Volume 5, Issue 3, Pages 569-579 (March 2000) Molecular and Genetic Analyses of the Silky1 Gene Reveal Conservation in Floral Organ Specification between Eudicots and Monocots  Barbara A. Ambrose, David R. Lerner, Pietro Ciceri, Christopher M. Padilla, Martin F. Yanofsky, Robert J. Schmidt  Molecular Cell  Volume 5, Issue 3, Pages 569-579 (March 2000) DOI: 10.1016/S1097-2765(00)80450-5

Figure 1 Phenotype of Wild-Type and si1-R Tassels and Ears (A) Anthers can be seen hanging from the opened spikelets of the wild-type tassel at anthesis. (B) A mature wild-type tassel spikelet. Two florets are contained within the inner (Gi) and outer (Go) glumes: the lower floret (LF) is to the left of the opened upper floret. The three stamens (S) and lodicules (Lo) are surrounded by the lemma (L) and palea (P). (C) A close up of the upper floret shown in (B). The lodicules are at the base of the staminal filaments and are surrounded by the lemma and palea. The inner glume is also noted. (D) A mature wild-type ear. (E) A close up of a spikelet pair from an immature ear. One silk can be seen emerging from each of the spikelets. (F) A mature si1-R tassel. Spikelets never open and have protruding silks. (G) A mature si1-R male spikelet manually opened to show the two florets within the inner and outer glumes. The upper floret is oriented to the right, while the lower floret bordered by the lemma and palea is on the left. The lemma and palea of the upper floret are shown enclosing the transformed stamens (TS) and transformed lodicules (TLo). The transformed stamens have silk-like projections extending from the midveins of palea/lemma-like tissue. (H) A close up of the upper floret shown in (G). The lemma and palea delineate the limits of the upper floret. The inner glume is labeled for reference. Two small palea/lemma-like organs arise at the position normally occupied by the lodicules. (I) The si1-R ear has more silks compared to the wild type shown in (D). (J) A spikelet pair from an immature si1-R ear. Three extra silks positioned around the central silk are seen emerging from each spikelet. Molecular Cell 2000 5, 569-579DOI: (10.1016/S1097-2765(00)80450-5)

Figure 2 SEM of Spikelet Development in Tassel and Ear of Wild Type and si1-R (A) A developing wild-type ear spikelet. In the upper floret, the pistil primordia (Pp) is surrounded by three stamen (S) primordia and two lodicule (Lo) primordia. The lower floret (LF) is separated from the upper floret by the palea (P). The upper and lower florets are enclosed within the inner and outer glumes (Gi and Go). A portion of the outer glume has been removed. (B) A developing wild-type ear spikelet at the time of silk initiation. In the upper floret, the silk primordia (Sp) is visible, while developing stamens begin assuming a tetralocular shape. The lower floret develops as a mirror image of the upper floret but is delayed developmentally. (C) Ear spikelet organogenesis continues with the extension of the silk primordia. The stamens have ceased to develop and will soon abort. (D) A developing wild-type tassel spikelet. The stamens of the upper floret have elongated while maintaining their tetralocular form, and the pistil has aborted. The details of the lower floret are not visible. (E) A close up of the base of the upper floret from a nearly mature tassel spikelet. The lodicules can be seen surrounded by the lemma and palea. (F) A si1-R ear spikelet at a similar developmental stage to the wild type shown in (A). (G) A developing si1-R ear spikelet at the time of silk initiation. The pistil develops as in the wild-type ear spikelet shown in (B), but transformed stamen (TS) primordia are recognizably distinct. (H) A more mature si1-R tassel spikelet showing a central silk primordia similar to wild type; however, the transformed stamens (TS) are now acquiring the shape of the central silk. (I) A developing si1-R tassel spikelet. The pistil of the floret has aborted as in wild type shown in (D); however, the stamens are transformed. The transformed lodicules (TLo) of the lower floret appear as long broadened ridges similar to the palea and lemma. (J) A nearly mature si1-R tassel spikelet manually opened to show the enclosed lower floret and the upper floret bound by the lemma and palea within which are transformed stamens and lodicules. (K) A light micrograph of a mature spikelet from the sil-5 mutant. The outer and inner glumes, palea, and lemma of the upper floret have been spread to reveal three pistils developing in place of the transformed stamens and a palea/lemma-like organ in place of the lodicule. All scale bars are 100 μm except in (J), where the scale bar is 500 μm. Molecular Cell 2000 5, 569-579DOI: (10.1016/S1097-2765(00)80450-5)

Figure 3 Genomic Structure of Si1 and Locations of Mu Element Insertions (A) The boxes depict Si1 exons, and the connecting lines indicate the introns. The open boxes 5′ to the ATG and 3′ to the stop codon represent untranslated regions. The coding sequence is composed of seven exons comprising the following nucleotides (nt): exon 1 (nt 1 to nt 188), exon 2 (nt 189 to nt 255), exon 3 (nt 256 to nt 317), exon 4 (nt 318 to nt 417), exon 5 (nt 418 to nt 459), exon 6 (nt 460 to nt 505), and exon 7 (nt 506 to nt 684). The filled box denotes the MADS domain, the hatched box represents the intervening region, and the stippled boxes represent the K domain. The locations of the Mu element insertions are represented by triangles. The si1-mum2 allele contains a MuDR element in the fourth intron between the coding sequences of the K box. The si1-mum3 allele contains a Mu element in the first intron after the MADS box, and the si1-mum4 allele contains a Mu element inserted after nt 198 in exon 2. (B) A comparison of the deduced amino acid sequence of SI1, AP3, PI, DEF, and GLO.Differences are indicated by the amino acid designation, and conservation is denoted by an asterisk. The MADS box (amino acids 2–57) and the K box (amino acids 89–154) are underlined in the SI1 amino acid sequence. A comparison of the amino acid sequence through the MADS, I, and K domains (MIK) of SI1 and DEF shows 60% identity and 74% similarity; SI1 and AP3 share 58% identity and 68% similarity. In comparison, SI1 has only 46% identity with the Antirrhinum GLO protein and 46% identity with the Arabidopsis PI protein through the MIK domains. Molecular Cell 2000 5, 569-579DOI: (10.1016/S1097-2765(00)80450-5)

Figure 4 Northern Analyses of Si1 (A) Si1 RNA accumulation in immature ears of wild-type or si1 mutants. Lane 1 contains RNA from wild-type ears (B73 inbred). Lane 2 contains RNA from plants homozygous for the si1-R allele (B73 background). Lane 3 contains RNA from plants homozygous for the si1-mum2 allele, and lane 4 contains RNA from plants heterozygous for the si1-mum2 allele. The arrow marks the position of a transcript visible in lane 3 upon longer exposure. The bottom panel shows the same blot hybridized with a probe for the maize ubiquitin gene. (B) Si1 expression in maize vegetative and reproductive tissues. Si1 RNA accumulation in young shoots (S), young roots (R), young leaves (L), immature tassel (T), immature ears (E), developing embryo (Em), and endosperm (En). The bottom panel shows the 28S rRNA from the same blot stained with methylene blue. Molecular Cell 2000 5, 569-579DOI: (10.1016/S1097-2765(00)80450-5)

Figure 5 Si1 RNA In Situ Hybridizations on Longitudinal Sections of Developing Tassel Spikelets (A) Two developing spikelets. Each is enclosed within an inner (Gi) and outer (Go) glume. Within the upper floret of the developing spikelet, Si1 expression is seen throughout the central portion of the floral meristem. (B) Only the upper floret of the spikelet, enclosed within the inner and outer glume, is visible in this longitudinal section. Here, Si1 expression is seen in the cells that will give rise to the stamen and lodicule primordia. (C) Si1 RNA is maintained in the developing stamen and lodicule primordia as they arise on the flanks of the floral meristem of the upper floret. (D) Si1 RNA persists in the developing stamen (S) and lodicule (Lo) of the upper floret as well as in the stamen and lodicule primordia of the lower floret (LF). No expression is detected in the developing palea (P) or glumes. (E) Si1 RNA is detected in the stamens and in the lodicule of the upper floret, which is flanked by its lemma (L) and palea. The lower floret primordia cannot be seen in this section; however, its lemma is visible. No expression above background is detected in the inner or outer glumes, lemma, palea, or in the aborted gynoecium (Pi). Molecular Cell 2000 5, 569-579DOI: (10.1016/S1097-2765(00)80450-5)

Figure 6 Tassel Spikelet of a si1-R zag1-mum1 Double Mutant (A) A young spikelet showing the inner and outer glumes (Gi and Go) enclosing the upper (UF) and lower florets (LF) of the double mutant. (B) Same spikelet as seen in (A) except that the lemma (L) and palea (P) of the upper floret have been opened to show the proliferating palea/lemma-like organs, some complete with silks. (C) SEM of developing mutant spikelet. Within the inner and outer glumes, the upper floral meristem (FM) can be seen proliferating palea/lemma-like organs as indicated by the arrows. A portion of the lower floret is visible below the palea. (D) A fully mature spikelet from the double mutant with the glumes manually spread to reveal the numerous palea/lemma-like organs that continue to be produced from the indeterminate floral meristem. Molecular Cell 2000 5, 569-579DOI: (10.1016/S1097-2765(00)80450-5)

Figure 7 The ABC Model in Eudicots and Maize (A) The eudicot ABC model. (B) The ABC model as it applies to maize. C function appears to be orchestrated by two genes, Zag1 and Zmm2, having partially redundant activities and distinct patterns of expression. B function is dictated by Si1 and likely by Zpi, the putative maize ortholog of PI. In maize, a combination of B and C function specifies stamens, a combination of B and (presumably) A function specifies lodicules, and C function specifies carpel development and determinacy of the floral meristem. Although no functional data yet exists, a presumed A function alone is shown responsible for formation of palea and possibly lemmas. (C) A diagram showing a trimerous arrangement of lodicules, stamens, and carpels of a radially symmetric grass floret. The anterior lodicule (closest to the palea) is suppressed in most grass species. The floret is bound by the palea, which has two vascular strands (o), and the lemma, which has a single vascular strand. If one considers the modern palea to be derived from the fusion of two separate organs, then the outer “whorl” of the ancestral grass floret could be interpreted as composed of three organs, alternating with the position of the lodicules. Molecular Cell 2000 5, 569-579DOI: (10.1016/S1097-2765(00)80450-5)