Arabidopsis CLP1-SIMILAR PROTEIN3, an ortholog of human polyadenylation factor CLP1, functions in gametophyte, embryo, and postembryonic development

Abstract

Polyadenylation factor CLP1 is essential for mRNA 3'-end processing in yeast and mammals. The Arabidopsis (Arabidopsis thaliana) CLP1-SIMILAR PROTEIN3 (CLPS3) is an ortholog of human hCLP1. CLPS3 was previously found to be a subunit in the affinity-purified PCFS4-TAP (tandem affinity purification) complex involved in the alternative polyadenylation of FCA and flowering time control in Arabidopsis. In this article, we further explored the components in the affinity-purified CLPS3-TAP complex, from which Arabidopsis cleavage and polyadenylation specificity factor (CPSF) subunits AtCPSF100 and AtCPSF160 were found. This result implies that CLPS3 may bridge CPSF to the PCFS4 complex. Characterization of the CLPS3 mutant revealed that CLPS3 was essential for embryo development and important for female gametophyte transmission. Overexpression of CLPS3-TAP fusion caused a range of postembryonic development abnormalities, including early flowering time, altered phyllotaxy, and abnormal numbers and shapes of flower organs. These phenotypes are associated with the altered gene expression levels of FCA, WUS, and CUC1. The decreased ratio of FCA-beta to FCA-gamma in the overexpression plants suggests that CLPS3 favored the usage of FCA regular poly(A) site over the alternative site. These observations indicate that Arabidopsis CLPS3 might be involved in the processing of pre-mRNAs encoded by a distinct subset of genes that are important in plant development.

Figure 1.

Phylogenetic relationship of yeast Clp1p homologs. The phylogenic tree was generated by parsimony and bootstrap programs within the PAUP 4.0 package based on the aligned amino acid sequences (Supplemental Fig. S1). The orthologs from plants are circled. Sc, Saccharomyces cerevisiae, NP_014893; Sp, Schizosaccharomyces pombe, NP_593741; Hs, Homo sapiens, NP_006822; Mm, Mus musculus, NP_598601; Gg, Gallus gallus, NP_001012292; Xl, Xenopus laevis, NP_001084787; Tn, Tetraodon nigroviridis, CAG04774; Ce, Caenorhabditis elegans, NP_001040858; Dm, Drosophila melanogaster, NP_610876; At3, At5, Arabidopsis thaliana, NP_187119, NP_198809; Os1, Os2, Oryza sativa, NP_001046299, EAZ22244; Ot, Ostreococcus tauri, CAL52668; Ol, Ostreococcus lucimarinus, XP_001416530.

Figure 2.

Protein components detected in the affinity-purified CLPS3:TAP complex. The affinity-purified protein complex from cultured suspension cells expressing 35S:CLPS3:TAP (lanes 2) or 35S:TAP (lanes 1 as a negative control) was resolved on 12% SDS-PAGE gel, transferred to membrane, and probed with designated antibodies separately. Two percent total protein was resolved and probed with peroxidase-conjugated antiperoxidase (α-Peroxidase) to show equal loading for the purified proteins. Arrows point to CPSF100, CPSF160, and FY, respectively. *, Nonspecific cross-reacting polypeptide. The low-M_r band in the CPSF100 and CPSF160 images is likely from degradation.

Figure 3.

Subcellular localization and expression domain of CLPS3. A, GFP fluorescence and fluorescence from chloroplasts were examined with enhanced GFP and PI filter in the guard cells of wild-type plants (Col), transgenic plants containing GFP (GFP), and CLPS3:GFP (CLPS3:GFP) protein fusion, respectively. The green fluorescence and PI pictures were merged to show the accurate localization of GFP fluorescence (Merged). Bar = 10 μm. B, Expression profiles of Arabidopsis CLPS3 examined by GUS activity in transgenic plants containing CLPS3_pro:GUS fusion in a variety of developmental stages and organs. a and b, Early stage (globular; a) and late stage (b) of embryo development; c to f, seedlings at 2, 4, 7, and 14 d postgermination; g and h, fully expanded rosette leaf and cauline leaf; i, inflorescence; j, open flower; k, anther. The arrow in a points to the embryo. Bar = 25 μm (a and b); bar = 1 mm (c–j).

Figure 4.

Mutation of CLPS3 causes aborted embryo development. A, Schematic gene structure of CLPS3 and the position of T-DNA insertion. The boxes and lines represent exons and introns, respectively. Gray boxes denote the coding regions. Inverted triangle points to the T-DNA insertion position. Arrows denote the annealing positions for a primer pair (p421/p499) used for PCR genotyping the CLPS3 allele. B, Seed segregation and abortion in siliques from wild-type plants W (+/+) (top), T-DNA insertion hemizygote plants H (t/+) (middle), and T-DNA insertion homozygote plants expressing 35S:CLPS3:TAP transgene, CLPS3:TAP (t/t) (bottom). C, Fluorescence microscopic examination of the embryo development. Siliques from T-DNA insertion hemizygote plants at a variety of postpollination stages were collected, fixed, cleared, and examined under the confocal microscope with the fluorescein isothiocyanate filter. Embryos in globular (top left), torpedo (top right), early mature (bottom right), and late mature (bottom left) stages are shown. White arrows point to the empty ovules presumably due to the developmental failure of embryos that are homozygotes for the T-DNA insertion. D, The existence and expression of CLPS3:TAP fusion in the transgenic plants were confirmed by PCR-based genotyping (using primers in A; left), western blotting with peroxidase-conjugated antiperoxidase (middle), and western blotting with antibodies against CLPS3 (right). Lanes 1, Wild-type plant; lanes 2, CLPS3:TAP (t/t) transgenic plants. *, Nonspecific cross-reaction.

Figure 5.

Altered morphology and development of the transgenic plants overexpressing CLPS3:TAP fusion protein. A, Loss of SAM after cotyledon expansion (a) or after bolting (b), branched (c), or multiple (d) main stems, altered shape for both rosette (e; bottom) and cauline (e; top) leaves, altered cauline leaf number at a single node (f), and altered phyllotaxy (g). f and g, Wild-type Col-0 at left and the CLPS3:TAP overexpression plant at right. Seedlings (a–d) are 35 days old. B, Altered flower morphology. a, Side view of the flowers; b, top view of the flowers with a variety of number of petals; c to f, altered shape of sepals, petals, and stamens. Each far-left image was from wild-type Col-0 and the other images from CLPS3:TAP overexpression plants. C, Photograph of 25-d-old CLPS3:TAP overexpression plant and wild-type Col-0 plant showing the earlier flowering phenotype. D, The reduced rosette leaf number of CLPS3:TAP overexpression plants growing in long-day (LD) and short-day (SD) conditions. [See online article for color version of this figure.]

Figure 6.

Expression of FCA and FLC in CLPS3:TAP overexpression plants. Semiquantitative RT-PCR was performed with DNase-treated total RNA extracted from 14-d-old seedlings. The expression of β-TUB6 was used as an internal control. A, FCA-γ, FCA-β, and FLC were amplified, respectively, with transcript-specific primer pairs. Shown is a representative of three independent experimental results. B, The band intensities in A were quantified with ImageQuant and the ratios of FCA-β to FCA-γ abundance normalized with β-TUB6 are presented. Ratios are expressed as means of three independent experiments.

Figure 7.

mRNA abundance of AS1, CUC1, WUS, and β-TUB6 in Col-0, transgenic plants containing TAP alone (TAP), and CLPS3:TAP (C3-TAP) overexpression plants. A, Semiquantitative RT-PCR was performed with DNase-treated total RNA extracted from shoots of 10-d-old seedlings. No amplifications were observed for the genes investigated in the non-RT control (data not shown). B, Band intensities in A were quantified with ImageQuant and normalized with β-TUB6. The abundance of the gene transcripts was presented relative to that in Col-0, which was arbitrarily set as 1. Data were means of three independent experimental results.