Michael Leunig A putative transcription co-repressor complex regulates floral organ identity and shape by Zhongchi Liu, University of Maryland, USA Dr. Seuss Gary Larson Michael Leunig

Floral homeotic gene AGAMOUS (AG) specifies stamen and carpel identity WT whorl 1 whorl 2 whorl 3 whorl 4 ag-1 AG expression in WT ag-1 Yanofsky et al., Nature, 1990; Drews et al., Cell, 1991

leunig (lug) mutation results in ectopic expression of AG wild-type lug lug-1 H S X B E3 GUS 2.98kb E2 Liu and Meyerowitz, Development (1995); Busch et al., Science (1999); Deyholos and Sieburth, Plant Cell (2000)

Structural Similarity to Transcription Co-repressors 1 931 LUG (Arabidopsis) Q-rich (89-184, 449-470) LUFS 7 WD 1 787 LUH (Arabidopsis) Q-rich LUFS 7 WD 1 706 Tup1 (Yeast) Ssn6 Binding Q-rich (97-118, 181-198) 7 WD 1 716 Groucho (Drosophila) Q-rich Ser-pro rich 7 WD Conner and Liu, PNAS, (2000)

The repression of a-cell-specific genes in S. cerevisiae a cells Tup1 Ssn6 a-cell specific genes a 2 MCM1 The repression of AG in first two whorls of Arabidopsis flower LUG ? ? AG exon ? AG second intron

seuss (seu) resembles lug and enhances lug lug seu

AG mRNA expression WT lug seu

SEU encodes a Q-rich protein with a conserved dimerization domain Q-rich domain LIM Interacting domain Frameshift (seu-2) Q to stop (seu-1) 1 877 SEUSS NLS 1 933 Rice EST 81% 1 748 Arabidopsis Homolog 55% 1 384 Zebra fish LIM domain- binding factor (Ldb1) 22% LID 1 375 Mouse Ldb1 21% LID

LUG LUFS domain interacts with full length SEU LUG-DB Constructs LUFS LUFS Q-rich (89-184, 449-470) LUFS Q-rich (89-184, 449-470) 7 WD Q-rich (89-184, 449-470) 7 WD

1. 2. 3. 4. 5. 6. Reporters 5x Gal4UAS CaMV 35S GUS 3’NOS LUC GAL4DB LUG SEU Effectors 3. 4. 5. 6. 2 4 6 8 10 12 14 16 18 GUS/LUC LUG-BD (1+2+4) SEU-BD (1+2+5) SEU No Effector 1+ 2 GAL4BD 1+2+3 1+2+ effector

LUG SEU ? AG exon ? AG second intron

larson-1 (lsn-1) enhances lug at 20oC ap2-2 lsn-1 lug-8 lsn-1 lug-8 lsn-1 lug-8

lsn-1 is temperature-sensitive lsn-1 at 20 oC lsn-1 at 29 oC

Ectopic AG expression in lsn-1 at 29oC AG second intron H S X B E3 GUS 2.98kb E2

LSN encodes a BEL1-like homeodomain protein 575 349 414 100% lsn-1 P>L lsn-2 R>L 173 228 295 391 456 85% BEL1 610 263 350 193 SKY domain BEL domain Homeodomain

LSN Belongs to The TALE Superclass Homeodomain Protein Helix 1 Helix 2 Helix 3 Cons. RELSKKRKRGKLPKEATQILRAWWPQHLKNPYPSEEEKEALAEETGLTLKQINNWFINARRRHWKPMIDMQ IRO VTYDLAARRKNATRESTSTLKAWLNEHKKNPYPTKGEKIMLAIITKMTLTQVSTWFANARRRLKKENKMTW PBC REQSRRRKRRNFNKQATEILNEYFYSHLSNPYPSEEAKEELAKKCGITVSQVSNWFGNKRIRYKKNIGKFQ KNOX LEFSKKKKKGKLPKEARQQLLEWWKGHYKWPYPTESDKISLAESTGLDPKQINNWFINQRKRHWKPSEHMQ CUP RRKQN-SGRSNLPKETVSILNTWLLNHLNNPYPTQQEKRELLIKTGLTKIQLSNWFINVRRRKIFSDYYTL TGIF GSGKR-RRRGNLPKESVQILRDWLYEHRYNAYPSEQEKALLSQQTHLSTLQVCNWFINARRRLLPDMLRKD MEIS KDKKRQKKRGIFPKVATNIMRAWLFQHLTHPYPSEEQKKQLAQDTGLTILQVNNWFINARRRIVQPMIDQS M-ATYP VVSPAVGCRNLSEDLPAYHMRKHFLHTLDNPYPTQEEKEGLVRLTNESTARVGSWFINARRRSGWSHILKK BEL HPQHAWRPQRGLPERSVSVLRAWLFEHFLHPYPKDSDKHMLARQTGLSRNQVSNWFINARVRLWKPMVEEM Classes L L LSN HHAPVWRPHRGLPERAVTVLRAWLFDHFLHPYPTDTDKLMLAKQTGLSRNQVSNWFINARVRVWKPMVEEI BELL1 ---HPWRPQRGLPERAVTTLRAWLFEHFLHPYPSDVDKHILARQTGLSRSQVSNWFINARVRLWKPMIEEM ATH1 KNHQIWRPQRGLPEKSVSVLRNWMFQNFLHPYPKDSEKHLLAIRSGLTRSQVSNWFINARVRLWKPMIEEM AAD51349 ---EAWRPQRGLPERSVNILRAWLFEHFLHPYPSDADKHLLARQTGLSRNQVSNWFINARVRLWKPMVEEM AAF18625 HHSNAWRPQRGLPEKAVSVLRSWLFEHFLHPYPRDLDKVMLAKQTGLTKSQVSNWFINARVRMWKPLVEEL AAK43834 ---EAWRPQRGLPERSVNILRAWLFEHFLNPYPSDADKHLLARQTGLSRNQVSNWFINARVRLWKPMVEEM AAK43836 PSNNAWRPQRGLPERAVSVLRAWLFEHFLHPYPKDSDKHMLAKQTGLTRSQVSNWFINARVRLWKPMVEEM AAL10487 ---HTWRPQRGLPDSSVLVLRAWLFEHFLHPYPKDSDKIMLARQTGLSRGQVSNWFINARVRLWKPMVEEM AAL32623 ----AWRPQRGLPENSVSILRAWLFEHFLHPYPKESEKIMLSKQTGLSKNQVANWFINARVRLWKPMIEEM NP180366 QQQHIWRPQRGLPERAVAVLRAWLFDHFLHPYPTDSDKQMLATQTGLSRNQVSNWFINARVRLWKPMVEEI NP195187 ---QAWRPQRGLPENSVLILRAWLFEHFLHPYPKDSDKIMLARQTGLSRGQVSNWFINARVRLWKPMVEEI ***:****: :* :** *:*::****** * :* :*: ::**:*:**:*********:****:* Arabidopsis Homologues

Structure of Hox B1-Pbx1 heterodimer bound to DNA Piper,D.E., Batchelor,A.H., Chang,C.P.,Cleary,M.L. and Wolberger,C. Cell 96 (4), 587-597 (1999)

Gal2p lacZ Gal7p ADE2 Gal1p HIS3 DB AD pJ69-4A yeast host LUG LSN D81 (three reporters) Gal2p lacZ LUG + Gal7p ADE2 + LSN Gal1p HIS3 + + LUG LSN D3 575 349 414 HD 81 pVA3 pTD1 LUG SEU

Binding of LSN to AG second intron 2985 AG GST-LSN GST

In whorls 1 and 2 of a flower A proposed model on how LUG and SEU repress AG In whorls 1 and 2 of a flower LUG SEU AP1? AG exon LSN AG second intron

seu and lug mutants develop narrow floral organs

SEU and LUG regulate organ shape independently of AG ag-1 seu-1 ag-1 ag-1 seu-1 lug-1 seu-1 lug-1 ag-1 seu-1 lug-1

The narrow organ shape may result from a loss of organ polarity ag-1 ag-1 seu-1 lug-1 PROBE PHABULOSA (Adaxial) YABBY1 (Abaxial) See poster # 14 by Robert Franks

In whorls 1 and 2 of a flower Organ shape LUG In whorls 1 and 2 of a flower SEU ? X PHB/YAB Floral organ identity LUG SEU AP1? AG exon LSN AG second intron

ACKNOWLEDGEMENT Postdoctoral fellows Joann A. Conner Robert G. Franks V.V. Sridhar Graduate students Alex Xiaozhong Bao Anandkumar Surendrarao Jayashree Sitaraman Black Chunxin Wang Joshua Levin, Syngenta John Bowman, UC Davis Steve Henikoff, FHCRC Our work is funded by USDA, DOE, NSF and a NIH postdoctoral fellowship to B. Franks