QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 26 January 2005
doi: 10.1242/dev.01642

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carles, C. C. Articles by Fletcher, J. C. Search for Related Content PubMed PubMed Citation Articles by Carles, C. C. Articles by Fletcher, J. C.

ULTRAPETALA1 encodes a SAND domain putative transcriptional regulator that controls shoot and floral meristem activity in Arabidopsis

Cristel C. Carles^1,2, Dan Choffnes-Inada^1,2, Keira Reville^1,2,*, Kvin Lertpiriyapong² and Jennifer C. Fletcher^1,2,

¹ Plant Gene Expression Center, USDA/UC Berkeley, 800 Buchanan Street, Albany, CA 94710 USA
² Plant and Microbial Biology Department, UC Berkeley, 111 Koshland Hall, Berkeley, CA 94720 USA

View larger version (116K): [in a new window]   Fig. 1. Inflorescence and flower phenotypes of ult1 mutants and complementation test. (A-D) Inflorescence meristems of (A) wild-type Ler, (B) ult1-1, (C) ult1-2 and (D) ult1-3 plants. (E-G) In situ expression analysis of STM in (E) Ler, (F) ult1-1 and (G) ult1-2 inflorescences. (H) Inflorescence and (inset) flower of ult1-1 plants transformed with the ULT1-214 construct containing a 2.7 kb ULT1 genomic fragment. (I-L) In situ expression analysis of WUS in (I) Ler, (J) ult1-1, (K) ult1-2 and (L) ult1-3 inflorescences. (M-O) Analysis of pSTM::uidA expression in (M) Ler, (N) ult1-1 and (O) ult1-2 inflorescences. (P) Quantification of WUS-expressing cells in Ler (WT) and all three ult1 alleles. The mean number of cells was calculated from the most central section of eight individual inflorescences for each genotype. For each section, the maximum number of cells found in one horizontal (width) and one vertical (height) cell file, as well as the total number of cells expressing WUS, was scored. The standard deviation is indicated. Scale bars: 50 µm.

View larger version (56K): [in a new window]   Fig. 2. ULTRAPETALA1 cloning and sequence analysis. (A) Schematic of the positional cloning of the ULT1 locus and the structure of the ULT1 gene. The region of chromosome 4 containing BACs T29A15 to F19B15 is represented. The CAPS markers designed for mapping ult1-2 are shown in black boxes and the frequency of recombinant chromosomes is indicated for each marker. The exon/intron structure of the ULT1 gene is shown along with the positions of the ult1-1 and ult1-2 mutations. (B) Alignment of the conceptual translation products of the Arabidopsis ULT1 and ULT2 genomic sequences with conceptually translated consensus EST sequences from four other plant species. The sequences compared are from Arabidopsis thaliana ULT1 (AtULT1, At4g28190), Arabidopsis thaliana ULT2 (AtULT2, At2g20825), Glycine max (GmULT, BM524875.1), Lycopersicon esculentum (LeULT, EST357945), Oryza sativa (OsULT, CA763280.1) and Triticum aestivum (TaULT, BG604592). Identical amino acids are boxed and blocks of similar amino acid residues are shaded. The positions of the mutations in ULT1 and ULT2 are shown above the sequences, the SAND domain is boxed in red and the B box-like motif is boxed in green. Stars indicate the amino acid substitution in the ult1-1 (C173T) and ult1-2 (S83F) alleles. Arrowheads denote the position of the T-DNA insertion in the ult1-3 and the ult2-1 allele. Arginine/lysine rich nuclear localization signal (NLS) candidate polypeptides are underlined in white. (C) Multiple sequence alignment of AtULT1, AtULT2 and animal SAND domains from CeC44F1.2 (Caenorhabditis elegans, Z49067), CeC25G4.4 (Caenorhabditis elegans, Z70680), HsSp100b (Homo sapiens, U36501), HsNucP41 (Homo sapiens, Q14976), HsNUDR (Homo sapiens, AF049459), DmDEAF-1 (Drosophila melanogaster, AAC47040, HsGMEB2 (Homo sapiens, NM031803), HsGMEB1 (Homo sapiens, NM006582), AIRE-1 (Homo sapiens, AB006682). The alignment was obtained with the ClustalW 1.82 program and manually refined using the calculated two-dimensional structure. Secondary structure elements are shown above the multiple alignment. Period, semicolon and asterisk mark partial to full residue conservation. Color-coding reflects the conservation of amino acid types. Background colors reveal their physiochemical properties (green: hydrophobic; red: positively charged residues; blue: negatively charged residues), while foreground colors mark identical (red) and similar (blue) amino acids. The amino acid corresponding to the position of ult1-2 mutation is underlined. (D) Alignment of the AtULT1 and AtULT2 B box-like domains with B-box proteins from animals: CeLIN-41 (Caenorhabditis elegans, NP492488), CeNCL-1 (Caenorhabditis elegans, P34611), DmDAPPLED (Drosophila melanogaster, Q9V4M2), HsTIF-1 (Homo sapiens, NP003843), HsPML (Homo sapiens, P29590). The conserved cysteine/histidine residues are boxed. Below the sequence alignment, the conserved spacing of the B2 B-box consensus (Torok and Etkin, 2000) and the ULT B-box consensus are compared.

View larger version (98K): [in a new window]   Fig. 3. Subcellular localization of the ULT proteins. (A) Dark-field exposure of an onion epidermal cell transiently expressing an ULT1-EGFP fusion protein. The GFP signal is detected in both the nucleus (arrowhead) and the cytosol. In the cytosol, the fusion protein appears to be distributed in cytoplasmic streams (arrows). (B) Confocal image of a root from a 35S::ULT1-EGFP T2 transgenic plant. (C,D) Confocal image of a petal from a 35S::ULT1-EGFP T2 transgenic plant. (D) DAPI staining of the nuclei and cell walls in the petal shown in C. (E) An immunoblot of protein extracts from inflorescence meristem tissue, using anti-GFP serum. WT, extract from a wild type Ler plant; EGFP, extract from a 35S:EGFP transgenic plant; ULT-EGFP, extract from a 35S::ULT1-EGFP T2 transgenic plant. (F,G) Dark-field exposures of onion epidermal cells transiently expressing the ULT1-GUS-EGFP fusion protein. The GFP signal is detected in the cytosol and the perinuclear region (F) or both in the cytosol and throughout the nucleus (G). N, nucleus; CS, cytoplasmic streams.

View larger version (56K): [in a new window]   Fig. 4. Expression profiles of the ULT1 and ULT2 genes. RT-PCR analysis was performed on RNA extracts from various wild type Ler tissues: roots (R), 8-day-old seedlings (S), mature rosette leaves (L), stems (St), inflorescence apices (In), pollen (P) and siliques (Si). ULT1 transcripts were amplified from all tissues examined, whereas ULT2 transcripts were detected only during the reproductive phase in inflorescences, pollen and siliques. EF1 was amplified as a control. In addition, control amplification reactions were run with each set of primers using genomic DNA (gDNA) as a template.

View larger version (162K): [in a new window]   Fig. 5. ULT1 and ULT2 mRNA expression patterns in inflorescence and flower tissues. RNA localization by in situ hybridization with ULT1 (A,D,H) and ULT2 (B,F,I) antisense probes hybridized to wild-type Ler tissues. (A-C) Longitudinal sections through the inflorescence meristem (ifm) and adjacent floral meristems (fm). ULT1 mRNA is localized throughout the inflorescence meristem. No signal was detected in stage 1 floral meristems (white arrowheads). ULT1 transcripts reappeared in late stage 2 floral primordia (black arrowheads). As soon as the sepals initiate (stage 3 flower), ULT1 expression becomes restricted to the center of the floral meristem. (C) Control hybridization with an ULT2 sense probe. (D-G) Longitudinal sections through stage 7-8 flowers. ULT1 (D) and ULT2 (F) mRNA was detected in stamen (St) and carpel (Ca) primordia. In both cases the signal appears stronger on the adaxial side of the carpels (arrows). (E,G) Control hybridizations with ULT1 and ULT2 sense probes. (H-J) Transverse sections through mature flowers. ULT1 (H) and ULT2 (I) mRNA was detected in ovules (white arrowheads) and tapetum tissue in the anthers (black arrowheads). (J) Control hybridization with an ULT1 sense probe. Scale bars: 50 µm.

View larger version (114K): [in a new window]   Fig. 6. ULT1 and ULT2 mRNA expression patterns in seedlings and embryos. RNA localization by in situ hybridization with ULT1 (A,D,J,K,N,P) and ULT2 (B,E,G,L,M,O) antisense probes hybridized to wild-type Ler seedlings (A-C) and embryos (D-Q). (A-C) Longitudinal sections through 7-day-old seedlings. (A) ULT1 transcripts are localized throughout the vegetative SAM and in young leaf primordia. (B) ULT2 transcripts are not detected in seedlings. (C) Control hybridization with an ULT1 sense probe. (D-H) Longitudinal sections through mature embryos. (D) ULT1 expression is restricted to the embryonic SAM (arrowhead). (E) ULT2 transcripts can be detected in the embryonic SAM and RAM (arrowheads). (G) Higher magnification of ULT2 expression in the RAM. (F,H) Control hybridization with ULT1 and ULT2 sense probes, respectively. (I-Q) Longitudinal sections through embryos at different stages of embryogenesis. (I) Early globular stage. (J) Eight-cell-stage embryo. (K) Late triangle stage. (L) Early heart stage. (M) Late heart stage. (N) Early torpedo stage. (O) Torpedo stage. (P,Q) Bending cotyledon stage. (I,Q) Control hybridizations with an ULT1 sense probe. Scale bars: 50 µm in A-F; 25 µm in G-Q.

View larger version (13K): [in a new window]   Fig. 7. Rescue of the ult1-1 mutant phenotype by an ULT2 transgene. (A) Floral organ number in wild-type Ler plants, ult1-1 plants and ult1-1 plants containing the d35S::ULT1 or d35S::ULT2 construct. Graph shows the mean number of organs in the first ten flowers of 10 plants (n=100 flowers), and the standard error is indicated. For the transgenic lines in the ult1-1 mutant background, the mean organ number was calculated from the first ten bolting T1 plants that did not show an overexpression phenotype. (B) Days to bolting after germination for Ler plants, ult1-1 plants and ult1-1 plants containing the d35S::ULT1 or d35S::ULT2 construct. The mean number of days to bolting was calculated from the same populations of plants that were used for the floral organ counts in (A) (n=10 plants), and the standard error is indicated.

View larger version (42K): [in a new window]   Fig. 8. ULT1 and ULT2 T-DNA insertion alleles. (A) RT-PCR on wild type Ler, ult1-3 and ult2-1 T-DNA insertion mutant inflorescences. ULT1 transcripts could be amplified from Ler (wild-type) plants but not from ult1-3 plants, while ULT2 transcripts were detected in wild-type Col-0 and ult2-1/+ heterozygous plants but not in ult2-1 homozygous plants after 40 cycles of PCR. However, after 45 cycles a faint signal corresponding to correctly spliced ULT2 transcript was detected in the ult2-1 homozygous lane. EF1 was amplified as a control. Additional control amplification reactions were run with each set of primers using genomic DNA (gDNA) as a template. (B) Floral organ number in Ler, ult1-1, ult1-2 and ult1-3 mutant plants. Graph shows the mean number of organs in the first ten flowers of 10 plants (n=100 flowers), and the standard error is indicated. (C) Mean days to bolting after germination for Ler, ult1-1, ult1-2 and ult1-3 plants (n=10 plants). The standard error is indicated. (D) Floral organ number in ult1-3 homozygous plants, ult1-1/+ heterozygous plants and ult1-1/ult1-3 plants. Graph shows the mean number of organs in the first ten flowers of four or six plants (n=40 flowers for ult1-3 and n=60 flowers for the other genotypes), and the standard error is indicated. (E) Mean days to bolting after germination for ult1-3 homozygous plants, ult1-1/+ heterozygous plants and ult1-1/ult1-3 plants (n=4 plants for ult1-3 and n=6 flowers for the other genotypes). The standard error is indicated.

View larger version (93K): [in a new window]   Fig. 9. Phenotypes of ULT antisense (AS) lines. (A) RT-PCR on inflorescences of the least affected ULT AS plants (those showing the flower phenotypes illustrated in G-I). The expression of both ULT1 and ULT2 is downregulated. EF1 was amplified as a control. (B) Vegetative phenotypes of the most severely affected ULT AS lines. The plants were grouped into three classes based on their SAM termination phenotypes (class 1 plants terminate the earliest). Shoot apex bright-field images (first row), SEM images (second row), and longitudinal sections (third row) are shown for 7-day-old wild-type Ler seedlings and 14-day-old ULT AS seedlings. (C-E) Reproductive phenotypes of the ULT AS lines. (C) Six-week-old Ler plants. (D,E) Six-week-old ULT AS plants showing reduced flower number and premature arrest of the axillary inflorescence meristems. (F-I) Flower phenotypes of the least affected ULT AS lines. (F) Ler flower. (G) ULT AS flower with extra sepals and petals. (H) Siliques from a Ler flower and from an ULT AS flower with extra carpels. (I) Silique from an ULT AS flower dissected open to reveal the presence of a fifth whorl carpeloid structure developing inside the fourth whorl (arrow). Scale bars: 20 µm.