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Ovule development in Arabidopsis: progress and challenge
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Ovule development in Arabidopsis: progress and challenge Dong-Qiao Shi and Wei-Cai Yang Female gametophyte, the central core of the ovule, is a simple seven-celled reproductive structure. Its stereotyped ontogeny provides a traceable model system to study mechanisms controlling cell growth, cell division, cell fate, pattern formation, and perhaps the function of essential genes in plants. An auxin concentration gradient was demonstrated for the first time in the embryo sac to control gametic cell fate. Mutant analysis also indicates a role of RNA processing in the mitotic progression of the gametophytic generation and cell fate determination in the embryo sac. Combined studies of genetics and transcriptome analysis revealed recently that epigenetic pathways play a critical role in female gametophyte development. In addition, the discovery that a large number of small secreted cysteine-rich proteins are enriched in embryo sac is of special interest. Except these insights and progresses, challenge ahead is to reveal the signaling pathways and their interactions that lead to the patterning of the female gametophyte. Address Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 1 West Beichen Road, Chaoyang District, Beijing 100101, China Corresponding author: Shi, Dong-Qiao ([email protected]) and Yang, Wei-Cai ([email protected])
Current Opinion in Plant Biology 2011, 14:74–80 This review comes from a themed issue on Growth and development Edited by Jiayang Li and Nam-Hai Chua Available online 29th September 2010 1369-5266/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2010.09.001
Introduction As the female sexual organ of higher plants, ovules provide the structural support or enclose of female gametophytes, and develop into seeds after fertilization. Ovules in Arabidopsis and most flowering plants are composed of four parts, the embryo sac, the nucellus, the integument, and the funiculus that connect the ovule to placenta. Female gametophytes, or embryo sacs, are embedded in the nucellus, which degenerates ultimately. The apexes of integuments form a mini-opening called micropyle where pollen tube enters. Both the ovule and the embryo sac are polarized organs. As the core of the Current Opinion in Plant Biology 2011, 14:74–80
ovule, female gametophyte in Arabidopsis originates from a single hypodermal cell – the archespore at the tip of the ovule primodium. The archespore directly differentiates into megasporocyte that undergoes further meiosis to produce four megaspores. Only the chalazal-most megaspore – the functional megaspore – undergoes three rounds of nuclear division to form a coenocytic, eightnucleated embryo sac. Subsequently, nuclear migration, polar nuclear fusion, and cellularization take place to yield ultimately a seven-celled embryo sac composed of two synergids, one egg cell, one central cell, and three antipodals (Figure 1). Ovules are essential reproductive organs that not only share features with vegetative organs, but also bear unique characters special for reproduction, with genes controlling ovule ontogeny overlapping with those required for both vegetative and sexual development. The genes involved in ovule development include those that function in polarity establishment, meristem maintenance, floral organ determination, ovule identity, and structure specification. Recent investigations revealed that integument and leaf may share common developmental pathways [1,2]. This review will focus mainly on female gametophyte development with an emphasis on gene expression, polarity, epigenetic control, and RNA processing in Arabidopsis. For more information on ovule development, readers are referred to excellent recent reviews [3,4,5,6].
Integument development and evolution As the focus of classic and molecular genetics in plant research, progress made in ovule and female gametophyte development benefited from the combinatorial approaches of mutants and high-throughput technology. Ovules are bitegmic in most angiosperms including Arabidopsis and unitegmic or ategmic in others such as Santalales and Gymnosperms. Integuments share many common features in morphology, development, and genetic program with leaves; it has been proposed that integuments and leaves are analogous structure [2]. Several genes, including AINTEGUMENTA (ANT), BELL1 (BEL1), and INNER NO OUTER (INO) that play a central role in integument development are also crucial for lateral organ development in Arabidopsis. Recently, the comparison of ANT and BEL1 gene expression pattern in Arabidopsis and Santalales suggests that, ategmic ovules are probably derived from a fusion of the integument with nucellus, or that the nucellus has acquired the characteristics confined to integuments in ancestral species [7]. Therefore, integument biogenesis is of interest for both ovule development and evolution. www.sciencedirect.com
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Figure 1
Ovule and embryo sac of Arabidopsis. (a) An electron scanning image of ovule, showing the ovule (ov), funiculus (fn), and micropyle (mp). (b) A confocal optical image showing a typical ovule structure with a seven-celled and eight-nucleate embryo sac. Gametophytic cells of the embryo sac are colored in red (egg), green (synergids), blue (central cell) and yellow (antipodals). The integuments are in purple. The polar nuclei (pn) of the central cell are not yet fused. (c) An optical image projection showing a mature ovule with a seven-celled embryo sac. The polar nuclei have fused to form a diploid central nucleus (cn). Three antipodal cells at the chalazal end of the embryo sac are under degeneration. an, antipodal nucleus; cn: central nucleus; en, egg nucleus; fn, funiculus; mp, micropyle; ov, ovule; pn, polar nucleus; sn, synergid nucleus; and vc, vacuole. Bars = 10 mm.
To identify genes controlling integument ontogeny in Arabidopsis, transcriptome analysis was performed between ino (which lacks the outer integument) and ant (which lacks both inner and outer integuments) [8–10]. The comparison of transcriptomes of ovules from the wildtype, ino and ant mutants revealed 207 genes predominantly expressed in integuments [7]. Among them, 25 genes are preferentially expressed in both inner and outer integuments, 132 putative genes in inner integument and 50 genes in outer integument. About 80% of the integument-expressed genes encode for proteins that are involved in metabolism, transcription, and proteins with unknown function. It is remarkable that transcription factors and DNA binding proteins increase to 20% in context of integument development, compared to 6–7% in the whole genome. These transcriptional factors are implicated in organ size control, auxin signaling, lateral organ development, growth, and transcription regulation. This implies the complexity of the regulation in the integuments although they are rather simple structures. Furthermore, these genes serve as candidates for further analysis for their roles in integument development, and would be valuable for the understanding of integument evolution as well.
Female gametophyte displays distinct transcriptome To identify genes expressed in female gametophyte, mutants without embryo sac are selected for transcriptome analysis. Mutant sporocyteless (spl) has no sporocyte and is www.sciencedirect.com
completely absent of embryo sac in ovules [11,12]. SPL gene encodes a novel transcription regulator that is required for the initiation of sporogenesis in both sexes and is sufficient to trigger microsporogenesis when activated ectopically [13]. Transcriptomic comparison between the wild-type and spl mutants with Affymetrix ATH1 chip revealed 225 ovule-expressed genes in total. 23.6% of them encode unknown proteins; the remaining ones are implicated in central metabolism, detoxification/ stress response, cell structure, transport, protein degradation, signal transduction, and transcription regulation [14]. Similarly, the comparison of expression profiles of male sterility1 (ms1) [15,16] and determinant infertile1 (dif1) (no embryo sac due to meiosis failure) [17] identifies 71 ovule-expressed genes [18]. 41 of them encode proteins with unknown function; the remaining ovule-expressed genes are involved in metabolism, electron transport, energy pathway, transport, signaling, cell organization, and transcriptional regulation. Conversely with the genes identified in integuments, transcription factors are much less abundant in female gametophyte. By comparing wildtype ovules with those of dif1 and myb98 (defective in synergid function) [19], with high-throughput sequencing and whole-genome tiling arrays, 382 and 77 genes were found down-regulated in ovules of dif1 and myb98, respectively [20]. Among the 382 dif1-down-regulated genes, 297 (78%) encode small secreted, cysteine-rich proteins (CRPs). These include antipathogenic peptides of the Defensin-Like (DEFL) family (74 genes) and thionin-like Current Opinion in Plant Biology 2011, 14:74–80
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family (11 genes), and the pistil-secreted SI proteins of Papaver Self-Incompatibility-Like (PSIL) family (22 genes). Among the 286 DEFL genes and 62 thionin-like genes in the Arabidopsis genome, 32% of the DEFL genes and 19% of the thionin-like genes respectively are embryo sac-expressed. In addition, many members of the DUF784 and DUF1278 gene families are also embryo sacexpressed, for example, 37 out of 40 DUF784 members are down-regulated in myb98. They are expressed specifically in synergids and potentially involved in synergid function or embryo sac maturation. Interestingly, many members of these families are encoded by tandemly arrayed, recently duplicated genes. This indicates that the DUF784 and DUF1278 families are embryo sacspecific and may have evolved with the embryo sac. Among the embryo sac-enriched genes, about 78% encode proteins with signal peptide and 66% encode peptides with molecular weight less than 20 kDa. This suggests that they may act as intercellular signaling molecules during embryo sac development and male–female interactions. For example, the PSILs inhibit growth and trigger cell death of self-pollen in Papaver, implying they may play a role in cell–cell communication during embryo sac–pollen tube interaction. Consistently, the maize CRP protein ZmEA1 and LUREs in Torenia act as pollen tube attractants [21,22]. Undoubtedly, further illustration of the function of the CRPs will shed light on the cell–cell signaling within the embryo sac, among the gametic cells, and between embryo sac and the pollen tube. To further identify genes expressed in a specific gametophytic cell type, Wuest et al. performed cell typespecific transcriptome analysis using laser-assisted micro-dissection [23]. After extensive validation, they estimate that about 43% (8850 genes) of the Arabidopsis genome are expressed in the embryo sac, with 7171 (36%) in egg, 7287 (35%) in central cell, and 5628 (27%) in synergids, respectively. 420 genes are significantly enriched in one of the three gametophytic cell types, these include previously identified genes such as MYB98, FIS2, DME, AGL26, AGL61, AGL80, and genes encoding CRPs. Interestingly, F-box and leucine-rich proteins are also enriched in the female gametophyte. These data show that gametophytic cells display distinct post-transcriptional and epigenetic regulatory mechanisms in regulating gene expression and metabolic pathways. Transcription factors with RWP-RK domain, MADS domain, and REM-TFs, are highly expressed in the female gametophyte. Moreover, genes encoding the double-stranded RNA-binding factors, or involved in various gene silencing pathways, are also abundant in the egg, or central cell. Intriguingly, genes involved in miRNA biogenesis including PAZ, PIWI domain and DUF1785 family are globally enriched in the egg cell. On the contrary, genes acting in gene silencing pathway are elevated in the central cell. This suggests an early Current Opinion in Plant Biology 2011, 14:74–80
differentiation in epigenetic regulatory machinery during female gametophyte development [23]. Nevertheless, the biological significance of these differences remains to be elucidated. Interestingly, components acting in auxin signaling and metabolism are also enriched in the embryo sac. This may further support a role of auxin gradient in patterning the female gametophyte [24]. It is no doubt that the data sets of the transcriptome profiling are invaluable to decipher the roles of auxin, small RNAs and RNA biogenesis in ovule and female gametophyte development.
Auxin gradient and the female gametophyte patterning Auxin is a mobile signaling molecule that is polarly transported from the site of synthesis to target cells via its influx and efflux carriers throughout the plant. It is inevitable that a gradient would be formed during polar auxin transport that is responsible for pattern formation during early embryogenesis, organogenesis and ovule development as well [24,25,26–29]. The female gametophyte is a polarized structure with four cell types. The positioning of nuclei, cells and manifestation of cell fate are concomitant with each other during embryo sac development. The establishment of cell identity coincides with the cellularization of the coenocytic embryo sac, the mis-positioning of nuclei often results in changes in cell identity as seen in eostre mutant [28]. Therefore positional or location-specific cues are expected to exist. Indeed, the auxin-dependent patterning and gamete specification in the embryo sac are elegantly demonstrated [4,24]. During early ovule development, the auxin concentration is high at the distal tip of the nucellus before and during meiosis, and maintains high in the nucellar tip at one-nucleate stage and later at the micropylar end of the embryo sac at twonucleate stage. The auxin concentration at the micropylar end is increased during further development of the embryo sac and remains at the maximum until eightnucleate stage. This polarized distribution of auxin is maintained up to cellularization stage and later it turns to less polarized in mature embryo sac. Thereby, an auxin gradient that declines along the micropylar to chalazal axis in the embryo sac is established. It seems that the initial auxin maximum at the nucellar tip is set up by polar auxin transport within the nucellar cells, and later by local de novo synthesis of auxin since only the expression of YUCCA, but not PIN1 has been detected in the embryo sac [24]. On the contrary, the nuclei are exposed to different auxin concentrations along the gradient before cellularization, implying that the production of auxin and its polarized accumulation act as the ‘prelude’ of cellularization and cell specification in the embryo sac. Furthermore, disturbance of auxin gradient leads to abnormal or defective gametic cell fate, or even the conversion of chalazal cell identities to micropylar cell www.sciencedirect.com
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identities [24]. All three micropylar cells adopt egg cell fate when AUXIN RESPONSE FACTORS (ARFs) ARF-18 and ARF-19 are knocked down by artificial microRNAs. This indicates that the cell fate determination of synergid, egg cell, central cell and antipodal that arranged from the micropylar to the chalazal end is somehow dependent on the auxin concentration gradient, which decreases along micropylar–chalazal axis. However, it is not known that whether auxin plays roles as the primary instructive signal, or through an undiscovered downstream factor [24]. It remains to be illustrated when and how the auxin gradient leads to the final patterning of the embryo sac. Since the auxin gradient is established early in embryo sac development, an obvious question is whether the eight nuclei in the coenocytic embryo sac respond differently or they are instructed by the concentration gradient to behave differently. More need to be done. The discovery of auxin signaling components, and the mechanism underlying their polarized distribution in embryo sac would be the targets for further investigation. And it is also to be elucidated whether these events happened in the embryo sac share common features with those in vegetative organs.
Small RNAs, new players in female gametophyte development Small RNAs produced from long double-stranded RNAs (dsRNAs) are a class of distinct RNAs that mediate almost every aspect of plant development or response to environment through transcriptional or post-transcriptional regulation [30]. Small RNAs are classified into three types according to their origin, namely microRNA (miRNA), small interfering RNA (siRNA, including trans-acting siRNA and natural antisense transcript siRNA), and piwi-interacting RNA (piRNA) [30,31,32]. miRNAs are produced from single-stranded RNAs with hairpin structures, siRNAs are derived from long dsRNAs. miRNAs and siRNAs are 20–24 or 18–25 nucleotide in length, respectively. piRNAs are a third type of longer small RNAs of 26–31 nucleotides that are only found in germline of animals currently [33]. Small RNAs are bound by ARGONAUTE (AGO) proteins, then loaded to RNA Silencing Complex (RISC), to guide to the target mRNA by sequence complementarities [34]. Small RNA populations have been unveiled through high-throughput sequencing. 33 different miRNA families were found in Arabidopsis mature pollens recently [35,36]. They are predicted to target transcripts of genes involved in epigenetic pathways and in hormone response such as ARFs in pollen [35]. The sperm-specific cis-nat-siRNA (natural cis-antisense siRNA) generated by reversely overlapped genes KOKOPELLI (KPL) and ARIADNE14 (ARI14) impairs double fertilization in kpl mutant [36]. However, small RNAs have not been investigated in the female gametophyte www.sciencedirect.com
using high-throughput sequencing due to the difficulties to obtain sufficient material. Recent reports indicate that some key components of small RNA pathways, such as AGO1, AGO9, and DICER-LIKE1, are involved in female or male gametophyte development [37,38]. These demonstrated the existence of small RNA regulatory network in female or male gametogenesis [39]. Moreover, as mentioned above, expression profiles of different cell types of the embryo sac indicate that genes encoding PAZ/Piwi proteins are up-regulated, suggesting that epigenetic regulation through small RNA networks occurs in the egg cell [23]. The involvement of small RNAs in female gametophyte development is further confirmed with investigation of AGO9 function recently [37]. AGO proteins are effectors of RNA silencing, they specifically bind small RNAs and mediate RNA cleavage, translation suppression, or DNA epigenetic modification [40–43]. AGO9 is highly expressed in ovule and anther of Arabidopsis. Mutations in AGO9, SUPPRESSOR OF GENE SILENCING3 and RNA-DEPENDENT RNA POLYMERASE6, form additional abnormal megasporocyte-like cells in ovule primordium in a dosage-dependent manner. Furthermore, these aberrant sub-epidermal cells in ago9 can acquire functional megaspore identity, or even develop into gametic cells and initiate gametogenesis without undergoing meiosis [37]. However, AGO9 expression is neither detected in megasporocyte, nor the megaspore or the subsequent female gametophyte, but present in the surrounding cells of the embryo sac. Consistently, it is not found in sperm cell as well, but vegetative cell of the male gametophyte. This indicates that a non-cell autonomous small RNA silencing pathway is involved in Arabidopsis ovule development, particularly in ensuring that a single megasporocyte forms in each ovule primordium and gametic cell fate in the male gametophyte [37]. AGO9 associates small RNAs derived from transposable elements (TEs) and is necessary for TE inactivation. Although the role of AGO9 associated TE inactivation in Arabidopsis ovule development is not clear, this study implies that an AGO9dependent, or to say the least, the small RNA-mediated pathway may act as a key regulatory pathway to prevent somatic cells from undergoing sexual reproductive or apomictic process in higher plants. Small RNAs have been shown to be mobile molecules that act in systemic gene silencing and leaf patterning. It was reported that a gradient of small RNA is created because of the intracellular movement of trans-acting siRNAs from the origin of biogenesis (adaxial) to the abaxial side of leaf [44]. Does the gradient of small RNAs, or some other factor participating in the small RNA pathway, exist in ovule or female gametophyte, just as that of auxin? Whether small RNA functions as a mobile messenger to ignite the regulation network in cell identity determination? These questions need to be addressed in the future. Current Opinion in Plant Biology 2011, 14:74–80
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RNA processing and female gametophyte development The functional megaspore undergoes three nuclear divisions without cytokinesis, resulting in an eightnucleate embryo sac. Therefore, the embryo sac is a giant cell with a large central vacuole before cellularization. Question remains to be addressed on how cell growth, cell division and differentiation are regulated. How are the transcriptional and translational activities coordinated in such a large cell? Recent studies suggest that nucleolar activities, such as RNA biogenesis and ribosome biogenesis, are crucial for embryo sac development in Arabidopsis. Mutations in genes involved in rRNA biogenesis, mRNA processing and ribosomal biogenesis, often block the mitotic progression of the gametophytic cell cycle. The first mutant reported is slow walker1 (swa1), which is defective in mitotic progression during female gametogenesis, resulting in complete female sterility in Arabidopsis [45]. SWA1 encodes a nucleolar WD40-domain protein with homology to the yeast UTP15 that is involved in prerRNA processing. Indeed, knockdown of SWA1 impairs 18S pre-rRNA biogenesis in Arabidopsis [45]. SWA2 is a nucleolar protein too, it is homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1/MAINTENANCE of KILLER21, and probably has conserved function in pre-ribosome biogenesis and nucleolar export [46]. SWA3/AtRH36 is a newly reported member of the DEAD-box RNA helicases that possibly acts in 18S rRNA biogenesis as well [47,48]. All three mutants of null mutations, which lead to complete loss of gene function, display slow progression of mitosis resulting in the arrest of embryo sac development at two-nucleate, four-nucleate, or eightnucleate stages, but have little or no effect on male gametophyte development. Consistently, loss-of-function mutations of GAMETOPHYTIC FACTOR1/ CLOTHO/VAJRA (GFA1/CLO/VAJ), homologous to the yeast U5 snRNP spliceosomal component Snu114p that regulates spliceosome dynamics, also arrest embryo sac development at these stages [49,50]. These data indicate that rRNA, or ribosomal biogenesis is crucial to cell cycle progression during female gametophyte development. In addition, MAGATAMA3 (MAA3), a plant homolog of the yeast Sen1, which is necessary to various aspects of RNA metabolism, such as rRNA/ tRNA processing, localization of nucleolar proteins, and pre-mRNA metabolism, functions in female gametophyte development as well. The mutant maa3 displays pleiotropical defects such as failure in polar nuclear fusion, small size of nucleoli of polar nuclei, partial fertility of the male gametophyte, and inability to attract pollen tube [51]. A simple explanation to this phenomenon is that these essential genes are required for rapid growth of the embryo sac, since they may also play a role in vegetative growth as shown for VAJ [52]. An alternative explanation is that there is a regulatory Current Opinion in Plant Biology 2011, 14:74–80
role of the nucleolar function for embryo sac development in Arabidopsis, for example, to coordinate transcriptional and translational activities during the rapid growth of the embryo sac. More intriguingly, RNA processing has been shown to be crucial for cell fate determination of the embryo sac. For example, in lachesis (lis) mutants, synergids and central cells adopt egg cell fate, whereas antipodal cells behave like central cells. LIS encodes an Arabidopsis homolog of the yeast splicing factor PRP4 [53]. Similarly, GFA1/ CLO/VAJ and ATROPOS (ATO), encoding Arabidopsis homolog of SF3a60 that is implicated in spliceosomal assembly [54], are also crucial for gametic cell fate specification in the embryo sac since mutations in these genes result in supernumerary egg cells or mis-specified gametic cells, indicating that they are key factors for gametic cell fate. These data suggest a key role of the spliceosomal components and related regulatory pathway in gametic cell fate in plants. Interesting questions arise: How could RNA splicing specifically regulate gametic cell fates in the embryo sac? Does it control the splicing of a specific subset of transcripts that is crucial for gametic cell fate? Or does it process all mRNAs in the embryo sac? Do they have functions other than RNA processing? These need to be investigated in the future.
Concluding remarks Female gametophyte in higher plants is a simple sevencelled reproductive structure. Its stereotyped ontogeny provides a traceable system to study mechanisms controlling cell growth, cell division, differentiation, cell fate, and perhaps the function of essential genes in plants. As discussed above, significant progresses have been made in understanding the patterning of the embryo sac and transcriptomes of each cell types, the demonstration of the auxin gradient is of great importance to developmental biology. Nevertheless, we are facing new questions and challenges, for example, how is the auxin gradient translated into positional information that patterns the embryo sac? What is the role of the epigenetic regulatory network in female gametophyte development? What is the role of nucleolus, such as RNA processing and ribosome biogenesis? How did the ovule and embryo sac evolve? Data generated by transcriptome analysis provide us many clues and candidate genes that are of special interest to address those questions. With the advancement of new technologies, such as live imaging, targeted mutagenesis, laserassisted techniques, and high-throughput sequencing, more progress on this miniature reproductive structure is yet to be made.
Acknowledgements We would like to thank the financial support from the Ministry of Science and Technology of China (2007CB947604) and the Natural Science Foundation of China (30921003). www.sciencedirect.com
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33. Kim VN: Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) in mammalian testes. Genes Dev 2006, 20:1993-1997. 34. Lu C, Tej SS, Luo S, Haudenschild CD, Meyers BC, Green PJ: Elucidation of the small RNA component of the transcriptome. Science 2005, 309:1567-1569. 35. Grant-Downton R, Le Trionnaire G, Schmid R, Rodriguez Enriquez J, Hafidh S, Mehdi S, Twell D, Dickinson H: MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana. BMC Genom 2009, 10:643. Micro RNA families were detected in mature pollen in this report. It is suggested that miRNA and trans-acting siRNA pathways are involved in pollen development. 36. Ron M, Saez MA, Williams LE, Fletcher JC, McCormick S: Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis. Genes Dev 2010, 24:1010-1021. KPL and ARI14 genes are inversely transcribed to generate a spermspecific nat-siRNA pair that targets ARI14. This nat-siRNA pair cannot be produced in kpl plants and results in activation of ARI14 gene in sperm. kpl mutant shows frequent single fertilization, instead of double fertilization in wild-type. This means that cis-nat-siRNA is essential for double fertilization. 37. Olmedo-Monfil V, Dura´n-Figueroa N, Arteaga-Va´zquez M, Demesa-Are´valo E, Autran D, Grimanelli D, Slotkin RK, Martienssen RA, Vielle-Calzada JP: Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 2010, 464:628-632. This is the first report of small RNA pathway in female gametophyte ontogenesis. AGO9 controls specification of MMC in a dosage-dependent manner. AGO9-dependent sRNA silencing in the ovule is crucial in plant gamete formation. 38. Grant-Downton R, Hafidh S, Twell D, Dickinson HG: Small RNA pathways are present and functional in the angiosperm male gametophyte. Mol Plant 2009, 2:500-512. 39. Le Trionnaire G, Twell D: Small RNAs in angiosperm gametophytes: from epigenetics to gamete development. Genes Dev 2010, 24:1081-1085. A review addressed the recent publications about small RNAs found in male or female gametophytes. The pathways of small RNAs uncovered recently are discussed. 40. Havecker ER, Wallbridge LM, Hardcastle TJ, Bush MS, Kelly KA, Dunn RM, Schwach F, Doonan JH, Baulcombe DC: The Arabidopsis RNA-Directed DNA methylation Argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 2010, 22:321-334. 41. Qi Y, Denli AM, Hannon GJ: Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 2005, 19:421-428. 42. Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL, Chapman EJ, Fahlgren N, Allen E, Carrington JC: Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 2008, 133:128-141. 43. Fahlgren N, Montgomery TA, Howell MD, Allen E, Dvorak SK, Alexander AL, Carrington JC: Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr Biol 2006, 16:939-944.
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44. Chitwood DH, Nogueira FTS, Howell MD, Montgomery TA, Carrington JC, Timmermans MCP: Pattern formation via small RNA mobility. Genes Dev 2009, 23:549-554. 45. Shi DQ, Liu J, Xiang YH, Ye D, Sundaresan V, Yang WC: SLOW WALKER1, essential for gametogenesis in Arabidopsis, encodes a WD40 protein involved in 18S ribosomal RNA biogenesis. Plant Cell 2005, 17:2340-2354. This report provides evidence that 18S pre-rRNA processing is crucial for the mitotic progression during female gametogenesis in Arabidopsis. 46. Li N, Yuan L, Liu N, Shi D, Li X, Tang Z, Liu J, Sundaresan V, Yang WC: SLOW WALKER2, a NOC1/MAK21 homologue, is essential for coordinated cell cycle progression during female gametophyte development in Arabidopsis. Plant Physiol 2009, 151:1486-1497. 47. Liu M, Shi DQ, Yuan L, Liu J, Yang WC: SLOW WALKER3, encoding a putative DEAD-box RNA helicase, is essential for female gametogenesis in Arabidopsis. J Integr Plant Biol 2010 doi: 10.1111/j.1744-7909.2010.00972.x. 48. Huang CK, Huang LF, Huang JJ, Wu SJ, Yeh CH, Lu CA: A DEADBox protein, AtRH36, is essential for female gametophyte development and is involved in rRNA biogenesis in Arabidopsis. Plant Cell Physiol 2010, 51:694-706. 49. Liu M, Yuan L, Liu NY, Shi DQ, Liu J, Yang WC: GAMETOPHYTIC FACTOR1, involved in pre-mRNA splicing, is essential for megagametogenesis and embryogenesis in Arabidopsis. J Integr Plant Biol 2009, 51:261-271. 50. Coury D, Zhang C, Ko A, Skaggs M, Christensen C, Drews GN, Feldmann A, Yadegari R: Segregation distortion in Arabidopsis gametophytic factor 1 (gfa1) mutants is caused by a deficiency of an essential RNA splicing factor. Sex Plant Reprod 2007, 20:87-97. 51. Shimizu KK, Ito T, Ishiguro S, Okada K: MAA3 (MAGATAMA3) helicase gene is required for female gametophyte development and pollen tube guidance in Arabidopsis thaliana. Plant Cell Physiol 2008, 49:1478-1483. The MAA3 is a helicase gene and possibly required for RNA metabolism. maa3 mutant is defective in polar nuclei fusion and micropylar pollen tube guidance. 52. Yagi N, Takeda S, Matsumoto N, Okada K: VAJ/GFA1/CLO is involved in the directional control of floral organ growth. Plant Cell Physiol 2009, 50:515-527. 53. Groß-Hardt R, Kagi C, Baumann N, Moore JM, Baskar R, Gagliano WB, Ju¨rgens G, Grossniklaus U: LACHESIS restricts gametic cell fate in the female gametophyte of Arabidopsis. PLoS Biol 2007, 5:494-500. LIS encodes homolog of yeast splicing factor PRP4. In lis mutant, the accessory cells can differentiate to gametic cells and leads to supernumerary egg cells. The investigation indicates that components of the splice apparatus participate in cell fate decisions. 54. Moll C, von Lyncker L, Zimmermann S, Kagi C, Baumann N, Twell D, Grossniklaus U, Gross-Hardt R: CLO/GFA1 and ATO are novel regulators of gametic cell fate in plants. Plant J 2008, 56:913-921. CLO/GFA1 and ATO encode homologs of spliceosomal components. Mutation of these genes results in mis-specification of cells in female gametophyte. This paper shows that core spliceosomal components are specially required for the female gametophyte patterning.
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Ovule development in Arabidopsis: progress and challenge Dong-Qiao Shi and Wei-Cai Yang Female gametophyte, the central core of the ovule, is a simple seven-celled reproductive structure. Its stereotyped ontogeny provides a traceable model system to study mechanisms controlling cell growth, cell division, cell fate, pattern formation, and perhaps the function of essential genes in plants. An auxin concentration gradient was demonstrated for the first time in the embryo sac to control gametic cell fate. Mutant analysis also indicates a role of RNA processing in the mitotic progression of the gametophytic generation and cell fate determination in the embryo sac. Combined studies of genetics and transcriptome analysis revealed recently that epigenetic pathways play a critical role in female gametophyte development. In addition, the discovery that a large number of small secreted cysteine-rich proteins are enriched in embryo sac is of special interest. Except these insights and progresses, challenge ahead is to reveal the signaling pathways and their interactions that lead to the patterning of the female gametophyte. Address Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 1 West Beichen Road, Chaoyang District, Beijing 100101, China Corresponding author: Shi, Dong-Qiao ([email protected]) and Yang, Wei-Cai ([email protected])
Current Opinion in Plant Biology 2011, 14:74–80 This review comes from a themed issue on Growth and development Edited by Jiayang Li and Nam-Hai Chua Available online 29th September 2010 1369-5266/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2010.09.001
Introduction As the female sexual organ of higher plants, ovules provide the structural support or enclose of female gametophytes, and develop into seeds after fertilization. Ovules in Arabidopsis and most flowering plants are composed of four parts, the embryo sac, the nucellus, the integument, and the funiculus that connect the ovule to placenta. Female gametophytes, or embryo sacs, are embedded in the nucellus, which degenerates ultimately. The apexes of integuments form a mini-opening called micropyle where pollen tube enters. Both the ovule and the embryo sac are polarized organs. As the core of the Current Opinion in Plant Biology 2011, 14:74–80
ovule, female gametophyte in Arabidopsis originates from a single hypodermal cell – the archespore at the tip of the ovule primodium. The archespore directly differentiates into megasporocyte that undergoes further meiosis to produce four megaspores. Only the chalazal-most megaspore – the functional megaspore – undergoes three rounds of nuclear division to form a coenocytic, eightnucleated embryo sac. Subsequently, nuclear migration, polar nuclear fusion, and cellularization take place to yield ultimately a seven-celled embryo sac composed of two synergids, one egg cell, one central cell, and three antipodals (Figure 1). Ovules are essential reproductive organs that not only share features with vegetative organs, but also bear unique characters special for reproduction, with genes controlling ovule ontogeny overlapping with those required for both vegetative and sexual development. The genes involved in ovule development include those that function in polarity establishment, meristem maintenance, floral organ determination, ovule identity, and structure specification. Recent investigations revealed that integument and leaf may share common developmental pathways [1,2]. This review will focus mainly on female gametophyte development with an emphasis on gene expression, polarity, epigenetic control, and RNA processing in Arabidopsis. For more information on ovule development, readers are referred to excellent recent reviews [3,4,5,6].
Integument development and evolution As the focus of classic and molecular genetics in plant research, progress made in ovule and female gametophyte development benefited from the combinatorial approaches of mutants and high-throughput technology. Ovules are bitegmic in most angiosperms including Arabidopsis and unitegmic or ategmic in others such as Santalales and Gymnosperms. Integuments share many common features in morphology, development, and genetic program with leaves; it has been proposed that integuments and leaves are analogous structure [2]. Several genes, including AINTEGUMENTA (ANT), BELL1 (BEL1), and INNER NO OUTER (INO) that play a central role in integument development are also crucial for lateral organ development in Arabidopsis. Recently, the comparison of ANT and BEL1 gene expression pattern in Arabidopsis and Santalales suggests that, ategmic ovules are probably derived from a fusion of the integument with nucellus, or that the nucellus has acquired the characteristics confined to integuments in ancestral species [7]. Therefore, integument biogenesis is of interest for both ovule development and evolution. www.sciencedirect.com
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Figure 1
Ovule and embryo sac of Arabidopsis. (a) An electron scanning image of ovule, showing the ovule (ov), funiculus (fn), and micropyle (mp). (b) A confocal optical image showing a typical ovule structure with a seven-celled and eight-nucleate embryo sac. Gametophytic cells of the embryo sac are colored in red (egg), green (synergids), blue (central cell) and yellow (antipodals). The integuments are in purple. The polar nuclei (pn) of the central cell are not yet fused. (c) An optical image projection showing a mature ovule with a seven-celled embryo sac. The polar nuclei have fused to form a diploid central nucleus (cn). Three antipodal cells at the chalazal end of the embryo sac are under degeneration. an, antipodal nucleus; cn: central nucleus; en, egg nucleus; fn, funiculus; mp, micropyle; ov, ovule; pn, polar nucleus; sn, synergid nucleus; and vc, vacuole. Bars = 10 mm.
To identify genes controlling integument ontogeny in Arabidopsis, transcriptome analysis was performed between ino (which lacks the outer integument) and ant (which lacks both inner and outer integuments) [8–10]. The comparison of transcriptomes of ovules from the wildtype, ino and ant mutants revealed 207 genes predominantly expressed in integuments [7]. Among them, 25 genes are preferentially expressed in both inner and outer integuments, 132 putative genes in inner integument and 50 genes in outer integument. About 80% of the integument-expressed genes encode for proteins that are involved in metabolism, transcription, and proteins with unknown function. It is remarkable that transcription factors and DNA binding proteins increase to 20% in context of integument development, compared to 6–7% in the whole genome. These transcriptional factors are implicated in organ size control, auxin signaling, lateral organ development, growth, and transcription regulation. This implies the complexity of the regulation in the integuments although they are rather simple structures. Furthermore, these genes serve as candidates for further analysis for their roles in integument development, and would be valuable for the understanding of integument evolution as well.
Female gametophyte displays distinct transcriptome To identify genes expressed in female gametophyte, mutants without embryo sac are selected for transcriptome analysis. Mutant sporocyteless (spl) has no sporocyte and is www.sciencedirect.com
completely absent of embryo sac in ovules [11,12]. SPL gene encodes a novel transcription regulator that is required for the initiation of sporogenesis in both sexes and is sufficient to trigger microsporogenesis when activated ectopically [13]. Transcriptomic comparison between the wild-type and spl mutants with Affymetrix ATH1 chip revealed 225 ovule-expressed genes in total. 23.6% of them encode unknown proteins; the remaining ones are implicated in central metabolism, detoxification/ stress response, cell structure, transport, protein degradation, signal transduction, and transcription regulation [14]. Similarly, the comparison of expression profiles of male sterility1 (ms1) [15,16] and determinant infertile1 (dif1) (no embryo sac due to meiosis failure) [17] identifies 71 ovule-expressed genes [18]. 41 of them encode proteins with unknown function; the remaining ovule-expressed genes are involved in metabolism, electron transport, energy pathway, transport, signaling, cell organization, and transcriptional regulation. Conversely with the genes identified in integuments, transcription factors are much less abundant in female gametophyte. By comparing wildtype ovules with those of dif1 and myb98 (defective in synergid function) [19], with high-throughput sequencing and whole-genome tiling arrays, 382 and 77 genes were found down-regulated in ovules of dif1 and myb98, respectively [20]. Among the 382 dif1-down-regulated genes, 297 (78%) encode small secreted, cysteine-rich proteins (CRPs). These include antipathogenic peptides of the Defensin-Like (DEFL) family (74 genes) and thionin-like Current Opinion in Plant Biology 2011, 14:74–80
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family (11 genes), and the pistil-secreted SI proteins of Papaver Self-Incompatibility-Like (PSIL) family (22 genes). Among the 286 DEFL genes and 62 thionin-like genes in the Arabidopsis genome, 32% of the DEFL genes and 19% of the thionin-like genes respectively are embryo sac-expressed. In addition, many members of the DUF784 and DUF1278 gene families are also embryo sacexpressed, for example, 37 out of 40 DUF784 members are down-regulated in myb98. They are expressed specifically in synergids and potentially involved in synergid function or embryo sac maturation. Interestingly, many members of these families are encoded by tandemly arrayed, recently duplicated genes. This indicates that the DUF784 and DUF1278 families are embryo sacspecific and may have evolved with the embryo sac. Among the embryo sac-enriched genes, about 78% encode proteins with signal peptide and 66% encode peptides with molecular weight less than 20 kDa. This suggests that they may act as intercellular signaling molecules during embryo sac development and male–female interactions. For example, the PSILs inhibit growth and trigger cell death of self-pollen in Papaver, implying they may play a role in cell–cell communication during embryo sac–pollen tube interaction. Consistently, the maize CRP protein ZmEA1 and LUREs in Torenia act as pollen tube attractants [21,22]. Undoubtedly, further illustration of the function of the CRPs will shed light on the cell–cell signaling within the embryo sac, among the gametic cells, and between embryo sac and the pollen tube. To further identify genes expressed in a specific gametophytic cell type, Wuest et al. performed cell typespecific transcriptome analysis using laser-assisted micro-dissection [23]. After extensive validation, they estimate that about 43% (8850 genes) of the Arabidopsis genome are expressed in the embryo sac, with 7171 (36%) in egg, 7287 (35%) in central cell, and 5628 (27%) in synergids, respectively. 420 genes are significantly enriched in one of the three gametophytic cell types, these include previously identified genes such as MYB98, FIS2, DME, AGL26, AGL61, AGL80, and genes encoding CRPs. Interestingly, F-box and leucine-rich proteins are also enriched in the female gametophyte. These data show that gametophytic cells display distinct post-transcriptional and epigenetic regulatory mechanisms in regulating gene expression and metabolic pathways. Transcription factors with RWP-RK domain, MADS domain, and REM-TFs, are highly expressed in the female gametophyte. Moreover, genes encoding the double-stranded RNA-binding factors, or involved in various gene silencing pathways, are also abundant in the egg, or central cell. Intriguingly, genes involved in miRNA biogenesis including PAZ, PIWI domain and DUF1785 family are globally enriched in the egg cell. On the contrary, genes acting in gene silencing pathway are elevated in the central cell. This suggests an early Current Opinion in Plant Biology 2011, 14:74–80
differentiation in epigenetic regulatory machinery during female gametophyte development [23]. Nevertheless, the biological significance of these differences remains to be elucidated. Interestingly, components acting in auxin signaling and metabolism are also enriched in the embryo sac. This may further support a role of auxin gradient in patterning the female gametophyte [24]. It is no doubt that the data sets of the transcriptome profiling are invaluable to decipher the roles of auxin, small RNAs and RNA biogenesis in ovule and female gametophyte development.
Auxin gradient and the female gametophyte patterning Auxin is a mobile signaling molecule that is polarly transported from the site of synthesis to target cells via its influx and efflux carriers throughout the plant. It is inevitable that a gradient would be formed during polar auxin transport that is responsible for pattern formation during early embryogenesis, organogenesis and ovule development as well [24,25,26–29]. The female gametophyte is a polarized structure with four cell types. The positioning of nuclei, cells and manifestation of cell fate are concomitant with each other during embryo sac development. The establishment of cell identity coincides with the cellularization of the coenocytic embryo sac, the mis-positioning of nuclei often results in changes in cell identity as seen in eostre mutant [28]. Therefore positional or location-specific cues are expected to exist. Indeed, the auxin-dependent patterning and gamete specification in the embryo sac are elegantly demonstrated [4,24]. During early ovule development, the auxin concentration is high at the distal tip of the nucellus before and during meiosis, and maintains high in the nucellar tip at one-nucleate stage and later at the micropylar end of the embryo sac at twonucleate stage. The auxin concentration at the micropylar end is increased during further development of the embryo sac and remains at the maximum until eightnucleate stage. This polarized distribution of auxin is maintained up to cellularization stage and later it turns to less polarized in mature embryo sac. Thereby, an auxin gradient that declines along the micropylar to chalazal axis in the embryo sac is established. It seems that the initial auxin maximum at the nucellar tip is set up by polar auxin transport within the nucellar cells, and later by local de novo synthesis of auxin since only the expression of YUCCA, but not PIN1 has been detected in the embryo sac [24]. On the contrary, the nuclei are exposed to different auxin concentrations along the gradient before cellularization, implying that the production of auxin and its polarized accumulation act as the ‘prelude’ of cellularization and cell specification in the embryo sac. Furthermore, disturbance of auxin gradient leads to abnormal or defective gametic cell fate, or even the conversion of chalazal cell identities to micropylar cell www.sciencedirect.com
Ovule development in Arabidopsis: progress and challenge Shi and Yang 77
identities [24]. All three micropylar cells adopt egg cell fate when AUXIN RESPONSE FACTORS (ARFs) ARF-18 and ARF-19 are knocked down by artificial microRNAs. This indicates that the cell fate determination of synergid, egg cell, central cell and antipodal that arranged from the micropylar to the chalazal end is somehow dependent on the auxin concentration gradient, which decreases along micropylar–chalazal axis. However, it is not known that whether auxin plays roles as the primary instructive signal, or through an undiscovered downstream factor [24]. It remains to be illustrated when and how the auxin gradient leads to the final patterning of the embryo sac. Since the auxin gradient is established early in embryo sac development, an obvious question is whether the eight nuclei in the coenocytic embryo sac respond differently or they are instructed by the concentration gradient to behave differently. More need to be done. The discovery of auxin signaling components, and the mechanism underlying their polarized distribution in embryo sac would be the targets for further investigation. And it is also to be elucidated whether these events happened in the embryo sac share common features with those in vegetative organs.
Small RNAs, new players in female gametophyte development Small RNAs produced from long double-stranded RNAs (dsRNAs) are a class of distinct RNAs that mediate almost every aspect of plant development or response to environment through transcriptional or post-transcriptional regulation [30]. Small RNAs are classified into three types according to their origin, namely microRNA (miRNA), small interfering RNA (siRNA, including trans-acting siRNA and natural antisense transcript siRNA), and piwi-interacting RNA (piRNA) [30,31,32]. miRNAs are produced from single-stranded RNAs with hairpin structures, siRNAs are derived from long dsRNAs. miRNAs and siRNAs are 20–24 or 18–25 nucleotide in length, respectively. piRNAs are a third type of longer small RNAs of 26–31 nucleotides that are only found in germline of animals currently [33]. Small RNAs are bound by ARGONAUTE (AGO) proteins, then loaded to RNA Silencing Complex (RISC), to guide to the target mRNA by sequence complementarities [34]. Small RNA populations have been unveiled through high-throughput sequencing. 33 different miRNA families were found in Arabidopsis mature pollens recently [35,36]. They are predicted to target transcripts of genes involved in epigenetic pathways and in hormone response such as ARFs in pollen [35]. The sperm-specific cis-nat-siRNA (natural cis-antisense siRNA) generated by reversely overlapped genes KOKOPELLI (KPL) and ARIADNE14 (ARI14) impairs double fertilization in kpl mutant [36]. However, small RNAs have not been investigated in the female gametophyte www.sciencedirect.com
using high-throughput sequencing due to the difficulties to obtain sufficient material. Recent reports indicate that some key components of small RNA pathways, such as AGO1, AGO9, and DICER-LIKE1, are involved in female or male gametophyte development [37,38]. These demonstrated the existence of small RNA regulatory network in female or male gametogenesis [39]. Moreover, as mentioned above, expression profiles of different cell types of the embryo sac indicate that genes encoding PAZ/Piwi proteins are up-regulated, suggesting that epigenetic regulation through small RNA networks occurs in the egg cell [23]. The involvement of small RNAs in female gametophyte development is further confirmed with investigation of AGO9 function recently [37]. AGO proteins are effectors of RNA silencing, they specifically bind small RNAs and mediate RNA cleavage, translation suppression, or DNA epigenetic modification [40–43]. AGO9 is highly expressed in ovule and anther of Arabidopsis. Mutations in AGO9, SUPPRESSOR OF GENE SILENCING3 and RNA-DEPENDENT RNA POLYMERASE6, form additional abnormal megasporocyte-like cells in ovule primordium in a dosage-dependent manner. Furthermore, these aberrant sub-epidermal cells in ago9 can acquire functional megaspore identity, or even develop into gametic cells and initiate gametogenesis without undergoing meiosis [37]. However, AGO9 expression is neither detected in megasporocyte, nor the megaspore or the subsequent female gametophyte, but present in the surrounding cells of the embryo sac. Consistently, it is not found in sperm cell as well, but vegetative cell of the male gametophyte. This indicates that a non-cell autonomous small RNA silencing pathway is involved in Arabidopsis ovule development, particularly in ensuring that a single megasporocyte forms in each ovule primordium and gametic cell fate in the male gametophyte [37]. AGO9 associates small RNAs derived from transposable elements (TEs) and is necessary for TE inactivation. Although the role of AGO9 associated TE inactivation in Arabidopsis ovule development is not clear, this study implies that an AGO9dependent, or to say the least, the small RNA-mediated pathway may act as a key regulatory pathway to prevent somatic cells from undergoing sexual reproductive or apomictic process in higher plants. Small RNAs have been shown to be mobile molecules that act in systemic gene silencing and leaf patterning. It was reported that a gradient of small RNA is created because of the intracellular movement of trans-acting siRNAs from the origin of biogenesis (adaxial) to the abaxial side of leaf [44]. Does the gradient of small RNAs, or some other factor participating in the small RNA pathway, exist in ovule or female gametophyte, just as that of auxin? Whether small RNA functions as a mobile messenger to ignite the regulation network in cell identity determination? These questions need to be addressed in the future. Current Opinion in Plant Biology 2011, 14:74–80
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RNA processing and female gametophyte development The functional megaspore undergoes three nuclear divisions without cytokinesis, resulting in an eightnucleate embryo sac. Therefore, the embryo sac is a giant cell with a large central vacuole before cellularization. Question remains to be addressed on how cell growth, cell division and differentiation are regulated. How are the transcriptional and translational activities coordinated in such a large cell? Recent studies suggest that nucleolar activities, such as RNA biogenesis and ribosome biogenesis, are crucial for embryo sac development in Arabidopsis. Mutations in genes involved in rRNA biogenesis, mRNA processing and ribosomal biogenesis, often block the mitotic progression of the gametophytic cell cycle. The first mutant reported is slow walker1 (swa1), which is defective in mitotic progression during female gametogenesis, resulting in complete female sterility in Arabidopsis [45]. SWA1 encodes a nucleolar WD40-domain protein with homology to the yeast UTP15 that is involved in prerRNA processing. Indeed, knockdown of SWA1 impairs 18S pre-rRNA biogenesis in Arabidopsis [45]. SWA2 is a nucleolar protein too, it is homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1/MAINTENANCE of KILLER21, and probably has conserved function in pre-ribosome biogenesis and nucleolar export [46]. SWA3/AtRH36 is a newly reported member of the DEAD-box RNA helicases that possibly acts in 18S rRNA biogenesis as well [47,48]. All three mutants of null mutations, which lead to complete loss of gene function, display slow progression of mitosis resulting in the arrest of embryo sac development at two-nucleate, four-nucleate, or eightnucleate stages, but have little or no effect on male gametophyte development. Consistently, loss-of-function mutations of GAMETOPHYTIC FACTOR1/ CLOTHO/VAJRA (GFA1/CLO/VAJ), homologous to the yeast U5 snRNP spliceosomal component Snu114p that regulates spliceosome dynamics, also arrest embryo sac development at these stages [49,50]. These data indicate that rRNA, or ribosomal biogenesis is crucial to cell cycle progression during female gametophyte development. In addition, MAGATAMA3 (MAA3), a plant homolog of the yeast Sen1, which is necessary to various aspects of RNA metabolism, such as rRNA/ tRNA processing, localization of nucleolar proteins, and pre-mRNA metabolism, functions in female gametophyte development as well. The mutant maa3 displays pleiotropical defects such as failure in polar nuclear fusion, small size of nucleoli of polar nuclei, partial fertility of the male gametophyte, and inability to attract pollen tube [51]. A simple explanation to this phenomenon is that these essential genes are required for rapid growth of the embryo sac, since they may also play a role in vegetative growth as shown for VAJ [52]. An alternative explanation is that there is a regulatory Current Opinion in Plant Biology 2011, 14:74–80
role of the nucleolar function for embryo sac development in Arabidopsis, for example, to coordinate transcriptional and translational activities during the rapid growth of the embryo sac. More intriguingly, RNA processing has been shown to be crucial for cell fate determination of the embryo sac. For example, in lachesis (lis) mutants, synergids and central cells adopt egg cell fate, whereas antipodal cells behave like central cells. LIS encodes an Arabidopsis homolog of the yeast splicing factor PRP4 [53]. Similarly, GFA1/ CLO/VAJ and ATROPOS (ATO), encoding Arabidopsis homolog of SF3a60 that is implicated in spliceosomal assembly [54], are also crucial for gametic cell fate specification in the embryo sac since mutations in these genes result in supernumerary egg cells or mis-specified gametic cells, indicating that they are key factors for gametic cell fate. These data suggest a key role of the spliceosomal components and related regulatory pathway in gametic cell fate in plants. Interesting questions arise: How could RNA splicing specifically regulate gametic cell fates in the embryo sac? Does it control the splicing of a specific subset of transcripts that is crucial for gametic cell fate? Or does it process all mRNAs in the embryo sac? Do they have functions other than RNA processing? These need to be investigated in the future.
Concluding remarks Female gametophyte in higher plants is a simple sevencelled reproductive structure. Its stereotyped ontogeny provides a traceable system to study mechanisms controlling cell growth, cell division, differentiation, cell fate, and perhaps the function of essential genes in plants. As discussed above, significant progresses have been made in understanding the patterning of the embryo sac and transcriptomes of each cell types, the demonstration of the auxin gradient is of great importance to developmental biology. Nevertheless, we are facing new questions and challenges, for example, how is the auxin gradient translated into positional information that patterns the embryo sac? What is the role of the epigenetic regulatory network in female gametophyte development? What is the role of nucleolus, such as RNA processing and ribosome biogenesis? How did the ovule and embryo sac evolve? Data generated by transcriptome analysis provide us many clues and candidate genes that are of special interest to address those questions. With the advancement of new technologies, such as live imaging, targeted mutagenesis, laserassisted techniques, and high-throughput sequencing, more progress on this miniature reproductive structure is yet to be made.
Acknowledgements We would like to thank the financial support from the Ministry of Science and Technology of China (2007CB947604) and the Natural Science Foundation of China (30921003). www.sciencedirect.com
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