MADS-complexes regulate transcriptome dynamics during pollen maturation

Abstract

Background: Differentiation processes are responsible for the diversity and functional specialization of the cell types that compose an organism. The outcome of these processes can be studied at molecular, physiologic, and biochemical levels by comparing different cell types, but the complexity and dynamics of the regulatory processes that specify the differentiation are largely unexplored.

Results: Here we identified the pollen-specific MIKC* class of MADS-domain transcription factors as major regulators of transcriptome dynamics during male reproductive cell development in Arabidopsis thaliana. Pollen transcript profiling of mutants deficient in different MIKC* protein complexes revealed that they control a transcriptional switch that directs pollen maturation and that is essential for pollen competitive ability. We resolved the functional redundancy among the MIKC* proteins and uncovered part of the underlying network by identifying the non-MIKC* MADS-box genes AGL18 and AGL29 as downstream regulators of a subset of the MIKC* MADS-controlled genes.

Conclusion: Our results provide a first, unique, and compelling insight into the complexity of a transcription factor network that directs cellular differentiation during pollen maturation, a process that is essential for male reproductive fitness in flowering plants.

Figure 1

AtMIKC* complexes regulate a transcriptional switch during pollen maturation. (a) Of the genes downregulated in agl65/66/104 triple mutant pollen, 83.4% exhibit peak expression at the mature pollen grain (MPG) stage of wild-type (WT) pollen development (according to Honys and Twell [11]), whereas 56.7% of the genes upregulated in this mutant peak during the immature tricellular stage (tricellular pollen [TCP]). In total, 83.5% of the upregulated genes peak during the three immature stages (unicellular microspores [UNM], BCP, and TCP). (b) The AtMIKC* complexes contribute quite significantly to the transcriptional changes that occur during pollen maturation. (c) We ranked all genes that were consistently called present in WT pollen according to their expression level in mature WT pollen, in descending order (the highest expressed gene received number 1). In this graph the ranking numbers of all genes upregulated and downregulated in triple mutant pollen are plotted, revealing that AtMIKC* complexes predominantly activate high-abundance and medium-abundance transcripts, while repressing low-abundance and medium-abundance transcripts. All calculations related to these graphs are included in Additional data file 2.

Figure 2

Functional redundancy is high among AtMIKC* complexes. (a) Overview of the presence (green), absence (red), and reduced abundance (orange) of the five MIKC* complexes in different mutant backgrounds. Values represent the number (upper row) and percentage (lower row) of AtMIKC*-controlled genes that were downregulated and upregulated in each of the mutants. (b) Graphical representation of the numbers of significantly affected genes shared by different mutants (based on the FIRe macro [38]); virtually all genes affected in agl65 and agl65/66 pollen are also affected in agl66/104 pollen.

Figure 3

Evolution of the AtMIKC* network. After duplication of one ancestral AGL30-like gene and one ancestral AGL66-like gene, the paralogs AGL30 and AGL65 maintained the ability to interact with AGL66 and AGL104, resulting in the complex AtMIKC* network that exists in pollen of extant Arabidopsis. Although AGL30, AGL66, and AGL104 expression is initiated early, starting from the unicellular microspore stage, AGL65 is only activated later, during the tricellular stage. This change in expression profile may have been responsible for the apparent loss of control over numerous genes by the AGL65 complexes (which was suggested by our analysis in Additional data file 4). BCP, bicellular pollen; MPG, mature pollen grains; TCP, tricellular pollen; UNM, unicellular microspores.

Figure 4

The sequential action of mutually interacting transcription factors directs transcriptome dynamics during pollen maturation. AtMIKC* complexes repress immature pollen specific transcription factors such as WRKY34, and activate mature pollen specific transcription factors such as AGL18 and AGL29. These in turn predominantly repress transcripts that peak in immature tricellular pollen (TCP). In addition, AtMIKC* complexes also repress AGL30 and AGL65 in a negative feedback loop, whereas AGL18 acts to fine tune the expression level of AGL29. For most genes displayed in this scheme, we confirmed the differential expression in wild-type and triple mutant pollen by reverse transcription polymerase chain reaction (Additional data file 5). In this scheme only MYB97 and WRKY34 are potential direct targets of the AtMIKC* complexes, as suggested by in silico analysis of their putative regulatory sequences (Additional data file 2). BCP, bicellular pollen; MPG, mature pollen grains; UNM, unicellular microspores.