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Proc Natl Acad Sci U S A. Sep 2, 2003; 100(18): 10558–10563.
Published online Aug 19, 2003. doi:  10.1073/pnas.1734110100
PMCID: PMC193600
Plant Biology

A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination

Abstract

Calmodulin (CaM), a ubiquitous multifunctional calcium sensor in all eukaryotes, mediates calcium action by regulating the activity/function of many unrelated proteins. Although calcium and CaM are known to play a crucial role in pollen germination and pollen tube growth, the proteins that mediate their action have not been identified. We isolated three closely related CaM-binding proteins (NPG1, NPGR1, and NPGR2) from Arabidopsis. NPG1 (No Pollen Germination1) is expressed only in pollen, whereas the NPG-related proteins (NPGR1 and NPGR2) are expressed in pollen and other tissues. The bacterially expressed NPG1 bound three isoforms of Arabidopsis CaM in a calcium-dependent manner. To analyze the function of NPG1, we performed a reverse genetics screen and isolated a mutant in which NPG1 is disrupted by a T-DNA insertion. Segregation and molecular analyses of the NPG1 knockout mutant and a cross with a male sterile mutant indicate that the mutated NPG1 is not transmitted through the male gametophyte. Expression of NPG1 in the knockout mutant complemented the mutant phenotype. Analysis of pollen development in the knockout mutant by light microscopy showed normal pollen development. Pollen from NPG1 mutant in the quartet background has confirmed that NPG1 is dispensable for pollen development. However, germination studies with pollen from the mutant in the quartet background indicate that pollen carrying a mutant allele does not germinate. Our genetic, histological, and pollen germination studies with the knockout mutant line indicate that NPG1 is not necessary for microsporogenesis and gametogenesis but is essential for pollen germination.

Sexual reproduction in flowering plants depends on pollination and fertilization. After landing on the stigma, pollen germinates and delivers sperm cells to the embryo sac by growing through the transmitting tissue in the style (1-4). The mechanisms controlling these processes are poorly understood (5, 6). Lipophilic molecules in the exine wall of pollen are believed to mediate pollen adhesion to stigma (4, 7). Components of the extracellular pollen coat such as tryphine have been shown to be important for pollen germination (8). Pollen lacking tryphine do not absorb water and fail to germinate in vivo (8). Flavanols, a specific class of flavanoids, also have a role in pollen germination of certain species (9, 10).

Numerous reports have implied a central role for calcium in pollen germination and pollen tube growth and guidance (2, 3, 11-15). Calcium is an essential constituent of in vitro pollen germination media, and a high calcium concentration has been reported near the germination aperture of hydrated pollen (16, 17). The requirement for calcium for pollen germination and tube growth has been shown by modulating its concentration by using chemicals that interfere with calcium transport (15, 17). Calcium measurement studies with fluorescent indicator dyes have demonstrated that cytosolic calcium forms a steep tip-focused gradient (18-21). The concentration of calcium reaches >3 μM at the tip and drops to <0.2 μM within 20 μm of the tip. The growth of the pollen tube is restricted to the tip of the tube, and the growth rate is positively correlated with changes in calcium at the tip (17, 21-23). Disruption of the calcium gradient at the tip results in inhibition of tube growth. Furthermore, the direction of pollen tube growth can be changed by manipulating the calcium gradient (20, 21). These studies indicate that the establishment of a precise tip-focused intracellular Ca2+ gradient and its oscillations are important for pollen tube elongation and directional growth. Recent studies have shown that Rho-related GTPases, also called ROPs, control pollen tube polar tip growth by modulating the generation of the tip-localized calcium gradient as well as the assembly of F-actin (23, 24). However, the mechanisms by which ROPs regulate these processes are not known.

Calmodulin (CaM), a universal calcium sensor in eukaryotes, is implicated in regulating diverse cellular process in plants (25-28), including pollen germination and tube growth (29-31), which are enhanced by exogenous application of CaM (32). Furthermore, CaM is localized to the germinal aperture and the cytoplasm near the germination bubble in hydrated pollen and in the cytosol of the pollen tube, where it forms a gradient similar to the tip-focused Ca2+ gradient (29). In another report, CaM showed uniform localization at the tip but formed a V-shaped collar behind the apical region, which dissipated on growth arrest (30). Despite numerous reports on the role of calcium and CaM in pollen germination and tube growth, the proteins that mediate calcium/CaM action have not been identified.

In this study, we cloned three closely related CaM-binding proteins (NPG1, NPGR1 and NPGR2) from Arabidopsis. NPG1 (NO POLLEN GERMINATION1) is expressed exclusively in pollen, whereas NPG-related genes (NPGR1 and -2) are expressed in pollen and several other tissues. The function of NPG1 was investigated by using an Arabidopsis mutant in which the gene is disrupted. Studies with the mutant show that NPG1 is not necessary for male meiosis and pollen development. However, NPG1 is essential for pollen germination, and the mutant allele is not transmitted through the male gametophyte.

Materials and Methods

Cloning of cDNAs. DNAse-treated RNA from pollen was used to synthesize first-strand cDNA with an oligo(dT) primer (33). Primers corresponding to 5′ and 3′ ends of predicted cDNAs were designed on the basis of genomic sequence in the TAIR database. The primers sets are 5′-ATGCTCGGGAATCAATCCGCGG-3′ (forward) and 5′-TTAAAGAATGGTTGAGAAGCTTTC-3′ (reverse) for NPG1 (At2g43040); 5′-GCAGAATTCATGTTGTGTGCTTGTTCAGGCG-3 (forward) and 5′-GCAGGATTCAAATGAAACTCTGTACCGGAG-3′ (reverse) for NPGR1 (At1g27460); and 5′-ATGAGGCATAGAGAAGAAGAAGACAAG-3′ (forward) and 5′-TCATCTGAATGGCTCCACTGG-3′ (reverse) for NPGR2 (gene At4g28600).

PCR was performed in a final volume of 50 μl by using the TaKaRa Ex TaqPCR (Takara Shuzo, Kyoto) system according to the manufacturer's instructions. The amplified products were resolved by electrophoresis, gel purified, cloned into pGEM-T Easy vector, and sequenced.

Bacterial Expression and Detection of NPG1. A 2.0-kb EcoRI fragment of NPG1 cDNA containing the entire coding region was subcloned into pET28a, and the orientation of the cDNA was confirmed by restriction analysis and sequencing. Induction of fusion protein, its purification on CaM Sepharose column, and detection with horseradish peroxidase CaM isoforms were performed essentially as described (34).

Expression Analysis of NPG1. Arabidopsis pollen was collected according to Huang et al. (35). RNA from pollen and tissues was isolated by using the TRIzol reagent according to the protocol provided by GIBCO/BRL with the following modifications for pollen RNA extraction. Five hundred microliters of TRIzol reagent plus 300 μl of glass beads (710-1,180 μM) were added to 200 μl of pollen and vortexed continuously at a high speed for 15 min, then another 500 μl of TRIzol reagent was added. RNA pellets were dissolved in either deionized formamide (for RNA gel blots) or RNAse-free water (for RT-PCR). Fifty micrograms of total RNA from each tissue was electrophoresed, blotted, hybridized with 32P-labeled cDNA, and washed under high-stringency conditions. Expression analysis of NPG1, NPGR1, and NPGR2 was performed by RT-PCR by using the first-strand cDNA prepared from DNAse-treated RNA as described.

Screening of T-DNA Insertion Lines for NPG1 Mutant. We screened 60,400 T-DNA-tagged Arabidopsis mutants generated at the University of Wisconsin Arabidopsis knockout facility (36) by using an NPG1-specific forward (5′-ACCAAAGAGGGAATTTAGAAGGCGCACTT-3′) or reverse (5′-TACGTAACTCCTAGCTAGGTTGTTTGGCT-3′) primer together with TDNA left or right border primer. Screening of superpools and pools was performed at the Arabidopsis knockout facility. Southern analysis of PCR-amplified products identified a T-DNA insertion in NPG1 with one primer combination (T-DNA left border and NPG1 reverse primers). We then reamplified the hybridizing band and sequenced the product to confirm that the insertion was in the coding region of NPG1. Final screening and identification of the NPG1 knockout line were performed at Colorado State University. We refer to NPG1/npg1 heterozygous plant as a “mutant” because of its pollen phenotype (no pollen germination).

Genetic Crosses. To determine whether the mutated gene is transmitted through pollen, we pollinated a male sterile (cer6-2) mutant with pollen from the NPG1/npg1 plant. Seeds from this cross were germinated on plates with or without kanamycin.

Tetrad analysis was performed by generating a double mutant. The pollen from qrt/qrt mutant (quartet1) (37) was used to pollinate emasculated flowers of the NPG1/npg1 plant. We selected the progeny on kanamycin plates for NPG1/npg1, QRT/qrt, and selfed. Seeds from selfing were grown on kanamycin plates, and plants that showed qrt phenotype (NPG1/npg1, qrt/qrt, one of every four kanamycin-resistant plants) were selected.

Microprojectile Bombardment of Pollen. The helium-driven PDS-1,000/He particle delivery system (Bio-Rad) was used for the biolistic transformation of midbicellular grains of tobacco pollen (Nicotiana tabaccum). Tobacco pollen was resuspended to 1 × 106 cells in 50-100 μl of germination medium (0.1 mg/ml H3BO3/0.71 mg/ml Ca(NO3)2 4H2O/0.2 mg/ml MgSO4 7H2O/0.1 mg/ml KNO3/10% sucrose/0.5 mg/ml Mes/1.0 mg/ml casein hydrolysate, pH 5.9) and placed in the middle of a 5-cm Petri dish, previously rinsed with the same buffer, to create an even monolayer of cells. Each pollen monolayer was bombarded at least three times, as described (38), and 5 ml of germination medium was added. The plates were then left in the dark without shaking for 12-24 h in a humid chamber at 21 ± 3°C. GFP-expressing pollen were initially scored on the plates and later transferred onto a glass slide for confocal microscopy.

Pollen Germination. In vitro germination of Arabidopsis pollen was performed in acid-washed depression slides in liquid medium as described (39). About 60-70% of pollen germinated in this medium. Pollen from qrt/qrt or NPG1/npg1, qrt/qrt flowers [stage 12 or 13 (40)] was carefully tapped onto the slides, and the medium was added. Slides were then incubated in a humid chamber at 21 ± 3°C without light for 10-24 h.

Generation and Analysis of Transgenic Plants. The following constructs were used for transient and/or stable expression. A 2.4-kb fragment (NP, NPG1 Promoter) upstream of the translation initiation codon of the NPG1 gene was amplified with forward (5′-CATGCCATGGTATGAGTCGAGTGTCTGACTT-3′) and reverse 5′-TCGCCATGGTTCTTCACCTTTTAGACTA-3′) primers carrying an NcoI site (underlined). The PCR fragment was cloned into pGEM-TE vector, sequenced, and used to replace the 35S promoter region (NcoI-NcoI) in a pBA002-GFP binary vector kindly provided by Nam-Hai Chua (The Rockefeller University, New York) to generate NP<GFP>Nos. For complementation of the NPG1 mutant, another construct was made by replacing the GFP fragment (NcoI-SmaI) with a full-length blunted SpeI-NcoI fragment of NPG1 cDNA in a pBA002-based construct NP<GFP>Nos to create NP<NPG1>Nos. The constructs were verified by sequencing, introduced into Agrobacterium GV3101 strain, and used to transform Arabidopsis plants by using the floral-dip method. Wild-type plants were transformed with NP<GFP>Nos, and NPG1/npg1 plants were transformed with NP<NPG1>Nos. Seeds from infiltrated plants were collected, surface-sterilized, stratified, and selected on appropriate selection plates.

Histology, Light Microscopy, and Confocal Laser Scanning Microscopy. Inflorescences were harvested, dehydrated in a standard ethanol series, fixed (formamide and glutaraldehyde solutions), and embedded in Paraplast tissue medium (41). Samples were sectioned and mounted on polyl-lysine-coated glass slides and then stained. Tissue sections were observed with a Leitz Laborlux S microscope. Images were captured by using Kodak Digital Science DC-120 zoom camera. For nucleus staining, 1 μl of 4′, 6-diamidino-2 phenylindole (DAPI) solution (1 mg/ml) was added to 1-2 ml of mounting buffer (0.1 M Tris·HCl, pH 9.0/50% glycerol), and the pollen was examined in UV light after 10-30 min. GFP images were captured by using a fluorescence microscope with an integrated confocal imaging (FVXIHRT Fluoview Confocal LSM) system from Olympus (Melville, NY). Images were acquired by using fluoview software provided by the manufacturer. Scanning electron microscopy was performed essentially as described (8). All images were processed with photoshop 5.0 software (Adobe Systems, Mountain View, CA).

Results and Discussion

Isolation of NPG1 and NPG-Related cDNAs from Arabidopsis Pollen. To investigate the role of CaM-binding proteins in pollen germination and tube growth, we identified three genes (At2g43040, At1g27460, and At4g28600) in the recently completed Arabidopsis genome sequence that are closely related to a gene encoding pollen-specific maize CaM-binding protein (34). We cloned the corresponding cDNAs from Arabidopsis pollen RNA by RT-PCR. An expected size fragment (≈2.3 kb) for each gene was obtained and verified by sequencing. Amplification of RNA itself did not yield any products, and amplification from genomic DNA with the corresponding primers yielded a larger-size product due to introns. Comparison of the At2g43040 (named NPG1 for no pollen germination; see below) cDNA sequence with the genomic sequence has revealed the presence of four introns in the coding region of the NPG1 gene (Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org). NPG1 encodes a protein of 704 aa (Fig. 6). The deduced amino acid sequence of NPG1 (GenBank accession no. AF474176) differs from the predicted protein in the Arabidopsis genome database at the C terminus due to inaccurate prediction of the last two introns. The predicted amino acid sequences of the two NPG-related proteins (NPGR1 coded by At1g27460 and NPGR2 coded by At4g28600) are the same as their predicted sequences in the database. NPG1, NPGR1, and NPGR2 share significant sequence similarity among themselves and with maize pollen-specific CaM-binding protein (39-56% identity and 56-70% similarity) (Fig. 7, which is published as supporting information on the PNAS web site). The NPG1 amino acid sequence showed 56% identity and 70% similarity to the maize CaM-binding protein. Analysis of the predicted amino acid sequence of Arabidopsis NPG1 and the related proteins using smart (http://smart.embl-heidelberg.de) revealed the presence of six tetratricopeptide repeats (TPRs) (Figs. 6 and 7). TPR domains consist of a degenerate 34-aa region and are implicated in protein-protein interaction (42, 43). The second TPR overlaps with the CaM-binding domain (CBD) (Fig. 6; see below). A homolog of NPG1 or NPGRs has not been found in any nonplant system, including the yeast, fly (Drosophila melanogaster), worm (Caenorhabditis elegans), and human (Homo sapiens) genomes, whose sequences have been completed, suggesting these represent a small family of plant-specific CaM-binding proteins.

NPG1 and NPGRs Are CaM-Binding Proteins. To demonstrate that NPG1 is a CaM-binding protein, we expressed the coding region of NPG1 as a T7-tag fusion by using an E. coli expression system and used the bacterially expressed protein in CaM-binding studies. The bacterially expressed NPG1 bound CaM-Sepharose beads in a calcium-dependent manner and eluted with EGTA-containing buffer (data not shown). The purified NPG1 bound three Arabidopsis CaM isoforms in a calcium-dependent manner in blot overlay assays (Fig. 1). These results confirm that NPG1 is a CaM-binding protein. The interaction of NPG1 with CaM only in the presence of calcium suggests that the function of NPG1 is modulated in vivo by calcium and CaM. To map the CBD, we expressed a truncated version (amino acids 448-496) of NPG1, which contains a putative CBD, in E. coli and tested for its ability to bind CaM. The truncated protein bound CaM in a manner similar to the full-length protein, indicating that the CBD is located between amino acids 448 and 496 (M.G., S.-B. Shin, and A.S.N.R., unpublished work). This region is highly conserved among these three proteins (see Fig. 7A). The proteins coded by the full-length cDNAs of NPGR1 and NPGR2 also bind CaM in a calcium-dependent manner (M.G., S.-B. Shin, and A.S.N.R., data not shown).

Fig. 1.
NPG1 is a CaM-binding protein. Binding of bacterially expressed and purified NPG1 (Coomassie) to horseradish peroxidase-labeled Arabidopsis CaM isoform-2 (CaM-2), -4 (CaM-4), and -6 (CaM-6) in the absence (-Ca2+) or presence of calcium (+Ca2+) is ...

NPG1 Is Expressed Specifically in Pollen. To determine the expression of NPG1, RNA from different tissues/organs was analyzed by Northern analysis. As shown in Fig. 2A, NPG1 transcripts were detected only in pollen. The transcript size (≈2.5 kb) is consistent with the expected size. Equal amounts of RNA on the blot were verified by hybridizing with ubiquitin (Fig. 2 A Lower). To eliminate the possibility that NPG1 may be expressed at very low levels, we performed RT-PCR analysis. The results from the RNA blot analysis were confirmed by RT-PCR, where the NPG1 transcript was detected only in pollen (Fig. 2B). The amplified transcript was verified by hybridization with NPG1 (data not shown). We have further confirmed pollen-specific expression of NPG1 by fusing its promoter to GFP and analyzing the promoter activity. In transient expression assays with tobacco pollen, a very strong expression of GFP was found in pollen tubes (Fig. 2C). In transgenic Arabidopsis plants, GFP was seen in mature and germinating pollen (Fig. 2D). The earliest expression of GFP was detectable in pollen at stages 12-13 of flower development (sepals open, petals can be seen between the sepals; data not shown) (40). During the later stages of pollen germination, the expression of GFP is mostly confined to the tube (Fig. 2E), GFP was not detected in any other tissues except pollen. To determine the expression of NPGRs, we performed RT-PCR analysis by using the first-strand cDNA from different tissues and gene-specific 5′ and 3′ primers for each gene. The amplified products were hybridized with corresponding cDNA to verify that the amplified products are real. Unlike NPG1, NPGR1 and NPGR2 are expressed in suspension culture cells, pollen, flowers, and fruits (Fig. 8, which is published as supporting information on the PNAS web site). In addition, NPGR2 is expressed in leaves also (Fig. 8). In the case of NPGRs, additional smaller bands were detected, which may have been the products of alternative splicing (data not shown). Together, these results clearly show that NPG1 is expressed only in pollen and pollen tubes.

Fig. 2.
NPG1 is expressed only in pollen. (A) Northern blot analysis. The blot was first hybridized with NPG1 cDNA (Upper) and then with Arabidopsis ubiquitin (Ubq) (Lower). (B) Amplification of NPG1 transcript in pollen and tissues by RT-PCR (Upper). Amplified ...

Isolation of an NPG1 Mutant. Because NPG1 is expressed exclusively in pollen, it is likely to function in one or more of the following processes: pollen development, germination, and/or pollen tube growth. To analyze the function of NPG1, we isolated a knockout mutant by screening a mutant library (60,400 independent kanamycin-resistant lines), which was generated by T-DNA insertion, by using a reverse genetics screen (36, 44). A plant with a T-DNA insertion in NPG1 was identified by PCR amplification of a 0.8-kb product by a reverse primer of NPG1 and T-DNA left border primer (Fig. 3 A and B). Sequence analysis of the PCR product amplified from the insertion allele revealed that the T-DNA interrupted the coding region (1,566 bp) after 461 aa in exon 3 (Fig. 3A). Any protein product from this allele would lack the CBD and five putative TPRs (Fig. 3A) known to be involved in protein-protein interaction. Amplification of both the wild-type and mutated genes from the plant suggested that it was a heterozygous plant (Fig. 3B). To confirm that the mutant plant is heterozygous and to determine the number of T-DNA insertions, we isolated DNA from wild type and mutant and digested with a restriction enzyme (BamHI), which does not cut NPG1 but has a single site in T-DNA. Therefore, it is expected that a Southern analysis probed with NPG1 should produce a single hybridizing band in wild type and three bands in the heterozygote. Furthermore, if there is a single insertion, then the Southern analysis with DNA from the mutant probed with the T-DNA should produce two hybridizing bands. As shown in Fig. 3C, Southern analysis with NPG1 and T-DNA probes confirmed that the plant was heterozygous with a single T-DNA insertion in the mutant allele (Fig. 3C).

Fig. 3.
NPG1 mutant characterization and complementation. (A) Schematic diagram of NPG1 gene showing the site of T-DNA insertion positioned at 1,566 bp. Numbered boxes represent exons. Coding region upstream of T-DNA is shown in green. The position of the ...

The Mutant Allele Is Not Transmitted Through Male Gametophyte. Heterozygous mutant plants were selfed to obtain homozygous plants. Analysis of the progeny from this cross revealed two significant findings. First, the ratio of kanamycin-resistant to kanamycin-sensitive plants was 1:1 (476:444) instead of the expected 3:1. Second, Southern and PCR analyses of the kanamycin-resistant progeny with primers that amplify the wild type and mutant allele showed that a subset of randomly selected kanamycin-resistant plants (50 plants) were heterozygous (data not shown). A distorted segregation ratio and an inability to produce homozygous plants, together with pollen-specific expression of NPG1, suggested that the mutated gene is not transmitted through the male gametophyte.

To further confirm that the npg1 allele is not transmitted through pollen, we used a convenient male sterile mutant (cer6-2) (8) to cross with an NPG1/npg1 plant (Fig. 3D). As expected, we did not obtain any kanamycin-resistant progeny from this cross (Fig. 3D). However, pollination of emasculated NPG1/npg1 flowers with pollen from wild-type plants produced the expected 1:1 kanamycin-resistant/sensitive progeny (data not shown) demonstrating that transmission of the npg1 allele through female gametes is not affected. These results confirm that the mutant allele is not transmitted through pollen.

Complementation of Mutant with NPG1 cDNA. To demonstrate that the mutant phenotype is caused by a disruption of the NPG1 gene, NPG1/npg1 plants were transformed with an NPG1 cDNA driven by the NPG1 promoter in a vector that confers resistance to BASTA. Plants resistant to both kanamycin and BASTA were selected. Pollen from 48 kanamycin- and BASTA-resistant plants was used to pollinate a male sterile (cer6-2) mutant to test transmission of the npg1 allele. From this cross, we obtained progeny resistant to both selective agents from 37 of 48 total crosses (an example is shown in Fig. 3E). The reason for not seeing complementation in some lines is most likely the variations in the expression of introduced NPG1 gene. The presence of introduced cDNA in the kanamycin- and BASTA-resistant progeny was verified by amplifying the cDNA from genomic DNA (data not shown). These results provide evidence that expression of the introduced NPG1 cDNA complements the mutant NPG1 allele and that the mutant phenotype is indeed due to the T-DNA insertion in the NPG1 gene.

Pollen Development Is Normal in the Mutant. Because the npg1 allele is not transmitted through pollen, we envisioned two possible scenarios by which the mutated allele (npg1) is not transmitted through pollen. One is that haploid cells that inherit the mutant allele after meiosis (half of the haploid cells) do not develop into pollen. Alternatively, the haploid cells carrying npg1 could develop normally but not be functional due to a defect in later events of pollen germination and/or tube growth.

To address the first possibility, we analyzed the pollen development in wild-type and NPG1/npg1 plants by light microscopy. We found that in the mutant normal tetrads are formed after meiosis, and the microspores developed into morphologically normal pollen (Fig. 4 A and B) (45). Pollen from NPG1/npg1 plants was indistinguishable from wild type in shape, size, and DAPI staining, which showed all three nuclei (a single diffusely staining vegetative nucleus and two condensed sperm nuclei per pollen) (Fig. 4C). These results indicate that pollen development is normal in NPG1/npg1 plants. To further confirm these results, we used the quartet1 (qrt/qrt) mutant of Arabidopsis. In the qrt1 mutant, the four microspores/pollen grains produced after male meiosis remain attached as tetrads due to fusion of the outer walls of the four meiotic products of the pollen mother cell (37). Therefore, any abnormalities in pollen development can be determined by analyzing the tetrads (46). We generated NPG1/npg1, qrt/qrt as described in Materials and Methods. On the basis of our earlier microscopic observations, we expect to see normal development of all four pollen grains, including those that carry the npg1 allele in the double mutant. The tetrads in the double mutant are normal in appearance and indistinguishable from tetrads of the qrt mutant (Fig. 5 A and B). Furthermore, DAPI staining revealed the presence of the vegetative and two sperm nuclei in each pollen grain in the tetrad (Fig. 5A). Scanning electron micrographs of tetrads from the double mutant show that all four pollen grains in a tetrad are indistinguishable in their morphology (Fig. 5B). These studies clearly indicate that NPG1 is not essential for male meiosis and pollen development.

Fig. 4.
Pollen development in NPG1 mutant. (A) Sections of Arabidopsis flower [stages 7-8 according to Sanders et al. (45)] from NPG1 mutant (Top), magnified view of the anther (Middle), and tetrad (Bottom). (B) Pollen development through stages 9 (Top), ...
Fig. 5.
Pollen development and germination in NPG/npg, qrt/qrt plant. (A) DAPI staining of a tetrad from an NPG1 mutant (NPG1/npg1) in the qrt background. White arrows point to the vegetative nucleus and two sperm nuclei in each pollen grain of a tetrad. ...

Pollen Carrying the Mutant Allele Does Not Germinate. To address the role of NPG1 in pollen germination and/or tube growth, we used the tetrads from NPG1/npg1, qrt/qrt, and qrt/qrt flowers (40) for in vitro germination (39). If pollen germination is affected in the double mutant, we expect to see germination of no more than two of the four pollen grains in a tetrad, whereas up to four pollen grains can germinate from qrt/qrt plants. The germination of pollen in tetrads from the qrt/qrt mutant ranged from one to four, whereas only one or two pollen grains germinated from tetrads from the double mutant (Fig. 5C). One of five germinating tetrads from qrt/qrt plants showed germination of three or four pollen grains. In contrast, all germinating tetrads from the double mutant showed germination of only one or two pollen grains (Fig. 5C). These results, together with our inability to obtain homozygous mutant and kanamycin-resistant plants from a cross between a male sterile mutant (cer6-2) and an NPG1/npg1 mutant, strongly suggest that pollen carrying the npg1 allele does not germinate, and NPG1 is a regulator of pollen germination.

Many sporophytic mutants that affect microsporogenesis have been isolated by screening for male sterility (8, 45). In contrast, isolation of gametophytic mutants affecting pollen development and/or function has been difficult for obvious reasons. Such mutants as heterozygotes produce 50% normal pollen and thus show no easily observable affect on fertility. Further, it would not be possible to obtain homozygous mutant plants. So far, a few male gametophytic mutants that affect pollen development and/or function have been identified by screening T-DNA insertion lines for distorted segregation ratios of antibiotic resistance or by screening mutant populations for abnormal pollen (47-50). To our knowledge, npg1 is the only gametophytic mutant for which the gene is cloned and the encoded protein is known to be required for pollen germination.

Although numerous reports suggest a critical role for calcium in pollen germination and pollen tube growth, nothing is known about the CaM target proteins and their function in pollen (15, 21, 29-31). Here, our data clearly indicate that NPG1, a calcium-dependent CaM-binding protein, is required for pollen germination and is likely to be a key protein in the calcium signaling pathway in mediating calcium changes during pollen germination and tip growth. At present, no other male gametophytic gene necessary for pollen germination has been identified, although a sporophytic mutation that affects pollen hydration and in vivo germination due to a deficiency in lipoidic tryphine has been reported (8). Because the NPG1 promoter was found to be active in growing pollen tubes as well as in mature pollen (Fig. 2 C-E), it is possible that NPG1 is required for pollen tube growth as well as germination. Our mutant does not permit the analysis of its role in pollen tube growth, because pollen germination is arrested, but blocking the expression of NPG1 in growing pollen tubes by using antisense oligonucleotides should help reveal the role of NPG1 in pollen tube growth. In the NPG1, there are six TPR motifs in addition to the CaM-binding domain (Fig. 6). TPR-containing proteins function through protein-protein interaction and modulate diverse cellular processes (42, 51, 52). Mutations in the TPRs have been shown to result in loss of function (42, 52). Hence, it is likely that NPG1 interacts with other proteins. Further studies on identification of proteins that interact with NPG1 should provide insights into its mode of action in controlling pollen germination.

Supplementary Material

Supporting Figures:

Acknowledgments

We thank Dr. Farida Safadi Chamberlain and Sung-Bong Shin for help with this project; Drs. Pat Bedinger, Ron Parsons, and Irene Day for helpful comments on the manuscript; Dr. Nam-Hai Chua for providing the GFP vectors; Drs. Alisher Touraev and Julia Barinova for help with the transient assay in tobacco pollen; Dr. Kurt Beam for the use of the Biolistic gun; Dr. Olga Kosaya for sectioning the plant material; the Arabidopsis Knockout Facility for the initial screening of T-DNA insertion library; and the Arabidopsis Biological Resource Center (Columbus, OH) for providing seeds of cer6-2 and quartet mutants. This work was supported by a grant from the National Science Foundation (to A.S.N.R.).

Notes

Abbreviations: CaM, calmodulin; CBD, CaM-binding domain; NPG, no pollen germination; TPR, tetratricopeptide repeat; DAPI, 4′,6-diamidino-2 phenylindole.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF474176).

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