The atapam2/+ Mutant is Defective in Mature Pollen Grains
We crossed the atapam2/+ plant with the quartet1 (qrt1) mutant to obtain the atapam2/+; qrt1/qrt1 plants for tetrad analysis. The qrt1 mutant caused the pollen grains not to separate from the tetrad, but had little effect on growth of the pollen tube . Therefore, the atapam2/+; qrt1/qrt1 plants produced a tetrad consisting of two qrt1 (representing wild type) pollen grains and two atapam2 pollen grains. Scanning electron microscopy (SEM) showed that all four pollen grains in the tetrad from qrt1 plants had normal morphology (Fig. 1a), yet the atapam2/+; qrt1/qrt1 plants had only two normal pollen grains (Fig. 1b). Alexander staining (Fig. 1c and 1d) and 4ʹ,6-diamidino–2-phenylindole (DAPI) staining showed the same results (Fig. 1e–1h). These results showed that the AtAPAM2 mutation affected male gametophytic function.
Because mature pollen grains from atapam2/+ were shrivelled and had no vitality, we were interested in how the mutation influenced development of pollen grains. The DAPI staining was used to investigate at which developmental stage pollen became abnormal (Fig. 2). Before the 12th anther development stage occurred, the developing nuclei of atapam2/+ pollen grains were of normal phenotype, as were wild-type pollen grains (Fig. 2a–2c and 2k–2m). However, when anthers developed to the 12th stage in which pollen grains had developed three nuclei, the pollen grains began to turn abnormal (Fig. 2d and 2n). The mutant grains were smaller than normal pollen grains in bright field image and their nuclei began to disappear under UV-light (Fig. 2d and 2n). By the 13th stage, the nuclei of mutant pollen grains had disappeared completely (Fig. 2e and 2o).
To further determine the results of DAPI staining, the different development stages of buds were embedded in resin. Then we obtained transverse sections of anthers from wild-type and atapam2/+ plants using the semi-thin section method (Fig. 3). The results showed that in the 11th stage of anther development or before, the appearance and size of pollen grains from mutants were the same as for wild type (Fig. 3a–3d). However, in the 12th stage of anther development, anthers were cracking and pollen grains were shed from anthers. Transverse sections of anthers showed that some pollen grains were of irregular appearance and were not as smooth as the wild type (Fig. 3e and 3f). Combining the results of DAPI staining with semi-thin sections, we conclude that pollen grains of atapam2/+ mutant began to turn abnormal at the 12th anther development stage.
Next, we used transmission electron microscopy (TEM) to observe differences in pollen wall structure between wild-type and atapam2 pollen (Fig. 4). Mature atapam2 pollen had characteristic wrinkled intine (Fig. 4c–4e), which was smooth in wild type and closely connected to the inner side of nexine (Fig. 4a and 4b). Mutant pollen also had deformed cytoplasm (Fig. 4c–4e).
At the 11th stage of anther development, pollen grains from atapam2/+ mutants were normal, with two sperm cell nuclei and a vegetative nucleus, suggesting that pollen grains had finished the second mitosis process. But at the 12th stage, pollen grains from atapam2/+ mutant began to turn abnormal. During this stage, the three nuclei pollen grains completed the process of dehydration and the mutation may have affected this process.
The Phenotype of atapam2/+ is Caused by a Ds Insertion in AtAPAM2
In a previous study, thermal asymmetric interlaced PCR was used to localise the mutation site in atapam2. Sequence analysis indicated that the Ds element was inserted in the 18th intron of At3g50590 . To confirm that the atapam2/+ mutant phenotype was caused by the Ds insertion in this gene, we subcloned the full-length genomic DNA fragment of At3g50590, including the promoter and 5ʹ and 3ʹ UTRs into the pCAMBIA1300 vector. Then, this construct was introduced into atapam2/+ mutant plants. Transgenic lines were screened by MS culture medium containing kanamycin and hygromycin. The self-pollinated T1 transgenic seeds were plated on MS culture medium containing kanamycin to determine the segregation ratio of Ds. The KanR:KanS segregation ratio was approximately 2:1 for most of the transformant lines (19 of 25). The complemented mutant plants produced offspring of atapam2/+ mutation homozygotes in T2 and subsequent generations. When pollen grains from the two independent complemented lines homozygous for the atapam2/+ mutation were observed by SEM, Alexander staining assay, and DAPI staining assay, the configuration of surface, vitality, and nuclei development were returned to the level of wild type (Fig. 5c, 5f, 5i, and 5l). At the same time, we calculated the abnormal rate of pollen grains from the two complemented lines, and found about 1.28% (8/624) and 1.42% (10/700) of pollen grains were abnormal, respectively (Fig. 5m). These results demonstrate that the defect in male gametophytic function in atapam2/+ plants was fully complemented by At3g50590 full-length genomic DNA.
To validate this result, atapam2–2 (SAIL_1288_C09) was obtained from the seed stocks of the Arabidopsis Biological Resource Center (ABRC). The atapam2–2 is a T-DNA insertion mutant of Columbia (Col) background. Using a T-DNA left-border primer Lba1 and a gene-specific primers (atapam2–2-F and atapam2–2-R) (Table 1) for PCR analysis, we confirmed that T-DNA was inserted in the 13th exon (Fig. 8a). The atapam2–2 mutant carried a BastaR-selective marker. The self-pollinated atapam2–2/+ progeny exhibited a BastaR:BastaS segregation ratio of approximately 1:1 (1590:1500) (Table 2) rather than 3:1. We also used SEM to observe the surface of pollen grains from atapam2–2/+, and found about 50% (134/271) of pollen grains were shrivelled (data not shown). These findings are consistent with previous results concerning atapam2/+ plants , and confirm that the defect in the male gametophyte in atapam2/+ was caused by mutation in At3g50590.
AtAPAM2 is Constitutively Expressed and Highly Expressed in Flowers and Pollen Grains
To understand the function of AtAPAM2, we used RT-PCR to assess its expression pattern. The RNA was isolated from various wild-type plant tissues including seedlings, roots, stems, leaves, inflorescences, and siliques. Then the RNA was reverse-transcribed to cDNA. The RT-PCR analyses showed that AtAPAM2 was ubiquitously expressed in many tissues, was constitutively expressed, and highly expressed in inflorescences (Fig. 6a).
To further study the expression pattern of AtAPAM2, the promoter fragment of AtAPAM2 was incorporated into GUS reporter gene and introduced into wild-type plants. In the T2 transgenic plants harbouring this fusion, GUS staining signals were detected in seedlings, roots, leaves, inflorescences, flowers, siliques, and pollen grains (Fig. 6b–6h). This further demonstrated that AtAPAM2 was expressed constitutively.
Subcellular Localisation of AtAPAM2 Protein
To further study the function of AtAPAM2 protein, the GFP–AtAPAM2 fusion protein under control of the 35S promoter (p35S:GFP–AtAPAM2) was constructed. This construct was transformed into leaves of Nicotiana benthamiana for transient expression and Arabidopsis wild-type plants for stable expression. Both in epidermal cells of N. benthamiana (Fig. 7a–7c) androots of Arabidopsis (Fig. 7d–7f), the GFP signals indicated AtAPAM2 localisation in plasma membrane and nucleus.
AtAPAM2 Encodes a Putative WD40-repeat Protein and is Evolutionarily Conserved
The AtAPAM2 mRNA encodes a putative WD40-repeat protein (Fig. 8b) of 1614 aa and contained three WD40-repeat domains (Fig. 8c). We used the entire aa sequence of AtAPAM2 to carry out a basic local alignment search tool (BLAST) search from NCBI. The results showed several proteins with >60% aa sequence similarity to AtAPAM2 in higher plants. Specifically, AtAPAM2 had 98% identity with EOA23389.1 of Capsella rubella, 96% with XP_002876038.1 of Arabidopsis lyrata subsp. lyrata, 77% with XP_002522312.1 of Ricinus communis, 76% with EOX91354.1 of Theobroma cacao, 76% with XP_004161728.1 of Cucumis sativus, 75% with XP_004232045.1 of Solanum lycopersicum, and 68% with EEC84558.1 of Oryza sativa (Fig. 8d). The above are WD40-repeat proteins, but of unknown function.
The AtAPAM2 has three WD40-repeats and in eukaryotes this domain usually functions as a protein–protein or protein–DNA/RNA interaction platform [1, 2]. In order to understand the function of this protein, we made use of the WD40 domain to carry out a yeast two-hybrid assay, with the aim of finding the protein that interacted with it; however, this was not successful.