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This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online reduces the time to publication by several weeks. The WD40-Repeat Proteins NFC101 and NFC102 Regulate Different Aspects of Maize Development through Chromatin Modification W Iride Mascheretti, Raffaella Battaglia,1 Davide Mainieri,2 Andrea Altana, Massimiliano Lauria,2 and Vincenzo Rossi3 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per la Maiscoltura, I-24126 Bergamo, Italy The maize (Zea mays) nucleosome remodeling factor complex component101 (nfc101) and nfc102 are putative paralogs encoding WD-repeat proteins with homology to plant and mammalian components of various chromatin modifying complexes. In this study, we generated transgenic lines with simultaneous nfc101 and nfc102 downregulation and analyzed phenotypic alterations, along with effects on RNA levels, the binding of NFC101/NFC102, and Rpd3-type histone deacetylases (HDACs), and histone modifications at selected targets. Direct NFC101/NFC102 binding and negative correlation with mRNA levels were observed for indeterminate1 (id1) and the florigen Zea mays CENTRORADIALIS8 (ZCN8), key activators of the floral transition. In addition, the abolition of NFC101/NFC102 association with repetitive sequences of different transposable elements (TEs) resulted in tissue-specific upregulation of nonpolyadenylated RNAs produced by these regions. All direct nfc101/nfc102 targets showed histone modification patterns linked to active chromatin in nfc101/nfc102 downregulation lines. However, different mechanisms may be involved because NFC101/NFC102 proteins mediate HDAC recruitment at id1 and TE repeats but not at ZCN8. These results, along with the pleiotropic effects observed in nfc101/nfc102 downregulation lines, suggest that NFC101 and NFC102 are components of distinct chromatin modifying complexes, which operate in different pathways and influence diverse aspects of maize development. INTRODUCTION The WD-repeat proteins have seven WD40-repeat motifs, with the conserved core of the repeat containing 44 to 60 residues that end with Trp and Asp. The repeats form a b-propeller fold, allowing formation of a highly stable structure that coordinates interactions with several other proteins (Stirnimann et al., 2010). The MSI1-like proteins are a particular class of WD40-repeat proteins, named for the founding member isolated in a screen for multicopy suppressors of the ira1 mutation in budding yeast (Saccharomyces cerevisiae; Ruggieri et al., 1989). This class contains mammalian retinoblastoma-associated proteins RbAp46/ 48 and their Drosophila melanogaster homolog p55, which are components of complexes involved in chromatin assembly and modification (Suganuma et al., 2008). In Arabidopsis thaliana, MSI proteins can be subdivided into three evolutionary distinct clades: MSI1, MSI2/MSI3, and FVE (MSI4)/MSI5 (Hennig et al., 2005). MSI1 is a component of various chromatin-remodeling and assembly complexes involved in sporophyte, gametophyte, and seed development (Kaya et al., 1 Current address: Università degli Studi di Milano, Dipartimento di Bioscienze, Via Celoria 26, 20133 Milan, Italy. 2 Current address: Consiglio Nazionale delle Ricerche, Istituto di Biologia e Biotecnologia Agraria, via Bassini 15, 20133 Milan, Italy. 3 Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Vincenzo Rossi (vincenzo. [email protected]). W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.112.107219 2001; Köhler et al., 2003; De Lucia et al., 2008). FVE was originally found to act in the autonomous flowering pathway and thus controls flowering independently of external signals (Koornneef et al., 1991). The fve mutants exhibit a late-flowering phenotype because they impair the recruitment of Rpd3-type histone deacetylases (HDACs) to the MADS box central floral repressor FLOWERING LOCUS C (FLC), resulting in histone hyperacetylation and overexpression of FLC (Ausín et al., 2004; Gu et al., 2011). The accumulation of histone H3 trimethylated at Lys 27 (H3K27me3), related to FVE interaction with Polycombrepressive complex 2 (PRC2) components, is also involved in the repression of FLC and of the FLOWERING LOCUS T (FT; Pazhouhandeh et al., 2011). FT encodes a protein with similarity to phosphatidylethanolamine binding–related kinase that acts as a florigen, a mobile floral inductive signal produced in leaves that migrates to the shoot apex. At the apex, the florigen interacts with the bZIP transcription factor encoded by FLOWERING LOCUS D (FD) to activate transcription of floral meristem identity genes, such as APETALA1 (Abe et al., 2005; Wigge et al., 2005; Corbesier et al., 2007; Turck et al., 2008). FVE activity is not limited to flowering regulation. Indeed, FVE is involved in coldinducible gene expression (Kim et al., 2004) and modulates silencing of transposable elements (TEs) and repeats, acting as an effector of the RNA-directed DNA methylation (RdDM) pathway (Bäurle and Dean, 2008; Gu et al., 2011). Maize (Zea mays) has five MSI family members (Hennig et al., 2005). These proteins were named NFC by the Plant Chromatin Database initiative (http://www.chromdb.org) because of similarity to RbAp46/48 NURF complex component, where NURF is the nucleosome remodeling factor (Clapier and Cairns, 2009). On the basis of amino acid sequence similarity, maize NFC103/ The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved. 1 of 17 2 of 17 The Plant Cell NFC108 and NFC104 belong to the Arabidopsis MSI1 and MSI2/ MSI3 clades, respectively (Hennig et al., 2005; http://www. chromdb.org). These two clades form a subgroup separate from the third clade, which is related to FVE/MSI5 and that contains maize NFC101 and NFC102. The nfc102 gene (originally named RbAp1) is expressed ubiquitously, and in in vitro assays, it was shown that the protein binds histones and interacts with the Retinoblastoma-related protein1, a key cell cycle regulator, and with Rpd3-type HDACs to promote transcriptional repression (Rossi et al., 2001, 2003; Varotto et al., 2003). These findings suggest that nfc102 possesses some of the features of Arabidopsis FVE. To test this hypothesis, we analyzed regulation of the recently identified maize homologs of Arabidopsis FVE/MSI5 targets. For example, it was demonstrated that, of the FT-like Zea mays CENTRORADIALIS (ZCN) gene family members, the ZCN8 gene possesses most of the attributes expected for maize florigen (Danilevskaya et al., 2008a; Lazakis et al., 2011; Meng et al., 2011). Moreover, although none of the several maize MADS box genes exhibits sequence or functional homology with Arabidopsis FLC (Becker and Theissen, 2003; Colasanti and Coneva, 2009), two genes with a major effect on flowering time were found. One of them is delayed flowering1, which is the Arabidopsis FD ortholog (Muszynski et al., 2006). The other is indeterminate1 (id1), which encodes a monocot specific zinc finger transcriptional regulator (Colasanti et al., 1998, 2006). id1 positively regulates ZCN8 expression in leaves (Lazakis et al., 2011; Meng et al., 2011); therefore, it acts as a master floral regulator in day-neutral maize, which relies almost exclusively on autonomous signals to control flowering. In this study, we generated nfc101/nfc102 transgenic lines and found that reduced activity of these genes has a pleiotropic effect with various developmental alterations. We provide evidence that NFC101/NFC102 proteins directly associate with the id1 and ZCN8 genes and with TEs and that binding always is correlated with altered chromatin modification patterns and transcriptional repression. The repressive effect is usually associated with Rpd3-type HDAC recruitment; however, HDACindependent mechanisms appear to be involved in ZCN8 regulation. Together, our results indicate that nfc101 and nfc102 regulate different aspects of plant development and provide new insights into the role of chromatin-mediated mechanisms in the maize flowering pathway. RESULTS Generation of nfc101/nfc102 Downregulation Transgenic Lines Maize NFC101 and NFC102 proteins have 99% amino acid sequence similarity to each other, and they share 92 and 86% sequence similarity with FVE and MSI5, respectively (see Supplemental Figure 1 online). This suggests that, similar to Arabidopsis FVE/MSI5 (Gu et al., 2011), the two maize proteins are functionally redundant. To simultaneously affect nfc101 and nfc102 activity, we generated maize transgenic lines with constitutive overexpression of nfc102 antisense transcript (AS lines). In addition, one line, with nfc101/nfc102 downregulation by RNA interference (R102 line), was obtained from the Plant Chromatin Database initiative (http://www.chromdb.org). Four lines, which displayed the highest reduction of nfc101/nfc102 RNA levels (AS15, AS36, AS63, and R102), were backcrossed to the B73 recurrent parent to minimize mixed genetic background influences and subsequently selfed to obtain homozygous and wildtype segregants used for molecular and phenotypic analysis (BC4-F3 generation; see Supplemental Figure 2 online). Both the B73 line and wild-type segregants were used as negative controls. DNA gel blot analysis revealed that two lines (AS15 and AS63) have a single transgene insertion, whereas the other two lines (AS36 and R102) have multiple copy transgenic insertions (see Supplemental Figure 3 online). Downregulation of nfc101/nfc102 expression was measured in leaf blades of the third and fourth fully extended leaf and in the seventh inner leaf of seedlings at the V3/V4 developmental stage (Figure 1A). The seventh inner leaf is enriched in meristematic tissues because it includes the shoot apical meristem (SAM) and axillary meristems along with the youngest leaf primordia surrounding the SAM; hence, it was named the meristematic enriched area (MA). Real-time quantitative RT-PCR (qRT-PCR) analysis revealed that the nfc101 and nfc102 RNA levels were reduced in leaf blade and MA tissues of all lines, while the expression of the nfc103 gene, related to a distinct MSI1-like clade, was unaffected (Figure 1B). Since a nfc102 sequence was used to generate the transgenic suppression lines, in all lines nfc102 mRNA was reduced more than nfc101 (ranging from 23.8- to 211.6-fold for nfc102 and from 22.4- to 26.5-fold for nfc101). Both genes showed the greatest downregulation in the R102 line. Phenotypic Alterations in the nfc101/nfc102 Downregulation Lines Phenotypic analysis was performed using BC4-F3 progeny of homozygous transgenic and wild-type segregant plants and the B73 inbred line (Figure 2). Variation for several quantitative traits was measured in two independent experiments. A phenotypic trait was considered altered in the transgenic line only if it showed a statistically significant difference in comparison with both BC4-F3 wild-type segregants and B73 lines and whether this occurred for at least three of the four nfc101/nfc102 mutant lines analyzed (Table 1). Using these criteria, variation was observed for the following phenotypes: percentage of germinated seeds, coleoptile emergence time, V2 stage seedling dry weight and first internode length, V2 seedling primary root dry weight and apical length, plant height, ear length, seed number for each ear, and flowering time for both male and female inflorescence. All these traits exhibited a quantitative reduction in the transgenic lines compared with wild-type controls, with the exception of germination and flowering time, which increased in nfc101/ nfc102 suppression lines. No statistically significant differences were reported for the other phenotypes analyzed: number of ears, 100 seed weight, tassel length, and leaf number at maturity. The latter observation suggests that the late-flowering phenotype was not associated with a delay in the developmental transition from vegetative to flowering stage. Accordingly, when the transition was estimated by the extent of apex elongation and appearance of branch meristems flanking the shoot apex nfc101/nfc102 Characterization 3 of 17 to first analyze whether putative maize homologs of FVE/MSI5 targets exhibited mRNA level variations in nfc101/nfc102 downregulation lines. All four transgenic lines, each representing a unique transgene insertion in distinct genomic locations, were used for this analysis to minimize positional effects and transgenic quality differences that are not specifically related to Figure 1. Expression of Different nfc Genes in nfc101/nfc102 Downregulation Lines. (A) Materials used for molecular analysis were extracted from the leaf blade (LB) of third and fourth leaf and from the MA of the seventh most inner leaf, including the SAM, after dissection of V3/V4 seedlings. (B) Real-time qRT-PCR quantification of nfc101, nfc102, and nfc103 mRNA in the wild type (wt) and four nfc101/nfc102 suppression lines. Bar diagrams represent the mean value of mRNA level obtained by two different cDNA preparations and three PCR replicates for each preparation. Standard errors are reported. The transcript levels were normalized to the mRNA amount of the gapc2 gene, which is unaffected in suppression lines (see Supplemental Figure 4 online). Relative expression to the wild type is presented. Asterisk indicates statistically significant change (P # 0.05) in NFC101/NFC102 lines versus the wild type. (Irish and Nelson, 1991), no obvious differences were observed in nfc101/nfc102 downregulation lines compared with the wild type (Figure 2F). Hence, the delay of flowering was likely due to a slow growth rate during plant development, which results in reduced seedling weight, adult plant height, and most of the other phenotypes described above. Together, these results provide evidence that perturbation of nfc101/nfc102 expression induces various phenotypic alterations that are indicative of pleiotropic effects, resulting in a general delay in plant growth. nfc101/nfc102 and Floral Transition Regulators Information available from studies of Arabidopsis FVE/MSI5 was used to assess whether they share common targets and mechanisms with nfc101/nfc102. Our experimental strategy was Figure 2. Phenotypic Alterations of nfc101/nfc102 Downregulation Lines. (A) to (E) Examples of visible phenotypes in nfc101/nfc102 downregulation lines (AS and R102 lines) compared with the wild type (wt), including the slow growth rate from early (A) to V6/V7 (B) developmental stages, which results in reduced plant height at maturity (C), smaller ears (D), and smaller young seedlings (E). (F) Light microscopy images of longitudinal section of SAM in wild-type and R102 plants. The timing of the vegetative to reproductive transition, evaluated by shoot apex elongation and appearance of lateral branches in plants harvested at the same time after sowing, was not obviously altered in the R102 suppression line compared with the wild type. 4 of 17 The Plant Cell Table 1. Phenotypic Variation of nfc101/nfc102 Downregulation Lines V2 Stage Seedlingsa Germination Adult Plantsb Root traits Maize Line % B73 R102 AS15 AS36 AS63 Wild-type R102 Wild-type AS15 Wild-type AS36 Wild-type AS63 86.2 68.0 63.0 72.2 71.8 / / / / Coleoptile Emergence (Days) 6 6 6 6 6 5.4* 3.1 4.7 3.3 3.5 6.6 11.5 12.7 9.7 10.5 / / / / 6 6 6 6 6 0.5* 1.6 1.7 1.6 1.7 Seedling Dry Weight (mg) 176 121 132 138 136 / / / / 6 6 6 6 6 28* 18 25 16 18 First Internode Length (cm) 5.8 3.8 4.6 4.2 4.7 / / / / 6 6 6 6 6 1.3* 0.7 1.3 0.8 1.2 Root Dry Weight (mg) 103 65 70 77 / / / / / 6 6 6 6 23* 13 23 25 Flowering Time Apical Root Length (cm) 18.7 11.6 12.1 / 13.6 / / / / 6 5.1* 6 2.7 6 3.3 6 2.2 Pollen Shed (GDD) 1093 1186 1154 1167 1150 / / / / 6 6 6 6 6 39* 35 37 39 15 Plant Height Silking (GDD) 1149 1229 1180 1190 1183 / / / / 6 6 6 6 6 Plant (cm) 38* 37 39 36 36 160.8 146.7 147.8 149.4 150.5 / 155.8 155.5 / 6 6 6 6 6 13.5* 13.4 16.2 17.7 14.1 6 13.9 6 13.9 Ear (cm) 73.5 66.3 67.1 67.8 68.5 / 71.7 71.6 / 6 6 6 6 6 9.6* 6.2 6.2 6.9 6.3 6 6.2 6 8.8 Ear Length (cm) 15.7 10.8 11.7 12.1 11.1 14.2 13.8 14.3 14.3 6 6 6 6 6 6 6 6 6 2.1* 1.1 2.0 2.3 2.4 2.4 2.1 1.9 1.9 Seed Number (One Ear) 327 99 131 190 177 273 276 277 280 6 6 6 6 6 6 6 6 6 68* 53 61 68 63 30 40 44 48 Flowering time is expressed in GDD. Plant and ear heights are measured from ground level to the base of the tassel and to the node bearing the uppermost ear, respectively. The data were obtained using a minimum of 60 plants for each genotype. Similar changes were observed in a second experiment, carried out using the same number of plants. For each mutant line, the homozygous transgenic and wild-type segregants from the BC4-F3 generation were used (see Supplemental Figure 2 online for the crossing scheme; e.g., R102 and wild-type R102 indicate the homozygous transgenic line and the wild type, respectively, of the R102 line BC4-F3 generation). All comparisons reported refer to the B73 recurrent parent (noted with asterisk). For clarity, only the values of traits exhibiting statistically significant difference compared to B73 by Student’s t test analysis (P # 0.05) are shown. A slash indicates no significant changes (see the main text for further details). a Phenotypic traits were measured when the phytotron-grown wild-type seedlings were at V2 stage. b Phenotypic traits were measured when all the greenhouse-grown plants were at maturity. Mean and se for each trait are reported. nfc101/nfc102 downregulation. The B73 line was used as the wild-type control. Second, genes exhibiting changes in mRNA levels in all four nfc101/nfc102 downregulation lines were analyzed for in vivo NFC101/NFC102 protein binding in wild-type plants to identify direct nfc101/nfc102 targets. Line R102 was chosen as the control for this analysis because it displayed the greatest reduction in nfc101/nfc102 expression (Figure 1) and exhibited all the phenotypic alterations observed in the other transgenic lines (Table 1). Finally, where NFC101/NFC102 binding was observed, the targets were further investigated for binding of Rpd3-type HDACs and histone modifications. The MADS box FLC gene is the best-characterized FVE/MSI5 target (He, 2009). Up to now, of the many maize MADS box genes, none have been proven to be a functional FLC homolog (Colasanti and Coneva, 2009). However, a list of 25 maize MADS box genes encoding flowering control–associated proteins (FLCPs) has been made available from the Plant Chromatin DB initiative (http://www.chromdb.org/; update of March, 2010). We employed primer combinations specific for these sequences (see Supplemental Table 1 online) to analyze their RNA levels in MA tissues of V3/V4 wild-type and transgenic seedlings (Figure 1A). These tissues contain SAM and leaf primordia and were sampled at a developmental stage preceding floral transition, which in the B73 temperate line occurs at stage V5 (Meng et al., 2011; Figure 2F). Therefore, a putative maize FLC homolog, acting as a general regulator of floral transition, might be expressed in these samples. Results indicate that none of the FLCP genes exhibited statistically significant variation of mRNA levels in nfc101/nfc102 suppression lines compared with the wild type (see Supplemental Figure 4 online), with the exception of FLCP109/ZMM15 and FLCP128/ZMM4, which showed mRNA reduction in the suppression lines (Figure 3A). These genes belong to the Arabidopsis APETALA1/FRUITFUL MADS box clade and regulate meristem floral identity, acting downstream of the floral activators dlf1 and id1 (Danilevskaya et al., 2008b; Meng et al., 2011). Binding of the NFC101/NFC102 proteins to FLCP109/ZMM15 and FLCP128/ZMM4 sequences was analyzed with chromatin immunoprecipitation (ChIP) assays, using an anti-FVE antibody previously validated for its ability to specifically recognize maize NFC101/NFC102 proteins (see Supplemental Figure 5 online). Immunoprecipitates were quantified by real-time quantitative PCR (qPCR). No signal above background was reported (Figure 4A), indicating that FLCP109/ZMM15 and FLCP128/ZMM4 genes are indirect nfc101/nfc102 targets. Since FLC is a central floral regulator, we also analyzed if perturbation of nfc101/nfc102 function affected expression of dlf1 and id1, which are the best-characterized maize genes displaying a major effect on floral transition (Colasanti and Coneva, 2009). The mRNA level of dlf1 was unaltered, whereas id1 overexpression was observed in the MA of the nfc101/ nfc102 suppression lines (Figure 3A). Since id1 expression is restricted to immature leaves (Colasanti et al., 1998), we assessed whether nfc101/nfc102 downregulation induced ectopic id1 expression in mature leaves (e.g., leaf blade tissues). However, no amplicons corresponding to id1 were observed via RT-PCR, even with a high cycle number (Figure 3B). Nevertheless, NFC101/NFC102 and Rpd3-type HDAC binding to the id1 59-end region, located upstream of the presumed ATG, was reported in both MA and leaf blade tissues of wild-type plants, but it was substantially abolished in the R102 suppression line (Figure 4B). No binding was observed in the id1 39-end region. FVE/MSI5 proteins interact with HDACs, Lys-specific histone demethylase encoded by FLOWERING LOCUS D, and PRC2 nfc101/nfc102 Characterization 5 of 17 and nfc101/nfc102-dependent Rpd3-type HDAC recruitment occurs at the 59-end of id1 in both MA and leaf blade tissues, the abolition of these interactions in transgenic lines induces id1 overexpression and H3ac/H3K4me2 accumulation only in MA. nfc101/nfc102 and ZCN8 Figure 3. Expression of Maize Flowering Pathway Regulators in nfc101/ nfc102 Downregulation Lines. (A) Real-time qRT-PCR quantification of four flowering regulatory genes in MA tissue from V3/V4 stage seedlings. Bar diagrams represent the mean value of mRNA level normalized to gapc2 mRNA, from two biological repetitions and three qRT-PCR replicates for each biological repetition. Data are expressed as fold changes (FC) relative to the wild type. Standard errors are reported. Asterisk indicates statistically significant change (P # 0.05). wt, the wild type. (B) Cycle-limited RT-PCR performed using RNA extracted from MA and leaf blade (LB) tissues of wild-type and R102 transgenic V3/V4 stage seedlings and with (+) or without (2) addition of reverse transcriptase (RT). complex (Gu et al., 2011; Pazhouhandeh et al., 2011). Accordingly, we investigated whether loss of NFC101/NFC102 binding in the id1 59-end domain caused alteration of the histone modification marks generated by these factors. Specifically, the following marks were analyzed: histone H3 acetylated at Lys 9 and 14 (H3ac), histone H3 dimethylated at Lys 4 (H3K4me2), and H3K27me3. The only variation observed was an increase of H3ac and H3K4me2 abundance in MA tissues of the nfc101/ nfc102 downregulation line (Figure 4C). In agreement with an id1 transcriptionally silent chromatin state, these histone marks were undetectable or very low in leaf blade. Together, our results indicate that, among the genes analyzed, the only floral transition regulator directly targeted by nfc101/nfc102 activity is id1. Although NFC101/NFC102 binding The Arabidopsis florigenic FT gene is an FVE target (Pazhouhandeh et al., 2011). Therefore, we investigated if nfc101/nfc102 downregulation affects expression of the maize putative florigenic gene ZCN8 (Lazakis et al., 2011; Meng et al., 2011). To this end, we first analyzed in more detail ZCN8 expression in the tissues selected for our study. In agreement with findings from a previous work (Danilevskaya et al., 2008a), we used RT-PCR to show that ZCN8 was expressed in leaf blade producing a mixture of spliced and unspliced transcripts, but only the unspliced form was detected in MA tissues (see Supplemental Figure 6A online). In addition, RNA gel blot analysis with strand-specific probes showed that the spliced transcript is represented by the sense RNA strand, but the unspliced transcripts correspond to both sense and antisense RNA strands, with the antisense strand representing the prevalent unspliced transcript form (see Supplemental Figure 6B online). Since the ZCN8 probes used in RNA gel blots can hybridize with other members of the ZCN family (see Supplemental Methods 1 online; Danilevskaya et al., 2008a), strand-specific RT-PCRs with locus-specific primers were performed to show that ZCN8 in fact produces all the three different RNAs: sense spliced mRNA, sense unspliced pre-mRNA, and antisense unspliced RNA (Figure 5A; see Supplemental Figure 6C online). The sequence of cDNA derived from the ZCN8 antisense RNA has short open reading frames that do not display sequence similarity with known proteins (see Supplemental Figure 7 online), suggesting that the antisense transcript represents a noncoding RNA. Previous finding indicates that id1 activates expression of ZCN8 spliced mRNA in mature leaf (Lazakis et al., 2011; Meng et al., 2011). Using strand-specific qRT-PCR, we showed that the amount of both spliced and unspliced sense ZCN8 transcripts was reduced in leaf blade tissues of the id1 mutant and that this occurs concomitantly with an increase of the unspliced antisense strand level (see Supplemental Figure 6D online). The observations described above allowed us to analyze the possible effect of nfc101/nfc102 downregulation on ZCN8 expression by considering the different transcripts produced by this gene. First, accumulation of ZCN8 spliced RNA was detected in leaf blade of all nfc101/nfc102 downregulation lines (Figure 5B). Second, the increase of spliced and unspliced sense ZCN8 transcripts in leaf blade tissues of the R102 suppression line occurred along with a reduction of unspliced antisense strand level (Figure 5C), which is the opposite to those observed in id1 mutants. The same pattern was reported in MA tissues of the R102 line, with the exception that the spliced sense RNA was not detected in these samples. Surprisingly, ChIP assays showed that NFC101/NFC102 binding occurs only in the 39-end region of ZCN8 (Figure 5D). The binding was reported in both MA and leaf blade tissues of wild-type plants, but Rpd3-type HDACs interaction with this gene was not detected. When we 6 of 17 The Plant Cell Figure 4. Binding of NFC101/NFC102 and Rpd3-Type HDACs and Histone Modifications at Flowering Pathway Genes in Wild-Type and nfc101/nfc102 Downregulation Lines. Bar diagrams represent real-time PCR quantification of ChIP DNA, reported as percentage of the chromatin input, from assays performed using the indicated antibodies (NFC101/NFC102, Rpd3, H3ac, H3K4me2, and H3K27me3) and chromatin extracted from MA or leaf blade (LB) tissues of wildtype (wt) and nfc101/nfc102 suppression line (R102) V3/V4 stage seedlings. The horizontal black line shows the background signal, measured by omitting antibody during the ChIP procedure. Data are average values from two independent ChIP assays and from three PCR repetitions for each ChIP assay. Standard errors are reported. Asterisk indicates statistically significant change (P # 0.05) in R102 line versus the wild type. In (A), data refer to binding of NFC101/NFC102 to the 59-end genomic region upstream of ATG of the indicated genes, but similar results were observed by analyzing 39end genomic region. The id1 genome sequence is diagrammed in (B), with black boxes and lines symbolizing exons and introns, respectively; positions of the 59-end and 39-end id1 regions analyzed are indicated. Bar graphs show NFC101/NFC102 and Rpd3 binding to the indicated id1 regions. In (C), the extent of different histone modifications in id1 59-end region from MA and leaf blades is shown. analyzed the distribution of histone modification marks, statistically significant increases of H3ac and H3K4me2 levels along the entire ZCN8 sequence was observed in MA tissues of nfc101/nfc102 downregulation lines compared with the wild type, whereas no differences were detected in leaf blade tissues (Figure 5E). Interestingly, H3K27me3 showed an accumulation in the ZCN8 59-end genomic region of nfc101/nfc102 downregulation line, and this accumulation was specifically detected only in leaf blade tissues. Comparison of H3K27me3 levels at the ZCN8 59-end in MA and leaf blade tissues of wild-type plants revealed that this histone mark is almost undetectable in MA, whereas it is relatively abundant in leaf blade tissues (Figure 5F), suggesting that its deposition occurs in a tissue-specific manner. Collectively, these results indicate that nfc101/nfc102 genes act oppositely to id1 and repress expression of ZCN8 sense transcripts. In both nfc101/nfc102 and id1 mutants, the expression of ZCN8 sense mRNA is negatively correlated with antisense strand production. Since NFC101/NFC102 can bind directly to the ZCN8 39-end genomic region without promotion of Rpd3-type HDAC recruitment, an nfc101/nfc102 role in controlling ZCN8 expression through HDAC-independent mechanisms can be envisaged. Analysis of histone modifications suggested that these mechanisms may be related to the regulation of H3K27me3, which specifically accumulated at the ZCN8 59-end genomic region and in leaf blade tissues. nfc101/nfc102 and TEs FVE and MSI5 are required for silencing of some Arabidopsis TEs and genomic repeats (Bäurle and Dean, 2008; Gu et al., 2011). Accordingly, we assessed whether the transcript level of maize TEs was affected in nfc101/nfc102 downregulation lines. Sequences representing repetitive regions of different TE classes were selected for this analysis, including the terminal inverted repeats of the Mutator (Mu) DNA transposon and the long terminal repeats (LTRs) of the following retrotransposons: a Prem2/Ji Copia-like TE, a Cinful/Zeon Gypsy-like TE, and the CRM2 centromeric TE. All these sequences are from high-copy nfc101/nfc102 Characterization 7 of 17 Figure 5. Analysis of ZCN8 Expression and NFC01/NFC102-Rpd3 Binding and Histone Modifications at ZCN8 in Wild-Type and nfc101/nfc102 Downregulation Lines. 8 of 17 The Plant Cell TEs, with copy number varying from hundreds (Mu and CRM2) to several thousand (Prem2/Ji and Cinful/Zeon; Baucom et al., 2009; http://maizetedb.org/). Therefore, the analysis of transcript levels of these repeats represents a broad sampling of these TEs in the genome, from a large number of distinct contexts and possible variation only provides an average of the effect of nfc101/nfc102 downregulation on these TEs. In agreement with a previous report indicating that high copy LTR retrotransposons are poorly represented in the polyadenylated RNA fraction (Hale et al., 2009), little to no transcript for any of the LTRs was detected when RT-PCR was performed using oligo(dT)-primed cDNA (see Supplemental Figure 8 online). Conversely, good amplification was achieved when RT-PCR was performed with random-primed cDNA, which allowed detection of nonpolyadenylated RNA (see Supplemental Figure 8 online). Using random-primed cDNA in qRT-PCR experiments, an increase of the RNA levels of TE repeats was observed in MA tissues of all the four nfc101/nfc102 downregulation lines analyzed (Figure 6A). Conversely, a weak upregulation of RNA from TE repeats was detected in leaf blade tissues and the variation was statistically significant only in some of the four nfc101/ nfc102 downregulation lines (Figure 6B). To rule out that TE upregulation was due to hybrid genomes formed during the generation of nfc101/nfc102 knockdown lines, the variation of TE repeat RNA levels in the mutant lines was compared with both B73 line and wild-type segregants with the same pedigree of the mutant lines (wild-type BC4-F3; see Supplemental Figure 2 online). No statistically significant variation was observed between B73 and BC4-F3 wild-type segregants, and similar changes in TE repeats RNA level were observed in nfc101/ nfc102 downregulation lines when statistical variation was estimated using B73 or the BC4-F3 wild types as controls (Figures 6A and 6B). This indicates that the increase of TE repeat RNA levels in MA tissues is in fact associated with nfc101/nfc102 downregulation and that the B73 line can be appropriately used as wild-type reference. It is worth noting that the level of RNA corresponding to TE repeats analyzed in this study is clearly more abundant in MA relative to leaf blade tissues of wild-type plants (see Supplemental Figure 9 online), suggesting that, in MA, the TE silencing is physiologically more leaky. Furthermore, when the polyadenylated RNA fraction was enriched by fractionating total RNA using oligo(dT) cellulose, no changes were detected in MA tissues of nfc101/nfc102 downregulation lines with respect to the wild type (see Supplemental Figure 10 online), indicating that the increased levels observed using random primed cDNA was due to an increase of the nonpolyadenylated fraction. Strand-specific RT-PCR showed that both sense and antisense RNA strands were produced for each TE repeat analyzed and that both strands appeared more abundant in the nfc101/nfc102 suppression line R102 (Figure 6D). To validate this observation, the strand-specific variation was quantified using qRT-PCR for all TE repeats, with the exception of Prem2/ Ji because, as previously reported (Hale et al., 2009), multiple antisense transcripts were detected, yielding their quantification by real-time PCR unreliable. The results confirmed that both RNA strands were upregulated in the MA of nfc101/nfc102 downregulation line relative to the wild type (Figure 6E). ChIP assays showed that the repeats of all four TEs analyzed were bound by NFC101/NFC102 proteins and Rpd3-type HDACs and that nfc101/nfc102 downregulation substantially abolished these interactions in both MA and leaf blade tissues (Figure 7A). Analysis of histone modifications indicated that the release of NFC101/NFC102 binding correlated with an accumulation of H3ac and H3K4me2, which are characteristic marks for transcriptionally active chromatin states (Lauria and Rossi, 2011), and with a reduction of the H3K9me2 heterochromatin mark (Figure 7B). Together, these results provide evidence that nfc101/nfc102 genes repress production of sense and antisense strands of nonpolyadenylated RNA, corresponding to repetitive sequences of high-copy-number TEs. This effect is related to direct NFC101/NFC102 binding and Rpd3-type HDAC recruitment at the target TE sequences, thus promoting histone modifications linked to a repressive chromatin environment. However, although NFC101/NFC102 binding occurs in both MA and leaf blade tissues, accumulation of TE nonpolyadenylated RNA is evident only in MA, suggesting tissue-specific mechanisms for the nfc101/nfc102-mediated regulation of TEs transcription. DISCUSSION In this study, we generated transgenic lines with simultaneous downregulation of nfc101 and nfc102 and used them for characterizing the biological function of these genes. Our results Figure 5. (continued). (A) Diagram of ZCN8 gene structure and the three RNAs produced by this gene. Black boxes and lines represent exons and introns, respectively; position of the forward (for) and reverse (rev) primers used for strand-specific reverse transcription and regions analyzed in ChIP assays (59-end, internal [int], and 39-end) are indicated. (B) and (C) Real-time qRT-PCR quantification of ZCN8 transcripts performed with oligo(dT)-primed cDNA (B) or cDNA from MA or leaf blade (LB) synthesized using strand- and locus-specific primers (C). Bar diagrams are the mean value of transcript amount normalized to gapc2 sense mRNA from two biological repetitions, with three replicates for each biological repetition. Data are expressed as fold changes (FC) relative to the wild type (wt). Standard errors are reported. Asterisk indicates statistically significant change (P # 0.05). (D) to (F) Bar diagrams represent qPCR results from ChIP assays performed on ZCN8 using the indicated antibodies and materials; data are reported as percentage of chromatin input and are average values from two independent ChIP assays and three PCR replicates for each assay. Standard errors are indicated. Horizontal black line shows the background signal, measured by omitting antibody during ChIP procedure. Asterisk indicates statistically significant change (P # 0.05). In (F), H3K27me3 data are plotted to compare its level in ZCN8 59-end region between leaf blade and MA tissues of wildtype plants. nfc101/nfc102 Characterization 9 of 17 Figure 6. TE Repeat Transcripts in Wild-Type and nfc101/nfc102 Downregulation Lines. Total RNA was extracted from MA ([A], [C], and [D]) or leaf blade (LB) (B) tissues of V3/V4 stage seedlings for the indicated genotypes. cDNA synthesis was performed using random hexamers ([A] and [B]) and sequence- and strand-specific primers ([C] and [D]). (A), (B), and (D) Bar diagrams represent the mean value of mRNA level normalized to gapc2 mRNA, from two biological repetitions and three real-time qRT-PCR replicates for each biological repetition. Standard errors are reported. Relative expression to the wild-type (wt) B73 line is presented. Asterisk 10 of 17 The Plant Cell Figure 7. Binding of NFC101/NFC102 and Rpd3-Type HDACs and Histone Modifications at TEs in MA Tissues of Wild-Type and nfc101/nfc102 Downregulation Lines. Bar diagrams represent real-time PCR quantification of ChIP DNA performed with the indicated antibodies and chromatin extracted from MA tissues of the wild type (wt) and nfc101/nfc102 suppression line (R102) V3/V4 stage seedlings. Data are reported as percentage of the chromatin input and are average values from two independent ChIP assays and from three PCR repetitions for each ChIP assay. Standard errors are indicated. Horizontal black line shows the background signal, measured by omitting antibody during ChIP procedure. Asterisk indicates statistically significant change (P # 0.05). indicate that NFC101/NFC102 proteins regulate expression of maize flowering pathway genes and affect production of TE repeat nonpolyadenylated RNA in a tissue-specific manner. In all their direct targets, NFC101/NFC102 proteins promote formation of histone modification patterns linked to repressive chromatin. This activity is related to Rpd3-type HDAC recruitment at id1 and TE repeats and to HDAC-independent mechanisms for ZCN8 regulation. In addition, various developmental defects are observed in nfc101/nfc102 downregulation lines. Therefore, these findings provide evidence that nfc101/nfc102 regulate multiple pathways during plant development, acting through distinct chromatin-related mechanisms. nfc101/nfc102 and the Maize Flowering Pathway Results from the analysis of id1 and ZCN8 in nfc101/nfc102 mutants allow us to formulate a molecular model that integrates the role of nfc101/nfc102 and chromatin modifications into the maize flowering pathway (Figure 8). The id1 gene is expressed in the immature leaf (partially corresponding to MA tissues used in Figure 6. (continued). indicates statistically significant change (P # 0.05). In (A) and (B), both B73 line (wt B73) and wild-type segregants, with the same pedigree of nfc101/ nfc102 knockdown lines (wt BC4-F3; see Supplemental Figure 2 online), were used as wild-type controls. Similar results were obtained when RNA variation was estimated using wild-type BC4-F3 as the wild-type control. In other panels the letters “wt” refers to B73 line. (C) Representative pictures of ethidium bromide–stained agarose gels from RT-PCRs. Omission of reverse transcriptase (RT) was used as a negative control. nfc101/nfc102 Characterization this study) in response to endogenous signals, and it indirectly activates expression of the florigen-encoding ZCN8 gene in the photosynthetically competent parts of leaf blade (the leaf blade tissues used in this study; Lazakis et al., 2011; Meng et al., 2011). ZCN8 protein then presumably migrates toward the shoot apex to interact with DLF1 to activate expression of floral identity genes, such as FLCP128/ZMM4 and FLCP109/ ZMM15, which initiate inflorescence development (Meng et al., 2011). We provide evidence that NFC101/NFC102 proteins directly bind id1 and ZCN8 and prevent accumulation of histone modifications linked to active chromatin to repress their expression. The NFC101/NFC102 binding mediates Rpd3-type HDAC recruitment at id1 59-end genomic region in both MA and leaf blade tissues. However, its abolition induces id1 overexpression and accumulation of H3ac and H3K4me2, which are linked to active chromatin, only in MA tissues. This indicates that NFC101/NFC102/Rpd3 proteins are dispensable for the id1 silencing observed in leaf blade tissues. Conversely, in MA tissues, the NFC101/NFC102/Rpd3-mediated repression of id1 might be required for maintaining ID1 protein concentration within a threshold level. In agreement with this scenario, the importance of transcription factor concentration for an optimized regulation of its targets has been clearly demonstrated in yeast (Kim and O’Shea, 2008). The NFC101/NFC102 proteins also repress ZCN8 transcription through HDAC-independent mechanisms. Our results suggest that this repression can be related to both direct and indirect nfc101/nfc102 activity, due to NFC101/NFC102 proteins binding at ZCN8 and to nfc101/nfc102-mediated id1 repression, respectively. Both mechanisms may operate in MA tissues, where nfc101/nfc102 downregulation correlates with ZCN8 sense mRNA overexpression and accumulation of H3ac and H3K4me2 in the ZCN8 chromatin. The NFC101/NFC102 proteins may stimulate the recruitment of histone modifiers distinct from HDACs. Alternatively, previous studies (Lazakis et al., 2011; Meng et al., 2011) have proposed that ID1 may promote formation of a transcriptionally competent chromatin state at ZCN8 in immature leaf (MA tissues), which is then maintained throughout leaf development to allow subsequent production of ZCN8 spliced mRNA in mature leaf (leaf blade tissues). Our study provides additional information that illuminates possible mechanisms by which NFC101/NFC102 direct ZCN8 repression. We showed that ZCN8 RNAs are constituted by a mixture of sense and antisense transcripts and that, in nfc101/ nfc102 and id1 mutants, the production of sense strand is negatively correlated with antisense strand synthesis. This suggests transcriptional or promoter interference via physical interactions between transcribing polymerases (Hongay et al., 2006) or mechanisms where the antisense RNA molecule itself can negatively influence transcription (e.g., by promoting recruitment of chromatin-modifying enzymes; De Lucia and Dean, 2011). Since NFC101/NFC102 proteins bind ZCN8 in the 39-end genomic region and because the ZCN8 antisense RNA level is reduced in nfc101/nfc102 knockdown lines, NFC101/NFC102 may somehow promote antisense RNA production, thus interfering with synthesis of the sense strand. In addition, we found that H3K27me3 is detected in the ZCN8 chromatin only in leaf blade 11 of 17 tissues and that its level is negatively regulated by nfc101/ nfc102. Interestingly, the ZCN8 sense mRNA in its spliced form is also detectable only in leaf blade tissues, very likely due to leaf blade–specific activation of the sense pre-mRNA splicing (Danilevskaya et al., 2008a). Since histone marks can be signals for spliceosomal components (Schwartz and Ast, 2010), the leaf blade–specific accumulation of H3K27me3 at ZCN8, which is prevented by nfc101/nfc102, may be related to ZCN8 premRNA processing. Nevertheless, it is worth mentioning that the nfc101/nfc102 control of H3K27me3 level may be linked to different mechanisms than splicing because this mark is also associated with Polycomb-mediated silencing and transcription initiation (Pal et al., 2011; Zheng and Chen, 2011). In any case, it is of interest to note that nfc101/nfc102 and their Arabidopsis counterpart FVE exhibit an opposite correlation with respect to regulation of the H3K27me3 level at florigenic genes because FVE promotes H3K27me3 accumulation at FT (Pazhouhandeh et al., 2011). Therefore, FVE- and nfc101/nfc102-mediated regulation of florigen expression involves different mechanisms, and these differences may be related to a peculiar feature of maize ZCN8 regulation at the RNA processing level. A further difference between FVE and nfc101/nfc102 is that FVE is directly involved in the repression of the floral transition regulator FLC, while nfc101/nfc102 indirectly activate the MADS box/FLC-like FLCP128/ZMM4 and FLCP109/ZMM15 floral identity genes, acting downstream of the flowering pathway. It is worth noting that the positive correlation between the expression of these genes and nfc101/nfc102 is an unexpected result because they are activated by id1/ZCN8 (Meng et al., 2011), which are, in turn, negatively regulated by nfc101/nfc102. This finding implies that additional and still unknown networks, involving nfc101/nfc102 and independent of id1/ZCN8 function, participate in the activation of FLCP128/ZMM4 and FLCP109/ ZMM15. In the nfc101/nfc102 downregulation lines, the effect of this network appears to be dominant with respect of the id1/ ZCN8 network. This may occur because FLCP128/ZMM4 and FLCP109/ZMM15 activation requires formation of the DLF1/ ZCN8 protein dimer (Muszynski et al., 2006), and only ZCN8 is upregulated in the nfc101/nfc102 suppression lines, while dlf1 expression is not affected, thus failing in achieving an appropriate stoichiometric ratio between the two dimer components. nfc101/nfc102 Activity in TE Transcription We reported that nonpolyadenylated RNA corresponding to repeats of representative maize TEs was upregulated in MA tissues of nfc101/nfc102 downregulation lines, concomitantly with the abolition of NFC101/NFC102 and Rpd3-type HDACs binding. In addition, TE repeats were enriched for H3ac and H3K4me2 and depleted of the H3K9me2 heterochromatin mark. These findings suggest that, similar to Arabidopsis FVE/MSI5/ HDAC proteins (Bäurle and Dean, 2008; Gu et al., 2011), the NFC101/NFC102/Rpd3 proteins are part of complexes that act as effectors of the RdDM pathway to form silenced chromatin at targeted TEs. Nevertheless, the level of RNA Polymerase II (Pol II)–derived polyadenylated RNA, which is the form required for TE activation, was unaffected in nfc101/nfc102 knockdown lines. This observation, along with the finding that both strands 12 of 17 The Plant Cell Figure 8. Molecular Model for nfc101/nfc102 Activity in the Maize Flowering Pathway. Schematic representation of nfc101/nfc102 involvement in flowering pathway. See the text for a detailed description of the model. For simplicity, only complexes containing NFC102 protein are depicted in the image; however, by considering the high sequence degree of conservation between NFC101 and NFC102 (see Supplemental Figure 1 online), the two proteins are likely interchangeable for their participation in the NFC101/NFC102 complexes. The gray area in the image represents the immature region of leaves that are hidden in the whorl, where id1 is expressed. This region is partially contained within the MA tissues described in this study, although MA tissues also includes shoot apex. The green area in the image is the photosynthetically competent parts of the leaf blade. ZCN8 spliced sense mRNA is specifically expressed in phloem of the vascular bundles of the leaf blade (red lines over the image), while its protein moves to shoot apex (blue line and blue arrow) to activate floral identity genes. R represents unknown factors required for id1 repression in the leaf blade; A represents unknown activators, the activity of which may be countered by NFC102/Rpd3 to maintain id1 expression in MA within a specific threshold level. Dotted lines indicate processes predicted from results of our and other studies, but for which the mechanism still must be clarified. The description of chromatin structure and the size of factors depicted in the image are schematic and do not accurately portray these structures. of TE repeat nonpolyadenylated RNA were upregulated in nfc101/nfc102 mutants, suggest that the nonpolyadenylated RNAs detected in our samples are primarily double-stranded RNAs, derived by RNA-dependent RNA polymerases (RDRs), which are other components of the RdDM pathway (Matzke et al., 2009). Although many of the Arabidopsis RdDM pathway components are conserved in maize, a previous work reported that production of polyadenylated RNA of the same TE repeats analyzed in our study was only affected in the absence of the RNA Polymerase IV (Pol IV) largest subunit (Hale et al., 2009). The authors suggested that, in maize, the competition of Pol IV with Pol II is the major factor responsible for TE transcriptional repression. In this context, the transcriptionally more permissive chromatin environment established in nfc101/nfc102 mutants may stimulate production of Pol IV–derived aberrant RNAs, which are subsequently processed by RDRs, without affecting the production of Pol II–derived polyadenylated TE transcripts. Therefore, our findings corroborate the hypothesis that, in presence of a functional Pol IV, the alteration of other RdDM pathway components, including NFC101/NFC102/HDAC proteins, seems to be not sufficient for silencing of certain maize TEs (Hale et al., 2009; Hollick, 2012). Our results show that upregulation of TE repeats nonpolyadenylated RNA is evident in MA but not in leaf blade tissues of nfc101/nfc102 downregulation lines. Li et al. (2010) reported that maize Mu killer-mediated silencing of active Mu element is reduced during the transition from vegetative to reproductive phase due to a transient loss of expression of a trans-acting nfc101/nfc102 Characterization small-interfering RNA (siRNA) pathway regulator. The authors suggested that this phase change might represent an opportunity for the genome to unmask potentially dangerous TEs, leading to production of siRNAs derived from TE transcripts in tissue adjacent to one that produces germ cells. Since siRNAs have been shown to move between tissues (Dunoyer et al., 2010; Molnar et al., 2010), they can migrate from vegetative tissues toward tissues that produce germ cells to enhance the TE silencing (Li et al., 2010; Lisch, 2012). The MA tissues used in our study were sampled immediately before the transition to reproductive phase and young leaf primordia surrounding the SAM are the major component of MA samples. In addition, it is known that for TE repeats, nonpolyadenylated and RDR-derived RNA are required for siRNA formation (Matzke et al., 2009). Therefore, in agreement with the hypothesis suggested by Li et al. (2010), the production of TE repeat nonpolyadenylated RNA may be higher and more sensitive to nfc101/nfc102 downregulation in MA compared with leaf blade tissues because, in MA, the leaf primordia would have the aim of protecting from TEs activation the adjacent reproductive region. nfc101/nfc102 Genes Have Multiple Functions during Plant Development Despite the nfc101/nfc102 involvement in regulating key players of the maize flowering pathway, phenotypic analysis did not show alteration of floral transition timing in nfc101/nfc102 downregulation lines. This result can be explained because the complete abolition of nfc101/nfc102 expression does not occur in knockdown lines, and it may be required to achieve an evident effect on floral transition. In this respect, is worth noting that maize transgenic lines with constitutive id1 overexpression exhibit only a minor alteration of flowering by showing a moderate early floral transition, even though a delay of tassel shed and ear silking were observed (Muszynski et al., 2000). In addition, the phenotypic analysis of the nfc101/nfc102 downregulation lines suggests that nfc101/nfc102 can simultaneously affect different pathways, even in an opposite manner within the same pathway (e.g., id1/ZCN8 repression and FLCP128/ZMM4 and FLCP109/ZMM15 activation). The result is a pleiotropic phenotypic effect that might mask a slight flowering phenotype. The involvement of nfc101/nfc102 in regulating different pathways, including TE transcription, G1/S cell cycle transition, and DNA repair, is supported by several lines of evidence from present and past studies (Rossi et al., 2003; Varotto et al., 2003; Casati et al., 2008; Campi et al., 2012). It is worth noting that all plant and mammalian MSI family members characterized to date are involved in multiple regulatory pathways, with the common theme that they are usually associated with chromatin modification and remodeling (Hennig et al., 2005; Suganuma et al., 2008). Altogether, these observations suggest that NFC101/NFC102 are components of distinct chromatin modifying complexes (i.e., HDAC dependent or independent), operating in different biological processes, and that the major role of these WDrepeat proteins is to facilitate and stabilize interactions between other complex components by means of their peculiar b-propeller architecture. 13 of 17 METHODS nfc101/nfc102 Downregulation Lines and Plant Materials A total of 30 independent AS T0 transformants were obtained by transforming maize (Zea mays) protoplasts with a plasmid that allow nfc102 antisense RNA overexpression, using the method previously described (Rossi et al., 2007). Details for plasmid preparation are reported in Supplemental Methods 1 online. The R102 RNA interference mutant line was obtained from the Plant Chromatin Database initiative (http:// www.chromdb.org/; McGinnis et al., 2007; T-MCG3480.04 locus; Maize Stock Center number 3201-07). Regenerated T0 plants were introgressed into the B73 recurrent parent. The crossing scheme adopted to achieve homozygosity for the transgene and wild-type segregant plants used for molecular and phenotypic analysis is described in Supplemental Figure 2 online. The presence of the transgene was validated for bialophos (BASTA) herbicide resistance and with PCR detection using the primer combinations reported by McGinnis et al. (2007) for the R102 line and the PUbi1 (59-TCGACGAGTCTAACGGACACC-39) plus RbAp35-3 (59-GTTGCCGATAACTCTGTCC-39) primers for the AS lines. Changes in RNA level of nfc101, nfc102, and nfc103 was assessed using real-time qRT-PCR with the following primer combinations: nfc101, NFC-1 (59GAGAGCACTGGTGGAGGTGG-39) plus NFC101-1 (59-GCCTACAGATTAGCCATCCAGC-39); nfc102, NFC-1 plus NFC102-1 (59-AGCTGGCCCACAGACATCCG-39); nfc103, NFC-2 (59-CGTCGCTGAAGACAACATCC39) plus NFC103-1 (59-CTACAGCCAACTCTCACTCG-39). RNA, DNA, and chromatin used for molecular analyses were extracted from a minimum of 10 seedlings harvested at the V3/V4 stage (full extension of the third/fourth leaf collar). For temperate B73 line, this stage is before the floral transition, which usually occurs at V5 stage (Meng et al., 2011; Figure 2F). Seeds were germinated directly in soil and seedlings grown in the phytotron with 16 h light at 27°C and 8 h dark at 22°C. Seedlings were manually dissected to obtain the MA and leaf blade tissues as illustrated in Figure 1A. The id1-m1 mutant allele used was backcrossed 10 times into the B73 background, and homozygous seedlings were genotyped as previously described (Lazakis et al., 2011). The ZCN8 transcript analysis in id1 mutant was performed by preparing total RNA from leaf blade of the seventh leaf of V5/V6 stage seedlings as described by Lazakis et al. (2011). Phenotypic and Morphological Analysis Phenotypic analysis was performed on greenhouse-grown plants in two different experiments. Homozygous transgenic and wild-type segregant plants from the BC4-F3 generation and the B73 inbred line were used (see Supplemental Figure 2 online). A minimum of 60 plants for each genotype and for each experiment were monitored daily for germination, plant traits, and onset of flowering. The days to flowering were calculated as growing degree days (GDDs): GDD = [(Tmax + Tmin)/2] – 10, measured from coleoptile emergence to onset of pollen shed and silk appearance. The average value of daily GDD accumulations, under growing conditions adopted in this study, was 16.5. Adult plant and V2stage seedlings traits were measured as previously reported (Rossi et al., 2007). Statistical analysis was performed by applying Student’s t tests. For morphological analysis of the meristem transition, SAMs from wildtype and nfc101/nfc102 knockdown lines were isolated and fixed for 14 h at 4°C in 10 volumes of freshly prepared FAA solution (50% ethanol, 10% acetic acid, and 5% formalin). Fixed tissue was dehydrated at 4°C in a graded series of ethanol (1 h each [v/v] 70, 85, 95, 100, and 100%) and embedded in (hydroxyl-ethyl) methacrylate Tecnovit 7100, according to the protocol of the manufacturer (Heraus-Kulzer Histo-Tec.). Technovit blocks were glued onto a mold and placed in a microtome. Sections (15 µm) were stained for 1 min with a solution consisting of 1% toluidine 14 of 17 The Plant Cell blue in distilled water and analyzed under bright-field illumination. Images were captured on an Axiocam MRc5 camera (Zeiss) using the Axiovision program (version 4.1). cDNA Synthesis Total RNA was extracted from all tissues with standard Trizol (Life Technologies) purification, and RNA quality and concentration were evaluated with a spectrophotometer. When indicated, the polyadenylated RNA fraction was purified from the total RNA using the Oligotex mRNA kit (Qiagen). Before cDNA synthesis, total RNA was treated with DNA TURBO DNA-free DNase (Ambion) to remove residual genomic DNA. First-strand cDNA synthesis was performed using 2 µg of DNase-treated RNA and Superscript III reverse transcriptase (Life Technologies), following the manufacturer’s instructions. For oligo(dT)-primed cDNA, reaction was performed in the presence of 500 ng of oligo(dT)17 in a final volume of 50 µL. For random-primed cDNA, 250 pmol of random hexamers (SigmaAldrich) were added in a 20-mL reaction. These reactions were incubated 1 h at 50°C. For strand-specific cDNA synthesis, reaction was performed by adding 2 pmol of the selected TE/locus-specific primer in the proper orientation, in a final volume of 20 mL and with an incubation of 45 min at 55°C. Reverse primers for the synthesis of glyceraldehyde-3-phosphate dehydrogenase2 (gapc2) and EF-1a cDNA produced by the sense RNA strand were also added in all strand-specific reactions to allow qRT-PCR data normalization. gapc2 and EF-1a can be used for normalization because their expression is unaffected in nfc101/nfc102 mutants (see Supplemental Figure 4 online). Sequences of TE/locus-specific primers used for strand-specific reverse transcription are reported in Supplemental Table 2 online. As negative controls, parallel reactions were performed by omitting addition of reverse transcriptase and, for the strand-specific reverse transcription, by adding the enzyme but by omitting the TE/locusspecific primer. RT-PCR The different cDNAs produced as described above were used as templates in RT-PCR analysis. The sequences of the primers and references for the amplified sequences are reported in Supplemental Table 1 online. The sequences of ZCN8 primers used for the PCR experiments illustrated in Supplemental Figure 6 online (ZCN8-1, ZCN8-2, ZCN8-3, and ZCN8-4) are reported in Supplemental Table 1 online. PCR conditions for amplification with ZCN8-1 plus ZCN8-2, located at the first and last exon, were as previously described by Danilevskaya et al. (2008a). Similar conditions were used also for all the other primer combinations, with the exception of the primer annealing temperature, which was 60°C. For strand-specific RT-PCR (ZCN8, TEs, and control genes primer combinations) the conditions were 96°C for 5 min, followed by an amplification cycle of 30 s at 94°C, 15 s at 60°C, and 45 s at 72°C. This cycle was repeated for a different number of times, for each primer combination and sample type, in order to obtain nonsaturating amplification conditions. The specificity of each primer combination was verified by cloning and sequencing the amplified fragments obtained from cDNA and/or genomic DNA as template. For locus-specific primer combinations (e.g., to distinguish between nfc101/nfc102, ZCN8/ZCN7, etc.) at least eight clones were sequenced, and the specificity was verified by checking for the presence in all eight clones of single nucleotide polymorphisms and small insertions/deletions characteristic of a given locus. Real-time qRT-PCR was performed as previously described (Rossi et al., 2007) using SYBR Green I (Sigma-Aldrich) and a real-time iCyclerIQ (Bio-Rad). Threshold cycles (TCs) were obtained for each sample. For each tissue/genotype, two cDNA preparations, from two distinct biological samples, were made and three replicates of qRT-PCR were performed for each cDNA preparation (a total of six replicates). To account for possible differences in cDNA synthesis and amplification efficiency, the data were normalized to the transcript amount of gapc2 in the samples analyzed. Similar results were obtained when the data were normalized to the transcript amount of EF-1a. Data were expressed as fold change in the downregulation line relative to the wild type, and their statistical significance (P # 0.05) was calculated using analysis of variance (Rossi et al., 2007). Specifically, each TC value (usually six values; see above) normalized to the TC mean value of gapc2 (or EF-1a) was used for analysis of variance, assuming biological sample replications as random effects and treatment effects (that is, effects due to genetic strains) considered as fixed effects. ChIP Assays MA and leaf blade samples used for chromatin extraction were obtained by manual dissection of V3/V4 seedlings, cut with a razor blade and immediately stored in MC buffer (10 mM NaPi, pH 7, 50 mM NaCl, and 0.1 M Suc) at 4°C for not more than 5 h before cross-linking. Usually, 1.5 g of sample was stored in a 50-mL Falcon tube containing 35 mL of MC buffer. To improve fixation of DNA-protein binding for proteins, such as NFC101/NFC102 and Rpd3, which do not bind DNA directly, a two-step fixation technique was adopted (Nowak et al., 2005). This procedure relies on a first step to cross-link the protein of interest to protein complexes and a second step, with formaldehyde treatment, to cross-link the complexes to DNA. Disuccinimidyl glutarate (Sigma-Aldrich) was freshly prepared at 0.5 M in DMSO immediately prior to use and was added to each 50-mL Falcon tube, containing samples in MC buffer, to 2 mM final concentration. Fixation was performed for 30 min with vacuum infiltration at room temperature, followed by three sequential washes with 30 mL of MC buffer at room temperature and with gentle rotation. After the final washing step, formaldehyde was added to 30 mL of fresh MC buffer at 1% final concentration, and fixation was done with 10 min of vacuum infiltration at room temperature. Fixation was stopped by adding Gly at 0.17 M final concentration, followed by 5 min of vacuum infiltration at room temperature. Fixed samples were washed three times with water at 4°C, dried with towels, frozen, and stored at 280°C. Chromatin was extracted, sonicated, and quantified as previously described (Locatelli et al., 2009), and the efficiency of the fixation procedure was monitored using phenol: chloroform extraction of chromatin from fixed and nonfixed samples as described by Haring et al. (2007). Usually, 12 and 5.5 µg of chromatin are obtained from 1 g of MA and leaf blade tissues, respectively. The two-step cross-linking method was also available to study histone modifications, since identical results were obtained when chromatin from the two-step or single-step fixation procedure (Locatelli et al., 2009) was used to analyze histone modifications. Immunoprecipitation was performed using 10 µg of chromatin, following the protocol described by Locatelli et al. (2009). Typically, the following amount of the different antibodies was used for immunoprecipitation: 10 mL of affinity-purified anti-MSI4 (Kenzior and Folk, 1998; Rossi et al., 2001); 18 mL of anti-ZmRpd3I (this antibody is expected to recognize members of class 1 of maize Rpd3-type HDACs, specifically hda101, hda102, and hda108 proteins; see Rossi et al., 2003; Varotto et al., 2003); 5 µg of a-H3ac (Millipore; 06-599); 8 µg of a-H3K4me2 (Millipore; 07-030); 10 µg of a-H3K27me3 (Millipore; 07-449); and 10 µg of a-H3K9me2 (Millipore; 07-441). A no-antibody negative control was performed with no antibody added during incubation. One microliter of ChIP DNAs and a 1:50 dilution of inputs were used for qPCR analysis. Two independent ChIP experiments were performed for each antibody and each plant material, and three repetitions of qPCR analysis were performed for each ChIP assay and for each sequence analyzed (a total of six qPCR replicates). Real-time qPCR was performed as described by Rossi et al. (2007), and the TC mean value and standard error were obtained for the six qPCR replicates. Data from ChIP assays were subjected to analysis nfc101/nfc102 Characterization 15 of 17 of variance to estimate statistically significant differences (P # 0.05). Specifically, the TC values from the three replicates for each of the two independent ChIP assays were used for calculating an F test in an analysis of variance, which was performed assuming biological sample replications as random effects and treatment effects (data from different genotypes) considered as fixed effects. The sequences of primers used for ChIP assays are reported in Supplemental Table 1 online. (Consiglio Nazionale delle Richerche) for the Epigenomics Flagship Project. I.M. has a PhD fellowship funded by the Research Agricultural Council (Consiglio per la Ricerca e la Sperimentazione in Agricoltura) and in agreement with the University of Padova, Italy. Research in V.R.’s lab is supported by institutional grants from Italian Ministry of Agriculture, Food, and Forestry Policies. Accession Numbers AUTHOR CONTRIBUTIONS Sequence data from this article can be found in the GenBank/EMBL databases under the following accession numbers:502087 (nfc101), 501934 (nfc102), and 542545 (nfc103). Gene ID numbers for all other sequences described in this article can be found in Supplemental Table 1 online. V.R. conceived and designed research. I.M., R.B., D.M., A.A., M.L., and V.R. performed research. I.M., R.B., M.L., and V.R. analyzed data. V.R. wrote the article. Supplemental Data Received November 6, 2012; revised January 18, 2013; accepted January 24, 2013; published February 19, 2013. The following materials are available in the online version of this article. Supplemental Figure 1. Alignment of Maize NFC101/NFC102 and Arabidopsis FVE/MSI5 Proteins. Supplemental Figure 2. Crossing Scheme for nfc101/nfc102 Downregulation Lines. Supplemental Figure 3. DNA Gel Blot Analysis of nfc101/nfc102 Downregulation Lines. Supplemental Figure 4. FLCP and Control Genes with Unaffected Expression in nfc101/nfc102 Downregulation Lines. Supplemental Figure 5. Validation That the Anti-MSI4 Antibody Specifically Recognizes NFC101/NFC102 in Maize Protein Extracts. Supplemental Figure 6. Analysis of ZCN8 Transcripts in Wild-Type and id1 Mutant Plants. Supplemental Figure 7. Nucleotide Sequence of the cDNA Produced by the ZCN8 Antisense RNA Strand. Supplemental Figure 8. RT-PCR Analysis of LTR-Type TE Transcripts Using Oligo(dT)-Primed and Random-Primed cDNAs. Supplemental Figure 9. RT-PCR Analysis of TE Repeat RNA Abundance in MA and Leaf Blade Tissues of Wild-Type Plants. Supplemental Figure 10. Analysis of TE Repeats Polyadenylated RNA Fraction. Supplemental Table 1. List of Sequences Analyzed and Primers. Supplemental Table 2. List of Primers Used for Strand-Specific Reverse Transcription. Supplemental Methods 1. 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The WD40-Repeat Proteins NFC101 and NFC102 Regulate Different Aspects of Maize Development through Chromatin Modification Iride Mascheretti, Raffaella Battaglia, Davide Mainieri, Andrea Altana, Massimiliano Lauria and Vincenzo Rossi Plant Cell; originally published online February 19, 2013; DOI 10.1105/tpc.112.107219 This information is current as of June 18, 2017 Supplemental Data /content/suppl/2013/01/29/tpc.112.107219.DC1.html Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY