Yue Lou

A Genetic Pathway for Tapetum Development and Function in Arabidopsis

In anther development, tapetal cells take part in complex processes, including endomitosis and apoptosis (programmed cell death). The tapetum provides many of the proteins, lipids, polysaccharides and other molecules necessary for pollen development. Several transcription factors, including DYT1, TDF1, AMS, MS188 and MS1, have been reported to be essential for tapetum development and function in Arabidopsis thaliana. Here, we present a detailed cytological analysis of knockout mutants for these genes, along with an in situ RNA hybridization experiment and double mutant analysis showing that these transcription factors form a genetic pathway in tapetum development. DYT1, TDF1 and AMS function in early tapetum development, while MS188 and MS1 are important for late tapetum development. The genetic pathway revealed in this work facilitates further investigation of the function and molecular mechanisms of tapetum development in Arabidopsis.

The tapetal AHL family protein TEK determines nexine formation in the pollen wall

The pollen wall, an essential structure for pollen function, consists of two layers, an inner intine and an outer exine. The latter is further divided into sexine and nexine. Many genes involved in sexine development have been reported, in which the MYB transcription factor Male Sterile 188 (MS188) specifies sexine in Arabidopsis. However, nexine formation remains poorly understood. Here we report the knockout of TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK) leads to nexine absence in Arabidopsis. TEK encodes an AT-hook nuclear localized family protein highly expressed in tapetum during the tetrad stage. Absence of nexine in tek disrupts the deposition of intine without affecting sexine formation. We find that ABORTED MICROSPORES directly regulates the expression of TEK and MS188 in tapetum for the nexine and sexine formation, respectively. Our data show that a transcriptional cascade in the tapetum specifies the development of pollen wall.

Arabidopsis AT-hook Protein TEK Positively Regulates the Expression of Arabinogalactan Proteins for Nexine Formation

Nexine is a conserved layer of the pollen wall. We previously reported that the nexine layer is absent in the knockout mutant of Arabidopsis TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK) gene. In this study, we investigated the molecular regulatory functions of TEK in pollen development and identified the genes encoding Arabinogalactan proteins (AGPs) as direct targets of TEK, which are essential for nexine formation. Phenotypic similarity between tek and the TEK-SRDX transgenic lines suggest that TEK plays a role in transcriptional activation in anther development. Microarray analysis identified a total of 661 genes downregulated in tek, including four genes encoding AGPs, AGP6, AGP11, AGP23, and AGP40. Electrophoretic mobility shift assays showed that TEK could directly bind the nuclear matrix attachment region (MAR) and the promoter of AGP6. Chromatin immunoprecipitation followed by PCR analysis demonstrated that TEK is enriched in the promoters of the four AGP genes. Expression of AGP6 driven by the TEK promoter in tek partially rescued both nexine formation and plant fertility. These results indicate that TEK directly regulates AGP expression in the anther to control nexine layer formation. We also proposed that glycoproteins might be essential components of the nexine layer in the pollen wall.

The transcription factors MS188 and AMS form a complex to activate the expression of CYP703A2 for sporopollenin biosynthesis in Arabidopsis thaliana

The sexine layer of pollen grain is mainly composed of sporopollenins. The sporophytic secretory tapetum is required for the biosynthesis of sporopollenin. Although several enzymes involved in sporopollenin biosynthesis have been reported, the regulatory mechanism of these enzymes in tapetal layer remains elusive. ABORTED MICROSPORES (AMS) and MALE STERILE 188/MYB103/MYB80 (MS188/MYB103/MYB80) are two tapetal cell-specific transcription factors required for pollen wall formation. AMS functions upstream of MS188. Here we report that AMS and MS188 target the CYP703A2 gene, which is involved in sporopollenin biosynthesis. We found that AMS and MS188 were localized in tapetum while CYP703A2 was localized in both tapetum and locule. Chromatin immunoprecipitation (ChIP) showed that MS188 directly bound to the promoter of CYP703A2 and luciferase-inducible assay showed that MS188 activated the expression of CYP703A2. Yeast two-hybrid and electrophoretic mobility shift assays (EMSAs) further demonstrated that MS188 complexed with AMS. The expression of CYP703A2 could be partially restored by the elevated levels of MS188 in the ams mutant. Therefore, our data reveal that MS188 coordinates with AMS to activate CYP703A2 in sporopollenin biosynthesis of plant tapetum.

Magnesium Transporter 5 plays an important role in Mg transport for male gametophyte development in Arabidopsis.

During anther development the male gametophyte develops inside the locule and the tapetal cells provide all nutrients for its development. Magnesium Transporter 5 (MGT5) is a member of the MGT family and has dual functions of Mg export and import. Here, we show that male gametophyte mitosis and intine formation are defective in a mgt5 mutant. The transient expression of GFP-MGT5 revealed that MGT5 is localized in the plasma membrane. These findings suggest that in the male gametophyte MGT5 plays a role in importing Mg from the locule and that Mg is essential for male gametophyte development. The expression of MGT5 in the knockout ABORTED MICROSPORES (AMS) mutant (AMS being an essential regulator of tapetum) is tremendously reduced. Chromatin immunoprecipitation and mobility shift assay experiments demonstrated that AMS can directly bind the promoter of MGT5. An immunoelectron microscopy assay revealed that MGT5-His is localized to the plasma membrane of the tapetum. These findings suggest that AMS directly regulates MGT5 in the tapetum and thus induces export of Mg into the locule. The mgt5 plant exhibits severe male sterility while the expression of MGT5 under the tapetum-specific promoter A9 partly rescued mgt5 fertility. mgt5 fertility was restored under high-Mg conditions. These findings suggest that the mgt5 tapetum still has the ability to export Mg and that a sufficient supply of Mg from the tapetum can improve the importation of Mg in the mgt5 male gametophyte. Therefore, MGT5 plays an important role in Mg transport from the tapetum to the microspore.

Molecular Cell Biology of Pollen Walls

The pollen wall comprises the outer exine and the inner intine layers. It plays important roles in protecting pollen from various environmental stresses including microbial attack and in cell-cell recognition during pollination. The exine is further divided into a sexine and a nexine layer. The material for the exine is provided directly by the tapetal cells. The pollen wall of each plant has its unique pattern. After meiosis, the four microspores are enwrapped by callose to form a tetrad. The pollen-wall pattern is determined at tetrad stage. In contrast, the intine is synthesized by the microspore itself. Many genes have been identified from male-sterile mutants in Arabidopsis thaliana and rice during recent years. The majority of these genes are involved in pollen-wall formation including tapetal development, sporopollenin biosynthesis and transport, callose wall and primexine deposition. This chapter introduces the recent advance of pollen-wall formation in genetic and molecular level.

Positive regulation of AMS by TDF1 and the formation of a TDF1–AMS complex are required for anther development in Arabidopsis thaliana

Tapetum development and pollen production are regulated by a complex transcriptional network that consists of a group of tapetum-specific Arabidopsis transcription factors (TFs). Among these TFs, DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION 1 (TDF1) encodes an R2R3 MYB factor, and ABORTED MICROSPORE (AMS) encodes a basic helix-loop-helix (bHLH) factor. However, knowledge regarding the regulatory role of TDF1 in anther development remains limited. Here, we discovered that TDF1 directly regulates AMS via an AACCT cis-element. We found the precocious AMS transcript and absence of AMS protein in ams-/- gpTDF1:AMS-FLAG lines, suggesting the timing of the TDF1-regulated AMS expression is a prerequisite for AMS functioning. We found that TDF1 interacts with AMS. Additionally, the TDF1-AMS complex additively promotes the expression of AMS-regulated genes, suggesting that TDF1 and AMS regulate the downstream genes through a feed-forward loop. EPXB5, encoding a beta-expansin family protein, is another direct target of TDF1, and it is highly expressed in the tapetum and pollen grains. The TDF1-AMS complex acts in concert to activate EXPB5 expression through a feed-forward loop. The identification of the regulatory pathway between TDF1 and AMS provides an interlocked feed-forward loop circuit that precisely regulates the transcriptional cascades that support anther development.

The regulation of sporopollenin biosynthesis genes for rapid pollen wall formation

A feed-forward loop facilitates the expression of the sporopollenin biosynthesis pathway genes, thereby promoting rapid sexine formation. Sporopollenin is the major component of the outer pollen wall (sexine). It is synthesized using a pathway of approximately eight genes in Arabidopsis (Arabidopsis thaliana). MALE STERILITY188 (MS188) and its direct upstream regulator ABORTED MICROSPORES (AMS) are two transcription factors essential for tapetum development. Here, we show that all the sporopollenin biosynthesis proteins are specifically expressed in the tapetum and are secreted into anther locules. MS188, a MYB transcription factor expressed in the tapetum, directly regulates the expression of POLYKETIDE SYNTHASE A (PKSA), PKSB, MALE STERILE2 (MS2), and a CYTOCHROME P450 gene (CYP703A2). By contrast, the expression of CYP704B1, ACYL-COA SYNTHETASE5 (ACOS5), TETRAKETIDE a-PYRONE REDUCTASE1 (TKPR1) and TKPR2 are significantly reduced in ams mutants but not affected in ms188 mutants. However, MS188 but not AMS can activate the expression of CYP704B1, ACOS5, and TKPR1. In ms188, dominant suppression of MS188 homologs reduced the expression of these genes, suggesting that MS188 and other MYB family members play redundant roles in activating their expression. The expression of some sporopollenin synthesis genes (PKSA, PKSB, TKPR2, CYP704B1, and ACOS5) was rescued when MS188 was expressed in ams. Therefore, MS188 is a key regulator for activation of sporopollenin synthesis, and AMS and MS188 may form a feed-forward loop that activates the expression of the sporopollenin biosynthesis pathway for rapid pollen wall formation.