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First published online 20 July 2005
doi: 10.1242/dev.01924

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An allelic series reveals essential roles for FY in plant development in addition to flowering-time control

Ian R. Henderson^*, Fuquan Liu, Sinead Drea, Gordon G. Simpson and Caroline Dean

Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK

View larger version (33K): [in a new window]   Fig. 1. Domain structure of proteins showing similarity to FY. BLAST searches identified eukaryotic proteins with amino acid similarity to FY (Altschul et al., 1990). The conserved WD repeats are shared between each of the proteins and are shown in blue. The divergent C-terminal domains are shown in grey with the presence of PPLPP motifs or collagen-like domains indicated. These C-terminal domains show little similarity between proteins, apart from the highlighted motifs. The sequences represented are: Saccharomyces cerevisae ScPfs2 (NP_014082), Schizosaccharomyces pombe SpPfs2 (S62544), Neurospora crassa NcPfs2 (XP_322123), Arabidopsis thaliana AtFY (NP_196852), Oryza sativa OsFY (BAB86205, Drosophila melanogaster DmPfs2 (NP_730982), Caenorhabditis elegans CePfs2 (NP_496985), Homo sapiens HsWDC146 (NP_060853.2), Mus musculus (BAC00776 and Tetraodon nigroviridis TnWDC146 (CAD27805.

View larger version (31K): [in a new window]   Fig. 2. In vitro interactions between FCA and FY. (A-D) The FY C-terminal PPLPP motifs interact with the FCA WW domain. (A) Autoradiograph of an acrylamide gel loaded with in-vitro translated, 35S labelled FY proteins present before and after binding to GST-FCA proteins. 10% input (IN) lanes were loaded to represent FCA pre-binding. The lanes adjacent to each input lane show FY expressed as either 7xWD repeats (WD) or the C-terminal domain (CT) that was retained after binding to either GST-FCA-WW (GST-WW) or GST-FCA-WF (GST-WF). (Right) A Coomassie Blue-stained acrylamide gel showing that GST-WW and GST-WF proteins were present in equivalent amounts. (B,D) The CT domain carries two PPLPP motifs (B), which were mutated to stretches of alanines (D). (C) CT domains with either PPLPP-1 (AA-1) or PPLPP-2 (AA-2) or both (AA-1,AA-2) mutated to alanines were tested for binding to GST-WW.

View larger version (30K): [in a new window]   Fig. 3. Analysis of the late-flowering fy and fca mutants. (A) Schematic diagram of FY gene and protein and position of fy mutations. The amino acid changes in fy-3 and fy-4 and nucleotide change in fy-1 are indicated in parentheses. Exons are represented as boxes and introns as lines. FY protein is represented as two protein domains, the WD repeats (7xWD) and C-terminal (CT) domain. (B) Phenotypes of late-flowering fy and fca mutations with (+VRN) and without (–VRN) 6-week vernalization treatments at 4°C. (B) The late-flowering mutants are grown alongside their wild-type, parental accession, either Ler (fy-1, fca-1) or Col (fy-2, fy-3, fca-9). (C) Northern blotting and hybridization analysis of FY, FLC and ß-TUB mRNA expression in wild-type, fy and fca mutant backgrounds. (D) Western blot analysis of FY accumulation in wild-type and fy mutant backgrounds. For northern and western blot analysis RNA and protein were extracted from non-vernalized, 10-day-old Arabidopsis seedlings.

View larger version (48K): [in a new window]   Fig. 4. Flowering time of fy flc double mutants. (A) Phenotypes of Col, flc-3, fy-2 and fy-2 flc-3 plants grown under long day (LD) conditions. (B) Average total number of leaves from 20 plants of each genotype, grown simultaneously under LD conditions (see Table 1). Bars indicate the standard error of the mean.

View larger version (44K): [in a new window]   Fig. 5. FY is an essential gene in Arabidopsis. (A-E) Phenotypic analysis of reproductive development in fy-4/FY plants. (A) Gross phenotype of a fy-4/FY silique showing healthy, green seeds alongside brown, aborted siblings (arrow). (B-E) DIC images of healthy and aborted seeds in fy-4/FY siliques; embryos are indicated with arrows. Healthy embryos are shown at globular (B) and heart (D) stage alongside aborted sibling embryos (C and E, respectively). Scale bars: 25 µm. (F-H) Phenotypic analysis of reproductive development in fy-1/FY, fpa-1/fpa-1 plants. (F) Gross phenotype of an fy-1/FY, fpa-1/fpa-1 silique. (G,H) DIC image of wild-type seed at globular stage of embryogenesis (G) and a sibling aborted seed (H) in an fy-1/FY, fpa-1/fpa-1 silique. White arrows indicate the embryo. Scale bars: 25 µm. (I) Quantification of seed abortion in fy, fpa and fca mutant backgrounds. (J) Complementation of fy-4 by the pFY::FY transgene is shown by dCAPs analysis. The lower DNA band on the ethidium bromide-stained gel is the XbaI-digested product from the fy-4 allele. Homozygote fy-4/fy-4 plants are only obtained after transformation with pFY::FY.

View larger version (48K): [in a new window]   Fig. 6. Virus-induced gene silencing (VIGS) of FCA and FY in Nicotiana benthamiana. (A,B) TRV-FCA (tobacco rattle virus-FCA) infection leads to asymptomatic silencing of FCA. Whole shoot (A) and leaf (B) phenotypes of Nicotiana plants infected with TRV-FCA. (C,D) TRV-FY infection leads to silencing of FY and associated yellowing of leaf tissues. Whole shoot (C) and leaf (D) phenotypes of Nicotiana plants infected with TRV-FY. (E) RNA was extracted from Nicotiana infected with either TRV-FCA, TRV-FY and TRV-00 (empty vector) and used to generate cDNA. The cDNA was then analysed by RT-PCR for expression of endogenous FCA, FY and ACTIN mRNA. The number of PCR cycles used is indicated above the gel and the final lane (–RT) is a minus RT control.

View larger version (113K): [in a new window]   Fig. 7. Expression pattern of FY. (A-C) FY::GUS expression during seed development. Seeds were histochemically stained for GUS activity which appears as a blue precipitate. Seed were stained at globular (A), heart (B) and mature (C) stages of embryogenesis. Scale bars: 50 µm. (D-F) FY::GUS expression in 12-day-old seedlings. Whole seedlings show GUS staining around the shoot meristem and in young leaves and in the vascular tissue of the cotyledons (D). The roots also show GUS staining in the meristematic regions. In roots, both the root apical meristem (E) and emerging lateral root meristems (F) show GUS expression. (G-J) Expression of the PFCA–FCAto exon5:GUS reporter transgene in Ler (G), fy-1 (J), fy-2 (I) and fca-1 (H) backgrounds. 12-day-old seedlings were grown on plates and histochemically stained for GUS expression. (K-R) FY expression was confirmed using in situ hybridization. Hybridization with an antisense FY probe to sectioned seed (K-M) or mature seedlings (O-Q) showed expression as blue staining. Scale bars: 50 µm. Hybridization with a sense probe to seeds or mature tissue shows no staining (N and R).

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