MIKC* MADS domain heterodimers are required for pollen maturation and tube growth in Arabidopsis

Abstract

MADS box genes encode transcription factors that play important regulatory roles at various stages in plant development. Transcripts encoding the MIKC*-type (for MADS DNA-binding domain, Intervening domain, Keratin-like domain, and C-terminal domain) factors, a divergent clade, are enriched in mature pollen. Previous studies have shown that these proteins bind DNA as heterodimers, which form between S- and P-class MIKC* proteins. In this study, Arabidopsis (Arabidopsis thaliana) pollen with little or no MIKC* activity was produced by combining strong loss-of-function alleles of the S-class proteins AGAMOUS-LIKE66 (AGL66) and AGL104. Double mutant plants produce pollen but have severely reduced fertility due to reduced pollen viability, delayed germination, and aberrant pollen tube growth. Microarray analysis of the mutant pollen revealed that the loss of MIKC* regulation has a major impact on pollen gene expression. Pollen competition assays involving various combinations of AGL65, AGL66, AGL104, and AGL94 mutant alleles provided genetic evidence that at least three heterodimers (AGL30-AGL104, AGL65-AGL104, and AGL30-AGL66) form and function in at least a partially redundant fashion in pollen. Analyses of transcript abundance in wild-type and mutant pollen indicated that AGL65-containing complexes are likely to be more abundant than the others and that accumulation of AGL30 and AGL94 transcripts increases in response to reductions in MIKC* activity. These results were combined to create a model to describe MIKC* heterodimer contributions in pollen.

Figure 1.

Analysis of agl66 agl104-2 pollen. A, A summary diagram describing how pollen competition assays were carried out. B, Competition assays show that agl66 agl104-2 pollen is significantly compromised relative to single mutant pollen. Shaded boxes illustrate which heterodimers (listed above the boxes) are present (black boxes), absent (white boxes), or reduced by the agl104-1 weak allele (gray boxes). Transmission of the lower mutant genotype (tr), χ² statistic (χ²), and P value (p) are also shown for each cross. Outcomes that differ significantly from the expected value of 50% (P < 0.01) are indicated with an asterisk. C, Fertility is significantly reduced when double or triple mutant pollen is placed onto a wild-type female, but fertility is not affected by the genotype of the female. n ≥ 13 siliques for each sample. Col, Ecotype Columbia. D, Double and triple mutant pollen had reduced viability relative to the wild type as assessed by a FDA viability assay. Fewer double and triple mutant pollen had visible nuclei after pollen were exposed to DAPI. E to H, Both wild-type pollen (E and F) and double mutant pollen (G and H) adhere to wild-type stigmas, but some mutant pollen in contact with papilla cells fail to fully rehydrate (arrows). Bars = 20 μm. I to L, Transmission electron microscopy images of mature wild-type pollen (I and K) and double mutant pollen (J and L). No ultrastructural changes are visible in the cytoplasm or cell wall of the double mutant pollen. Bars = 5 μm in I and J and 1 μm in K and L.

Figure 2.

Aniline blue staining of pollen tubes in vivo. Pollen were placed onto wild-type stigmas and stained to visualize pollen tube growth defects in MIKC* mutant backgrounds. Pollen tube growth was inhibited in agl66 agl104-2 pollen, while the growth of agl65 agl66 agl104-1 pollen tubes was not consistently distinguishable from that of the wild type. Red arrows indicate agl66 agl104-2 pollen tubes. Bars = 0.25 mm for all images. Col, Ecotype Columbia.

Figure 3.

Analysis of MIKC* target genes. A, Diagrams show the number of differentially expressed genes in agl65 agl66 agl104-1 and agl66 agl104-2 mutant pollen relative to the wild type, according to microarray analysis. While many of the same genes show changes in both mutants, a larger number of genes are affected in the double mutant, and targets are both up- and down-regulated. Numbers in parentheses correspond to categories in Supplemental Data File S1. B, Comparison of target genes in the triple mutant and agl65 agl66 agl94 agl104-1 pollen. C, Differential expression of selected non-MIKC* MADS box genes was confirmed by quantitative RT-PCR. The y axis values and numbers above the bars indicate fold change relative to the wild type.

Figure 4.

Pollen competition assays in higher order MIKC* mutants. Competitions were carried out in various mutant backgrounds to eliminate functional redundancy between MIKC* heterodimers and examine the contribution of specific heterodimers. Shaded boxes illustrate which heterodimers (listed above the boxes) are present (black boxes), absent (white boxes), or reduced by the agl104-1 weak allele (gray boxes). Transmission of the lower mutant genotype (tr), χ² statistic (χ²), and P value (p) are also shown for each cross. Outcomes that are significantly different from 50% (P < 0.01) are indicated with an asterisk. The crosses that demonstrate AGL65-AGL104 (A), AGL30-AGL104 (B), and AGL30-AGL66 (C) activity contribute positively to pollen competitive ability.

Figure 5.

Quantitative RT-PCR analysis of MIKC* transcript accumulation. A, Expression of MIKC* genes in wild-type mature pollen. Numbers above the bars indicate fold differences in the transcript levels of various MIKC* genes relative to AGL66 transcript levels. Note the logarithmic scale on the y axis. B, Expression of P-class MIKC* genes in agl66 agl104-2 mutant pollen. Numbers above the bars indicate fold change relative to wild type. The y axis is a linear scale. Col, Ecotype Columbia.

Figure 6.

Model showing the relative contributions of different MIKC* heterodimers in Arabidopsis pollen. AGL65-AGL104, AGL30-AGL104, and AGL30-AGL66 were each shown to contribute to pollen function. AGL94 may not play a major role in pollen when other P-class proteins are present. Redundancy exists between MIKC* heterodimers, and differential levels of P-class transcript abundance likely affect the contribution of each heterodimer.