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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00622

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Novel as1 and as2 defects in leaf adaxial-abaxial polarity reveal the requirement for ASYMMETRIC LEAVES1 and 2 and ERECTA functions in specifying leaf adaxial identity

Lin Xu1,2, Yi Xu1, Aiwu Dong3, Yue Sun4, Limin Pi1, Yuquan Xu2 and Hai Huang1,*

1 National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
2 College of Life Science and Biotechnology, Shanghai Jiao Tong University, 1954 Hua Shan Road, Shanghai 200030, China
3 College of Life Sciences, Fudan University, 220 Han Dan Road, Shanghai 200433, China
4 College of Life Sciences, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China



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  		Fig. 1. The lotus-leaf structure of as1-101 and as2-101 mutants in the Ler genetic background. (A) Ler; (B) as1-101 and (C) as2-101 seedlings. Note that first pairs of rosette leaves in the as1 and as2 mutants often show the lotus-leaf structure (arrows). c, cotyledon. (D-L) Each panel shows one of the first pairs of rosette leaves, all of similar ages, and photos were taken from an adaxial view. (D) A Ler rosette leaf with an asymmetric petiole in the adaxial-abaxial axis. (E,F) as2-101 rosette leaves showing petioles with radially symmetric portions that vary in length (arrowheads). Note that the petiole portions below arrowheads are radially symmetric. (G-I) as2-101 rosette leaves with a varying degree of severity of the lotus-leaf structure. (J) A needle-like structure of an as2-101 seedling. (K) An as2-1 rosette leaf with the radially symmetric portion in the petiole (arrowhead). (L) An as1-1 petiole. Scale bars:1 mm (A-C); 0.2 mm (D-L).

 


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  		Fig. 2. Transverse sections showing the effects of the as2 mutation on petiole anatomy. (A) Ler petiole. (B) Lotus-leaf petiole of as2-101. (C) Non-lotus-leaf petiole of as2-101. Note that three vascular bundles were seen in the mutant. d, adaxial epidermis between two arrowheads; b, abaxial epidermis between two small arrows; pa, parenchyma; vs, vascular bundle. The small and dense epidermal cells between the arrowheads and small arrow are the petiole margin epidermis in the Ler plant. Scale bars: 0.1 mm.

 


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  		Fig. 3. Phenotypes of 35S::AS1 and 35S::AS2 transgenic plants. (A) Seedling carrying 35S::AS2 in the Ler background. (B) A plant containing 35S::AS2 in the as2-101 mutant background. (C) Plant carrying the 35S::AS1 in the Ler background showing dark green leaves. (D-F) Adaxial lamina epidermis of (D) Ler, (E) 35S::AS2/Ler and (F) 35S::AS1/Ler. Note that since leaves of 35S::AS2/Ler seedlings are curled upwards, only the central portion of the adaxial surface is visible. (G-I) Abaxial lamina epidermis of (G) Ler, (H) 35S::AS2 and (I) 35S::AS1. First pairs of rosette leaves were used for the SEM images. t, three-branched trichome. Scale bars: 1 mm (A-C); 0.05 mm (D-I).

 


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  		Fig. 4. SEM of epidermal cells of Ler, as2-101 and 35S::AS2/Ler leaves. (A) Adaxial surface of the proximal part of a Ler lamina. (B) The boxed region of A showing a close-up of two types of epidermal cells. (C) Adaxial epidermal cells on a Ler petiole. Arrowhead, margin cells; arrow, adaxial cells. (D) The proximal part of the adaxial surface of an as2-101 lamina. (E) The boxed region of D showing a close-up of the long and narrow epidermal cells. (F) Adaxial epidermal cells on an as2-101 petiole, showing that all epidermal cells on the petiole are long and narrow. Note that these cells show a very similar pattern to those found on the proximal part of the as2-101 leaf lamina in D, and are also similar to petiole margin epidermal cells of Ler. (G) Epidermal cells of an adaxial 35S::AS2 petiole. (H) The boxed region of G showing the uniformly shaped adaxial epidermis (white arrowhead) and the thick midvein-like epidermis (black arrowhead). (I) Abaxial side of a 35S::AS2 petiole with mosaic adaxial and abaxial patches. Scale bars: 0.2 mm (A,D); 0.1 mm (B,C,E-G,I); 0.05 mm (H).

 


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  		Fig. 5. SEM of needle-like leaves in as2-101 and 35S::AS2//Ler plants. (A) A first as2-101 rosette leaf with needle-like structure. (B-D) The bottom, middle and top boxed areas of A, respectively. (B) The epidermal cells of the proximal region of this structure mimic those of the petiole abaxial cells in Ler plants, and are also similar to those of phan-607 needle-like leaves (Waites and Hudson, 1995Go). (C) In the more distal portion, cells differentiate into abaxial epidermis (white arrowhead) with some intermediate cells between the jigsaw-shaped abaxial epidermal cells and the petiole abaxial cells in the Ler (black arrowhead). (D) At the tip of the needle-like organ, all cells are abaxilized. (E) A first appearing 35S::AS2//Ler leaf with needle-like structure. (F-H) The bottom, middle and top boxed areas of E, respectively. Note that the epidermal pattern of this structure is similar to that of the needle-like leaves in the phb-1d mutant (McConnell and Barton, 1998Go). Scale bars: 0.2 mm (A,E); 0.1 mm (B,C,F,G); 0.05 mm (D,H).

 


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  		Fig. 6. Overexpression of AS2 suppresses KNAT1 expression. (A-F) Same-stage inflorescences. (A) Ler, (B) as1-101, (C) 35S::AS1/Ler, (D) as2-101, (E) 35S::AS2/Ler, (F) bp. Note that 35S::AS2 transgenic plants and bp mutant plants exhibit similar altered inflorescence architecture, with downward-pointing siliques. (G) RT-PCR. RNA was extracted from the primary inflorescence, and the amplified DNA fragments were separated by electrophoresis on an agarose gel and visualized by staining with ethidium bromide. Scale bars: 1 mm (A-F).

 


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  		Fig. 7. Phenotypes of 35S::AS1/as2 and 35S::AS2/as1 transgenic plants. (A) 35S::AS1/as2-101 seedling that is similar to the as2 mutant. (B) 35S::AS2/as1-101 seedling that resembles the as1 mutant. Note, the curled leaves in 35S::AS2/Ler were not seen in the 35S::AS2/as1-101 plants. (C) 35S::AS2/Ler inflorescence with a downward-pointing flower. (D) 35S::AS2/as1-101 inflorescence. Flowers are not downward-pointing. Scale bars: 1 mm.

 


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  		Fig. 8. AS1 and AS2 interaction in yeast and in ELISA. (A,B) Yeast two-hybrid analyses by coexpression of AS1 and AS2, showing that the interaction of AS1 and AS2 turns on the reporter genes lacZ (A) and HIS3 and ADE2 activities (B). (C) Recombinant purified proteins His-AS1 and GSTAS2 were analyzed by SDS-PAGE and stained by Coomassie Blue R-250. Note, sizes of the recombinant His-AS1 and GST-AS2 proteins agreed with their predicted molecular masses. M, molecular mass marker (in kDa); GST, glutathione S-transferase control; GST-AS2, GST tag and AS2 fusion protein; His-AS1, His tag and AS1 fusion protein. (D) ELISA was performed showing protein-protein interaction between the His-AS1 and GST-AS2.

 


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  		Fig. 9. A genetic model for AS1, AS2 and ER actions in establishment of the leaf adaxial-abaxial polarity. lp, leaf primodium; ad, adaxial side; and ab, abaxial side.

 

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© The Company of Biologists Ltd 2003