Plant and Cell Physiology Advance Access originally published online on November 28, 2007
Plant and Cell Physiology 2008 49(1):58-67; doi:10.1093/pcp/pcm167
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Ultrastructural Characterization of Exine Development of the transient defective exine 1 Mutant Suggests the Existence of a Factor Involved in Constructing Reticulate Exine Architecture from Sporopollenin Aggregates
1Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555 Japan
2Vegetable Breeding Research Team, National Institute of Vegetable and Tea Science, Ano, 514-2392 Japan
3Department of Plant Genome Research, Kazusa DNA Research Institute, Kisarazu, 292-0818 Japan
*Corresponding author: E-mail, torikin{at}bios.tohoku.ac.jp; Fax, +81-22-717-8834.
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Abstract |
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A male-sterile mutant of Arabidopsis thaliana, in which filament elongation was defective although pollen fertility was normal, was isolated by means of T-DNA tagging. Transmission electron microscopy (TEM) analysis revealed that primexine synthesis and probacula formation, which are thought to be the initial steps of exine formation, were defective, and that globular sporopollenin aggregation was randomly deposited onto the microspore at the early uninucleate microspore stage. Sporopollenin aggregation, which failed to anchor to the microspore plasma membrane, was deposited on the locule wall and in the locule at the uninucleate microspore stage. However, visually normal exine with a basic reticulate structure was observed at the middle uninucleate microspore stage, indicating that the exine formation was restored in the mutant. Thus, the mutant was designated transient defective exine 1 (tde1). These results indicated that tde1 mutation affects the initial process of the exine formation, but does not impair any critical processes. Our results also suggest the existence of a certain factor responsible for exine patterning in A. thaliana. The TDE1 gene was found to be identical to the DE-ETIOLATED 2 gene known to be involved in brassinosteroid (BR) biosynthesis, and the tde1 probacula-defective phenotypes were recovered in the presence of BR application. These results suggest that BRs control the rate or efficiency of initial process of exine pattern formation.
Keywords: Arabidopsis thaliana - Exine formation - Male sterility - Probacula
Abbreviations: BAC, bacterial artificial chromosome; BR, brassinosteroid; CV, coated vesicle; ER, endoplasmic reticulum; FAA, formalin/alcohol/acetic acid; qRT–PCR, quantitative RT–PCR; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
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Introduction |
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Pollen is surrounded by an outer sculptured thick wall, the exine. The exine functions as a protector of pollen from various environmental stresses and bacterial attack when pollen moves from anther to stigma. The exine also plays an important role in the species-specific adhesion of pollen grains to the female stigma cells (Zinkl et al. 1999
, reviewed by Scott 1994, Piffanelli et al. 1998, Scott et al. 2004). It has been demonstrated that exine is made of sequential polymerization of sporopollenin, which consists of phenols and fatty acid derivatives (Osthoff and Wiermann 1987, Wilmesmeier and Wilermann 1995, Ahlers et al. 1999, Meuter-Gerhards et al. 1999).
During the tetrad stage, sporopollenin deposits onto the primexine, which is formed between the microspore plasma membrane and callose wall (Owen and Makaroff 1995, Rhee and Somerville 1998) and acts as a scaffold of sporopollenin deposition. This sporopollenin deposition within the primexine produces a columnae-like structure of exine, the probacula. Further sporopollenin deposition produces a roof-like structure of exine, the tectum. The probacula and tectum continue to increase in size when microspores are released into the locule after the degeneration of the callose wall by the β-1,3-glucanase secreted from tapetum. By the time microspores undergo mitotic division at the bicellular pollen stage, the reticulate pattern of the exine is complete in Arabidopsis (Paxson-sowders et al. 1997, Ariizumi et al. 2004). Then tryphine, which mainly consists of lipid-abundant organelles, fills the interstices of the exine.
There are several Arabidopsis mutants defective in exine formation, and they have been characterized in detail. The absence of normal sporopollenin deposition in the male sterility2 (ms2) mutant has been demonstrated by an acetolysis experiment (Aarts et al. 1997). Acetolysis causes the pollen grains of the ms2 mutant to be completely lysed, while wild-type pollen grains remain intact. It is suggested that the MS2 protein encodes a fatty acyl reductase, which might reduce very long chain fatty acids to fatty alcohol, and it is postulated that this reaction is one of the steps in the formation of sporopollenin (Aarts et al. 1997). In the flp1 mutant, many parts of the exine appeared to have broken apart after formalin/alcohol/acetic acid (FAA) treatment, although the microspores and their exine were visually normal without this treatment (Ariizumi et al. 2003). The FLP1 protein was suggested to be involved in fatty acid biosynthesis as a transporter or a catalytic enzyme (Ariizumi et al. 2003). Recently, it has been reported that the Arabidopsis cyp703a2 mutant shows impaired pollen development without exine (Morant et al. 2007). A heterologous expression experiment of CYP703A2 in yeast cells has demonstrated the ability of CYP703A2 to catalyze the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids with hydroxylation of lauric acid. Therefore, it is suggested that medium-chain hydroxy fatty acids are essential building blocks for sporopollenin synthesis (Morant et al. 2007). The defective in exine formation 1 (dex1) mutant is characterized by an abnormal random deposition of sporopollenin on the microspore plasma membrane, which results in pollen degeneration (Paxson-Sowders et al. 1997, Paxson-Sowders et al. 2001). It has been suggested that the DEX1 protein is a component of either the primexine matrix or the endoplasmic reticulum (ER) and is involved in the assembly of primexine (Paxson-Sowders et al. 2001). Sporopollenin is produced in the no exine formation 1 (nef1) mutant, but is not deposited onto the membrane due to the coarsely developed primexine (Ariizumi et al. 2004). It is predicted that the NEF1 encodes a membrane protein that maintains the envelope integrity in the plastids (Ariizumi et al. 2004). The callose wall formation in the callose synthase5 (cals5)/less adherent1 (lap1) mutant is dramatically reduced, and the sexine consisting of bacula and tectum is missing, whereas the tryphine forms aggregates on the outer layer of the cals5 microspores (Zinkl and Preuss 2000, Dong et al. 2005, Nishikawa et al. 2005). The CALS5/LAP1 gene encodes a membrane protein acting as a callose synthase, and may interact with other components of the callose synthase complex (Dong et al. 2005). Though the hkm mutant produces primexine that partially plays a role in sporopollenin deposition, sporopollenin was deposited onto the microspores and an exine-like hackly structure was observed on the microspores (Ariizumi et al. 2005). The HKM gene is shown to be identical to the MS1 gene encoding a transcription factor that regulates tapetum differentiation (Wilson et al. 2001, Ito and Shinozaki 2002, Ariizumi et al. 2005). These mutant analyses have demonstrated that the sporopollenin, the primexine and the callose wall play critical roles in exine formation (reviewed by Ariizumi and Toriyama 2007). However, factors that are responsible for species-specific exine patterning are still poorly understood.
Male sterility has also been reported in many of the mutants that lack hormones such as brassinosteroid (BR) (Clouse and Sasse 1998) and gibberellin (Cheng et al. 2004). Transgenic plants in which cytokinine levels are disturbed have been reported to show male sterility (Huang et al. 2003). Though BRs were originally identified from pollen in Brassica napus in 1979 (Clouse and Sasse 1998), how BRs regulate pollen development has been poorly understood. It has not yet been characterized whether these hormone-defective plants are also defective in exine formation.
By screening T-DNA-tagged lines for male sterility, we isolated a male-sterile mutant tde1. Transmission electron microscopy (TEM) analysis revealed that the tde1 mutant produced microspores with defective sporopollenin aggregation at the early uninucleate microspore stage. However, normal reticulate exine was formed at the later stages. Our results suggest that the tde1 mutant lacks a factor required for the rapid and efficient formation of exine. As a result tde1 mutants show formation of an unusual sporopollenin aggregate prior to formation of a fundamental reticulate exine structure. Our findings provide a new view to gain insight into the early process of pollen exine patterning with the commitment of BRs.
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Mutant isolation
Based on macroscopic screening of about 3,000 lines of Arabidopsis thaliana ecotype Columbia (Col), mutagenized with T-DNA, we identified a sterile mutant (KE-1903) that had completely empty siliques. In order to examine the segregation ratio of the male sterility phenotype, the mutant plant was backcrossed twice with wild-type pollen and then self-pollinated. The resulting seeds were sown for a genetic analysis of two generations (F3–F4) derived from heterozygous plants. The segregation ratio of fertile to male sterile was fitted at a theoretical ratio of 3 : 1 in both mutant generations (Table 1;
2 = 0.05, 2 = 0.5; 1.0 > P > 0.5). These results indicated that a single recessive mutation caused male sterility. The phenotype of male sterility, however, was not linked to the T-DNA (data not shown, see below), thus it is considered that the mutation was introduced during the transformation process. The mutant was named transient defective exine 1 (tde1) as described below.
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Microspore development
To gain information about pollen development in the tde1 mutant, TEM analysis was carried out with anther sections at each stage from the tetrad stage to the tricellular pollen stage (Fig. 1). Tetrads enclosed by a thick callose wall were visible in both the wild type and the tde1 mutant (Fig. 1A, D). Aniline blue staining also confirmed the presence of the callose wall in both the wild type and the tde1 mutant (data not shown). Sporopollenin deposits, which were probably secreted from tapetum, were visible on the callose wall in the wild type and the tde1 mutant (Fig. 1A, D; white arrows). When the individual microspores were released from tetrads, reticulate exine was visible in the wild type (Fig. 1B). Microspore release from tetrads was also observed in the tde1 mutant. However, microspores with no exine, but globular sporopollenin aggregation were observed in the tde1 mutant (Fig. 1E). Such globular sporopollenin aggregation was visible on the mutant microspore and in the locule (Fig. 1E; white arrows), but never observed in the wild type. All microspores in the locules of the tde1 mutant were aberrant, indicating that the phenotype was uniform. At the middle unicellular microspore stage when microspores are more rounded than in the previous stage, microspores with almost completely reticulate exine structure were produced in the wild type (Fig. 1C). Surprisingly, microspores with a reticulate exine structure were also observed in tde1 at the same stage (Fig. 1F), indicating that the defect in exine formation was transient. Thus, the mutant was designated transient defective exine 1 (tde1). Wild-type and tde1 microspore development was similar from this stage onward. Microspores in both the wild type and tde1 produced bicellular and tricellular pollen in a similar manner (Fig. 1H–J, L–N). When mature pollen grains were observed by scanning electron microscopy (SEM), no structural differences were found in the reticulate pattern of exine between the wild type and tde1 (data not shown).
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Pollen wall development in tde1
A detailed observation of pollen wall development was also carried out using TEM. At the tetrad stage, electron-dense deposits of sporopollenin were visible on the callose wall both in the wild type and in tde1 (Fig. 2A, F). At this stage, the probacula was visible in the wild type (Fig. 2A; white arrows). An undulant surface of the microspore plasma membrane structure was also evident. On the other hand, in the tde1 mutant, the primexine formation was significantly reduced (Fig. 2F; black arrows), and no probacula formation was found. However, the undulant membrane structure looked similar to that in the wild type. At the early uninucleate microspore stage in the wild type, the probacula became elongated and expanded to form the bacula and tectum (Fig. 2B). Nexine and intine formation also commenced at this stage, and reticulate patterning was almost completed. In the tde1 mutant, the globular sporopollenin aggregation was randomly deposited on the plasma membrane (Fig. 2G). Neither bacula nor tectum was observed. On the other hand, intine formation appeared similar to that in the wild type. The microspore plasma membrane was also similar in structure to that of the wild type. At the middle uninucleate microspore stage, bacula and tectum expanded further and microspores with a reticulate patterned exine were visible in the wild type (Fig. 2C), while in the tde1 mutant the exine with a complete pattern was also observed at this stage (Fig. 2H). The pollen wall of the tde1 mutant developed in a similar manner to that of the wild type from this stage to the mature stage, as shown in Fig. 2D, E, I, J.
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Sporopollenin deposition on locule wall
When the anther sections at the early uninucleate microspore stage (Fig. 3A, C) and in the middle uninucleate microspore stage were observed (Fig. 3B, D), numerous electron-dense granules were found on the locule wall only in the tde1 mutant (Fig. 3C, D; white arrows). These deposits are considered to be sporopollenin that failed to attach properly to the microspore plasma membrane, as previously observed in the dex1 and the nef1 mutants (Paxson-Sowders et al. 1997
, Ariizumi et al. 2004). Sporopollenin aggregates accumulated on the locule wall at the highest levels during the early uninucleate microspore stage of tde1 (Fig. 3C; arrows). The amount of sporopollenin aggregation decreased at the middle uninucleate stage (Fig. 3D) compared with the early uninucleate microspore stage. Next, tapetum structure was observed by TEM from the tetrad stage to the late bicellular pollen stage (Supplementary Fig. S1). However, no obvious differences were found between the wild type and the tde1 mutant.
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Flower development in tde1
Several defects were observed in whole tde1 plants. The first visual characteristic of tde1 was a dwarf phenotype and dark colored leaves (data not shown). Next we examined the reproductive tissues at anthesis in the tde1 mutant. The sepal, petal and stamen were clearly shorter than those in the wild type (Fig. 4A, E). In particular, the ultimate stamen filament was much shorter than the mature stamens of the wild type. The filament length was 5.0 ± 0.14 mm for the wild type and 3.0 ± 0.1 mm for tde1. Light microscopic observation indicated that the arrest of stamen filament growth was due to reduced cell length (Fig. 4B, F). The anther size in the tde1 mutant was smaller than that in the wild type (Fig. 4C, G). In order to confirm the pollen viability of the tde1 mutant, pollen tube elongation was observed on the pistils at anthesis after aniline blue staining. Significant pollen tube elongation was observed in the wild type (Fig. 4D), whereas neither pollen attachment nor germination was visible on the tde1 papilla cells (Fig. 4H). When the tde1 pollen grains were artificially applied to their pistil (self-pollination), the tde1 pollen grains germinated well, as shown in Fig. 4I. This indicates that the tde1 pollen grains are viable, but may not reach the stigmatic surface due to short filament length. The tde1 mutant produced siliques with a normal number of seeds when artificially pollinated with wild-type pollen, as the number of seeds per siliques was 40.4 ± 2.2 for the wild type and 37.5 ± 2.5 for tde1, showing that the female was fertile and the male was sterile. These results indicated that the male sterility in the tde1 mutant was due to the arrest of stamen filament growth rather than to lack of pollen viability.
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TDE1 is an allele of the DET2 gene
Although the tde1 mutant was identified from T-DNA insertion lines, the phenotype of the male sterility was not linked to the T-DNA. Thus the TDE1 gene was cloned using a map-based and candidate gene approach. The tde1/tde1 mutant in the Col ecotype was crossed to wild-type ecotype Landsberg erecta (Ler) to generate an F2 mapping population of tde1/tde1 plants segregating for Ler- and Col-specific markers. Based on the phenotype of male sterility, the TDE1 gene was mapped between an InDel marker CER449022 on a bacterial artificial chromosome (BAC) clone F12M22 and an InDel marker CER449589 on an adjacent BAC clone F16M14. We suspected that the tde1 mutant would be defective in BR biosynthesis or its signal transduction because the phenotypes including dark green leaves, dwarf and reduced fertility are characteristics observed in other BR mutants (Clouse and Sasse 1998
). On the candidate region of chromosome 2, there was only a single gene known to be involved in BR synthesis, the DE-ETIOLATED 2 (DET2) gene. After determining the entire sequence of the DET2 gene of the tde1 mutant, we found a 7 bp deletion around nucleotides 794–801 from the first ATG codon (Fig. 5A). This deleted mutation caused a frameshift, resulting in creation of a distinct sequence of 26 amino acids at the C-terminal end. Next, to determine if the TDE1 gene is allelic to the DET2 gene, complementation experiments were carried out with pollen from heterozygous det2 (DET2/det2) plants applied to male-sterile tde1 plants. If all the progeny are fertile, the two lines represent mutations at different gene loci, and if half of the progeny are fertile and the other half sterile, the two lines represent mutations at the same locus. As we expected, the progeny segregated into 42 fertile plants and 38 male-sterile plants (2 = 0.2, 1.0 > P > 0.5). This result confirmed that TDE1 is the DET2 gene. Complementation of the male-sterile phenotype with the wild-type DET2 gene was also examined. A complementation vector, pBIN-DET2-Ubi-bar, which included the entire DET2-coding region, 3.0 kbp of the 5' promoter region, 1.4 kbp of the 3' downstream region, and the bar gene, was introduced into heterozygous tde1 plants (TDE1/tde1) using floral dip methods. The plants containing the complementation vector were selected based on bialaphos resistance. Based on the Southern blot analysis, we identified that three plants were homozygous for the deletion at the TDE1 locus. They thus contained the disrupted endogenous TDE1 gene and the complementation vector. All the three plants were phenotypically indistinguishable from wild-type plants in terms of restored fertility. The successful complementation test indicated that DET2 was responsible for the tde1 phenotype. Next, the tde1 mutant was grown in the presence of brassinolide to determine whether the phenotypes were recovered. This experiment demonstrated that brassinolide application rescued the reproductive phenotypes as well as the vegetative phenotypes, including primexine and probacula formation and male sterility (Supplementary Fig. S2). Further, the globular sporopollenin aggregation was not observed on the microspores at the early uninucleate microspore stage. These successful complementation experiments indicate that the phenotypes observed during the early process of exine formation in the tde1 mutant are dependent on BR synthesis.
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Reverse transcription–PCR (RT–PCR) was performed in various tissues that included open flowers, early buds, middle buds, late buds, rosette leaves, cauline leaves, stems, siliques and roots. Excluding the siliques, 330 bp of the DET2/TDE1 gene product was detected in all of the tissues tested (Fig. 5B), suggesting that BRs are synthesized in developing microspores. We also confirmed the same level of DET2/TDE1 mRNA expression in the tde1 mutant (data not shown), suggesting that mutant phenotypes are due to the defect in DET2/TDE1 protein function rather than its transcription. In order to determine if the defective phenotype (e.g. reduced primexine formation and probacula formation) of the det2/tde1 mutant is due to reduced mRNA accumulation of previously known exine-associated genes [MS1/HKM, ADENINE PHOSPHORIBOSYLTRANSFERASE1 (APT1), MS2, NEF1 and DEX1], the expression level of these genes was determined by quantitative RT–PCR (qRT–PCR; Supplementary Fig. S3). However, there was no obvious difference in the transcription levels of these genes between the wild type and the tde1 mutant.
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TEM analysis demonstrated that the tde1 mutant showed reduced primexine formation and failure to produce probacula at the tetrad stage, and that the globular sporopollenin aggregation was randomly deposited on the plasma membrane at the early stage of pollen development (Fig. 2). Excess sporopollenin aggregation was observed on the locule wall and in the locule during exine development in the tde1 mutant (Fig. 3). Previous work suggests that such aggregation is most probably due to failure of normal sporopollenin deposition onto the microspore plasma membrane (Paxson-Sowders et al. 2001
, Ariizumi et al. 2004). In spite of these defects, reticulate exine was clearly formed at the later stage in the tde1 mutant. It is known that previously isolated mutants showing defects in primexine formation never form normal exine at the later stages. In the dex1 mutant, the thinner primexine is formed and sporopollenin is randomly deposited on the primexine, but sporopollenin never anchors to the microspore plasma membrane (Paxson-Sowders et al. 1997, Paxson-Sowders et al. 2001). In the nef1 mutant, the coarse primexine is formed, but sporopollenin never deposits on the primexine (Ariizumi et al. 2004). Finally, microspores without exine are formed in these mutants. In the hkm mutant, the thinner primexine is formed, and sporopollenin eventually anchors to the plasma membrane. Finally, microspores with aberrant exine are formed in the hkm mutant (Ariizumi et al. 2005). However, these defective microspores eventually degrade in the locule. These results suggest that the tde1 mutation does have a defect in some initial processes of exine pattern formation, but does not have a defect in the critical step. Another possibility is that the tde1 mutation causes a delay in the development of primexine and probacula formation. Although primexine formation is considered to be essential for exine formation (Paxson-Sowders et al. 2001, Ariizumi et al. 2004), the tde1 mutant formed the thin primexine. While probacula formation was never seen in tde1, exine did eventually form. It may be that probacula and primexine actually and normally formed briefly between the tetrad stage and the early uninucleate microspore stage, and so went undetected. In this case, the tde1 mutation might affect the timing for their development. Further analysis of these processes in tde1 mutants may improve our understanding of the processes involved in exine development.
This study of tde1 pollen development suggests the existence of factors controlling the rate or efficiency of exine pattern formation. It is likely that these factors act on exine patterning from the early uninucleate to the middle uninucleate microspore stage, when the exine formation was restored in the tde1 mutant. It is possible that the tde1 mutant was able to form normal exine structure later because other factors remain functional. Several factors have been shown to control exine patterning. It has been demonstrated that exine pattern formation is disturbed by early dissolution of the callose wall in transgenic tobacco and rice with β-1,3-glucanase under the tapetum-specific promoter A9 and Osg6B promoters (Worall et al. 1992, Tsuchiya et al. 1994). Also, it has been shown that lack of the callose synthase gene results in failure to form exine as in the cals5/lap1 mutant (Dong et al. 2005, Nishikawa et al. 2005). These results indicate the importance of callose synthesis for exine patterning. It is possible that the callose wall participated in changing the sporopollenin aggregates on the plasma membrane into a reticulate exine in late uninucleate stage tde1 mutants because callose wall synthesis was observed. In this case, callose wall might support or provide spatial and temporal spaces for additional factors to work on exine patterning. However, callose wall degradation starts before the sporopollenin aggregates appeared on the plasma membrane in the tde1 mutant, and these aggregates transformed into exine in the absence of callose wall. Therefore, it remains unclear if callose wall was directly associated with exine restoration in the tde1 mutant.
A factor that determines pollen wall patterning might be located in the plasma membrane. Sheldon and Dickson (1983) employed a centrifugation experiment that enables displacement of cytoplasmic components of meiocytes by the centrifugation of developing anthers. This centrifugation experiment suggested that a family of coated vesicles (CVs), which are present throughout the meiocyte cytoplasm, are likely to be associated with large assemblages of smooth ER and with plasma membrane, and that the CVs are responsible for reticulate patterning (Sheldon and Dickson 1983). The four previously isolated mutants (dex1, nef1, cals5/lap1 and hkm) have something in common in terms of their abnormal membrane structure. It has been shown that the plasma membrane in the dex1 and nef1 mutants is shallower than that in the wild type (Paxson-Sowders et al. 2001, Ariizumi et al. 2004), and the hkm membrane is deeper than that in the wild type (Ariizumi et al. 2005). The membrane of the cals5/lap1 has irregular waves compared with that in the wild type (Dong et al. 2005). In contrast, the tde1 membrane structure was similar to that in the wild type (Fig. 2). These results suggested that these CVs were functional in the tde1 plasma membrane, which leads to the production of microspores with restored reticulate exine patterning at the middle uninucleate microspore stage in the tde1 mutant.
The det2 mutant was originally identified as a mutant that has characteristics of light-grown plants even when grown in the dark (Clouse and Sasse 1998). It has been shown that loss-of-function mutations in the DET2 gene have pleiotropic effects including male sterility, dwarf, darker green leaves, reduced cell size in hypocotyls, cotyledons and leaves, and reduced apical dominance (Li et al. 1996). On the other hand, ultrastructural characterization of exine patterning in the det2 mutants has not yet been reported. In our current study, we found that the TDE1 gene was identical to the DET2 gene, and that the tde1 primexine- and probacula-defective phenotypes were BR dependent. Our results suggest that BRs are involved in the primexine and probacula formation during the early process of exine patterning.
In order to confirm if the tde1 mutation affected the expression of genes involved in the pollen exine formation, mRNA accumulation of these genes was determined by qRT–PCR (Supplementary Fig. S3). However, this experiment showed no differences in these mRNA expression levels between the wild type and the tde1 mutant, suggesting that BRs support exine pattern formation in a distinct pathway. Further study is needed to identify factors involved in constructing reticulate exine architecture from sporopollenin aggregates.
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Mutant isolation
A male-sterile mutant (KE-1903) was screened from approximately 3,000 T-DNA mutagenized lines (KE-501 to KE-3600) of Arabidopsis ecotype Col, which were generated at the Kazusa DNA Research Institute (Ariizumi et al. 2004
). Anther filaments and pollen grains were observed under a light microscope. Aniline blue staining was performed according to Ariizumi et al. (2003).
Cytological analysis using microscopy
For preparing sections, flower buds of the wild-type and tde1 plants were fixed in 3% glutaraldehyde in 100 mM phosphate buffer (pH 7.0), rinsed overnight in 100 mM phosphate buffer (pH 7.0) containing 140 mM saccharose, post-fixed in a solution containing 1% osmium tetroxide (EM Sciences, Fort Washington, PA, USA), 100 mM phosphate buffer (pH 7.0) and 210 mM saccharose for 2 h, and washed in a dehydrated ethanol series for 30 min at each step (70%, 80%, 90%, 100% x2). The dehydrated tissues were soaked in a mixture of 100% ethanol and propylene oxide (1 : 1, v/v) for 30 min, 100% propylene oxide for 30 min twice, then incubated in propylene oxide and Spurr's resin (1 : 2, v/v) for 12 h. Subsequently, tissues were embedded in Spurr's resin for 3 d at 60°C. Thin sections, 60–90 nm, were prepared and stained in 4% uranyl acetate for 20 min and in lead citrate for 3 min. For TEM analysis, these specimens were analyzed with a TEM (H-8100, HITACHI). SEM analysis was performed according to Ariizumi et al. (2003). Two independent experiments were carried out using flower buds from independent plants, and at least four independent locules were observed in each stage.
Identification of the TDE1 gene
For map-based cloning, the tde1 mutant (Col) was crossed with Ler. The genotypes of the resulting F2 plants were determined using InDel markers in the Cereron Arabidopsis polymorphism collection (http://www.arabidopsis.org/Cereron/index.html). To examine the possibility of the TDE1 gene being allelic to the DET2 gene, we crossed the tde1 mutant with the det2-1 mutant, which was kindly provided by The Arabidopsis Information Resource (TAIR) (http://www.arabidopsis.org/info/aboutarabidopsis.jsp). The tde1 pistils were pollinated by the pollen grains of a heterozygous det2-1 plant. We subsequently examined the segregation ratio of male fertility and sterility of the progeny. For the complementation test, the BAC clone T8P21, obtained from TAIR (http://www.arabidopsis.org/), was confirmed to include the wild-type DET2 gene. A 5,180 bp fragment containing the entire coding sequence, 3,300 bp of the 5' promoter sequence and 500 bp of the 3' sequence was generated by HindIII digestion. This fragment was introduced into the HindIII site of pBIN19-Ubi-bar, in which the cauliflower mosaic virus 35S promoter and β-glucuronidase (GUS) gene of pBI121 (Jefferson et al. 1987) was replaced by the maize ubiquitin promoter and bar gene (Christensen et al. 1992), and generated pBIN-DET2-Ubi-bar. This pBIN-DET2-Ubi-bar vector was transferred to Agrobacterium tumefacience strain GV3101 using the freeze–thaw method (An et al. 1988). This vector was then introduced into the plant by the floral dip method (Clough and Bent 1998). Transgenic plants were selected based on bialaphos resistance (10 mg l–1). To test the recovery of the phenotypes observed in tde1, mutant seeds were sown on the media including brassinolide according to the procedure reported by Li et al. (1996) and Fujioka et al. (1997).
Expression analysis
For the RT–PCR analysis, mRNA was isolated from open flowers, early buds that corresponded to buds 11–17 of the inflorescence (buds prior to the tetrad stage), middle buds that represented buds 5–10 of the inflorescence (uninucleate microspore and bicellular pollen stages), late buds that represented buds 1 and 4 (unopened petals visible, tricellular pollen stage), open flowers, rosette leaves, cauline leaves, stems, siliques and roots using the Dynabeads mRNA DIRECTTM Kit (Dynal, Smestad, Norway). The isolated 160 ng of mRNA was reverse-transcribed to synthesize first-strand cDNA using a First-Strand cDNA Synthesis Kit (Amersham-Pharmacia, Uppsala, Sweden). The cDNA was then used as a template for PCR amplification with the F2-F (5'-CCGGTAAAAACGGATTTCCG-3') and F3-R (5'-CGGACAGCTTACCAACTCGAA-3') primers. The ACTIN2 (ACT2) gene was amplified as a positive control following the method of Steiner-Lange et al. (2003) (Supplementary Table S1).
For qRT–PCR, mRNA was isolated from flower buds in the wild type and the tde1 mutant, and then cDNA was synthesized as described above. The cDNA was used as a template for the qRT–PCR. Each cDNA sample was diluted 1 : 30 in water, and 5 µl of this dilution was used as template for the qRT–PCR. This qRT–PCR was performed by the Lightcycler FastStart DNA master SYBY Green I kit (Roche) on a Roche LightCycler real-time PCR machine according to the manufacturer's instruction. The ACT2 gene was used as a control in qRT–RCR. Gene-specific primers used are listed in Supplementary Table S1. Three independent experiments were performed.
Supplementary material
Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oxfordjournals.org.
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Funding |
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Grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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We are grateful to Dr. Camille Steber of Washington State University for critical reading of this manuscript.
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4Present address: Department of Crop and Soil Science, Washington State University, Pullman, WA 99164-6420, USA.
5These authors contributed equally to this work.
6Present address: Forestry Research Institute, Oji Paper Company Co. Ltd, Kameyama, 519-0212 Japan.
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Aarts MGM, Hodge R, Kalantidis K, Florack D, Wilson ZA, Mulligan B, Stiekema WJ, Scott R, Pereira A. The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes. Plant J (1997) 12:615–623.[CrossRef][ISI][Medline]
Ahlers H, Thom I, Lambert J, Kuckuk R, Wiermann R. 1H NMR analysis of sporopollenin from. Typha angustifolia. Phytochemistry (1999) 50:1095–1098.
An G, Ebert PR, Mitra A, Ha SB. Binary vectors. In: Plant Molecular Biology Manual.—Gelvin SB, Schilpeoort RA, eds. (1988) Dordrecht, The Netherlands: Kluwer Academic Publishers. 1–19.
Ariizumi T, Hatakeyama K, Hinata K, Sato S, Kato T, Tabata S, Toriyama K. A novel male-sterile mutant of Arabidopsis thaliana, faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine. Plant Mol. Biol (2003) 53:107–116.[CrossRef][ISI][Medline]
Ariizumi T, Hatakeyama K, Hinata K, Inatsugi R, Nishida I, Sato S, Kato T, Tabata S, Toriyama K. Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen, resulting in male sterility in Arabidopsis thaliana. Plant J (2004) 39:170–181.[CrossRef][ISI][Medline]
Ariizumi T, Hatakeyama K, Hinata K, Sato S, Kato T, Tabata S, Toriyama K. The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation. Sex. Plant Reprod (2005) 18:1–7.[CrossRef]
Ariizumi T, Toriyama K. Pollen exine pattern formation is dependent on three major developmental processes in Arabidopsis thaliana. Int. J. Plant Dev. Biol (2007) 1:106–115.
Cheng H, Qin L, Lee S, Fu X, Richards DE, Cao D, Luo D, Harberd NP, Peng J. Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function. Development (2003) 131:1055–1064.[CrossRef][ISI]
Christensen AH, Sharrock RA, Quail PH. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol (1992) 18:675–689.[CrossRef][ISI][Medline]
Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J (1998) 16:735–743.[CrossRef][ISI][Medline]
Clouse SD, Sasse JM. BRASSINOSTEROIDS: essential regulators of plant growth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol (1998) 49:427–451.[CrossRef][ISI]
Dong X, Hong Z, Sivaramakrishnan M, Mahfouz M, Verma DP. Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis. Plant J (2005) 42:315–328.[CrossRef][ISI][Medline]
Fujioka S, Li J, Choi YH, Seto H, Takatsuto S, Noguchi T, Watanabe T, Kuriyama H, Yokota T, Chory J, Sakurai A. The Arabidopsis deetiolated2 mutant is blocked early in brassinosteroid biosynthesis. Plant Cell (1997) 9:1951–1962.[Abstract]
Huang S, Cerny RE, Qi Y, Bhat D, Aydt CM, Hanson DD, Malloy KP, Ness LA. Transgenic studies on the involvement of cytokinin and gibberellin in male development. Plant Physiol (2003) 131:1270–1282.
Ito T, Shinozaki K. The male sterility1 gene of Arabidopsis, encoding a nuclear protein with a PHD-finger motif, is expressed in tapetal cells and is required for pollen maturation. Plant Cell Physiol (2002) 45:1285–1292.
Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J (1987) 6:3901–3907.[ISI][Medline]
Li JM, Nagpal P, Vitart V, McMorris TC, Chory J. A role for brassinosteroids in light-dependent development of Arabidopsis. Science (1996) 272:398–401.[Abstract]
Meuter-Gerhards A, Riegart S, Wiermann R. Studies on sporopollenin biosynthesis in Curcurbita maxima (DUCH)-II: the involvement of aliphatic metabolism. J. Plant Physiol (1999) 154:431–436.[ISI]
Morant M, Jorgensen K, Schaller H, Pinot F, Moller BD, Werck-Reichhart D, Bak S. CYP703 is an ancient cytochrome P450 in land plants catalyzing in-chain hydroxylation of lauric acid to provide building blocks for sporopollenin synthesis in pollen. Plant Cell (2007) 19:1473–1487.
Nishikawa S, Zinkl GM, Swanson RJ, Maruyama D, Preuss D. Callose (β-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth. BMC Plant Biol (2005) 5:22.[CrossRef][Medline]
Osthoff KS, Wiermann R. Phenols as integrated compounds of sporopollenin from Pinus pollen. J. Plant Physiol (1987) 131:5–15.[ISI]
Owen HA, Makaroff CA. Ultrastructure of microsporogenesis and microgametogenesis in Arabidopsis thaliana (L.) Heynh. Ecotype Wassilewskija (Brassicaceae). Protoplasma (1995) 185:7–21.[CrossRef][ISI]
Paxson-Sowders DM, Dodrill CH, Owen HA, Makaroff CA. DEX1, a novel plant protein is required for exine pattern formation during development in Arabidopsis. Plant Physiol (2001) 127:1739–1749.
Paxson-Sowders DM, Owen HA, Makaroff CA. A comparative ultrastructural analysis of exine pattern development in wild-type Arabidopsis and a mutant defective in pattern formation. Protoplasma (1997) 198:53–65.[CrossRef][ISI]
Piffanelli P, Ross JHE, Murphy DJ. Biogenesis and function of the lipidic structures of pollen grains. Sex. Plant Reprod (1998) 11:65–80.[CrossRef]
Rhee SY, Somerville CR. Tetrad pollen formation in quarted mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall. Plant J (1998) 15:79–88.[CrossRef][ISI][Medline]
Scott RJ. Pollen exine: the sporopollenin enigma and the physics of pattern. In: Molecular and Cellular Aspects of Plant Reproduction.—Scott RJ, Stead MA, eds. (1994) Cambridge: Cambridge University Press. 49–81.
Scott RJ, Spielman M, Dickinson HG. Stamen structure and function. Plant Cell (2004) 16:S46–S60.
Sheldon JM, Dickson HG. Determination of patterning in the pollen wall of Lilium henryi. J. Cell Sci (1983) 63:191–208.[Abstract]
Steiner-Lange S, Unte US, Eckstein L, Yang C, Wilson ZA, Schmelzer E, Dekker K, Saedler H. Disruption of Arabidopsis thaliana MYB26 results in male sterility due to non-dehiscent anthers. Plant J (2003) 34:519–528.[CrossRef][ISI][Medline]
Tsuchiya T, Toriyama K, Yoshikawa M, Ejiri S, Hinata K. Tapetum-specific expression of the gene for an endo-β-1,3-glucanase causes male sterility in transgenic tobacco. Plant Cell Physiol (1995) 36:487–494.
Wilmesmeier S, Wilermann R. Influence of EPTC (A-ethyl-dipropyl-thiocarbamate) on the composition of surface waxes and sporopollenin structure in Zea mays. J. Plant Physiol (1995) 146:22–28.[ISI]
Wilson ZA, Morroll AM, Dawson J, Swarup R, Tighe PJ. The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. Plant J (2001) 28:27–39.[CrossRef][ISI][Medline]
Worall D, Hird DL, Hodge R, Paul W, Draper J, Scott R. Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco. Plant Cell (1992) 4:759–771.
Zinkl GM, Preuss D. Dissecting Arabidopsis pollen–stigma interactions reveals novel mechanisms that confer mating specificity. Ann. Bot (2000) 85:15–21.
Zinkl GM, Zwiebel BI, Grier DG, Preuss D. Pollen–stigma adhesion in Arabidopsis: a species-specific interaction mediated by lipophilic molecules in the pollen exine. Development (1999) 126:5431–5440.[Abstract]
(Received September 23, 2007; Accepted November 25, 2007)
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