Molecular Plant Advance Access published online on June 25, 2008
Molecular Plant, doi:10.1093/mp/ssn027
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Microscopy and Bioinformatic Analyses of Lipid Metabolism Implicate a Sporophytic Signaling Network Supporting Pollen Development in Arabidopsis
a Department of Botany, Oklahoma State University, 104 Life Sciences East, Stillwater, OK 74078, USA
b College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
1 To whom correspondence should be addressed. E-mail ming.yang{at}okstate.edu, fax 405–744–7074, tel. 405–744–9508.
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Abstract |
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The Arabidopsis sporophytic tapetum undergoes a programmed degeneration process to secrete lipid and other materials to support pollen development. However, the molecular mechanism regulating the degeneration process is unknown. To gain insight into this molecular mechanism, we first determined that the most critical period for tapetal secretion to support pollen development is from the vacuolate microspore stage to the early binucleate pollen stage. We then analyzed the expression of enzymes responsible for lipid biosynthesis and degradation with available in-silico data. The genes for these enzymes that are expressed in the stamen but not in the concurrent uninucleate microspore and binucleate pollen are of particular interest, as they presumably hold the clues to unique molecular processes in the sporophytic tissues compared to the gametophytic tissue. No gene for lipid biosynthesis but a single gene encoding a patatin-like protein likely for lipid mobilization was identified based on the selection criterion. A search for genes co-expressed with this gene identified additional genes encoding typical signal transduction components such as a leucine-rich repeat receptor kinase, an extra-large G-protein, other protein kinases, and transcription factors. In addition, proteases, cell wall degradation enzymes, and other proteins were also identified. These proteins thus may be components of a signaling network leading to degradation of a broad range of cellular components. Since a broad range of degradation activities is expected to occur only in the tapetal degeneration process at this stage in the stamen, it is further hypothesized that the signaling network acts in the tapetal degeneration process.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTS AND DISCUSSIONMETHODSFUNDING |
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The sporophytic tapetum in plants undergoes a programmed degeneration process that exhibits some characteristics of apoptosis (also called programmed cell death) in animals. Although it is still under debate whether the tapetal degeneration process is a bona fide example of apoptosis (Frank and Barr, 2003; Kawanabe et al., 2006; to conform to the current thoughts, the phrase ‘programmed cell death’ and the word ‘apoptosis’ are deliberately avoided when describing the tapetal degeneration process in this report), the lipid and other materials mobilized from the degenerating tapetum are crucial for pollen development, as consistently demonstrated in many post-meiotic male sterile mutants. If a signaling network for mobilizing lipid and other materials to support pollen development exists, the major components of the signaling network are expected to act in the tapetum, not in the microspores or pollen. Such components have not been identified.
The connection between lipid metabolism in the tapetum and pollen fertility has been consistently demonstrated. The tapetum-specific MS2 protein in Arabidopsis is predicted to be involved in lipid metabolism, and its loss of function leads to male sterility (Aarts et al., 1997). The Arabidopsis NEF protein, which is predicted to be a plastid integral membrane protein, is required for lipid accumulation in the tapetum and for male fertility (Ariizumi et al., 2004). The loss-of-function mutations in the Arabidopsis AtGPAT1 gene cause perturbed tapetal degeneration and reduced secretion from the tapetum prior to and during pollen mitosis, leading to much reduced male fertility (Zhen et al., 2003). AtGPAT1 is predicted to mediate the initial step of glycerolipid biosynthesis in the extraplastidic compartments by its glycerol-3-phosphate acyltransferase activity. Male sterility is also linked with disruptions of lipid-rich structures in the tobacco tapetum when a mitochondrial pyruvate dehydrogenase is down-regulated by an antisense approach (Yui et al., 2003). Mutants in the rice Wax-deficient anther1 gene that is predicted to encode an integral membrane protein for wax and cuticle production exhibit abnormal lipid content in tissues including the tapetum and aborted pollen with defective exine (Jung et al., 2006).
Apparently, due to the nurturing role of the tapetum for pollen development, mutations in genes regulating the timing or progression of tapetal degeneration can have devastating effects on pollen development. It has been demonstrated that certain post-meiotically and tapetum-exclusively (comparing to other anther tissues) expressed transcription factors such as TAZ1 (Kapoor et al., 2002) in petunia and AtMYB103 (Li et al., 2007; Zhang et al., 2007) and ABORTED MICROSPORES (Sorensen et al., 2003) in Arabidopsis are required for male fertility; functional knockout or knockdown of these genes results in premature degeneration of the tapetum with impaired secretion and pollen degeneration prior to pollen mitosis. The association between a premature onset of tapetal degeneration and pollen abortion has also been observed in Brassica napus with the knockdown of a CCAAT-binding factor in the tapetum (Lévesque-Lemay et al., 2003) and in a rice thermosensitive male sterile mutant (Ku et al., 2003). In an opposite scenario, delayed tapetal degeneration in the rice tdr mutant defective in a basic helix-loop-helix protein seems to cause male sterility (Li et al., 2006). The MS1 protein in Arabidopsis is a transcription factor possibly specifying the programmed degeneration process in the tapetum, which affects pollen wall formation and fertility (Vizcay-Barrena and Wilson, 2006; Ito et al., 2007; Yang et al., 2007). Although its molecular function is unknown, the rice tapetum-specific RTS gene, whose promoter directs similar expression patterns in both monocotyledonous and dicotyledonous plants, is involved in tapetal development, which, in turn, affects pollen development (Luo et al., 2006).
In addition to the studies of individual gene functions, many genes in the anther walls and/or pollen have been identified by transcriptomic (Honys and Twell, 2004; Wijeratne et al., 2007) and proteomic (Holmes-Davis et al., 2005) approaches in Arabidopsis. Transcriptomic studies have also been carried out with rice (Wang et al., 2005) and maize (Ma et al., 2007) anthers. The Arabidopsis database (www.arabidopsis.org) provides readily searchable microarray data for genes expressed in organs at a number of developmental stages, including stamens at the floral stage 12 (Schmid et al., 2005). The data generated by Honys and Twell (2004) are particularly useful for detection of genes expressed in microspores and binucleate pollen, and the data by Schmid et al. (2005) can be used to detect genes expressed in stage 12 stamens.
In this report, we present evidence that the period from vacuolate microspores to early binucleate pollen is the period of peak-level secretion by the tapetum, and describe the screening for genes directly involved in lipid metabolism and their co-expressed genes that are expressed in stage 12 stamens but not in uninucleate microspores and binucleate pollen. The results suggest the presence of a signaling network participating in mobilizing lipid and other materials in the tapetum during early pollen development.
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RESULTS AND DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTS AND DISCUSSIONMETHODSFUNDING |
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The Peak of Tapetal Secretion during Early Pollen Development Occurs from the Vacuolate Microspore Stage to the Early Binucleate Pollen Stage
Although previous studies have determined that tapetal secretion occurs during pollen mitosis in Arabidopsis (Owen and Makaroff, 1995; Zhang et al., 2002), the dynamics of the secretion have not been precisely described. To determine the period of the secretion peak, which is deemed most critical for early pollen development, we examined the morphology of the anther wall and male reproductive cells in a small developmental window prior to and during the first pollen mitosis by transmission electron microscopy. The anther locule appeared empty at the uninucleate microspore stage, when the large vacuole in the microspore had not been formed (Figure 1A), indicating that the tapetal secretion was not occurring or was occurring at a very low level. At this stage, the tapetum appeared to contain a large amount of aggregated lipid globules with both high and medium levels of electron density. The exine layer of the microspore was substantially developed but the intine layer was inconspicuous. When the microspore reached the vacuolate stage, however, the locule was filled with an electron-dense material, presumably secreted from the tapetum (Figure 1B). The lipid globules in the tapetum appeared less aggregated than the previous stage but still maintained the distinctive shape, and the intine of the microspore became obvious. Just after the first pollen mitosis as indicated by the peripheral location of the generative cell, the secreted material was still at a high level in the locule, while the lipid globules in the tapetum became less distinctive than at the previous stage (Figure 1C). By the time the generative cells migrated away from the pollen periphery, the amount of the secreted material was already diminished and the internal structures in the tapetum were obscure (Figure 1D). Light microscopy studies of the anther cross-sections also revealed the same dynamics of the tapetal secretion (not shown). These observations indicate that the peak of the tapetal secretion coincides with the period from the vacuolate microspore stage to the early binucleate pollen stage.
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The above observations suggest that the vacuolate microspore stage to the early binucleate pollen stage is the most critical period for tapetal contribution to pollen development. A search for a signaling network supporting pollen development but expressed only in the sporophytic anther tissues was thus focused on the period of uninucleate microspore and binucleate pollen, as described in the following sections.
Genes of All Major Enzymes for Lipid Biosynthesis Are Expressed in Microspores and Binucleate Pollen
Because of the assumed dependence of pollen development on lipid material from the tapetum, we first examined whether some of the genes for lipid biosynthesis are not expressed in the uninucleate microspores and binucleate pollen. Genes of all major types of enzymes documented by Mekhedov et al. (2000) were identified in the Arabidopsis genome according to the Arabidopsis Gene Initiative (www.arabidopsis.org) and their expression in the uninucleate microspores and binucleate pollen was searched with the data from Honys and Twell (2004). Moreover, the same analysis was also carried out with genes for sulfolipid and cyclopropane fatty acid biosynthesis. As indicated in Table 1, one or more homologues of all the genes examined are expressed in the uninucleate microspores and/or binucleate pollen. This result suggests that the uninucleate microspores and binucleate pollen are not deficient in primary enzymes for lipid biosynthesis. It then raises the possibility that the development of the gametophyte relies on the tapetum for providing ‘raw’ lipid-building material, not the enzymes for lipid biosynthesis.
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Stamens Have but Uninucleate Microspores and Binucleate Pollen Lack the Expression of a PATATIN-LIKE PROTEIN Gene Likely Functioning in Mobilizing Lipid Reserves
Given that ‘raw’ lipid-building material from the tapetum is likely needed for the developing gametophyte, we next searched for genes that encode enzymes for lipid degradation. A search of the Arabidopsis genome using the key word ‘lipid’ produced 485 hits, which included various genes for both lipid biosynthesis and degradation, and other lipid-binding proteins. This list of genes was further expanded by searching for their homologs in the genome. We then searched the AtGenExpress microarray data for the genes of lipid-degradation enzymes expressed in the stage 12 stamen samples followed by a second search for the absence of their expression in the uninucleate microspores and binucleate pollen according to Honys and Twell (2004). We reasoned that certain lipid-degradation enzymes expressed in the stamen but not in the uninucleate microspores and/or binucleate pollen are likely responsible for providing the lipid-building material for the early gametophyte development. Here, it is assumed that the samples of the stage 12 stamens for collecting the microarray data included stamens in which the first pollen mitosis was occurring. Only one group of genes encoding the patatin-like proteins (PLPs) was found not to be expressed in the uninucleate microspores and binucleate pollen. This group of PLP genes consists of nine members (Table 2) that are predicted to be lipases involved in mobilizing lipid reserves in plants (Andrews et al., 1988; May et al., 1998) and animals (Zechner et al., 2005). Of the nine members, only PLP6 was found to be expressed in stage 12 stamens (and stage 10–11 flowers) while three others are expressed in stage 10–11 flowers but not in stage 12 stamens. Therefore, PLP6 is most likely among the PLPs to be responsible for mobilizing lipid in the sporophytic anther tissues to support early gametophyte development.
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Expression of PLPs Is Consistently Associated with the Expression of Signaling Components and Other Degradation Enzymes
If a PLP participates in the tapetal degeneration process, it is anticipated that it will be co-expressed with other enzymes for a broad range of degradation activities and with other signaling factors. To test this idea, genes co-expressed with PLP6 were analyzed using ATTED-II, a tool for identification of co-expressed genes (Obayashi et al., 2007). Because the goal of this investigation is only to identify some of the major components of a signaling network in the tapetum, the first 300 PLP6-co-expressed genes identified in ATTED-II were further reduced to 29 genes after excluding the ones either not expressed in stage 12 stamens or expressed in uninucleate microspores or binucleate pollen (Table 3). Some of these genes are known or predicted to encode signaling molecules, such as a leucine-rich repeat receptor kinase, an extra-large G-protein-related protein, other kinases, and transcription factors. In addition, genes encoding enzymes for degradation of proteins and cell wall components, cytoskeleton-related proteins, and other proteins are also represented in this gene group. These co-expressed proteins can potentially fit very well into a signaling network linking extracellular signal perception to intracellular signal relay and amplification to transcriptional regulation of genes for degradation enzymes. The co-expression of different types of degradation enzymes is consistent with the notion that the signaling network brings about a broad range of degradation activities. Because a broad range of degradation activities can be envisioned only during the tapetal degeneration process during early gametophyte development in the stamen, it is further hypothesized that the signaling network acts in the tapetal degeneration process.
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The above co-expression of the signaling molecules and degradation enzymes is unlikely coincidental because co-expression of similar genes was found with all the other PLPs. To illustrate this point, the genes co-expressed with the three PLPs that are expressed in stage 10–11 flowers but not in stage 12 stamens, uninucleate microspores, and binucleate pollen are shown in Tables 4
–6, respectively. It is interesting that Tables 5 and 6 contain genes (At2g23700 and At3g58780) expressed in the fourth whorl of the flower, which suggests that the corresponding signaling networks may act in other programmed degeneration processes in the silique.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTS AND DISCUSSIONMETHODSFUNDING |
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Plant Material and Microscopy Analysis
Arabidopsis thaliana Landsberg erecta ecotype was used for the microscopy analysis. The plant growth condition and the transmission electron microscopy and light microscopy methods were as previously described (Wang et al., 2004; Wu and Yang, 2005).
Bioinformatic Analysis
AtGenExpress searches and keyword searches were conducted in www.arabidopsis.org. Additional functional studies (BLASTP searches) and alignment of protein sequences were conducted in both www.arabidopsis.org and www.ncbi.nlm.nih.gov/blast/Blast.cgi. Co-expression analysis was carried out in www.atted.bio.titech.ac.jp/. The expression data by Honys and Twell (2004) were downloaded to an MS Excel file and searched with the AGI loci numbers.
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This work was supported by funds from Oklahoma State University.
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Acknowledgements |
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We thank Steven Cadenhead and Michael Cobbs for critically reading this manuscript.
No conflict of interest declared.
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