Volume 13, Issue 8, Pages 627-637 (April 2003) ATX-1, an Arabidopsis Homolog of Trithorax, Activates Flower Homeotic Genes  Raul Alvarez-Venegas, Stephane Pien, Monther Sadder, Xiaohong Witmer, Ueli Grossniklaus, Zoya Avramova  Current Biology  Volume 13, Issue 8, Pages 627-637 (April 2003) DOI: 10.1016/S0960-9822(03)00243-4

Figure 1 Phenotypes of atx1 Mutant Plants (A) Molecular structure of ATX1. The site of the T-DNA integration is shown as a triangle (between exons 15 and 16), truncating the transcribed message. Filled boxes represent exons, and empty boxes represent introns. The black lines below outline the regions coding for the various architectural motifs. The detailed molecular structure of the gene has been established in our previous work [16]. (B) RT-PCR analysis with mRNA isolated from wild-type and atx1-1 tissues as template and specific primers abutting two architectural domains, DAST and PHD. Lower panels show the transcription of elF4, as a control. (C) Whole 32-day-old plants grown under long day (14 hr light, 10 hr dark) conditions. Wild-type (Wassilevskija) and atx1-1 mutant plants are shown on the left- and the right-hand sides, respectively. (D) Mature atx1 flower, with severely defective architecture. (E) Rescue of the growth phenotypes in 3-week-old seedling mutants transformed with wild-type ATX1. a noncomplemented mutant atx1-1 plant is shown on the right, and a mutant transformed with 35S::ATX1 is shown on the left. (F) A flower from a mutant plant transformed with 35S::ATX1. Current Biology 2003 13, 627-637DOI: (10.1016/S0960-9822(03)00243-4)

Figure 2 In Situ Hybridization with ATX1 The numbers indicate the stage of flower development [49]. (A–E) In situ localization of the ATX1 transcript in wild-type (WT) inflorescence and more mature flowers. (A) ATX1 transcripts accumulate in the inflorescence meristem (im) and in stage-2 flowers. (B) By stage 4, transcripts are present in the petal primordia. (C) ATX1 expression is detected in the immature stamens (st) and in the gynecium cylinder (gy) in stage-8 flowers. (D) A stage-10 flower displays ATX1 expression in the gynecium in association with ovule primordium initiation. (E) By stage 11, ATX1 remains expressed in the unfertilized ovules (ov). (F–H) Sense control in situ hybridization for ATX1 gives no signal in stage-2, -4, -8, and -10 flowers. (I–K) Antisense in situ hybridization for ATX1 in atx1-1 plants (atx1), no transcript could be detected in any of the stages depicted. (L) In situ localization of the ATX1 transcript in 35S::ATX1 inflorescence (35S); the expression pattern is similar to the one observed in the wild-type, but the signal is stronger in the sepals (se), petals (pe), and stamens (st). Current Biology 2003 13, 627-637DOI: (10.1016/S0960-9822(03)00243-4)

Figure 3 Scanning Microscope Pictures of atx1-1 Flowers (A and B) Seven-week-old buds that have remained closed, showing undeveloped gynoecia and short stamens. (C) Mature flowers with aberrant-shaped gynoecia. The arrowhead points to a fifth petal, while the long arrow shows a stamen-like organ. (D) A closeup of this stamen-like organ is shown. It did not produce pollen and had atypical hexagonal cells at the top. (E) A mutant atx1-1 flower with short stamens. (F) The arrow points to a petal that has lost its identity, which is shown in a closeup. This second-whorl organ has a stamen-like stem supporting a petal-like formation. Two arrows, one longer and one shorter, point to the stem. (G) A carpeloid, third-whorl organ from a 32-day-old atx1-1 mutant flower. The scale bars represent 100 μm. (H) Cobbled-like appearance of wild-type petal cells from the abaxial (outer) side petal tissue. (I) Abaxial tissue of mutant petals with stamen-like cells. Stomata may be seen. The scale bars represent 10 μm. (J) Scanning microscope pictures of atx1-1 inflorescence. A 4-week-old bud with asymmetrically assembled sepals. Trichomes are missing. (K) Defective sepal initiation. Only two primordia have been initiated. (L) Inflorescence with buds at different developmental stages according to Smyth et al. [51]. The long arrow points to a bud with apparently normal sepal initiation. The arrowhead points to a bud with aberrant organ initiation. Note the file of flat cells in the middle of the central zone. (M) A mutant bud with defective sepal initiation (arrowhead) is in the same inflorescence with an apparently normal bud at stage 3 (long arrow). (N) Eight-week-old atx1-1 inflorescence. Arrow 1 points to a bud with asymmetrically assembled sepals. A mutant bud shows a different type of sepal aberration (arrow 2). Note the cells lacking the rough striation pattern of the other buds in the bunch. The bud behind (arrow 3) has failed to initiate organs altogether. The scale bars represent 100 μm. Current Biology 2003 13, 627-637DOI: (10.1016/S0960-9822(03)00243-4)

Figure 4 Expression Levels of Flower Homeotic Genes in Wild-Type and atx1-1 Tissues The left-hand panel in each pair shows data for bud (b), while the right-hand panels show mature flower (f) expression levels for all tested genes. RNA samples were isolated as described in the Experimental Procedures. The AG panels show the levels of AG mRNA in buds (b) and flowers (f) of wild-type, atx1-1, and clf plants. Current Biology 2003 13, 627-637DOI: (10.1016/S0960-9822(03)00243-4)

Figure 5 In Situ Hybridization for Class A Genes The numbers indicate the stage of flower development. (A and B) Antisense in situ hybridization for AP1 in wild-type (WT) plants. (A) AP1 transcripts are present in the flower primordia (stage 2) and in the initial cells for the sepal, and they remain detectable in more mature sepals of stage-5 and -6 flowers. (B) By stage 9 flowers, AP1 transcripts are detected in the sepals and petals. (C) No signal with sense control. (D and E) Antisense in situ hybridization for AP1 in 8-week-old atx1 plants (atx1). (D) Transcripts do not accumulate in stage-2 and stage-5 flowers. (E) Same as in (D) for stage-6 flowers. (F and G) Antisense in situ hybridization for AP1 in 35S::ATX1 plants (35S). (F) Plants overexpressing ATX1 show a normal pattern of AP1 expression, though the signal seems weaker in stage-2, -3, and -5 flowers. (G) By stage 8, AP1 transcript accumulation is strongly reduced in the sepals. (H and I) Antisense in situ hybridization for AP2 in wild-type inflorescence. (H) AP2 expression is detected in flower primordia (stage 2) and in sepals (se) of stage-4 flowers. (I) A stage-6 flower displays a signal in the sepal (se) and stamen primordia (st). (J) No signal is seen with a sense probe for AP2 in wild-type flowers. (K and L) Antisense in situ hybridization for AP2 in 8-week-old atx1-1 plants (atx1). (K) The AP2 expression is no longer detected in stage-5 and -6 flower buds. (L) Same as in (K) for stage-8 flowers. Reduced expression of AP2 in the atx1 mutant background may specify the formation of stamenoid petals. (M and N) In situ hybridization for AP2 in 35S::ATX1 flowers (35S). (M) The AP2 signal is extremely strong in early stage-2 flower primordial, as it is in sepals of stage-4 and -5 flowers. (N) The signal remains strong in stage-8 flower stamens. (O) Note the AP2 ectopic expression in the gynoecium (gy) of a stage-11 flower. No signal could be detected with a sense hybridization probe for AP2 in 35S::ATX1 flowers (data not shown). Current Biology 2003 13, 627-637DOI: (10.1016/S0960-9822(03)00243-4)

Figure 6 In Situ Hybridization for a Class C Gene (A–L) The numbers indicate the stage of flower development. (A, B, D, and E) Antisense in situ hybridization in wild-type flowers (WT). (A) An antisense probe shows the characteristic expression pattern for AG in central regions of floral meristem and stamen (st) of stage-4 to stage-6 flowers. (B) A stage-7 flower shows an accumulation of AG transcripts in the gynecium cylinder (gy). (D and E) By stage 11 and 15, AG transcripts are present in the ovule primordium (op) and in the immature ovule (ov) before fertilization. (C and F) Sense control with wild-type inflorescence gives no signal. (G and H) In situ hybridization with AG antisense probes in atx1-1 plants (atx1). (G) In atx1-1 mutant 8-week-old inflorescence, the accumulation of the AG transcript is strongly reduced in stage-4 and stage-6 flower buds. (H) The transcript level decreases dramatically in stamens in stage-8 flowers. (I, J, and K) Antisense in situ hybridization in 35S::ATX1 plants (35S). (I) The AG expression pattern is similar to the wild-type. (J) AG, in addition to its normal expression pattern, is ectopically expressed in sepals of stage-9 flowers. (K) In stage-11 flowers, the expression level is higher in ovule primordia (ov). (L) No signal could be detected with the AG sense probe in 35S::ATX1 inflorescence. Current Biology 2003 13, 627-637DOI: (10.1016/S0960-9822(03)00243-4)

Figure 7 Recombinantly Expressed Proteins after Affinity Column Purification and Reaction with Either the Anti-GST-HRP Conjugate or His-Monoclonal Antibody Molecular weights of the fusion proteins are shown on the left. Products of the methyltransferase activity (assayed, as described in [20]), run on 18% SDS gels, stained with Coomassie blue, and autoradiographed are shown in the upper and the lower gels. The histone H3 tail peptides of wild-type, of the mutated H3R9, and of H3R4 were tested as substrates for the recombinant SET proteins. At the end of the incubation, the reaction mix was spotted on Phosphocellulose filter paper P-81 (Whatman), washed three times for 15 min in 50 mM sodium carbonate (pH 9.2), dried, and counted in a scintillation counter. The standard deviation error is ±9%. Current Biology 2003 13, 627-637DOI: (10.1016/S0960-9822(03)00243-4)