Fig 1.
lsq lines are tetraploid and have ectopic pollen apertures.
(A-D) Karyotypes of microspore mother cells undergoing meiosis I. (A) A wild-type Columbia plant (2n); (B) lsq6 plant (4n); (C, D) F1 progeny of a cross between Columbia and lsq6 (3n). (E-F’) Pollen with 3 apertures from wild-type Columbia (E, E’) and with four apertures from lsq6 (F, F’). Front and back views of the same pollen grains are shown. Scale bars = 5 μm in (A-D) and 10 μm in (E-F’). (G) Percentages of pollen grains having indicated number of apertures in pollen populations from wild-type Col plants and from different mutants and accessions used in this study. n = 150–250 pollen grains.
Fig 2.
Changes in pollen ploidy correlate with changes in pollen aperture numbers in different Arabidopsis ecotypes and in Brassica species, but not in maize.
(A-C’) Front and back views of pollen grains with four apertures from tetraploid Columbia (A, A’), tetraploid Landsberg erecta (B, B’), and tetraploid Warschau (C, C’) accessions of Arabidopsis. (D, E) Pollen from both diploid (D) and tetraploid (E) maize has a single pore-like aperture. Scale bars = 10 μm. (F-I’) Pollen from a diploid B. oleracea (F, F’) and B. rapa species has 3 apertures, whereas pollen from their allotetraploid hybrid B. napus often has more than 3 apertures. Front and back views of B. napus pollen with four apertures (H, H’) and with six apertures (I, I’). Plant ploidy is indicated on all images.
Fig 3.
lsq plants have normal tetrahedral arrangement of microspores in a tetrad.
Some of the developing apertures in lsq6 tetrads are not aligned with any apertures in sister microspores. (A-D) Tetrads from the wild-type (A, C) and lsq6 (B, D) plants exhibit similar tetrahedral morphology. (A, B) Single optical sections. Membrane structures are stained with CellMask Deep Red (magenta) and callose walls are stained with calcofluor white (blue). Scale bars = 5 μm. (C, D) 3-D reconstructions from confocal z-stacks of the tetrads shown in (A) and (B). Each of the four microspores in a tetrad is labeled with a number. (E-F’) 3-D reconstruction and surface rendering from confocal z-stacks of a wild-type (INP1pr:INP1-YFP) (E) and a lsq6 (F, F’) tetrad of microspores allows visualizing apertures developing on microspore surfaces (magenta). Microspore surfaces were transiently labeled with DAPI (green) and rendered with IMARIS (E) or Nikon Elements (F, F’). Developing apertures (indicated by arrowheads) are visible due to underlying INP1-YFP fluorescence (E, magenta) or with the help of the membrane stain CellMask Deep Red (F, F’, magenta). (F) A view of a lsq6 tetrad that shows alignment between apertures on sister microspores (arrowheads). (F’) A different view of the same tetrad that shows an aperture (arrow) not aligned with any apertures in a sister microspore. See also S1 and S2 Movies.
Fig 4.
Positions of the last points of contact in tetrads of wild type and lsq6 and in dyads of 2n and 4n osd1 plants.
(A, C, E, G) Single optical sections through tetrads and dyads. Gaps in callose walls and last points of contact are labeled with arrows. Membrane structures are stained with CellMask Deep Red (magenta) and callose walls are stained with calcofluor white (blue). Scale bars = 5 μm. (B, D, F, H) 3-D reconstructions from confocal z-stacks of the tetrads and dyads shown in (A, C, E, G). Positions of cytoplasmic bridges at the last points of contact are indicated by arrowheads.
Fig 5.
Diploid pollen in osd1 and tam-2 mutants has predominantly four or six apertures, some of which develop without any contact with intersporal callose wall.
(A, B’) Front and back views of osd1 diploid pollen with four (A, A’) or six (B, B’) apertures. (C-D’) Front and back views of tam-2 diploid pollen with four (C, C’) or six (D, D’) apertures. (E) 3D reconstruction of a diploid osd1 dyad at the stage when developing apertures become visible on the surfaces of microspores. Two apertures that develop at the distal side of one of the microspores, away from the intersporal callose wall (CW), are indicated by arrowheads. Microspore surfaces were stained with DAPI and callose wall was stained with calcofluor white (both blue). (F-I) Dyads of mature pollen from the osd1; qrt1 plants showing examples of dyads with 4/4 (F), 6/6 (G), 8/6 (H), and 4/6 (I) aperture configurations. Scale bars = 10 μm.
Fig 6.
The majority of haploid pollen in 1n MiMe plants has three normal apertures.
(A) A diagram of microsporogenesis that occurs in the haploid MiMe plants and leads to the formation of haploid dyads of microspores. MMC–microspore mother cell, S–DNA synthesis, CK–cytokinesis. (B-D) Haploid MiMe microspores in a dyad (C) are similar in size to haploid wild-type microspores in a tetrad (Col, B) and are smaller than diploid osd1 microspores in a dyad (D). (E-E’) 67% of pollen produced by the haploid MiMe plants had three normally arranged apertures. (F-F’) In ~ 25% of cases, 1n MiMe pollen had six apertures. Scale bars = 10 μm.
Fig 7.
Pollen ploidy higher than 2n is accompanied by changes in aperture morphology.
(A-F’) Ring-shaped apertures are common in tetraploid pollen from osd1 (A-B’), tam-2 (C- D’), and tes (E-F’). Front and back views are shown for each pollen grain (e.g. A and A’). (G-H’) Tetraploid pollen from tam-2; osd1 mutants often has abnormal exine patterns that make recognizing apertures difficult. Still, ring-shaped apertures are sometimes visible in tam-2; osd1 pollen (H, H’, arrowheads). Scale bars = 10 μm.
Fig 8.
Pollen grains of the 4n plants generated from the 2n lines with reduced levels of INP1 expression and shortened apertures develop four shortened apertures.
(A) Schematic description of the experiment to distinguish between possible mechanisms involved in specifying aperture number. Tetraploid plants generated from the diploid short-aperture line by colchicine treatment are expected to produce 2n pollen with 3 longer apertures if INP1 level is the factor that determines the number of apertures. 2n pollen with 4 short apertures is expected if other factors (e.g. levels of another gene or changes in cell geometry) dictate aperture number. (B-C’) An INP1pr::INP1-myc; inp1 S line produces three short apertures in haploid pollen (B-B’) and four short apertures in diploid pollen (C-C’). An INP1pr::INP1-myc; inp1 M line produces three medium-size apertures in haploid pollen (D-D’) and four medium-size apertures in diploid pollen (E-E’). Apertures are indicated by arrowheads. Scale bars = 10 μm.
Fig 9.
Higher levels of INP1 expression do not result in increased number of apertures.
(A-B’) Front and back views of pollen from the INP1pr:INP1-YFP; inp1 line (A, A’) and the DMC1pr:INP1-YFP; inp1 line (B, B). Scale bars = 10 μm. (C, D) Representative images of optical sections through tetrads that were used to measure the mean intensity of diffused YFP fluorescence in the INP1pr:INP1-YFP; inp1 (C) and the DMC1pr:INP1-YFP; inp1 (D) lines. (E) Quantification of mean YFP fluorescence in INP1pr:INP1-YFP and DMC1pr:INP1-YFP tetrad-stage microspores (a.u. = arbitrary units). Error bars indicate SD. (F) INP1-YFP transcript levels were measured by qRT-PCR and normalized to the levels of the endogenous inp1-1 transcript. Error bars indicate SE (n = 4 biological replicates).
Fig 10.
INP1 localizes to the positions of ectopic apertures and is required for their formation.
(A-D) Tetrads or dyads of microspores expressing INP1pr:INP1-YFP viewed from the distal pole of a microspore. Ploidy of a microspore is indicated. INP1-YFP forms three punctate lines per microspore in haploid tetrads of wild-type 2n plants (A), but forms ectopic lines (four or more) in tetrads of diploid microspores of 4n plants (B) or in diploid or tetraploid dyads of 2n (C) or 4n (D) osd1 plants. Positions of INP1-YFP lines are indicated by arrowheads. Note the four lines of INP1-YFP puncta assembling into a ring-like structure on a distal surface of a microspore in the 4n osd1 dyad (D, arrowheads). (E) Pollen grains in the osd1; inp1 double mutant lack apertures. Scale bars = 5 μm.