ETTIN Patterns the Arabidopsis Floral Meristem and Reproductive Organs
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Development 124, 4481-4491 (1997) 4481 Printed in Great Britain © The Company of Biologists Limited 1997 DEV0134 ETTIN patterns the Arabidopsis floral meristem and reproductive organs Allen Sessions1,†, Jennifer L. Nemhauser1, Andy McCall 1,‡, Judith L. Roe1, Ken A. Feldmann2 and Patricia C. Zambryski1,* 1Department of Plant and Microbiology, 111 Koshland Hall, University of California at Berkeley, Berkeley, CA 94720, USA 2Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA †Present address: Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, CA 92093-0116, USA ‡Present address: Department of Biology, Carleton College, Northfield, MN 55057-4000, USA *Author for correspondence SUMMARY ettin (ett) mutations have pleiotropic effects on Arabidopsis involved in prepatterning apical and basal boundaries in flower development, causing increases in perianth organ the gynoecium primordium. Double mutant analyses and number, decreases in stamen number and anther expression studies show that although ETT transcriptional formation, and apical-basal patterning defects in the activation occurs independently of the meristem and organ gynoecium. The ETTIN gene was cloned and encodes a identity genes LEAFY, APETELA1, APETELA2 and protein with homology to DNA binding proteins which bind AGAMOUS, the functioning of these genes is necessary for to auxin response elements. ETT transcript is expressed ETT activity. Double mutant analyses also demonstrate throughout stage 1 floral meristems and subsequently that ETT functions independently of the ‘b’ class genes resolves to a complex pattern within petal, stamen and APETELA3 and PISTILLATA. Lastly, double mutant carpel primordia. The data suggest that ETT functions to analyses suggest that ETT control of floral organ number impart regional identity in floral meristems that affects acts independently of CLAVATA loci and redundantly with perianth organ number spacing, stamen formation, and PERIANTHIA. regional differentiation in stamens and the gynoecium. During stage 5, ETT expression appears in a ring at the top of the floral meristem before morphological appearance of Key words: Arabidopsis, flower development, ETTIN, positional the gynoecium, consistent with the proposal that ETT is information INTRODUCTION and 1997; Running and Meyerowitz, 1996; Sessions and Zambryski, 1995; Sessions, 1997). These genes likely act to In higher plants, proper flo ral development re q u i res the coor- pattern growth, cell division and regional differentiation during d i n ated activity of a number of genes that control the pat t e rn- the acquisition of identity within the developing flower. CLV1 ing of organ type, organ number and organ fo rm (We i ge l , functions partially redundantly with meristem identity genes, 1995; Ya n o f s ky, 1995). In A rab i d o p s i s, these genes have been whereas PAN and TSL function independently of the meristem cl a s s i fied into those functioning early in the establishment of and organ identity genes (Clark et al., 1993; Roe et al, 1997; flo ral meristem identity such as L E A F Y (L F Y), A P E T E L A 1 Running and Meyerowitz, 1996). (A P 1), and APETELA2 (A P 2), and those acting later duri n g H e re we describe the isolation of the E T T gene and its the pat t e rning of organ identity such as A P E T E L A 3 (A P 3) , ex p ression pat t e rn during early flo ral development. E T T P I S T I L L ATA (P I) and AG A M O U S (AG) (We i gel and encodes a protein that is predicted to be nu clear localized and M eye rowitz, 1994). Mutations in these genes affect the flora l is homologous to DNA binding proteins wh i ch bind to auxin ve rsus inflo resence identity of flo ral shoots and/or the identity response elements (AREs). E T T m R NA is detected in a of the individual flo ral organs. Most of these genes are thought c o m p l ex pat t e rn throughout early flower development and is to encode tra n s c ription fa c t o rs (We i gel, 1995; Ya n o f s ky, consistent with the proposed role for E T T in pat t e rning: (i) 1995). p e rianth organ nu m b e r, and (ii) stamen and carpel fo rm. Early There are a number of other mutations which affect organ E T T ex p ression in the gynoecium suggests that the pro t e i n number, organ shape and regional differentiation within floral p a rt i c i p ates in the pat t e rning of abaxial tissues and the estab- organs without changes in the identity of either the floral shoot lishment of apical and basal boundaries in the pri m o rd i u m . or its component organs. For example, mutations in CLAVATA1 I n t e re s t i n g ly, the tra n s c riptional activation of E T T is inde- (CLV1), CLAVATA3 (CLV3), ETTIN (ETT), and PERIANTHIA pendent of known meristem and organ identity functions. (PAN) increase organ number within the flower, whereas L a s t ly, genetic analyses indicate that E T T functions indep e n- mutations in TOUSLED (TSL) decrease organ number and d e n t ly of C LV and re d u n d a n t ly with PA N to control flo ra l organ size (Clark et al., 1993, 1995, and 1997; Roe et al, 1993 o rgan nu m b e r. 4482 A. Sessions and others MATERIALS AND METHODS WsO ecotype. With the exception of lfy-1 and ag-5 which are in the Columbia (ColO) ecotype, the remaining mutant alleles (ap1-1, ap2- Plant material 2, pi-1, ap3-1, ag-1, clv1-1, clv3-1) are in LaO. Control outcrosses of ett alleles and plant growth conditions were similar to those described ett-1 identified ecotype specific suppressors of ett-1 in ColO and LaO, by Sessions and Zambryski (1995). ag-5 was isolated and provided which segregate as single recessive loci in F2 outcross populations: by Eva Huala (Roe et al., 1997). All other mutant alleles were 25% of F2 ett-1 plants have a suppressed gynoecium phenotype, obtained from the laboratory of Elliot Meyerowitz (California allowing more valve formation and some seed set. The loci imparting Institute of Technology). suppression of ett-1 in LaO and ColO have not been mapped. In F2 Scanning electron microscopy (SEM) double mutant populations these suppressor loci segregated indepen- dently of ett-1 and all other mutations with the exception of lfy-1 and Fixation, drying and viewing are the same as Sessions (1997). pi-1, which both reside on chromosome 5. Suppressed phenotypes in Identification and isolation of the ETT gene double mutant populations were distinct and additive, and were not Plasmids, strains and cloning details, as well as data not shown are scored in the phenotypic analyses presented. available upon request. ETT was isolated using the ett-1 allele. ett-1 was ett-1 was crossed as a male onto the different homozygous identified in a large screen of T-DNA mutagenized seed (#7581; mutants. Individuals doubly homozygous for ett-1 and the other Feldmann, 1991; Azpiroz-Leehan and Feldmann, 1997) and shown by mutation of interest were identified in five different ways: (i) as a Southern analysis with T-DNA border-specific probes to contain a single new/additive phenotype segregating 1/16 in F2 populations (ap2-2 T-DNA insert encoding kanamyacin resistance. The ett mutant ett-1, ap3-1 ett-1, ett-1 pi-1, ett-1 pan-1); (ii) by Southern analysis phenotype was completely linked to the T-DNA and kanamyacin resis- of individual F2 mutants to detect ett-1 specific T-DNA induced RFLPs (ett-1 lfy-1, ag-1 ett-1, ag-5 ett-1); (iii) in F3 populations from tance (0 recombinants in 50 homozygous ett-1/ett-1 plants) in F2 backcross populations. Left border plasmid rescue of the ett-1 T-DNA F2 suppressed ett-1 individuals that were heterozygous for the other border generated a plant specific 0.4 kb StyI/EcoRI fragment that was mutation of interest (ett-1 lfy-1, ag-1 ett-1); (iv) in F3 populations used to screen a Wassilewskija (WsO) lambda genomic library (Roe et from F2 mutants that were heterozygous for ett-1 (ap1-1 ett-1, clv1- al., 1993). Five clones spanning 25 kb were isolated. Southern analysis 1 ett-1, clv3-1 ett-1, ett-1 pan-1); and (v) by testcrosses (ett-1 pan- mapped the 0.4 kb StyI/EcoRI border fragment to the center of the 12 1). The ap1-1 enhancing cauliflower-1 (cal-1) mutation resident in kb clone ASL2. Clone ASL2 was inserted into transformation vector WsO (Bowman, 1993) was not present in the families segregating for pSLJ6991 (Jones et al., 1992) as a SacI fragment and introduced directly ap1-1 ett-1 used here. into a suppressed ett-1 line (see below) by vacuum mediated transfor- mation. ASL2 rescued the ett-1 phenotype and subsequently rescued ett- 2 and ett-3 in crosses, demonstrating that ETT resides within ASL2. Two RESULTS overlapping fragments from ASL2, one spanning the predicted insertion site in ett-1, were used to screen a Landsberg erecta (LaO) floral and infloresence specific lambda ZAP cDNA library (Weigel et al., 1992). ett phenotypes The longest (2.1 kb) of three similar clones, designated number 5, was Wild-type Arabidopsis flowers are composed of four sepals, purified as a pBluescript plasmid (pAS13), and sequenced in full four petals, six stamens and a bicarpelate gynoecium (Fig.