Cis-Regulatory Elements Determine Germline
Specificity and Expression Level of an
Isopentenyltransferase Gene in Sperm
Cells of Arabidopsis1[OPEN]
Jinghua Zhang, Tong Yuan, Xiaomeng Duan, Xiaoping Wei 2, Tao Shi, Jia Li, Scott D. Russell, and
Xiaoping Gou
Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences,
Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology
and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.)
ORCID IDs: 0000-0002-0542-0704 (J.Z.); 0000-0002-6340-266X (T.S.); 0000-0001-8302-4160 (S.D.R.); 0000-0002-8391-0258 (X.G.).
Flowering plant sperm cells transcribe a divergent and complex complement of genes. To examine promoter function, we chose
an isopentenyltransferase gene known as PzIPT1. This gene is highly selectively transcribed in one sperm cell morphotype of
Plumbago zeylanica, which preferentially fuses with the central cell during fertilization and is thus a founding cell of the primary
endosperm. In transgenic Arabidopsis (Arabidopsis thaliana), PzIPT1 promoter displays activity in both sperm cells and upon
progressive promoter truncation from the 59-end results in a progressive decrease in reporter production, consistent with
occurrence of multiple enhancer sites. Cytokinin-dependent protein binding motifs are identified in the promoter sequence,
which respond with stimulation by cytokinin. Expression of PzIPT1 promoter in sperm cells confers specificity independently of
previously reported Germline Restrictive Silencer Factor binding sequence. Instead, a cis-acting regulatory region consisting of
two duplicated 6-bp Male Gamete Selective Activation (MGSA) motifs occurs near the site of transcription initiation. Disruption
of this sequence-specific site inactivates expression of a GFP reporter gene in sperm cells. Multiple copies of the MGSA motif
fused with the minimal CaMV35S promoter elements confer reporter gene expression in sperm cells. Similar duplicated MGSA
motifs are also identified from promoter sequences of sperm cell-expressed genes in Arabidopsis, suggesting selective activation
is possibly a common mechanism for regulation of gene expression in sperm cells of flowering plants.
In angiosperms, the meiotic division of microsporocytes produces microspores that establish the male
germ lineage through asymmetric mitotic division of
the microspore, which forms as its products a large
vegetative cell and a small generative cell that is the
founder cell of the male germ lineage (Boavida et al.,
2005; Ma, 2005; Borg et al., 2009). In bicellular pollen,
1
This work was supported by National Natural Science Foundation
of China grants 31471402 and 31270229 (to X.G.); start-up fund from
Lanzhou University (to X.G.); Fundamental Research Funds for the Central Universities grant lzujbky-2014-251 (to J.Z.); State Administration of
Foreign Expert Affairs grant MS2010LZDX077 (to J.L.); and US National
Science Foundation (IOS 1128145) and University of Oklahoma (to S.R.).
2
Present address: Monsanto Company, 700 Chesterfield Parkway
West, Chesterfield, MO 63017.
* Address correspondence to Xiaoping Gou ([email protected])
and Scott D. Russell ([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.plantphysiol.org) is: Xiaoping
Gou ([email protected]).
X.G., J.L., and S.D.R. conceived and designed the experiments;
X.G., J.Z., T.Y., X.D., and X.W. performed the experiments; X.G.,
J.Z., and T.S. analyzed the data; and X.G. and S.D.R. wrote the paper.
[OPEN]
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1524
the generative cell divides to form two sperm cells
within the germinated elongating pollen tube, whereas
in tricellular pollen such as Arabidopsis (Arabidopsis
thaliana) and Plumbago zeylanica, the two sperm cells are
produced precociously, prior to anthesis. Regardless of
cellular condition at the time of pollination, successful
transit of the cells of the male germ lineage in the
elongating pollen tube will transfer two sperm cells into
the embryo sac. One sperm cell fuses with the egg cell to
form the zygote, establishing the next generation of
plants, and the other sperm cell fuses with the central
cell, establishing the endosperm, which provides nutrition during embryo development. In P. zeylanica, the
two sperm cells display cytoplasmic dimorphism in
which sperm cells display differential abundance of
heritable organelles and follow different fusion fates
during double fertilization. The sperm cell associated
with the vegetative nucleus (Svn) contains the majority
of mitochondria and rare plastids and preferentially
fuses with the central cell, whereas the sperm cell unassociated with the vegetative nucleus (Sua) contains
abundant plastids and few mitochondria and preferentially fuses with the egg cell (Russell, 1984, 1985).
That P. zeylanica undergoes preferential fertilization
makes this plant uniquely suitable for studying the
regulation of gene expression in paired sperm cells and
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Regulation of Gene Expression in Plant Male Gamete
examining cell-to-cell recognition during double fertilization. Correspondingly, promoters unique to each
sperm type appear to be activated in order to achieve
this uniquely distinct pattern of gene expression in the
Sua and Svn, corresponding to their unique fates (Gou
et al., 2009).
Some male germline-expressed transcripts have been
characterized that are vital for sperm cell function,
fertilization, and embryo development (Bayer et al.,
2009; Ron et al., 2010; Stoeckius et al., 2014), suggesting
that sperm cell-expressed genes may possess a distinct
role in early stages of postfertilization development.
Several promoters have been isolated and studied in
flowering plants in the context of male germlinespecific gene expression. LILY GENERATIVE CELLSPECIFIC 1 (LGC1), isolated from a lily generative cell
cDNA library, encodes a plasma membrane-localized
protein that is probably involved in cell-to-cell recognition during fertilization (Xu et al., 1999). The expression of LGC1 is exclusively restricted in male gamete
cells (Xu et al., 1999). Another lily gene, GCS1, and its
Arabidopsis homolog HAP2 are expressed in sperm
cells and are essential for fertilization (Mori et al., 2006;
von Besser et al., 2006). Sperm cell-expressed genes
GAMETE EXPRESSED 1 (GEX1) and GEX2 were
identified in maize sperm cell-specific transcripts, and
homologous Arabidopsis genes were designated
AtGEX1 and AtGEX2 (Engel et al., 2005). AtGEX1 is
expressed in sperm cells of mature pollen in Arabidopsis. AtGEX2 was observed in generative cells and
sperm cells, but not in any other tissues. The rice
homolog of AtGEX2, OsGEX2, also confers sperm
cell expression (Cook and Thilmony, 2012). DUO
POLLEN 1 (DUO1) encodes a MYB transcription
factor that is expressed specifically in generative cell
and sperm cells and serves a key regulatory function
for generative cell division and sperm cell differentiation in Arabidopsis (Rotman et al., 2005;
Brownfield et al., 2009a; Borg et al., 2011), whereas
DUO3 functions to activate expression of target
germline genes of DUO1 and is required for cell cycle
progression, sperm cell specification, fertilization,
and embryogenesis (Brownfield et al., 2009b). Male
gamete-expressed histone variants have also been
identified from lily (Lilium longiflorum) and Arabidopsis (Ingouff et al., 2007; Okada et al., 2005a,
2005b). In Arabidopsis, AtMGH3/HTR10 encodes a
variant histone H3 detected in the generative cell of
late bicellular pollen and sperm cells of anthesis
pollen. At the genome scale, sperm cell-expressed
genes in Arabidopsis were identified by microarray
analysis using FACS-purified sperm cells (Borges
et al., 2008).
Specific gene expression in a given organ or cell is
achieved by recruiting specific transcription factors to
corresponding cis-regulatory elements (CREs) that are
functional DNA sequences carried by the gene itself. In
efforts to identify CREs controlling gene expression in
the germ lineage, promoter sequences of LGC1 and
DUO1 have already been analyzed. The promoter
sequence of LGC1 was cloned by uneven PCR, and its
specific transcription activity was verified in lily and
tobacco generative cell in transient and stable transformation experiments (Singh et al., 2003). Truncation analysis of LGC1 promoter identified a repressor
binding site that suppresses the expression of LGC1
in sporophytic tissues (Singh et al., 2003). A related
Germline Restrictive Silencing Factor (GRSF) encoding
a 24-kD DNA-binding repressor protein is expressed
ubiquitously in all plant tissues except the male germ
lineage. Chromatin immunoprecipitation assays
demonstrated that GRSF interacts with a specific
regulatory element in the promoter region of LGC1,
which was confirmed by using a synthesized competitor to release somatic cells from the repressive
GRSF. Presence of GRSF binding sequences in other
male gamete-expressed genes suggested widespread
control of male gamete gene expression by this
functionally conserved sequence (Haerizadeh et al.,
2006). DUO1 functions in another way, by directly
binding to MYB sites to activate its target genes
DUO1-ACTIVATED ZINC FINGER1 (DAZ1) and
DAZ2, which encode transacting transcriptional
repressors (Rotman et al., 2005; Brownfield et al.,
2009a; Borg et al., 2011, 2014). A putative GRSF
binding site was predicted in the DUO1 promoter
(Haerizadeh et al., 2006). However, when the predicted GRSF binding site was mutated, the expression specificity of DUO1 in germline was not affected.
Truncated DUO1 promoters, excluding the putative
GRSF site, were sufficient to drive expression of
H2B::GFP in sperm cells (Brownfield et al., 2009a). To
identify putative CREs controlling sperm cell-specific
gene expression in rice, Sharma et al. (2011) performed
in silico analyses of promoter sequence motifs of 40 rice
sperm cell-expressed genes. Although the authors
identified some possible CREs for gene expression in
sperm cells, experimental validation will be needed to
examine the functions of these identified motifs in living plants.
Only a few sperm-expressed promoters have been
investigated in detail, and limited information is
available about the regulation of gene expression in
sperm cells. Efforts to identify more CREs regulating
gene expression in sperm cells are needed to understand more fully how expression in the male germ lineage is controlled. In previous studies, we identified
an isopentenyltransferase gene termed PzIPT1 that
is exclusively expressed in Svn sperm cells of P. zeylanica,
confirmed by quantitative reverse transcriptase
(RT)-PCR and whole-mount in situ hybridization (Gou
et al., 2009). The corresponding promoter sequence
was cloned from P. zeylanica, and its expression in
sperm cells was confirmed with GFP reporter gene in
Arabidopsis (Ge et al., 2011). In this study, we show
that a cis-regulatory region for Male Gamete Selective
Activation (MGSA) determines the expression of PzIPT1
in sperm cells, and its expression strength can be enhanced by cytokinin via a cytokinin-dependent protein
binding (CPB) site.
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Zhang et al.
RESULTS
The PzIPT1 Promoter Confers Sperm Cell-Specific
Expression in Transgenic Arabidopsis Pollen
A 1.1-kb 59-upstream DNA fragment of PzIPT1 was
isolated and fused to GUS and GFP or nuclear localized
YFP reporter genes in order to view the expression
patterns of PzIPT1 promoter in .20 independent
transgenic lines. All of them showed very similar expression patterns. GUS expression could be detected in
whole seedlings just after germination (Fig. 1A) with
strong GUS signals evident in flowers, especially in
ovaries and anthers (Fig. 1B). At higher magnification,
GUS expression could be detected in embryo sacs, with
a strong signal at the micropylar end (Fig. 1C), revealing localization in the egg cell and synergid cells by a
nuclear-localized YFP reporter (Fig. 1E). A strong GFP
signal was detected in paired sperm cells of mature
pollen with negligible GFP expression in the background pollen cytoplasm (Fig. 1D), consistent with a
GUS signal (Supplemental Fig. S1). When gametogenesis of transgenic plants was examined, no detectable
signal of GFP was observed in microspores and bicellular pollen (Supplemental Fig. S2). Our previous study
showed that PzIPT1 is highly up-regulated in the Svn
sperm cell in P. zeylanica, and no obvious signals could
be detected in other investigated organs or cells (Gou
et al., 2009). This difference may reflect the speciesspecific expression of PzIPT1 promoter.
PzIPT1 Expression in Sperm Cells Is Not Regulated by
Transcriptional Repression
To understand how PzIPT1 is activated to express in
sperm cells, its promoter sequence was analyzed to find
CREs by searching the Plant cis-Acting Regulatory DNA
Elements database (Higo et al., 1999). A typical TATA
box is located at -29 from the putative transcription start
site in the PzIPT1 promoter (Fig. 2A). Several abundant
motifs were identified, such as DOF, ARR1, GT1, MYB,
and CPBCSPOR (CPB). Four typical CPB motifs for
cytokinin-dependent protein binding containing the
characteristic TATTAG nucleotide sequence (Fusada
et al., 2005) locate at positions -12 to -17, -89 to -94, -304
to -309, and -433 to -438 (Fig. 2A).
To identify potential CREs controlling sperm cell
expression and potential GRSF-like repressor binding
sites in PzIPT1 promoter, ten 59-deletional PzIPT1 promoters were fused with GFP, transformed, and examined in transgenic Arabidopsis to view their expression
patterns (Fig. 2B). At least twelve T1 transgenic plants
were analyzed for each promoter, and all of them
showed very similar expression patterns. Results from
T3 transgenic lines for each construct are shown in
Figure 2, C and D. The Δ761 promoter showed the same
expression level of GFP as the original 1.1-kb PzIPT1
promoter. The following three deletions (Δ531, Δ368,
Δ255) each showed gradually decreased but still strong
GFP expression in both sperm cells compared with the
Δ761 promoter (Fig. 2, C and D). Expression levels of
GFP decreased in successive deletion constructs of
Δ154, Δ130, and Δ105, although the signals were strong
enough to be detected easily in sperm cells (Fig. 2, C and
D). The signal of GFP in deletion Δ87, however, was
very weak, and it was difficult to observe whether
sperm cells were positive for GFP expression in this
deletion, although expression was conspicuous in
sperm cells of mature pollen harboring the intact promoter sequence (Fig. 2, C and D). Such progressive
depletion in sperm cell-restricted expression of PzIPT1
is inconsistent with a GRSF-like repressive expression
Figure 1. Representative expression patterns of
PzIPT1 promoter in transgenic Arabidopsis.
Bright field microscopy of GUS expression in
3-d-old seedlings (A), flowers (B), and embryo
sac (C). D, Fluorescence microscopy of GFP
expression in two sperm cells. E, Confocal laser
scanning microscopy of nuclear-localized YFP
in synergids and the egg cell. Bars: A, B, 1 mm;
C, 100 mm; D, 10 mm; E, 25 mm.
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Regulation of Gene Expression in Plant Male Gamete
Figure 2. PzIPT1 promoter structure, truncation, and deletion analysis. A, Predicted cis-acting regulatory elements by PLACE are
shown for DOF (▲), ARR1 (●), GT1 (◊), MYB1 (oval), and CPB (orange oval). Black thick lines represent PzIPT1 promoter.
Numbers indicate positions from putative transcription start site. Sequence between -1 and -105 is shown, with two identified
MGSA motifs, two predicted CPB sites in orange, and a TATA box in blue. B, Schematic showing PzIPT1 deletions used to drive
GFP expression in sperm cells of transgenic Arabidopsis. Expression pattern of each construct in mature pollen is summarized on
the right. Numbers refer to the 59 end of the deletions from the transcription start site and the positions of the CPB sites (orange
ovals) and the TATA box (blue ovals). C, Confocal images of expression patterns generated by truncated PzIPT1 promoters in
sperm cells of transgenic Arabidopsis captured with identical parameters. Bars: 5 mm. D, Measured expression levels of each
construct by ImageJ. Values are means of 120 pollen grains from six independent T3 transgenic lines (20 pollen/line) for each
construct; error bars represent SD.
system, as male germ lineage expression was never lost
and the vegetative cell was not labeled. No GFP signals
were detected in deletions Δ63 and Δ39, which indicates
that the sequence between -87 and -40 of the PzIPT1
promoter is critical for sperm cell expression of PzIPT1
(Fig. 2, C and D).
in the PzIPT1 promoter, the expression of GFP was
drastically decreased to a level similar to that of the Δ130
construct (Fig. 3A). These data provide evidence that
the CPB motif-containing region between -94 and -89
positively regulates the expression level of PzIPT1 in
sperm cells.
A CPB Motif-Containing Region Regulates the Expression
Level of PzIPT1 in Sperm Cells
Expression of PzIPT1 in Sperm Cells Can Be Enhanced by
Exogenously Applied Cytokinin
Progressive 59-deletional analyses showed that the
expression of PzIPT1 decreased dramatically in Δ87
compared with Δ105 (Fig. 2D), suggesting that the sequence between -105 and -87 of the PzIPT1 promoter is
critical for the expression strength of PzIPT1. Sequence
analysis revealed one CPB motif located between -94
and -89 (Fig. 2A). To examine whether this CPB motif
regulates the expression level of PzIPT1, a mutated
promoter construct Δ88-95 was created by deleting sequences between -95 and -88 and transformed into
Arabidopsis (Fig. 2B). Although only 8 bp were deleted
The construct Δ88-95, with a typical CPB site excised,
significantly impaired the expression level of PzIPT1
promoter, suggesting regulation of gene expression
in response to cytokinin may exist in the wild-type
PzIPT1 promoter. To test this hypothesis, transcription of PzIPT1 was evaluated in P. zeylanica pollen.
When treated with 100 nM 6-Benzylaminopurine (6-BA),
PzIPT1 transcription was enhanced dramatically
(Fig. 3B). To test whether expression enhancement also
occurred in Arabidopsis, transgenic pollen with Δ154
and Δ130 constructs was treated with 100 nM 6-BA to
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Zhang et al.
Figure 3. PzIPT1 promoter responses to
exogenously applied cytokinin. A, GFP
expression in Δ761 (left) and Δ88-95
(right). Bars: 5 mm. B, RT-PCR analysis
of PzIPT1 in P. zeylanica pollen treated
or untreated (+/2) with 100 nM cytokinin
(CK). HIS3.3 was used as the control. C,
Expression of PzIPT1 promoter after addition of 100 nM exogenous CK in transgenic
pollen grains of Δ154, Δ130, and Δ88-95 at
0, 30, 45, 60, and 120 min after treatment,
then retreated, and observed at 120+15,
120+30, 120+45, and 120+60 min. D,
Expression of PzIPT1 promoter in pollen
tubes of Δ130, Δ154, and Δ88-95 using
confocal (left), bright field (middle), and
mixed confocal/bright field microscopy
(right) at 0 to 120 min after treatment.
Bars: 5 mm. Photographs in C and D are
not sequential images of same pollen
grains or pollen tubes. E, Relative GFP
signal intensity of Δ130, Δ154, and Δ88-95
in transgenic pollen treated with or without CK (n = 20 pollen grains; error bars
represent SD).
examine its response to exogenously applied cytokinin.
GFP signals in mature pollen of both Δ154 and Δ130
transgenic plants were enhanced 15 min after treatment
with cytokinin. Signal strength reached a peak 60 min
after treatment and remained conspicuous for approximately another 60 min before decreasing gradually to
background florescence levels (Fig. 3, C and E). Thus,
cytokinin treatment appears to elevate the GFP level
above background for a total of approximately 2 h. No
obvious change of GFP signal was observed in the
mock treatment without cytokinin, nor in the Δ88-95
transgenic pollen with or without cytokinin treatment
(Fig. 3, C and E). After three rinses in medium without
cytokinin for 1 h, the cytokinin-pretreated pollen of
Δ154 and Δ130 plants was retreated with cytokinin.
Dramatically enhanced GFP signals were again observed approximately 15 min after treatment; the signal gradually weakened when incubated for longer
periods (Fig. 3, C and E). These data also indicate that
the CPB motif located between -88 and -95 in PzIPT1
promoter could be activated in response to a cytokinin
pulse. Similar results were obtained when germinated pollen tubes were treated with cytokinin, i.e.
the GFP signal in sperm cells became much stronger
in cytokinin-treated pollen tubes of Δ154 and Δ130
constructs, and no obvious changes were observed
in sperm cells of cytokinin-treated Δ88-95 pollen
tubes (Fig. 3D).
A Cis-Acting Region Is Required to Activate the PzIPT1
Promoter in Sperm Cells
59-Deletional analyses showed that neither Δ63 nor
Δ39 constructs could drive expression of GFP in sperm
cells, suggesting that this region may determine sperm
cell expression specificity of the PzIPT1 promoter (Fig.
2, B-D). We therefore created two deletion constructs
(Δ64-87 and Δ40-63) excising sequences corresponding
to -64 to -87 and -40 to -63 from the intact promoter and
transformed them into Arabidopsis (Fig. 2B). Neither
construct could drive expression of GFP in sperm cells
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Regulation of Gene Expression in Plant Male Gamete
(Fig. 4, B and C; Supplemental Fig. S3, A, B, G and H).
Since both fragments, -64 to -87 and -40 to -63, are
required for sperm cell expression of PzIPT1, we hypothesized that basal promoter activity may require
elements harbored in these two constructs. To test
this hypothesis, we examined GUS expression patterns in transgenic seedlings. The original PzIPT1
promoter could drive GUS expression in seedlings
(Fig. 1A). If essential basal promoter activities were
impaired in constructs Δ64-87 and Δ40-63, no GUS
signal would be expected in the corresponding
transgenic seedlings. As shown in Figure 4, F and G,
however, these two constructs still retained their
capability to drive GUS expression in seedlings, indicating that the deleted sequences did not affect the basal
activities of the PzIPT1 promoter. Thus, the entire region from -40 to -87 was required for sperm-specific
expression.
To further test whether the whole or part of the
region between -40 to -87 is sufficient to drive the
expression of PzIPT1 promoter in sperm cells, constructs were created fusing the whole region or the
region of -64 to -87 or -40 to -63 to minimal CaMV35S
promoter elements linked with a TMV leader sequence from a DR5 construct (Ulmasov et al., 1997)
to examine their expression patterns in transgenic
Figure 4. Cis-acting region determines expression specificity of PzIPT1 in sperm cells.
At least 12 T1 transgenic plants were observed
for each construct, and representative data are
shown. A,GFP expression is evident in Δ761,
but expression is absent in deletions Δ64-87
(B) and Δ40-63 (C) and in mutated promoters
Δ761m1 (D) and Δ761m2 (E). F, Promoter activity in contrast remains intact in seedlings of
Δ64-87 and Δ40-63 deletions using GUS expression. Relative GUS activity is shown in G.
H, Synthetic promoters used to test GFP expression in sperm cells. 35Smini, the minimal
CaMV35S promoter elements and the TMV 59
leader sequence. (40-63), (64-87), and (4087), cis-acting regions from the PzIPT1 promoter. I-P, Expression patterns of synthetic
promoters. I, Minimal CaMV35S promoter
elements, or cis-acting regions 40-63 (J) and
64-87 (K), and 13 MGSA motif fused with
minimal CaMV35S promoter elements cannot
drive GFP expression in sperm cells, whereas
40-87 (L), 23(40-63) (M), and 23(64-87) (N)
cis-acting regions and 43MGSA motifs (P)
fused with minimal CaMV35S promoter elements can activate GFP expression in sperm
cells. Bars: A-E, 5 mm; F, 2 mm; I-P, 5 mm.
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Zhang et al.
pollen (Fig. 4H). The minimal CaMV35S promoter by
itself did not activate GFP in sperm cells (Fig. 4I;
Supplemental Fig. S3, E and K), nor did intact CaMV35S
promoter in a prior study (Singh et al., 2003). When the
regions of -40 to -63 and -64 to -87 were fused to the
minimal CaMV35S promoter, the synthetic promoters
could not activate GFP expression in sperm cells (Fig.
4, J and K; Supplemental Fig. S3, F, L, M and S). However, the region of -40 to -87 could activate GFP expression in sperm cells (Fig. 4L; Supplemental Fig. S3,
N and T). GFP was detected in sperm cells harboring both regions of -64 to -87 and -40 to -63. All these
data support the conclusion that the cis-acting region
-40 to -87 confers activation of the PzIPT1 promoter in
sperm cells.
MGSA Sites Determine Selective Activation of PzIPT1
Promoter in Sperm Cells
When either sequence from -64 to -87 or -40 to -63 was
excised, expression in sperm cells was lost (Fig. 4, B and
C), although expression in seedlings was unchanged
(Fig. 4, F and G), suggesting that both of these sequences
harbor motifs important for expression of PzIPT1 promoter in sperm cells and neither one by itself was sufficient for sperm cell expression. However, synthetic
promoters containing duplicated -64 to -87 or -40 to -63
sequences fused with the minimal CaMV35S promoter
elements could drive GFP expression in sperm cells (Fig.
4, M and N; Supplemental Figure S3, O, U, P and V).
Further sequence analysis identified two identical motifs
of GAAACG at -69 to -74 and -49 to -54 (Fig. 2A),
designated herein as MGSA motifs. When either MGSA
motif of PzIPT1 promoter was mutated, no GFP signal
could be detected in transgenic pollen (Fig. 4, D and E;
Supplemental Fig. S3, C, I, D and J).
To examine whether this motif is capable of driving
gene expression in sperm cells, a synthetic promoter
containing multiple copies of MGSA and the minimal
CaMV35S promoter elements fused to GFP was introduced into Arabidopsis (Fig. 4H) and its expression
was observed in sperm cells (Fig. 4P; Supplemental
Fig. S3, R and X). No GFP signal was detected in
sperm cells when only one copy of MGSA motif was
employed (Fig. 4O; Supplemental Fig. S3, Q and W).
These data together support the conclusion that at
least two copies of MGSA motif are needed and are
sufficient to determine gene expression in sperm
cells.
Arabidopsis 500-bp upstream promoter sequences
were retrieved from TAIR and screened for the presence of MGSA motif in previously identified sperm
and pollen-expressed genes (Borges et al., 2008). A
total of 277 sperm cell-expressed genes were identified
with one MGSA motif and 37 genes have at least two
MGSA motifs (Supplemental Table S1; Supplemental
Table S2). The promoter activity of six genes with
at least two MGSA motifs, At1g07910, At1g23060,
At1g31010, At2g36660, At3g27540, and At3g46230, has
already been confirmed in sperm cells (Fig. 5, A-F;
Supplemental Table S2). When either one of the two
MGSA motifs close to transcription start site was mutated, expression of these genes in sperm cells was
abolished (Fig. 5, G-R).
Figure 5. MGSA motifs function in sperm active promoters of Arabidopsis. A-F, MGSA motif-containing promoters can drive GFP
expression in sperm cells. G-R, Mutation of the upstream (m1) or downstream (m2) MGSA motif close to transcription start site
abolishes GFP expression in sperm cells. A, G, M, At1g07910; B, H, N, At1g23060; C, I, O, At1g31010; D, J, P, At2g36660; E, K, Q,
At3g27540; F, L, R, At3g46230. Bars: 5 mm.
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Regulation of Gene Expression in Plant Male Gamete
DISCUSSION
Conservation of Sperm Cell Expression of
PzIPT1 Promoter
Sperm cells produce a complex complement of messenger RNAs contributing to their development, differentiation and fertilization (Gou et al., 2001, 2009;
Engel et al., 2003; Borges et al., 2008; Xin et al., 2011). A
number of these mRNAs encode key regulators that are
required for sperm cell control, identity, and function.
For example, DUO1 is a MYB transcription factor that is
essential to male germline transcription and identity,
with specific expression in generative and sperm cells of
Arabidopsis (Rotman et al., 2005; Brownfield et al.,
2009a; Borg et al., 2011). The highly conserved GCS1/
HAP2 gene encoding a secreted membrane protein is
essential for fertilization in a myriad of other eukaryotes as well (Mori et al., 2006; von Besser et al., 2006; Liu
et al., 2008). Additional male gamete-selective genes
have been identified and characterized in lily and
Arabidopsis (Engel et al., 2005; Okada et al., 2005a,
2005b; Brownfield et al., 2009a, 2009b).
A sperm type-selective gene PzIPT1 of P. zeylanica
was identified using suppression subtractive hybridization and in situ hybridization as having differential
expression in the Svn sperm cell (Gou et al., 2009). Since
transformation in P. zeylanica has proven refractory
(Wei et al., 2006), we examined transcriptional activity
of the promoter in transgenic Arabidopsis. Expression
of this gene was confined to the sperm cells in pollen of
Arabidopsis with no detectable signal in pollen cytoplasm (Fig. 1D), suggesting that expression of PzIPT1
promoter in sperm cells is conserved, but without detectable differential response between sperm cell types.
This diverged significantly from the differential transcription observed in P. zeylanica. Thus, either the
Arabidopsis sperm cells lack a similar pattern of dimorphism or sperm type-specific expression is species
dependent.
A cis-acting regulatory region for expression activation in sperm cells was identified in the DUO1 promoter. Although a putative GRSF binding site was
predicted in the promoter region of DUO1 (Haerizadeh
et al., 2006), a truncated version of DUO1 promoter
excluding the putative GRSF binding site did not prevent germline-specific expression, nor did it result in
constitutive expression (Brownfield et al., 2009a). On
the other hand, MYB binding sites were overrepresented in the promoters of DUO1-activated target
(DAT) genes (Borg et al., 2011, 2014). DUO1 directly
regulates the expression of DATs through binding to
the MYB sites in the promoter regions. For example,
DUO1 binds to the MYB consensus sequences of the
promoters of AtMGH3/HTR10, DAZ1, and DAZ2 by its
MYB domain to directly activate their expression (Borg
et al., 2011). No GRSF binding site was identified in
PzIPT1 promoter sequences, and moreover truncation
results also demonstrated that no GRSF binding site
exists in the promoter. Truncation analyses instead
suggested that there were positive regulatory motifs for
sperm specificity in the region of -40 to -87 of the PzIPT1
promoter (Fig. 4), as is observed in the case of DUO1mediated male germline gene expression (Fig. 6).
Our studies further identified two identical MGSA
motifs that could activate PzIPT1 promoter in sperm
cells (Fig. 4). Moreover, repeated MGSA motifs were
also identified in the promoters of 37 Arabidopsis
sperm-expressed genes (Supplemental Table S2) and
their importance for the activation of six genes in sperm
cells was confirmed (Fig. 5), suggesting that this type
of gene activation in sperm cells may be conserved.
We noticed that a total of 250 genes was identified
with at least two MGSA motifs in the Arabidopsis
genome (Supplemental Table S1), but only 37 of them
Gene Expression in Male Germline Cells Is Regulated by
Diverse Mechanisms of Activation and Repression
Several different patterns of gene expression in the
male germ lineage have been elucidated. Expression of
LGC1 is restricted to generative cell and sperm cells (Xu
et al., 1999). When LGC1 promoter was truncated, male
germline specificity was lost and expression became
constitutive (Singh et al., 2003). Further experiments
resulted in the identification of the GRSF repressor
protein ubiquitously expressed in sporophytic cells and
the corresponding silencing sequence in the LGC1
promoter. In this model, transcriptional repression was
mediated by a specific repressor and corresponding
binding element based on similar sequences in a number of male germ expressed genes, and thus this was
proposed as a possible general regulatory mechanism
for the expression of male germline-specific genes
(Haerizadeh et al., 2006).
Figure 6. Regulatory mechanisms for PzIPT1 promoter expression in
sperm cells of Arabidopsis. Male germ cell-expressed positive factors
activate or enhance germline genes in male gametes. Orange and light
blue lines represent the 59-upstream CBP and MGSA sites, respectively.
Orange squares and light blue pentagons represent the corresponding
positive activators. Gene expression is shown in green. VN, vegetative
nucleus.
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Zhang et al.
had a present call in sperm cell transcriptomic data
(Supplemental Table S2; Borges et al., 2008), indicating
that not all genes with two MGSA motifs in their promoters will express in sperm cell. Some other factors,
such as the positions, distance, and flanking sequences
of the MGSA motifs, may affect their function. On the
other hand, we also noticed that many known spermexpressed genes, such as DUO3, AtMGH3/HTR10,
DAZ1, and DAZ2, have no MGSA motifs in their promoters, suggesting that other regulatory mechanisms
for sperm gene expression function simultaneously, as
revealed by DUO1-activated expression of DAT genes
(Borg et al., 2011). Collectively, the current available
data suggest multiple regulation mechanisms for gene
expression in male germ cells, represented by LGC1,
DUO1, and PzIPT1. The regulatory mechanisms of sperm
type-specific gene expression, however, will need to
await a suitable transformation system for heteromorphic male germ cells. Similar mechanisms still may
be employed to distinguish gene expression between a
pair of sperm cells.
PzIPT1 Promoter Responds to Changes of
Cytokinin Concentration
In P. zeylanica, PzIPT1 expression is restricted to the
Svn sperm cell, which is known to fuse with the central
cell during preferential fertilization (Russell, 1985),
suggesting a targeted function of the Svn PzIPT1 protein
during double fertilization and endosperm development. Control of early development by paternally
encoded transcripts, as shown by the postfertilization
control of cell fate in the two cell embryo through SSP
function in Arabidopsis (Bayer et al., 2009), may have a
counterpart in the control of the early endosperm as
well. The possible function of the cytokinin regulating
double fertilization, embryo and endosperm development of P. zeylanica has not been elucidated yet. That
the GFP signal could be enhanced dramatically upon
cytokinin treatment and again by reapplication (Fig. 3)
suggests that this gene may participate in a feedforward mechanism that enhances cytokinin in the
fusion product of the Svn. Our analyses indicate that
PzIPT1 promoter could respond to cytokinin pulsing,
and this effect is supported by the presence of the CPB
sites. The CPB site was first identified in the cucumber
POR (NADPH-protochlorophyllide reductase) gene
promoter that is critical for cytokinin-dependent protein
binding in vitro (Fusada et al., 2005). It is already known
that the levels of cytokinin and isopentenyltransferase
are strongly increased during endosperm induction and
development (Miyawaki et al., 2004; Day et al., 2008). It
is effective to control the cytokinin levels by regulating
the expression of the key cytokinin biosynthase, PzIPT1.
When PzIPT1 promoter is exposed to a significant
elevated cytokinin level after double fertilization, it
may respond to this change and produce more cytokinin via a possible positive feedback mechanism to
stimulate the endosperm development nursing the
embryo. On the other hand, the concentration of
cytokinin must be finely regulated, and the expression
of PzIPT1 will be down-regulated when the required
cytokinin is produced, which is reflected by the decreased expression of GFP after pollen was treated
with cytokinin for 2 h. There are nine IPT genes in
Arabidopsis (Miyawaki et al., 2004). However, no
significant up-regulation of these IPTs is observed
when treated with cytokinin according to expression
data from Genevestigator (http://genevestigator.
com), suggesting a different mechanism for regulation
of PzIPT1 expression. The possible transcriptional
activator responding to cytokinin may play a vital role
during double fertilization and endosperm and embryo development. The transcriptional regulators
controlling PzIPT1 expression in sperm cells have not
been revealed yet. Identification and functional analysis
of both of the transcriptional factors involved in fine
regulation of PzIPT1 expression will provide more insights into regulation of gene expression in male germline cells. It is also interesting and possible to utilize
PzIPT1 promoter or its components to develop a system
for gene expression induction in sperm cells by using
cytokinin as an external chemical.
MATERIALS AND METHODS
Plant Growth and Transformation
Plumbago zeylanica plants were grown in greenhouses of the University of
Oklahoma and Lanzhou University. Plants of Arabidopsis (Arabidopsis thaliana)
wild-type Columbia-0 were grown in growth rooms with 16 h light and 8 h dark
at 22°C. Agrobacterium-mediated transformation was performed to generate
transgenic Arabidopsis plants by floral dip method (Clough and Bent, 1998). At
least 10 transgenic lines were observed.
Plasmid Constructs for Arabidopsis Transformation
The promoter of PzIPT1 was inserted into a binary vector pBIB-BASTA-GUS
modified from pBIB vector (Becker, 1990) at the HindIII and SalI sites to make
pPzIPT1::GUS. The PzIPT1 promoter was also recombined into the Gatewaycompatible pFYTAG binary vector to drive the expression of fused coding
regions of histone 2A (HTA6; At5g59870) and enhanced YFP (EYFP; Zhang
et al., 2005). The binary vector pBIB-BASTA-GFP (Ge et al., 2011) was modified
to a Gateway-compatible destination vector, pBIB-BASTA-GFP-GWR, by
inserting the Gateway module at HindIII and XbaI sites for promoter analyses.
Deletion fragments were amplified by PCR from cloned PzIPT1 promoter
and transferred into pDONR/Zeo vector by Gateway in vitro DNA recombination for sequencing analysis. Following sequence verification, these truncated
promoter fragments were in vitro recombined into pBIB-BASTA-GFP-GWR and
pBIB-BASTA-GUS-GWR (Yuan et al., 2007) to create final binary transformation
constructs. IPTPromPB2 was used as a reverse primer for all ten 59-deletions.
Forward primers (Δ761PB1, Δ531PB1, Δ368PB1, Δ255PB1, Δ154PB1, Δ130PB1,
Δ105PB1, Δ87PB1, Δ63PB1, Δ39PB1) were designed according to the positions of
the deletions in the PzIPT1 promoter. The deletion constructs Δ88-95, Δ64-87,
and Δ40-63 were created according to the manual of QuikChange SiteDirected Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) using
primers Δ88-95F, Δ88-95R, Δ64-87F, Δ64-87R, Δ40-63F, and Δ40-63R.
To clone promoters of sperm active genes in Arabidopsis, the following
Gateway-compatible primers were used: P07910-F, P07910-R for At1g07910;
P23060-F, P23060-R for At1g23060; P31010-F, P31010-R for At1g31010; P36660-F,
P36660-R for At2g36660; P27540-F, P27540-R for At3g27540; P46230-F, P46230-R
for At3g46230. The following primers were used to mutate the two putative
MGSA sites close to the transcription start site in these promoters according to the
manual of the QuikChange Site-Directed Mutagenesis Kit: Δ761m1F, Δ761m1R,
Δ761m2F, Δ761m2R for Δ761; 07910m1F, 07910m1R, 07910m2F, 07910m2R for
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Regulation of Gene Expression in Plant Male Gamete
At1g07910; 23060m1F, 23060m1R, 23060m2F, 23060m2R for At1g23060;
31010m1F, 31010m1R, 31010m2F, 31010m2R for At1g31010; 36660m1F,
36660m1R, 36660m2F, 36660m2R for At2g36660; 27540m1F, 27540m1R,
27540m2F, 27540m2R for At3g27540; 46230m1F, 46230m1R, 46230m2F,
46230m2R for At3g46230.
Sequence of the synthetic DR5 promoter (Ulmasov et al., 1997) containing
seven copies of DR5, the -46 CaMV35S promoter, and a TMV 59 leader was
PCR-amplified from a DR5::GUS construct provided by Dr. Guilfoyle (University of Missouri, Columbia) with primers DR5PB1 and DR5PB2 and cloned
into pDONR/Zeo vector for site-directed mutagenesis and in vitro DNA recombination with the Gateway destination vector pBIB-BASTA-GFP-GWR.
Site-directed mutagenesis was performed to make the synthetic promoters
with the following primers: 35SminiF, 35SminiR for 35Smini; (40-63)F, (40-63)R
for 40-63; (64-87)F, (64-87)R for 64-87; (40-87)F, (40-87)R for 40-87; 23(40-63)F,
23(40-63)R for 23(40-63); 23(64-87)F, 23(64-87)R for 23(64-87); 13MGSAF,
13MGSAR for 13MGSA; 43MGSAF, 43MGSAR for 43MGSA.
Designing of Gateway-compatible primers and gateway cloning were
conducted according to the Gateway Technology manual (Invitrogen, http://
www.invitrogen.com). All primer sequences are listed in Supplemental
Table S3.
used for statistical analysis of GFP signal, which was performed by ImageJ
analysis software from the US National Institutes of Health (http://rsbweb.
nih.gov/ij) according to Burgess et al. (2010).
Accession Numbers
Accession number for PzIPT1 promoter sequence: JN665068.
SUPPLEMENTAL DATA
The following supplemental materials are available.
Supplemental Figure S1. PzIPT1 promoter drives GUS expression in mature pollen of transgenic Arabidopsis.
Supplemental Figure S2. Expression patterns of PzIPT1 promoter during
Arabidopsis male gametogenesis.
Supplemental Figure S3. Cis-acting region analyses of PzIPT1 in Arabidopsis sperm cells.
Supplemental Table S1. MGSA elements in Arabidopsis promoters.
Pollen Collection, Pollen Tube Culture, and
Cytokinin Treatment
Arabidopsis pollen from freshly opened flowers was harvested from a
representative transgenic line for each construct and spread on solidified medium on a glass microscope slide for germination and observation (Wang and
Jiang, 2011). Collected pollen and germinated pollen tubes were incubated in
pollen germination medium containing 100 nM 6-BA (Boavida and McCormick,
2007; Müller and Sheen, 2008) at room temperature for durations of 5, 15, 30, 45,
60, 90, and 120 min. Pollen grains treated for 120 min were suspended and
incubated in three changes of germination medium over 60 min to remove
exogenously applied cytokinin. Washed pollen was resuspended in germination medium with 100 nM 6-BA for intervals of 15, 30, 45, and 60 min. The experiment was repeated three times.
Supplemental Table S2. Summary of 37 genes with putative MGSA
motifs.
Supplemental Table S3. Primers used in the study.
ACKNOWLEDGMENTS
We thank Dr. Tom J. Guilfoyle (University of Missouri, Columbia) for
providing the construct of DR5 promoter. We are grateful to Liping Guan, Yang
Zhao, and Liang Peng for their technical assistance.
Received September 28, 2015; accepted January 4, 2016; published January 6,
2016.
LITERATURE CITED
RT-PCR Analysis
Gene expression differences of pollen treated with or without cytokinin were
examined by reverse transcriptase-PCR reactions. Pollen from P. zeylanica was
treated with 100 nM 6-BA for 60 min in 70% glycerol (Southworth et al., 1997).
Total RNA of treated and untreated pollen was isolated using RNAprep Pure
Plant Kit with on-column DNase-treatment (Tiangen Biotech, http://www.
tiangen.com). Then 1 mg total RNA of each sample was reverse transcribed in a
50-mL volume using a Moloney murine leukemia virus reverse transcriptase
(Invitrogen, http://www.invitrogen.com). The RT product of 100 ng total
RNA was used as PCR template for one reaction. Different cycles (20, 22, 24,
26) were used to amplify PzIPT1 and HIS3.3 (as a control) of P. zeylanica with
primers PzIPT1-F (59-TCATACTGAAGGCAGGTCGTCT-39), PzIPT1-R (59CCTTGAACCTCCGTATCTTGGA-39), HIS3.3-F (59-GAGGAAAGGCTCCTAGAAAGCAA-39), and HIS3.3-R (59-CGGTGGTGGGAGCAGACTT-39). PCR
products were separated by 1% agarose gel electrophoresis and photographed.
GUS Staining, MUG Assay, and Microscopic Analysis
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detection of GUS activity (Robatzek and Somssich, 2001). Plant tissues
were infiltrated in GUS staining solution containing 50 m M NaPO 4 , 0.5 m M
K3Fe(CN)6, 0.5mM K4Fe(CN)6, 0.1% Triton X-100, 5 mM EDTA, and 1 mg mL21
X-Gluc, incubated at 37°C overnight, and destained several times in 70%
ethanol and then photographed using a Leica M165C Stereo microscope (Leica
Microsystems, http://www.leica-microsystems.com). Columbia-0, Δ761, Δ40-63,
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scanning microscope or Leica DM6000 epifluorescence microscope equipped
with a GFP or YFP filter set. To evaluate GFP signal intensity, all samples were
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