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Genes Dev. 2010 May 15; 24(10): 1010–1021.
PMCID: PMC2867206

Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis


Natural cis-antisense siRNAs (cis-nat-siRNAs) are a recently characterized class of small regulatory RNAs that are widespread in eukaryotes. Despite their abundance, the importance of their regulatory activity is largely unknown. The only functional role for eukaryotic cis-nat-siRNAs that has been described to date is in environmental stress responses in plants. Here we demonstrate that cis-nat-siRNA-based regulation plays key roles in Arabidopsis reproductive function, as it facilitates gametophyte formation and double fertilization, a developmental process of enormous agricultural value. We show that male gametophytic kokopelli (kpl) mutants display frequent single-fertilization events, and that KPL and a inversely transcribed gene, ARIADNE14 (ARI14), which encodes a putative ubiquitin E3 ligase, generate a sperm-specific nat-siRNA pair. In the absence of KPL, ARI14 RNA levels in sperm are increased and fertilization is impaired. Furthermore, ARI14 transcripts accumulate in several siRNA biogenesis pathway mutants, and overexpression of ARI14 in sperm phenocopies the reduced seed set of the kokopelli mutants. These results extend the regulatory capacity of cis-nat-siRNAs to development by identifying a role for cis-nat-siRNAs in controlling sperm function during double fertilization.

Keywords: ARIADNE, single fertilization, embryo, endosperm, small RNA, ubiquitin ligase

Some small RNAs regulate gene expression through post-transcriptional gene silencing, translational inhibition, and heterochromatin modification (Vaucheret 2006). siRNAs, which include trans-acting siRNAs (tasiRNAs) and natural cis-antisense siRNAs (cis-nat-siRNAs), are derived from long, perfectly dsRNAs (Xie and Qi 2008). Although specific siRNAs have been implicated in regulation of development, adaptive responses to biotic and abiotic stresses, genome stability, and transposon movement (Vaucheret 2006), the extent of their regulatory importance remains unclear (Ghildiyal and Zamore 2009).

nat-siRNAs are derived from the overlapping transcripts of two adjacent genes located on opposite strands (Borsani et al. 2005). The overlapping transcripts are thought to form a dsRNA that is cleaved by a DICER endonuclease to produce small 21- to 24-nucleotide (nt) RNAs (Kim 2005). The small RNA then is loaded into a RNA-induced silencing complex (RISC) and directs cleavage of target RNAs by sequence complementarity (Vazquez 2006). Recent genome-wide analyses demonstrated the widespread existence of cis-nat-siRNAs in eukaryotic genomes, which raises the possibility that cis-nat-siRNAs could be major effectors of gene regulation (Jin et al. 2008; Ghildiyal and Zamore 2009). Approximately 26% of human genes, 22% of mouse genes, 16% of Drosophila genes (Zhang et al. 2006), and 9% of Arabidopsis genes (Jen et al. 2005; Jin et al. 2008) are overlapping and may form cis-nat-siRNAs. Yet despite their abundance in animal genomes, to date there is no direct evidence showing the gene regulation capacity of cis-nat-siRNAs (Okamura and Lai 2008; Werner et al. 2009). In plants, there are two studies demonstrating regulatory roles of cis-nat-siRNAs generated from two coding RNAs. In each case, one transcript of the cis-nat-siRNA pair was constitutively expressed and the other was induced by stress, either abiotic (Borsani et al. 2005) or biotic (Katiyar-Agarwal et al. 2006). In both cases, down-regulation of the target gene by specific cis-nat-siRNAs conferred tolerance to the inductive stress.

In flowering plants, the male gametophyte (pollen grain) contains three cells: a vegetative cell that forms the pollen tube, and two sperm enclosed within the vegetative cell cytoplasm (McCormick 2004). The female gametophyte (embryo sac) develops within the ovule and consists of seven cells of four types: three antipodals, two synergids, one egg, and one central cell (Yadegari and Drews 2004). The fertilization process starts when a pollen grain lands on the stigma, rehydrates, and begins germination. The pollen tube grows through female tissue until it reaches an available ovule, enters one of the synergids, and releases the sperm. Double fertilization occurs when one sperm fuses with the egg to produce the zygote and the other sperm fuses with the central cell to form the primary endosperm cell. Double fertilization is one of the most important developmental processes in higher plants, because the embryo and endosperm constitute the edible parts of many seeds and grains used in human and animal nutrition. Understanding the fertilization process therefore has great relevance for genetic engineering aimed at improving the nutritional quality of crop plants. Because they have condensed chromatin, sperm were thought to be relatively transcriptionally quiescent, but several studies showed that sperm express a distinct and diverse set of genes (Engel et al. 2003; Okada et al. 2007; Borges et al. 2008).

Kokopelli is a Native American male fertility deity. Here we describe analyses of the kokopelli (kpl) mutant of Arabidopsis thaliana that implicate a regulatory role for cis-nat-siRNAs in sperm function. We show that KPL and an inversely transcribed gene, ARIADNE14 (ARI14), which encodes a putative ubiquitin E3 ligase, have inverse RNA expression patterns, and that, in the absence of KPL transcripts, ARI14 RNA levels in sperm were increased and fertilization was impaired. We show that KPL and ARI14 represent a sperm-specific cis-nat-siRNA pair. ARI14 is transcribed both in the vegetative cell and in sperm, but a KPL-ARI14-nat-siRNA is generated only in sperm, resulting in depletion of ARI14 in sperm. kpl plants showed frequent single-fertilization events, in which only the egg or the central cell was fertilized, causing seed abortion. We also show that overexpression of ARI14 in sperm phenocopies the reduced seed set of kpl plants. Our data demonstrate that cell-specific nat-siRNA activity is required for fertilization, thus showing that nat-siRNAs can spatially regulate a key developmental process.


T-DNA insertions in KOKOPELLI affect the male gametophyte

The kokopelli-1 (kpl-1) allele has a T-DNA insertion in the third exon of At5g63720 (Fig. 1A). Plants heterozygous for kpl-1 showed atypical segregation of the T-DNA-linked kanamycin resistance gene in self-pollinated progeny (Supplemental Table S1), which caught our interest. The KanR to KanS segregation ratio of 1.6:1 deviates significantly from the expected 3:1 ratio for a recessive sporophytic mutation (χ2 = 57.6, P < 0.0001) and from the expected 1:1 ratio for a gametophytic mutation (χ2 = 30.9, P < 0.0001), indicating abnormal transmission through one or both parents. The reduced seed set seen in self-pollinated plants containing kpl-1 was variable from plant to plant and from silique to silique, but, on average, kpl-1/+ plants had ∼32% reduced seed set (aborted, 2.4% ± 1.9%; undeveloped, 29.5% ± 7.5%) ,while kpl-1/kpl-1 plants had ∼70% reduced seed set (aborted, 7.7% ± 4.2%; undeveloped, 61.9% ± 17.1%) (Fig. 1B,C). Reciprocal crosses between kpl/+ or kpl/kpl with wild type showed reduced transmission through the male (Fig. 1D,E; Supplemental Table S1), indicating that the reduced seed set was due to a defect in the male gametophyte. Note that the kpl-1 defect is not fully penetrant, since homozygous plants were obtained.

Figure 1.
kokopelli is a male gametophytic mutant that shows reduced seed set. (A) Schematic representation of the KPL and ARI14 genomic region. Thick and thin solid lines represent exons and introns, respectively. Dotted lines represent 3′ UTRs. Dashed ...

To determine the step causing reduced kpl-1 transmission, we checked for morphological abnormalities in kpl-1 pollen. Mature kpl-1 pollen grains were tricellular when stained with DAPI (Supplemental Fig. S1C), indicating that male gametophyte development was not affected, and kpl-1 pollen showed no differences from wild-type pollen (Supplemental Fig. S1A,B) when germinated in vitro (Boavida and McCormick 2007). In addition, when we examined young siliques (2–3 d after pollination, DAP) of kpl-1/kpl-1 plants harboring the LAT52:GUS pollen-specific transgene (Twell et al. 1990), we detected GUS in almost all ovules (92%, n = 531), indicating that pollen tube guidance was not affected (Supplemental Fig. S1D). Because kpl-1 pollen development, germination, elongation, and ovule penetration appeared normal, the defect causing the reduced seed set likely occurs during later fertilization events. To confirm the involvement of KPL, we obtained a second T-DNA allele, kpl-2 (Fig. 1A), which also had reduced seed set. On average, kpl-2/+ plants had ∼27% reduced seed set (aborted, 7.2% ± 3.5%; undeveloped, 20.2% ± 8.3%), while kpl-2/kpl-2 plants had ∼45% reduced seed set (aborted, 15.4% ± 5.9%; undeveloped, 29.9% ± 4.9%) (Supplemental Table S1; Supplemental Fig. S2).

Undeveloped ovules of kpl plants show frequent single-fertilization events

Two different phenotypes together account for the reduced seed set in siliques of self-pollinated kpl-1/+ and kpl-1/kpl-1 plants: undeveloped ovules and aborted seeds. To further characterize the undeveloped ovules, we examined 1- to 2-DAP pistils of self-pollinated kpl-1/kpl-1 plants. We observed ovules that had not been fertilized (Fig. 2A), and ovules that had been fertilized and were developing correctly, with a developing embryo and proliferating endosperm (Fig. 2B). Surprisingly, we also observed ovules in which only one fertilization event had occurred: Either the egg had been fertilized and a developing embryo was evident, but the central cell was unfertilized (Fig. 2C), or the central cell had been fertilized and endosperm nuclear divisions were evident, but the egg was unfertilized (Fig. 2D). Ovules from the kpl-2/kpl-2 mutant displayed similar phenotypes (Supplemental Fig. S3).

Figure 2.
Defects in ovules receiving kpl-1pollen. (A–D) Siliques from a kpl-1/kpl-1 plant were dissected 1–2 DAP, and then the ovules were cleared and examined using differential interference contrast (DIC) microscopy. The frequency of each phenotype ...

To assess whether both sperm were delivered to ovules, we introgressed the reporter construct HTR10pro:HTR10-mRFP, which is expressed specifically in sperm nuclei (Berger et al. 2008), into kpl-1/kpl-1 plants. At 1–2 DAP, in ovules receiving wild-type pollen, most will have been double-fertilized, and therefore no RFP signal from sperm nuclei should be observed at the micropylar pole. In wild-type plants harboring HTR10pro:HTR10-mRFP, only 14 of 416 (3.3%) ovules had two sperm nuclei visible (Supplemental Fig. S4A–D). These presumably reflect failed fertilization events, because this incidence is similar to the incidence of undeveloped ovules in wild-type plants. In self-pollinated kpl-1 plants, 59 of 312 ovules (18.9%) had either one or two sperm nuclei visible in the region of the degenerated synergid (Fig. 2E,G, respectively), although all the pollen grains from these plants contained two sperm (Supplemental Fig. S4E). In some ovules in which only one sperm nucleus was detected, there was also a developing embryo (Fig. 2F), indicating that kpl-1/kpl-1 pollen had delivered two sperm to the embryo sac but double fertilization was impaired. These ratios are different from the expected ∼65% ovules with one or no fertilization events, due to technical difficulties in detecting the red sperm nuclei; nonetheless, the difference from wild type is significant (χ2 = 238.2, P < 0.0001).

To confirm that the embryo or endosperm in ovules with a single-fertilization event resulted from fertilization and were not spontaneous, we crossed kpl-1 with a plant carrying a FAC1pro:GUS reporter. FAC1 (Embryonic Factor 1) is expressed from the paternal allele in both the developing embryo and endosperm (Xu et al. 2005). We used pollen from a kpl-1/+; FAC1pro:GUS/+ F1 plant to pollinate wild-type plants, and assessed expression of FAC1pro:GUS in ovules at 2.5 DAP. We detected three different GUS expression patterns. One had GUS in both the embryo and endosperm, indicating that the ovules had been doubly fertilized (Fig. 2H), while the other two had GUS in ovules containing either only an embryo (Fig. 2I) or only endosperm (Fig. 2J), indicating that these ovules had indeed been fertilized.

The KPL and ARI14 transcripts have the potential to generate nat-siRNAs in sperm

At5g63720 (KPL) is in a reverse orientation to the adjacent gene At5g63730 (ARI14) (Fig. 1A), and the distance between their stop codons is 82 base pairs (bp). The Arabidopsis Information Resource (TAIR; http://www.arabidopsis.org) annotations for KPL and ARI14 lack 3′ untranslated regions (UTRs), explaining their absence in the Jin et al. (2008) list of gene pairs potentially generating nat-siRNA pairs. Using BLAST, we identified ESTs (e.g., AYAOO64TF) representing the 3′ end of KPL, which overlaps 72 bp with the last exon of ARI14. KPL has no recognizable protein domain signatures, but contains a putative nuclear localization signal. ARI14 is a member of the gene family encoding ARIADNE 1-like proteins, a family of putative ubiquitin E3 ligases with RING finger domains (Mladek et al. 2003). When we examined KPL and ARI14 expression in different tissues using RT–PCR, both were detected only in closed flower buds, open flowers, and pollen (Fig. 1F). In addition, KPL and ARI14 expression throughout male gametophyte development (Honys and Twell 2004) showed inverse patterns (Fig. 1G). According to microarray data (Borges et al. 2008), ARI14 transcripts are called “present” in pollen and “absent” in sperm, while KPL transcripts are sperm-enriched; they are called “present” in both pollen and sperm, but the signal in pollen is coming from the sperm. Given these observations, we hypothesized that KPL and ARI14 generate a pair of cis-nat-siRNA, and that the transcripts of KPL post-transcriptionally down-regulate the level of ARI14 in sperm.

KPL and ARI14 expression patterns

To test our hypothesis, we used RT–PCR and quantitative real-time PCR (qPCR) assays to determine relative KPL and ARI14 expression levels. KPL transcripts were readily detected in wild-type open flowers, but the level of ARI14 transcripts was very low (Fig. 3A,B). In homozygous kpl mutants, the level of KPL transcripts was reduced, while the level of ARI14 transcripts was substantially increased (Fig. 3A,B), suggesting inverse regulation of the two transcripts. To test their cell specificity, we generated transgenic reporter lines harboring constructs in which expression of the tdTomato fluorescent protein was driven by either the KPL or ARI14 promoters. In all lines examined, the KPL promoter was active specifically in sperm (Fig. 3C), while the ARI14 promoter was active in both the vegetative cell and the sperm (Fig. 3C; Supplemental Fig. S5). Because ARI14 transcripts were called “absent” from sperm (Borges et al. 2008), we determined the expression pattern of a translational fusion, in which the entire upstream region and coding sequence of ARI14 was fused to the mCherry fluorescent protein. In all lines examined, the mCherry-ARI14 fusion protein was absent from sperm and was in speckles within the vegetative cell cytoplasm (Fig. 3D–F; Supplemental Fig. S6). These results support the hypothesis that KPL and ARI14 mRNAs are both transcribed in sperm, but that ARI14 transcript levels are unstable in the presence of KPL transcripts.

Figure 3.
Gene expression of KPL and ARI14 in wild-type and kpl pollen. (A,B) Relative expression levels of KPL and ARI14. Total RNA was extracted from 1-d-old open flowers of kpl homozygous mutants and their corresponding wild-type ecotypes. (A) RT–PCR ...

Coexpression of ARI14 and KPL induces ARI14-specific small RNAs

To test the hypothesis that ARI14 is regulated post-transcriptionally, and to be able to detect ARI14-specific small RNAs, we generated stable transgenic Arabidopsis plants harboring either a 35Spro:GFP-ARI14-30C fusion construct or a 35Spro:KPL construct. The expression levels in parental plants containing these constructs were verified by GFP imaging or RT–PCR, respectively. We generated F1 plants that coexpressed both GFP-ARI14-30C and KPL and compared their GFP expression levels with those of parental plants that contained only the GFP-ARI14-30C fusion (Fig. 4). The F1 plants had less GFP than the GFP-ARI14-30C parent (Fig. 4A,B), indicating that the GFP-ARI14-30C fusion protein was reduced. Due to chlorophyll autofluorescence and reduced GFP, these F1 plants looked red when imaged, and are referred to as “Red” plants (Fig. 4B). F2 seeds from self-pollinated individual Red plants were doubly selected for seedlings harboring both constructs, and then KPL and GFP expression was evaluated by qPCR (Fig. 4C,D, respectively). This analysis confirmed lower GFP expression levels in plants coexpressing both constructs than in plants expressing only GFP-ARI14-30C. The reduction of KPL expression in Red plants (32%–36%) (Fig. 4C) compared with the parent is likely explained by a dosage effect of different copy numbers, as seeds from several resistant plants were pooled together and were likely a mix of heterozygous and homozygous plants. Nevertheless, the reduction of GFP expression (68%–82%) (Fig. 4D) was greater and cannot be explained solely by a copy number difference, therefore indicating that the overlapping region of ARI14 and KPL is sufficient to reduce GFP-ARI14-30C transcript levels. As a control, we also crossed 35Spro:GFP-ARI14-30C plants with 35Spro:PIF3 plants. When imaged, the GFP-ARI14-30C parent and the F1 plants showed equivalent fluorescence intensities (Supplemental Fig. S7), indicating that the reduction of GFP expression in the Red plants was due to the presence of the KPL transcript and not due to GFP copy number or cosuppression of the two 35Spro-containing transgenes.

Figure 4.
ARI14 is regulated post-transcriptionally by KPL. (A,B) GFP expression in leaf epidermal cells of transgenic plants expressing the 35Spro:GFP-ARI14 construct (A) or both the 35Spro:KPL and 35Spro:GFP-ARI14 constructs (B). (C,D) qPCR analyses of KPL ( ...

To further characterize the interaction between GFP-ARI14-30C and KPL transcripts, we isolated total RNA, including small RNA, from pooled F2 seedlings that contained both constructs. We used a mixture of six ARI14-specific oligomers as probes (Supplemental Fig. S8A) and detected a 21-nt RNA species only in F2 plants that contained both KPL and GFP-ARI14-30C, while no similar RNA species were detected in parental plants containing only the GFP-ARI14-30C fusion (Fig. 4E). These probes are from the ARI14 sequence and can recognize small RNAs representing the KPL sequence, indicating that ARI14 was being down-regulated post-transcriptionally. We repeated this experiment using subsets of these six oligomers as probes, and were able to delimit the hybridizing sequences to a 60-nt region of ARI14 (probe set 2 + 3) (Supplemental Fig. S8B), which indicated that there was a specific target in the ARI14 mRNA.

To test whether the 21-nt species possibly targeted ARI14 mRNA for endonucleolytic cleavage in wild-type sperm, we performed a 5′ rapid amplification of cDNA ends (RACE) assay using polyadenylated RNA isolated from wild-type pollen to map potential cleavage sites. Among the cleavage sites found in the ARI14 mRNA, 14 out of 28 mapped to one site at the overlap between the KPL 3′UTR and the ARI14 exon (at position 1455 of the ARI14 mRNA). This site is within the 60-nt region that detected a 21-nt species (Supplemental Fig. S8A). Two additional cleavage sites were mapped upstream (at positions 1393 and 1203 of ARI14 mRNA) (Fig. 4F). Furthermore, in a database of small RNAs from wild-type Arabidopsis sperm (Slotkin et al. 2009), we identified one 21-nt RNA that could target ARI14. This small RNA represents position 1426–1446 of the KPL mRNA, and could potentially target cleavage of ARI14 at position 1647 (Fig. 4F). These positions are not in the predicted overlap between KPL and ARI14 mRNAs, suggesting that the primary siRNAs are generated from a longer overlap between the two transcripts, probably in the pre-mRNA, before polyA addition.

To confirm that this KPL small RNA species indeed targeted ARI14, we first used 3′ RACE to show that the polyA addition site was at position 1604 in both the F2 Red plants and wild-type pollen (Fig. 4F), at the STOP codon of KPL. To detect ARI14 small RNAs, we used low-molecular-weight-enriched RNA from wild-type pollen and Red plants, probes based on the 21-nt RNA sequence from the sperm small RNA library (probe 1647), and probes 2, 4, 5, and 6 used in Figure 4E. Probe 2 detected a 21-nt RNA only in RNA from Red plants, while probes 4, 5, and 6 did not detect any small RNA species (as in Supplemental Fig. S8B). In addition, probe 1647 detected a 22-nt siRNA (Supplemental Fig. S8C). These results confirm that the pre-mRNA of KPL and ARI14 form dsRNA in the nucleus, and that this dsRNA is processed to generate small RNAs that target ARI14.

Biogenesis pathway of KPL-ARI14 nat-siRNA

We wanted to determine which RNAi pathway components might be involved in biogenesis of the KPL-ARI14 nat-siRNA. We did not detect any KPL-ARI14 nat-siRNA species with small RNA isolated from pollen (Supplemental Fig. S8), probably because such a species is expected to be sperm-specific and would be diluted in pollen RNA. Therefore, we used qPCR to examine the ARI14 expression level in several small RNA biogenesis mutants and in their corresponding wild-type ecotypes (Fig. 5). ARI14 transcript levels were increased in dcl1, indicating that DCL1 was the only DICER-like protein that had any effect. Higher levels of ARI14 RNA were also detected in the dsRNA-binding protein mutant hyl1, in the RNA methyltransferase mutant hen1, in the RNA polymerase IV mutant sde4, and in sgs3. A mutation in RDR6 (SDE1) had no effect on ARI14 transcript levels, while a mutation in RDR2 showed a slight increase. These results implicate a biogenesis pathway in which KPL-ARI14 nat-siRNA might be processed by the DCL1-HYL1 complex; stabilized by HEN1-mediated methylation; and amplified by RDR2, SGS3-mediated, and RNA polymerase IV-mediated reactions; and are consistent with findings for the salt-induced nat-siRNAs (Borsani et al. 2005) and pathogen-induced nat-siRNAs (Katiyar-Agarwal et al. 2006).

Figure 5.
Accumulation of ARI14 mRNA in different siRNA biogenesis mutants. (A–D) qPCR analysis of ARI14 expression in 1-d-old open flowers of various small RNA biosynthesis mutants and their corresponding wild-type controls. The RNA level of the wild-type ...

Overexpression of ARI14 in sperm results in a reduced seed set phenotype

We hypothesized that the reduced seed set phenotype in the kpl mutants was a result of the failure to clear ARI14 transcripts, leading to higher levels of ARI14 in sperm. To test this hypothesis, we generated transgenic plants overexpressing ARI14 under the control of the sperm-specific KPL promoter. Because endogenous KPL transcripts might overlap with the ARI14 transgene, which could result in suppression of the transgene, we constructed a siRNA-resistant form of ARI14. We obtained 20 independent T1 lines, and verified by RT–PCR that they overexpressed the resistant form of ARI14. The siliques of these lines showed a reduced seed set similar to that of the kpl mutants (data not shown). We obtained T2 progenies of six T1 lines and grew them without selection, allowing the T-DNA to segregate. The T-DNA cassette carrying the resistant form of ARI14 also contained a pollen-specific marker (LAT52:tdTomato), which we used to identify heterozygous plants. From each progeny, we analyzed two heterozygous plants and one plant from which the T-DNA had segregated away. We measured the relative expression levels of ARI14 RNA in these plants by qPCR, and compared those levels with those in wild-type and kpl-1/kpl-1 plants (Fig. 6A). We also scored the seed set (Fig. 6B,C). In all T2 progenies tested, only the heterozygous plants carrying the transgene showed higher ARI14 expression levels (4.2-fold to 19.7-fold, compared with 3.7-fold in kpl-1/kpl-1) (Fig. 6A). These plants also had, on average, ∼50% reduced seed set (Fig. 6B), indicating that high ARI14 levels in sperm impair fertilization. Notably, no single-fertilization events were observed in these plants, but only unfertilized ovules.

Figure 6.
Overexpression of ARI14 in sperm phenocopies the reduced seed set observed in kpl mutants. (A) Total RNA was extracted from 1-d-old open flowers of T2 progenies from six independent T1 lines (nos. 28, 39, 45, 85, 92, and 93) that ectopically expressed ...

The KPL-ARI14 nat-siRNA pair appears to be of recent origin

The ARIADNE proteins are members of an ancient RING protein family, with homologs in animals, fungi, and plants (Eisenhaber et al. 2007). In contrast, we could identify only KPL homologs from Arabidopsis lyrata, Arabidopsis arenosa, Capsella rubella, and Brassica rapa. KPL and ARI14 are adjacent only in Arabidopsis and C. rubella (Supplemental Fig. S9). Most sequenced genomes have only a few ARI genes, but Arabidopsis has 16 members; probably as a result of several genome duplications (Mladek et al. 2003). In Brassica, there is only one gene representing the ARI13/14/15 cluster (Supplemental Fig. S10). Alignments of a bacterial artificial chromosome (BAC) of B. rapa subsp. pekinesis (accession no. AC189659.2) with A. thaliana and A. lyrata, using the CoGe server (Lyons and Freeling 2008), show full synteny in the region (Supplemental Fig. S10) except for KPL and three upstream genes, although presumably KPL exists elsewhere in Brassica genomes, as there are homologs of KPL in Brassica ESTs (EST identifier JCVI_14548 and EE509369, from Brassica oleracea and Brassica napus, respectively). KPL was likely inserted into this genomic region in the Brassicaceae lineage, because no homologs are found in the syntenic regions of papaya, the most closely related outgroup in the Order Brassicales, or in grape, a more distantly related outgroup of basal eurosids. Thus, the KPL-ARI14 nat-siRNA is likely to have originated recently within the Brassicaceae lineage of eurosid plants.


KPL plays a key role in double fertilization

We provide evidence that post-transcriptional regulation, via a cis-nat-siRNA corresponding to the KPL and ARI14 transcript pair, plays an important role in fertilization. Although double fertilization in angiosperms was described more than a century ago (McCormick 2004), the molecular basis underlying the mechanism that ensures double fertilization is still largely unknown. It is still unresolved as to whether both sperm are equally capable of fertilizing either the egg or central cell. Plants employ a rapid polyspermy block on the egg, but not on the central cell, to prevent both sperm from fertilizing the egg (Scott et al. 2008). Several Arabidopsis mutants have been described that have a single sperm-like cell, rather than two sperm, but there are differing findings as to whether such single sperm-like cells have preferred fusion partners (Iwakawa et al. 2006; Nowack et al. 2006; Chen et al. 2008; Kim et al. 2008; Frank and Johnson 2009; Gusti et al. 2009). Furthermore, when two wild-type sperm are delivered to a mutant female gametophyte that produces two eggs rather than one, both eggs can be fertilized, suggesting that both sperm have the capacity to fuse with an egg (Pagnussat et al. 2007; Ingouff et al. 2009). Interestingly, Plumbago zeylanica has morphologically distinct sperm that preferentially fertilize either the egg or the central cell (Russell 1985), and expression studies in these dimorphic sperm indicate that there might be promoters that are specific to one of the two sperm (Gou et al. 2009). However, the KPL promoter and all other promoters thus far tested in Arabidopsis (Alandete-Saez et al. 2008; Berger et al. 2008) drive expression in both sperm.

To date, kpl is the only mutant that shows prevalent single fertilizations, in which ∼40% of the ovules show either only embryo or only endosperm development, even though the pollen tubes of kpl deliver two normal-looking sperm into the embryo sac. These data suggest that the KPL-ARI cis-nat-siRNA regulates an intrinsic component in the sperm that is essential for fertilization. For this reason, kpl, in combination with recently developed tools to image the fertilization process (Berger et al. 2008), offers a unique opportunity to further explore the molecular mechanisms of double fertilization.

KPL and ARI14 generate a sperm-specific cis-nat-siRNA pair

Our data indicate that KPL and ARI14 generate a previously undiscovered cis-nat-siRNA pair. We showed that KPL and ARI14 have anti-correlated expression patterns throughout pollen development (Fig. 1), and when comparing wild-type and kpl pollen (Fig. 3). We also demonstrated that coexpression of ARI14 and KPL in transgenic plants induces the formation of ARI14-specific small RNAs (Fig. 4), and that the Arabidopsis KPL-ARI14 nat-siRNAs are generated by small RNA biogenesis pathway components (Fig. 5). Genes involved in plant siRNA biosynthesis have pleiotropic phenotypes that have been studied extensively (Chen et al. 2002; Han et al. 2004; Peragine et al. 2004; Mosher et al. 2009). Homozygous mutants of dcl1, hyl1, and hen1 show severe fertility defects among other developmental phenotypes. Male fertility is reduced in hen1-1 and hen1-2 plants (Chen et al. 2002). Although we are not aware of other published studies that directly address this issue, male transmission defects could certainly contribute to the overall reduced fertility of the dcl1 and hyl1 mutants. Other homozygous mutants like sde4, rdr2, and sgs3 show either very subtle or no morphological or fertility differences, although their roles in siRNA biosynthesis are already well established (Peragine et al. 2004; Xie et al. 2004; Mosher et al. 2009). These observations suggest the possibility of redundancy between different proteins in the siRNA biosynthesis pathway, which might mask any effects on male transmission in the single mutants.

The reduced seed set phenotype seen when ARI14 is overexpressed in sperm supports our hypothesis that high levels of ARI14 transcripts in sperm impair fertilization (Fig. 6). In addition, the single fertilization events observed in kpl plants (Fig. 2) indicate that each sperm operates independently, depending on its level of KPL and ARI14 transcripts. This interpretation is consistent with the variable (i.e., from silique to silique) seed set phenotypes (Fig. 1).

High-throughput sequencing of small RNAs (Backman et al. 2008) and genome-wide analyses of Arabidopsis potential cis-NAT pairs (Henz et al. 2007; Jin et al. 2008) have identified small RNAs that potentially target many protein-coding genes in Arabidopsis. However, genes that are expressed at very low levels, in a specific tissue, or under specific conditions, as well as genes that are not accurately annotated might have been missed in these studies. KPL and ARI14 could be such genes due to their restricted expression within the male gametophyte. In animals, cis-nat-siRNAs are specially abundant in testis, and large-scale genomic approaches suggest that nat-siRNA overexpression in testis is a general phenomenon and not restricted to specific genes (Okada et al. 2008; Werner et al. 2009). This may suggest that cis-nat-siRNAs are a common regulatory mechanism for gene expression in gametes.

KPL can probably be defined as a new gene because its expression is sperm-specific, it is found only in some species of the Brassicaceae, and there are no homologs outside of this family. Sperm specificity and restricted species distributions are characteristic of some new genes in Drosophila species (Dorus et al. 2008). Furthermore, it appears that KPL was inserted recently in the genomic region adjacent to ARI14 (Supplemental Fig. S10), suggesting that regulation of ARI14 via a KPL-ARI14 nat-siRNA is a novel evolutionary feature. Because KPL encodes a sperm-specific protein, it is plausible that it might play a role in fertilization, and that down-regulation of KPL in kpl mutants also might contribute to the reduced seed set phenotype.

Model for regulation of fertilization by KPL-ARI14 nat-siRNA

Ubiquitination has been implicated in pollen development, gametogenesis, and seed development, and our results suggest that it is also important for proper fertilization. Ubiquitination of a protein requires a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) (Hershko and Ciechanover 1998); specificity is largely determined by the interactions between different E2s and E3s and their target proteins (Kumar et al. 1997). Transcripts encoding proteins involved in the ubiquitin-mediated degradation pathway were highly represented in sperm (Singh et al. 2002; Engel et al. 2003; Okada et al. 2007; Borges et al. 2008), suggesting that ubiquitin-mediated proteolysis might play an important role in male gametogenesis and fertilization.

ARI14 is a member of the ARIADNE gene family that encodes putative E3 ubiquitin ligases (Tan et al. 2000; Qiu and Fay 2006). Arabidopsis ARI8 was shown to exhibit E3 ubiquitin ligase activity (Stone et al. 2005), thus it was presumed that other members of the ARIADNE-like family in Arabidopsis had similar activities. ARI14 is highly similar to ARI13 and ARI15, and they, together with ARI16, are in the same subgroup of ARIADNE-like proteins in Arabidopsis (Mladek et al. 2003). ARI13 is expressed in sperm and pollen, ARI15 is ubiquitously expressed, including in sperm, and ARI16 is expressed in siliques (Zimmermann et al. 2004; Borges et al. 2008). ARI13/14/15 are located in a cluster on chromosome 5—probably a result of a late duplication occurring ∼50 million years ago (Mladek et al. 2003). ARI14 is missing four conserved cysteines in the first RING domain (Supplemental Fig. S11) that were shown to be crucial for binding to E2 ubiquitin-conjugating enzymes (Aguilera et al. 2000), so it is likely that ARI14 lacks E3 ubiquitin ligase activity. It is plausible that ARI13 and/or ARI15 are active E3 ubiquitin ligases in sperm, because they have the required cysteines (Supplemental Fig. S11).

We propose a model for the generation of the KPL-ARI14 nat-siRNA pair and its role in regulating fertilization (Fig. 7). Our model is based on the premise that ARI14 is not an active E3 ubiquitin ligase and that it might interfere with the activity of bona fide E3 ubiquitin ligases in sperm, such as ARI13 (used as an example since it is most similar to ARI14). In wild type, ARI14 is expressed in both the vegetative and the sperm cells, while in sperm, ARI13 and KPL are also expressed (Fig. 3). In the presence of KPL transcripts (i.e., in sperm), ARI14 and KPL pre-mRNAs generate dsRNAs due to their transcript complementarity (Supplemental Fig. S8). These dsRNAs are recognized and processed by DCL1 and HYL1 to generate several small RNA products. Strand-specific small RNAs representing KPL sequences are incorporated into a RISC complex, which targets and cleaves ARI14 transcripts in sperm, resulting in down-regulation of ARI14. In the absence of ARI14, ARI13, which is a putative active E3 ubiquitin ligase, can bind to its substrate and target it for degradation, leading to proper fertilization. In the kpl mutants, the level of full-length KPL transcripts is reduced, so that less dsRNA is produced, allowing some expression of ARI14 in sperm. Although ARI14 is unlikely to have enzymatic activity, it is possible that it can bind to a substrate without tagging it with ubiquitin. Thus, we propose that, in kpl mutants, ARI14 can bind to a substrate and prevent it from being tagged for degradation, resulting in a higher level of the substrate in sperm, and that the activity of this substrate impairs fertilization.

Figure 7.
Model for biogenesis of KPL-ARI14 nat-siRNAs and their proposed role in fertilization. In wild-type sperm, the KPL-ARI14 nat-siRNAs lead to down-regulation of ARI14. Reduced KPL transcripts in kpl mutants result in accumulation of ARI14 in the sperm. ...

In this scenario, one might expect kpl mutants that misexpress ARI14 in sperm to have only unfertilized ovules, since both sperm are affected. However, they show frequent single-fertilization events. This could be due to a dosage effect; that is, in kpl sperm, there could be competition for the substrate between the inactive ARI14 and the active ARI13, thus affecting the level of the substrate and therefore determining the fate of each sperm. It will be important to identify proteins with which ARI14 interacts to test this hypothesis. Nonetheless, because each sperm regulates its KPL and ARI14 expression levels independently of the other sperm, single fertilizations are possible. The idea of a dosage effect is also consistent with the phenotypes caused by overexpression of ARI14 in sperm (Fig. 6); ARI14 expression was driven by the strong KPL promoter, which resulted in higher levels of ARI14 in sperm than found in the kpl mutants. Thus, the balance is shifted toward a situation in which ARI14 protects the substrate from being degraded. In these transgenic lines, reduced seed sets were associated only with unfertilized ovules, and no single-fertilization events were observed. Taken together, our results demonstrate a key role for a cis-nat-siRNA in the regulation of double fertilization in plants, and open new avenues for investigating the molecular mechanisms that control this critical developmental process.

Materials and methods

Plant material and growth conditions

The A. thaliana mutants rdr2-1, dcl2-1, dcl3-1, and dcl4-2 were provided by Jim Carrington (Oregon State University), dcl1-9 and hen1-1 were provided by Xuemei Chen (University of California at Riverside), sde1(rdr6) and sde4/nrpd1a was provided by David Baulcombe (Sainsbury Laboratory, Norwich, United Kingdom), sgs3 was provided by Herve Vaucheret (Institut National de la Recherche Agronomique, Versailles, France), and hyl1 was provided by Nina Fedoroff (Pennsylvania State University). These mutants were in the Columbia (Col-0), Landsberg erecta (Ler), No-0, or C24 genetic backgrounds, as indicated in the text and figures. FAC1pro:eGFP-GUS seeds were from Chun-Ming Liu (Institute of Botany, Chinese Academy of Sciences, Beijing, China). Growth conditions are described in the Supplemental Material.

T-DNA lines

The kpl-1 mutant, carrying a T-DNA inserted in the third exon of At5g63720 (FST 184H02), was obtained from the INRA Versailles T-DNA mutant collection (WS4 ecotype) (Samson et al. 2002). The kpl-2 mutant (Ler ecotype), carrying a gene trap transposon insertion (GT_5_38351) was obtained from the Arabidopsis Biological Resource Center (ABRC) Stock Center. We used a PCR-based approach to confirm both T-DNA insertions. See the Supplemental Material for technical details and primer information.

Phenotypic analysis

Seed set analyses, GUS and DAPI staining, and imaging of unfertilized and fertilized ovules were performed as described in Alandete-Saez et al. (2008). See the Supplemental Material for details.

Statistical analysis

χ2 was calculated using GraphPad QuickCalcs (http://www.graphpad.com/quickcalcs/chisquared1.cfm).

RNA analyses

High-molecular-weight RNA was extracted using RNeasy Kits (Qiagen). Low-molecular-weight RNA was extracted using TRIzol reagent (Invitrogen) or a PureLink miRNA isolation kit (Invitrogen). See the Supplemental Material for details and primers.

RLM 5′ RACE and 3′ RACE

A modified procedure for RNA ligase-mediated RACE (RLM 5′ RACE) and 3′ RACE were performed using the GeneRacer kit (Invitrogen). See the Supplemental Material for details and primers.


We thank Feng Li for assistance and advice on small RNA analyses, Leonor Boavida for discussions and advice on phenotypic characterizations, Eric Lyons for advice on evolution and synteny, and Jay Hollick, Jorge Muschietti, Binglian Zheng, Miltos Tsiantis, and Mona Monfared for comments on the manuscript. We thank Roger Tsien for tdTomato and mCherry, Fred Berger for HTR10pro:HTR10-mRFP seeds, Luca Comai for A. arenosa sequences, Peter Quail for 35S:PIF3 seeds, David Hantz and Julie Calfas for excellent greenhouse assistance, and undergraduates Sam Leiboff, Marian Wahba, Jamison Smith, Artur Karasyov, Chih-Han Lee, Frazier Philips, Laura Mahoney, and Allison Clark for technical assistance. This work was supported by NSF Plant Genome program grant 0211742, and by U.S. Department of Agriculture Current Research Information System 5335-21000-030-00D. M.R. was partially supported by a Vaadia-BARD Post-doctoral Fellowship Award number FI-391-2006 from BARD, the United States–Israel Binational Agricultural Research and Development Fund.


Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1882810.

Supplemental material is available at http://www.genesdev.org.


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