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HUA ENHANCER2, a putative DExH-box RNA helicase, maintains homeotic B and C gene expression in Arabidopsis

Tamara L. Western^*, Yulan Cheng, Jun Liu and Xuemei Chen

Waksman Institute, Rutgers University, 190 Frelinghuysen Rd., Piscataway, NJ 08854, USA
^* Present address: Department of Botany and Biotechnology Laboratory, University of British Columbia, Vancouver, BC V6T 1Z4, Canada

View larger version (66K): [in a new window]   Fig. 1. Inflorescence and floral phenotypes of hen2-1 mutants. (A) Ler; (B) hua1-1 hua2-1; (C) hen2-1 hua1-1 hua2-1, petals are small and third whorl organs are a mosaic of sepal-petal tissue (arrows). (D-G) Side view of inflorescences, all at same magnification. (D) Ler. (E) hen2-1, note increased number of siliques and altered phyllotaxy (arrows). (F) hua1-1 hua2-1. (G) hen2-1 hua1-1 hua2-1, note reduction in internode length. (H) Ler inflorescence. (I) hen2-1 inflorescence, note the increased number of flowers and buds. (J) hen2-1 flower, note crowding of a lateral sepal with a medial sepal (arrows) and an extra petal (arrowhead). (K) hen2-1 hua1-1 flower, exhibiting an extra petal (arrowhead). (L) hen2-1 hua2-1 flower, note the short, apparently immature stamens. (M) Comparison of hen2-1 hua1-1 hua2-1 third whorl organs (five organs on the right), with sepal, petal and stamen from a hua1-1 hua2-1 flower. (N-S) Isolated gynoecia. (N) Ler. (O) hua1-1 hua2-1, note broadening at top of gynoecium. (P) hen2-1 hua1-1 hua2-1, gynoecium is small and bulging, with reduced stigmatic papillae. (Q) hen2-1, note lack of bulge. (R) hen2-1 hua1-1, the gynoecium bulges at the top. (S) hen2-1 hua2-1, note broadening at top.

View larger version (145K): [in a new window]   Fig. 2. Scanning electron micrographs of mature and developing flowers of wild-type and hen2-1 single, and double and triple mutants with hua1-1 and hua2-1. (A) Ler inflorescence meristem. (B) hen2-1 inflorescence meristem, note crowding of lateral sepals towards the adaxial sepal in top developing flower (arrows). (C) Ler at stage 4-5 with four evenly spaced sepal primordia. (D) hen2-1 at stage 4-5, note extra sepals and disrupted spacing of sepals. (E) Ler abaxial sepal cells, note epicuticular striations and the long cell. (F) Ler adaxial petal cells. (G) Ler abaxial petal cells. (H) Ler stamen cells. (I) A hen2-1 hua1-1 hua2-1 third whorl organ. (J) Higher magnification of part of I, showing patches of abaxial petal (arrow) and sepal-like cells (arrowhead). (K) Detail of a hen2-1 hua1-1 hua2-1 third whorl organ showing both petal (arrow) and stamen-like cell types (arrowhead). (L) Ler ovary valve cells. (M) The top, lateral region of a hua1-1 hua2-1 ovary showing presence of ovary (arrow) and sepal-like cells (arrowhead). (N) The top, lateral area of a hen2-1 hua1-1 ovary, note cells with both ovary (arrow) and sepal characteristics (arrowhead). (O) The top, lateral portion of a hen2-1 hua2-1 ovary with both ovary (arrow) and sepal-like cells (arrowhead). (P) hen2-1 hua1-1 hua2-1 ovary cells, all have sepal-like features. (Q) hua1-1 hua2-1 at stage 7-8, note stalk of developing stamen (arrow). (R) hen2-1 hua1-1 hua2-1 at stage 7-8 showing large, flat third whorl organs. Scale bars, 10 µm (C,D,E-H,J-P), 50 µm (I), 100 µm (A,B,Q,R).

View larger version (42K): [in a new window]   Fig. 3. Floral phenotypes of hua1-1 hua2-1 triple mutants and hen2-1 hua1-1 hua2-1 quadruple mutants with A, B and C class mutations. (A) hua1-1 hua2-1 ap1-1. (B) hen2-1 hua1-1 hua2-1 ap1-1, note stamens replaced by green organs (arrow). (C) hua1-1 hua2-1 ap2-2. (D) hen2-1 hua1-1 hua2-1 ap2-2 with leafy organs in all whorls. (E) hua1-1 hua2-1 ap3-3. (F) hen2-1 hua1-1 hua2-1 ap3-3 with third whorl sepals (arrow). (G) hua1-1 hua2-1 pi-3. (H) hen2-1 hua1-1 hua2-1 pi-3 with third whorl sepals. (I) hua1-1 hua2-1 ag-4, note third whorl petals/petal-stamens and internal flower. (J) hen2-1 hua1-1 hua2-1 ag-4, the third whorl organs are narrow petals.

View larger version (139K): [in a new window]   Fig. 4. In situ localization of RNAs from floral homeotic genes in hen2-1 hua1-1 hua2-1 and hua1-1 hua2-1 developing flowers. (A-C) Hybridization with AP1 antisense probe. (A) hen2-1 hua1-1 hua2-1 at stage 4. (B) hen2-1 hua1-1 hua2-1 at stage 8-9 showing strong patches of AP1 expression in the gynoecium (arrowhead) and one third whorl organ (arrow). (C) Stage 8-9 hua1-1 hua2-1 from the same slide as (B) with weak expression of AP1 in the third and fourth whorls. (D-F) Hybridization with AP3 antisense probe. (D) Stage 3 hen2-1 hua1-1 hua2-1. (E) Stage 8-9 hen2-1 hua1-1 hua2-1 with patchy AP3 expression in the third whorl organs. (F) hua1-1 hua2-1 at stage 8-9. (G-I) Hybridization with PI antisense probe. (G) Stage 3 hen2-1 hua1-1 hua2-1. (H) hen2-1 hua1-1 hua2-1 at stage 8-9, note PI expression in only one of the visible third whorl organs (arrow). (I) Stage 8-9 hua1-1 hua2-1. (J-L) Hybridization with AG antisense probe. (J) Stage 3 hen2-1 hua1-1 hua2-1. (K) hen2-1 hua1-1 hua2-1 at stage 8 with low expression in the third and fourth whorl organs, note strong expression of AG in a stage 5 flower at the bottom of the panel. (L) Stage 8 hua1-1 hua2-1 flower from the same slide as (K). Scale bars, 50 µm (A,D,G,J), 100 µm (B,C,E,F,H,I,K,L).

View larger version (44K): [in a new window]   Fig. 5. Cloning of HEN2. (A) Mapping of the HEN2 locus to six BACs at the top of chromosome 2. Numbers represent the number of recombinant chromosomes in a total of 1390 chromosomes. The schematic below shows the basic structure of HEN2 with exons and introns represented by boxes and lines, respectively. The nucleotide substitution in exon 3 found in hen2-1 is marked. (B-D) Molecular complementation of hen2-1 hua1-1 hua2-1. (B) A flower from a hen2-1 hua1-1 hua2-1 plant containing HEN2g, showing rescue of the third whorl and gynoecium phenotypes. (C) A flower from a hen2-1 hua1-1 hua2-1 plant containing the vector alone. (D) A flower from a hen2-1 hua1-1 hua2-1 plant containing mHEN2g. Note lack of rescue in C and D.

View larger version (34K): [in a new window]   Fig. 6. HEN2 protein sequence and similarity of HEN2 to putative DExH-box helicases in diverse eukaryotes. (A) The protein sequence of HEN2. The conserved DExH motifs are marked with double underlines. The sequences were identified according to the Ski2p family consensus as defined by de la Cruz et al. (de la Cruz et al., 1999). The nuclear localization signal is marked with a single underline. The proline to leucine substitution found in the protein encoded by hen2-1 is in bold. (B) A schematic diagram of HEN2 showing the location of the helicase motifs (black bars) and the position of the amino acid substitution in the protein encoded by hen2-1 (asterisk). Below is a list of the homologs from various eukaryotes. The numbers represent percentage identity and similarity (in parentheses) within the respective domains. Within the actual helicase motifs, HEN2 has 95% identity (39/41 amino acids) with the consensus of the proteins listed. At, Arabidopsis thaliana; Hs, Homo sapiens; Dm, Drosophila melanogaster; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ce, Caenorhabditis elegans.

View larger version (46K): [in a new window]   Fig. 7. RNA filter hybridization of Ler tissues probed with HEN2. UBQ5 was used to indicate the amount of RNA in each sample. The intensity of the signals was quantified with a phosphorimager and the relative abundance of HEN2 among the samples is as indicated.

View larger version (187K): [in a new window]   Fig. 8. In situ localization of HEN2 RNA in Ler inflorescences. (A-G) Antisense probe. (A) Low magnification of an Ler inflorescence showing strong expression of HEN2 in developing flowers of various stages. (B) An Ler inflorescence showing expression throughout the inflorescence meristem and stage 2 and 3 floral primordia. (C) A stage 3 flower with HEN2 expressed throughout. (D) A stage 5 flower, note HEN2 is no longer expressed in sepals. (E) A stage 7 flower. (F) A stage 8 flower. (G) A stage 10 flower, HEN2 is still expressed at a low level in the petals and gynoecium and strongly expressed in the developing ovules. (H,I) Hybridization with sense probe to Ler inflorescence (H) and stage 6 flower (I), showing lack of strong signal. Scale bars, 50 µm (B-F,I), 100 µm (A,G,H).