The immunophilin-interacting protein AtFIP37 from Arabidopsis is essential for plant development and is involved in trichome endoreduplication

Abstract

The FKBP12 (FK506-binding protein 12 kD) immunophilin interacts with several protein partners in mammals and is a physiological regulator of the cell cycle. In Arabidopsis, only one specific partner of AtFKBP12, namely AtFIP37 (FKBP12 interacting protein 37 kD), has been identified but its function in plant development is not known. We present here the functional analysis of AtFIP37 in Arabidopsis. Knockout mutants of AtFIP37 show an embryo-lethal phenotype that is caused by a strong delay in endosperm development and embryo arrest. AtFIP37 promoter::beta-glucuronidase reporter gene constructs show that the gene is expressed during embryogenesis and throughout plant development, in undifferentiating cells such as meristem or embryonic cells as well as highly differentiating cells such as trichomes. A translational fusion with the enhanced yellow fluorescent protein indicates that AtFIP37 is a nuclear protein localized in multiple subnuclear foci that show a speckled distribution pattern. Overexpression of AtFIP37 in transgenic lines induces the formation of large trichome cells with up to six branches. These large trichomes have a DNA content up to 256C, implying that these cells have undergone extra rounds of endoreduplication. Altogether, these data show that AtFIP37 is critical for life in Arabidopsis and implies a role for AtFIP37 in the regulation of the cell cycle as shown for FKBP12 and TOR (target of rapamycin) in mammals.

Figure 1.

T-DNA insertions and phenotype of the AtFIP37/atfip37 mutants. A, Representation of the T-DNA insertion sites in atfip37-1, atfip37-2, and atfip37-3 mutant lines. The left borders of T-DNAs are represented by arrowheads. Black boxes represent exons and white boxes represent 5′ and 3′ untranslated regions. White triangles beneath the AtFIP37 locus indicate the location of forward (F) and reverse (R) primers used for screening the University of Wisconsin at Madison mutant collection. Numbers are given relative to the transcription start site located 111 bp upstream of the translational start site (Faure et al., 1998). B, A heterozygous silique (10 d after pollination [DAP]) from an AtFIP37-1/atfip37-1 plant.

Figure 2.

Endosperm and embryo development in wild-type and atfip37 mutants. A and E, wild-type and atfip37-1 seeds, respectively, showing early endosperm with fewer nuclei in mutant endosperm; wild-type endosperm is at stage VI (24–28 nuclei) while mutant endosperm is at stage V (16 nuclei). The nucleolus of each nucleus is clearly visible (black arrow); P, posterior pole with two larger nuclei (white arrows) representing the posterior mitotic domain in wild type that is not differentiated yet in the atfip37-1 endosperm. The embryo in A is not visible in this focal plane. B and F, wild-type and atfip37-1 seeds, respectively, showing the reduced size of the atfip37-seeds relative to the wild type. The embryo in the wild type is at the octant stage while the embryo in atfip37-1 is only at the two cell stage. C and G, wild-type and atfip37-1 seeds, respectively, with proper posterior pole differentiation in both wild-type and atfip37-1 endosperm. The posterior pole differentiates properly. c, cyst; n, nodules. D and H, wild-type and atfip37-1 embryos, respectively, showing a late globular stage wild-type embryo while the atfip37-1 embryo has only reached the early globular stage. I and M, wild-type and atfip37-1 embryos, respectively, showing a late heart stage wild-type embryo and an arrested atfip37-1 embryo. Cell division in atfip37-1 is still active as shown (white arrowhead showing early anaphase). Some cells in the mutant embryo have collapsed (black arrowheads) and lead to alteration of the overall embryo pattern. J and N, wild-type and atfip37-1 seeds, respectively, showing the cellularized wild-type endosperm (embryo at early torpedo stage) while atfip37-1 endosperm remains at syncytial stage VIII. K and O, wild-type and atfip37-1 endosperm, respectively, showing a fully cellularized wild-type endosperm while the mutant endosperm shows only some cellularization. L and P, wild-type and atfip37-1 mature seeds showing the reduced size of the mutant seed. A–C, E–G, J, and N are micrographs of whole mount seeds cleared with Hoyer's medium and observed with Normarski optics. D, H, I, K, M, and O are confocal sections of whole mount seeds. L and P are micrographs of seeds observed with a stereomicroscope. Bars = 20 μm (A and E), 40 μm (B, C, F, G, J, and N), 15 μm (D, H, I, K, M, and O), and 160 μm (L and P).

Figure 3.

Expression pattern of the AtFIP37 gene and protein. A, Representation of the fusion between AtFIP37 and the uidA (GUS) reporter gene. The HindIII fragment of AtFIP37 locus extending from 1.3 kb upstream of the transcription start site to the beginning of the second exon of AtFIP37 coding sequence was fused in-frame to the GUS coding sequence. The arrow indicates the transcriptional start site. Other symbols are as in Figure 1. B, Embryos (from left to right) at walking-stick, bent-cotyledon, early-mature, and late-mature embryo stages. C, Root apex of a primary root; pr, primary root; ez, elongation zone. D, Lateral root primordium emerging from a primary root; lrp, lateral root primordium; pr, primary root. E, Close-up of a leaf edge showing vasculature and a mature three-branch trichome cell. F, Entire 2-week-old seedling. G, Flower at postanthesis stage. H, Real time quantitative RT-PCR in roots (Ro), rosette leaves (R), stems (S), floral buds (FB), flowers (F), and 9–10 DAP young siliques (YS), old siliques (OS) about 14 DAP, calli (Ca), and cells (Ce). I, Western-blot analysis using a polyclonal antibody raised against the entire AtFIP37 protein. A polyclonal antibody against the β-tubulin was used as a loading control; abbreviations are like in H. Bars = 500 μm in A, B, E, F, and G and 250 μm in C and D.

Figure 4.

Protein sequence similarities and cellular localization of AtFIP37. A, Alignment of AtFIP37 (accession AAC72922) with the speckle-localized HsWTAP (accession CAC10188) and the putative splicing factor DmFL(2)D (accession NP523732). Positions at which a residue occurs in AtFIP37 and at least in one other sequence are shown by dark boxes. Positions at which a conservative substitution occurs (such as RK, IVL, ED, FY, and ST) are shown by shaded boxes. Bars above AtFIP37 sequence indicate potential coiled-coiled structures common to all three sequences as predicted with the coiled-coiled prediction software at http://npsa-pbil.ibcp.fr. In the consensus line, capital letters designate identical residues found in all three sequences while lowercase letters designate identical residues found in two sequences with a conservative substitution in the third sequence. B, Cellular localization of the AtFIP37::eYFP protein in biolistically bombarded leaf cells of Arabidopsis. The top row shows the eYFP fluorescence signal; the bottom row shows the corresponding DAPI fluorescence signal to visualize the nucleus. B1, Nuclear control of an eYFP fused to the lexA::NLS sequence (Kato et al., 2002). B2–4, Localization of the AtFIP37::eYFP protein in speckles. B5, Diffuse nuclear localization of the AtFIP37::eYFP protein. At least 100 nuclei were observed in each experiment. Bars = 10 μm. NLS, nuclear localization signal.

Figure 5.

Overexpression of AtFIP37 and increased trichome branching in transgenic Arabidopsis plants. A, Northern blot performed with 30 μg of total RNA extracted from rosettes of wild-type (Col) and two 35S::AtFIP37 lines. Endogenous AtFIP37 mRNA is barely detectable in wild-type rosettes or other organs (data not shown). Ethidium bromide-stained 25S ribosomal RNA is shown as the gel-loading control. B, Illustration of the increase in trichome number and trichome branching in 35S::AtFIP37 lines; top row, leaf 2 from wild-type rosette (left), three-branch (center), and four-branch (right) wild-type trichomes; bottom row, leaf 2 of 35S::AtFIP37, five-branch (center), and six-branch (right) trichomes. C, Distribution of trichome populations on rosette leaves. Trichomes were counted on the first pair of rosette leaves of 10 plants for each genotype. Numbers on top of histogram bars give the total number of trichomes counted on leaf 1 and 2. Bars = 1 mm (leaves) and 150 μm (trichomes).

Figure 6.

Increased DNA content in trichomes of 35S::AtFIP37 plants. A, DAPI staining of a wild-type three-branch trichome and a 35S::AtFIP37 overbranched trichome. B, Quantification of the DNA content of wild-type and 35S::AtFIP37 trichomes. The most highly branched trichomes were collected from wild-type (three- and four-branch trichomes) and 35S::AtFIP37 plants (five- and six-branch trichomes) and stained with DAPI. DNA level of three- and four-branch trichomes from 35S::AtFIP37 plants was similar to wild-type trichomes. The DNA content was quantified under a microscope with a CCD camera using the SAMBA device (Perazza et al., 1999). Bars = 150 μm.