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First published online 20 July 2005
doi: 10.1242/dev.01942

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Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166g and its AtHD-ZIP target genes

Leor Williams¹, Stephen P. Grigg^1,*, Mingtang Xie², Sioux Christensen² and Jennifer C. Fletcher^1,

¹ Plant Gene Expression Center, USDA/UC Berkeley, 800 Buchanan Street, Albany, CA 94710, USA
² Dept of Molecular, Cellular and Developmental Biology, UCLA, Los Angeles, CA 90095, USA

View larger version (98K): [in a new window]   Fig. 1. jba-1D morphological phenotypes are dose dependent. (A) Wild-type Columbia (Col) inflorescence stem. (B) Fasciated jba-1D/+ stem. (C) Severely fasciated jba-1D stem. (D) SEM of a wild-type inflorescence meristem. Inset: an enlargement of the shoot tip (the boxed region). (E) SEM of a fasciated jba-1D inflorescence meristem. (F) Wild-type silique consisting of two fused carpels. (G) Reduced and filamentous jba-1D/+ siliques. (H) Filamentous jba-1D silique. (I) Radialized leaf projecting outward from the center of a jba-1D inflorescence meristem. (J) Wild-type rosette leaf, viewed from the abaxial side. (K) Slightly downward curled jba-1D/+ rosette leaf. (L) Severely downward curled jba-1D rosette leaf. (M) Completely radialized rosette leaf from a jba-1D seedling. Scale bars: 60 µm (D,E).

View larger version (107K): [in a new window]   Fig. 2. jba-1D shoot apical meristem phenotypes. (A) Wild-type inflorescence meristem at the transition to flowering. (B) jba-1D/+ inflorescence meristem that is both taller and wider than normal. (C) jba-1D inflorescence meristem that has split into multiple independent meristems (*), each generating floral meristem primordia on its flanks. (D) Wild-type seedling SAM after 11 days of vegetative growth. (E) Enlarged jba-1D seedling SAM. (F) Mean width and height of Col (n=12) and jba-1D (n=11) SAMs from 11-day-old seedlings. (G) Confocal micrograph of a wild-type mature embryo SAM. (H) Confocal micrograph of an enlarged jba-1D mature embryo SAM. (I) GUS expression from a pSTM::GUS reporter construct in the peripheral region (arrows) of a wild-type seedling SAM. (J) pSTM::GUS expression in a jba-1D seedling delimits two well-defined meristems, while the leaf primordium developing between them is radialized (arrowhead). Scale bars: 80 µm (A-E); 20 µm (G-H).

View larger version (76K): [in a new window]   Fig. 3. WUS and CLV3 expression in jba-1D shoot apical meristems. (A) GUS expression from a pWUS::GUS reporter construct in a wild-type seedling SAM, after 1 hour incubation in X-Gluc substrate. (B) No pWUS::GUS activity is detected in a wild-type seedling SAM after 20 minute incubation in X-Gluc substrate. (C) pWUS::GUS activity is readily detected in a jba-1D seedling SAM after 20 minute incubation in X-Gluc substrate. The WUS expression domain also expands laterally in the jba-1D SAM. (D) In some jba-1D seedlings, two independent foci of WUS expression are observed (arrows). (E) GUS expression from a pCLV3::GUS reporter construct in a wild-type seedling SAM. (F) The CLV3 expression domain expands laterally in a jba-1D seedling SAM, coordinate with the expansion of the WUS expression domain. All seedlings are 11 days old. (G) CLV3 and WUS mRNA transcription levels in Col and jba-1D inflorescences and floral meristems as determined by real-time qRT-PCR. Scale bars: 40 µm.

View larger version (118K): [in a new window]   Fig. 4. Defective jba-1D inflorescence stem vasculature and lateral organ polarity. (A) Cross section through a wild-type stem. (B) Higher magnification of the boxed region in A. The vascular bundle consists of xylem (xy) located on the inner side of the bundle and phloem (ph) located on the outer side of the bundle. (C) Cross section through a jba-1D stem showing additional vascular bundles at the periphery and ectopic vascular bundles in the center of the stem. (D,F) Higher magnification of the boxed region in C and the boxed region in D, respectively. Xylem elements can be detected ectopically on the outer side of the bundle. (E) Higher magnification of the vascular bundles located at the center of the jba-1D stem (smaller box in C). The vascular bundles are radialized, with xylem cells (arrowheads) surrounding phloem cells. (G,H) Cross section through a radialized jba-1D leaf. Higher magnification the vascular bundles (H) shows a polarity in the vascular bundle, with phloem cells at one side of the bundle and xylem cells protruding to the other side. Scale bars: 500 µm (A,C); 50 µm (B,D,E,H,F); 100 µm (G).

View larger version (56K): [in a new window]   Fig. 5. Elevated expression of miR166 in jba-1D plants. (A) Schematic of the insertion site of jba-1D relative to annotated genes. Green triangles indicate the 35S enhancers on the T-DNA vector. The orange line indicates the region of genomic DNA used to recapitulate the jba-1D phenotype. (B) Alignment of miR166 with its putative Arabidopsis class III HD-ZIP gene target sequences. The blue box indicates the miR166 sequence complementarity to the HD-ZIP genes sequences. (C) miR166 expression. Blot of low molecular mass RNA extracted from seedlings and inflorescences of wild-type and jba plants. The relative amount of miR166 accumulation in the various tissues is indicated below the autoradiograph. The ethidium bromide-stained gel is shown as loading control (bottom).

View larger version (110K): [in a new window]   Fig. 6. Expression patterns of miR166 and REV during embryogenesis. In situ localization of miR166 in wild-type (A-E) and jba-1D/+ (G-L) embryos. (A,G) At the late globular stage, miR166 is expressed in the peripheral region of the hypocotyl and at the tips of the initiating cotyledons (arrows). (B-D,H-J) During the heart and torpedo stages, miR166 expression expands to include the abaxial region and the distal tips of the cotyledons. (E,K) In mature embryos, miR166 expression becomes localized to the SAM, the adaxial region of the cotyledons and the provascular tissues. (F,L) Control hybridization with a miR166 sense probe, at the torpedo stage. (M-Q) REV expression in wild-type embryos. (M) REV is expressed in the central apical region of late globular stage embryos. (N) During the heart stage the REV expression expands to the adaxial region of the cotyledons and the central provascular tissues of the hypocotyl. (O,P) REV expression in early and late torpedo stages is similar to the heart stage. (Q) In mature embryos, REV is localized to the SAM, the adaxial regions of the cotyledons and the central provascular tissues. Scale bars: 500 µm.

View larger version (76K): [in a new window]   Fig. 7. Alteration of miR166g target gene expression levels in jba-1D plants. RT-PCR analysis of the class III HD-ZIP genes REV, PHB, PHV, CNA and ATHB8 was performed on RNA extracts from whole seedlings and inflorescences (IFMs) of wild-type and jba-1D plants. EF1 was amplified as a control.

View larger version (104K): [in a new window]   Fig. 8. REV, CNA and PHB expression patterns in wild-type and jba-1D seedlings. In situ localization of (A,B) REV, (C,D) CNA, and (E,F) PHB mRNA in longitudinal sections through 9-day-old seedlings. (A) Wild-type and (B) jba-1D seedlings showing REV expression in the central zone of the SAM, the adaxial regions of leaf primordia and the vascular tissues. (C) Wild-type seedling showing CNA expression in the central, interior cells of the SAM, the adaxial region of leaf primordia, and the vascular tissues. (D) In jba-1D seedlings, CNA is expressed at a low level in the adaxial region of initiating leaf primordia (arrow). CNA transcripts were not detected in the center of the SAM or in the vascular tissues. (E) Wild-type seedling showing PHB expression in the SAM, the adaxial region of leaf primordia, and the vascular tissues. (F) In jba-1D seedlings, PHB is expressed in the adaxial region of initiating leaf primordia (right arrow) and at a low level in the vascular tissues (left arrow). PHB transcripts were not detected in the SAM proper. For each probe, tissues from wild-type and jba-1D seedlings were analyzed on the same slide. Scale bars: 80 µm.

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