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First published online 22 March 2006
doi: 10.1242/dev.02331

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The LEAFY target LMI1 is a meristem identity regulator and acts together with LEAFY to regulate expression of CAULIFLOWER

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.

View larger version (29K): [in a new window]   Fig. 1. T-DNA insertion alleles of LMI1. (A) Map of the LMI1 locus. Exons are shown as rectangles, introns as black lines. The homeodomain is indicated in red, the adjacent leucine zipper motif in blue. The four SALK insertion lines analyzed are indicated as triangles. The distance between the lines connecting each insertion with the sequence indicates the size of the deletion in each insertion. The lmi1 allele designation and SALK number are indicated. (B) Semi-quantitative RT-PCR analysis of LMI1 expression in four-day-old seedlings of the insertion lines compared with wild type (Col). PCR was performed with primers flanking the insertions (LMI1; primers L5' and L3' in A) or upstream of the lmi1-1 insertion (LMI1up; primers Lu5' and Lu3' in A). The eukaryotic translation initiation factor 4A (EIF4) was amplified as an internal control.

View larger version (78K): [in a new window]   Fig. 2. lmi1 lfy-10 meristem identity phenotypes. (A-C) Meristem identity and flowering time of lmi1 lfy-10 double mutants compared with the weak lfy-10 allele, the intermediate lfy-9 allele, and the lfy-1 null allele. Secondary inflorescence (A) and bract numbers (B) were counted, as well as the number of rosette leaves formed (C). Shown are the mean values±standard error (s.e.m.) of at least 20 plants counted (10 for lfy-1). Asterisks denote statistical significance in comparison to values of lfy-10 (Student's t-test, P<0.0088). Arrow identifies the lfy-10 mutant in A-C. (D) Meristem identity transition in lfy-10 (left) and lmi1-1 lfy-10 (right). The arrowhead indicates the position of the first flower formed. (E) Flowers of lfy-10 (left) and lmi1-1 lfy-10 (right), with floral homeotic defects typical of weak lfy mutants, such as a reduction in the number of petals and stamens formed.

View larger version (80K): [in a new window]   Fig. 3. lmi1 lfy-1 meristem identity phenotypes. (A-C) Meristem identity and flowering time were assayed in lmi1 lfy-1 double mutants compared with the lfy-1 null allele. Secondary inflorescence (A) and bract numbers (B) were counted, as well as the number of rosette leaves formed (C). Shown are the mean values ±s.e.m. of at least 10 plants counted. Asterisks denote statistical significance in comparison to values of lfy-1 (Student's t-test, P<0.0065). (D) Primary inflorescence branches of lfy-1 (top) lmi1-1 lfy-1 (center) and lmi1-4 lfy-1 (bottom). Small arrows point to lfy-1-looking flowers that are subtended by bracts.

View larger version (47K): [in a new window]   Fig. 4. Delayed AP1 induction and reduced CAL expression in lmi1-1 lfy-10. (A) Quantitative real-time RT-PCR analysis of the expression of the known direct LFY targets AP1, CAL, and LMI1 through LMI5 in eleven-day-old lmi1-1 lfy-10 compared with lfy-10. The values were normalized using eukaryotic translation initiation factor 4A (EIF4). Expression levels in lfy-10 lmi1-1 were set to one to facilitate comparison of the expression of the different genes. The mean and standard error of three technical replicates is shown. Similar results were obtained using an independent biological replicate (not shown). (B) Quantitative real-time PCR amplification of CAL and AP1. For CAL expression, lmi1-1 lfy-10, lmi1-4 lfy-10 and lfy-10 seedlings were assayed at 7, 9, 11 or 13 days of age. For AP1 expression, lmi1-1 lfy-10 and lfy-10 seedlings were assayed at 9, 11 or 13 days of age. Similar results were obtained for both CAL and AP1 using an independent biological replicate (not shown). Normalization was as in A. (C) Transverse sections through the inflorescence meristems of fixed fifteen-day-old lfy-10 (top) and lmi1-1 lfy-10 (bottom) seedlings. Flowers (F) and secondary inflorescences (I) are indicated. Scale bars: 100 µm.

View larger version (16K): [in a new window]   Fig. 5. LMI1 binds CAL promoter proximal elements. (A) Map of the promoter proximal region of LMI1. The pseudo-palindromic class I HD-ZIP-binding site (CAATNATTG) and the LFY-binding site (CCANTG) are indicated above the diagram, as is the CAL translation start site (long arrow). Below the diagram is the region amplified by chromatin immunoprecipitation by William et al. (William et al., 2004) and in B. (B) Quantitative real-time PCR amplification of regulatory regions upstream of CAL and AP1, before (input) and after chromatin immunoprecipitation (ChIP). Plants expressing MYC-LMI1 (+) were compared with those not expressing MYC-LMI1 (-). Shown is the mean±s.e.m. of the ChIP signal normalized by the input signal. Two technical replicates were performed. Similar results were obtained using an independent biological replicate (not shown).

View larger version (83K): [in a new window]   Fig. 6. LMI1 expression in sectioned apices based on reporter fusion. (A-H,K,L) Dark-field and (I,J) bright-field images of 8 µm sections of wild-type plants (A-I,K) and lfy-1 null mutants (J,L), showing ß-glucuronidase (GUS) reporter activity controlled by LMI1 regulatory regions. Seedling stages assayed were vegetative (A), bolting (B), first flower primordium formed (C) and reproductive (D-G,I-L). A stage 10 flower is shown in H. se, sepal; pe, petal; st, stamen; F, flower; I, secondary inflorescence. Arrows point to staining in floral organs; arrowheads highlight staining in the abaxial side of young flowers. All sections were longitudinal. except for E. Scale bar: 100 µm.

View larger version (82K): [in a new window]   Fig. 7. LMI1:GUS expression in whole-mount samples. (A-H) Bright-field (A,C-G) and differential interference contrast (B,H) images of ß-glucuronidase (GUS) reporter activity controlled by LMI1 regulatory regions. Stages assayed were young seedlings (A,B), older seedlings (C,D), young inflorescence with secondary inflorescences (E), and older inflorescences (F,G). A heart-stage embryo is shown in H. The inset in H depicts a torpedo-stage embryo. Hy, hypocotyls; L(y), young leaves; L(e), elongating leaves; Br, bracts; Sp, stipules; S, sepal; P, petal; St, stamen; Ca, carpel. Scale bars: white, 1 mm; black, 100 µm.

View larger version (58K): [in a new window]   Fig. 8. Leaf morphogenesis defects in lmi1 mutants. (A,B) ß-glucuronidase (GUS) expression driven by LMI1 regulatory regions in (A) wild-type and (B) lfy-1 null mutant. Scale bar: 1 mm. (C) Images of rosette leaves in lmi1-1 compared with Col. Shown are early (leaf 9, top), intermediate (leaf 13, center) and later (leaf 19, bottom) rosette leaves. Plants were grown in short-day conditions. (D) Leaves grown and selected as in C from lmi1-1 lfy-1 mutants. (E) Quantitative real-time PCR analysis of BREVIPEDICELLUS (BP) expression in expanding leaves one and two of 11-day-old lmi1-1 compared with Col seedlings. Three technical replicates of two biological replicates were each normalized using ß-TUBULIN. Mean±s.e.m. is shown.

View larger version (24K): [in a new window]   Fig. 9. Model of meristem identity transition. The transcription factor LFY directly activates three known meristem identity regulators: AP1, CAL and LMI1. LFY and LMI1 together activate CAL directly in a feed-forward loop network motif. CAL is known to act upstream of AP1. LMI1 is upregulated by another unknown factor (indicated as X) in addition to LFY. LMI1 also has a LFY-independent role in meristem identity (dashed arrow from LMI1). Additional direct LFY targets are known (arrow from LFY) that may also play a role in meristem identity. Dashed arrows indicate direct or indirect effects, solid arrows indicate a direct effect. Developmental time is indicated along the vertical axis.

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