Download Control of female gamete formation by a small RNA pathway in

Vol 464 | 25 March 2010 | doi:10.1038/nature08828
LETTERS
Control of female gamete formation by a small RNA
pathway in Arabidopsis
Vianey Olmedo-Monfil1, Noé Durán-Figueroa1, Mario Arteaga-Vázquez1{, Edgar Demesa-Arévalo1,
Daphné Autran2, Daniel Grimanelli2, R. Keith Slotkin3{, Robert A. Martienssen3 & Jean-Philippe Vielle-Calzada1
In the ovules of most sexual flowering plants female gametogenesis is
initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore
mother cell (MMC)1,2. Because some mutants and certain sexual species exhibit more than one MMC2–4, and many others are able to form
gametes without meiosis (by apomixis)5, it has been suggested that
somatic cells in the ovule are competent to respond to a local signal
likely to have an important function in determination6. Here we show
that the Arabidopsis protein ARGONAUTE 9 (AGO9) controls
female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple
gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in
cytoplasmic foci of somatic companion cells. Mutations in
SUPPRESSOR OF GENE SILENCING 3 and RNA-DEPENDENT
RNA POLYMERASE 6 exhibit an identical defect to ago9 mutants,
indicating that the movement of small RNA (sRNAs) silencing out of
somatic companion cells is necessary for controlling the specification
of gametic cells. AGO9 preferentially interacts with 24-nucleotide
sRNAs derived from transposable elements (TEs), and its activity is
necessary to silence TEs in female gametes and their accessory cells.
Our results show that AGO9-dependent sRNA silencing is crucial to
specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA–dependent
silencing in plant gametes.
Large-scale transcriptional analysis indicated that a gene encoding
an ARGONAUTE (AGO) protein (At5g21150 or ARGONAUTE 9) is
highly expressed in ovules and anthers of Arabidopsis (Supplementary
Fig. 1). The in situ pattern of expression confirmed that AGO9 messenger RNA is localized in ovules throughout development
(Supplementary Fig. 2). In both plants and animals, AGO proteins
are known to cleave endogenous mRNAs during either microRNA
(miRNA) or short interfering RNA (siRNA)-guided post-transcriptional silencing7–9. To elucidate the function of AGO9 in Arabidopsis,
individuals from three independent insertional lines harbouring
T-DNA elements within the coding region of the AGO9 gene were
phenotypically analysed at all stages of ovule development10 (Fig. 1a
and Table 1). Whereas 94.2% of pre-meiotic ovules showed a single
MMC in wild-type plants (Fig. 1b and Supplementary Fig. 3), 5.8%
ago9-2 ago9-4
a
5′UTR
3′UTR
Ex18
PAZ domain
b
ago9-3
c
Figure 1 | Phenotypes of ago9 insertional mutants before meiosis.
a, Genomic structure of the AGO9 gene in Arabidopsis; the location of
T-DNA insertions and the gene length (nucleotides) are indicated. UTR,
untranslated region. b, Pre-meiotic wild-type (WT) ovules showing a single
subepidermal MMC, adjacent to L1 cells. c, Pre-meiotic wild-type ovule
Ex22
+5326
PIWI domain
d
e
showing two MMCs. d, Pre-meiotic ago9-2 mutant ovule showing two larger
(black arrows) and two smaller (white arrows) abnormal cells. e, Pre-meiotic
ago9-3 ovule showing abnormally enlarged cells (arrows); one of them has
initiated a nuclear division. Scale bars, 10 mm.
1
Grupo de Desarrollo Reproductivo y Apomixis, Laboratorio Nacional de Genómica para la Biodiversidad y Departamento de Ingenierı́a Genética de Plantas, Cinvestav Irapuato
CP36500 Guanajuato, México. 2Institut de Recherche pour le Développement, UMR 5096, 34394 Montpellier, France. 3Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
11724, USA. {Present addresses: BIO5 Institute, University of Arizona, Arizona 85721-0240, USA (M.A.-V.); Department of Molecular Genetics, The Ohio State University, Columbus,
Ohio 43210, USA (R.K.S.).
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©2010 Macmillan Publishers Limited. All rights reserved
LETTERS
NATURE | Vol 464 | 25 March 2010
of undergoing meiosis, we analysed callose deposition in wild-type
and homozygous ago9-3 ovules. In agreement with previous descriptions, wild-type ovules showed patches of callose in the MMC before
the initiation of meiosis (Fig. 2a). After meiosis, callose was deposited
in transverse walls between the functional megaspore and its degenerated sister cells (Fig. 2b). In pre-meiotic ago9-3 ovules, less than
10% of abnormally enlarged cells showed patches of callose deposits
(Fig. 2c, d). During meiosis, callose was only detected in the intermediate walls of a single cell and the degenerated neighbouring cells,
but not in the closely associated abnormally enlarged cells (Fig. 2e, f).
This pattern persisted following meiosis (Fig. 2g, h). These results show
that several enlarged cells differentiate before meiosis in ago9-3 ovules,
but that a single one undergoes meiosis and gives rise to a functional
haploid megaspore, indicating that the activity of AGO9 is necessary to
restrict differentiation to a single sub-epidermal cell in the pre-meiotic
ovule.
After meiosis, ago9-3 ovules showed persistent enlarged cells adjacent
to meiotic products, including the three degenerated megaspores and
the functional megaspore (Fig. 2i–k). To determine the identity and
assess the developmental potential of extranumerary enlarged cells in
mutant ovules, we examined the expression of pFM2, a marker
expressed in the functional megaspore and the developing female
gametophyte, but not in the MMC or in the three meiotically-derived
degenerated megaspores (Fig. 2l, m). In ago9-3 ovules, pFM2 expression was initially observed following meiosis in the functional
megaspore, but also in a cluster of adjacent cells that forms the nucellus
and includes the abnormal gamete precursors (Fig. 2n). In all ago9-3
ovules observed, more than four cells showed strong reporter gene
expression (b-glucuronidase or GUS) at post-meiotic stages, indicating
that at least some of the cells that express pFM2 have a somatic origin.
pFM2 expression was absent at pre-meiotic stages, indicating that defective ago9-3 individuals differentiate other cells that persist in the
Table 1 | Genetic analysis of insertional ago9 mutants in Arabidopsis.
Allele
Genotype
Single MMC
Abnormally enlarged cells
ago9-3
ago9-3/ago9-3
ago9-3/1
ago9-3m/1p/1p
1m/1m/ago9-3p
ago9-4/ago9-4
ago9-2/ago9-2
1/1
208
214
286
241
139
162
292
123 (37.16%)
93 (30.29%)
47 (14.11%)
74 (23.49%)
118 (45.9%)
148 (47.7%)
18 (5.8%)
ago9-4
ago9-2
Wild type
exhibited two MMCs (Fig. 1c); however, only one of the latter underwent gametogenesis as twin female gametophytes were never observed.
All ago9 insertional lines were fertile and did not show signs of ovule or
seed abortion; however, in contrast to wild-type plants, the pre-meiotic
ovule primordia of heterozygous ago9/1 individuals—including allele
ago9-2 that was previously reported as having no defective phenotype11—showed several abnormally enlarged sub-epidermal cells
(Fig. 1d, e). In ago9/1 individuals, the ovules exhibited up to six cells
containing a conspicuous nucleus and nucleolus at a frequency of
30.29%, indicating that ago9 alleles are dominant and affect early cell
differentiation in the developing ovule. In homozygous ago9/ago9 individuals, the percentage of ovule primordia showing more than one
enlarged cell was of 37.16% to 47.7%, depending on the allelic variant
(Table 1). Triploid (3n) individuals that had two wild-type and one
mutant ago9-3 allele showed 14.11% to 23.49% of abnormal ovules, a
value intermediate between diploid plants carrying a single ago9-3
allele and wild type (Table 1). These results indicate that a dosagedependent mechanism is responsible for the mutant ago9 phenotype.
No molecular marker exclusively expressed in the MMC has been
reported, but the pattern of callose deposition is a reliable method to
determine cell identity at pre-meiotic stages12. To determine whether
one or several of the enlarged cells present in ago9-3 ovules are capable
a
i
o
c
b
j
d
e
k
g
f
l
m
h
n
p
Figure 2 | Meiotic and post-meiotic phenotype of the ago9 mutant.
a–h, Callose deposition (a, b, d, f, h) and morphology (c, e, g) of wild-type
and ago9-3 ovules. i–k, ago9-3 post-meiotic ovules showing abnormal
gametic cells (AGC) adjacent to degenerated (asterisk and DM) and
functional (FM) megaspores. l, Absence of pFM2-driven GUS expression in
a pre-meiotic wild-type ovule. m, pFM2-driven GUS expression in the
functional megaspore (dashed) of a post-meiotic wild-type ovule. n, pFM2driven GUS expression in the functional megaspore and adjacent cells of a
post-meiotic ago9-3 ovule. o, ago9-3 ovule containing a 2-nuclear (2NFG)
and a 1-nuclear (1NFG) female gametophyte. p, pFM2-driven GUS
expression in an ago9-3 ovule containing two female gametophytes. Scale
bars: a–h, 5 mm; i–n, 10 mm; o and p, 25 mm.
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©2010 Macmillan Publishers Limited. All rights reserved
LETTERS
NATURE | Vol 464 | 25 March 2010
developing ovule adjacent to the meiotic products and subsequently
acquire a functional megaspore identity without undergoing meiosis.
At subsequent stages of development, ago9-3 individuals exhibited an
unusual phenotype of two independent female gametophytes developing in the same ovule at a frequency of 44.03% (n 5 243; Fig. 2o).
Crosses of ago9-3 plants with individuals expressing the pFM1 (ref. 13)
or pFM2 marker showed that both acquire a female gametophyte
identity (Fig. 2p and Supplementary Fig. 4). These results indicate
that abnormal somatic cells are able to differentiate into gametic cells
and initiate gametogenesis without undergoing meiosis.
Immunoblots hybridized with a polyclonal antibody against AGO9
detected a protein of the expected 100.5 kDa size in developing wildtype gynoecia but not in 1-week-old seedlings, developing rosette
leaves or developing siliques (Fig. 3a). Immunolocalizations showed
that the AGO9 protein was initially expressed in somatic cells of the
epidermal (L1) layer located in the apical region of the pre-meiotic
ovule, but not in the MMC (Fig. 3b). Interestingly, we observed AGO9
in cytoplasmic foci reminiscent of P-bodies or stress granules present
in the cytoplasm of animal cells (Fig. 3c–e). AGO9 did not localize in
the haploid megaspores or the developing female gametophyte before
of after cellularization. In ovules containing a female gametophyte at
the four-nuclear stage, AGO9 was localized in the outer integumentary
cells, but also in the periphery of the endothelium, at the sporophytegametophyte cellular boundary (Fig. 3f). In anthers, AGO9 was localized in the cytoplasm of microsporocytes following meiosis, and later
in the cytoplasm of the vegetative cell but not in the sperm cells
(Supplementary Fig. 5a–d). Ovules or pollen of ago9-3 individuals
did not show AGO9 expression (Supplementary Fig. 5d, e), confirming that the antibody exclusively recognized AGO9. Overall, these
results indicate that AGO9 is preferentially expressed in reproductive
companion cells but not in the associated male or female gametes or
their precursors.
In Arabidopsis, trans-acting siRNAs (ta-siRNAs) are known to
move as signal molecules and cause gene silencing beyond their cellular sites of initiation14–16. Their biogenesis depends on transcription
by RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) that converts
their single-stranded RNA precursors into double-stranded RNA in a
pathway that is also dependent on the function of the putative RNA
binding protein SUPRESSOR OF GENE SILENCING 3 (SGS3)17,18.
The extent of gene silencing movement outside their site of initiation
also depends on the activity of RDR6 (ref. 19). To determine whether
the function of AGO9 could be associated with a non-cell-autonomous pathway, we examined ovule development in homozygous sgs311 and rdr6-11 individuals. Although both sgs3 and rdr6 mutants
show seedling and floral defects characterized by leaf curling and
limited stamen elongation17, their possible role during gamete formation has not been investigated. Both sgs3-11 and rdr6-11 plants
showed an identical phenotype to ago9 mutants with additional gametic cells differentiating in the pre-meiotic ovule (Fig. 4a–d). In rdr611 plants, post-meiotic ovules showed two independently developing
female gametophytes at a frequency of 43.3% (n 5 224). Crosses of
a
kDa
130
95
1
2
3
4
b
c
d
rdr6-11 plants to individuals expressing the pFM2 marker indicate
that both acquire a female gametophyte identity (Fig. 4e). These
results support the hypothesis that AGO9 controls gametic cell commitment by acting in a non-cell-autonomous sRNA-dependent pathway in the developing ovule of Arabidopsis.
To identify the nature of AGO9-associated sRNAs, wild-type
developing gynoecia were isolated and used for total protein extraction, immunoprecipitation with the AGO9 antibody, and elution of
the associated sRNA fraction. After sequencing, 2,508 sRNA sequences
(98% of total) could be mapped to the Arabidopsis nuclear genome and
categorized based on their location and function (Supplementary Tables 1 and 2). Although most are 24 nucleotides in length
(79.1%), 8.9% are 21 to 22 nucleotides long. Most 24-nucleotide
sequences derive from TEs belonging to distinct families of retrotransposons: Gypsy (23%) Athila (9.3%), CACTA (5.5%), and less frequently LINE or Mutator. All sequences mapping to Gypsy TEs
belong to the AtGP1 sub-family, and 3% of all sequences mapping
to retrotransposons correspond to siRNAs shown to be dependent
on RNA polymerase IV (PolIV) for their biogenesis20. A further
17.4% of the total maps to genomic signatures assigned to other families containing nested components of Gypsy, Athila or CACTA TEs. In
contrast, 21-nucleotide sRNAs preferentially derive from previously
characterized miRNAs (3.2%), including MIR167 that is known to
act in the ovule21, and protein-coding genes (14.5%). These results
show that primary targets of AGO9-dependent silencing in the ovule
of Arabidopsis are TEs.
Previous studies have shown that some TEs that are active in mature
pollen grains are not expressed in developing or fully differentiated
ovules of Arabidopsis22. To determine whether AGO9 is necessary for
the inactivation of these TEs in the ovule, we crossed lines containing
enhancer traps that tagged specific TEs to homozygous ago9 individuals. In agreement with previous results, no GUS expression was
observed in the ovule of enhancer trap lines present in a wild-type
genetic background (Fig. 4f). By contrast, heterozygous ago9/1 individuals containing an enhancer trap within either an Athila, LINE or
Atlantys retrotransposon showed strong GUS staining in the egg and
synergid cells of the mature female gametophyte before pollination
(Fig. 4g–i). These results not only confirm that AGO9 is necessary for
TE inactivation in the ovule, but also show that one of its targets is the
egg and synergid cells (the egg apparatus) before fertilization.
The ago9 phenotype was also identified in homozygous mutants
for RNA-dependent RNA polymerase 2 (rdr2), dicer-like 3 (dcl3), and
the double mutant nrpd1a nrpd1b that is defective in the activity of
both polymerase IV and polymerase V, but not in dicer-like 1 and
dicer-like 4 (dcl4) that are essential for the generation of miRNAs and
ta-siRNAs, respectively, indicating that AGO9-dependent TE inactivation restricts female gametogenesis to a single gametic cell
through an endogenous 24-nucleotide siRNA biosynthetic pathway23
(Supplementary Figs 6 and 7). The consistent identification of a
single cell undergoing meiosis and several cells acquiring a functional
megaspore identity in the post-meiotic ovule, combined to the
e
DM
L1 f
FM
130
95
72
55
43
Figure 3 | AGO9 protein expression in developing ovules. a, Immunoblot
analysis of AGO9 in wild-type seedlings (1), developing gynoecia (2) leaves
(3), and 7-day-old siliques (4). b–d, AGO9 is expressed in cytoplasmic foci
(green) of companion cells but absent from the MMC (outlined in b) or the
functional megaspore (arrow in nuclei are counterstained with
49,6-diamidino-2-phenylindole (DAPI) (b and d). e, Diagram showing
AGO9 localization (green) in a wild-type ovule at the end of meiosis. f, AGO9
expression in companion somatic cells but not within a 4-nuclear female
gametophyte (FG). Scale bars: b, 5 mm; c and d, 10 mm; f, 20 mm.
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LETTERS
NATURE | Vol 464 | 25 March 2010
a
f
c
b
e
d
g
h
i
Figure 4 | Phenotype of the rdr6 mutant and activation of transposable
elements in the ago9 mutant. a–d, Ovules of rdr6-11 or sgs3-11 showing
abnormal gametic cells adjacent to the functional megaspore and two female
gametophytes (arrows) separated by the L1 cell layer or degenerated cells
(arrowheads). e, pFM2-driven GUS expression in a rdr6-11 ovule.
f, Enhancer trap ET13889 inserted in a AtLINE3 transposon shows no GUS
expression in mature wild-type ovules. g–i, GUS expression conferred by
enhancer traps ET13889 (g), ET11075 (h) and ET10306 (i) in the egg
apparatus of ago9-3/1 ovules. Scale bars: a, 10 mm; b–i, 25 mm.
presence of two developing female gametophytes separated by several
somatic cells, provides strong evidence for the initiation of female
gametophytes from two non-sister cells, one of which is somatic in
origin.
By preferentially interacting with sRNAs derived from TEs and
silencing their activity in the female gametophyte, the function of
AGO9 is reminiscent of the PIWI subclass of ARGONAUTE proteins
that are necessary to maintain transposon silencing in the germline
genome of invertebrates and mammals24. Some maternal siRNA
sequences found in the endosperm20 and 24-nucleotide siRNA found
in pollen22 resemble AGO9-interacting sRNAs, raising the possibility
that AGO9 may also contribute to these populations in a nonautonomous way. The ago9 mutant phenotype is reminiscent of
apospory, a component of asexual reproduction through seeds (apomixis) prevailing in many flowering species that produce unreduced
female gametes from somatic cells5. Our findings provide opportunities to investigate the genetic basis and molecular mechanisms that
control cell fate, offering new possibilities to explore the epigenetic
induction of apomixis in sexual plants.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 16 September 2009; accepted 11 January 2010.
Published online 7 March; corrected 25 March 2010 (see full-text HTML version for
details).
1.
2.
3.
4.
5.
6.
7.
8.
METHODS SUMMARY
Material and growth conditions. We used Arabidopsis thaliana of ecotype
Columbia 0 (Col-0). Insertional mutant lines were ago9-2 (SALK_112059),
ago9-3 (SAIL_34_G10) and ago9-4 (SAIL_260_A03) (ago9-1 showed an identical
phenotype but was not quantified). Seeds were sterilized with 100% ethanol and
germinated under stable long day (16 h light/8 h dark) conditions at 22 uC.
Seedlings were planted and grown under controlled greenhouse conditions
(24 uC). For a detailed description of mutant stocks, enhancer trap lines, transgenic
lines and DNA constructs, see Methods.
Histological analysis. Cleared ovules and histochemical GUS analysis was performed as described25. Callose analysis was performed as described26 with minor
modifications described in Methods.
Immunoblot and immunoprecipitation. Amino acids N-SSRNHAGNDTNDA
DRK were used to generate a specific AGO9 antibody (Invitrogen). Immunopurification of AGO9–sRNA complexes was performed as described27, with
modifications described in Methods.
Cloning and genomic analysis of small RNAs. After sequencing, sRNA reads
were filtered and sequences were mapped to the Arabidopsis genome (http://
www.arabidopsis.org). Details of the sRNA annotation procedure are provided
in Methods.
9.
10.
11.
12.
13.
14.
15.
16.
Walbot, V. & Evans, M. M. S. Unique features of the plant life cycle and their
consequences. Nature Rev. Genet. 4, 369–379 (2003).
Maheswari, P. An Introduction to the Embryology of the Angiosperms (McGraw-Hill,
1950).
Sheridan, W. F., Avalkina, N. A., Shamrov, I. I., Batygina, T. B. & Golubovskaya, I. N.
The mac1 gene: controlling the commitment to the meiotic pathway in maize.
Genetics 142, 1009–1020 (1996).
Nonomura, K. et al. The MSP1 gene is necessary to restrict the number of cells
entering into male and female sporogenesis and to initiate anther wall formation
in rice. Plant Cell 15, 1728–1739 (2003).
Bicknell, R. A. & Koltunow, A. M. Understanding apomixis: recent advances and
remaining conundrums. Plant Cell 16, S228–S245 (2004).
Grossniklaus, U. & Schneitz, K. The molecular and genetic basis of ovule and
megagametophyte development. Semin. Cell Dev. Biol. 9, 227–238 (1998).
Baumberger, N. & Baulcombe, D. C. Arabidopsis ARGONAUTE1 is an RNA Slicer
that selectively recruits microRNAs and short interfering RNAs. Proc. Natl Acad.
Sci. USA 102, 11928–11933 (2005).
Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell
115, 199–208 (2003).
Pham, J. W., Pellino, J. L., Lee, Y. S., Carthew, R. W. & Sontheimer, E. J. A. Dicer-2dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila. Cell
117, 83–94 (2004).
Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana.
Science 301, 653–657 (2003).
Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M. & Watanabe, Y. The mechanism
selecting the guide strand from small RNA duplexes is different among Argonaute
proteins. Plant Cell Physiol. 49, 493–500 (2008).
Webb, M. C. & Gunning, B. E. S. Embryo sac development in Arabidopsis thaliana. I.
Megasporogenesis, including the microtubular cytoskeleton. Sex. Plant Reprod. 3,
244–256 (1990).
Huanca-Mamani, W., Garcia-Aguilar, M., León-Martı́nez, G., Grossniklaus, U. &
Vielle-Calzada, J. P. CHR11, a chromatin-remodeling factor essential for nuclear
proliferation during female gametogenesis in Arabidopsis thaliana. Proc. Natl Acad.
Sci. USA 102, 17231–17236 (2005).
Chitwood, D. H. et al. Pattern formation via small RNA mobility. Genes Dev. 23,
549–554 (2009).
Schwab, R. et al. Endogenous tasiRNAs mediate non-cell autonomous effects on
gene regulation in Arabidopsis thaliana. PLoS One 4, e5980 (2009).
Voinnet, O. Non-cell autonomous RNA silencing. FEBS Lett. 579, 5858–5871
(2005).
631
©2010 Macmillan Publishers Limited. All rights reserved
LETTERS
NATURE | Vol 464 | 25 March 2010
17. Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L. & Poethig, R. S. SGS3 and
SGS2/SDE1/RDR6 are required for juvenile development and the production of
trans-acting siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379 (2004).
18. Yoshikawa, M., Peragine, A., Park, M. Y. & Poethig, R. S. A pathway for the
biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19, 2164–2175 (2005).
19. Himber, C., Dunoyer, P., Moissiard, G., Ritzenthaler, C. & Voinnet, O. Transitivitydependent and -independent cell-to-cell movement of RNA silencing. EMBO J. 22,
4523–4533 (2003).
20. Mosher, R. A. et al. Uniparental expression of PolIV-dependent siRNAs in
developing endosperm of Arabidopsis. Nature 460, 283–286 (2009).
21. Wu, M. F., Tian, Q. & Reed, J. W. Arabidopsis microRNA167 controls patterns of
ARF6 and ARF8 expression, and regulates both female and male reproduction.
Development 133, 4211–4218 (2006).
22. Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of
transposable elements in pollen. Cell 136, 461–472 (2009).
23. Chen, X. Small RNAs and their roles in plant development. Annu. Rev. Cell Dev. Biol.
25, 21–44 (2009).
24. Klattenhoff, C. & Theurkauf W.. Biogenesis and germline functions of piRNAs.
Development 135, 3–9 (2008).
25. Vielle-Calzada, J. P., Baskar, R. & Grossniklaus, U. Delayed activation of the
paternal genome during seed development. Nature 404, 91–94 (2000).
26. Siddiqi, I., Ganesh, G., Grossniklaus, U. & Subbiah, V. The dyad gene is required for
progression through female meiosis in Arabidopsis. Development 127, 197–207
(2000).
27. Qi, Y., Denli, A. M. & Hannon, G. J. Biochemical specialization within Arabidopsis
RNA silencing pathways. Mol. Cell 19, 421–428 (2005).
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank N. Sánchez for sharing the pFM2 marker,
E. Demunck for technical assistance during cloning and sequencing, S. Poethig,
J. Carrington, T. Lagrange and the Arabidopsis Stock Center for providing mutants,
J. Mendiola and C. Alvarez for help with genetic and bioinformatic analysis, and
R. Jorgensen for critically reading the manuscript. This work was supported by
IRD-France and ANR (D.A. and D.G.), NIH and NSF (R.K.S. and R.A.M), Consejo
Nacional de Ciencia y Tecnologı́a (V.O.-M., N.D.-F., M.A.-V., E.D.-A. and
J.-P.V.-C.), Consejo Estatal de Ciencia y Tecnologı́a de Guanajuato (J.-P.V.-C.), and
the Howard Hughes Medical Institute (J.-P.V.-C.).
Author Contributions J.-P.V.-C. and V.O.-M. designed the research, V.O.-M.
generated the phenotypic analysis, performed the histological and expression
analysis, and conducted the genetic experiments, N.D.-F. designed the antibody
and performed the immunoprecipitations and sRNA analysis, M.A.-V. conducted
the bioinformatic expression analysis, E.D.-A. performed immunolocalization
experiments, D.G. contributed ideas and performed immunolocalization
experiments, R.K.S. and D.A. provided unpublished materials, R.A.M. contributed
ideas and J.-P.V.-C. wrote the paper.
Author Information sRNA sequences are deposited in the EMBL Nucleotide
Sequence Database (FN649764 to FN650107). Reprints and permissions
information is available at www.nature.com/reprints. The authors declare no
competing financial interests. Correspondence and requests for materials should
be addressed to J.P.V.-C. ([email protected]).
632
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08828
METHODS
Plant material and growth conditions. We used A. thaliana ecotype Columbia
(Col-0) for wild-type plants, chemical homozygous mutant sgs3-11 and insertional lines CS24285 (rdr6-11), SALK005512 (dcl3-1) (ref. 28), GABI160G05
(dcl4-2) (ref. 29), SAIL-1277H08 (rdr2-1) (ref. 28), a double mutant nrpd1a-2
nrpd1b-11 (SALK_128428 and SALK_029919, respectively), SAIL_34_G10
(ago9-3), SAIL_260_A03 (ago9-4), SALK_112059 (ago9-2) and A. thaliana ecotype Landsberg erecta (Ler) for Enhancer Trap lines ET13889, ET11075 and
ET10306. Seeds were surface-sterilized by washing three times with 100%
ethanol and plated on Murashige and Skoog (MS) medium. The pFM2 plasmid
construction was generated by amplifying the pFM2 genomic regulatory region
using primers 59-GCGTGACACGCCACTACAACACACCAA-39 (sense) and
59-GCGGATCCAGGAAGCCATCGTCAGACAG-39 (antisense); a 564-basepair (bp) genomic fragment was subsequently cloned in front of the uidA gene
using the pBI101.2 plasmid. Transformation was in Col-0. In all cases MS medium plates containing seeds were placed in full darkness for 3 days at 4 uC, and
subsequently germinated in a growth chamber at 22 uC under a 16 h light/8 h
dark photoperiod, transferred to soil, and grown in the greenhouse under longday 16 h light/8 h dark controlled conditions.
In situ hybridization. A specific 149-bp fragment corresponding to the AGO9
39UTR was PCR amplified by using primers ago9isS2 (59-TCCAGTCCAC
ACGATAGCT-39) and ago9isAS2 (59-ATTCTGTCGGTTTTTGTGGG-39) and
cloned in TOPO-PCRII (Invitrogen). The resulting plasmid was linearized with
BamH1 (sense) and NotI (antisense) and used for generating digoxigeninlabelled RNA-probes (DIG RNA labelling kit SP6/T7; Roche). Developing flower
buds were fixed in 4% paraformaldehyde and embedded in tissue-prep paraffin
(Fisher Scientific). Sections of 10–12-mm thickness were generated using a Leica
microtome and mounted on ProbeOnPlus slides (Fisher Biotech). Hybridization
was performed as described30; for whole mount in situ hybridization, anthers and
ovules were fixed, mounted in acrylamide-covered slides, and hybridized as
described31.
PCR with reverse transcription (RT–PCR). Total RNA was extracted from
leaves, roots, stems, inflorescences, mature flowers, gynoecia, ovules and seedlings with TRIzol (Invitrogen). Total RNA (2 mg) from each tissue was used to
synthesize first-strand cDNA by using 20mer-oligo dT (Sigma), 0.25 mM dNTPs
and SuperScript III Reverse Transcriptase (Invitrogen), and incubating at 42 uC
for 2 h. cDNA (100 ng) was used to amplify a 369-bp AGO9 fragment by using
primers gntpS2 (59-TCCCCAATCAAAGGAAAATGG-39) and gntpAS2 (59-TC
TTGGAATTGTGACTCAGTGCA-39). Amplification of a 96-bp fragment corresponding to ACTIN2 mRNA was used as a control32. PCR was performed with
an initial denaturation step at 94 uC for 1 min 30 s, followed by 30 cycles of
denaturation at 94 uC for 30 s, annealing at 60 uC, and extension at 72 uC for 30 s.
Histological analysis. For phenotypic analysis of ovules, inflorescences from
wild-type and mutant plants were fixed in FAA (formaldehyde 10%, acetic acid
5%, ethanol 50%), for 12 h and subsequently dehydrated in 70% ethanol.
Gynoecia at different developing stages were dissected with hypodermic needles
(1-ml insulin syringes), cleared in Herr’s solution (phenol:chloral hydrate:85%
lactic acid:xylene:clove oil in a 1:1:1:0.5:1 proportion), and observed using a
DMR Leica microscope with Nomarski optics. Histochemical localization of
GUS activity was performed by incubating dissected gynoecia in GUS staining
solution (10 mM EDTA, 0.1% Triton X-100, 0.5 mM potassium ferrocyanide,
0.5 mM potassium ferricyanide and 1 mg ml21 5-bromo-4-chloro-3-indolyl-bD-glucoronic acid in 50 mM sodium phosphate buffer, pH 7.0) for 24 h as
described25. Callose was detected by incubating floral buds in aniline blue staining solution (0.1% aniline blue, 100 mM Na2HPO4, pH 7.4) for 12–24 h in
darkness; for each developmental stage and sample, at least 100 ovules were
dissected on a slide and mounted in 30% glycerol. Observations were conducted
in an Olympus BX60 (Model BX60F5; Olympus Optical) microscope using
epifluorescence ultraviolet filters (365 nm excitation, 420 nm emission).
Micrographs were acquired using Image Pro-Plus Software, version 4.0.
Protein analysis and immunoblots. The amino-terminal sequence of 16
N-SSRNHAGNDTNDADRK-C (16 amino acids) was selected after threedimensional modelling (HHpred, available at http://toolkit.tuebingen.mpg.de)
to generate a rabbit polyclonal peptide antibody (Invitrogen). After affinity
purification, the same antibody was used for Immunoblot analysis (1:500 dilution) or immunolocalization (1:100 dilution). Immunoblots were generated with
the WesternBreeze Chemiluminescent Detection Kit (Invitrogen) using 5 mg of total
protein for each assay (1-week-old seedlings, developing gynoecia, developing rosette leaves, and siliques 7 days after pollination). Proteins were stained with SYPRO
Ruby protein gel stain (Invitrogen) and with a Silver Staining Kit (Invitrogen).
Immunolocalization in sectioned specimens. For immunolocalization experiments, flowers at different developmental stages were fixed in 4% paraformaldehyde in PBS (10 mM KH2PO4, 150 mM NaCl, pH 7) for 12 h at room
temperature, gradually dehydrated in an ethanol series (10%), and embedded
in LR White Resin (Electron Microscopy Sciences). Sections (0.5 mm) were
generated with a ultramicrotome (Leica Ultracut R) and placed on
ProbeOnPlus (Fisher Biotech) slides. After washing twice with PBS, sections
were blocked for 2 h with 5% BSA and 0.05% Tween 20 in PBS, and incubated
with the AGO9 antibody (1:100 in 0.1% BSA in PBS) for 2 h at room temperature. After washing with PBS, slides were incubated with Alexa Fluor 488
goat anti-rabbit (Invitrogen) 1:50 dilution during 2 h at room temperature,
washed with PBS and counterstained with 1 mg ml21 DAPI (Sigma). The slides
were mounted with ProLong Gold antifade reagent (Invitrogen). Fluorescence
was visualized using a Leica DM 6000B epifluorescence microscope, using filter
cubes I3 (excitation 450–490 nm, emission 510 nm) and ultraviolet filter A
(excitation 340–380 nm, emission 400 nm). Images were acquired by using
Leica QWin Standard V3.4.0 (Leica Microsystems).
Immunolocalization in whole-mounted specimens. Pistils and siliques at
various developmental stages were fixed overnight at 4 uC in 4%
paraformaldehyde:PBS:2% Triton fixative, washed three times in PBS, and dissected to isolate the ovules and early seeds. The dissected ovules and seeds were
embedded in acrylamide as described33 to facilitate manipulation and maintain
the three-dimensional architecture of the tissues. Samples were digested in an
enzymatic solution (1% driselase, 0.5% cellulase, 1% pectolyase, 1% BSA, all
from Sigma) for 25 min to 1 h at 37 uC, depending on the developmental stage,
subsequently rinsed three times in PBS, and permeabilized for 2 h in PBS:2%
Triton. They were then incubated overnight at 4 uC with primary antibodies used
at a dilution of 1:100 for AGO9 and 1:400 otherwise. The slides were washed for a
day in PBS:0.2% Triton, and coated overnight at 4 uC with secondary antibodies
(Alexa Fluor 488 or 568 conjugate, Molecular Probes) used at 1:400 dilution.
After washing in PBS:0.2% Triton for a minimum of 6 h, the slides were incubated with DAPI (1 mg ml21 in PBS) for 1 h, washed for 2 h in PBS, and mounted
in PROLONG medium (Molecular Probes). Complete 3D ovule or seed images
were captured on a laser scanning confocal microscope (Leica SP2) equipped
with 405 nm (DAPI), 488 nm (green) or 568 nm (red) excitation and either 340
or 363 objectives. Projections of selected optical sections were generated for this
report, and edited using Graphic Converter (LemkeSOFT). At least 50 ovules
were scored for each developmental stage.
Immunoprecipitation and analysis of small RNAs. Immunopurification
AGO9 and its associated sRNAs was conducted as described34, with some modifications. Owing to extremely low protein yields obtained in preliminary experiments conducted with hundreds of female reproductive organs, protein
extraction was conducted with a total of 12,000 wild-type developing gynoecia
containing ovules at mixed developmental stages (four-nuclear stage of gametogenesis to unpollinated mature). Protein extract (0.5 ml) from 12,000 wildtype gynoecia was pre-cleared by incubation with 10 ml Protein A-Sepharose
(Invitrogen, Cat. No. 10-1041) at 4 uC for 30 min. Pre-cleared extracts were then
incubated either with AGO9 antibody or AGO9 pre-immune serum as a negative
control, and 30 ml Protein A-Sepharose at 4 uC overnight. The immunoprecipitates were washed three times (15 min each) in extraction buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.2% NP-40, 5 mM MgCl2, 5 mM
dithiothreitol, one tablet of Roche Protease Inhibitor Cocktail for each 10 ml).
Commercial columns (Ambion) were used to isolate sRNAs from the purified
AGO9 complex. Small RNAs were resolved on a 12.5% denaturing PAGE 7 M
urea gel, and stained with SYBR-gold (Invitrogen). Before cloning, gel slices
within the range of 18–30 nucleotides were excised, and the RNAs were eluted
and purified using DTR Gel Filtration Cartridges (EdgeBio). A detailed protocol
of the immunoprecipitation and elution procedure is available on request.
After elution and gel-purification, sRNAs were ligated with adaptors at their 59
and 39 ends, converted to cDNA products, and subsequently cloned and
sequenced by Sanger methods. Whereas immunopurifications conducted with
the pre-immune serum did not yield any bacterial clones containing endogenous
Arabidopsis sequences, we obtained a total 2,552 sequences representing 344 distinct small RNAs with the AGO9 antibody. Cloning of small RNAs was performed
with the miRCat Small RNA Cloning Kit (Integrated DNA Technologies) following manufacturer instructions. Individually cloned products were sequenced with
the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) in a 3730xl
DNA Analyzer (Applied Biosystems,). Sequences were quality-checked with
sequence Scanner 1.0 (Applied Biosystems). Sequences were filtered and mapped
to the Arabidopsis genome (http://www.arabidopsis.org). Annotation of sRNAs
was performed using information from TAIR9 (ftp://ftp.arabidopsis.org/
Sequences/blast_datasets/TAIR9_blastsets), and miRBase (http://microrna.sanger.
ac.uk/sequences).
28. Xie, Z. et al. Genetic and functional diversification of RNA pathways in plants. PLoS
Biol. 2, e104 (2004).
©2010 Macmillan Publishers Limited. All rights reserved
doi:10.1038/nature08828
29. Xie, Z., Allen, E., Wilken, A. & Carrington J. C.. DICER-LIKE 4 functions in transacting small interfering RNA biogenesis and vegetative phase change in
Arabidopsis thaliana. Proc Natl Acad Sci USA 102, 12984–12989 (2005).
30. Vielle-Calzada, J.-P. et al. Maintenance of genomic imprinting at the Arabidopsis
medea locus requires zygotic DDM1 activity. Genes Dev. 13, 2971–2982 (1999).
31. Garcı́a-Aguilar, M., Dorantes-Acosta, A., Pérez-España, V. & Vielle-Calzada, J.-P.
Whole-mount in situ mRNA localization in developing ovules and seeds of
Arabidopsis. Plant Mol. Biol. Rep. 23, 279–289 (2005).
32. Kasahara, R. D., Portereiko, M. F., Sandaklie-Nikolova, L., Rabiger, D. S. & Drews,
G. N. MYB98 is required for pollen tube guidance and synergid cell differentiation
in Arabidopsis. Plant Cell 17, 2981–2992 (2005).
33. Bass, H. W. et al. Evidence for the coincident initiation of homolog pairing and
synapsis during the telomere-clustering (bouquet) stage of meiotic prophase. J.
Cell Sci. 113, 1033–1042 (2000).
34. Qi, Y., Denli, A. M. & Hannon, G. J. Biochemical specialization within Arabidopsis
RNA silencing pathways. Mol. Cell 19, 421–428 (2005).
©2010 Macmillan Publishers Limited. All rights reserved