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First published online February 11, 2009; 10.1104/pp.109.135806 Plant Physiology 149:1713-1723 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
MIKC* MADS Domain Heterodimers Are Required for Pollen Maturation and Tube Growth in Arabidopsis1,[W],[OA]Department of Botany, University of Wisconsin, Madison, Wisconsin 53706–1381
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.
The male gametophyte in angiosperms is highly specialized. During anther development, a microspore mother cell divides meiotically, giving rise to four haploid microspores, and each microspore then divides mitotically to create a vegetative cell and a generative cell. In Arabidopsis (Arabidopsis thaliana), the generative cell will divide once again prior to pollen maturity to create two sperm cells, which are enclosed within the larger vegetative cell (Boavida et al., 2005  ). Mature pollen has the unique characteristic of being able to respond to specific environmental cues with carefully regulated polar growth. Upon landing on a papilla cell, a pollen grain will adhere, rehydrate, and expand asymmetrically to form a pollen tube. The pollen tube must then navigate through the female reproductive tissues and into an unfertilized ovule. This activity is coordinated with the movement of the sperm cells, which travel down the pollen tube to reach the egg and central cells of the female gametophyte (Wilhelmi and Preuss, 1997; Edlund et al., 2004). Much of the regulatory machinery that controls this carefully timed sequence of events has not been fully characterized.
The regulation of gene expression is likely to contribute to these processes. Recently, several groups have used microarray analyses to characterize the mRNA expression profiles of Arabidopsis pollen. These studies have revealed that the pollen transcriptome is distinct from that of sporophytic tissues (Pina et al., 2005
The MADS box gene family in Arabidopsis is large and diverse and consists of at least 109 members (Kofuji et al., 2003
A relatively small subset of the MADS domain gene family is expressed in pollen. Five of the six MIKC* factors are pollen enriched (i.e. their transcripts accumulate at higher levels in pollen than at any other stage of the life cycle). We previously reported that AGAMOUS-LIKE67 (AGL67; At1g77950) is preferentially expressed in embryonic tissue and that AGL30 (At2g03060), AGL65 (At1g18750), AGL66 (At1g77980), AGL94 (At1g69540), and AGL104 (At1g22130) transcripts could be detected in inflorescence tissues (Lehti-Shiu et al., 2005
The Arabidopsis MIKC* genes can be further subdivided into two groups, which represent monophyletic lineages. Proteins homologous to AGL66, AGL67, and AGL104 in Arabidopsis have been designated as S-class proteins, while those homologous to AGL30, AGL65, and AGL94 have been designated as P-class proteins. Analysis of rice (Oryza sativa), tobacco (Nicotiana tabacum), and Arabidopsis sequences has revealed that proteins in the same class (either S or P) in different species are more closely related than two proteins from different classes within the same species (Nam et al., 2004
MIKC* heterodimers may be major regulators of pollen maturation programs. Previous work has shown that 83.4% of transcripts that are enriched in mature pollen are down-regulated when the levels of MIKC* heterodimers are reduced (Verelst et al., 2007b
Loss of MIKC* Activity Affects Pollen Performance To investigate the contributions of MIKC* factors in vivo, T-DNA alleles were isolated for four of the five pollen-expressed MIKC* genes. All of the MIKC* loci, with the exception of AGL30, are located on chromosome I. A mutant allele could not be obtained for AGL30. One mutant allele each was isolated for AGL65, AGL66, and AGL94, and two alleles were isolated for AGL104 (agl104-1 and agl104-2). Reverse transcription (RT)-PCR using primers flanking the T-DNA insertion sites revealed that full-length transcripts accumulate at reduced levels in agl104-1 pollen, while full-length transcripts could not be detected in the agl104-2, agl65, agl66, and agl94 backgrounds. Transcript could be detected, however, using primers upstream of each T-DNA insertion. Additional information about the mutant alleles is provided in Supplemental Figures S1 and S2.
Each single mutant allele should result in the reduction or loss of two or more of the five predicted functional heterodimers: AGL66-AGL30, AGL66-AGL65, AGL66-AGL94, AGL104-AGL30, and AGL104-AGL65 (Verelst et al., 2007a
The simultaneous removal of AGL66 and AGL104 should eliminate all of the predicted MIKC* complexes in pollen. To test this, competition assays were performed with pollen from a plant that was heterozygous for agl104-2 and homozygous for agl66 and pollen from a plant that was heterozygous for agl66 and homozygous for agl104-2. In both cases, transmission of the double mutant combination was significantly lower than the expected rate of 50% (Fig. 1B). Transmission was reduced to 12% in one case (n = 50) and not detected in the other (n = 29). Thus, the loss of all MIKC* complexes has a severe effect on pollen performance, but at least some of the double mutant pollen appear to be capable of successful fertilization.
Although the agl66 agl104-2 double mutant combination can be transmitted through pollen at rates as high as 12% on wild-type females, recovering double homozygous plants following self-pollination of plants that were homozygous for agl66 and heterozygous for agl104-2 proved to be difficult. We were successful only when a limited quantity of pollen was placed onto the stigma of an emasculated parent, thereby minimizing pollen competition. Several homozygous double mutant plants were recovered, and each produced less than one seed per silique on average when allowed to set seed without intervention (Supplemental Fig. S4). Aside from the severely reduced fertility, homozygous plants were indistinguishable from wild-type plants. To further quantify the fertility defect, wild-type plants were emasculated and hand-pollinated with saturating amounts of wild-type and agl66 agl104-2 pollen. Seed yield was reduced by 98% in the double mutant relative to the wild type. For comparison, the same test was performed using pollen from the previously described agl65 agl66 agl104-1 mutant (Verelst et al., 2007b To determine the basis of the defects in the agl66 agl104-2 pollen, various assays were performed. A fluorescein diacetate (FDA) assay was used to examine pollen viability (Fig. 1D). Double and triple mutant pollen viability was reduced by 48% and 12% relative to the wild type, respectively. Pollen were also exposed to 4',6-diamidino-2-phenylindole (DAPI) in order to visualize nuclei in the mutant pollen. Approximately 40% of the double mutant pollen had at least one visible nucleus, while one or more nuclei were visible in approximately 70% of wild-type pollen (Fig. 1D). To examine pollen hydration on the stigma surface, scanning electron microscopy was used (Fig. 1, E–H). Pollen from wild-type individuals were able to adhere and rehydrate when in contact with a papilla cell, but double mutant pollen frequently failed to rehydrate.
These observations suggest that defects in pollen development occur prior to or during the final stage of maturation in agl66 agl104-2 pollen. To determine whether there was a consistent cellular basis for the defect, stage 11 anthers (Smyth et al., 1990 Pollen tube growth was examined in vivo by clearing fertilized flowers and staining with aniline blue. agl66 agl104-2, agl65 agl66 agl104-1, and wild-type pollen were each placed onto wild-type stigmas and given various lengths of time, up to 34 h, to germinate and elongate before being processed (Fig. 2 ). By 1 h after pollination, both wild-type and triple mutant pollen had produced a large number of pollen tubes. A few pollen tubes could be seen 3 h after pollination with double mutant pollen; however, very few pollen had germinated and the tubes rarely extended beyond the papillae cells. At 12 h after pollination, a few elongated double mutant pollen tubes were seen in the transmitting tract of some gynoecia, although most did not have any visible pollen tubes. In pollinations with wild-type and triple mutant pollen, on the other hand, the transmitting tract was packed with pollen tubes after only 3 h. We conclude that limited and delayed pollen tube growth in the double mutant is a likely basis for the reduced fertility of the double mutant and the low rate of allele transmission in the presence of competing pollen.
Analysis of Target Genes
The agl66 agl104-2 double mutant provided us with a useful tool to identify genes that are indirect or direct targets of MIKC* regulatory activity. RNA was extracted from wild-type and mutant pollen, and cDNA was hybridized to Nimblegen whole-genome microarray chips. We also included agl65 agl66 agl104-1 pollen in the analysis so our data could be directly compared with the Affymetrix chip results of Verelst et al. (2007b) There was significant overlap between the double, triple, and quadruple mutant data sets, and a unique set of regulated genes also appeared in each (Fig. 3, A and B ). A higher number of genes were significantly affected in the double mutant than in the triple mutant. However, of the 1,783 genes significantly up-regulated in both backgrounds, 54% had a stronger fold change in the triple mutant than in the double mutant. Of the 1,326 genes that were down-regulated, 68% were more strongly affected in the triple mutant than in the double mutant. While the basis for this is unknown, one possibility is that compromised but viable pollen represent a larger portion of the population in the triple mutant samples. In the double mutant, severely compromised pollen die and do not contribute to the RNA pool. In comparing the triple and quadruple mutant data sets, with the exception of AGL94 itself, very few genes showed a strong fold change between the triple and quadruple mutant samples. A summary of differentially expressed genes is provided in Supplemental Data File S1.
Among the target genes discovered by microarray analysis were 11 non-MIKC* MADS box genes. Probes for two of them, AGL52 and AGL97, are not available on the ATH1 Affymetrix array, and their expression in pollen has not been previously reported. Four others, AGL18, AGL19, AGL21, and AGL29, were examined by quantitative PCR (Fig. 3C). AGL18, AGL21, and AGL29 were strongly down-regulated in the double mutant, and AGL18 and AGL29 were similarly affected, but to a lesser degree, in triple and quadruple mutant backgrounds. AGL19, which shows little or no expression in wild-type pollen, is significantly up-regulated in the double mutant and may be affected to a lesser degree in the triple and quadruple mutant backgrounds. These data show that a broader MADS domain regulatory network exists in pollen and that at least some of its components are more strongly affected in the double mutant than in the triple mutant.
The disruption of all MIKC* activity has a severe effect on pollen performance, but no single MIKC* protein appears to play an essential role. To probe the degree of redundancy and to determine whether particular heterodimers are functional in vivo, we compared the pollen performance of various mutant combinations through competition assays. Although there is no direct evidence that AGL94-AGL104 complexes form, this sixth heterodimer was also considered in our analysis because AGL104 and AGL66 are closely related and AGL94 has been shown to interact with AGL66. The weak agl104-1 allele was used to determine whether quantitative differences in MIKC* levels in pollen are also important. To minimize the effect of linkage and maximize the sensitivity, the competition assays were set up such that only one allele was segregating and there were only two possible genotypes in the progeny. According to this strategy, any outcome that is statistically different from 50% is significant. In the first set of experiments (Fig. 4A ), the effect of removing AGL65-AGL104 complexes was examined. For this, competitions were carried out between agl66 and agl65 agl66 pollen as well as between agl66 agl104-1 and agl65 agl66 agl104-1 pollen. In each case, the competing pollen differ with regard to the presence of AGL65-AGL104, but the inclusion of the weak agl104-1 allele in the second experiment also reduces the levels of AGL30-AGL104 and AGL94-AGL104 heterodimers. We found that loss of AGL65-AGL104 has a significant effect on pollen performance, but only when the other AGL104-containing complexes are reduced. This was further confirmed by a competition with agl66 pollen and agl65 agl66 agl104-1 pollen (Fig. 4A). In this case, the transmission of the triple mutant combination was also severely compromised relative to the single mutant. These results suggest that the AGL104-containing complexes act in a partially redundant fashion, but the experiments also demonstrate that AGL65-AGL104 is functional and likely to regulate downstream targets that are important for pollen activity.
Next, the roles of AGL30-AGL104 and AGL94-AGL104 were examined by comparing agl66 agl104-2 double mutant pollen with agl65 agl66 agl104-1 triple mutant pollen, which contain only these two heterodimers, at lower levels than in the wild type. In this case, the agl104-2 T-DNA was transmitted at a frequency of 11%, suggesting that one or both of these two heterodimers control genes that confer a competitive advantage (Fig. 4B). An additional assay comparing agl65 agl66 agl104-1 and agl65 agl66 pollen showed that simply reducing the levels of these heterodimers is sufficient to reduce competitive ability. In this case, the agl104-1 allele had a 9% transmission frequency, suggesting that these heterodimers do not need to be completely eliminated in order to see an impact on pollen function.
Although physical interactions between AGL94 and AGL104 have not been detected previously (Verelst et al., 2007b
A previous competition experiment had shown that agl65 agl66 agl104-1 pollen is compromised relative to agl65 agl104-1 pollen (Verelst et al., 2007b Taken together, these pollen competition experiments provide evidence for in vivo function for at least three MIKC* heterodimers. They also show that MIKC* heterodimers contribute quantitatively and in a partially redundant fashion in Arabidopsis pollen.
Given that functional redundancy exists between MIKC* heterodimers, their relative abundance may be an important factor that determines the strength of their contributions in pollen. Quantitative RT-PCR was used to measure mRNA transcript levels for the five pollen-expressed MIKC* genes in mature wild-type pollen (Fig. 5A ). Less than a 20% difference in transcript accumulation was detected for the S-class proteins AGL66 and AGL104. Thus, if translation and turnover rates are equal, the abundance of AGL66- and AGL104-containing heterodimers should be similar. In contrast, transcript levels differ by greater than 2 orders of magnitude between the different P-class factors. AGL65 transcripts are more abundant than AGL30 transcripts, and both are more abundant than AGL66 and AGL104. AGL94, however, accumulates at much lower levels than other MIKC* transcripts in pollen.
The effect of loss of MIKC* activity on P-class transcript accumulation was also examined using quantitative RT-PCR (Fig. 5B). Changes in S-class transcript accumulation could not be examined because the mutant alleles changed the nature of the transcripts. In the pollen of agl66 agl104-2 double mutants, AGL65 transcript accumulation was essentially unchanged relative to the wild type. AGL30 transcripts increased to almost twice the level found in the wild type. AGL94 transcripts became much more abundant, increasing 36-fold, but still below the AGL30 and AGL65 transcript levels in wild-type pollen. A similar up-regulation of AGL94 was also seen in agl65 agl66 agl104-1 triple mutant pollen (data not shown).
MIKC* Activity Is Required for Pollen Germination and Tube Growth
MIKC* proteins have been proposed as major regulators of pollen maturation (Verelst et al., 2007b We have created plants that carry the stronger agl104-2 allele along with the agl66 allele, and severe defects in pollen function are apparent. This mutant combination should result in disruption of all of the predicted MIKC* heterodimers, including AGL30-AGL104. Homozygous double mutant plants produce few seeds, and aniline blue staining showed that, even on wild-type females, pollen germination is delayed and limited and few pollen tubes extend into the transmitting tract. The mutant pollen that are capable of growing tubes may correspond to those that show few cellular alterations relative to the wild type. This suggests that the mutations are not fully penetrant, which is perhaps fortunate, because we would have failed to recover homozygous double mutant plants otherwise. This may be due to remaining, but not readily detected, full-length or truncated transcripts in the mutant backgrounds. Although the defects appear to be exclusively on the male side, it is interesting that the mutant pollen appears to function somewhat better in mixed pollinations, as in a competition assay, than when pollen from a homozygous plant is used. The double mutant combination is transmitted to up to 12% of the progeny when both single mutant and double mutant pollen are present (Fig. 1B), and we were able to recover homozygous double mutant plants after pollinations with a limited pollen load. However, it is extremely difficult to obtain seeds when a homozygous mutant self-pollinates or is used to pollinate a wild-type female plant. This suggests that the wild-type pollen may condition the transmitting tract in a way that facilitates the growth of mutant pollen. This may involve the release of signaling molecules from the pollen or female tissues or both.
Verelst et al. (2007b)
Pollen performance was only inhibited in mutant backgrounds in which both AGL66 and AGL104 were disrupted, indicating that only one S-class protein is required for normal pollen activity. AGL66 and AGL104 represent a duplicated pair, which originated following a large-scale duplication of a portion of chromosome 1 (Blanc et al., 2000 Because the system is highly redundant, loss of individual complexes will only have a measurable impact on pollen performance in genetic backgrounds in which most of the other heterodimers are disrupted. By disrupting functionally redundant heterodimers, three MIKC* heterodimers (AGL65-AGL104, AGL30-AGL104, and AGL30-AGL66) were identified as likely contributors to the regulation of pollen development. If protein abundance reflects transcript abundance and the capacity for heterodimer formation is similar for all three P-class factors, we can expect complexes containing AGL65 to be at least 10 times more abundant than AGL30-containing complexes and over 600 times more abundant than AGL94-containing complexes. Because AGL104 and AGL66 transcripts are not as abundant as either AGL65 or AGL30 transcripts, the availability of S-class binding partners may limit complex formation in the system. Because there is potentially a large excess of AGL65 and a smaller excess of AGL30 in the system, elimination of either one would affect the composition of complexes but might not have a large impact on the total number of MIKC* complexes in the cell. While AGL65 heterodimers likely regulate the most targets in wild-type pollen, AGL30 heterodimers may take over some of those regulatory roles when AGL65 heterodimers are absent, as in agl65 mutants. The feedback regulation of AGL30 and AGL94 potentially adds to the capacity of the system for compensation. When MIKC* activity is limited (agl66 agl104-2 or agl65 agl66 agl104-1 mutants), AGL30 and AGL94 transcripts, and presumably proteins, become more abundant. What role does AGL94 play in this regulatory system? If AGL94 heterodimers contribute, their low abundance may explain why AGL94 removal does not affect pollen performance in a measurable way when AGL30 heterodimers are present. Without a loss-of-function allele for AGL30, a definitive genetic test cannot be set up. Pollen competition assays showed that agl66 agl94 double mutant pollen was comparable to either agl66 or agl94 single mutant pollen (data not shown) and agl65 agl66 agl104-1 triple mutant pollen was comparable to agl65 agl66 agl94 agl104-1 quadruple mutant pollen (Fig. 4B; Supplemental Fig. S5). Although these experiments do not provide evidence that AGL94 heterodimers form and function, neither do they preclude this. Heterodimers that include AGL94 may simply be functionally redundant with other MIKC* heterodimers. The up-regulation of AGL94 in the double and triple mutants is not likely to have a negative impact, because quadruple mutant pollen performance was comparable to that of the triple mutant. The differences seen between quadruple and triple mutant regulatory targets could be due in part to the elevated expression of AGL94 in the triple mutant. Based on the redundancy and relative expression levels of the MIKC* genes, we propose a model for the relative contribution of MIKC* heterodimers in pollen (Fig. 6 ). In this model, four of the five predicted heterodimers act in a partially redundant and quantitative manner to regulate pollen development. Any possible heterodimers that require AGL94 have little or no contribution in pollen. From this, we would predict that pollen from an agl30 agl65 double mutant, if it could be obtained, would be comparable to agl66 agl104-2 pollen. If this is not the case, it would provide evidence that AGL94 complexes play important regulatory roles.
MIKC* Regulation Affects a Large Suite of Genes in Pollen
The dramatic phenotypic effects seen in the double mutant may be due to changes in a small number of pivotal genes or they may reflect the collective effect of changes in many genes. Of the 4,415 genes that show changes, only 654 (14.8%) contain at least one of the MEF2 or N9 CArG box target sequences that MIKC* factors prefer (Verelst et al., 2007a
The double mutant also shows differential regulation of 11 non-MIKC*-type MADS box genes, suggesting that a broader MADS domain regulatory network is active in pollen. Within this set, quantitative RT-PCR showed that some MADS box genes, such as AGL18, AGL19, AGL21, and AGL29, may be more strongly affected in the double mutant (Fig. 3C). Mutant lines have been characterized for AGL18 (Adamczyk et al., 2007 Analysis of mutant pollen has shown that regulation by MIKC* heterodimers is necessary for successful pollen development and function. Like some well-characterized MIKCC factors, MIKC* proteins appear to act near the top of a regulatory hierarchy and their activity influences the expression of many downstream genes. This study has highlighted the importance of transcriptional regulation in pollen, and future work will be aimed at further characterizing additional players in the transcriptional network.
Plant Material
Experiments were conducted using Arabidopsis (Arabidopsis thaliana) plants of the Columbia-0 ecotype. All mutant lines were obtained from the SALK T-DNA collection (Alonso et al., 2003
RNA was extracted from hydrated mature pollen according to the phenol extraction method described previously (Lehti-Shiu et al., 2005
For each pollen competition assay, stage 12 wild-type flower buds (Smyth et al., 1990
Pollen viability was assessed using a 20 µM FDA treatment (Heslop-Harrison and Heslop-Harrison, 1970
To examine pollen at the subcellular level, anthers from stage 12 flowers (Smyth et al., 1990
RNA was collected and extracted from mature pollen samples, as described above, from wild-type, agl65 agl66 agl104-1, agl66 agl104-2, and agl65 agl66 agl94 agl104-1 homozygous plants. Three biological replicates were collected for each genotype, and the quality of each sample was assessed using the Agilent Bioanalyzer 2100. RNA was reverse transcribed, and amino-allyl labeling was carried out using the Ambion Amino Allyl MessageAmp kit (Ambion) according to the manufacturer's protocol. Labeled cDNA was hybridized to three Nimblegen ATH6 60mer expr X4 four-plex arrays (Nimblegen-Roche) and scanned with an Axon400B scanner (Axon Systems). Robust multichip average normalization was performed using NimbleScan version 2.3 software. CYBER-T (Baldi and Long, 2001
The following materials are available in the online version of this article.
The authors thank John Beirne, Connie Vo, Kelsey Weis, and Pamela Hall for help with plant genotyping, Dr. Ravi Palanivelu for advice on pollen imaging, and Dr. Peter Dowd for helpful comments on the manuscript. Seed stocks for the mutant lines were obtained from the Arabidopsis Biological Resource Center. Imaging was performed at the Plant Imaging Center, Department of Botany, University of Wisconsin-Madison. Received January 21, 2009; accepted February 6, 2009; published February 11, 2009.
1 This work was supported by the University of Wisconsin-Madison Graduate School, the United States Department of Agriculture National Research Initiative Competitive Grants Program (grant no. 2001–35304–10887), and the National Science Foundation (grant no. IOS–0718598).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Donna E. Fernandez (dfernand{at}wisc.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.135806 * Corresponding author; e-mail dfernand{at}wisc.edu.
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ABSTRACT
RESULTS
) genes based on the inferred evolutionary history of the family (Henschel et al., 2002
2 statistic (
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.
