- © 2012 American Society of Plant Biologists. All rights reserved.
Abstract
Primexine deposition and plasma membrane undulation are the initial steps of pollen wall formation. However, little is known about the genes involved in this important biological process. Here, we report a novel gene, NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU), which functions in the early stage of pollen wall development in Arabidopsis (Arabidopsis thaliana). Loss of NPU function causes male sterility due to a defect in callose synthesis and sporopollenin deposition, resulting in disrupted pollen in npu mutants. Transmission electronic microscopy observation demonstrated that primexine deposition and plasma membrane undulation are completely absent in the npu mutants. NPU encodes a membrane protein with two transmembrane domains and one intracellular domain. In situ hybridization analysis revealed that NPU is strongly expressed in microspores and the tapetum during the tetrad stage. All these results together indicate that NPU plays a vital role in primexine deposition and plasma membrane undulation during early pollen wall development.
The pollen wall plays a pivotal role in pollen-stigma recognition, pollen hydration, and pollen protection on the stigma and pollen tube elongation (Zinkl et al., 1999; Edlund et al., 2004; Scott et al., 2004). The pollen wall is organized in a highly complex manner, with three major layers: exine, intine, and tryphine (Heslop-Harrison, 1971; Piffanelli et al., 1998). The exine is the outer layer of the pollen wall and includes the sexine (tectum and bacula) and nexine (foot layer). Located between the exine layer and plasma membrane, the intine is mainly comprised of cellulose, pectin, and proteins (Brett and Waldron, 1990). Tryphine is secreted from the tapetal cells and deposited onto the exine layer during the later stages of pollen development.
Pollen wall development is a complicated process in which a set of elaborate and coordinated mechanisms are carried out by both the microspores and tapetum (Blackmore et al., 2007). It is initiated along with the termination of meiosis (Paxson-Sowders et al., 1997; Piffanelli et al., 1998). Close to the end of meiosis, the microspores are enclosed by the callose wall, which serves as a mold for primexine patterning and also protects the meiocytes and tetrads (Waterkeyn and Bienfait, 1970; Worrall et al., 1992; Dong et al., 2005). At the tetrad stage, the primexine is deposited between the plasma membrane and the inner callose wall (Fitzgerald and Knox, 1995; Paxson-Sowders et al., 1997), and the plasma membrane starts to invaginate and form the undulations that are common to various species (Dahl, 1986; Dickinson and Sheldon, 1986; Takahashi, 1989; Fitzgerald and Knox, 1995). On the peaks of the undulations, probaculae are deposited in a well-regulated fashion and eventually form the mature pollen exine layer (Paxson-Sowders et al., 1997). Therefore, the primexine deposition and timely undulation of the plasma membrane both play important roles in early pollen wall formation (Rowley and Skvarla 1975; Takahashi and Skvarla, 1991; Fitzgerald and Knox, 1995).
At present, a number of mutants have been identified that are involved in primexine formation and microspore membrane undulation in Arabidopsis (Arabidopsis thaliana). In the no exine formation1(nef1) mutant, the primexine is coarsely developed, and some parts of the plasma membrane of the microspore are disrupted at the later stages. The NEF1 protein is predicted to be a plastid integral membrane protein (Ariizumi et al., 2004). In the mutant defective in exine formation1 (dex1) the primexine deposition is delayed and reduced in thickness, and the undulation of the microspore plasma membrane is scant (Paxson-Sowders et al., 1997, 2001). As a result, the exine pattern is not formed properly, and the pollen wall does not form. The DEX1 protein is predicted to be a membrane-associated protein that has at least two calcium-binding ligands (Paxson-Sowders et al., 2001). In the ruptured pollen grain1 (rpg1) mutant, the primexine is irregularly deposited, and as a result only a spotted, irregular exine layer forms. The microspores are ruptured, with cytoplasmic leakage. The RPG1 protein is a membrane protein of the MtN3/saliva family that functions as a sugar transporter (Guan et al., 2008; Chen et al., 2010). These reported proteins are helpful in understanding the importance of primexine deposition and plasma membrane undulation on a molecular basis.
In this article, we report on the gene NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU), which encodes a transmembrane protein in Arabidopsis. The callose synthesis is severely disrupted, and the absence of primexine deposition and plasma membrane undulation leads to the defects of microsporogenesis and male sterility in the npu mutants. Functional analysis of NPU indicates that it plays an important role in primexine deposition and plasma membrane undulation during pollen wall formation in Arabidopsis.
RESULTS
The npu-1 Mutant Exhibits a Male Sterility Phenotype
The npu-1 mutant was isolated from a pool of T-DNA-tagged lines (Li et al., 2005). The mutant plants exhibited reduced fertility with normal vegetative growth (Fig. 1A). The average seed yield of the mutant plants was approximately 0.43% of the wild type. Backcrossing with wild-type pollen grains resulted in F1 plants with normal fertility. This result indicated that male fertility was hampered and female fertility was unaffected in the npu-1 mutant. The progeny of the self-pollinated heterozygous plants segregated wild-type and mutant phenotypes at a 3:1 (215:74) ratio, which indicates that the phenotype of npu-1 is controlled by a single recessive locus.
Characterization of the npu mutants. A, The wild type (Columbia [Col] and Landsberg erecta [Ler]), the npu allele mutant, and the NPU-complemented plants. B, Alexander staining of the wild-type and npu anthers. SEM examination of dehiscent anthers and pollen grains of wild-type (C and E) and npu-3 (D and F) plants. C and D, Dehiscent anthers. The wild-type anther contains numerous pollen grains, while the npu-3 anther is filled with degenerated microspores. Bars = 100 μm. E, Wild-type pollen grains with a regular reticulate exine pattern. Bar = 5 μm. F, Ruptured npu-3 microspores of stages 9 and 10 with defective exine pattern formation. Bar = 2 μm.
Molecular Cloning of the NPU Gene
To identify the corresponding gene of the npu-1 mutant, we performed thermal asymmetric interlaced (TAIL)-PCR (Liu et al., 1995). A genomic DNA fragment that flanked the left border of T-DNA was obtained. Sequencing of the TAIL-PCR products indicated that the T-DNA was inserted into the seventh exon of a predicted open reading frame of At3g51610 (Fig. 2A). PCR analysis using T-DNA and genome-specific primers showed that all of the mutant plants analyzed (n > 80) were homozygous for the insertion (data not shown). Therefore, the phenotype of the npu-1 mutants is evidently linked with the T-DNA insertion. To ensure that NPU is in fact At3g51610, a complementation experiment was conducted. A 4.7-kb DNA fragment containing the genomic sequence of NPU, a sequence 1.5-kb upstream from the translation initiation codon and 800 bp downstream from the stop codon, was cloned from wild-type Arabidopsis and introduced into the homozygous mutant plants. A total of 40 transgenic plants were obtained and all of them exhibited normal fertility (Fig. 1A). This demonstrated that At3g51610 is the NPU gene, and the 4.7-kb genomic DNA fragment contains sufficient information for normal NPU function.
Characterization of the NPU gene. A, Gene structure of NPU and the T-DNA insertion. Black boxes, exons; gray lines, introns; light-gray lines, untranslated regions. npu-1, npu-2, and npu-3 are allele mutant lines. Left (LB) and right (RB) borders of T-DNA sequences are shown. DNF and DNR, The left border and right border of the DNA fragment. B, Predicted protein structure of NPU. Gray box, plasma membrane; black bar, transmembrane regions. C. Subcellular localization of 35S:GFP inflorescence in transgenic wild-type protoplast. Green fluorescence indicates the localization of 35S:GFP protein. Bar = 20 μm. D, Subcellular localization of NPU:GFP inflorescence in transgenic wild-type protoplast. Green fluorescence indicates the localization of NPU:GFP protein; red fluorescence is the autofluorescence of chloroplasts. Bar = 20 μm.
We also obtained two additional npu mutant alleles. The T-DNA-tagged line SALK-062174 (npu-2) was obtained from the SIGnAL collection at the Arabidopsis Biological Resource Center (Fig. 1A). PCR analysis confirmed the T-DNA insertion into the second exon of NPU (Fig. 2A). The npu-3 mutant was screened from an ethyl methanesulfonate-induced population in our laboratory. Sequence analysis revealed a point mutation from G to A, in 246th base after the start codon of the NPU genomic sequence in the mutant. This transition causes a premature termination at the second exon of At3g51610 (Fig. 2A). The phenotypes of both npu-2 and npu-3 were similar to npu-1 (Fig. 1A), except that they are completely male sterile.
Microsporogenesis of the npu Mutant Is Disrupted after Meiosis
To analyze the male fertility defects of the npu mutant, Alexander staining (Alexander, 1969) was performed. As shown in Figure 1B, wild-type pollen grains were stained purple. In the locules of the npu-1 mutant, there were a few purple-stained pollen grains. However, no normal pollen was observed in the locules of either the npu-2 or npu-3 mutant. These observations are in agreement with their fertility patterns, in that npu-1 is partially sterile, and npu-2 and npu-3 are completely sterile.
We then performed anther cross sections to compare anther development in the wild-type and npu-3 plants (Fig. 3, A–L). According to the 14 well-ordered anther development stages in Arabidopsis reported by Sanders et al. (1999), the anther development in npu-3 was similar to that of the wild type from stage 1 to stage 6. However, at stage 7, the tetrads of npu-3 seemed to be slightly different from that of wild type. The microspores were more closely wrapped inside the tetrad of npu-3 mutant than that of wild type (Fig. 3, A and B). At stage 8, the wild-type microspores were angular in shape, while the mutant microspores were round in appearance (Fig. 3, C and D). At stage 9, the wild-type microspores became vacuolated and developed the basis of a regular exine wall (Fig. 3E), while the npu-3 microspores began to degenerate (Fig. 3F). During stages 10 and 11, most microspores in npu-3 degenerated (Fig. 3, H and J), while the wild-type locules were filled with well-shaped microspores (Fig. 3, G and I). At stage 12, the microspores of the wild type were released from the locules following anther dehiscence (Fig. 3K). However, the microspores in npu-3 were completely aborted (Fig. 3L). These results showed that the microspore development started to be abnormal from stage 7. To further examine the defect of microspore development at stage 7, aniline blue was used to stain callose. The result showed that the callose wall between microspores in the tetrads of npu-3 was much thinner than that of the wild type (Fig. 3M). It was reported that CalS5 was related with callose synthesis, and A6 was proposed to be related with callose dissolution in Arabidopsis (Hird et al., 1993; Dong et al., 2005). The expression of CalS5 was largely down-regulated in npu-3, while the expression level of A6 was similar to that of the wild type (Fig. 3, N and O). This demonstrated that NPU affected the callose synthesis in anther development.
Anther development, callose wall, and expression analyses of A6 and CalS5 in wild-type and npu-3 mutant plants. A and B, Anthers of tetrad stage. npu-3 tetrads (A) seemed to be closely compacted compared with those of the wild type (B). C and D, Anthers at stage 8. npu-3 microspores (C) are round compared with wild-type microspores (D). E and F, Anthers at stage 9. npu-3 microspores began to be degenerated (F). G and H, Anthers at stage 10. Cytoplasm of npu-3 microspores was disintegrated (H). I and J, Anthers at stage 11. Most npu-3 microspores were degenerated (J). K and L, Anthers at stage 12. Remnants of microspores were observed in npu-3 anther locule (L). M, Callose wall of npu-3 mutant was thinner around the microspores compared with that of the wild type. N, Semiquantitative RT-PCR analyses of A6 and CalS5 in wild-type and npu-3 floral buds. TUB, TUBULIN expression as a control. O, Real-time PCR analysis of CalS5 in wild-type and npu-3 floral buds. E, epidermis; En, endothecium; MSp, microspore; PG, pollen grain; RM, remnants of microspores; T, tapetum; Tds, tetrads. Bars = 10 μm.
Primexine Deposition or Microspore Plasma Membrane Undulation Was Not Observed in npu-3
To further elucidate the cause of pollen degeneration in npu plants, we compared the ultrastructure of the microspore development in the wild-type and npu-3 plants by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As shown in Figure 1C, the wild-type anther was filled with mature pollen grains after dehiscence, and the pollen grains were plump in shape. By contrast, broken pollen and pollen remnants were observed in the npu-3 anther (Fig. 1D). The mature wild-type pollen grains were covered with orderly reticulate pollen exine (Fig. 1E). Nevertheless, the reticulate exine pattern was seldom observed on the surface of the npu-3 pollen grains (Fig. 1F). These results indicate that the exine pattern formation and the sporopollenin deposition were defective in npu-3 pollen.
To further clarify the details of abnormal exine development of npu-3 pollen, we performed TEM observation. At the early tetrad stage in the wild type, both the primexine deposition and regular plasma membrane undulation were observed (n = 25), and the probaculae was observed growing on the membrane undulation peaks (Fig. 4, A and C). By contrast, neither primexine deposition nor regular undulation of the microspore membrane occurred around the microspores of npu-3 (n = 28), and the globular sporopollennin was observed on the surface of plasma membrane (Fig. 4, B and D). After the microspores released from tetrads, the primexine was still visible, and the exine layer formed a regular pattern in the wild type (Fig. 4E). By contrast, the exine pattern was not observed in the npu-3 mutants, and the globular sporopollenin was randomly deposited on the plasma membrane (Fig. 4F). Later, the primexine disappeared and the typical exine pattern successfully formed, including the tectum and the foot layer in the wild type (Fig. 4G). However, much sporopollenin was accumulated irregularly on the pollen surface of the npu-3 mutant plants instead of the typical exine pattern (Fig. 4H). These TEM observations showed that primexine was not deposited on the plasma membrane and the undulation did not form in npu-3 during microsporogenesis, which resulted in the altered exine pattern formation in the npu-3 mutants.
Ultrastructure of the pollen wall development in wild-type (A, C, E, and G) and npu-3 (B, D, F, and H) plants. A and C, Tetrad stage. Primexine was deposited between the plasma membrane and the inner callose wall, and the probaculae was growing on the peaks of the undulation. Arrowheads show the microspore membrane undulation. B and D, Tetrad stage. Arrow indicates the absence of primexine deposition and microspore membrane undulation. E and F, Released microspore stage. Baculae and tectum were formed in wild-type microspores; no baculae or tectum was formed on npu-3 microspores, only globular sporopollenin. G and H, Stage 9 microspore. Sporopollenin was randomly accumulated around the degenerated npu-3 microspores. Ba, baculae; CW, callose wall; DMsp, degenerated microspore; Msp, microspore; Pe, primexine; Pm, plasma membrane; Pb, probaculae; SP, spotted sporopollenin; Tc, tectum. Bars = 1 μm.
NPU Encodes an Unknown Protein Localized to the Plasma Membrane
The NPU gene encodes a functionally unknown protein of 230 amino acids that is predicted to localize to the plasma membrane. Residues 35 to 177 were predicted to be on the cytoplasmic side of the membrane with residues 1 to 14 and 201 to 230 being extracellular. To confirm the subcellular localization of the NPU protein, we fused this gene with GFP driven by the Cauliflower mosaic virus 35S promoter. The construct was transformed to the protoplast of the wild type. As shown in Figure 2C, the GFP signal could only be detected on the plasma membrane. This result confirmed that NPU is a plasma membrane protein.
Pfam database (http://www.sanger.ac.uk/Software/Pfam/) analysis showed that there was only one copy of the NPU gene in Arabidopsis. Furthermore, homologs of the NPU protein were identified in various plant species by BLASTp and tBLASTn searches in the National Center for Biotechnology Information database (Fig. 5A) and The Institute for Genomic Research Functional Genome Database (Fig. 5B). No significant sequence similarity was found outside the plant kingdom (Fig. 5). The homologs of NPU showed strong conservation in plants, including a rice (Oryza sativa) protein with 70% identity, a grape (Vitis vinifera) protein with 81% identity, a poplar (Populus trichocarpa) protein with 77% identity, a maize (Zea mays) protein with 69% identity, a castor bean (Ricinus communis) protein with 75% identity, a sorghum protein (Sorghum bicolor) with 70% identity, a moss (Physcomitrella patens ssp.) protein with 38% identity, and a green algae (Chlamydomonas reinhardtii) with 38% identity to NPU protein. However, the function of these proteins is presently unknown.
Phylogenetic analysis of NPU and homologous proteins. A, Multiple alignments of NPU and its homologs. Black bars, putative transmembrane regions; thin black line, predicted intracellular region; black curved sweeping lines, predicted extracellular domains. Protein sequence of NPU homologs in plants are as follows: Sh, Sorghum bicolor, SORBIDRAFT_09g005000; Zm, Zea mays, LOC100278212; Os, Oryza sativa, Os05g0168400; Rc, Ricinus communis, XP_002532862.1; Pp, Populus trichocarpa, XP_002323065.1; Vv, Vitis vinifera, XP_002270471.1; Ph, Physcomitrella patens, XP_001784831.1; Ch, Chlamydomonas reinhardtii, XP_001690235.1. B, Unrooted phylogenetic tree of NPU and its homologous proteins. Protein sequences of NPU and its homologs were analyzed with the neighbor-joining method by MEGA 4.0 software. The numbers at the nodes represent percentage bootstrap values based on 1,000 replications.
NPU Is Highly Expressed in Microspores and Tapetal Cells
We performed semiquantitative reverse transcription (RT)-PCR to analyze the expression levels in roots, stems, leaves, inflorescences, and 10-d-old seedlings. The results showed that NPU was expressed in all of these organs, with the strongest expression in buds (Fig. 6A). To obtain the precise expression pattern of NPU during anther development, RNA in situ hybridization experiments were performed. The hybridization signal was first detected in the microsporocytes and tapetal cells at stage 5 (Fig. 6B) and became stronger at stage 6 (Fig. 6C). The signal was the strongest in tetrads and tapetal cells at stage 7 (Fig. 6D) and decreased significantly during stages 8 and 9 in developing microspores and tapetal cells (Fig. 6, E and F). These results were in accordance with the TEM observations of npu-3 and together suggested that NPU functions mainly at the tetrad stage.
Expression analysis of NPU. A, Semiquantitative RT-PCR of RNA isolated from various tissues with NPU and β-TUBULIN-specific primer sets. Inf, Inflorescence; R, root; S, stem; L, leaf; B, buds; Sdl, seedling. B, In situ hybridization of the NPU transcript in a stage 5 anther with an antisense probe. C, In situ hybridization of the NPU transcript in a stage 6 anther with an antisense probe. D, In situ hybridization of the NPU transcript in a stage 7 anther with an antisense probe. F, In situ hybridization of the NPU transcript in a stage 8 anther with an antisense probe. G, In situ hybridization of the NPU transcript in a stage 9 anther with an antisense probe. H, In situ hybridization of the NPU transcript in a stage 7 anther with a sense probe.
DISCUSSION
npu Is a Novel Male Sterile Mutant with a Defect in Sporopollenin Deposition
In Arabidopsis, many male-sterile mutants have been reported. In this study, we identified a new male-sterile mutant of npu. Of the three alleles, the weak allele npu-1 exhibits reduced male fertility. However, both npu-2 and npu-3 are completely male sterile. This shows that the NPU gene is essential for male fertility. In npu-1, T-DNA was inserted in the 3′ terminus of this gene, while the T-DNA insertion in npu-2 and the point mutation in npu-3 were located in the 5′ terminus (Fig. 2A). The position of the T-DNA insertion in this gene was consistent with their fertility phenotype. Cytological analysis revealed that male sterility is caused by the defect in pollen wall formation (Fig. 3). Callose synthesis, callose dissolution, sporopollenin synthesis, and deposition play important roles in pollen wall formation during anther development (Worrall et al., 1992; Dong et al., 2005; de Azevedo Souza et al., 2009). Our research showed that callose synthesis and sporopollenin deposition were abnormal in npu mutants. The high expression of NPU at stage 7 (Fig. 6D), the defective tetrads in the npu mutants (Fig. 4, G and I), and the largely down-regulated expression of CalS5 in npu buds indicate that NPU functions at the early stages of pollen wall formation. Besides, primexine deposition and plasma membrane undulation were defective in npu mutants, which led to the defect in sporopollenin deposition (Fig. 4). This further confirms that NPU is responsible for the early stages of pollen wall formation.
Primexine and Plasma Membrane Undulation
Fitzgerald and Knox (1995) reported the events in early pollen wall development using rapid freeze-substitution technology. The microspores in the tetrads were initially surrounded by a callose wall after meiosis. After the primexine matrix had assembled between the microspore plasma membrane and callose wall, the plasma membrane gradually undulated and formed crypts. Several mutants were reported to affect the early events of pollen wall development. In the dex1 mutant, the primexine matrix assembly is both reduced and delayed, and the plasma membrane undulation of dex1 fails to form (Paxson-Sowders et al., 1997, 2001). In the nef1 mutant, the primexine matrix assembly appears to be coarser than that of the wild type, and the undulation of the nef1 microspore plasma membrane appears to be abnormal (Ariizumi et al., 2004). NPU is a critical protein in primexine matrix assembly, as the primexine matrix is completely absent in npu mutants (Fig. 4).
Southworth and Jernstedt (1995) proposed a Tensegrity model to explain the processes of exine patterning. After the primexine matrix is secreted from microspores, the hydrated primexine matrix exerts osmotic pressure on the microspore cell surface, resulting in a change in the cytoskeletal tension. Subsequently, the plasma membrane begins to undulate and the exine layer is assembled. In npu-3, the primexine matrix is completely absent and plasma membrane is not undulated (Fig. 4). Based on this model, we believe that the plasma membrane undulation is dependent on the primexine matrix during primexine formation. Due to the lack of primexine matrix, the osmotic pressure is not exerted on the microspore surface, and the plasma membrane fails to undulate in the npu mutants.
The Putative Role of NPU
The NPU protein was predicted to have two extracellular regions, two transmembrane regions, and one intracellular region (Fig. 2B). Its subcellular localization in the plasma membrane was confirmed in our research (Fig. 2C). NPU functions sporophytically in anther development because the wild type and npu-3 male sterile phenotype segregated in F2 population with 3:1. It is in agreement with its expression at transcription level in microsporocytes and tapetal cells in anther development (Fig. 6D). In the knockout mutant of NPU (npu-3), primexine could not form (Fig. 4). It was reported that the synthesis, secretion, and siting of primexine were all controlled by individual microspores (Fitzgerald and Knox, 1995; Perez-Munoz et al., 1993a, 1993b). Therefore, the NPU protein is likely to be located on the membrane of microspore. It is believed that primexine is composed of polysaccharide material (Heslop-Harrison, 1968). Another membrane integral protein RPG1, which is involved in the exine patterning in Arabidopsis, has recently been demonstrated to be a sugar transporter (Guan et al., 2008; Chen et al., 2010). Therefore, the NPU protein may also function as a sugar transporter on the microspore membrane, which is responsible for transporting the polysaccharide material essential for primexine matrix formation.
Callose is composed of a polysaccharide (β-1,3 glucan), and CalS5 was reported to be responsible for the synthesis of callose deposited at the primary cell wall of meiocyte and tetrads (Dong et al., 2005). In situ hybridization results also show that NPU initially expresses at meiocytes with its highest expression at tetrad stage (Fig. 6). The expression pattern of NPU is similar to Cals5 in anther development. It was reported that plant cells maintain a relatively stable level of sugar concentration, and a high or low level of sugar may inhibit or activate some other gene expressions (Koch, 1996). In the npu mutant, the block of primexine matrix material might lead to a high level of sugar inside microspores/microsporocytes, which inhibits the expression of Cals5 in the mutant. However, there might be other mechanisms that demonstrate how the knockout of NPU affects Cals5 expression. The downregulated expression of CalS5 further affects the callose synthesis, as shown in Figure 3. Therefore, the low callose accumulation in the npu mutant might be a side effect of the block of primexine matrix formation.
MATERIALS AND METHODS
Plant Material
Arabidopsis (Arabidopsis thaliana) plants used in this study are in the ecotype Columbia-0 background. The npu mutant was isolated from a population of T-DNA-tagged transformants provided by Dr. Zuhua He (Li et al., 2005). Plants were grown under long-day conditions (16 h of light/8 h of dark) in an approximately 22°C growth room. The herbicide Basta was used to monitor the segregation of the T-DNA inserts.
Microscopy
Plants were photographed with a Nikon digital camera (Coolpix 4500). Flower images were taken using an Olympus dissection microscope with an Olympus digital camera (BX51). Alexander solution was used as described (Alexander, 1969). Plant material for the semithin sections was prepared and embedded in Spurr’s resin as previously described (Owen and Makaroff, 1995). For SEM examination, fresh stamens and pollen grains were coated with 8 nm of gold and observed under a JSM-840 microscope (JEOL). For TEM observation, Arabidopsis floral buds from the inflorescence were fixed and embedded as described (Zhang et al., 2007).
Protein Structure Prediction and Phylogenetic Analysis
The SOSUI, TMHMM, and SMART programs were run to predict the transmembrane, intracellular, and extracellular regions, respectively, of NPU. The NPU protein sequence was used to search for NPU homologs using the BLASTp and tBLASTn programs. Multiple sequence alignment of full-length protein sequences was performed using ClustalX 2.0 and was displayed using BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html). Phylogenetic trees were constructed and tested by MEGA 4.0 based on the neighbor-joining method (http://www.megasoftware.net/).
Identification and Complementation of the NPU Gene
The identification of the T-DNA insertion was performed using primers that specifically amplify the BAR gene of T-DNA: Bar-F (5′-GCACCATCGTCAACCACTAC-3′) and Bar-R (5′-TGCCAGAAACCCACGTCAT-3′). For TAIL-PCR, T-DNA left border primers AtLB1 (5′-ATACGACGGATCGTAATTTGTC-3′), AtLB2 (5′-TAATAACGCTGCGGACATCTAC-3′), and AtLB3 (5′-TTGACCATCATACTCATTGCTG-3′) and genomic DNA of the mutant plants were used. The TAIL-PCR procedure and arbitrary degenerate primers were as described (Liu et al., 1995). Close linkage of the T-DNA insertion site and mutant phenotype were analyzed with primer AtLB3 and plant-specific primers, LP (5′-GACTAGGTAATTGATATTGAACC-3′) and RP (5′-GGTTATGTTAATAGTGGCTTTG-3′). A DNA fragment of 4.7 kb, including 1.5-kb upstream and 800-bp downstream sequences, was amplified using KOD polymerase (Takara Biotechnology) with primers CMP-F (5′-GAATTCATTTAGAAACAACGACCAGCAT-3′) and CMP-R (5′-GTCGACGGGTAAGAGATCCTAACACGG-3′). The fragment was verified by sequencing and was cloned into a pCAMBIA1300 binary vector (Cambia). The plasmids were transformed into Agrobacterium tumefaciens GV3101 and then introduced into the homozygous mutant plants. Seeds were selected using 20 mg/L hygromycin for transformants that were fertile with a homozygous background. The primers used for the verification of the background of the transformants were AtLB3 and plant-specific primers CMPV-F (5′-ATGAAAACCCTTCAACGTCTAT-3′) and CMPV-R (5′-TGTCTGTCTGGTGGCATTACTA-3′).
Expression Analysis
The full-length cDNA of the wild-type plants without the stop codon was cloned for the GFP fusion with the primers GFP-F (5′-CCCGGGATGGCGGGCATGGCTGCGGTG-3′) and GFP-R (5′-GGTACCCCATCCCCCTTGCCAAATTCTCC-3′). The cDNA was cloned into the pMON530 vector with enhanced GFP. The primers used for the expression analysis of CalS5 and A6 genes were as follows: CalS5 primers, forward, 5′-ATTATTGCAGCTGCTAGAGATG-3′, and reverse, 5′-CTTGTTCAGAGGTTCTGGCTT-3′; A6 primers, forward, 5′-TACCTAAACCGACGAACA-3′, and reverse, 5′-ATGCCAATAAATGGAGAC-3′. Semiquantitative RT-PCR with 30 cycles was used to analyze the expression level of the NPU gene. The primers were as follows: RT-F, 5′-CAGGATTACGACCGTGGAAC-3′, and RT-R, 5′-CATTCATGCTGCTGCTCTCC-3′. The DIG (for digoxigenin) RNA labeling kit (Roche) and PCR DIG probe synthesis kit (Roche) were used for the RNA in situ hybridization experiment. An NPU-specific cDNA fragment of 304 bp was amplified and cloned into the pSK vector. Antisense and sense DIG-labeled probes were prepared as described (Zhu et al., 2008).
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers SORBIDRAFT_09g005000, LOC100278212, Os05g0168400, XP_002532862.1, XP_002323065.1, XP_002270471.1, XP_001784831.1, and XP_001690235.1.
Acknowledgments
We thank Dr. Zu-hua He from the Shanghai Institute of Plant Physiology and Ecology for providing the T-DNA-tagged lines and Hui-qi Zhang and Nai-ying Yang from Shanghai Normal University for their help with SEM and TEM. We also thank Pacific Edit for reviewing the manuscript prior to submission.
Footnotes
The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Sen Zhang (senzhang{at}shnu.edu.cn) and Zhong-Nan Yang (znyang{at}shnu.edu.cn).
↵1 This work was supported by the National Natural Science Foundation of China (grant nos. 30925007 and 30971553) and the National Basic Research Program of China (grant no. 2007CB947600) and by the Leading Academic Discipline Project of Shanghai Municipal Education Commission (grant no. J50401). This work was also supported by the National Key Laboratory of Plant Molecular Genetics of China.
↵2 These authors contributed equally to the article.
↵3 These authors contributed equally to the article.
↵[OA] Open Access articles can be viewed online without a subscription.
- Received August 5, 2011.
- Accepted November 15, 2011.
- Published November 18, 2011.
REFERENCES
