

View larger version (162K): [in a new window]   Fig. 2. KANADI loss-of-function anatomical phenotypes. (A,B) Longitudinal and transverse sections of 12-day-old wild-type seedlings demonstrate that young leaf primordia exhibit polarity along the ab/ad axis immediately after their separation from the shoot apical meristem. Polarity is evident both by their crescent shape and the appearance of vacuolated cells (arrows) on the abaxial side first (B). (C) In differentiating leaves, asymmetric anatomy along the ab/ad axis is evident in the shape of the adaxial palisade mesophyll versus the abaxial spongy mesophyll. Anticlinal cell divisions characterize the L1 and L2 cell layers both adaxially and abaxially (insert; arrow). (D,E) In contrast, leaf primordia of kan1-2 kan2-1 12-day-old seedlings appear radial (arrows), with all cells maintaining their densely cytoplasmic appearance for a prolonged period. (F) Many more cell layers are found in differentiating leaves, resulting from abnormal periclinal divisions at the abaxial side (insert; arrows). (G-I) Leaf primordia of kan1-2 kan2-1 kan3-1 seedlings (G,H) are also radial at inception (arrows), but do exhibit some asymmetric growth later in development such that the leaves are also thicker than those of wild type (I). The transverse sections of the leaves in C, F, and I are from the proximal region of expanding leaves of 12-day-old seedlings. *, stipules; ab, abaxial; ad, adaxial; lp, leaf primordia; m, meristem; pm, palisade mesophyll; sm, spongy mesophyll. Scale bars: 50 µm.

View larger version (97K): [in a new window]   Fig. 3. Gene expression patterns in kan1 kan2 kan3 and AS1>>KAN2 leaves. (A) GUS activity, due to an enhancer trap in the YAB3 gene, is localized to the abaxial regions of developing leaves (arrowheads) in yab3-2 plants. (B) In contrast, no detectable GUS activity is detected in kan1-2 kan2-1 kan3-1 yab3-2 plants. (F) In wild-type plants PHB expression is localized to the apical meristem and the adaxial regions of lateral organs. (C,D) In a kan1 kan2 kan3 background, PHB expression is no longer adaxially confined in developing leaves, but still displays a gradient with the highest level adaxially. (E) When KANADI activity is expressed uniformly throughout leaves, such as when KAN2 is driven by the AS1 promoter, the leaves are both radialized with no lamina formation and abaxialized. (G,F) PHB expression is not detected in AS1>>KAN2 leaves (G) whereas PHB is expressed adaxially in wild-type leaves (F). (H) FIL is expressed abaxially in wild-type leaf primordia, and its expression becomes localized to the abaxial marginal regions as leaves differentiate. (I) In contrast, FIL is only transiently expressed in the radial abaxialized AS1>>KAN2 leaf primordia. ab, abaxial; ad, adaxial; lp, leaf primordium; m, meristem.

View larger version (102K): [in a new window]   Fig. 4. The nature of the kan1 kan2 blade outgrowths. (A) The outgrowths developing on the abaxial side of kan1-2 kan2-1 leaves appear nearly radial, with cell types normally found on leaf margins positioned around their entire circumference. (B) In wild-type seedlings, these cells show blue staining when the GUS reporter is driven by the enhancer trap YJ158. (C) This reporter drives GUS throughout the epidermis of the kan1 kan2 blade outgrowths and in scattered cells on the abaxial leaf surface. (D) Transverse section through a mature wild-type leaf displaying an asymmetric anatomy both within the leaf and in the vascular bundle. (E,F) In mature kan1 kan2 leaves, numerous vascular bundles are formed, connecting the outgrowths to leaf main bundles (E). The radial outgrowths have nearly radial anatomy, with a large bundle (arrow) found in their center (F). (G) Close-up of a wild-type minor leaf bundle showing xylem vessel members (arrow) positioned adaxially, while phloem sieve tube elements (arrow) are located abaxially. (H) In kan1 kan2 the central bundles of the outgrowths have clusters of phloem tissue surrounding xylem vessel members. (I,J) The prolonged period of cell division in the abaxial region of kan1 kan2 leaves is responsible for the formation of outgrowths, reflected by the maintenance of the densely cytoplasmic appearance of these cells (I), and correlated with prolonged localized FIL mRNA expression (J). (K) Earlier in leaf development, high levels of FIL expression demarcate presumptive outgrowths (arrow) while a lower level of FIL expression is throughout the abaxial region of the leaves. ab, abaxial; ad, adaxial; ph, phloem; xy, xylem. Scale bars: 1 mm (A), 50 µm (B-K).

View larger version (145K): [in a new window]   Fig. 5. Simultaneous loss of both KANADI and YABBY functions. (A) In contrast to the clear asymmetric development of kan1 kan2 leaves, kan1 kan2 fil yab3 quadruple mutants initiate nearly radial first leaves. The two genotypes, kan1-2 kan2-1 fil-5 yab3-1 and kan1-2 kan2-1 fil-8 yab3-2, are indistinguishable in morphology. (B) The leaves have trichomes on all sides, an adaxial characteristic of the first leaves, and expand laterally only at their distal end. Later-formed leaves are short, thick and lack organized plane of expansion. (C-E) The epidermal surfaces of these leaves (E) appear similar on both sides, and resemble those of adaxial leaf surfaces (C), rather than the abaxial leaf surfaces (D) of wild-type leaves. (F,K) Flower organs of the quadruple mutants are completely radialized (F), lack epidermal characteristics of wild-type floral organs, and resemble the flowers of adaxialized homozygous phb-1d mutants (K). (G) Longitudinal section through a developing third leaf of a kan1-2 kan2-1 fil-5 yab3-1 quadruple mutant reveals anatomical symmetry. (H,I) This symmetry is also apparent in by transverse sections of either young leaf primordia (H) or a differentiating leaf (I). (J) In a similar fashion to its expression in wild-type leaves, FIL mRNA is detected in the margins of the quadruple mutant leaf where prolonged cell divisions occurs, yet this expression is no longer abaxial as seen in this transverse section through two leaves above the level of the meristem. (L) Leaves of fil-5 yab3-1 show dramatic loss in their polarity as expressed by trichome distribution on both sides of the first two leaves, when the activity of the KANADI proteins is partially compromised. This phenotype, is in sharp contrast to the normal distribution of trichomes in fil-5 yab3-1 alone (inset; first three leaves removed). (M) Besides the distribution of cell types along the ab/ad leaf axes, all the described mutations have a strong effect on growth along the proximal/distal axis. In general, increasing losses of asymmetric development are accompanied by reduced leaf elongation. All leaves shown, except for no. 5 are fully expanded 4th or 5th leaves of the respective genotypes. (1, wild type; 2, kan1 kan2 adaxial surface; 3, kan1 kan2 abaxial surface; 4, fil5 yab3-1; 5, kan1-2 kan2-1 fil-5 yab3-1 first leaf; 6, kan1-2 kan2-1 fil-5 yab3-1 5th leaf; 7, phb-1d. (Bars to the left of each leaf: 5 mm.) ab, abaxial; ad, adaxial; lp, leaf primordium; m, meristem; wt, wild type. Scale bars: 100 µm (C-E and G-I).

View larger version (153K): [in a new window]   Fig. 6. YABBY and lamina expansion. (A) In kan1-2 kan2-1 syd-2 leaves, no abaxial outgrowths form, and the leaves are radialized. (B) PHB, normally confined to the adaxial regions of the developing leaves, is expressed throughout kan1 kan2 syd leaves. (C,D) Consistent with YABBY gene expression being responsible for the abaxial outgrowths on kan1-2 kan2-1 leaves, FIL expression is greatly reduced in kan1-2 kan2-1 syd-2 triple mutants as seen in both longitudinal (C) and transverse (D) sections of developing leaves. (E,F) In developing Solanum tuberosum leaves (E), YABBY1 expression is associated with abaxial regions in which cells are cytoplasmically dense (F). These regions include the entire abaxial domain in young leaf primordia (*), and in differentiating leaves, the marginal abaxial regions (arrows) and towards the center of the leaf, along the boundary between the abaxial and adaxial domains (arrowhead). lp, leaf primordium; m, meristem.