Glycerol-3-Phosphate Acyltransferase 6 (GPAT6) Is Important for Tapetum Development in Arabidopsis and Plays Multiple Roles in Plant Fertility

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1 Molecular Plant Volume 5 Number 1 Pages January 2012 RESEARCH ARTICLE Glycerol-3-Phosphate Acyltransferase 6 (GPAT6) Is Important for Tapetum Development in Arabidopsis and Plays Multiple Roles in Plant Fertility Xiao-Chuan Li 2, Jun Zhu 2, Jun Yang, Guo-Rui Zhang, Wei-Feng Xing, Sen Zhang and Zhong-Nan Yang 1 College of Life and Environmental Sciences, Shanghai Normal University, Shanghai , China ABSTRACT Glycerol-3-phosphate acyltransferase (GPAT) mediates the initial synthetic step for the formation of glycerolipids, which act as the major components of biological membranes and the principal stored forms of energy. GPAT6 is a member of the Arabidopsis GPAT family, which is crucial for cutin biosynthesis in sepals and petals. In this work, a functional analysis of GPAT6 in anther development and plant fertility was performed. GPAT6 was highly expressed in the tapetum and microspores during anther development. The knockout mutant, gpat6, caused a massive reduction in seed production. This report shows that the ablation of GPAT6 caused defective tapetum development with reduced endoplasmic reticulum (ER) profiles in the tapetum, which largely led to the abortion of pollen grains and defective pollen wall formation. In addition, pollen germination and pollen tube elongation were affected in the mutant plants. Furthermore, the double mutant analysis showed that GPAT6 and GPAT1 make joint effects on the release of microspores from tetrads and stamen filament elongation. This work shows that GPAT6 plays multiple roles in stamen development and fertility in Arabidopsis. Key words: glycerolipid. Arabidopsis GPAT6; stamen development; tapetum; endoplasmic reticulum; exine and pollen coat; INTRODUCTION In Arabidopsis (Arabidopsis thaliana), stamen development involves the formation of an anther with four somatic layers (from the exterior to the interior: epidermis, endothecium, middle layer, and tapetum). These house microspores and a filament that supports the anther for nutrient transport and optimal self-pollination (Goldberg et al., 1995; Alvarez-Buylla et al., 2010). Both sporophytic and gametophytic tissues are responsible for the development of microspores, which can be readily monitored by reference to the defined stages (Owen and Makaroff, 1995; Sanders et al., 1999; Wang et al., 2008; Alvarez-Buylla et al., 2010). The tapetum layer in Arabidopsis plays a vital role in microspore development through providing diverse substances (Scott et al., 2004). At the tetrad stage, the tapetal cells transit to the secretory type, which accompany the first obvious appearance of the extensive endoplasmic reticulum layers (ERs). The microspores are released from tetrads by tapetum-produced callases during the tetrad stage (Stieglitz and Stern, 1973; Alvarez-Buylla et al., 2010). During and after that, tapetum ERs are enlarged. In late stage 8, the tapetal cells are characterized by a large stack of extensive ER. Simultaneously, a large amount of fine fibrillar materials, which are thought to be the exine wall precursors, are secreted into the locule from the tapetal cells (Owen and Makaroff, 1995; Scott et al., 2004). When the exine layer is essentially formed, one of specialized plastids the elaioplasts and the tapetosomes accumulate large amounts of lipids and are generally present within the tapetal cells (Hernandez-Pinzon et al., 1999). Following the second mitotic cycle of the pollen grains, the tapetum ruptures, and the lipids in elaioplasts and tapetosomes are 1 To whom correspondence should be addressed. znyang@shnu. edu.cn, tel , fax These authors contributed equally to this work. ª The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: /mp/ssr057, Advance Access publication 10 July 2011 Received 24 April 2011; accepted 3 June 2011

2 132 Li et al. d GPAT6 for Stamen Development deposited on the exine wall to form the pollen coat, which is also called the tryphine (Piffanelli et al., 1998; Hernandez- Pinzon et al., 1999). In anther development, the extensive intracellular membrane system and biosynthesis of storage oil bodies are crucial for tapetal function and pollen maturation (Dorne et al., 1988; Piffanelli et al., 1998). The phospholipids are the main structural component of biological membranes, such as the cellular plasma membranes and the intracellular membranes of organelles. The triacylglycerols are a major form of energy storage for many organisms. Phospholipids and triacylglycerols are two types of glycerolipids, which are formed by esterification of glycerol with fatty acids (Coleman and Lee, 2004; Athenstaedt and Daum, 2006; van Meer et al., 2008). Heterotrophs can absorb glycerolipids for their needs, but glycerolipids can also be synthesized de novo through the glycerol phosphate pathway in most organisms (Gimeno and Cao, 2008). Most plants are autotrophs, so glycerolipid biosynthesis is considered to be essential for cellular processes that include membrane biogenesis, the maintenance of membrane integrity, energy storage, and other biological processes (Gimeno and Cao, 2008). In the glycerol phosphate pathway, glycerol-3-phosphate acyltransferase (GPAT) catalyzes the committed step in glycerolipid biosynthesis (Gimeno and Cao, 2008). Plants contain three types of GPAT that belong to different glycerolipid biosynthetic pathways, which are compartmentalized in the mitochondria, ER, or plastid (Browse et al., 1986; Kunst et al., 1988; Xu et al., 2006). The ERs are the major site for glycerolipid biosynthesis, but the mechanisms of the coordination and exchange of glycerolipids between intracellular compartments have also been discovered (Browse et al., 1986; Kunst et al., 1988; Murata and Tasaka, 1997; Benning, 2008). The GPAT family in Arabidopsis has eight conserved, membrane-bound GPATs (Zheng et al., 2003; Beisson et al., 2007). In the GPAT family, GPAT1 is a mitochondrial imported protein that characteristically plays a pivotal role in pollen development. Microscopic examinations of gpat1 mutant plants show perturbed degeneration of the tapetum, which is associated with altered ER profiles and reduced secretion. The ER profiles and lipid contents are also reduced in gpat1 pollen grains (Zheng et al., 2003). In a previous report, GPAT6 was considered to be crucial for cutin biosynthesis in sepal and petal formation (Li-Beisson et al., 2009). In this work, we have studied the roles of GPAT6 in the reproductive processes of Arabidopsis. We provide evidence that GPAT6 plays crucial roles in tapetum and pollen development. This manuscript also shows that the GPAT6 and GPAT1 have joint effects on the process of microspore releasing and stamen filament elongation. RESULTS GPAT6 Is Mainly Expressed in Inflorescence Previous investigations have shown that GPAT6 is dominantly expressed in the inflorescence (Zheng et al., 2003; Beisson et al., 2007). To examine the expression pattern of GPAT6 further, we made a construct with the b-glucuronidase (GUS) reporter driven by the GPAT6 promoter and transfected it into Arabidopsis plants. GUS activity was detected within the flowers, including in the sepals, stigma, filaments, and anthers, but not in carpels (Figure 1A, 1B, and 1D). GUS staining was also observed in mature pollen grains and pollen tubes during pollen germination (Figure 1C). In vegetative tissues, GUS activity could also be detected in trichomes (Supplemental Figure 1). As the fertility of the gpat6 mutant declined (see below), RNA in-situ hybridization experiments were performed to study the detailed expression pattern of GPAT6 during anther development. The GPAT6 mrna transcript was initially detected in microspore mother cells and tapetal cells at the premeiosis stage (Figure 1E). The signal increased significantly in tapetal cells and tetrads during stage 7 (Figure 1F). During microgametogenesis, the expression of GPAT6 was detected specifically in the tapetum and microspores (Figure 1G and 1H), which suggests that GPAT6 may be involved in anther development. Knockout of GPAT6 Causes Reduced Fertility We isolated a semi-sterile mutant from a population of Arabidopsis ecotype Columbia-0 (Col-0) mutagenized with the insertion of T-DNA, which contains a kanamycin resistance gene (Qin et al., 2003; Figure 2A). The fertility of the mutant varied, depending on different development stages: in the siliques that formed early, very few seeds were observed (gpat6 in Figure 2A); seed yield gradually increased as the development of the inflorescence progressed (L-gpat6 in Figure 2A). In total, the mutant plant contained approximately 2.4 seeds per silique compared with 50 seeds per silique on average in the wild-type. Siliques with a normal seed yield were produced by crosspollination of the mutant stigma with wild-type pollen, which indicated that the mutations did not affect female fertility. In the reciprocal crosses using the heterozygous pollen grains to pollinate the wild-type plants, the ratio of kanamycin resistance to sensitivity was 0.9 (538:597), which indicated that the development of the mutant male gametophytes might be altered. Progeny of these heterozygous mutant plants segregated fertile and mutant plants with a ratio of 3.6 (727:202; 2 = 4.78, P, 0.05). These results indicated that the mutation was caused by a single recessive nuclear mutation and the development of the male gametophyte was slightly affected. Thermal asymmetric interlaced (TAIL) PCR amplification (Liu et al., 1995) and sequencing of the T-DNA flanking sequence showed that the T-DNA was inserted into the intron of GPAT6 (At2g38110; Figure 2B). PCR analysis with T-DNA and genome-specific primers indicated that all mutant plants analyzed (n = 60) were homozygous for the insertion (data not shown). Reverse transcription (RT) PCR showed that the At2g38110 was not expressed in mutant plants inflorescences (Supplemental Figure 2). This indicated that the sterile phenotype was genetically linked with GPAT6.

3 Li et al. d GPAT6 for Stamen Development 133 Figure 1. Expression Analysis of GPAT6 and GPAT1. (A D) Promoter GUS staining of (A) inflorescence, (B) stamen, (C) pollen tube, (D) pistil, where the GUS staining signal is found specifically in the stigma, but not in the carpels. Scale bar = 200 lm. (E I) In-situ hybridization of GPAT6 transcript with antisense probe within the (E) stage 5 anther, (F) stage 7 anther, (G) stage 8 anther, (H) stage 11 anther. (I) In-situ hybridization of GPAT6 transcript with sense probe. The signal is detected initially in stage 5, peaking in stages 7 and 8. (J N) In-situ hybridization of GPAT1 transcript with antisense probe within the (J) stage 5 anther, (K) stage 7 anther, (L) stage 8 anther, (M) stage 11 anther. (N) In-situ hybridization of GPAT1 transcript with sense probe. The signal of GPAT1 transcript is similar with GPAT6 transcript. Bar = 20 lm. A genetic complementation experiment was performed to verify the sterile phenotype results from the T-DNA insertion in GPAT6. The genomic fragment that contained the entire GPAT6 gene, the putative upstream promoter region, and the downstream region was cloned and introduced into homozygous mutant plants. Ten transgenic lines were obtained and all of these plants showed normal fertility (C-gpat6 in Figure 2A). These results confirmed that GPAT6 was responsible for the mutant phenotype. As two of the knockout mutants (gpat6-1 and gpat6-2) were previously reported (Li-Beisson et al., 2009), this mutant was designated as gpat6-3. We also clarified whether the fertility recovery of gpat6-3 in the later-formed siliques was related to the expression variation of other GPATs. The total inflorescence RNA of wild-type and gpat6-3 from these two stages was isolated and semiquantitative RT PCR was performed. The results showed that the expression levels of GPAT4 and GPAT8 were increased in the later stages of inflorescence of gpat6-3 (Supplemental Figure 2), but did not change obviously in wild-type (data not shown). In a previous report, GPAT6 was considered to be crucial for sepal and petal surface cutin monomer biosynthesis (Li-Beisson et al., 2009). The ablation of GPAT6 produced abnormal flowers, most of which displayed organ fusion and did not open (Li-Beisson et al., 2009). This phenotype was also detected in gpat6-3. Furthermore, more normally opened flowers in gpat6-3 were found in the flowers that formed later. Toluidine blue permeability tests of gpat6-3 sepal epidermis indicated that the cutin layer of sepals in the flowers formed later were partially rescued (Supplemental Figure 3). Microspore Development Is Aberrant in the gpat6 Mutants Alexander s staining (Alexander, 1969) showed that approximately 50% of pollen grains were degraded in gpat6-3 anthers (Figure 3G). A similar pollen abortion phenotype was also observed in gpat6-1 anthers (Supplemental Figure 4). To determine whether there was any defect in male mitosis of the gpat6 pollen grains, 4,6-diamidino-2-phenylindole (DAPI) was used to stain pollen grains from mutants (Ross et al., 1996). Two sperms and one vegetative nucleus were observed in the gpat6-3 pollen grains, which is similar to that in the wildtype; this indicated that mitosis is not affected in gpat6 pollen (Supplemental Figure 5). To determine the defects of anther development in the mutants, semi-cross-sections of gpat6-3 and wild-type anthers were examined by light microscopy. In Arabidopsis, anther development can be divided into 14 well-ordered stages according to the morphological characteristics (Sanders et al., 1999). From stage 1 to stage 7, no detectable differences in anther development were observed between the gpat6-3 and the wild-type plants. Microspore mother cells were able to complete meiosis and form tetrads in the gpat6-3 anthers (Figure 4B and 4G). However, when the callose wall of the tetrads

4 134 Li et al. d GPAT6 for Stamen Development Figure 2. Characterization of the Wild-Type (Col) and gpat6-3 Mutant. (A) Phenotypes of the 35-day-old wild-type Arabidopsis ecotype Columbia (WT) with normal fertility; 35-day-old gpat6-3 mutant (gpat6) with very small siliques (white arrow head) containing no seeds; gpat6-3 reproductive organs at the 55-day-old development stage (L-gpat6) with some siliques rescued and bearing seeds; and a gpat6-3 plant containing the GPAT6 transgene (C-gpat6) with normal fertility. (B) Gene structure of GPAT6 and T-DNA insertion. The left (LB) and right (RB) borders of the T-DNA sequences are shown. The T-DNA contains a kanamycin resistance (KanR) gene. Black box, exon; gray line, intron; light gray line, untranslated regions. L-TEST F and L-TEST R primers used for linkage analysis. degenerated, the gpat6-3 tapetal cells were more obviously expanded than the wild-type tapetal cells (Figure 4C and 4H). At stage 10, the wild-type microspores were densely stained, whereas some pollen grains in the gpat6-3 mutant underwent degradation (Figure 4D and 4I). However, the degradation of the tapetum and the dehiscence program of the anther were not affected (Figure 4E and 4J). The ERs in Tapetum and the Loading of the Pollen Wall Are Altered in gpat6 To elucidate the mechanism of sterility in gpat6 further, we compared the ultrastructure of pollen grains in both wild-type and gpat6-3 homozygous plants by scanning electron microscopy (SEM). SEM analysis showed that some of gpat6-3 pollen grains were collapsed (Figure 5A and 5B). As the altered pollen structure might be related to defective pollen wall formation, we further examined the exine of gpat6-3. A regular reticulate pattern was observed in wild-type mature pollen grains (Figure 5B and 5E). However, the surface of gpat6-3 pollen grains exhibited partially incomplete or flawed patterns that suggested pollen wall formation was aberrant in the mutant (Figure 5D, 5F, and 5G). Since the tapetal cells play critical roles in pollen development, we examined the ultrastructural changes in the mutant tapetal cells by transmission electron microscopy (TEM). Until anther development stage 7, the ultrastructures in gpat6-3 tapetal cells appeared to be comparable with the wild-type (Figure 6A and 6B). In stage 8, the tapetal cells contained large

5 Li et al. d GPAT6 for Stamen Development 135 Figure 3. Examination of Stamens in Wild-Type, gpat1, gpat6-3, and gpat1 gpat6 Double Mutant. (A E) The length of stamen filaments in (A) wild-type, (B) gpat6-3, (C) gpat1, and (D, E) gpat1 gpat6 double mutant. (A) The stamen filaments in the wild-type are slightly longer than the pistils. (B, C) In gpat1 and gpat6-3, the stamen filaments are shorter than the pistils. (D) In the gpat1 gpat6 double mutant, distinctively short stamen filaments are generated with shrunken anthers (arrow head). (E) This phenotype was partly rescued in later-generated flowers. (F J) Examination of pollen production in (F) wild-type, (G) gpat6-3, (H) gpat1, and (I, J) gpat1 gpat6 double mutant anthers by Alexander s staining. The production of pollen grains in gpat1 and gpat6-3 are diminished compared to wild-type pollen production, and no pollen grains can be detected in the double mutant in both early (I) and late (J) generated flowers. S, stigma; SF, stamen filament; PGs, pollen grains. Bar = 50 lm. stacks of ER with electron-dense contents (Figure 6C and 6D), which is a feature generally considered to be closely related to the secretory function of tapetal cells. However, ER dilation in the mutant was barely detectable (Figure 6E and 6F). In Arabidopsis, tapetal cells accumulated lipid materials at the bicellular pollen grain stages to form tapetosomes and elaioplasts for pollen coat deposition (Owen and Makaroff, 1995). Therefore, very few lipid bodies are deposited in wild-type tapetum during stage 8 (Figure 6C). In contrast, at the same stage, osmophilic material, which is usually considered to be lipids accumulated in the plastids, and osmophilic material droplets, which are morphologically similar to tapetosomes, accumulated in the tapetal cells of gpat6-3 (Figure 6E) (Owen and Makaroff, 1995). However, the tapetosomes and elaioplasts in both wild-type and gpat6-3 tapetum appeared with no visible difference at stage 10 (Figure 6G and 6H). These results indicated that tapetum development was abnormal in gpat6. TEM also demonstrated exine deposition of both wild-type and gpat6-3. At the tetrad stage, exine was regularly deposited outside the microspore in gpat6-3, which was similar to the wild-type (Figure 6I and 6J). After microspores were released, the exine monomers were polymerized on the surface of microspores, forming the characteristic bacula and tectum (Figure 6K). In the mutant, exine deposition was irregular and partial structures of the exine were absent on some microspores (Figure 6L). In mature pollen grains, aberrant exine formation was also observed in gpat6-3 (Figure 6M 6P), although the intine layer was regularly formed in wild-type and gpat6-3 plants (Figure 6M 6P). When the pollen grains matured, the pollen coat filled the spaces in between the baculae in the wild-type (Figure 6M and 6O), whereas the osmophilic material deposited on the exine surface in the gpat6-3 was reduced, which indicated that the loading of the pollen coat was flawed (Figure 6N and 6P). Furthermore, wild-type pollen grains normally contain numerous electron-dense and electron-transparent small storage bodies that are encircled by the intracellular membrane (Figure 6Q). However, the stack of ER was hardly detectable and many irregularly shaped vacuoles were found in the gpat6-3 survival pollen grains (Figure 6R), which suggested that the contents of gpat6 pollen grains were also affected. The Germination and Growth of Pollen Grains Are Affected in gpat6 Due to the aberrant contents of gpat6-3 pollen, the pollen germination and the growth of the pollen tubes were examined under in vitro conditions. The germination assay showed that the germination rate of pollen grains from gpat6-3 (46%) was lower than that of the wild-type pollen grains (62%; Figure 7A). In addition, the length of the gpat6-3 pollen tubes was decreased compared with that of the wild-type tubes after

6 136 Li et al. d GPAT6 for Stamen Development Figure 4. Anther Development from Stage 5 to Stage 12 in the Wild-Type (Col), the gpat6-3 Mutant, and the gpat1 gpat6 Double Mutant. Anther sections: (A E, Q, R), wild-type; (F J, S, T), gpat6-3 mutant; (L P, U, V), gpat1 gpat6 double mutant. (A, F, L; anthers during stage 6) No differences are detected between these three lines. (B, G; anthers during stage 7) No differences are detected between the wild-type and gpat6-3 mutant lines. (M; anthers during stage 7) The tapetal cells of the double mutant become swollen, highly vacuolated, and the tetrads begin to collapse. (N; after phase m) The locule of the double mutant begins to shrink and the tapetum and tetrads are abolished. (C, H; anthers during stage 8) The gpat6-3 tapetum is more obviously expanded than wild-type. (O; anthers during stage 8) The locule of the gpat1 gpat6 double mutant is shrunken and no microspores can be detected. (D, I; anthers during stage 10) Defective pollen grains have emerged in gpat6-3. (E, J; anthers during stage 12) Both malformed and healthy pollen grains are observed in the gpat6-3 anther locule. (P; anthers during stage 12) The locule of the gpat1 gpat6 double mutant resembles the locule of the double mutant in stage 8. (Q V) Aniline blue staining of the anthers. Aniline blue fluorescence (arrow head) remains detectable in the (V) double mutant anthers after (U) tetrad stages. Aniline blue fluorescence, detectable in the tetrad stage (Q, S), cannot be detected in either (R) wild-type or (T) gpat6-3 anthers. E, epidermis; En, endothecium; ML, middle layer; Ms, meiocytes; Msp, microspores; SL, shrunken locule; T, tapetum; Tds, tetrads. Bar = 20 lm. germinating for 8 h. The data showed that 43% of gpat6-3 pollen tubes were in the range of lm, whereas 75% of wild-type pollen tubes were between 200 and 500 lm in length (Figure 7B). The growth of pollen tubes was further investigated in vivo by pollinating the pre-emasculated wildtype flowers with either wild-type or mutant pollen grains. The pollen tubes were stained with aniline blue and viewed under UV illumination 3 h after pollination. The results showed that the growth of the gpat6-3 pollen tubes was much slower than the wild-type (Figure 7C and 7D). However, after germination for 24 h, the pollen tubes of gpat6-3 could reach to the bottom of the transmitting tract, just like the wild-type tubes (data not shown). The Double Mutant, gpat1 gpat6, Exhibits Completely Male Sterile and Short Filament Phenotypes GPAT1 has been shown to be involved in pollen fertility by mediating glycerolipid assembly in tapetum and microspores (Zheng et al., 2003). Most of the pollen grains were abolished in the gpat1 mutant, as shown by Alexander s stain (Figure 3H). Through RNA in-situ hybridization, we observed that, in anther development, GPAT1 exhibited a similar expression pattern to GPAT6 (Figure 1N 1Q). As a result of the similar functions of GPAT6 and GPAT1, we constructed a double mutant (gpat1 gpat6) to investigate the deficient phenotype in anther development. The gpat1 gpat6 double mutant exhibited a completely male sterile phenotype (Figure 3I and 3J). Semi-thick sections of anthers were generated to compare development of anthers in the wild-type, gpat6-3, and gpat1 gpat6. Until stage 6, the double mutant appeared to be comparable with the wild-type and gpat6-3 plants in tapetal differentiation and meiosis (Figure 4A, 4F, and 4L). However, early in stage 7, the tapetal cells of the double mutant became swollen, vacuolated, and enlarged, and invaded the spaces of the locules (Figure 4M). Subsequently, the locules of the double mutant began to shrink and the microspores could not be released from the tetrads (Figure 4N). In the later stages, the locules shrank to a linear, dark staining gap and no individual microspores were detected in the gpat1 gpat6 double mutant (Figure 4O and 4P). In addition, aniline blue staining was performed to test the callose degradation in wild-type, gpat6-3, and gpat1 gpat6 anthers. At tetrad stage, callose fluorescence could be clearly observed in these three types of locules (Figure 4Q, 4S, and 4U). By stage 8, callose fluorescence was not detected in the wild-type and gpat6-3 locules (Figure 4R and 4T), which suggested that the callose was completely degraded. However, we found that the fluorescence of callose residue was still detectable in the shrunken locules by utilizing aniline blue staining (Figure 4V). As the callose is digested by tapetum-produced callase, these results implied that the

7 Li et al. d GPAT6 for Stamen Development 137 Figure 5. SEM Examination of Dehiscent Anthers and Pollen Grains. Pollen grains of wild-type (A, B, E) and gpat6-3 (B, D, F, G) plants. (A, B) Emasculated pollen grains. Several gpat6-3 pollen grains are collapsed and aborted. Bar = 5 lm. (C, D) Higher magnification of pollen grains. (D) Defective pollen grains of gpat6-3. Bar = 5 lm. (E) Wild-type pollen grains with a regular reticulate exine pattern. (F, G) The gpat6-3 pollen grains with impaired or flawed exine pattern (white arrow head). Bar = 2 lm. production of the callase was affected in the double mutant and the functions of GPAT1 and GPAT6 were largely redundant in tapetal development during stage 7. In normal flower development, an opened flower contains stamens, which are generally slightly longer than the pistils (Figure 3A). This allows the anther to release pollen onto the stigma, which results in optimal pollination efficiency (Edlund et al., 2004). The length of stamen filament examination showed that the stamens of the gpat6-3 or gpat1 were slightly shorter than the pistils (Figure 3B and 3C). In the gpat1 gpat6 double mutant, markedly short stamen filaments with shrunken anthers were generated, although this phenotype was partly rescued in the flowers generated later in the process as the development of the inflorescence progressed (Figure 3D and 3E). This phenotype suggested that ablation of GPAT1 or GPAT6 caused defective stamen filament elongation. DISCUSSION GPAT6 Is Important for ER Assembly in the Tapetum The tapetal cells of the Arabidopsis anther play essential roles in the development of pollen grains from microspores (Scott et al., 2004). The ER is crucial for normal functioning of tapetal cells (Zheng et al., 2003) and glycerolipids are the major components of ER, as they form the phospholipid bilayer (van Meer et al., 2008). Glycerolipids are synthesized de novo through the glycerol phosphate pathway, in which GPATs catalyze the committed step (Gimeno and Cao, 2008). It has been reported that GPAT6 transfers acyl groups to either the sn-1 or the sn-2 position of glycerol-3-phosphate (Yang et al., 2010). Wild-type tapetal cells were highly active after the stage of meiosis, with large stacks of intensive ER (Figure 6A and 6C; Owen and Makaroff, 1995). However, ER profiles in the gpat6-3 tapetum were greatly reduced (Figure 6B), which indicated that GPAT6 is important for ER assembly in the tapetum. The high level of expression of GPAT6 in tapetal cells at this stage also supports the participation of GPAT6 in ER assembly in the tapetum (Figure 1J and 1K). In the GPAT family, GPAT1 and GPAT8 were identified to be localized sub-cellularly in the mitochondria or ER, respectively (Zheng et al., 2003; Gidda et al., 2009). GPAT6 shares a similar amino acid sequence with GPAT8 and a verified ER retrieval signal exists for both GPAT6 and GPAT8 (Beisson et al., 2007; Gidda et al., 2009; Li et al., 2007). Therefore, GPAT6 is likely to be located in the ER. Furthermore, a previous study showed that GPAT1 also participated in the assembly of tapetum ER (Zheng et al., 2003). GPAT6 shared a similar expression pattern to GPAT1 in anther development (Figure 1E 1N). The ablation of either GPAT1 or GPAT6 exhibited reduced ER profiles in the tapetum. These showed that, in the tapetum, both GPAT1 and GPAT6 are essential for ER profile assembly and each of them alone was not sufficient for ER assembly in the tapetum. The timely and adequate degradation of the callose by tapetum-produced callase is important for microspore development (Scott et al., 2004; Wu and Yang, 2005; Zhu et al., 2008). In anther development, the synthesized callase enzymes first target the ER in tapetal cells and are secreted into the locule to digest the callose of the tetrads (Melchers et al., 1993; Carter et al., 2004). In the male sterile32 Arabidopsis mutant, callose degradation occurs before meiosis and is associated with the early formation of large stacks of ER in tapetal cells (Fei and Sawhney, 1999). Therefore, callose degradation is linked to the ER formation in tapetal cells after meiosis. Although the ER profiles were reduced in the gpat1 and gpat6 single mutant, the microspores were released normally. This indicated that the ER system still had the required functions to support callase synthesis. However, the callose wall of tetrads could not be dissolved and microspores were aborted in the gpat1 gpat6 double mutant (Figure 4U and 4V). Therefore, we presume that the ER system in the gpat1 gpat6 double mutant was severely impaired and lost the functions required to support callase synthesis. GPAT6 Exerts Multiple Effects on Plant Fertility GPAT6 was reported to participate in the formation of floral cutin in Arabidopsis. The gpat6 mutant produces abnormal flowers, many of which display organ fusion and do not open (Li-Beisson et al., 2009). In this work, our results show that GPAT6 is involved in anther development and plant fertility in Arabidopsis. Ingpat6-3, the exine deposition was partially disordered and the pollen coat was inadequately loaded (Figure 6N and 6P). The tapetum is believed to provide the materials for the formation of exine and pollen coat loading.

8 138 Li et al. d GPAT6 for Stamen Development Figure 6. TEM Examination of Anthers and Pollen Grains. (A, C, D, G) Tapetum of wild-type and (B, E, F, H) gpat6-3 mutant plants. During stage 7, the ultrastructure of (B) gpat6-3 tapetal cells are similar to (A) wild-type. (C) By late stage 8, little osmophilic material is accumulated in the normal wild-type tapetum, which contains (D) large stacks of endoplasmic reticulum (ER). (E) During late stage 8 in gpat6-3 anthers, large amounts of osmophilic material has accumulated in the tapetal cells and (F) little ER is detectable. The tapetosomes and elaioplasts in both (G) wild-type and (H) gpat6-3 tapetum exhibit no visible difference during stage 10. (I, K, M, O) Pollen wall of wild-type and (J, L, N, P) gpat6-3 mutant. At stage 7, the exine of (J) gpat6-3 tapetal cells are similar to (I) wild-type. Aberrant exine formation is observed in (K)

9 Li et al. d GPAT6 for Stamen Development 139 The exine monomers are created, modified, and exported by tapetum; pollen coat material is accumulated in the tapetosome and elaioplast compartments of the tapetum and deposited onto pollen when the tapetal cells rupture (Ma, 2005). In gpat6-3 tapetum, ER profiles were reduced and the development of the tapetum was defective. Therefore, the defective exine and pollen coat in gpat6-3 may result from the impaired tapetum. In gpat6-3, approximately half of the pollen grains were degraded, and the plants with reduced pollen content showed lower germination rates than the wild-type. In addition, the mutant showed unopened flowers and short stamen filaments, which may affect efficient self-pollination in gpat6-3. In eight members of the GPAT family, GPAT6 shows high and specific expression in flowers (Zheng et al., 2003; Beisson et al., 2007), which is consistent with the results of this present study. Therefore, GPAT6 appears to play multiple roles during plant reproductive development. GPAT is considered to mediate the initial step of synthesis of glycerolipids, which act as the principal stored forms of energy and the major components of biological membranes. The multiple roles of GPAT6 in plant reproductive development suggest a function for it in glycerolipid synthesis. GPAT6 was reported to esterify acyl groups to the sn-2 position of G3P to produce sn-2 monoacylglycerol (2-MAG), which may act as an intermediate for cutin biosynthesis (Yang et al., 2010). Cutin and exine may share some common metabolic synthetic pathways, and GPAT6 is known to be crucial for cutin synthesis (Scott et al., 2004; Pollard et al., 2008; Li-Beisson et al., 2009; Li et al., 2010). Therefore, it could be possible that the GPAT6 directly participates in exine formation. METHODS Plant Growth and Mutant Isolation The Arabidopsis plants used in this study are in the Columbia- 0 background. Seeds were sown on vermiculite and allowed to imbibe for 3 d at 4 C. Plants were grown under long-day conditions (16 h of light/8 h of darkness) in a growth room that was kept at approximately 22 C. The gpat6-3 mutant was characterized by the pski15 activation-tagging T-DNA mutant pools (Qin et al., 2003). The SALK mutants of SALK for GPAT6 and SALK for GPAT1 were bought from ABRC. Figure 7. In Vitro and In Vivo Germination of Both Wild-Type and gpat6-3 Pollen Grains. (A) Statistics of the in vitro germination rate of pollen grains from wild-type and gpat6-3 (means of at least 1000 pollen grains from three independent assays). (B) Statistics of length distribution for wild-type and gpat6-3 pollen tubes after in vitro germination for 8 h (means of at least 600 pollen grains from three independent assays). (C, D) In vivo germination of pollen grains of (C) wild-type and (D) gpat6-3 mutant in wild-type pistil for 3 h stained by aniline blue, which shows that the pollen grains of gpat6-3 slow pollen tube elongation. PTs, pollen tubes. Bar = 50 lm. Phenotypic Characterization and Microscopy Plants were photographed with a Canon digital camera (Powershot-A710IS). Flower images were taken using an Olympus dissection microscope with an Olympus digital camera. Alexander solution and DAPI staining were performed as described (Alexander, 1969; Ross et al., 1996). The epidermal permeability of sepals was performed as previously described (Li-Beisson et al., 2009). Photography was performed with an Olympus BX-51 microscope. For semi-thick light microscopy, the processes of fixation, dehydration, embedding, sectioning, and staining were performed as described by Zhang et al. (2007). The germination of pollen grains in vitro was performed as described by Boavida and McCormick (2007). The pollen germination rate was measured using light microscopy after incubation at C for 8 h. To assess growth of the pollen tubes in vivo, the pre-emasculated, matured wild-type gpat6-3 microspores compared to (L) the wild-type. (N and P) The exine of gpat6-3 pollen grain is irregularly loaded compared to (M, O) the wild-type. (N, P) The gpat6-3 pollen coat loading is inadequate. (N, M) are part of pollen present in (P, O), respectively. In (R), gpat6-3 pollen grains, some mis-shaped vacuoles are detected and hardly any ER can be observed. In (Q), wild-type pollen grains, numerous small storage bodies, and extensive intracellular membrane (arrow head) can be observed. Ba, bacula; E, exine; El, elaioplast; ER, endoplasmic reticulum; In, intine; P, plastid; PC, pollen coat; PR, prime bacula; T, tapetosomes; Te, tectum. V, vacuole. Bar = 2 lm.

10 140 Li et al. d GPAT6 for Stamen Development flowers were pollinated with pollen grains from gpat6-3 or wild-type plants. After 3 h, the pollinated pistils were collected and stained as described by Chaiwongsar et al. (2009). An Olympus BX51 microscope (Olympus, Japan) with an Olympus DX51 digital camera was used for cytological and pollen tube observations. For SEM examination, fresh stamens and pollen grains were coated with 8 nm of gold and observed using a JSM-840 microscope (JEOL). For TEM examination, Arabidopsis buds from the inflorescence were fixed and embedded as described by Zhang et al. (2007). Ultrathin sections ( nm thick) were observed with a JEM-1230 transmission electron microscope (JEOL). TAIL PCR and Isolation of Single and Double T-DNA Knockout Mutants The presence of the T-DNA inserted into the mutant was confirmed as described by Guan et al. (2008). Co-segregation of the T-DNA insertion site with the mutant phenotype was analyzed with AtLB3 (5#-TTGACCATCATACTCATTGCTG-3#)andthe plant-specific primers LP (5 -AAACCAGGTATTCTCGTCG-3 ) and RP (5 -ATAAAAGTTTGTAATGGCTGAC-3 ). For complementation, a DNA fragment of 4.5 kb, including 1.5-kb upstream and 0.5-kb downstream sequences, was amplified using PRIME-STAR Taq polymerase (Takara Biotechnology) with the following primers: CMP-F (5 -TcccgggGCACTTGATTAGCACCTTTCG-3 ) and CMP-R (5 -TggatccTGTCAATCAACTTCGTCGTCA-3 ). After verification by sequencing, the fragment was cloned into the pcambia1300 binary vector (CAMBIA; and then introduced into homozygous mutant plants using the infiltration method with Agrobacterium tumefaciens strain LBA4404 (Zhang et al., 2007). The transformants were selected on 1/2 MS culture medium with 20 mg L 1 hygromycin, and then screened for fertile plants. For SALK mutant identification, the T-DNA insertion site was verified as described above with the prok2 vectorspecific primer prok2-lb3 (5 -GACCGCTTGCTGCAACTCT-3 ) and genome-specific primers as follows: SALK LP (5 -CGTGACGTCGTTTTGAGAGA-3 ), SALK RP (5 - GTTGTAACGGGCGATACGTT-3 ), SALK LP (5 -GACCC- TATCTTCTTCCTC-3 ), and SALK RP (5 -GATGCCAAT- GAATCAACC-3 ). The mutant lines of SALK and gpat6-3 were crossed to generate double mutant gpat1 gpat6 plants. The homozygous double knockout plants were identified in a segregating F 2 population first by phenotype and then the genotype was confirmed by PCR. Expression Analysis For the expression analysis of GPATs, RNA was extracted by TRizol (Invitrogen) from inflorescences of mutant and wild-type plants from both early and late stages as the development of the inflorescence progressed. Semi-quantitative RT PCR for 30 cycles was used to assess the levels of expression of the GPATs, using the primer sets shown in Supplemental Table 1. For the promoter GUS fusion, the 1.7-kb promoter fragment upstream from the start ATG of GPAT6 was obtained by PCR with the following primers: Promoter-R (5 -TgaattcCAACGC- CACATAAGACAACTA-3 ) and Promoter-F (5 -TctgcagGAATG- GATATGGATTGGTGAA-3 ). After verification by sequencing, the fragment was cloned into pcambia1301 without the cauliflower mosaic virus 35S promoter binary vector (CAM- BIA; and transformation was performed as described above. GUS histochemical staining was performed as described by Li et al. (2008). In-situ hybridization was performed with the DIG (for digoxigenin) RNA Labeling Kit (Roche) and the DIG Probe Synthesis Kit (Roche). A GPAT6 or GPAT1-specific cdna fragment was amplified and cloned into the Psk vector. Antisense and sense digoxigeninlabeled probes were prepared with SalI orbamhi digestion and in vitro transcription using T3 and T7 RNA polymerases, respectively. SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online. 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