The ABORTED MICROSPORES regulatory network is required for postmeiotic male reproductive development in Arabidopsis thaliana

Abstract

The Arabidopsis thaliana ABORTED MICROSPORES (AMS) gene encodes a basic helix-loop-helix (bHLH) transcription factor that is required for tapetal cell development and postmeiotic microspore formation. However, the regulatory role of AMS in anther and pollen development has not been fully defined. Here, we show by microarray analysis that the expression of 549 anther-expressed genes was altered in ams buds and that these genes are associated with tapetal function and pollen wall formation. We demonstrate that AMS has the ability to bind in vitro to DNA containing a 6-bp consensus motif, CANNTG. Moreover, 13 genes involved in transportation of lipids, oligopeptides, and ions, fatty acid synthesis and metabolism, flavonol accumulation, substrate oxidation, methyl-modification, and pectin dynamics were identified as direct targets of AMS by chromatin immunoprecipitation. The functional importance of the AMS regulatory pathway was further demonstrated by analysis of an insertional mutant of one of these downstream AMS targets, an ABC transporter, White-Brown Complex homolog, which fails to undergo pollen development and is male sterile. Yeast two-hybrid screens and pull-down assays revealed that AMS has the ability to interact with two bHLH proteins (AtbHLH089 and AtbHLH091) and the ATA20 protein. These results provide insight into the regulatory role of the AMS network during anther development.

Figure 1.

The 549 Genes That Showed Altered Expression in the ams Mutant Were Clustered into 10 Groups by K-Mean Clustering. Columns from left to right in each cluster are expression profiles of the meiosis and earlier stages (Mei), the pollen mitosis I stage (PMI), the bicellular stage (Bi), and the pollen mitosis II stage (PMII), respectively. The y-axes show relative expression compared with wild-type expression. Cluster 1: Genes were slightly upregulated at the meiosis stage, greatly in the mitosis I and bicellular stages, and less at the mitosis II stage in the ams mutant. Cluster 2: Downregulated genes, slightly at the mitosis I stage but greatly at bicellular and mitosis II stages. Cluster 3: Genes upregulated at meiosis, mitosis I, and bicellular stages but slightly downregulated at the mitosis II stage. Cluster 4: Genes downregulated at all four stages. Cluster 5: Genes downregulated only at the mitosis I stage. Cluster 6: Genes downregulated only at the bicellular stage. Cluster 7: Genes downregulated at meiosis, mitosis I, and bicellular stages but no change at the mitosis II stage. Cluster 8: Genes upregulated only at the mitosis II stage. Cluster 9: Genes upregulated only at the bicellular stage. Cluster 10: Genes downregulated only at the mitosis II stage.

Figure 2.

Assay of AMS Binding Sites. (A) Schematic representation of the AMS-GST fusion construct. The XhoI and BamHI sites in pGEX-4T-GST were used as cloning sites for insertion of the AMS gene. (B) Random sequence oligonucleotides (RS-Oligo) used for target binding site selection. Oligo F is a sequence-specific sense primer, and Oligo R is a sequence-specific antisense primer, which were used for PCR amplification of AMS-selected oligonucleotides. (C) Canonical AMS binding motif analyzed by WebLogo (Web-based sequence logo generating application; Weblogo.berkeley.edu).

Figure 3.

qChIP-PCR Analysis of the Enrichment of AMS Regulatory Targets and the Predicted E-Boxes in Their Promoter Regions. Fold enrichment calculations from qPCR assays in three independent ChIP experiments. The predicted E-boxes are indicated as vertical black lines in the promoter regions of the AMS targets, and the PCR amplicons used for ChIP-qPCR containing the E-boxes are underlined. Fold enrichment data were analyzed to calculate the fold change between the ChIP (anti-AMS immune serum) and no-antibody control, and all of the targets tested were present at higher amounts in the sample with anti-AMS immune serum than in the no-AMS antibody preimmune precipitation.

Figure 4.

Analysis of ABC Transporter WBC27 Knockout. (A) and (C) Columbia wild-type plant with normal silique development (arrows). (B) and (D) The wbc27/SALK_062317 homozygous mutant, showing reduced silique elongation (arrows). (E) Wild-type flower containing dehisced anthers. (F) Mature wbc27/SALK_062317 mutant flower containing shrivelled anthers that fail to dehisce. (G) Immature wild-type anther containing newly released microspores. (H) Immature wbc27/SALK_062317 mutant anther containing abnormal immature microspores, which lack the defined shape of the wild-type microspores. Excessive amounts of material are visible in the locule, suggesting abnormal tapetal development. (I) Alexander-stained mature wild-type anther showing viable pollen grains. (J) Alexander-stained maturewbc27/SALK_062317 mutant anther lacking viable pollen. (K) qRT-PCR of WBC27 expression in the Columbia wild-type and wbc27/SALK_062317 mutant inflorescences, indicating that only trace levels of expression were observed in the wbc27 mutant. Expression was normalized to ACTIN7 and presented relative to wild-type expression levels and was based upon a minimum of two biological replicates; error bars represent sd. (L) SALK-062317 T-DNA insertion position in At3g13220. Blue arrow represents coding sequence of At3g13220, gray boxes represent introns, black arrow shows sequenced T-DNA flanking region, black arrowhead shows approximate T-DNA insertion point, and red and green arrows show primers used for qRT-PCR. Bars in (G) to (J) = 50 μ m.

Figure 5.

Proteins Interacting with AMS in Yeast and in Vitro. (A) Schematic representations of the AMS-pGBKT7, ATA20-pGADT7, AtbHLH089-pGADT7, and AtbHLH091-pGADT7 fusion constructs. (B) Interaction in yeast AH109 cells. ATA20, AtbHLH089, and AtbHLH091 were able to activate the expression of the His⁺ and β -Gal reporter genes. (C) Protein gel blot analysis of in vitro–translated full-length ATA20, AtbHLH089, and AtbHLH091 pulled down with bacterially expressed GST-AMS. GST alone was used as a negative control and showed no interaction with the His-tag.

Figure 6.

Model for the Role of AMS during Anther and Pollen Development. AMS regulates a number of direct and downstream regulatory targets related to tapetal PCD and pollen wall formation and interacts with a number of proteins associated with anther and pollen development. This model provides an indication of the regulatory network for anther development. LTP, lipid transfer protein; CAT, carbohydrate transport and metabolism; AAT, amino acid transport and metabolism. Arrows indicate regulation; lines with diamond ends indicate interaction.