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• W2087268291 abstract "Pollen formation, while critical for the success of plant reproduction, also represents an important paradigm for differential cellular development within small groups of cells. In Arabidopsis thaliana pollen, the male meiotic product first divides asymmetrically to form a vegetative and a generative (germ) cell, the latter then dividing to generate two sperm cells. Here we have used artificial microRNAs to study small RNA processing in the different pollen cell types. Our data suggest that translational repression by small RNAs is enhanced in the sperm. This work also provides insights into germline RNA movement and the cell-autonomous action of microRNAs. Pollen formation, while critical for the success of plant reproduction, also represents an important paradigm for differential cellular development within small groups of cells. In Arabidopsis thaliana pollen, the male meiotic product first divides asymmetrically to form a vegetative and a generative (germ) cell, the latter then dividing to generate two sperm cells. Here we have used artificial microRNAs to study small RNA processing in the different pollen cell types. Our data suggest that translational repression by small RNAs is enhanced in the sperm. This work also provides insights into germline RNA movement and the cell-autonomous action of microRNAs. Pollen development is accompanied by changes in the expression of many non-coding small RNA biogenesis genes [1Grant-Downton R. Hafidh S. Twell D. Dickinson H.G. Small RNA pathways are present and functional in the angiosperm male gametophyte.Mol. Plant. 2009; 2: 500-512Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 2Borges F. Gomes G. Gardner R. Moreno N. McCormick S. Feijo J.A. BEcker J.D. Comparative transcriptomics of Arabidopsis sperm cells.Plant Physiol. 2008; 148: 1168-1181Crossref PubMed Scopus (298) Google Scholar] and in target cleavage by diverse microRNAs [1Grant-Downton R. Hafidh S. Twell D. Dickinson H.G. Small RNA pathways are present and functional in the angiosperm male gametophyte.Mol. Plant. 2009; 2: 500-512Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]. To determine whether the vegetative cell and the germline within the pollen grain differ in microRNA processing and target silencing, we generated artificial microRNA (amiR) constructs driven by cell type-specific promoters. AmiRs are effective tools for gene silencing in plants; by exploiting a non-coding RNA backbone that forms a precursor ‘hairpin’, the region excised by DICER-LIKE1 to form a mature microRNA can be engineered to specifically target RNAs through ARGONAUTE-mediated interactions [3Schwab R. Ossowski S. Riester M. Warthmann N. Weigel D. Highly specific gene silencing by artificial microRNAs in Arabidopsis.Plant Cell. 2006; 18: 1121-1133Crossref PubMed Scopus (1031) Google Scholar]. Thus, specific RNAs can be ‘knocked-down’, generally by cleavage, the predominant mode of microRNA action in plants. First, to test our experimental system we designed an artificial microRNA construct (amiRGFP) to silence a GFP marker expressed specifically either in the germline (sperm cells) or in the accessory vegetative cell of pollen. To express the amiRGFP we chose the tomato LAT52 promoter, as it is effective in Arabidopsis and, when driving the GUS reporter, it is first expressed in late microspore development and subsequently accumulates to a higher level in the vegetative cell [4Eady C. Lindsey K. Twell D. Differential activation and conserved vegetative-cell-specific activity of a late pollen promoter in species with bi- and tricellular pollen.Plant J. 1994; 5: 543-550Crossref Scopus (3) Google Scholar]. The expression of our PromLAT52:H2B-GFP construct in Arabidopsis followed this pattern, but also revealed that GFP was inherited from the microspore nucleus into the generative cell — the plant male germline (Supplemental Figure S1A–E). Importantly, in this work we also utilised a novel promoter that is specific to the vegetative cell. Analysis of expression data of At2g24370, encoding a putative serine/threonine protein kinase, revealed that it is expressed late in male gametophyte development, but not in sperm cells [2Borges F. Gomes G. Gardner R. Moreno N. McCormick S. Feijo J.A. BEcker J.D. Comparative transcriptomics of Arabidopsis sperm cells.Plant Physiol. 2008; 148: 1168-1181Crossref PubMed Scopus (298) Google Scholar]. Analysis of GFP expression under its promoter established that expression was restricted to the later stages of vegetative cell development (Supplemental Figure S1F–J), hence we named this PromVCK1 (vegetative cell kinase1). We also used the promoter of the MGH3 gene, a histone H3 variant gene with well-characterised generative and sperm cell-specific expression at high levels [5Brownfield L. Hafidh S. Borg M. Sidorova A. Mori T. Twell D. A plant germ cell-specific integrator of cell cycle progression and sperm specification.PLoS Genet. 2009; 5: e1000430Crossref PubMed Scopus (132) Google Scholar]. The ability of these pollen-specific promoters to permit generation of mature artificial microRNAs is shown in Supplemental Figure S1K. Knockdown of vegetative cell-specific GFP expression under PromVCK1 control, by the amiRGFP under the LAT52 promoter, revealed efficient reduction of mRNA and protein levels (Table 1). By contrast, knockdown of GFP under PromMGH3 control, by amiRGFP under the MGH3 promoter, led to a significant reduction in GFP protein levels, but without a concomitant reduction in mRNA levels. This result indicates that in the sperm the amiRGFP is working primarily by translational repression, as opposed to the canonical cleavage-induced mRNA turnover in the vegetative cell.Table 1Analysis of GFP protein and transcript levels in Prom:H2B-GFP target reporter lines harbouring Prom:amiR effector constructs (see also Figure S1)amiR effectorTarget reporter1Number of T1 lines analysed.T1 (n)2Proportion of T1 lines with reduced GFP signal.%KDamiR effector in target reporter lineTarget reporter line alone7Mean GFP signal in amiR effector line/mean GFP signal in segregating target reporter line without amiR effector construct X 100.amiR KD efficiency GFP protein (%)8Mean GFP transcript level in amiR effector line/mean GFP transcript level in segregating target reporter line.amiR KD efficiency GFP transcript (%)3ID of lines homozygous for amiRGFP and target reporter constructs.ID4Mean GFP fluorescence in pollen nuclei.GFP protein±SE5Mean GFP transcript level by q-RT-PCR.GFP transcript±SE6ID of lines homozygous for target reporter constructs.ID4Mean GFP fluorescence in pollen nuclei.GFP protein±SE5Mean GFP transcript level by q-RT-PCR.GFP transcript±SEProLAT52 :amiRGFPProVCK :H2B-GFP3678A20.2±0.03.4±0.1A625.4±1.621.2±0.099.485.8B60.2±0.03.1±0.0B338.0±1.224.5±0.2ProLAT52 :amiRGFPProMGH3 :H2B-GFP2673C10.2±0.12.8±0.1B42.1±0.14.1±0.090.819.2C60.2±0.03.5±0.0B62.1±0.13.7±0.0ProMGH3 :amiRGFPProMGH3 :H2B-GFP4139B50.5±0.05.0±0.1A13.3±0.23.9±0.179.615.9NSNot significantly different from control target reporter line (P < 0.05).D20.9±0.14.5±0.2D53.2±0.27.4±0.2ProVCK :amiRGFPProMGH3 :H2B-GFP200B41.9±0.14.4±0.1A12.1±0.14.7±0.19.8NSNot significantly different from control target reporter line (P < 0.05).-2.5NSNot significantly different from control target reporter line (P < 0.05).B52.2±0.13.8±0.1D32.3±0.13.3±0.11 Number of T1 lines analysed.2 Proportion of T1 lines with reduced GFP signal.3 ID of lines homozygous for amiRGFP and target reporter constructs.4 Mean GFP fluorescence in pollen nuclei.5 Mean GFP transcript level by q-RT-PCR.6 ID of lines homozygous for target reporter constructs.7 Mean GFP signal in amiR effector line/mean GFP signal in segregating target reporter line without amiR effector construct X 100.8 Mean GFP transcript level in amiR effector line/mean GFP transcript level in segregating target reporter line.NS Not significantly different from control target reporter line (P < 0.05). Open table in a new tab To investigate further the translational control in sperm cells we analysed transcriptome data for SUO, which encodes a GW-repeat protein involved in promoting translational repression by microRNAs [6Yang L. Wu G. Poethig R.S. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2012; 109: 315-320Crossref PubMed Scopus (121) Google Scholar] and found both SUO and its close paralogue (At3g48060) to be at their highest levels in sperm when compared with the vegetative cell or the sporophytic phase of plant development [2Borges F. Gomes G. Gardner R. Moreno N. McCormick S. Feijo J.A. BEcker J.D. Comparative transcriptomics of Arabidopsis sperm cells.Plant Physiol. 2008; 148: 1168-1181Crossref PubMed Scopus (298) Google Scholar] (Supplemental Table S1). Translational repression is sensitive to SUO dosage [6Yang L. Wu G. Poethig R.S. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2012; 109: 315-320Crossref PubMed Scopus (121) Google Scholar] and thus SUO upregulation constitutes a second strand of evidence supporting the existence of active translational repression in Arabidopsis sperm. A further component recently shown to be important for translational repression of plant microRNAs on the rough endoplasmic reticulum [7Li S. Liu L. Zhuang X. Yu Y. Liu X. Cui X. Ji L. Pan Z. Cao X. Mo B. et al.MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis.Cell. 2013; 153: 562-574Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar], ALTERED MERISTEM PROGRAM1 (AMP1), encodes a homologue of human glutamate carboxypeptidase II, which is also enriched in sperm cells compared with the vegetative cell of mature pollen (Supplemental Table S1). Translational repression is known to be important in plant reproduction as paternally derived SHORT SUSPENSOR transcripts, essential for regulation of the first asymmetric division of the zygote, are translationally repressed in sperm and only translated upon fertilisation [8Bayer M. Nawy T. Giglione C. Galli M. Meinnel T. Lukowitz W. Paternal control of embryonic patterning in Arabidopsis thaliana.Science. 2009; 323: 1485-1488Crossref PubMed Scopus (259) Google Scholar]. Previous experiments using artificial microRNAs targeting pollen gene expression led to the development of a model for small RNA function in the male gametophyte [9Slotkin R.K. Vaughn M. Borges F. Tanurdzic M. Becker J.D. et al.Epigenetic reprogramming and small RNA silencing of transposable elements in pollen.Cell. 2009; 136: 461-472Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar]. In this work, Slotkin et al. used PromLAT52 to direct amiR expression combined with sperm-specific GFP expression directed by PromGEX2. This resulted in knockdown of GFP in sperm, and given that PromLAT52 is highly expressed in the vegetative cell, it was proposed that knockdown arises through small RNA transfer from the vegetative cell to the germline. These data were used to support the notion that transposable element (TE)-derived siRNAs, produced by epigenomic changes in the vegetative cell, move to the germline where they reinforce TE silencing by RNA-dependent DNA methylation [9Slotkin R.K. Vaughn M. Borges F. Tanurdzic M. Becker J.D. et al.Epigenetic reprogramming and small RNA silencing of transposable elements in pollen.Cell. 2009; 136: 461-472Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar]. Our experiments using PromLAT52 resulted in silencing of germline GFP (as in Slotkin et al. [9Slotkin R.K. Vaughn M. Borges F. Tanurdzic M. Becker J.D. et al.Epigenetic reprogramming and small RNA silencing of transposable elements in pollen.Cell. 2009; 136: 461-472Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar]) but, against expectation, expression of the amiRGFP using PromVCK1 failed to silence germline-specific GFP expression directed by PromMGH3 (Table 1). Although we cannot rule out that continuity may exist transiently between the vegetative cell and generative cell prior to PromVCK1 activation, an equally reasonable explanation for germline silencing directed by PromLAT52 is that amiRNAs, derived from early PromLAT52 expression in the microspore [4Eady C. Lindsey K. Twell D. Differential activation and conserved vegetative-cell-specific activity of a late pollen promoter in species with bi- and tricellular pollen.Plant J. 1994; 5: 543-550Crossref Scopus (3) Google Scholar] (Supplemental Figure S1A–E), are inherited by the germline. Furthermore, the observation that sperm cells display a microRNA profile distinct from whole mature pollen [10Borges F. Pereira P.A. Slotkin R.K. Martienssen R.A. Becker J.D. MicroRNA activity in the Arabidopsis male germline.J. Exp. Bot. 2011; 62: 1611-1620Crossref PubMed Scopus (105) Google Scholar] is consistent with our finding that miRNAs do not appear to act across the vegetative/sperm cell interface. Our data clearly reveal the presence of different and cell-autonomous differences in miRNA activity in the mature pollen grain of Arabidopsis. They further highlight the importance of promoter specificity when using amiRs to target mRNAs in rapidly dividing and differentiating systems, and the caution required when interpreting cell-to-cell transfer data from amiR experiments. This work was supported by the Biology and Biotechnology Research Council (BB/F008694/1 to H.G.D. and BB/F007558/1 to D.T.). Download .pdf (.17 MB) Help with pdf files Document S1. Figure S1, Table S1 and Experimental Procedures" @default.
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• W2087268291 title "Artificial microRNAs reveal cell-specific differences in small RNA activity in pollen" @default.
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