Plant Physiology Preview. Published on January 6, 2016, as DOI:10.1104/pp.15.01510
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Running head: Regulation of gene expression in plant male gamete
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Xiaoping Gou
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222 South Tianshui Road, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, China
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Tel: 86-13519610667
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Email: [email protected]
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Scott D. Russell
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770 Van Vleet Oval, Department of Microbiology and Plant Biology, University of Oklahoma, Norman,
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OK 73019
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Tel: 405-325-4391
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Email: [email protected]
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Research areas:
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Genes, Development and Evolution
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Cell Biology
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Copyright 2016 by the American Society of Plant Biologists
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Cis-regulatory elements determine germline specificity and expression level of an
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isopentenyltransferase gene in sperm cells of Arabidopsis
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Jinghua Zhang, Tong Yuan, Xiaomeng Duan, Xiaoping Wei, Tao Shi, Jia Li, Scott D. Russell, Xiaoping
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Gou
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Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life
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Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.);
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Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK 73019 (T.Y.,
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X.W., S.D.R.)
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One-sentence summary:
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A regulatory region consisting of duplicated motifs activates gene expression in plant male gamete.
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Author Contributions：
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Xiaoping Gou, Jia Li and Scott D. Russell conceived and designed the experiments. Xiaoping Gou,
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Jinghua Zhang, Tong Yuan, Xiaomeng Duan and Xiaoping Wei performed the experiments. Xiaoping
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Gou, Jinghua Zhang and Tao Shi analyzed the data. Xiaoping Gou and Scott D. Russell wrote the paper.
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Financial source:
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This work was supported by National Natural Science Foundation of China grants 31471402 and
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31270229 (to X.G.), the start-up fund from Lanzhou University (to X.G.); the Fundamental Research
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Funds for the Central Universities grant lzujbky-2014-251 (to J.Z.); the State Administration of Foreign
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Expert Affairs grant MS2010LZDX077 (to J.L.); US National Science Foundation (IOS 1128145) and
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University of Oklahoma (to S.R.)
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Present address for Xiaoping Wei:
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Monsanto Company
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700 Chesterfield Parkway West,
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Chesterfield, MO 63017, USA
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Corresponding authors:
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Xiaoping Gou, [email protected]
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Scott D. Russell, [email protected]
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ABSTRACT
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Flowering plant sperm cells transcribe a divergent and complex complement of genes. To examine
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promoter function, we chose an isopentenyltransferase gene known as PzIPT1. This gene is highly
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selectively transcribed in one sperm cell morphotype (Svn) of Plumbago zeylanica, which preferentially
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fuses with the central cell during fertilization and is thus a founding cell of the primary endosperm. In
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transgenic Arabidopsis, PzIPT1 promoter displays activity in both sperm cells and upon progressive
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promoter truncation from the 5’-end results in a progressive decrease in reporter production, consistent
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with occurrence of multiple enhancer sites. Cytokinin-dependent protein binding (CPB) motifs are
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identified in the promoter sequence, which respond with stimulation by cytokinin. Expression of PzIPT1
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promoter in sperm cells confers specificity independently of previously reported Germline Restrictive
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Silencer Factor (GRSF) binding sequence. Instead, a cis-acting regulatory region consisting of two
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duplicated 6-bp Male Gamete Selective Activation (MGSA) motifs occurs near the site of transcription
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initiation. Disruption of this sequence-specific site inactivates expression of a GFP reporter gene in
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sperm cells. Multiple copies of the MGSA motif fused with the minimal CaMV35S promoter elements
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confer reporter gene expression in sperm cells. Similar duplicated MGSA motifs are also identified from
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promoter sequences of sperm cell-expressed genes in Arabidopsis, suggesting selective activation is
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possibly a common mechanism for regulation of gene expression in sperm cells of flowering plants.
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Key words: Angiosperm sperm cells, cytokinin, isopentenyltransferase, male gamete expression, sperm
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promoter
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INTRODUCTION
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In angiosperms, the meiotic division of microsporocytes produces microspores that establish the male
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germ lineage through asymmetric mitotic division of the microspore, which forms as its products a large
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vegetative cell and a small generative cell that is the founder cell of the male germ lineage (Boavida et
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al., 2005; Ma, 2005; Borg et al., 2009). In bicellular pollen, the generative cell divides to form two
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sperm cells within the germinated elongating pollen tube, whereas in tricellular pollen such as
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Arabidopsis and Plumbago zeylanica, the two sperm cells are produced precociously, prior to anthesis.
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Regardless of cellular condition at the time of pollination, successful transit of the cells of the male germ
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lineage in the elongating pollen tube will transfer two sperm cells into embryo sac. One sperm cell fuses
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with the egg cell to form the zygote, establishing the next generation of plants, and the other sperm cell
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fuses with the central cell, establishing the endosperm, which provides nutrition during embryo
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development. In Plumbago zeylanica, the two sperm cells display cytoplasmic dimorphism in which
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sperm cells display differential abundance of heritable organelles and follow different fusion fates
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during double fertilization. The sperm cell associated with the vegetative nucleus (Svn) contains the
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majority of mitochondria and rare plastids and preferentially fuses with the central cell, whereas the
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sperm cell unassociated with the vegetative nucleus (Sua) contains abundant plastids and few
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mitochondria and preferentially fuses with the egg cell (Russell, 1984; Russell, 1985). That Plumbago
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zeylanica undergoes preferential fertilization makes this plant uniquely suitable for studying the
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regulation of gene expression in paired sperm cells and examining cell-to-cell recognition during double
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fertilization. Correspondingly, promoters unique to each sperm type appear to be activated in order to
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achieve this uniquely distinct pattern of gene expression in the Sua and Svn, corresponding to their unique
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fates (Gou et al., 2009).
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Some male germline-expressed transcripts have been characterized which are vital for sperm cell
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function, fertilization and embryo development (Bayer et al., 2009; Ron et al., 2010; Stoeckius et al.,
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2014), suggesting that sperm cell-expressed genes may possess a distinct role in early stages of post-
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fertilization development. Several promoters have been isolated and studied in flowering plants in the
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context of male germline-specific gene expression. LILY GENERATIVE CELL-SPECIFIC 1 (LGC1),
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isolated from a lily generative cell cDNA library, encodes a plasma membrane-localized protein that is
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probably involved in cell-to-cell recognition during fertilization (Xu et al., 1999). The expression of
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LGC1 is exclusively restricted in male gamete cells (Xu et al., 1999). Another lily gene, GCS1, and its
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Arabidopsis homolog HAP2 are expressed in sperm cells, and are essential for fertilization (Mori et al.,
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2006; von Besser et al., 2006). Sperm cell-expressed genes GAMETE EXPRESSED 1 (GEX1) and GEX2
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were identified in maize sperm cell-specific transcripts and homologous Arabidopsis genes were
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designated AtGEX1 and AtGEX2 (Engel et al., 2005). AtGEX1 is expressed in sperm cells of mature
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pollen in Arabidopsis. AtGEX2 was observed in generative cells and sperm cells, but not in any other
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tissues. The rice homolog of AtGEX2, OsGEX2, also confers sperm cell expression (Cook and Thilmony,
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2012). DUO POLLEN 1 (DUO1) encodes a MYB transcription factor that is expressed specifically in
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generative cell and sperm cells and serves a key regulatory function for generative cell division and
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sperm cell differentiation in Arabidopsis (Rotman et al., 2005; Brownfield et al., 2009a; Borg et al.,
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2011), whereas DUO3 functions to activate expression of target germline genes of DUO1, and is
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required for cell cycle progression, sperm cell specification, fertilization, and embryogenesis
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(Brownfield et al., 2009b). Male gamete-expressed histone variants have also been identified from lily
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(Lilium longiflorum) and Arabidopsis (Ingouff et al., 2007; Okada et al., 2005a; Okada et al., 2005b). In
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Arabidopsis, AtMGH3/HTR10 encodes a variant histone H3 detected in the generative cell of late
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bicellular pollen and sperm cells of anthesis pollen. At the genome scale, sperm cell-expressed genes in
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Arabidopsis were identified by microarray analysis using FACS-purified sperm cells (Borges et al.,
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2008).
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Specific gene expression in a given organ or cell is achieved by recruiting specific transcription
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factors to corresponding cis-regulatory elements (CREs) that are functional DNA sequences carried by
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the gene itself. In efforts to identify CREs controlling gene expression in the germ lineage, promoter
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sequences of LGC1 and DUO1 have already been analyzed. The promoter sequence of LGC1 was
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cloned by uneven PCR and its specific transcription activity was verified in lily and tobacco generative
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cell in transient and stable transformation experiments (Singh et al., 2003). Truncation analysis of LGC1
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promoter identified a repressor binding site that suppresses the expression of LGC1 in sporophytic
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tissues (Singh et al., 2003). A related Germline Restrictive Silencing Factor (GRSF) encoding a novel
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24-kDa DNA-binding repressor protein is expressed ubiquitously in all plant tissues except the male
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germ lineage. Chromatin immunoprecipitation assays demonstrated that GRSF interacts with a specific
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regulatory element in the promoter region of LGC1, which was confirmed by using a synthesized
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competitor to release somatic cells from the repressive GRSF. Presence of GRSF binding sequences in
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other male gamete-expressed genes suggested widespread control of male gamete gene expression by
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this functionally conserved sequence (Haerizadeh et al., 2006). DUO1 functions in another way, by
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directly binding to MYB sites to activate its target genes DUO1-ACTIVATED ZINC FINGER1 (DAZ1)
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and DAZ2, which encode trans-acting transcriptional repressors (Rotman et al., 2005; Brownfield et al.,
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2009a; Borg et al., 2011; Borg et al., 2014). A putative GRSF binding site was predicted in the DUO1
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promoter (Haerizadeh et al., 2006). However, when the predicted GRSF binding site was mutated, the
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expression specificity of DUO1 in germline was not affected. Truncated DUO1 promoters, excluding
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the putative GRSF site, were sufficient to drive expression of H2B::GFP in sperm cells (Brownfield et
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al., 2009a). To identify putative CREs controlling sperm cell-specific gene expression in rice, Sharma et
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al. performed in silico analyses of promoter sequence motifs of 40 rice sperm cell-expressed genes
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(Sharma et al., 2011). Although the authors identified some possible CREs for gene expression in sperm
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cells, experimental validation will be needed to examine the functions of these identified motifs in living
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plants.
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Only a few sperm-expressed promoters have been investigated in detail and limited information
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is available about the regulation of gene expression in sperm cells. Efforts to identify more CREs
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regulating gene expression in sperm cells are needed to understand more fully how expression in the
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male germ lineage is controlled. In previous studies, we identified an isopentenyltransferase gene termed
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PzIPT1 that is exclusively expressed in Svn sperm cells of Plumbago zeylanica, confirmed by qRT-PCR
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and whole-mount in situ hybridization (Gou et al., 2009). The corresponding promoter sequence was
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cloned from Plumbago zeylanica, and its expression in sperm cells was confirmed with GFP reporter
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gene in Arabidopsis (Ge et al., 2011). In this study, we show that a cis-regulatory region for Male
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Gamete Selective Activation (MGSA) determines the expression of PzIPT1 in sperm cells and its
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expression strength can be enhanced by cytokinin via a cytokinin-dependent protein binding (CPB) site.
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RESULTS
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The PzIPT1 Promoter Confers Sperm Cell Specific Expression in Transgenic Arabidopsis Pollen
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A 1.1 Kb 5’-upstream DNA fragment of PzIPT1 was isolated and fused to GUS and GFP or nuclear
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localized YFP reporter genes in order to view the expression patterns of PzIPT1 promoter in >20
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independent transgenic lines. All of them showed very similar expression patterns. GUS expression
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could be detected in whole seedlings just after germination (Fig. 1A) with strong GUS signals evident in
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flowers, especially in ovaries and anthers (Fig. 1B). At higher magnification, GUS expression could be
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detected in embryo sacs, with a strong signal at the micropylar end (Fig. 1C), revealing localization in
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the egg cell and synergid cells by a nuclear-localized YFP reporter (Fig. 1E). A strong GFP signal was
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detected in paired sperm cells of mature pollen with negligible GFP expression in the background pollen
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cytoplasm (Fig. 1D), consistent with a GUS signal (Supplemental Fig. S1). When gametogenesis of
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transgenic plants was examined, no detectable signal of GFP was observed in microspores and bicellular
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pollen (Supplemental Fig. S2). Our previous study showed that PzIPT1 is highly up-regulated in the Svn
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sperm cell in Plumbago zeylanica, and no obvious signals could be detected in other investigated organs
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or cells (Gou et al., 2009). This difference may reflect the species-specific expression of PzIPT1
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promoter.
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PzIPT1 Expression in Sperm Cells Is Not Regulated by Transcriptional Repression
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To understand how PzIPT1 is activated to express in sperm cells, its promoter sequence was analyzed to
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find cis-regulatory elements by searching the Plant cis-Acting Regulatory DNA Elements (PLACE)
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database (Higo et al., 1999). A typical TATA box is located at -29 from the putative transcription start
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site in the PzIPT1 promoter (Fig. 2A). Several abundant motifs were identified, such as DOF, ARR1,
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GT1, MYB, and CPBCSPOR (CPB). Four typical CPB motifs for cytokinin-dependent protein binding
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containing the characteristic TATTAG nucleotide sequence (Fusada et al., 2005) locate at positions -12
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to -17, -89 to -94, -304 to -309, and -433 to -438 (Fig. 2A).
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To identify potential cis-regulatory elements controlling sperm cell expression and potential
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GRSF-like repressor binding sites in PzIPT1 promoter, ten 5’-deletional PzIPT1 promoters were fused
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with GFP, transformed and examined in transgenic Arabidopsis to view their expression patterns (Fig.
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2B). At least twelve T1 transgenic plants were analyzed for each promoter and all of them showed very
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similar expression patterns. Results from T3 transgenic lines for each construct are shown in Fig. 2, C
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and D. The ∆761 promoter showed the same expression level of GFP as the original 1.1 Kb PzIPT1
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promoter. The following three deletions (∆531, ∆368, ∆255) each showed gradually decreased but still
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strong GFP expression in both sperm cells compared to the ∆761 promoter (Fig. 2, C and D). Expression
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levels of GFP decreased in successive deletion constructs of ∆154, ∆130 and ∆105, although the signals
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were strong enough to be detected easily in sperm cells (Fig. 2, C and D). The signal of GFP in deletion
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∆87, however, was very weak and it was difficult to observe whether sperm cells were positive for GFP
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expression in this deletion, although expression was conspicuous in sperm cells of mature pollen
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harboring the intact promoter sequence (Fig. 2, C and D). Such progressive depletion in sperm cell-
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restricted expression of PzIPT1 is inconsistent with a GRSF-like repressive expression system, as male
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germ lineage expression was never lost and the vegetative cell was not labeled. No GFP signals were
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detected in deletions ∆63 and ∆39, which indicates that the sequence between -87 and -40 of the PzIPT1
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promoter is critical for sperm cell expression of PzIPT1 (Fig. 2, C and D).
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A CPB Motif-Containing Region Regulates the Expression Level of PzIPT1 in Sperm Cells
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Progressive 5’-deletional analyses showed that the expression of PzIPT1 decreased dramatically in ∆87
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compared to ∆105 (Fig. 2D), suggesting that the sequence between -105 and -87 of the PzIPT1 promoter
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is critical for the expression strength of PzIPT1. Sequence analysis revealed one CPB motif located
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between -94 and -89 (Fig. 2A). To examine whether this CPB motif regulates the expression level of
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PzIPT1, a mutated promoter construct ∆88-95 was created by deleting sequences between -95 and -88
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and transformed into Arabidopsis (Fig. 2B). Although only eight base pairs were deleted in the PzIPT1
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promoter, the expression of GFP was drastically decreased to a level similar to that of the ∆130
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construct (Fig. 3A). These data provide evidence that the CPB motif-containing region between -94 and
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-89 positively regulates the expression level of PzIPT1 in sperm cells.
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Expression of PzIPT1 in Sperm Cells Can Be Enhanced by Exogenously Applied Cytokinin
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The construct ∆88-95, with a typical CPB site excised, significantly impaired the expression level of
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PzIPT1 promoter, suggesting regulation of gene expression in response to cytokinin may exist in the
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wild type PzIPT1 promoter. To test this hypothesis, transcription of PzIPT1 was evaluated in Plumbago
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zeylanica pollen. When treated with 100 nM 6-BA, PzIPT1 transcription was enhanced dramatically
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(Fig. 3B). To test whether expression enhancement also occurred in Arabidopsis, transgenic pollen with
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∆154 and ∆130 constructs were treated with 100 nM 6-BA to examine their response to exogenously
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applied cytokinin. GFP signals in mature pollen of both ∆154 and ∆130 transgenic plants were enhanced
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15 min after treatment with cytokinin. Signal strength reached a peak 60 min after treatment and
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remained conspicuous for approximately another 60 min before decreasing gradually to background
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florescence levels (Fig. 3, C and E). Thus, cytokinin treatment appears to elevate GFP level above
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background for a total of approximately two hours. No obvious change of GFP signal was observed in
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the mock treatment without cytokinin, nor in the ∆88-95 transgenic pollen with or without cytokinin
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treatment (Fig. 3, C and E). After three rinses in medium without cytokinin for one hour, the cytokinin
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pre-treated pollen of ∆154 and ∆130 plants were retreated with cytokinin. Dramatically enhanced GFP
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signals were again observed approximately 15 min after treatment; the signal gradually weakened when
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incubated for longer periods (Fig. 3, C and E). These data also indicate that the CPB motif located
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between -88 and -95 in PzIPT1 promoter could be activated in response to a cytokinin pulse. Similar
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results were obtained when germinated pollen tubes were treated with cytokinin, i.e., the GFP signal in
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sperm cells became much stronger in cytokinin-treated pollen tubes of ∆154 and ∆130 constructs, and
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no obvious changes were observed in sperm cells of cytokinin-treated ∆88-95 pollen tubes (Fig. 3D).
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A Cis-Acting Region Is Required to Activate the PzIPT1 Promoter in Sperm Cells
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5’-deletional analyses showed that neither ∆63 nor ∆39 constructs could drive expression of GFP in
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sperm cells, suggesting that this region may determine sperm cell expression specificity of the PzIPT1
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promoter (Fig. 2, B-D). We therefore created two deletion constructs (∆64-87, ∆40-63) excising
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sequences corresponding to -64 to -87 and -40 to -63 from the intact promoter and transformed them
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into Arabidopsis (Fig. 2B). Neither construct could drive expression of GFP in sperm cells (Fig. 4, B
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and C; Supplemental Fig. S3, A, B, G and H). Since both fragments, -64 to -87 and -40 to -63, are
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required for sperm cell expression of PzIPT1, we hypothesized that basal promoter activity may require
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elements harbored in these two constructs. To test this hypothesis, we examined GUS expression
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patterns in transgenic seedlings. The original PzIPT1 promoter could drive GUS expression in seedlings
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(Fig. 1A). If essential basal promoter activities were impaired in constructs ∆64-87 and ∆40-63, no GUS
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signal would be expected in the corresponding transgenic seedlings. As shown in Fig. 4, F and G,
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however, these two constructs still retained their capability to drive GUS expression in seedlings,
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indicating that the deleted sequences did not affect the basal activities of the PzIPT1 promoter. Thus, the
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entire region from -40 to -87 was required for sperm specific expression.
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To further test whether the whole or part of the region between -40 to -87 is sufficient to drive
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the expression of PzIPT1 promoter in sperm cells, constructs were created fusing the whole region or the
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region of -64 to -87 or -40 to -63 to minimal CaMV35S promoter elements linked with a TMV leader
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sequence from a DR5 construct (Ulmasov et al., 1997) to examine their expression patterns in transgenic
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pollen (Fig. 4H). The minimal CaMV35S promoter by itself did not activate GFP in sperm cells (Fig. 4I;
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Supplemental Fig. S3, E and K), nor did intact CaMV35S promoter in a prior study (Singh et al., 2003).
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When the regions of -40 to -63 and -64 to -87 were fused to the minimal CaMV35S promoter, the
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synthetic promoters could not activate GFP expression in sperm cells (Fig. 4, J and K; Supplemental Fig.
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S3, F, L, M and S). However, the region of -40 to -87 could activate GFP expression in sperm cells (Fig.
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4L; Supplemental Fig. S3, N and T). GFP was detected in sperm cells harboring both regions of -64 to 13
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87 and -40 to -63. All these data support the conclusion that the cis-acting region -40 to -87 confers
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activation of the PzIPT1 promoter in sperm cells.
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MGSA Sites Determine Selective Activation of PzIPT1 Promoter in Sperm Cells
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When either sequence from -64 to -87 or -40 to -63 was excised, expression in sperm cells was lost (Fig.
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4, B and C) although expression in seedlings was unchanged (Fig. 4, F and G), suggesting that both of
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these sequences harbor motifs important for expression of PzIPT1 promoter in sperm cells and neither
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one by itself was sufficient for sperm cell expression. However, synthetic promoters containing
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duplicated -64 to -87 or -40 to -63 sequences fused with the minimal CaMV35S promoter elements could
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drive GFP expression in sperm cells (Fig. 4, M and N; Supplemental Fig. S3, O, U, P and V). Further
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sequence analysis identified two identical motifs of GAAACG at -69 to -74 and -49 to -54 (Fig. 2A),
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designated herein as Male Gamete Selective Activation (MGSA) motifs. When either MGSA motif of
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PzIPT1 promoter was mutated, no GFP signal could be detected in transgenic pollen (Fig. 4, D and E;
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Supplemental Fig. S3, C, I, D and J).
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To examine whether this motif is capable of driving gene expression in sperm cells, a synthetic
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promoter containing multiple copies of MGSA and the minimal CaMV35S promoter elements fused to
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GFP was introduced into Arabidopsis (Fig. 4H) and its expression was observed in sperm cells (Fig. 4P;
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Supplemental Fig. S3, R and X). No GFP signal was detected in sperm cells when only one copy of
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MGSA motif was employed (Fig. 4O; Supplemental Fig. S3, Q and W). These data together support the
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conclusion that at least two copies of MGSA motif are needed, and are sufficient to determine gene
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expression in sperm cells.
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Arabidopsis 500 bp upstream promoter sequences were retrieved from TAIR and screened for
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the presence of MGSA motif in previously identified sperm and pollen-expressed genes (Borges et al.,
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2008). A total of 277 sperm cell-expressed genes were identified with one MGSA motif and 37 genes
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have at least two MGSA motifs (Supplemental Table S1; Supplemental Table S2). The promoter activity
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of six genes with at least two MGSA motifs, At1g07910, At1g23060, At1g31010, At2g36660, At3g27540
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and At3g46230, has already been confirmed in sperm cells (Fig. 5, A-F; Supplemental Table S2). When
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either one of the two MGSA motifs close to transcription start site was mutated, expression of these
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genes in sperm cells was abolished (Fig. 5, G-R).
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DISCUSSION
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Conservation of Sperm Cell Expression of PzIPT1 Promoter
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Sperm cells produce a complex complement of messenger RNAs contributing to their development,
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differentiation and fertilization (Gou et al., 2001; Engel et al., 2003; Borges et al., 2008; Gou et al., 2009;
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Xin et al., 2011). A number of these mRNAs encode key regulators that are required for sperm cell
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control, identity and function. For example, DUO1 is a MYB transcription factor that is essential to male
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germline transcription and identity, with specific expression in generative and sperm cells of
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Arabidopsis (Rotman et al., 2005; Brownfield et al., 2009a; Borg et al., 2011). The highly conserved
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GCS1/HAP2 gene encoding a secreted membrane protein is essential for fertilization in a myriad of
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other eukaryotes as well (Mori et al., 2006; von Besser et al., 2006; Liu et al., 2008). Additional male
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gamete-selective genes have been identified and characterized in lily and Arabidopsis (Engel et al., 2005;
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Okada et al., 2005a; Okada et al., 2005b; Brownfield et al., 2009a, 2009b).
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A sperm type-selective gene PzIPT1 of Plumbago zeylanica was identified using suppression
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subtractive hybridization and in situ hybridization as having differential expression in the Svn sperm cell
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(Gou et al., 2009). Since transformation in Plumbago zeylanica has proven refractory (Wei et al., 2006),
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we examined transcriptional activity of the promoter in transgenic Arabidopsis. Expression of this gene
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was confined to the sperm cells in pollen of Arabidopsis with no detectable signal in pollen cytoplasm
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(Fig. 1D), suggesting that expression of PzIPT1 promoter in sperm cells is conserved, but without
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detectable differential response between sperm cell types. This diverged significantly from the
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differential transcription observed in Plumbago zeylanica. Thus, either the Arabidopsis sperm cells lack
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a similar pattern of dimorphism or sperm type-specific expression is species-dependent.
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Gene Expression in Male Germline Cells Is Regulated by Diverse Mechanisms of Activation and
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Repression
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Several different patterns of gene expression in the male germ lineage have been elucidated. Expression
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of LGC1 is restricted to generative cell and sperm cells (Xu et al., 1999). When LGC1 promoter was
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truncated, male germline specificity was lost and expression became constitutive (Singh et al., 2003).
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Further experiments resulted in the identification of the GRSF repressor protein ubiquitously expressed
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in sporophytic cells and the corresponding silencing sequence in the LGC1 promoter. In this model,
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transcriptional repression was mediated by a specific repressor and corresponding binding element;
336
based on similar sequences in a number of male germ expressed genes and thus this was proposed as a
337
possible general regulatory mechanism for the expression of male germline-specific genes (Haerizadeh
338
et al., 2006).
339
A cis-acting regulatory region for expression activation in sperm cells was identified in the
340
DUO1 promoter. Although a putative GRSF binding site was predicted in the promoter region of DUO1
341
(Haerizadeh et al., 2006), a truncated version of DUO1 promoter excluding the putative GRSF binding
342
site did not prevent germline-specific expression nor did it result in constitutive expression (Brownfield
343
et al., 2009a). On the other hand, MYB binding sites were overrepresented in the promoters of DUO1-
344
activated target (DAT) genes (Borg et al., 2011; Borg et al., 2014). DUO1 directly regulates the
345
expression of DATs through binding to the MYB sites in the promoter regions. For example, DUO1
346
binds to the MYB consensus sequences of the promoters of AtMGH3/HTR10, DAZ1 and DAZ2 by its
347
MYB domain to directly activate their expression (Borg et al., 2011). No GRSF binding site was
348
identified in PzIPT1 promoter sequences and moreover truncation results also demonstrated that no
349
GRSF binding site exists in the promoter. Truncation analyses instead suggested that there were positive
350
regulatory motifs for sperm specificity in the region of -40 to -87 of the PzIPT1 promoter (Fig. 4), as is
351
observed in the case of DUO1-mediated male germline gene expression (Fig. 6).
352
Our studies further identified two identical MGSA motifs that could activate PzIPT1 promoter in
353
sperm cells (Fig. 4). Moreover, repeated MGSA motifs were also identified in the promoters of 37
354
Arabidopsis sperm-expressed genes (Supplemental Table S2), and their importance for the activation of
355
six genes in sperm cells was confirmed (Fig. 5), suggesting that this type of gene activation in sperm
356
cells may be conserved. We noticed that a total of 250 genes were identified with at least two MGSA
357
motifs in Arabidopsis genome (Supplemental Table S1), but only 37 of them had a present call in sperm
358
cell transcriptomic data (Supplemental Table S2) (Borges et al., 2008), indicating that not all genes with
359
two MGSA motifs in their promoters will express in sperm cell. Some other factors, such as the
360
positions, the distance and the flanking sequences of the MGSA motifs, may affect their function. On
361
the other hand, we also noticed that many known sperm-expressed genes, such as DUO3,
16
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
362
AtMGH3/HTR10, DAZ1 and DAZ2, have no MGSA motifs in their promoters, suggesting that other
363
regulatory mechanisms for sperm gene expression function simultaneously, as revealed by DUO1-
364
activated expression of DAT genes (Borg et al., 2011). Collectively, the current available data suggest
365
multiple regulation mechanisms for gene expression in male germ cells, represented by LGC1, DUO1
366
and PzIPT1. The regulatory mechanisms of sperm type-specific gene expression, however, will need to
367
await a suitable transformation system for heteromorphic male germ cells. Similar mechanisms still may
368
369
be employed to distinct gene expression between a pair of sperm cells.
370
PzIPT1 Promoter Responds to Changes of Cytokinin Concentration
371
In Plumbago zeylanica, PzIPT1 expression is restricted to the Svn sperm cell, which is known to fuse
372
with the central cell during preferential fertilization (Russell, 1985), suggesting a targeted function of the
373
Svn PzIPT1 protein during double fertilization and endosperm development. Control of early
374
development by paternally encoded transcripts, as shown by the post-fertilization control of cell fate in
375
the two cell embryo though SSP function in Arabidopsis (Bayer et al., 2009) may have a counterpart in
376
the control of the early endosperm as well. The possible function of the cytokinin regulating double
377
fertilization, embryo and endosperm development of Plumbago zeylanica has not been elucidated yet.
378
That GFP signal could be enhanced dramatically upon cytokinin treatment and enhanced again by
379
reapplication (Fig. 3) suggests that this gene may participate in a feed-forward mechanism that enhances
380
cytokinin in the fusion product of the Svn. Our analyses indicate that PzIPT1 promoter could respond to
381
cytokinin pulsing, and this effect is supported by the presence of the CPB sites. CPB site was first
382
identified in the cucumber POR (NADPH-protochlorophyllide reductase) gene promoter that is critical
383
for cytokinin-dependent protein binding in vitro (Fusada et al., 2005). It is already known that the levels
384
of cytokinin and isopentenyltransferase are strongly increased during endosperm induction and
385
development (Miyawaki et al., 2004; Day et al., 2008). It is effective to control the cytokinin levels by
386
regulating the expression of the key cytokinin biosynthase, PzIPT1. When PzIPT1 promoter is exposed
387
to a significant elevated cytokinin level after double fertilization, it may respond to this change and
388
produce more cytokinin via a possible positive feedback mechanism to stimulate the endosperm
389
development nursing the embryo. On the other hand, the concentration of cytokinin must be finely
390
regulated and the expression of PzIPT1 will be down regulated when required cytokinin is produced,
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391
which is reflected by the decreased expression of GFP after pollen was treated with cytokinin for two
392
hours. There are 9 IPT genes in Arabidopsis (Miyawaki et al., 2004). However, no significant
393
upregulation of these IPTs is observed when treated with cytokinin according to expression data from
394
Genevestigator (http://genevestigator.com), suggesting a different mechanism for regulation of PzIPT1
395
expression. The possible transcriptional activator responding to cytokinin may play a vital role during
396
double fertilization, endosperm and embryo development. The transcriptional regulators controlling
397
PzIPT1 expression in sperm cells have not been revealed yet. Identification and functional analysis of
398
both of the transcriptional factors involved in fine regulation of PzIPT1 expression will provide more
399
insights into regulation of gene expression in male germline cells. It is also interesting and possible to
400
utilize PzIPT1 promoter or its components to develop a system for gene expression induction in sperm
401
cells by using cytokinin as an external chemical.
402
403
404
MATERIALS AND METHODS
405
406
Plant Growth and Transformation
407
Plumbago zeylanica L. plants were grown in greenhouses of the University of Oklahoma and Lanzhou
408
University. Plants of Arabidopsis thaliana wild type Columbia-0 (Col-0) were grown in growth rooms
409
with 16 hr light and 8 hr dark at 22°C. Agrobacterium-mediated transformation was performed to
410
generate transgenic Arabidopsis plants by floral dip method (Clough and Bent, 1998). At least 10
411
transgenic lines were observed.
412
413
Plasmid Constructs for Arabidopsis Transformation
414
The promoter of PzIPT1 was inserted into a binary vector pBIB-BASTA-GUS modified from pBIB
415
vector (Becker, 1990) at the Hind III and Sal I sites to make pPzIPT1::GUS. The PzIPT1 promoter was
416
also recombined into the Gateway-compatible pFYTAG binary vector to drive the expression of fused
417
coding regions of histone 2A (HTA6; At5g59870) and enhanced YFP (EYFP) (Zhang et al., 2005). The
418
binary vector pBIB-BASTA-GFP (Ge et al., 2011) was modified to a Gateway-compatible destination
419
vector, pBIB-BASTA-GFP-GWR, by inserting the Gateway module at Hind III and Xba I sites for
420
promoter analyses.
421
Deletion fragments were amplified by PCR from cloned PzIPT1 promoter and transferred into
422
pDONR/Zeo vector by Gateway in vitro DNA recombination for sequencing analysis. Following
423
sequence verification these truncated promoter fragments were in vitro recombined into pBIB-BASTA-
424
GFP-GWR and pBIB-BASTA-GUS-GWR (Yuan et al., 2007) to create final binary transformation
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
425
constructs. IPTPromPB2 was used as a reverse primer for all ten 5’-deletions. Forward primers
426
(∆761PB1, ∆531PB1, ∆368PB1, ∆255PB1, ∆154PB1, ∆130PB1, ∆105PB1, ∆87PB1, ∆63PB1, ∆39PB1)
427
were designed according to the positions of the deletions in the PzIPT1 promoter. The deletion
428
constructs ∆88-95, ∆64-87, and ∆40-63 were created according to the manual of QuikChange Site-
429
Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) using primers ∆88-95F, ∆88-95R,
430
∆64-87F, ∆64-87R, ∆40-63F, and ∆40-63R.
431
To clone promoters of sperm active genes in Arabidopsis, the following Gateway-compatible
432
primers were used: P07910-F, P07910-R for At1g07910; P23060-F, P23060-R for At1g23060; P31010-
433
F, P31010-R for At1g31010; P36660-F, P36660-R for At2g36660; P27540-F, P27540-R for At3g27540;
434
P46230-F, P46230-R for At3g46230. The following primers were used to mutate the two putative
435
MGSA sites close to the transcription start site in these promoters according to the manual of
436
QuikChange Site-Directed Mutagenesis Kit: ∆761m1F, ∆761m1R, ∆761m2F, ∆761m2R for ∆761;
437
07910m1F, 07910m1R, 07910m2F, 07910m2R for At1g07910; 23060m1F, 23060m1R, 23060m2F,
438
23060m2R for At1g23060; 31010m1F, 31010m1R, 31010m2F, 31010m2R for At1g31010; 36660m1F,
439
36660m1R, 36660m2F, 36660m2R for At2g36660; 27540m1F, 27540m1R, 27540m2F, 27540m2R for
440
At3g27540; 46230m1F, 46230m1R, 46230m2F, 46230m2R for At3g46230.
441
Sequence of the synthetic DR5 promoter (Ulmasov et al., 1997) containing seven copies of DR5,
442
the -46 CaMV35S promoter, and a TMV 5' leader was PCR-amplified from a DR5::GUS construct
443
provided by Dr. Guilfoyle (University of Missouri, Columbia) with primers DR5PB1 and DR5PB2, and
444
cloned into pDONR/Zeo vector for site-directed mutagenesis and in vitro DNA recombination with the
445
Gateway destination vector pBIB-BASTA-GFP-GWR. Site-directed mutagenesis was performed to
446
make the synthetic promoters with the following primers: 35SminiF, 35SminiR for 35Smini; (40-63)F,
447
(40-63)R for 40-63; (64-87)F, (64-87)R for 64-87; (40-87)F, (40-87)R for 40-87; 2×(40-63)F, 2×(40-
448
63)R for 2×(40-63); 2×(64-87)F, 2×(64-87)R for 2×(64-87); 1×MGSAF, 1×MGSAR for 1×MGSA;
449
4×MGSAF, 4×MGSAR for 4×MGSA.
450
Designing of Gateway-compatible primers and gateway cloning were conducted according to the
451
Gateway® Technology manual (Invitrogen, http://www.invitrogen.com). All primer sequences are listed
452
in Supplemental Table S3.
453
454
Pollen Collection, Pollen Tube Culture, and Cytokinin Treatment
455
Arabidopsis pollen from freshly-opened flowers was harvested from a representative transgenic line for
456
each construct, spread on solidified medium on a glass microscope slide for germination and
457
observation (Wang and Jiang, 2011). Collected pollen and germinated pollen tubes were incubated in
19
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
458
pollen germination medium containing 100 nM 6-Benzylaminopurine (6-BA) (Boavida and McCormick,
459
2007; Muller and Sheen, 2008) at room temperature for durations of 5, 15, 30, 45, 60, 90, and 120 min.
460
Pollen grains treated for 120 min were suspended and incubated in 3 changes of germination medium
461
over 60 min to remove exogenously applied cytokinin. Washed pollen was resuspended in germination
462
medium with 100 nM 6-BA for intervals of 15, 30, 45, and 60 min. The experiment was repeated for
463
three times.
464
465
Reverse Transcriptase-PCR Analysis
466
Gene expression differences of pollen treated with or without cytokinin were examined by reverse
467
transcriptase-PCR reactions. Pollen from Plumbago zeylanica was treated with 100 nM 6-BA for 60 min
468
in 70% glycerol (Southworth et al., 1997). Total RNA of treated and untreated pollen was isolated using
469
RNAprep Pure Plant Kit with on-column DNase-treatment (Tiangen Biotech, http://www.tiangen.com/).
470
Then 1 μg total RNA of each sample was reverse transcribed in a 50 μl volume using an M-MLV
471
reverse transcriptase (Invitrogen, http://www.invitrogen.com). RT product of 100 ng total RNA was
472
used as PCR template for one reaction. Different cycles (20, 22, 24, 26) were used to amplify PzIPT1
473
and
474
TCATACTGAAGGCAGGTCGTCT-3’),
475
HIS3.3-F
476
CGGTGGTGGGAGCAGACTT-3’). PCR products were separated by 1% agarose gel electrophoresis
477
and photographed.
HIS3.3
(as
a
control)
of
Plumbago
PzIPT1-R
zeylanica
with
primers
PzIPT1-F
(5’-
(5’-CCTTGAACCTCCGTATCTTGGA-3’),
(5’-GAGGAAAGGCTCCTAGAAAGCAA-3’)
and
HIS3.3-R
(5’-
478
479
GUS Staining, MUG assay and Microscopic Analysis
480
Transgenic plants harboring pIPT1-GUS were used for histochemical detection of GUS activity
481
(Robatzek and Somssich, 2001). Plant tissues were infiltrated in GUS staining solution containing 50
482
mM NaPO4, 0.5 mM K3Fe(CN)6, 0.5mM K4Fe(CN)6, 0.1% Triton X-100, 5 mM EDTA and 1mg ml-1
483
X-Gluc, incubated at 37°C overnight and destained several times in 70% ethanol and then photographed
484
using a Leica M165C Stereo microscope (Leica Microsystems, http://www.leica-microsystems.com/).
485
Col-0, ∆761, ∆40-63 and ∆64-87 were grown on ½ MS medium for 3 days and 20 seedlings for each
486
sample were harvested for quantitative measurement of GUS activity (4-methylumbelliferyl-beta-D-
487
glucuronide, MUG assays) according to Hornitschek et al., 2012. Three transgenic lines for each
488
construct were used for MUG assays, and the experiment was repeated for three times. Reproductive
489
organs with GFP or YFP reporter were observed using a Leica TCS SP5 confocal laser scanning
490
microscope or Leica DM6000 epifluorescence microscope equipped with a GFP or YFP filter set. To
20
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
491
evaluate GFP signal intensity, all samples were photographed with the same parameter settings. At least
492
20 pollen grains were used for statistical analysis of GFP signal, which was performed by ImageJ
493
analysis software from the US National Institutes of Health (http://rsbweb.nih.gov/ij/) according to
494
Burgess et al (Burgess et al., 2010).
495
496
Accession number for PzIPT1 promoter sequence: JN665068.
497
498
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
499
SUPPLEMENTAL DATA
500
501
The following supplemental materials are available.
502
Supplemental Figure S1. PzIPT1 promoter drives GUS expression in mature pollen of transgenic
503
Arabidopsis.
504
Supplemental Figure S2. Expression patterns of PzIPT1 promoter during Arabidopsis male
505
gametogenesis.
506
Supplemental Figure S3. Cis-acting region analyses of PzIPT1 in Arabidopsis sperm cells.
507
Supplemental Table S1. MGSA elements in Arabidopsis promoters.
508
Supplemental Table S2. Summary of 37 genes with putative MGSA motifs.
509
Supplemental Table S3. Primers used in the study.
510
511
512
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
513
ACKNOWLEDGEMENTS
514
The authors thank Dr. Tom J. Guilfoyle (University of Missouri, Columbia) for providing the construct
515
of DR5 promoter. We are grateful to Liping Guan, Yang Zhao, Liang Peng for their technical assistance.
516
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
517
FIGURE LEGENDS
518
519
Figure 1. Representative expression patterns of PzIPT1 promoter in transgenic Arabidopsis. Brightfield
520
microscopy of GUS expression (A) in 3-day old seedlings, (B) flowers, and (C) embryo sac. (D)
521
Fluorescence microscopy of GFP expression in two sperm cells. (E) Confocal laser scanning
522
microscopy of nuclear-localized YFP in synergids and the egg cell. Bars: A, B, 1 mm; C, 100 µm; D, 10
523
µm; E, 25 µm.
524
525
Figure 2. PzIPT1 promoter structure, truncation and deletion analysis. (A) Predicted cis-acting
526
regulatory elements by PLACE are shown for DOF (▲), ARR1 (●), GT1 (◊), MYB1 (oval), and CPB
527
(orange oval). Black thick lines represent PzIPT1 promoter. Numbers indicate positions from putative
528
transcription start site. Sequence between -1 and -105 is shown, with two identified MGSA motifs, two
529
predicted CPB sites in orange and a TATA box in blue. (B) Schematic showing PzIPT1 deletions used
530
to drive GFP expression in sperm cells of transgenic Arabidopsis. Expression pattern of each construct
531
in mature pollen is summarized on the right. Numbers refer to the 5’ end of the deletions from the
532
transcription start site and the positions of the CPB sites (orange ovals) and the TATA box (blue ovals).
533
(C) Confocal images of expression patterns generated by truncated PzIPT1 promoters in sperm cells of
534
transgenic Arabidopsis captured with identical parameters. Bars: 5µm. (D) Measured expression levels
535
of each construct by ImageJ. Values are means of 120 pollen grains from six independent T3 transgenic
536
lines (20 pollen/line) for each construct; error bars represent standard deviation.
537
538
Figure 3. PzIPT1 promoter responses to exogenously applied cytokinin. (A) GFP expression in ∆761
539
(left) and ∆88-95 (right). Bars: 5 µm. (B) RT-PCR analysis of PzIPT1 in Plumbago zeylanica pollen
540
treated or untreated (+/-) with 100 nM cytokinin (CK) and HIS3.3 used as the control. (C) Expression of
541
PzIPT1 promoter after addition of 100 nM exogenous CK in transgenic pollen grains of ∆154, ∆130 and
542
∆88-95 at 0, 30, 45, 60 and 120 min after treatment, then re-treated and observed at 120+15, 120+30,
543
120+45 and 120+60 min. (D) Expression of PzIPT1 promoter in pollen tubes of ∆130, ∆154 and ∆88-95
544
using confocal (left), bright field (middle) and mixed confocal/bright field microscopy (right) at 0 to 120
545
min. Bars: 5 µm. Photographs in C and D are not sequential images of same pollen grains or pollen
546
tubes. (E) Relative GFP signal intensity of ∆130, ∆154 and ∆88-95 in transgenic pollen treated with or
547
without CK (n=20 pollen grains; error bars represent standard deviation).
548
24
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
549
Figure 4. Cis-acting region determines expression specificity of PzIPT1 in sperm cells. At least 12 T1
550
transgenic plants were observed for each construct, and representative data are shown. (A) GFP
551
expression is evident in ∆761, but expression is absent in deletions (B) ∆64-87 and (C) ∆40-63, and in
552
mutated promoters (D) ∆761m1 and (E) ∆761m2. (F) Promoter activity in contrast remains intact in
553
seedlings of ∆64-87 and (E) ∆40-63 deletions using GUS expression. Relative GUS activity is shown in
554
(G). (H) Synthetic promoters used to test GFP expression in sperm cells. 35Smini, the minimal
555
CaMV35S promoter elements and the TMV 5’ leader sequence. (40-63), (64-87) and (40-87), cis-acting
556
regions from the PzIPT1 promoter. MGSA, Male Gamete Selective Activation motifs. I-P, Expression
557
patterns of synthetic promoters. (I) Minimal CaMV35S promoter elements, or cis-acting regions (J) 40-
558
63 and (K) 64-87, and 1×MGSA motif fused with minimal CaMV35S promoter elements cannot drive
559
GFP expression in sperm cells, whereas (L) 40-87, (M) 2×(40-63), (N) 2×(64-87) cis-acting regions and
560
(P) 4×MGSA motifs fused with minimal CaMV35S promoter elements can activate GFP expression in
561
sperm cells. Bars: A-E, 5 µm; F, 2 mm; I-P, 5 µm.
562
563
Figure 5. MGSA motifs function in sperm active promoters of Arabidopsis. A-F, MGSA motifs-
564
containing promoters can drive GFP expression in sperm cells. G-R, Mutation of the upstream (m1) or
565
downstream (m2) MGSA motif close to transcription start site abolishes GFP expression in sperm cells.
566
(A, G, M) At1g07910; (B, H, N) At1g23060; (C, I, O) At1g31010; (D, J, P) At2g36660; (E, K, Q)
567
At3g27540; (F, L, R) At3g46230. Bars: 5 µm.
568
569
Figure 6. Regulatory mechanisms for PzIPT1 promoter expression in sperm cells of Arabidopsis. Male
570
germ cell-expressed positive factors activate or enhance germline genes in male gametes. Orange and
571
light blue lines represent the 5’-upstream CBP and MGSA sites, respectively. Orange squares and light
572
blue pentagons represent the corresponding positive activators. Gene expression is shown in green. Sua,
573
sperm cell unassociated with the vegetative nucleus; Svn, sperm cell associated with the vegetative
574
nucleus; VN, vegetative nucleus.
575
576
Supplemental Figure S1. PzIPT1 promoter drives GUS expression in mature pollen of transgenic
577
Arabidopsis. Bar: 10 µm.
578
579
Supplemental Figure S2. Expression patterns of PzIPT1 promoter during Arabidopsis male
580
gametogenesis. A-I, Wildtype Col-0; J-R, Transgenic pollen of GFP driven by PzIPT1 promoter. (A-C,
25
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
581
J-L) Microspores; (D-F, M-O) Bicellular pollen; (G-I, P-R) Mature pollen. (A, D, G, J, M, P) Stained
582
with DAPI; (B, E, H, K, N, Q) GFP expression; (C, F, I, L, O, R) DIC microscopy. Bars: 5 µm.
583
584
Supplemental Figure S3. Cis-acting region analyses of PzIPT1 in Arabidopsis sperm cells. Results
585
from two more transgenic lines (L1 and L2) for each construct are shown. GFP expression is absent in
586
deletions (A, G) ∆40-63 and (B, H) ∆64-87, and in mutated promoters (C, I) ∆761m1 and (D, J)
587
∆761m2. (E, K) Minimal CaMV35S promoter elements, or cis-acting regions (F, L) 40-63 and (M, S)
588
64-87, and 1×MGSA motif fused with minimal CaMV35S promoter elements cannot drive GFP
589
expression in sperm cells, whereas (N, T) 40-87, (O, U) 2×(40-63), (P, V) 2×(64-87) cis-acting regions
590
and (P, X) 4×MGSA motifs fused with minimal CaMV35S promoter elements can activate GFP
591
expression in sperm cells. 35Smini, the minimal CaMV35S promoter elements and the TMV 5’ leader
592
sequence. 40-63, 64-87 and 40-87, cis-acting regions from the PzIPT1 promoter. MGSA, Male Gamete
593
Selective Activation motifs. Bars: 5 µm.
594
595
Supplemental Table S1. MGSA elements in Arabidopsis promoters.
596
597
Supplemental Table S2. Summary of 37 genes with putative MGSA motifs.
598
599
Supplemental Table S3. Primers used in the study.
600
26
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