Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER

Cristina Ferrndiz 1 Qing Gu 0 2 Robert Martienssen 0 Martin F. Yanofsky 1 0 Cold Spring Harbor Laboratory , PO Box 100, Cold Spring Harbor, NY 11724 , USA 1 Department of Biology, University of California at San Diego , La Jolla, CA 92093-0116 , USA 2 Present address: M409-WSL, Center for Legume Research and Division of Biology, University of Tennessee , Knoxville, TN 37996-0845 , USA SUMMARY The transition from vegetative to reproductive phases during Arabidopsis development is the result of a complex interaction of environmental and endogenous factors. One of the key regulators of this transition is LEAFY (LFY), whose threshold levels of activity are proposed to mediate the initiation of flowers. The closely related APETALA1 (AP1) and CAULIFLOWER (CAL) meristem identity genes are also important for flower initiation, in part because of their roles in upregulating LFY expression. We have found that mutations in the FRUITFULL (FUL) MADS-box gene, when combined with mutations in AP1 and CAL, lead to a dramatic non-flowering phenotype in which plants continuously elaborate leafy shoots in place of flowers. We Postembryonic development in Arabidopsis proceeds through a series of phases, each characterized by the identity of the lateral primordia produced by the shoot apical meristem (SAM) (Poethig, 1990). During the vegetative phase, the SAM produces closely spaced leaf primordia, each subtending a secondary shoot meristem, to form a rosette. During the reproductive, or inflorescence (I) phase, the SAM produces determinate floral meristems on its flanks. The last few vegetative leaves produced are referred to as cauline leaves and become separated along the inflorescence stem by longer internode distances. Thus, the production of leaves can be considered to occur within two distinct subphases, V1 (rosette) and V2 (cauline). Genes that promote flowering in Arabidopsis were identified as mutations that extend the duration of the V phase, increasing the number of leaves formed before the development of flowers, but generally not affecting the fate of the lateral primordia produced during the I phase (reviewed by Pieiro and Coupland, 1998). Another group of genes, including TERMINAL FLOWER1 (TFL1), act by delaying phase change and preventing the normally indeterminate SAM from becoming a flower (Alvarez et al., 1992; Shannon and MeeksWagner, 1991). In addition, several meristem-identity genes are responsible for conferring floral characteristics to the lateral demonstrate that this phenotype is caused both by the lack of LFY upregulation and by the ectopic expression of the TERMINAL FLOWER1 (TFL1) gene. Our results suggest that the FUL, AP1 and CAL genes act redundantly to control inflorescence architecture by affecting the domains of LFY and TFL1 expression as well as the relative levels of their activities. primordia produced by the SAM during the I phase. Mutations in floral meristem identity genes cause primordia that would develop into flowers to instead develop shoot characteristics. The best characterized of these genes are LEAFY (LFY), APETALA1 (AP1), APETALA2 (AP2) and CAULIFLOWER (CAL) (for review, see Yanofsky, 1995). Only lfy and ap1 mutants show dramatic flower-to-shoot phenotypes, especially in the most basal nodes. Furthermore, the nearly complete conversion of flowers into shoots observed in lfy ap1 double mutants reveals that they act redundantly to specify meristem fate (Bowman et al., 1993; Huala and Sussex, 1992; Irish and Sussex, 1990; Schultz and Haughn, 1991; Shannon and MeeksWagner, 1993; Weigel et al., 1992). Together, the LFY, AP1, CAL and AP2 genes appear to reinforce each others activities leading to a sharp transition from vegetative to reproductive development. The FRUITFULL (FUL) gene encodes a MADS-box protein that has previously been shown to be required for carpel and fruit development (Gu et al., 1998; Mandel and Yanofsky, 1995a). However, in addition to its expression domain during carpel and fruit development, the FUL gene is upregulated in the SAM at around the transition to flowering, suggesting that it may also play a role during this transition (Mandel and Yanofsky, 1995a; Hempel et al., 1997). FUL is closely related to the meristem identity genes AP1 and CAL, suggesting the possibility of functionally redundant activities. In this work we have undertaken a molecular genetic approach to uncover the possible roles of FUL in the transition to flowering as well as its interactions with different meristem identity genes. We have found that in addition to its role during carpel and fruit development, FUL acts as a flowering-time and meristem-identity gene. These studies provide new insights into the functional redundancy of MADS-box genes during the transition to flowering and on the upregulation of the LFY meristem identity gene. MATERIALS AND METHODS Plant material and growth conditions The ap1-1, ful-1, tfl1-2 and lfy-26 alleles have been described previously (Bradley et al., 1997; Gu et al., 1998; Lee et al., 1997; Mandel et al., 1992). The cal-5 allele was generated in a g -irradiation mutagenesis experiment and contains a single base-pair deletion 33 bp downstream of the translation initiation codon that causes a frame shift and introduces a STOP codon 19 amino acids later (Savidge, 1996). 35S::LFY lines (DW151.2.5, in Landsberg erecta background; Weigel and Nilsson, 1995) and LFY::GUS (DW150.209, in Columbia; Blzquez et al., 1997) were kindly provided by Detlef Weigel. The 35S::AG line was obtained from Hong Ma (Mizukami and Ma, 1992). For all experiments, seeds were vernalized for 3-5 days at 4C, then germinated and grown at 22-24C under continuous light conditions. Characterization of the molecular lesions in the ful alleles For ful-2, ful-4, ful-5 and ful-6, genomic DNAs were amplified by PCR with the primers OAM25 (5 -GGTCATTTCAGGGTTGTCGGTT-3 ) and OAM14 (5 -AATCATTACCAAGATATGAA-3 ), which hybridize respectively 59 ncl upstream of the initiation codon and 202 ncl downstream of the STOP codon of the FUL gene. The amplification products of two independent reactions were sequenced and compared with the wild-type sequence for each allele. For ful-5, since the sequencing of the FUL genomic DNA only showed a silent change in the coding region, we analyzed the sequence of the transcribed RNA by performing a reverse transcription of the ful-5 RNA using OAM14 as a primer, coupled with a PCR amplification using OAM25 and OAM14 as primers. GUS activity measurements For quantitative measurements of GUS activity in LFY::GUS ful-2 plants, the assay described by Blzquez et al. (1997) was used. In situ hybridizations For in situ experiments at day 12 in ap1 cal and ful ap1 cal plants, genotyping for the presence of the ful-1 allele was necessary since double and triple mutants were indistinguishable (see below). Tissue was fixed for 2 hours at room temperature in FAE solution (ethanol:acetic acid:formaldehyde:water, 50:5:3.5:41.5, v/ (...truncated)