Download The WD40-Repeat Proteins NFC101 and NFC102

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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/
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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
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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.
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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
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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
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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
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Figure 5. Analysis of ZCN8 Expression and NFC01/NFC102-Rpd3 Binding and Histone Modifications at ZCN8 in Wild-Type and nfc101/nfc102
Downregulation Lines.
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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
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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
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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
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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
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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.
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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
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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.
ACKNOWLEDGMENTS
We thank Joseph Colasanti, Hans Hartings, Alexandra Lusser, and
Michael Muszynski for critical reading of the manuscript and Guenter
Donn, Sabrina Locatelli, and Vania Michelotti for contribution in generating nfc101/nfc102 mutants and assistance in phenotypic analysis. We
also thank William Folk for providing anti-FVE antibody, Peter Loidl for
the anti-Rpd3 antibody, and Joseph Colasanti for id1 mutant seeds. This
work was principally supported by special grants from the European
Commission (FP7 Project KBBE 2009 226477 - “AENEAS”: Acquired Environmental Epigenetics Advances: from Arabidopsis to maize). Additional funds
were from special grants from the Italian Ministry of Education,
University, and Research and the National Research Council of Italy
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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
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