HUA1 and HUA2 Are Two Members of the Floral Homeotic AGAMOUS Pathway Xuemei Chen, Elliot M Meyerowitz  Molecular Cell  Volume 3, Issue 3, Pages 349-360 (March 1999) DOI: 10.1016/S1097-2765(00)80462-1

Figure 1 Phenotypic Effects of hua1 and hua2 in Flowers of Various Genetic Backgrounds (A) Wild-type. (B) ag-3. (C) ag-4. (D) ag-4 hua1 hua2. (E–I) Dissected third whorl organs of ag-4 hua1 hua2 (E), ag-4 hua1 (F), ag-4 hua2 (G), ag-4/ag-4 hua1/hua1 hua2/+ (H), and ag-4/ag-4 hua1/+ hua2/hua2 (I) flowers. (J) hua1. (K) hua2. (L–M) An early and a late hua1 hua2 flower, respectively. (N) 35S::AP1. Numbers indicate the positions of the whorls. Arrows indicate third whorl organs, stamens in (A) and (C), petals in (B) and (D), and petaloid stamens in (L). Arrowheads indicate bulges at the tips of carpels. Se, sepals; P, petals; St, stamens; C, carpels; l and m, lateral and medial third whorl organs, respectively. Molecular Cell 1999 3, 349-360DOI: (10.1016/S1097-2765(00)80462-1)

Figure 2 Phenotypes of Triple or Quadruple Mutants of hua1, hua2, and Various Homeotic Mutations (A–B) An ag-1/+ and an ag-1/+ hua1/hua1 hua2/hua2 flower, respectively. (C) Dissected third whorl organs of the flower in (B). St, a wild-type stamen; P, a wild-type petal; l and m, lateral and medial third whorl organs, respectively. The medial third whorl organs show anther characteristics (arrows). (D–G) ag-4/+ (D), ag-4/+ hua1/hua1 hua2/hua2 (E), ag-4/+ hua1/hua1 hua2/hua2 ap1-1/ap1-1 (F), and ag-4/+ ap1-1/ap1-1 (G) flowers. Arrows indicate third whorl organs, petaloid stamens in (E), and stamens in (F). (H–I) Dissected flowers of ap1-1 and ap1-1 hua1 hua2, respectively, to show the morphology of the gynoecia. (J–K) Flowers of ap2-2 and ap2-2 hua1 hua2, respectively. (L) An ag-1 hua1 hua2 flower. (M–N) Flowers of pi-3 and pi-3 hua1 hua2, respectively. (O–P) Flowers of ap3-3 and ap3-3 hua1 hua2, respectively. Molecular Cell 1999 3, 349-360DOI: (10.1016/S1097-2765(00)80462-1)

Figure 4 Localization of AP1 RNA by In Situ Hybridization (A and B) Longitudinal sections of a wild-type and a hua1 hua2 stage 10 or older flower, respectively. (C and D) Light and dark field photographs of a longitudinal section of a stage 6 ag-4/+ flower. (E and F) Light and dark field photographs of a stage 6 ag-4/+ hua1/hua1 hua2/hua2 flower. (G and H) Light and dark field photographs of a longitudinal section of a stage 10 or older ag-4/+ hua1/hua1 hua2/hua2 flower. The arrow indicates the additional floral meristem inside the fourth whorl. (I and J) Light and dark field photographs of a longitudinal section of an ag-4 stage 3–4 floral primordium. (K and L) Light and dark field photographs of a longitudinal section of an ag-4 hua1 hua2 inflorescence showing a stage 3–4 flower in the center. Se, sepal; C, carpel. Numbers indicate the positions of floral whorls. Size bars, 50 μm. Molecular Cell 1999 3, 349-360DOI: (10.1016/S1097-2765(00)80462-1)

Figure 3 Scanning Electron Micrographs of Petals and Stamens of Various Genotypes (A and B) Adaxial and abaxial surfaces of a wild-type petal, respectively. (C) Abaxial surface of an ag-3 third whorl petal. (D and E) Abaxial and adaxial surfaces of ag-4 stamens, respectively. (F and G) Low and high magnification views, respectively, of the adaxial side of an ag-4 hua1 anther. (H and I) Low and high magnification views, respectively, of the abaxial side of an ag-4 hua1 anther. (J and K) Low and high magnification views, respectively, of the adaxial side of an ag-4 hua2 anther. (L and M) Low and high magnification views, respectively, of the abaxial side of an ag-4 hua2 anther. (N) A low magnification view of a third whorl organ in an ag-4/+ hua1/hua1 hua2/hua2 flower. (O and P) High magnification views of the abaxial and adaxial sides, respectively, of the organ shown in (N). (Q) A low magnification view of a third whorl organ in an ag-4 hua1 hua2 flower. 3, a third whorl organ. (R) A high magnification view of the adaxial side of a third whorl organ in an ag-4 hua1 hua2 flower. (S and T) Two high magnification views of the abaxial side of the third whorl organ in (Q). Arrows indicate the organs in low magnification view that were examined under high magnification. Arrowheads point to stomatal guard cells. Thicker size bars (F, H, J, L, N, and Q), 100 μm; thinner size bars (A–E, G, I, K, M, O, P, and R–T), 10 μm. Molecular Cell 1999 3, 349-360DOI: (10.1016/S1097-2765(00)80462-1)

Figure 7 Localization of HUA2 and AG RNAs by In Situ Hybridization (A and B) Light and dark field photographs of a longitudinal section of a wild-type inflorescence hybridized with a HUA2 antisense probe common to the two forms of HUA2 RNAs. (C and D) Light and dark field photographs of a longitudinal section of an ag-3 inflorescence hybridized with the same HUA2 antisense probe. (E and F) Longitudinal sections of an ag-4 and an ag-4 hua1 hua2 inflorescence, respectively, hybridized with an AG antisense probe. All slides were exposed to autoradiography emulsion (NTB2 from Kodak) for 2 weeks. The numbers indicate floral stages. Size bars, 50 μm. Molecular Cell 1999 3, 349-360DOI: (10.1016/S1097-2765(00)80462-1)

Figure 5 Cloning of HUA2 by Chromosome Walking (A) A schematic of the HUA2 region on chromosome 5. HUA2 maps in between NIT4 and nga139, a region containing some mapped P1 clones (MRN17, MYJ24, MKD15, and MRO11). The numbers above or below the P1 clones indicate the number of recombination break points among the number of chromosomes assayed. The fourteen lines at the bottom represent overlapping plasmid subclones in the plant transformation vector pPZP211. The thicker line represents the clone (pPZP211-S7) that rescued the hua1 hua2 mutant phenotype. (B) Phenotypic rescue of hua1 hua2. hua1 hua2 plants were transformed with the above-mentioned 14 plasmids and the vector pPZP211. 1, a flower of a T1 plant containing the pPZP211 vector alone. The carpel bulging phenotype is not rescued. 2, a flower of a T1 transformant carrying pPZP211-S7. The gynoecium is phenotypically wild-type. 3, mature siliques of a hua1 hua2 plant (the two on the left) and a hua1 hua2 T1 transformant carrying pPZP211-S7 (the two on the right). Molecular Cell 1999 3, 349-360DOI: (10.1016/S1097-2765(00)80462-1)

Figure 6 The HUA2 Gene (A) Schematic diagrams of the HUA2 genomic region, depicting the two forms of HUA2 cDNAs. The triangles represent introns, the boxes represent exons. Black boxes represent exons shared by the two cDNAs, whereas gray and white boxes represent exons unique to each cDNA. The hua2 mutation is a G to A change in the splice acceptor site of the sixth intron. (B) Sequence of the HUA2 protein. The dotted line indicates the domain (hhath domain) similar to part of the hath domain in the hepatoma-derived growth factor (HDGF) family of proteins. Nuclear localization signals are underlined. The boxed amino acids are identical or closely related to the LXXLL motif known to mediate protein–protein interactions. (C) Alignment of the hath and/or hhath domains among ten genes/ESTs represented by their GenBank accession numbers. The top six sequences are similar over the entire hath domain, whereas the bottom four sequences are similar to each other and to the top six sequences only over half of the hath domain, the hhath domain. Amino acids that are identical among the top six or all ten sequences are boxed; positions where four out of six (for the top six sequences) or eight out of ten (for all sequences) amino acids are either identical or display conservative changes are shaded. Among the 98 amino acids initially included in the hath domain (Izumoto et al. 1997), only 91 are shown since the others do not show similarity among the proteins. P51859, mouse HDGF; P51858, human HDGF; AF063020, human lens epithelium-derived growth factor; W64700, a mouse EST; AA964144, a rat EST; AA984020, a human EST; AA942551 and AA979251, two Drosophila ESTs; and U97193, a C. elegans gene. Molecular Cell 1999 3, 349-360DOI: (10.1016/S1097-2765(00)80462-1)