Isolation of Ethyl Methanesulfonate-Induced Gametophytic Mutants in Arabidopsis thaliana by a Segregation Distortion Assay Using the Multimarker Chromosome 1

RESULTS

Rationale of the screening strategy: Our screening strategy is based on the idea that a male or female gametophytic mutation on one chromosome should result in a higher apparent transmission frequency of the homologous chromosome. If the latter chromosome carried a recessive visible marker closely linked to the locus of the gametophytic mutation, the proportion of progeny plants showing the marker phenotype would increase from 25 to 50% [Figure 1, see only the parental classes (boldface square)]. However, meiotic recombination between the two loci would limit the increase in marker frequency and as the distance from the gametophytic locus increases, the apparent marker frequency would gradually decrease to the normal value of 25%. Thus, this strategy is limited by the distance at which a gametophytic mutation still causes a recognizable increase in marker frequency. We chose a marker frequency of 40% as our lower operational limit under screening conditions. This corresponds to a distance of 25 cM (for calculation see Figure 1). We used a multiply marked chromosome 1 (mm1), which carries five visible recessive markers that cover the whole chromosome (Figure 2). Figure 2 shows the relative distances as calculated from the recombination frequencies between the five markers. On the basis of the above estimations any induced gametophytic defective mutation on chromosome 1 should result in an increased frequency of two neighboring markers. This has two important consequences: first, it reduces the probability of false positives and second, this strategy enables the newly identified gametophytic mutants to be mapped relative to the flanking markers.

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Figure 1.

–Screening strategy. Detection of gametophytic mutations by increased frequency of recessive marker plants among progeny of selfed mutagenized plants. (Top) Transheterozygous plant carrying a male gametophytic defective (mad) mutation. (Middle) Progeny classes resulting from meiotic recombination and random fertilization of functional gametes (nonfunctional male gametophytes shaded). Parental classes are indicated as a boldface square. (Bottom) Proportion of recessive marker plants as a function of recombination. Distance (p) between marker (m) and mad locus. mad, mad⁺: male gametophytic mutation and the corresponding wild-type locus. m,m+: recessive morphological marker and the corresponding wild-type locus. . If the marker frequency = 0.4 the recombination distance (p is ; p = 0.2. This corresponds to a genetic distance in centimorgans: 50 ln(1/(1 – 2p)) = 25 cM).

Isolation and genetic characterization of gametophytic mutants: Out of 200 single lines screened we have isolated 7 lines that showed an increased frequency of two neighboring markers. Reciprocal backcrosses with mm1 enabled us to determine whether the male [male gametophytic defective (mad)], the female [female gametophytic defective (fad)], or both the male and female gametes [both male and female gametophytic defective (bod)] are affected (Table 1). Transmission through the male gametes is specifically affected in 4 mutant lines, mad1, mad2, mad3, and mad4. Three lines, bod1, bod2, and bod3, turned out to be impaired in the transmission through both the male and the female gametes (Table 1).

The map position of each gene was calculated from the marker frequencies obtained for each line (Table 2, Figure 2). To take into account that the marker frequencies may be reduced by incomplete penetrance of the mutations, we calculated the RGD to the two flanking markers (Table 2 and Figure 2; for details see materials and methods).

Gametophytic mutations affecting the development of the male gametophyte: The male gametophyte (pollen) develops within the anther (microgametogenesis). After meiosis, the four haploid microspores begin to differentiate. The microspores enlarge and form a prominent vacuole (Figure 3, A and D, B and E). Subsequently, the nucleus moves to the pole and the microspore undergoes an asymmetric cell division, producing a large vegetative and a small generative cell (Figure 3, C and F). In whole-mount DAPI stainings the vegetative nucleus can easily be recognized as a large diffusely stained nucleus, while the generative nucleus appears smaller and more intensely stained. The generative cell is initially closely attached to the outer cell wall (bicellular stage I; Figure 3, C and F). Slightly later, this cell is detached from the outer wall and surrounded by the vegetative cell (bicellular stage II; Figure 3, G and J). The generative cell then divides to give rise to two sperm cells (tricellular stage; Figure 3, H and K; Mascarenhas 1989; McCormick 1993; Twellet al. 1998).

mad1: In heterozygous mad1 plants ∼18% of the pollen at the tricellular stage showed no nuclear or cytoplasmic staining in whole-mount DAPI preparations and ∼37% had only two nuclei (n = 618). In the latter class the vegetative nucleus always appeared normal in size and shape. The generative nucleus showed a variable phenotype: 62% were indistinguishable from wild type (Figure 4A), 8% were drastically increased in size (Figure 4B), and 27% had a speckled appearance (Figure 4C). Occasionally we found pollen carrying only one irregularly shaped enlarged nucleus (3%, Figure 4D). Some pollen of the two nuclear classes appeared to be viable. In in vitro germination experiments we occasionally found pollen tubes with one vegetative and one enlarged generative nucleus (Figure 4, E and F). DAPI stainings of pollen at the mononuclear stage revealed no deviation from wild type (n = 400). At the bicellular I stage ∼19% of the pollen were unstained and 5% showed diffuse staining (n = 280). At the transmission electron microscopy (TEM) level the mutant phenotypes at the bicellular II stage are likely to correspond to aborted pollen (Figure 4G, left) and pollen with retarded growth (Figure 4G, right). Approximately 5% of all pollen exhibited a thick cell wall subdividing the pollen into two large cells with the smaller cell encompassing approximately one-third of the total area in individual sections (Figure 4G, left pollen, and 4H). We did not observe nuclei in these cells (n = 20), suggesting that cytokinesis occurred in the absence of a nuclear division. In addition, we found pollen with one normal vegetative nucleus and one smaller nucleus that otherwise resembled the vegetative nucleus (Figure 4I). Although the smaller nucleus is surrounded by a plasma membrane similar to a typical generative cell at the bicellular I stage, no small vacuoles accumulate around this cell and the nucleolus is clearly condensated (Figure 4I). At the tricellular stage several pollen, in which the two generative cells and sometimes also the vegetative cell are closely associated, were found (Figure 4, J and K). These complexes looked like aggregations of nuclear material and cell membranes without a clear distinction between individual cells (Figure 4, J and K) and they may correspond to the class of mutants that appeared speckled in DAPI stainings. In summary, the range of phenotypic aspects in mad1 mutants suggests that MAD1 is involved in the cell and/or nuclear division during mitosis II of pollen development.

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Figure 2.

–Schematic representation of the multiple marker chromosome 1 (mm1). The mm1 chromosome carries five visible markers: angustifolia (an), distorted-1 (dis1), eceriferum-5 (cer5), apetala-1 (ap1), and glabra-2 (gl2) as described in Meyerowitz and Ma (1994). The genetic distances between the five morphological markers have been calculated from the F₁ progeny of selfed heterozygous mm1 plants and the reciprocal crosses are shown in the lower part of this figure. The total number of F₁ plants analyzed (n) are shown in brackets. Map positions are from the Unified Genetic Map, Unified-1 (AtDB). The map positions of gametophytic mutations are indicated relative to their flanking markers. For mapping details see materials and methods.

mad2: In DAPI stainings of mature pollen of heterozygous mad2 plants, ∼49% (n = 503) of all pollen showed morphological aberrations of different strength: 17% showed no nuclear or cytoplasmic staining, 17% had one generative and one vegetative nucleus, in 14% the generative nucleus had a speckled appearance, 1% showed only one highly enlarged nucleus (Figure 5D), and 1% had two diffuse, large nuclei that both resembled the vegetative nucleus (Figure 5C). The frequency of the latter phenotype was variable, ranging between 1 and 10% in single anther preparations even in the same plant. The earliest deviation from wild type was seen at the bicellular I stage. Approximately 9% of all pollen had two small nuclei (Figure 5A). In addition, 1% exhibited two equally sized diffuse nuclei, suggesting that the failure of the generative nucleus to differentiate can be traced back to this stage (Figure 5B). A more detailed analysis of slightly older pollen at the bicellular II stage by TEM provided evidence for cell differentiation defects of the generative cell. We frequently found pollen in which the generative cell was still attached to the pollen wall and exhibited three characteristics of a vegetative cell (Figure 5, E and F). First, the generative cell appeared to be surrounded by two intact plasma membranes but lacked the accumulation of small vacuoles. Second, no cell wall material/intine accumulates around the generative cell. Third, we often found relatively large vacuoles in the generative cell that are normally found only in the vegetative cell. In summary, this suggests that mad2 pollen are affected in the differentiation of the generative cell. This interpretation would also be in agreement with the late phenotypes found in DAPI stainings, which all affect the differentiation of the generative cell.

mad3: In heterozygous mad3 plants ∼49% of the pollen are reduced in size and are often collapsed (n = 918; Figure 6, A and C). DAPI staining of mature pollen revealed three phenotypic classes: The most frequent aberrant class has one vegetative and one generative nucleus (53%; Figure 6A). The generative nucleus appeared to be larger and more intensely stained than generative nuclei in wild type (compare left and right pollen in Figure 6A), suggesting that the generative nucleus has undergone DNA replication but failed to divide. This visual impression was confirmed by cytophotometric measurements of nuclear DNA amounts. For determination of the relative DNA content the relative fluorescence intensity of whole-mount DAPI-stained pollen was compared between mutant and wild-type pollen within the same sample preparation. While generative nuclei of wild-type pollen showed a relative staining intensity of 5.5 ± 0.97 (n = 50), mutant generative nuclei exhibited 9.9 ± 1.25 (n = 50). Thus, mutant generative nuclei have approximately twice the DNA content as wild type. In a second class (31%), only one vegetative nucleus was found (Figure 6B). A small fraction of pollen showed no nuclear or cytoplasmic staining (16%). The earliest deviation from wild-type development was observed at the bicellular II stage. Of all pollen, 49% was significantly smaller and could be divided into three classes (n = 912): In ∼74% the generative nucleus was still attached to the cell wall (Figure 6E, similar to the bicellular I stage). In 22% the generative nucleus was found in the center of the pollen (similar to bicellular II stage, Figure 6D) and 4% of the pollen showed no staining. This finding suggests that a significant fraction of mutant pollen is delayed in development. This view is supported by the observation that mutant pollen that appeared to be arrested in the bicellular I stage still had extranuclear DNA that is normally not visible at bicellular II stage (compare Figures 6E and 3, C and G). A closer inspection by TEM revealed that virtually no intine layers were detectable in mad3 mutant pollen at bicellular I stage. The generative cell, when still attached to the outer pollen wall, clearly showed a cell membrane but lacked the intine layers (compare Figure 6, G and H). After separation from the pollen wall the generative cell appeared to be intact; however, no cell wall and no vesicle accumulation around the generative cell was observed (compare Figure 6, I–J and K–L). Thus, by morphological criteria, the primary defect appears to be in the formation of the intine layers. This suggests that an intact intine layer is important for further differentiation of the generative cell.

Gametophytic mutations affecting the development of the male and female gametophyte: Three mutant lines, bod1, bod2, and bod3, showed a reduced transmission of the gametophytic mutation via both the male and the female side (Table 1). This suggests that the corresponding genes are involved in more general functions that are required for the development of both gametophytes. The penetrance of the mutant phenotype was generally low, as expected from the transmission frequencies in reciprocal backcrosses (Table 1). In particular the penetrance of female gametophytic defects was fairly low. In the following we therefore focus primarily on the description of male gametophytic defects. The female gametophytic defects are described separately thereafter.

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Figure 3.

–Microspore development in wild type. (A–C, G and H) Fluorescent micrographs of DAPI-stained whole-mount pollen. (D–F and J–K) Transmission electron micrographs of sectioned anthers. (I) Scanning electron micrograph of a mature pollen grain. Bar, 3 μm unless indicated otherwise. (A and D) Released microspores. The nucleus is large and centrally located. At this stage extrachromosomal DNA is found around the nucleus. (B and E) Vacuolated microspore. (C and F) Bicellular I pollen after pollen mitosis I (PMI). Note that the generative cell is in immediate contact with the pollen wall and surrounded by a cell wall continuous with the intine layer of the outer wall. (G and J) Bicellular II pollen. The generative cell is disengaged from the intine wall and migrated to a central position. Note accumulation of vesicles at the surface of the generative cell. (H and K) Tricellular pollen after pollen mitosis II (PMII). The generative cell has divided to produce two sperm cells. (I and L) Mature pollen. Note the cytoplasm is more dense than in earlier stages. n, nucleus; gn, generative nucleus; vn, vegetative nucleus; vc, vegetative cell; gc, generative cell; v, vacuole.

bod1: In heterozygous bod1 plants 48% of the mature pollen showed a mutant phenotype (n = 817). The majority exhibited no nuclear or cytoplasmic staining (42%). A total of 24% showed one vegetative and one smaller nucleus with a speckled appearance and ∼30% contained two generative nuclei and one additional small and intensely stained nucleus (Figure 7B). This suggests that in this mutant class the vegetative cell is either defective or has adopted the identity of a generative cell. To distinguish between these two possibilities we introduced a nuclear-targeted vegetative cell-specific marker, Lat52::GUS/NIA, into the bod1 background (Twell 1992). Mutant pollen displaying three generative-like nuclei were selected in DAPI stainings and inspected for the expression of the GUS/NIA marker gene. While wild-type pollen showed strong GUS staining in the vegetative nucleus (Figure 7D) most mutant pollen of this class did not show any localized GUS staining. A few pollen that showed a spot of GUS expression inside a large nucleus were found (Figure 7C). This observation suggests a defect in the nuclear integrity rather than a switch in cell fate. In accord with this a closer inspection at the TEM level revealed a highly aberrant morphology of the vegetative nucleus (Figure 7, J and K). The vegetative nucleus looked lobed rather than round and appeared fragmented inside (Figure 7K). Despite the apparent defects of the vegetative nucleus, vegetative cell-specific functions still appear to be present because pollen of this mutant class are able to germinate and to grow a pollen tube in vitro (Figure 7, G and H). A second phenotypic aspect we frequently observed associated with this mutant class was that the two generative nuclei appeared to be located together in a distinct compartment (compare Figure 7E with 7F). At the TEM level this phenotype is likely to correspond to generative cells that contain two separate nuclei (Figure 7, I and L). This phenotype suggests that nuclear division has taken place but cytokinesis failed. A third phenotypic aspect was noted when analyzing sections at the TEM level. We often found large densely stained structures that were surrounded by stacks of membranes (Figure 7, L and M). To determine the earliest stage in which bod1 mutant pollen differs from wild type we analyzed pollen at earlier stages. Pollen at the one nuclear stage (n = 600) and the bicellular II stage (n = 1011) revealed no deviation from wild type, suggesting that BOD1 is not required before the second mitotic division. Although the wide range of phenotypes does not allow one to draw conclusions on the role of the BOD1 gene, it is conceivable that defects of the vegetative nucleus and thus the vegetative cell is a primary event, which in turn may affect the division of the generative cell.

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Figure 4.

–Phenotype of mad1 pollen. (A–D) Fluorescent micrographs of DAPI-stained whole-mount pollen. (E–F) Fluorescent micrographs of DAPI and Aniline Blue double-stained pollen tubes of in vitro germinated pollen. (G–K) Transmission electron micrographs of sectioned anthers. Bar, 3 μm if not indicated otherwise. (A–D) Pollen from mature anthers. (A) Comparison of wild-type (left) and mutant pollen (right). The mutant pollen shows one apparently normal generative nucleus. (B) Mutant pollen with large generative nucleus. Compare size of generative nucleus in A. (C) Generative nucleus with speckled appearance. (D) Pollen with one large nucleus. (E–F) Migrating nuclei in wild-type (E) and mutant (F) pollen tubes. The mutant pollen tube shows one large generative nucleus (arrowhead, compare with wild type). (G–I) Mutant and wild-type pollen at bicellular II stage. (G) Comparison of wild-type and mutant pollen. Left, pollen with internal wall (arrow); middle, wild-type pollen; right, pollen arrested at vacuolate stage. Note difference in cytoplasmic density. (H) Mutant pollen with internal cell wall (arrowhead). (I) High magnification of the generative and vegetative nuclei in mutant pollen (compare with wild type, Figure 3J). The nucleolus of the generative nucleus appears more dense than in wild type and the nucleus is surrounded by cell membranes (arrowhead). Bar, 1 μm. (J and K) Pollen from mature anthers. Note that cell membranes do not clearly separate distinct cells. (J) Two closely attached generative cells. (K) Two generative cells in close contact with vegetative nucleus. Bar, 1 μm. gn, generative nucleus; vn, vegetative nucleus; vc, vegetative cell; gc, generative cell; v, vacuole; mt, mitochondrion.

bod2: Of the mature pollen, 38% of heterozygous bod2 plants shows morphological defects (n = 1522). Mutant pollen show different size classes with smaller as well as larger pollen (Figure 8, A, B, and D). The nuclear phenotype is quite variable and the relative frequency of different classes differed in separate anthers of a single flower. Pollen often showed no nuclear or cytoplasmic staining or only one generative and one vegetative nucleus. Compared to wild type, the most striking difference was that mature pollen frequently still had a large vacuole (Figure 8, A and B). Some of these pollen appeared to be viable, as we found some that germinated and grew a pollen tube in in vitro germination assays (Figure 8C). The first deviation from wild-type development could be traced back to the bicellular II stage where 40% of the microspores were highly vacuolated and often showed no nuclear staining (n = 486). The TEM analysis of mutant microspores at the bicellular II stage revealed a large fraction that appeared delayed in development compared to the surrounding wild-type pollen. The cytoplasm was less dense (Figure 8F), which is typical for earlier stages of wild-type development, and we often found a prominent vacuole (Figure 8E). In addition we observed differentiation defects of the vegetative and generative nuclei/cells. The generative cell did not show an accumulation of small vacuoles at the cell membrane and the cytoplasm was darker than that in wild type. The nucleolus of the vegetative nucleus often showed a round light area that, in serial sections, appeared like an invagination. In summary, the wide range of general defects in bod2 mutant pollen suggests that BOD1 is involved in general cellular processes rather than in the regulation of distinct differentiation processes.

bod3: In plants heterozygous for bod3 ∼37% of the mature pollen showed no nuclear or cytoplasmic staining (n = 880). However, slightly younger three-celled pollen exhibited a clear defect in the development of the generative cell (n = 518). A total of 25% displayed five distinct intensely DAPI-stained spots (Figure 9B). The number of these spots was always five, suggesting that these spots represent condensed chromosomes. Of the pollen 12% showed one vegetative and only one enlarged generative nucleus (Figure 9A). DNA measurements revealed that the enlarged nuclei had approximately twice the DNA content of generative cells of wild-type pollen in the same sample preparation [relative staining intensities: 18 ± 2.97 (n = 50) vs. 9.4 ± 2.27 (n = 50)]. The two phenotypic aspects, the “chromosome phenotype” and the enlarged DNA content of the generative cell, suggest that bod3 mutants undergo replication, but fail to complete cell division at different stages. Consistent with this interpretation is the finding that no deviation from wild-type development was found in DAPI stainings at the bicellular II stage. Our ultrastructural analysis at the TEM level generally supported this interpretation. The earliest deviation from wild type was found at the bicellular I stage. Mutant pollen were found with unusually large and irregularly shaped generative cells that were still attached to the pollen wall (Figure 9C). At the bicellular II stage generative cells frequently did not accumulate vacuoles at the cell membrane (Figure 9D). In mature pollen we often found very elongated generative cells (Figure 9, E and G) or up to three small generative cells (Figure 9, F and H). It is, however, unclear whether the latter correspond to the chromosome-like DAPI phenotype or the class of enlarged generative nuclei.

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Figure 5.

–Phenotype of mad2 pollen. (A–D) Fluorescent micrographs of DAPI-stained whole-mount pollen. (E–F) Transmission electron micrographs of sectioned anthers. Bar, 3 μm if not indicated otherwise. (A) Comparison of wild-type (right) and mutant pollen (left) in anthers at bicellular I stage. Mutant pollen shows two equally stained regions (arrows). (B) Mutant pollen from same anther as A. Two vegetative-like nuclei are visible (arrowheads). (C–D) Mutant pollen from mature anthers. (C) Two vegetative-like nuclei. (D) One large intensely stained nucleus. (E) Bicellular II stage. Note prominent nucleolus in generative nucleus (arrowhead). Small vacuoles normally surrounding the generative cell are absent (compare Figure 3J). Large vacuoles are found in the vegetative cell. (F) High magnification of the generative cell in E. The plasma membranes around the generative cell are intact (arrowheads). Accumulation of intine at the pollen wall appears to be normal while no cell wall material and/or intine was found around the generative cell (compare Figure 3F). Nuclear membrane is indicated by arrows. Bar, 200 nm. (×40.000). gn, generative nucleus; vn, vegetative nucleus; vc, vegetative cell; gc, generative cell; int, intine; v, vacuole; mt, mitochondrion.

Female gametophytic development in bod1, bod2, and bod3 mutants: In reciprocal backcrosses the three lines, bod1, bod2, and bod3, showed a weak reduction in the transmission of the gametophytic mutation via the female gametes (Table 1). To determine whether embryo sac development is affected in these mutants we studied cleared whole-mount preparations of mature ovules (Schneitzet al. 1995). The development of the female gametophyte (megagametogenesis) takes place inside the ovules. Megagametogenesis consists of four mitotic cycles that yield an eight-nucleate, seven-celled embryo sac (Webb and Gunning 1990; Reiser and Fischer 1993; Grossniklaus and Schneitz 1998). Eventually the embryo sac differentiates to produce an egg cell, two synergids, three antipodal cells, and a large central cell (Figure 10A). Female gametogenesis was found to be moderately affected in all three mutant lines (Table 3). bod2 appeared to express the weakest phenotype. Of all embryo sacs ∼17% either were reduced in size (Figure 10D) or appeared to be collapsed (Figure 10E). In plants heterozygous for bod3 ∼12% of all ovules showed a collapsed embryo sac (Figure 10C). bod1 mutants exhibited the strongest phenotype. On average, 23% of all mature ovules appeared to lack an embryo sac completely (Figure 10B). A more detailed analysis of mutant phenotypes in earlier stages was not attempted because the frequency of mutant phenotypes was low and also varied considerably even between different flowers from the same plant.

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Figure 6.

–Phenotype of mad3 pollen. (A, B, D, and E) Fluorescent micrographs of DAPI-stained whole-mount pollen. (C) Scanning electron micrograph of mutant pollen. (F–L) Transmission electron micrographs of pollen and sectioned anthers. Bar, 3 μm if not indicated otherwise. (A) Comparison of wild-type (left) and mutant pollen (right) in mature anthers. The mutant pollen is significantly smaller than wild type. The generative nucleus is larger and more intensely stained. (B) Mature pollen with one diffusely stained nucleus. (C) Scanning electron micrograph of a collapsed mutant pollen (compare Figure 3G). (D) Bicellular II stage, comparison of mutant (right) and wild-type pollen (left). Mutant pollen is smaller. (E) Mutant pollen at bicellular I stage. Generative cell is attached to the pollen wall (arrowhead, compare Figure 6D). Note extrachromosomal DNA (compare Figure 3D). (F) Mutant pollen collapsed. (G and H) Bicellular I stage. Comparison of generative cells in mutant (G) and wild type (H; compare Figure 3I). No intine or cell wall is seen in the mutant (see arrowheads). Note vegetative and generative cytoplasm is less dense in the mutant. Bar, 1 μm. (I–L) Bicellular II stage. Comparison of mutant (I and J) and wild type (K and L). The cytoplasm of mutant pollen is less dense than in wild type. (J and L) Higher magnification of generative cells in mutant and wild type. Nuclear membranes and cell membranes appear to be intact in mutant pollen; however, no accumulation of lipid bodies and small vacuoles around the generative cell surface is observed. Bar, 1 μm. n, nucleus; gn, generative nucleus; vn, vegetative nucleus; vc, vegetative cell; gc, generative cell; int, intine; v, vacuole.

mad4, a gametophytic mutant affecting pollen tube growth: In mad4 mutants transmission is specifically impaired via the pollen. mad4 does not show any obvious defect during pollen development (Table 4). Thus, this mutant affects the function of the pollen or pollen tube during the fertilization process. To examine the stage at which the mutant pollen/pollen tube fails to proceed, we analyzed different steps during the fertilization process. The first steps, the recognition of the pollen grain as judged by the hydration of pollen grains and the germination rate of pollen from mad4, were indistinguishable from wild type (Table 4). Further pollen tube growth, however, was retarded relative to wild type. In pollination experiments with wild-type pollen we found that after 24 hr pollen tubes of 98% of all pollen applied to the stigma had arrived in the ovary (Table 4). In contrast only 51% of the pollen tubes from mad4 mutant plants had traveled down to the ovary in the same time period (Table 4). We often observed pollen tubes that had stopped growth even before penetrating the stigma surface (data not shown). These findings indicate that in mad4 mutants, pollen tube growth is affected.

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Figure 7.

–Phenotype of bod1 pollen. (A–B) Fluorescent micrographs of DAPI-stained whole-mount pollen. (C–D) DIC micrographs of GUS-stained whole-mount pollen. (E–F) Fluorescent/DIC micrographs of DAPI-stained whole-mount pollen. (G–H) Fluorescent micrographs of DAPI/Aniline Blue double-stained pollen tubes from in vitro germinated pollen. (I–M) Transmission electron micrographs of sectioned anthers. Bar, 3 μm if not indicated otherwise. (A–F) Pollen from mature anthers. (A–B) Comparison of wild-type (A) and mutant pollen (B). The mutant vegetative nucleus is smaller and more condensed (arrows; C–D). Comparison of GUS-stained pollen from heterozygous Lat52::GUS/Nia plants (D) and heterozygous Lat52::GUS/Nia bod1-plants (C). Note the nucleus in the mutant pollen is clearly visible, but GUS staining is restricted to a small region (arrowhead). (E–F) Comparison of wild type (F) and mutant (E). The generative nuclei in the mutant seems to reside in a common compartment (compared to wild type in 7F, arrowheads). The vegetative nucleus in the mutant is not in the focal plane. (G–H) Migrating nuclei in in vitro germinated wild-type (G) and bod1 (H) pollen tubes. Compare vegetative nuclei (arrowheads). (I–M) Mutant pollen at the tricellular stage. (I) High magnification of one generative cell with two nuclei. The two nuclei were clearly separate in serial sections. Bar, 1 μm. (J–K) Mutant pollen with degenerated vegetative nucleus (J) and high magnification of the vegetative nucleus (K). The vegetative nuclear membrane appears to be intact (arrows). (L) Mutant pollen with large generative cell. (M) High magnification of L. Note densely stained region surrounded by several membrane layers (arrowheads). Bar, 200 nm. gn, generative nucleus; vn, vegetative nucleus; vc, vegetative cell; gc, generative cell; mt, mitochondrion.