for pollen viability in Arabidopsis thaliana

The Plant Journal (2006) 48, 193 205 doi: 10.1111/j.1365-313X.2006.02863.x The protein kinase genes MAP3Ke1 and MAP3Ke2 are required for pollen viability in Arabidopsis thaliana Suraphon Chaiwongsar 1, Marisa S. Otegui 2, Peter J. Jester 1, Sean S. Monson 1 and Patrick J. Krysan 1,* 1 Horticulture Department and Genome Center of Wisconsin, University of Wisconsin, Madison, WI 53706, USA, and 2 Department of Botany, University of Wisconsin, Madison, WI 53706, USA Received 31 March 2006; revised 21 June 2006; accepted 22 June 2006. *For correspondence (fax þ1 608 262 4743; e-mail fpat@biotech.wisc.edu). Summary We have used reverse-genetic analysis to investigate the function of MAP3Ke1 and MAP3Ke2, a pair of closely related Arabidopsis thaliana genes that encode protein kinases. Plants homozygous for either map3ke1 or map3ke2 displayed no apparent mutant phenotype, whereas the double-mutant combination caused pollen lethality. Transmission of the double-mutant combination through the female gametophyte was normal. Tetrad analysis performed using the Arabidopsis quartet mutation demonstrated that the pollen-lethal phenotype segregated at meiosis with the map3ke1;map3ke2 genotype. We used transmission electron microscopy to determine that double-mutant pollen grains develop plasma membrane irregularities following pollen mitosis I. Analysis of the subcellular localization of a yellow fluorescent protein (YFP):MAP3Ke1 fusion protein using confocal microscopy and biochemical fractionation indicated that a substantial portion of the MAP3Ke1 present in Arabidopsis cells is localized to the plasma membrane. Taken together, our results suggest that MAP3Ke1 is required for the normal functioning of the plasma membrane in developing Arabidopsis pollen. Keywords: Map3K-epsilon, pollen, plasma membrane, Arabidopsis, reverse genetics. Introduction The genome of Arabidopsis thaliana encodes over 1000 protein kinases (Wang et al., 2003). One approach to understanding the function of these proteins is to analyze plants in which the corresponding genes have been mutated. In the present study we have applied this strategy to a pair of Arabidopsis protein kinase genes named MAP3Ke1 and MAP3Ke2 (Jouannic et al., 2001). Although these two genes have historically been characterized as members of the MAP kinase kinase kinase (MAP3K) gene family (MAPK Group, 2002), phylogenetic analysis has indicated that the most closely related non-plant relatives of these genes are CDC7 from Schizosaccharomyces pombe and CDC15 from Saccharomyces cerevisiae, neither of which is a MAP3K (Jouannic et al., 2001). Cdc7p is a component of the septation initiation network (SIN), which regulates the formation of the septum after chromosome segregation has been completed (Gould and Simanis, 1997). Cdc15p is a component of the mitotic exit network, which is a structure that promotes the release of the protein phosphatase Cdc14p from the nucleolus and is essential for the exit from mitosis in S. cerevisiae (Bardin et al., 2003). BnMAP3Ke1, the Brassica napus homolog of MAP3Ke1, has been shown through in vitro phosphorylation studies to encode an active protein kinase (Jouannic et al., 2001). BnMAP3Ke1 was also found to partially complement the S. pombe Cdc7 mutant (Jouannic et al., 2001). In addition, BnMAP3Ke1 has been shown to interact with AtSGP1 in yeast two-hybrid experiments (Champion et al., 2004a). AtSGP1 is a homolog of the S. pombe protein Spg1p, which encodes a GTPase protein that recruits Cdc7p to the poles of the mitotic spindles in the yeast SIN pathway (Champion et al., 2004a). It has been shown that MAP3Ke1 and MAP3Ke2 are expressed in all tissues of Arabidopsis, with the highest expression observed in reproductive organs (Champion et al., 2004a,b; Charrier et al., 2002; Jouannic et al., 2001). In addition, in situ hybridization analyses of BnMAP3Ke1 and MAP3Ke1 transcripts revealed that both genes are expressed 193 Journal compilation ª 2006 Blackwell Publishing Ltd

194 Suraphon Chaiwongsar et al. in sporophytic and gametophytic tissues (Jouannic et al., 2001). Using Arabidopsis suspension culture cells that had been synchronized with aphidicolin, it was determined that MAP3Ke1 and MAP3Ke2 expression is cell-cycle regulated, with the highest level of expression seen in the G2 and M phases of the cell cycle. It has therefore been suggested that MAP3Ke1 and MAP3Ke2 may be involved in a signal transduction pathway(s) that acts during late stages of the cell cycle (Jouannic et al., 2001). In the present study, we have investigated the function of MAP3Ke1 and MAP3Ke2 by studying Arabidopsis plants in which these genes have been disrupted by T-DNA insertional mutagenesis (Alonso et al., 2003). Through genetic transmission studies, microscopic analysis and functional complementation we have determined that MAP3Ke1 and MAP3Ke2 are functionally redundant genes that are required for pollen development but are not essential for the function of the female gametophyte (for a review of pollen development see Twell et al., 1998). Analysis of pollen ultrastructure indicated that one of the first phenotypic defects to arise in double-mutant pollen was the appearance of irregularities in the plasma membrane. We also observed that a significant portion of the MAP3Ke1 protein present in Arabidopsis cells was localized to the plasma membrane. These results suggest that the protein encoded by MAP3Ke1 may be involved in regulating a process that is required for the normal functioning of the plasma membrane in Arabidopsis pollen. Results Genetic analysis Insertional mutations within MAP3Ke1 and MAP3Ke2 were obtained from the Salk Institute s collection of T-DNA transformed Arabidopsis lines (Alonso et al., 2003). We used DNA sequencing to determine the precise locations of the T-DNA insertions in the mutant lines. In map3ke1, the T-DNA was located in exon 8; in map3ke2 the T-DNA was located in the intron between exon 6 and exon 7 (Figure 1). Reverse-transcriptase PCR (RT-PCR) was used to determine the effect that these T-DNA insertions had on the messenger RNA levels of these two genes. Total RNA from plants homozygous for either map3ke1 or map3ke2 was analyzed using primers that amplify a region of the wild-type transcript that spans the location of the T-DNA insertion in each gene (Figure 1). Messenger RNA was detected in wild-type Columbia plants for both genes. By contrast, no MAP3Ke1 message was detected in the map3ke1 homozygous plants, and no MAP3Ke2 message was detected in the map3ke2 homozygotes (Figure 1). These results indicated that no full-length transcript is produced in these T-DNA lines. To further characterize map3ke1 and map3ke2 we also performed quantitative, real-time RT-PCR using primer pairs located downstream of each T-DNA insertion site. Using these primers, we detected a truncated message in map3ke1 plants at a level about 10-fold lower than that of the fulllength message made by wild-type plants. No detectable downstream RNA was produced in the map3ke2 plants, suggesting that map3ke2 constitutes a null allele. We have observed in our studies of other SALK T-DNA lines that the left border region often has the capacity to serve as a promoter driving expression of downstream sequences (data not shown). The genetic analysis that we report later in this study provides conclusive evidence that the map3ke1;- map3ke2 double-mutant combination causes pollen lethality. This result indicates that the map3ke1 mutation severely compromises the function of the MAP3Ke1 gene. Because an ectopic copy of the wild-type MAP3Ke1 is able to rescue this (a) (b) (c) Figure 1. Mutant alleles of MAP3Ke1 and MAP3Ke2. (a) Genomic structure of MAP3Ke1 and MAP3Ke2. Exons are indicated by thick lines. The locations of the T-DNA insertions are indicated with triangles. Approximate locations of the primers used for reverse-transcriptase PCR (RT-PCR) analysis are indicated with arrows. The heads of the arrows indicate the primer positions. The scale bar indicates 1 kilobase. Expression of MAP3Ke1 (b) and MAP3Ke2 (c) in wild-type and homozygous single-mutants by RT-PCR. The RT-PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. The lane labeled wt corresponds to wild-type Columbia. Lanes labeled 1 and 2 represent two independent homozygous plants. HIS2A, control RT-PCR reactions performed using primers specific for a histone gene.

Map3K-epsilon and pollen viability 195 Table 1 Progeny of self-pollinated plants Parental genotype Progeny genotype M3Ke1/M3Ke1 M3Ke1/m3ke1 m3ke1/m3ke1 m3ke1/m3ke1 a 90 (25%) d 185 (51%) 89 (24%) M3Ke2/M3Ke2 M3Ke2/m3ke2 m3ke2/m3ke2 m3ke2/m3ke2 b 46 (26%) 85 (49%) 45 (26%) M3Ke1/M3Ke1 M3Ke1/m3ke1 m3ke1/m3ke1 m3ke2/m3ke2 m3ke2/m3ke2 m3ke2/m3ke2 m3ke1/m3ke1 102 (51%) 98 (49%) 0 m3ke2/m3ke2 c Genotypes of a 364 progeny, b 175 progeny and c 200 progeny were determined by PCR. d Percentage of total progeny. pollen lethality, map3ke1 constitutes a recessive, loss-offunction allele. We did not observe any abnormal phenotypes in plants homozygous for either map3ke1 or map3ke2 when grown under standard laboratory conditions. In addition, each of these mutant alleles displayed a normal Mendelian segregation ratio (Table 1). As MAP3Ke1 and MAP3Ke2 are closely related genes, it seemed likely that functional redundancy could be masking the appearance of a mutant phenotype in these homozygous lines (Liljegren et al., 2000). Therefore, map3ke1 and map3ke2 plants were crossed to create double mutants. One of the resulting double-heterozygous lines was then allowed to self-fertilize, and more than 400 individuals from the resulting population were genotyped using PCR. No homozygous double mutants were found. However, plants homozygous-mutant at one locus and heterozygous at the other were found. One such plant with the genotype map3ke1/map3ke1;map3ke2/map3ke2 was allowed to self-pollinate, and although we genotyped 201 of its progeny, we still failed to recover any homozygous double mutants (Table 1). In addition, the segregation ratio displayed by the map3ke1 mutation in this population was 1:1, suggesting that a gametophyte-lethal mutation was segregating (Table 1). We next performed reciprocal crosses between a map3ke1/map3ke1;map3ke2/map3ke2 plant and a wild-type Columbia plant to test for gametophytic defects. Progeny from the reciprocal crosses were genotyped by PCR to test for transmission of the map3ke1 mutant allele. It was determined that the map3ke1;map3ke2 combination could not be transmitted through pollen (Table 2). Transmission through the female gametophyte was normal. Molecular complementation of map3ke1 Table 2 Transmission of the map3ke1;map3ke2 double-mutant combination through male and female gametes Gamete Gamete frequency M3Ke1;m3ke2 m3ke1;m3ke2 Female 112 (51%) a 106 (49%) Male 152 (100%) 0 (0%) Reciprocal crosses were performed between wild-type and map3ke/ MAP3Ke1;map3ke2;map3ke2 parents. The genotypes of 370 progeny from these crosses were determined by PCR and used to infer the genotype of the gamete contributed by the mutant parent in each cross. a The number in parentheses indicates the percentage of gametes with the given genotype. We used genetic complementation to confirm that the inability of map3ke1;map3ke2 to be transmitted by pollen was due to mutation of the map3ke genes. An approximately 11-kb fragment of genomic DNA encompassing the wild-type MAP3Ke1 locus was PCR amplified, cloned into a binary vector and introduced into map3ke1/map3ke1;- map3ke2/map3ke2 plants via Agrobacterium-mediated transformation. Progeny of the primary transformants were screened by PCR to identify plants that were homozygous double mutant at the native MAP3Ke1 and MAP3Ke2 loci. For this analysis we made use of PCR primers that can distinguish the endogenous MAP3Ke1 locus from the introduced copy of MAP3Ke1. Using this strategy, we were able to generate map3ke1/map3ke1;map3ke2/map3ke2 plants only when the lines also carried an ectopic copy of MAP3Ke1. Controls transformed using the binary vector alone did not yield any homozygous double-mutant progeny. These results indicated that the segregating mutation responsible for pollen lethality is map3ke1. The pollen lethality caused by map3ke1 is only observed in plants that are also homozygous for map3ke2, presumably due to functional redundancy. In order to further confirm these molecular complementation results, we performed genetic analysis of map3ke1/ map3ke1;map3ke2/map3ke2 plants that were hemizygous for an ectopic copy of MAP3Ke1 by performing reciprocal crosses between these plants and wild-type Columbia. As the T-DNA vector used to introduce the ectopic copy of MAP3Ke1 also encodes resistance to the herbicide Basta, transmission of the ectopic MAP3Ke1 construct was measured by plating the progeny of these crosses onto growth media containing Basta. When plants segregating the ectopic MAP3Ke1 construct were used as the pollen donor, 100% of the progeny were Basta resistant, indicating that the MAP3Ke1 locus is required for pollen function (Table 3). By contrast, when plants segregating the ectopic MAP3Ke1 construct were fertilized with wild-type pollen about 50% of the progeny were Basta sensitive and about 50% were Basta resistant (Table 3), confirming that the MAP3Ke1 locus is not essential for the function of the female gametophyte.

196 Suraphon Chaiwongsar et al. Table 3 Transmission of an ectopic copy of MAP3Ke1 through the male and female gametes of double-mutant plants Basta selection (a) (b) Gamete Basta S Basta R Female 102 (49%) c 108 (51%) Male 0 (0%) 154 (100%) Reciprocal crosses were performed between wild-type and rescued mutant lines (map3kemap3ke1;map3ke2;map3ke2;qrt1-2/qrt1-2 with an ectopic copy of MAP3Ke1). Transmission of the ectopic copy of MAP3Ke1 was monitored by scoring resistance of the seedlings to the herbicide Basta. The rescued mutant line was used as either the female or male parent in the crosses. c The number in parentheses indicates the percentage of progeny in the given category. (c) (d) Pollen viability The genetic analyses described above indicated that the map3ke1;map3ke2 combination cannot be transmitted through the male gamete. Failure to transmit mutant alleles through the pollen can be caused by defects in pollen viability, germination, pollen tube growth or fertilization. In order to decide between these possibilities, we began by testing for pollen viability using Alexander s staining (Alexander, 1969). Mature pollen grains from wild-type plants appear as full, round, red-stained grains when treated with Alexander s stain. As seen in Figure 2(a,b), pollen isolated from map3ke1/map3ke1;map3ke2/map3ke2 and map3ke1/map3- Ke1;map3ke2/map3ke2 plants was composed of a mixture of viable and dead pollen, suggesting that the double-mutant combination may cause pollen lethality. In order to determine whether a mutation segregating at meiosis was responsible for this pollen lethality, we made use of the Arabidopsis quartet mutation. Plants homozygous for the quartet mutation produce pollen in which the four products of meiosis remain physically stuck together, resulting in quartets of pollen grains (Preuss et al., 1994). By segregating a mutation in the quartet background, it is possible to examine the four products of meiosis. We therefore crossed a map3ke1/map3ke1;map3ke2/map3ke2 plant to a qrt1-2/ qrt1-2 plant and selected progeny in subsequent generations by genotype at the MAP3Ke1 and MAP3Ke2 loci and by phenotype at the QRT locus. When the quartets of pollen produced by a map3ke1/map3ke1;map3ke2/map3ke2;qrt1-2/ qrt1-2 plant were analyzed by Alexander s staining, we always observed two viable pollen grains and two dead pollen grains (n ¼ 620 quartets). By contrast, pollen from a qrt1-2/ qrt1-2 plant always produced quartets in which all four pollen grains were viable (Figure 2c,d). These data, in conjunction with the additional genetic analyses described above, indicated that segregation of the map3ke1 mutation at meiosis was responsible for the pollen lethality observed in map3ke1/map3ke1;map3ke2/map3ke2;qrt1-2/qrt1-2 Figure 2. Mature pollen stained with Alexander s viability stain. Pollen from plants with the following genotypes was analyzed: (a) map3ke1/ map3ke1;map3ke2/map3ke2, (b) map3ke1/map3ke1;map3ke2/map3ke2, (c) qrt1-2/qrt1-2 and (d) qrt1-2/qrt1-2;map3ke1/map3ke1;map3ke2/map3ke2. Arrows indicate examples of dead pollen. Scale bar (a, b) ¼ 15 lm and (c, d) ¼ 10 lm. plants. As shown earlier, the pollen lethality of map3ke1 is dependent on the presence of the map3ke2 mutation. Nuclear development in mutant pollen grains In order to determine the developmental stage at which pollen lethality occurred, we observed pollen isolated from map3ke1/map3ke1;map3ke2/map3ke2;qrt1-2/qrt1-2 plants at various stages of floral development. By using the quartet background we were able to directly compare wild-type and mutant pollen derived from the same microsporocyte. Pollen was isolated from flowers ranging from stage )3 to stage þ2, where stage þ1 corresponds to the first opened flower. Lower numbers represent flower buds before the opened stage and higher numbers indicate flowers after the opened stage. The fluorescent stain 4,6-diamidino-2-phenylindole (DAPI) is an effective method of monitoring the nuclei of pollen because of the stain s preference for binding to nucleic acids. At flower stage )3, the quartet of microspores has completed meiosis, and a single nucleus can be seen in each of the four members of the quartet (Figure 3a). No differences can be seen between the four pollen grains at this stage, suggesting that the double-mutant combination does not affect meiosis or early pollen development.

Map3K-epsilon and pollen viability 197 Figure 3. Showing 4,6-diamidino-2-phenylindole (DAPI) staining to observe pollen nuclei. Quartets of developing pollen were isolated from a map3ke1/map3ke1;map3ke2/map3ke2;qrt1-2/ qrt1-2 plant at various developmental stages, stained with DAPI and viewed with either white light (Brightfield) or ultraviolet light (DAPI). (a) A quartet from floral stage )3. (b) A quartet from floral stage 0. The same quartet was imaged in two different focal planes under ultraviolet light in order to capture all of the nuclear staining present in the pollen. Asterisks indicate sperm nuclei, which appear as compact, brightly staining spots. The vegetative nuclei are larger and more diffusely stained. Arrows in (b) indicate a pollen grain with only one sperm nucleus visible. Scale bar (a) ¼ 5mm and (b) ¼ 10 mm. (a) (b) Pollen from stage 0 flowers is expected to have completed the second mitotic division. Each wild-type pollen grain should therefore contain two sperm nuclei and one vegetative nucleus. When stained with DAPI, sperm nuclei appear as compact, bright spots of blue, while the vegetative nucleus appears as a diffuse patch of blue, which is sometimes difficult to observe. We often observed that three of the four pollen grains from stage 0 flowers contained two sperm nuclei, while the fourth grain contained only one (Figure 3b). These results indicated that one of the mutant pollen grains was able to proceed through pollen mitosis II, while the other appeared to have stopped development sometime between pollen mitosis I and II. When DAPI staining was performed on quartets of mature pollen, we always observed two normal, wild-type pollen grains and two dead pollen grains with no visible DAPI staining (data not shown). These observations are consistent with the results we obtained using Alexander s stain. Our analysis of the DAPI staining pattern of pollen from various developmental stages indicated that pollen lethality occurs after the completion of pollen mitosis I, and in many cases not until after the completion of pollen mitosis II. Pollen isolated from map3ke1/map3ke1;map3ke2/map3- Ke2;qrt1-2/qrt1-2 plants gave the same results when analyzed by DAPI staining. Pollen ultrastructure In order to better understand the phenotype of the doublemutant pollen, we attempted to identify the first structural defects that arise during the course of map3ke1;map3ke2 pollen development. Based on our preliminary analysis using DAPI staining, we focused these studies on the stages of development between pollen mitosis I and pollen mitosis II. Transmission electron microscope analysis of large numbers of pollen grains collected from map3ke1/ MAP3Ke1;map3ke2/map3ke2 anthers revealed that mutant phenotypes were detectable only following the completion of pollen mitosis I. Two distinct classes of mutant pollen were observed, and examples of each are shown in Figure 4. Class I mutant pollen has three characteristic features: irregularity of the plasma membrane, a thickened intine layer and degenerating mitochondria. A representative class I mutant is shown in Figure 4. The plasma membranes of these pollen grains have an irregular shape when compared with wild type and feature unusual invaginations that protrude into the cytoplasm (Figure 4h). In addition, the intine layer of the mutants appears thicker than wild type (Figure 4e,h). The intine layers of three representative wildtype and mutant pollen grains were measured at 24 positions around the perimeter of each pollen grain in order to determine the average intine thickness. The average intine thickness was 97 nm for wild type and 368 nm for the class I mutant. The final characteristic of class I mutant pollen is mitochondrial degeneration. Compared to wildtype mitochondria (Figure 4d,g), mitochondria in class I mutants are swollen, with a lightly stained matrix and reduced cristae (Figure 4e,h). Despite these abnormalities, however, class I mutant pollen contains an intact generative cell (Figure 4k), suggesting that pollen mitosis I has completed normally in these mutants.

198 Suraphon Chaiwongsar et al. Class II mutant pollen has three characteristic features: plasma membrane irregularity, a thickened intine layer and unusual vacuole-like structures. These first two characteristics are shared with class I mutants. The unique features of class II mutants are that they have normal mitochondria but abnormal vacuole-like structures. These vacuole-like struc- (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m)

Map3K-epsilon and pollen viability 199 tures are not found in wild-type pollen at this stage of development. Representative class II mutant pollen grains are shown in Figure 4. Despite the abnormal plasma membrane present in this pollen, class II mutants are able to complete pollen mitosis II, as indicated by the presence of the two sperm cells in the cytoplasm of the vegetative cell (Figure 4c,l,m). These sperm cells appeared to have a normal structure and organization, suggesting that the mutations do not directly affect mitosis or cell division. Instead, the main effects appear to be a disruption of the plasma membrane, thickening of the intine and the appearance of unusual vacuole-like structures. Transmission electron microscope analysis of mature pollen collected from map3ke1/map3ke1;map3ke2/map3ke2 anthers revealed that about 50% of the pollen population was fully collapsed with full degradation of cellular structures (data not shown). These observations indicated that the two distinct classes of mutant pollen share the same fate and are indistinguishable at the mature pollen stage. The analysis described above indicated that class II mutant pollen grains develop unusual structures that have the appearance of vacuoles when viewed using TEM. In order to determine whether these unusual structures were indeed vacuoles, we made use of neutral red, which serves as a vacuole-specific fluorescent dye. Neutral red has been shown to accumulate in vacuoles and vesicles, and its fluorescence is affected by the ph of the vacuoles (Clarke et al., 2002; Mahlberg, 1972; Regan and Moffatt, 1990). By choosing appropriate excitation and emission filters, neutral red-specific fluorescence of vacuoles can be detected in pollen using confocal microscopy. Pollen grains were collected from the flowers of a map3ke1/map3ke1;map3ke2/map3ke2;qrt1-2/qrt1-2 plant at the stage in development where the unusual vacuole-like structures appear in class II mutants. This developmental stage is the same as that used for the TEM experiments displayed in Figure 4. At this stage of development wild-type pollen does not contain any large vacuole structures (Yamamoto et al., 2003). By using the quartet background for this experiment, we were able to directly compare wildtype pollen grains and mutant pollen grains in the same tetrad. As a control, pollen from a qrt1-2 plant was also analyzed. In this case, all four members of the quartet should display a wild-type phenotype. As seen in Figure 5(a), the four wild-type pollen grains show only a low level of vacuole-specific neutral red fluorescence (Figure 5a, upper panel). The merged image in the lower panel overlays the vacuole-specific fluorescence collected in the red channel with cell wall autofluorescence collected in the green channel. Cell wall autofluorescence was used to demarcate the positions of the four members of the tetrad. (a) (b) Analysis of vacuoles in mutant pollen Figure 5. Neutral red staining to observe vacuoles. Tetrads of pollen were collected from stage 0 flowers, stained with neutral red, and viewed with a confocal microscope. (a) Tetrad collected from a qrt1-2 flower. (b) Tetrad collected from a map3ke1/map3ke1;map3ke2/map3ke2;qrt1-2/qrt1-2 flower. Upper panel: vacuole-specific neutral red fluorescence. Image is a Z- series projection of the emission detected at 590 640 nm (red channel). Lower panel: image from the upper panel merged with and a Z-series projection of the same pollen tetrads collected at 550 580 nm (green channel) to detect autofluorescence of the pollen cell wall. The green channel provides an outline of the four pollen grains composing each tetrad, while the red channel indicates vacuole-specific fluorescence. The asterisk indicates a mutant pollen grain with a high level of vacuole-specific fluorescent signal. Figure 4. Transmission electron microscope analysis of mutant and wild-type pollen grains. All pollen grains were collected from the same anther of a map3ke1/map3ke1;map3ke2/map3ke2 plant. (a c) Whole-cell views of wild-type and mutant pollen grains: (a) wild type, (b) class I mutant, (c) class II mutant. (d f) Outer regions of wild-type and mutant pollen grains. (d) Wild-type pollen with normal mitochondria and intine. (e) Class I mutant with abnormal mitochondria and thickened intine. Plasma membrane irregularities indicated by an arrow. (f) Class two mutant with abnormal vacuoles. (g i) Plasma membrane of mutant and wild-type pollen. Arrows in (h) and (i) indicate irregularities of the plasma membrane in the mutant pollen. (j) Generative cell of normal pollen grain after pollen mitosis I. (k) Generative cell of the mutant pollen grain shown in (b). (l, m) Sperm cells of the mutant pollen grain shown in (c). EX, exine; GC, generative cell; IN, intine; M, mitochondria; PM, plasma membrane; V, vacuole; VN, vegetative nucleus. Scale bar (a c) ¼ 5 lm, (d f) ¼ 3 lm, (g i) ¼ 0.5 lm, (j m) ¼ 1 lm.

200 Suraphon Chaiwongsar et al. When pollen from a map3ke1/map3ke1;map3ke2/map3- ke2;qrt1-2/qrt1-2 plant was analyzed using this same procedure, it was often observed that one of the mutant pollen grains present in the quartet displayed strong vacuolespecific neutral red fluorescence (Figure 5b). This result suggests that the large vacuole-like structures observed via TEM in the class II mutants are indeed vacuoles as opposed to other cellular structures. Because class I and class II mutant pollen grains occur with approximately equal frequency, and only the class II mutants show the large vacuole structures under TEM analysis, it is expected that only about 50% of the mutant pollen grains should generate vacuolespecific fluorescence. Consistent with this prediction, our analysis of several dozen quartets indicated that only one of the mutant pollen grains in a given quartet displayed strong vacuole-specific, neutral red fluorescence. Subcellular localization of MAP3Ke1 The TEM analysis described above indicated that one of the phenotypic consequences of mutating MAP3Ke1 and MAP3Ke2 was a disruption of the normal functioning of the plasma membrane. In order to better understand how directly MAP3Ke1 and MAP3Ke2 might affect the plasma membrane, we analyzed the subcellular localization of MAP3Ke1. To accomplish this goal we constructed a binary vector encoding an N-terminal translational fusion between MAP3Ke1 and the YFP under the transcriptional control of the native MAP3Ke1 promoter. This construct was stably introduced into map3ke1/map3ke1;map3ke2/map3ke2 plants via Agrobacterium transformation. Progeny from the primary transformants were screened by PCR to identify homozygous double-mutant plants. Using this strategy we were able to generate map3ke1/map3ke1;map3ke2/map3ke2 plants only when the lines also carried an ectopic copy of the YFP MAP3Ke1 construct, indicating that the fusion protein was functional because of its ability to rescue the pollenlethal phenotype. Thirty independent transgenic lines were screened for the presence of YFP fluorescence. Twenty five of the lines expressed the YFP fusion protein at a high enough level to be detected by fluorescence microscopy. Analysis of young seedlings revealed that YFP MAP3Ke1 was most highly expressed in newly emerging leaves and near the root tip (data not shown). All 25 transgenic lines displayed a similar expression pattern. These observations are consistent with previous reports that MAP3Ke1 is most highly expressed in tissues containing dividing cells (Champion et al., 2004a,b; Charrier et al., 2002; Jouannic et al., 2001). We attempted to visualize YFP MAP3Ke1 in pollen, but were not able to reproducibly observe a fluorescent signal, which is likely to be due to the high level of autofluorescence displayed by the pollen cell wall. In order to determine the subcellular localization of MAP3Ke1, we used confocal microscopy to observe the cells of young leaves of Arabidopsis plants expressing YFP MAP3Ke1. This tissue was chosen because it displayed the highest level of expression of YFP MAP3Ke1. The confocal microscope settings used for these experiments resulted in no autofluorescence when the cells of untransformed wildtype plants were observed for YFP excitation/emission (data not shown). The merged confocal images collected from two representative young leaves expressing YFP MAP3Ke1 are shown in Figure 6(a,b). A majority of the fluorescent signal present in these images is located at the periphery of the cell. A low level of fluorescence was seen in the cytoplasm, but none was detected in the nuclei. For comparison, a merged confocal image of a similar young leaf from a 35S:GFP control plant is shown in Figure 6(c). The fluorescent signal in the 35S:GFP plants is observed throughout the cytoplasm and nucleus. Fluorescence associated with the cell perimeter could be due to YFP MAP3Ke1 localization to the cell wall, the plasma membrane or both. To distinguish between these possibilities we performed plasmolysis experiments using plants expressing YFP MAP3Ke1. The YFP fluorescent signal in these plants localized with the displaced membrane in plasmolysed cells, indicating that the peripheral localization of YFP MAP3Ke1 corresponds to the plasma membrane rather than the cell wall (data not shown). MAP3Ke1 associates with the membrane compartment The confocal microscopy results described above suggested that MAP3Ke1 may be associated with the plasma membrane. In order to further investigate this possibility, we (a) (b) (c) Figure 6. Subcellular localization of MAP3Ke1. (a, b) Z-series projection of yellow fluorescent protein (YFP) fluorescence in two newly emerged leaves from transgenic line YFP20 expressing YFP MAP3Ke1 from the MAP3Ke1 native promoter. (c) Z-series projection of GFP fluorescence in a newly emerged leaf of a transgenic line expressing soluble GFP via the cauliflower mosaic virus 35S promoter.

Map3K-epsilon and pollen viability 201 performed a biochemical fractionation experiment to separate cellular proteins into cytosolic and membrane fractions. Preliminary experiments revealed that the MAP3Ke1 native promoter was not able to generate a level of protein expression that was high enough to be detectable in the fractionation assay. In order to overcome this problem we constructed an expression vector in which the YFP MAP3Ke1 fusion construct was under the transcriptional control of an alcohol-inducible promoter and used this construct to generate transgenic Arabidopsis lines (Caddick et al., 1998). Using this ethanol-inducible system we were able to generate a level of YFP MAP3Ke1 expression that was sufficient for detection in the fractionation assay. Three different Arabidopsis lines were used in the fractionation experiment: wild-type Columbia, a 35S:GFP control line and plants expressing the ethanol-inducible YFP MAP3Ke1. Three days prior to protein extraction the ethanol-inducible YFP MAP3Ke1 plants were watered with 1% ethanol to induce YFP MAP3Ke1 expression. Tissue was harvested from leaf and inflorescence tissues of soil-grown plants, and protein extracts were separated into post-nuclear supernatant (S1), membrane-free cytosolic (S150) and cytosol-free microsomal membrane (P150) fractions by highspeed centrifugation. In order to verify the success of the fractionation, immunoblotting was performed with antibodies that recognize well-characterized cytosolic and membrane-associated proteins. An antibody against the plant UBX domain-containing (PUX) protein was used as the cytosolic marker (Rancour et al., 2004), and an antibody against syntaxin 31 (SYP31) was the membrane compartment marker (Rancour et al., 2002). These controls confirmed the identity and purity of each subcellular fraction (Figure 7c,d). We next attempted to visualize YFP MAP3Ke1 by immunoblotting with an anti-gfp antibody but were not able to detect this fusion protein by Western blot analysis. This difficultly could be related to the relatively large size of the MAP3Ke1 protein. We therefore chose to use an alternative method for quantifying the abundance of YFP MAP3Ke1 in each of the protein fractions. For this analysis, a fluorescence plate reader was used to measure the amount of YFP or GFP fluorescence present in each sample. This approach is analogous to the well-established practice of using enzyme activities to follow specific proteins through biochemical fractionation experiments. In our case, the activity that we were measuring was YFP fluorescence rather than an enzymatic reaction. This activity is highly specific for the YFP and GFP proteins present in our samples, as demon- (a) (b) (c) (d) Figure 7. Membrane localization of MAP3Ke1. (a) Protein extracts from wild-type Columbia or transgenic Arabidopsis plants expressing YFP MAP3Ke1 via an ethanol-inducible promoter were separated into subcellular fractions. To quantitatively measure YFP MAP3Ke1 protein levels in each fraction, 50 lg of total protein for each fraction was scanned for YFP fluorescence using a microplate reader. Relative fluorescence units were calculated relative to a buffer-only control. Each bar represents an independent protein sample. Values for wild-type Columbia represent the background level of autofluorescence. S1, post-nuclear supernatant; S150, membrane-free cytosolic; P150, cytosol-free microsomal membrane. (b) Protein extracts from wild-type Columbia or transgenic Arabidopsis plants expressing soluble GFP via cauliflower mosaic virus 35S promoter were analyzed for GFP fluorescence as described above. (c, d) The protein extracts used in (a) and (b) were resolved by SDS PAGE and analyzed by immunoblotting with antibodies to SYP31, a membrane-specific control (c) or PUX, a cytosol-specific control (d).

202 Suraphon Chaiwongsar et al. strated by the low level of background fluorescence detected in the wild-type Columbia controls. The optimum combination of excitation wavelength, emission wavelength and cutoff filter settings were used for YFP fluorescence in the YFP MAP3Ke1 samples and GFP fluorescence in the 35S:GFP samples. In each case wild-type Columbia samples were also analyzed using the same parameters in order to determine the level of background fluorescence for each combination of filter settings. As shown in Figure 7(a,b), the 35S:GFP and YFP MAP3Ke1 protein extracts produced fluorescent signals that were well above the background level observed in wild-type Columbia. The relative fluorescence units shown in these graphs are normalized to a buffer-only control. The data presented in Figure 7(a) indicate that the YFP MAP3Ke1 protein is more abundant in the P150 membrane fraction than it is in the S150 cytosolic fraction. For comparison, Figure 7(b) presents the results obtained with protein extracted from the 35S:GFP plants. In this case the majority of the GFP protein was observed in the S150 cytosolic fraction. This result is consistent with the fact that GFP is a soluble protein that is known to be distributed throughout the cytoplasm and nucleus of plant cells. Taken together, these results indicate that a significant portion of the YFP MAP3Ke1 protein present in Arabidopsis cells is associated with the membrane compartment. The fluorescence readings in Figure 7 were collected from samples that had been adjusted to contain equal amounts of total protein. The S1, S150 and P150 samples were all adjusted to 1 mg ml )1 of total protein prior to analysis using the plate reader. This normalization strategy explains why the amount of fluorescent signal observed in a subcellular fraction is higher than the amount of signal present in the corresponding S1 sample prior to fractionation. Discussion Using a reverse-genetic approach we have shown that MAP3Ke1 and MAP3Ke2 are a functionally redundant pair of genes that are required for pollen viability. These results are supported by previously published work demonstrating that BnMAP3Ke1, the B. napus homolog of MAP3Ke1, is expressed in B. napus microspores (Jouannic et al., 2001). Charrier et al. (2002) have shown that MAP3Ke1 and MAP3Ke2 are expressed in all tissues of Arabidopsis, and we have observed that the MAP3Ke1 protein is expressed in both root and shoots. These data suggest that MAP3Ke1 and MAP3Ke2 are likely to play a role in general cellular function, rather than a specialized role limited only to pollen development. The ultrastructural data that we have presented indicate that the first structural defects to appear in mutant pollen arise after the completion of pollen mitosis I. The most consistent early phenotype that we observed was the presence of irregularities at the plasma membrane and a thickening of the intine layer. These features were present in all examples of mutant pollen that we observed, suggesting that these phenotypes may be directly caused by the doublemutant combination. By contrast, the unusual vacuole-like structures and degenerating mitochondria present in some of the mutant pollen grains were not a universal feature of the mutant condition, suggesting that these effects are not a primary consequence of the mutations and may represent secondary effects related to the progression of cell death. Protein localization studies performed using confocal microscopy as well as biochemical fractionation indicated that a majority of the MAP3Ke1 protein present in Arabidopsis plants is localized to the plasma membrane. This localization pattern suggests that the protein may be involved in regulating a process that is important for the normal functioning of the plasma membrane, a possibility that is consistent with our observation of structural abnormalities in the plasma membrane of the mutant pollen. Because MAP3Ke1 does not appear to contain any transmembrane domains, it most likely represents a peripheral membrane protein that associates with the plasma membrane through its interaction with a membrane-bound protein. Champion et al. (2004a) have reported that BnMAP3Ke1, the Brassica homolog of MAP3Ke1, localizes almost exclusively to the nucleolus when transiently expressed in tobacco protoplasts. This expression pattern contrasts sharply with our observation that YFP MAP3Ke1 localizes to the plasma membrane. Because our confocal experiments utilized YFP MAP3Ke1 expressed using the MAP3Ke1 native promoter in stably transformed Arabidopsis lines, it seems likely that the localization pattern that we have reported may more closely reflect the distribution of the native MAP3Ke1 protein in Arabidopsis plants. Phylogenetic analysis indicates that the closest homologs of MAP3Ke1 and MAP3Ke2 are cdc7p from S. pombe and cdc15p from S. cerevisiae (Jouannic et al., 2001). In addition to this structural similarity, functional conservation has been demonstrated by the observation that BnMAP3Ke1 is able to partially complement the S. pombe cdc7 mutant (Jouannic et al., 2001). The function of cdc7p in yeast cells is to regulate the formation of the septum after chromosome segregation has been completed through the activity of the septum initiation network (SIN) (Gould and Simanis, 1997). More recently it has been reported that two more components of the SIN pathway, spg1p and gid1p, have Arabidopsis homologs that are also able to complement the corresponding yeast mutations (Champion et al., 2004a). Although both symmetrical and asymmetrical mitotic division occur during pollen development to generate tricellular pollen, we have found no evidence to support a role for MAP3Ke1 and MAP3Ke2 in the regulation of cell division during pollen development. We observed that

Map3K-epsilon and pollen viability 203 mutant pollen was able to complete mitosis II and establish two intact sperm cells with apparently normal structures. The defects that were observed in these pollen grains affected the plasma membrane, intine and vacuoles. If cell division was the primary target of MAP3Ke1 function then one would have expected to see evidence of failed division of the two sperm cells. The fact that Arabidopsis proteins are able to functionally complement the yeast SIN pathway mutants indicates that these homologous proteins have retained significant structural similarity. Our data suggest, however, that the pathways in which these proteins are utilized in their native organisms may have diverged. Jouannic et al. (2001) reported that MAP3Ke1 and MAP3Ke2 expression is cell-cycle regulated, with the highest level of transcription seen during late stages of the cell cycle (Jouannic et al., 2001). As we did not observe any obvious defects in mitosis or cell division in double-mutant pollen, the possibility should be considered that these proteins function in some cell-cycle-related process that does not directly affect mitosis or cytokinesis. After the first asymmetric mitotic division of the microspore, the vegetative cell and the generative cell are produced. These cells have different cell fates: the vegetative cell exits the cell cycle while the generative cell undergoes a second, symmetric mitotic division to create the two sperm cells (McCormick, 1993; Twell et al., 1998). Our observation that structural abnormalities in the mutant pollen only arise after the completion of pollen mitosis I could indicate that MAP3Ke1 and MAP3Ke2 are involved in maintaining cell integrity following the completion of the cell cycle or are involved in the cell-cycle exit pathway for the vegetative cell. Because the cell divisions involved in pollen development have many unique characteristics that are not shared with the cell divisions that occur in the sporophyte, analysis of MAP3Ke1 and MAP3Ke2 function in sporophytic tissue will be needed to better understand the functional significance of the cellcycle regulation of transcription that has been observed for MAP3Ke1 and MAP3Ke2. We have demonstrated that absence of the MAP3Ke1 and MAP3Ke1 genes results in pollen lethality. It was therefore surprising that the double-mutant combination had no detrimental affect on the female gametophyte. There are several explanations that could account for this observation. One possibility is that MAP3Ke1 and MAP3Ke2 may regulate a process that is unique to pollen development. If this were the case, it would be difficult to explain why these proteins are also expressed in root and shoot tissues of the sporophyte. Another explanation of the wild-type function of double-mutant female gametophytes is that residual levels of MAP3Ke1 from the sporophytic tissue may be present in sufficient quantity to allow survival of the double mutants. It is also possible that an alternative mechanism is active in the female gametophyte that is able to compensate for the absence of the MAP3Ke1 and MAP3Ke2 proteins. Future studies will be needed to pinpoint the specific cellular process that is responsible for lethality in the doublemutant pollen grains. The data that we have presented in this study suggest that this process may be required for the normal functioning of the plasma membrane. As MAP3Ke1 and MAP3Ke2 are known to be expressed in root and shoot tissues, it seems likely that these proteins have functions outside of pollen development. Future experiments that make use of conditional-rescue constructs should provide the means to investigate this possibility. Experimental procedures T-DNA mutants Arabidopsis plants carrying T-DNA insertions within MAP3Ke1 (At3g13530; SALK_01724) and MAP3Ke2 (At3g07980; SALK_150512) were obtained from the Salk Institute Genomic Analysis Laboratory (Alonso et al., 2003). The following PCR primers were used to genotype plants carrying these T-DNA insertions. MAP3Ke1-F: 5 - CACTCTGGACTATGGACGGGAAACTCAAG-3 ; MAP3Ke1-R: 5 -AC- CCAAGGACATTACAGGTTCAGTGGATG-3 ; MAP3Ke2-F: 5 -TTATG- ACTTTTGAGCGCAGATGCTAGGAG-3 ; MAP3Ke2-R: 5 -TCAGAAG- CAGCACAAACTCCTGACAATTC-3 ; T-DNA left border, 5 -CAAACT- GGAACAACACTCAACCCTATCTC-3. DNA sequencing of the genomic DNA flanking the T-DNA insertion sites confirmed the identity of each gene and the precise locations of the T-DNA insertions. All of the T-DNA mutants and wild-type plants in this study were from the Columbia ecotype Col-0. Reverse-transcriptase PCR Ribonucleic acid was isolated from map3ke1/map3ke1 plants, map3ke2/map3ke2 plants and wild-type plants using the Qiagen Rneasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Real-time, quantitative RT-PCR was carried out using the QuantiTect SYBR Green RT-PCR Kit (Qiagen GmbH, Hilden, Germany), with H2A (histone H2A-like protein, At4g27320) as a control. Polymerase chain reaction primers specific to the predicted cdna sequences of each gene were used for this analysis: e1-rt-a1, 5 -AAAAACATTGTGAAGTATCTT- GGGTCGTC-3 ; e1-rt-a2, 5 -GCTTCTTTACGAATTTCGCGAGAACG- ATC-3 ; e2-rt-a1, 5 -AAAACATTGTCAAGTATCTCGGATCGTTG-3 ; e2-rt-a2, 5 -TTCTCGTGATCGATCCCTCTCAAAACCAG-3 ; H2A-1, 5 -AACAACTTGGATCTGGTGCAGCGAAGAAG-3 ; and H2A-2, 5 - ACTATACGGGTCTTCTTGTTGTCTCTCGC-3. Reactions were carried out on an icycler iq TM real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Genetic complementation The PCR primers 5 -CCGTGTTCACCACCAAAGACATTG-3 and 5 - CTGACCACTTTTCCTCTATGCATC-3 were used to amplify an 11 190-bp long fragment of genomic DNA that included the entire MAP3Ke1 coding region plus about 2 kb of upstream sequences and about 1.3 kb of downstream sequences. This PCR product was ligated into pcambia3300s (Krysan et al., 2002), a spectinomycinresistant derivative of pcambia3300. The resulting binary vector containing the MAP3Ke1 gene was introduced into map3ke1/ MAP3Ke1;map3ke2/map3ke2 plants using Agrobacterium-mediated transformation (Clough and Bent, 1998). Transformed plants were

204 Suraphon Chaiwongsar et al. selected in soil in the next generation by spraying seedlings with the herbicide Basta. In order to create the YFP MAP3Ke1 fusion construct, the wildtype MAP3Ke1 genomic clone described above was modified using site-directed mutagenesis in order to add two restriction sites immediately after the start codon. Recognitions sites for the enzymes AvrII and AgeI were introduced. The YFP coding sequence (CDS) was then PCR amplified from a plasmid vector using PCR primers that added a NheI site to the 5 end of the CDS and an AgeI site to the 3 end. This PCR-amplified fragment containing the YFP CDS was then ligated into the modified MAP3Ke1 clone using sticky ends generated by AvrII, AgeI and NheI cleavage. NheI sticky ends are identical to those produced by AvrII. The resulting construct contains the YFP CDS fused in frame to the 5 end of the MAP3Ke1 coding region. The resulting plasmid was introduced into map3ke1/ MAP3Ke1;map3ke2/map3ke2;qrt1-2/qrt1-2 plants using Agrobacterium-mediated transformation as described above. In order to construct the ethanol-inducible version of the YFP MAP3Ke1 fusion protein, the YFP MAP3Ke1 coding region described above was moved into the binary vector developed by Caddick et al. (1998) that carries an ethanol-inducible promoter system and the vector was introduced into Arabidopsis plants via Agrobacterium-mediated transformation. Light microscopy To monitor pollen viability, pollen was collected by squashing anthers containing mature pollen onto microscope slides. Alexander s staining solution (Alexander, 1969) was added and pollen grains were viewed with bright-field illumination. To visualize pollen nuclei, pollen was applied to a microscope slide, stained with 1 mm DAPI solution [1 lg ml )1 DAPI, 100 mm NaPO 4,1 mm EDTA and 0.1% (w/v) Triton], covered with a cover slip and viewed with a UV light. High-pressure freezing and freezing substitution Whole developing anthers were removed from flower buds and immediately loaded into sample holders filled with 0.1 M sucrose. The samples were frozen in a Baltec HPM 010 high-pressure freezer (Technotrade, Manchester, NH, USA) and transferred into liquid nitrogen for storage. Substitution and sample embedding were performed as described in Otegui and Staehelin (2004). Thin sections were cut with a Reichert Jung Ultracut model E microtome (Reichert Jung, Vienna, Austria) and images were collected using a Philips CM120 scanning transmission electron microscope (Philips, Eindhoven, the Netherlands). Confocal laser scanning microscopy and fluorescence microscopy Live-cell imaging transgenic plants was performed on 3-day-old seedlings grown on 0.7% agarose (w/v) media containing 0.5 Murashige and Skoog salt mixture or young leaves isolated from soil-grown plants. Confocal imaging was performed with a Zeiss Axiovert 100 M inverted microscope equipped with Bio-Rad MR1024 laser scanning. For higher magnification, a high numerical aperture (1.4) oil immersion objective (60 ) was employed. A 488- nm or a 514-nm laser line from an argon ion laser was used to excite GFP and YFP, while the fluorescence emission was collected by a broad band-pass filter (480 550 nm). For neutral red staining, pollen grains were soaked in 0.1% neutral red for 30 min. To detect fluorescence, a 543-nm laser band was used for excitation, and emission was split by the use of a 500 570-nm filter to detect pollen cell wall autofluorescence (green channel) and a 590 640-nm filter to detect vacuole-specific, neutral red fluorescence (red channel) (Clarke et al., 2002). Cellular fractionation Soluble and membrane fractions were prepared from 4-week-old soil-grown Arabidopsis plants. Whole plants, not including root tissue, were ground with liquid nitrogen and suspended in modified MIB buffer [20 mm HEPES-KOH, ph 7.0; 50 mm potassium acetate; 1mMmagnesium acetate; 250 mm sorbitol; 1 mm DTT; 1 mm phenylmethylsulfonyl fluoride; 5 lg ml )1 protease inhibitor cocktail (Sigma catalog no. 9599)]. A post-nuclear supernatant designated as S1 was prepared by centrifugation at 1000 g for 10 min at 4 C. Microsomal membranes were prepared by centrifugation of the S1 at 150 000 g in a TLA100.3 rotor (Beckman Coulter, Fullerton, CA, USA) for 30 min at 4 C. The supernatant (S150) was transferred to a new tube, and the pellet (P150) was resuspended in the modified MIB buffer described above using a glass dounce homogenizer (Kontes Glass Co., Vineland, NJ, USA). Aliquots were made of each fraction, snap frozen in liquid nitrogen, and stored at )80 C. The protein content of each fraction was determined using the Bio-Rad DC Protein Assay Kit and BSA as a standard. To verify the success of protein fractionation, S1, S150 and P150 fractions were analyzed by immunoblotting using PUX1 (Rancour et al., 2004) and SYP31 antibody (Rancour et al., 2002). Before immunoblotting, the membrane was analyzed by PonceauS staining to confirm protein recovery and equal loading. Analysis of MAP3Ke1 subcellular localization To induce YFP MAP3Ke1 expression, plants were watered with 1% ethanol every 3 days. Leaf and inflorescence tissue from 4-week-old soil-grown wild-type plants, plants expressing 35S-GFP, and plants expressing ethanol-inducible YFP MAP3Ke1 was collected, and protein samples were fractionated as described above to generate S1, S150 and P150 samples. The protein concentration of each sample following fractionation was adjusted to approximately 1.0 mg ml )1. Fifty microliters of each adjusted sample was then loaded into FluoroNunc TM /LumiNunc TM 96-well polystyrene plates (Nunc, Rochester, NY, USA), and the fluorescence of each fraction was detected by the SpectraMax Gemini (EM) microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA). For reading YFP fluorescence, the excitation wavelength was 510 nm, the emission wavelength was 560 nm, and the auto cutoff was 550 nm. For GFP fluorescence, the excitation wavelength was 490 nm, the emission wavelength was 510 nm and the auto cutoff was 495 nm. All readings were performed in triplicate. Acknowledgements We thank Daphne Preuss for supplying quartet seeds, the Salk Institute for T-DNA mutant lines and Sebastian Bednarek for helpful discussions. This project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2003 35304 13265. References Alexander, M.P. (1969) Differential staining of aborted and nonaborted pollen. Stain. Technol. 44, 117 122.