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Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis

Valérie Gaudin^1,*, Marc Libault¹, Sylvie Pouteau¹, Trine Juul¹, Gengchun Zhao^1,, Delphine Lefebvre¹ and Olivier Grandjean²

¹ Laboratoire de Biologie Cellulaire,
² Laboratoire de Génétique, INRA, route de St Cyr, 78026 Versailles cedex, France
Present address: Northeast Normal University, Changchun, China

View larger version (121K): [in a new window]   Fig. 1. Arabidopsis lhp1 mutant phenotype. (A) Six-week-old wild-type Arabidopsis plant (right) and the lhp1-1 mutant (left) under SD. (B) Close-up view of an lhp1-1 inflorescence showing small and upwardly curled cauline leaves, and normal inflorescences and flowers. (C) Close-up view of an lhp1-1 rosette with small, narrow leaves downwardly curled along the longitudinal axis of the leaf. (D,E) Scanning electron micrographs of the upper epidermis of a third rosette leaf from 33-day-old lhp1-1 (D) and wild-type (E) plants. Scale bars, 100 µm.

View larger version (22K): [in a new window]   Fig. 2. Structure of the lhp1-1 locus. (A) Localisation of the 1.2 kb deletion on the genomic MVA3 P1 clone which accompanied the T-DNA region insertion in lhp1-1. Database searches indicated two putative genes in this region, LHP1 and TMP. The plasmids, pCaS, pCaES and pCaSSP, were used for complementation experiments. (B) Detail of the T-DNA insertion in lhp1-1. Insertion occurs at position 11954, 22 bp upstream of the 5' end of the isolated cDNA (11932). At the T-DNA right-border::genomic DNA junction, the integration was accompanied by deletions of the 24 bp RB repeat and the 21 bp adjacent to it. A small 47 bp insertion with no identified homology was also detected. The T-DNA LB was better conserved than the RB as commonly observed during T-DNA integration. Numbers refer to the sequence of the MVA3 P1 clone (accession number AB006706).

View larger version (93K): [in a new window]   Fig. 3. Sequence of LHP1(GenBank accession no. AF387639). Nuclear localisation signals (NLS) are in bold and underlined. The bipartite NLS is marked with stars. The N-terminal acidic domain is indicated by a wavy underline. The chromo domain (108-159 aa) is boxed and framed with two dots. The chromo shadow domain (379-441 aa) is in bold and boxed. The sequence is highly conserved between two different ecotypes, Ler and Col-0, with only one substitution from E22 (Col-0) to D (Ler), and a change at nt 96 from C (Col-0) to A (Ler).

View larger version (38K): [in a new window]   Fig. 4. Sequence comparisons of the chromo (A) and chromo shadow domains (B). Arabidopsis AtLHP1 (present work), B. rapa BrLHP1 (the EST sequence is likely truncated at the 5' end), Daucus carota DcCB1 (D83719), Drosophila melanogaster HP1 (DmHP1; AAA28620) and Polycomb (DmPC; A38565), Homo sapiens HP1 (HuHP1, P45973) and Mus musculus HP1 (MmMOD1; P23197). The positions of the regions used for alignments and identity scores between AtLHP1 and different CD/CSD are indicated. Based on NMR studies, ß-sheet and -helix secondary structures are indicated above the alignments (Ball et al., 1997; Brasher et al., 2000). The residues that form the hydrophobic core are highlighted in yellow. (A) Residues glycine 34 and 44 and proline 54, playing a crucial role in the tertiary CD structure, are indicated (green star). A black star indicates the position of the Y24F mutation in DmHP1. (B) Residues involved in dimerisation (with the main contacts being at MmMOD1 I161, Y164 and L168) are highlighted in purple.

View larger version (71K): [in a new window]   Fig. 5. LHP1 homodimerisation in the yeast two-hybrid system. (A) Schematic representations of LHP1 and truncated LHP1 proteins. LHP1N: N-terminal region (aa 1-194). LHP1C: long C-terminal region (aa 162-445). LHP1CSD: short C-terminal region (aa 378-445). (B,C) Growth of different yeast strains containing combinations of fusion proteins. Selective media lacking particular amino acids (L: leu, W: trp, H: his) or rich YPD medium lacking adenine are indicated under each plate. On medium lacking adenine, interacting proteins result in white colonies, non-interacting proteins results in red colonies. X-Gal plates show staining for ß-galactosidase activity. 1, positive control. The plasmids pTD1-1 and pVA3-1 encoding proteins p53 and SV40 are known to interact in vivo. 2, pBD-LHP1 and pAD-LHP1. 3, pBD-LHP1 and pTD1-1. 4, pAD-LHP1 and pVA3-1. 5, pBD-LHP1N and pAD-LHP1N. 6, pBD-LHP1C and pAD-LHP1C. 7, pBD-LHP1CSD and pAD-LHP1CSD.

View larger version (37K): [in a new window]   Fig. 6. RT-PCR transcriptional analyses of LHP1 (A) and CONSTANS (B) in the wild type and lhp1-1 mutant. (A) LHP1 expression is ubiquitous during development in wild-type plants whereas it is downregulated in the mutant. (B) CO expression is upregulated in lhp1 at an early vegetative stage. Expression of the constitutive APT1 gene was used as a positive control and to normalise the amounts of cDNA. Different organs or tissues were collected from plants grown in LD, at the following developmental stages: S-2c (two cotyledons), S-6rl (6 rosette leaves), S-eb (early bolting), S-of (first open flower). The tissues were WP (whole plants), AP (aerial parts), R (roots), FB (floral buds), F (mature flowers) and YS (young siliques).

View larger version (57K): [in a new window]   Fig. 7. LHP1 has a specific subnuclear localisation in tobacco mesophyll protoplasts, in transient assays. Chloroplasts appear red and GFP fluorescence is green; when the two fluorescences overlap, the yellow colour appears. (A,B) Protoplasts expressing GFP alone (pAVA121 plasmid). (A) Projection. (B) Section. (C,D) Protoplast expressing GFP-VirD2NLS. (C) Projection. (D) Section. The GFP fluorescence is uniformly distributed throughout the nucleus. (E-H) Protoplasts electroporated with the LHP1-GFP construct. A diffuse nucleoplasmic distribution and discrete particles are observed. (E) Projection of several protoplasts. One protoplast expresses the LHP1-GFP fusion in the nucleus, the others are not transformed. (F) Close-up view of the nucleus (projection). (G-H) Sections. Scale bar, (A-E) 10 µm; (F-H) 2 µm.

View larger version (44K): [in a new window]   Fig. 8. Sequential confocal optical sections through a nucleus expressing the LHP1-GFP fusion. A diffuse fluorescence was detected in the nucleoplasm but was excluded from the nucleolus. Discrete nuclear bodies were observed which tended to accumulate around the nucleolus.