First published online July 10, 2003; 10.1104/pp.103.023192
Plant Physiology 132:1884-1891 (2003)
© 2003 American Society of Plant Biologists
DEVELOPMENT AND HORMONE ACTION
Expression of U1 Small Nuclear Ribonucleoprotein 70K Antisense Transcript Using APETALA3 Promoter Suppresses the Development of Sepals and Petals1
Maxim Golovkin and
Anireddy S.N. Reddy*
Department of Biology and Program in Cell and Molecular Biology, Colorado
State University, Fort Collins, Colorado 80523
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ABSTRACT
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U1 small nuclear ribonucleoprotein (snRNP)-70K (U1-70K), a U1
snRNP-specific protein, is involved in the early stages of spliceosome
formation. In non-plant systems, it is involved in constitutive and
alternative splicing. It has been shown that U1snRNP is dispensable for in
vitro splicing of some animal pre-mRNAs, and inactivation of U1-70K
in yeast (Saccharomyces cerevisiae) is not lethal. As in yeast and
humans (Homo sapiens), plant U1-70K is coded by a single gene. In
this study, we blocked the expression of Arabidopsis U1-70K in petals
and stamens by expressing U1-70K antisense transcript using the
AP3 (APETALA3) promoter specific to these floral organs.
Flowers of transgenic Arabidopsis plants expressing U1-70K antisense
transcript showed partially developed stamens and petals that are arrested at
different stages of development. In some transgenic lines, flowers have
rudimentary petals and stamens and are male sterile. The severity of the
phenotype is correlated with the level of the antisense transcript. Molecular
analysis of transgenic plants has confirmed that the observed phenotype is not
due to disruption of whorl-specific homeotic genes, AP3 or
PISTILLATA, responsible for petal and stamen development. The
AP3 transcript was not detected in transgenic flowers with severe
phenotype. Flowers of Arabidopsis plants transformed with a reporter gene
driven by the same promoter showed no abnormalities. These results show that
U1-70K is necessary for the development of sepals and petals and is an
essential gene in plants.
Most eukaryotic nuclear genes contain non-coding intervening sequences
(introns; Goodall et al.,
1991 ; Sharp,
1994). The introns in the precursor mRNAs are removed, and exons
are joined in the nucleus by a process called pre-mRNA splicing to generate
functional mRNAs (Sharp,
1994). In the recently completed Arabidopsis genome, it is
predicted that 79% of nuclear genes contain one or more introns
(Arabidopsis Genome Initiative,
2000; Reddy,
2001). Although plant and animal introns have some common
structural features, plant introns differ from non-plant introns in many
aspects, suggesting that the early events of spliceosome formation, especially
the mechanisms involved in intron recognition in plants, may not be similar to
yeast (Saccharomyces cerevisiae) and animals
(Luehrsen et al., 1994;
Schuler, 1998;
Lorkovic et al., 2000;
Reddy, 2001). Some pre-mRNAs
with multiple introns display complex patterns of alternative splicing,
thereby contributing to the proteomic complexity and an additional level of
control of gene expression (Smith et al.,
1989; Lorkovic et al.,
2000; Reddy,
2001). In mammalian genomes, about 30% to 60% of genes produce
multiple transcripts by alternative splicing
(Hanke et al., 1999;
Brett et al., 2000;
Lee et al., 2003).
The splicing of nuclear pre-mRNA takes place in a large RNA-protein complex
called the spliceosome. In most metazoans, and most likely in plants, there
are two (major and recently discovered minor) types of spliceosomes that
differ in their composition (for review, see
Burge et al., 1999;
Reddy, 2001). The major
spliceosome contains four small nuclear ribonucleoprotein (snRNP) particles
(U1, U2, U4/6, and U5) and a number of non-snRNP proteins
(Will and Luhrmann, 1997;
Staley and Guthrie, 1998;
Burge et al., 1999). The
assembly of the spliceosome on the pre-mRNA is an orderly process that
involves a series of complex RNA-RNA, RNA-protein, and protein-protein
interactions (Fu, 1995;
Burge et al., 1999). The U1
snRNP recognizes the 5' splice site in an ATP-independent manner to form
a complex called early complex in mammalian cells or commitment complex in
yeast that commits the pre-mRNA to spliceosome assembly. This complex
formation involves base pairing between conserved nucleotides at the 5'
end of U1 snRNA and the 5' splice site of pre-mRNA
(Mount et al., 1983;
Burge et al., 1999). In
metazoans, U1 snRNP contains one U1 snRNA molecule and at least 11 proteins
including three U1snRNP-specific proteins (U1-70K, U1-A, and U1-C), whereas
the yeast U1snRNP contains an additional six U1snRNP-specific proteins
(Fabrizio et al., 1994;
Gottschalk et al., 1998). U1
snRNP-specific proteins are required for efficient formation of a complex
between U1 snRNA and the 5' splice site junction
(Mount et al., 1983;
Heinrichs et al., 1990;
Rosbash and Séraphin,
1991).
U1 snRNP-specific protein, U1-70K, is involved in both basic and
alternative splicing of pre-mRNAs in animals
(Manley and Tacke, 1996;
Will and Luhrmann, 1997).
U1-70K interacts directly with splicing factors (ASF/SF2 and SC-35) of the SR
family that are involved in splice site selection
(Wu and Maniatis, 1993;
Kohtz et al., 1994;
Manley and Tacke, 1996;
Will and Luhrmann, 1997).
Furthermore, overexpression of U1-70K in cultured animal cells inhibits
splicing and nucleocytoplasmic transport
(Romac and Keene, 1995).
However, Crispino et al.
(1994) have shown that nuclear
extracts depleted of U1 snRNP could be reconstituted by a high level of
Ser/Arg-rich proteins. In another study, SR proteins or purified SC35 protein
complemented splicing in extracts where the interaction of U1 snRNP with the
5' splice site is blocked by antisense RNA
(Tarn and Steitz, 1994). The
yeast U1-70K has only 30% amino acid identity to human (Homo sapiens)
U1-70K and lacks the Arg-rich region
(Smith and Barrell, 1991;
Kao and Siliciano, 1992). The
yeast U1-70K was initially reported to be essential for yeast
viability (Smith et al.,
1989). Later, it was shown that the lack of U1-70K does
not cause lethality; instead, it makes the mutant temperature sensitive and
defective in pre-mRNA splicing but still viable
(Hilleren et al., 1995). By
rescuing the mutant phenotype, the most important functional region of yeast
U1-70K was mapped to the amino-terminal part (1–97 amino acids). The
other part of U1-70K containing the RNA-binding domain (also called RNA
recognition motif [RRM]) and Gly-rich domain was unable to complement the
mutant.
In plants, U1-70K has been characterized only from Arabidopsis. It
is coded by a single gene, which produces two (short and long) transcripts by
inclusion or exclusion of a 910-bp intron
(Golovkin and Reddy, 1996).
Both U1-70K transcripts are expressed in all tissues, and the level
of the transcripts varied in different organs. The deduced amino acid sequence
from the short transcript is similar to the animal U1-70K protein and contains
an RRM, a Gly hinge, and an Arg-rich region characteristic of the animal
U1-70K protein (Golovkin and Reddy,
1996). The long transcript has an in-frame translational
termination codon within the 910-bp included intron and produces a truncated
protein containing 204 amino acids with part of the RRM containing only RNP2.
Plant U1-70K shares some characteristic features with animal U1-70K but
differs in others and interacts with novel plant Ser/Arg-rich proteins
(Golovkin and Reddy, 1998;
1999). Because the U1snRNP is
dispensable in in vitro splicing of some pre-mRNAs and inactivation of
U1-70K in yeast does not cause lethality
(Crispino et al., 1994;
Tarn and Steitz, 1994;
Hilleren et al., 1995), we
tested to see if it is dispensable in plants. To this end, we blocked the
expression of U1-70K using an antisense construct driven by a strong
floral organ-specific promoter (AP3 promoter) that is active only in
two whorls (petals and stamens) of flowers. Here, we present the evidence that
expression of U1-70K antisense transcript in sepals and petals aborts
their development, suggesting that U1-70K is essential for the development of
these organs.
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RESULTS
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Expression of U1-70K Antisense with AP3 Promoter Suppressed
the Development of Petals and Stamens
To determine if the U1-70K gene is necessary for development, we
chose to block the expression of U1-70K in two floral organs (petals
and stamens) using the AP3 promoter that is specific to these organs.
The expression of AP3, a floral homeotic gene that is required for
specifying petal and stamen identities, begins in young flowers in the
precursor cells for the petals and stamens and continues in these organs
throughout most of the flower development
(Bowman et al., 1989;
Jack et al., 1992). It has
been shown that the fusion of the 3.7 kb of 5' AP3 promoter
sequence to green fluorescent protein, together with 1.5 kb of 3'
AP3 sequence downstream of GUS, results in expression of the reporter
gene specifically in stamens and petals
(Jack et al., 1994). In this
work, we have used a shorter promoter sequence (approximately 700 bp) of AP3
gene without any 3' sequence. To determine the activity of this 700-bp
region of the AP3 promoter, we transformed Arabidopsis plants with
 -glucuronidase (GUS) driven by this promoter
(Fig. 1A). As shown in
Figure 1B, the expression of
reporter gene driven by the 700-bp promoter of AP3 is confined to
only petals and stamens. However, the level of reporter gene expression in
these organs varied among independent transgenic lines (see
Fig. 1B). For example, in one
plant GUS expression was very strong in petals and anther filaments with
little expression in anther (see Fig. 1B,
2), whereas another line showed strong GUS expression in anthers
and low expression in petals and anther filaments
(Fig. 1B, 3). The strength of
the AP3 promoter in these organs is comparable with the constitutive
cauliflower mosaic virus 35 promoter (data not shown). Recently, Tilly et al.
(1998) have shown that
cis-elements necessary for petal and stamen expression reside within the
700-bp promoter region used in this study. It is worth mentioning that in over
200 transgenic plants containing the GUS reporter driven by the AP3
promoter, we did not observe any flower abnormalities.
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Figure 1. Analysis of 700-bp AP3 promoter activity in transgenic plants. A,
Schematic diagram of the promoter-reporter fusion construct. AP3, 700-bp
promoter region of AP3 gene; Term, NOS3 terminator. B, Flowers from
wild type and three independent transgenic lines were stained for GUS
activity. The arrows indicate the parts of the stamens with little or no GUS
expression.
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To block the expression of U1-70K efficiently, we fused the
AP3 promoter (approximately 700-bp region) with the longest cDNA of
Arabidopsis U1-70K comprising the entire coding region, 910-bp
alternative intron, and extended untranslated region
(Golovkin and Reddy, 1996) and
used this construct to transform Arabidopsis
(Fig. 2A). More than 100
kanamycin-resistant transgenic plants were obtained. Among these, 11% of the
plants revealed abnormal flowers that had either rudimentary or partially
developed petals and stamens arrested at different stages of their development
(Figs. 2 and
3). The number and position of
the sepals and carpel remained normal in all transgenic flowers. In seven
independent transgenic plants with severe flower phenotype, only sepals and
carpel were visible in mature flowers. The inflorescence and a single flower
from wild type and one such transgenic plant are shown in
Figure 2B. The lines with
severe phenotype were completely male sterile. As shown in
Figure 3A, in some of the
transgenic lines, underdeveloped petal and stamens were present but hidden
under sepals. However, flowers of transgenic plants rarely had carpel-like
features in place of stamens (Fig.
3B). Transgenic lines with severe phenotype (male sterile)
produced viable seeds when pollinated with pollen from wild-type plants, and
the kanamycin-resistant plants from such crosses also produced abnormal
flowers with rudimentary petals and stamens.
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Figure 2. Targeted expression of U1-70K antisense transcript using the
AP3 promoter. A, Schematic diagram of U1-70K antisense
cassette used to generate transgenic plants. AP3, Promoter (700 bp) from
AP3 gene; U1-70K antisense, U1-70K long cDNA in
antisense orientation; Term, NOS3 terminator. B, Expression of U1-70K
antisense transcript results in flowers with rudimentary petals and stamens.
Left, Inflorescence of a wild-type plant (top) and wild-type flower. Right,
Inflorescence of a transgenic plant expressing U1-70K antisense
transcript (top) and flower from a transgenic plant with rudimentary petals
and stamens (bottom).
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Figure 3. Variations in the phenotype of flowers expressing U1-70K antisense
transcript. A, Opened flowers from two different U1-70K antisense
transgenic plants showing variations in stamen development. B, Scanning
electron micrograph showing a rarely observed flower phenotype from a
transgenic plant with carpel-like structures in place of stamens. Stigmatic
papillae on stamens are circled. C, Carpel; St, stamen.
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The Severity of Flower Phenotype Is Correlated with the Level of
U1-70K Antisense Transcript
To determine the number of copies of the introduced gene, we performed
Southern analysis with independent transgenic lines. The genomic DNA was
digested with BamHI, which does not cut the endogenous gene and the
introduced AP3/U1-70K antisense cassette. As shown in
Figure 4A, the wild-type and
transgenic lines yielded the expected size endogenous band. In addition, each
transgenic line carried multiple copies of the introduced gene and the number
of copies varied significantly from plant to plant (from two in line 6 to
several in line 1). To test if the observed flower phenotype correlated with
the level of the antisense transcript, we monitored the level of the
expression of the introduced gene in five lines. Of these, transgenic line 1
showed severe flower phenotype; lines 2, 3, and 4 showed partially developed
stamens; and line 5 showed visibly normal flowers. RNA from flowers and flower
buds from wild-type and transgenic plants was probed with a 910-bp fragment
that is specific to the long transcript and, therefore, is expected to detect
the endogenous long transcript and AP3-driven antisense transcript.
The level of the antisense transcript varied considerably between different
transgenic plants (Fig. 4B).
Plant 1 with severe phenotype (very rudimentary petals and stamens in mature
flowers) has a very high level of antisense transcript, whereas the flowers
with less severe phenotype (lanes 2–4) showed lower levels of antisense
transcript. Flowers from line 5 with visibly normal flowers showed barely
detectable levels of antisense transcript. These results show a strong
correlation between the level of U1-70K antisense overexpression with
the phenotype severity. The flowers of transgenic progeny in the T1
and T2 generations that showed abnormal flower phenotype also
showed high levels of antisense transcript (data not shown). Transgenic lines
expressing a very high level of U1-70K antisense transcript showed
additional smaller size transcripts, including a transcript corresponding to
the endogenous transcript (see Fig.
4B, lane 1), which are likely due to the degradation of
overexpressed transcript.
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Figure 4. Molecular characterization of transgenic plants. A, DNA gel-blot analysis.
Genomic DNA from the wild type (Wt) and six independent transgenic plants was
digested with BamHI and probed with a U1-70K. Arrow
indicates the endogenous U1-70K gene. B, Expression of antisense
U1-70K transcript in flowers of transgenic plants. RNA from wild type
(Wt) and five transgenic plants was hybridized with the 910-bp
"alternative intron" fragment that is present only in the large
transcript of plant U1-70K (top). A hollow arrow indicates expression
of endogenous U1-70K. Solid arrow indicates the level of antisense
transcript. Stained gel with ribosomal RNA is shown in the bottom panel.
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The Observed Flower Phenotypes Are Not Due to the Disruption of
Homeotic Genes, AP3 or PI (PISTILLATA), That Control the Development of Petals
and Stamens
Floral organ identity is based on the combinatorial activities of three
classes (ABC) of homeotic genes (Coen and
Meyerowitz, 1991; Clark and
Meyerowitz, 1994). Each class of genes acts in two adjacent whorls
in a combinatorial fashion to specify organ identity. Class A genes are active
in sepals and petals, Class B genes control petals and stamens development,
and Class C genes are active in stamens and carpels. Expression of A and B
leads to petal development, whereas the expression of B and C leads to stamen
development. There are two class B genes (AP3 and PI) in
Arabidopsis (Bowman et al.,
1991; Coen and Meyerowitz,
1991). The expression of Class B genes, AP3 and
PI, is restricted to petals and stamens
(Hill and Lord, 1989;
Jack et al., 1992). Mutations
in Class B genes result in flowers that contain sepals in place of petals and
carpels in place of stamens (Bowman et al.,
1991). Thus, the disruption of the normal function of the B class
genes (AP3 or PI) leads to the development of abnormal
flowers. It is possible but very unlikely that the integration of introduced
gene into either AP3 or PI could have caused the phenotype
that we observed. To rule out this possibility, we verified the integrity of
AP3 and PI genes. We hybridized BamHI-digested
genomic DNA of the individual transgenic plants with the 7.5-kb BamHI
DNA fragment of the AP3 gene, which contains the entire gene and its
promoter or PI cDNA. As shown in
Figure 5A, a single expected
size fragment hybridized in both wild-type and transgenic plants with both
probes, suggesting that AP3 and PI genes are intact in the
transgenic plants.
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Figure 5. Analysis of AP3 and PI genes in plants expressing
U1-70K antisense transcript. A, DNA-blot analysis of transgenic
plants with AP3 and PI genes. Genomic DNA from wild type
(Wt) and four transgenic plants (lanes 1–4) was digested by
BamHI and hybridized with AP3 and PI genes. The
size of the BamHI fragment is about 7.5 kb with the APETALA3
probe and 12 kb with the PISTILLATA probe. Based on the bacterial
artificial chromosome sequence in this region, the hybridizing bands
correspond to the expected sizes and contain the whole gene and the promoter.
B, Expression of AP3 in transgenic plants. Total RNA from buds and
flowers of wild type (Wt) and transgenic plants with severe phenotype
(sterile) and fertile (fertile) were used for reverse transcriptase (RT)-PCR
analysis using AP3-specific forward and reverse primers corresponding
to exons one and seven. Top, Stained gel of amplified products. Middle,
Hybridization of a blot prepared from the top gel with 32P-labeled
AP3-specific probe. Bottom, Amplification of cyclophilin (Cycl) by
RT-PCR showing the presence of template in all reactions.
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Petal- and Stamen-Specific Expression of U1-70K Antisense
Transcript Affected the Level of AP3 mRNA in These Organs
Because the AP3 gene is intact in the transgenic lines, we expect
it to be expressed in flowers with partially developed petals and stamens. It
has been shown previously that the AP3 promoter is active throughout
the development of petals and stamens
(Jack et al., 1994;
Tilly et al., 1998).
Furthermore, the maintenance of AP3 expression is dependent on its
own transcription (autoregulation; Goto
and Meyerowitz, 1994; Jack et
al., 1994). In rudimentary petals and stamens of transgenic plants
with high levels of antisense transcript, we expect severe impairment of
splicing due to low or no U1-70K, resulting in little or no processed mRNA.
Hence, we tested the level of AP3 mRNA in these transgenic floral
tissues using RT-PCR analysis. AP3 pre-mRNA contains six introns that
are spliced to produce functional mRNA in petals and stamens. Using primers
corresponding to the first and seventh exons of the AP3 gene, we
detected the expected size (about 400 bp) PCR-amplified product in the RT
sample from the RNA of the wild type and the transgenic flowers with less
severe (fertile) phenotype (Fig.
5B). However, in RT samples obtained from the floral RNA of severe
phenotype (sterile), no PCR products were detected
(Fig. 5B). Hybridization with
the same AP3-specific probe (7.5-kb BamHI fragment) confirmed these
results (Fig. 5B, middle).
These data suggest that there is no mature mRNA from AP3 gene present
in transgenic flowers with severe phenotype, whereas there is a high level of
AP3-driven U1-70K antisense product. The presence of first
strand cDNA in all samples was verified by amplifying cyclophilin
(Fig. 5B, bottom;
Golovkin and Reddy, 1999).
These results show a strong correlation between the disappearance of mature
AP3 transcripts in flowers with a severe phenotype and a high level
of antisense U1-70K transcript. The probable reason for the lack of
AP3 transcripts in flowers with severe phenotype is that the unspliced AP3
mRNA is unstable and undergoes degradation. Alternatively, it is possible that
the AP3 promoter of the transgenic construct may compete with the
endogenous AP3 promoter for the trans-acting factors such as AP1,
AP3, PI, and AG that are known to interact with the AP3 promoter
(Hill et al., 1998), resulting
in no expression of AP3 mRNA. The fact that we did not observe
ap3 mutant phenotype in about 200 transgenic plants transformed with
GUS reporter driven by AP3 promoter indicates that the second
possibility is very unlikely.
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DISCUSSION
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In metazoans, U1-70K has been shown to interact with Ser/Arg-rich splicing
factors of the SR family (SC35, ASF/SF2, and Sip1) and control both basic and
alternative splicing (Wu and Maniatis,
1993; Kohtz et al.,
1994; Manley and Tacke,
1996; Zhang and Wu,
1998). The yeast U1-70K also interacts with a large number of
proteins (Fromont-Racine et al.,
1997). The Arabidopsis U1-70K shares significant similarity with
human and metazoan U1-70K and interacts with a novel set of proteins including
SR45, a plant-specific SR protein, two 9G8-like splicing factors, and an
SC35-like protein (Golovkin and Reddy,
1998,
1999;
Lopato et al., 1999),
suggesting the plant U1-70K is also likely to regulate basic and alternative
splicing of pre-mRNAs. Because U1-70K interacts with several other proteins
involved in splicing, it was suggested that U1-70K serves as an assembly focus
for the functional components of the splicing/transport machinery (such as SR
proteins) and plays a key regulatory role in early events of spliceosome
assembly (Romac and Keene,
1995; Golovkin and Reddy,
1999; Lopato et al.,
1999). Hence, it is reasonable to expect that expression of
U1-70K antisense transcript would inhibit splicing of many pre-mRNAs
and the production of corresponding proteins resulting in adverse affects on
cellular processes. In fact, transient overexpression of full-length and the
truncated versions of U1-70K in cultured animal cells affected the nuclear
distribution of splicing factor SC35 and led to the inhibition of both
splicing and nucleocytoplasmic transport of mRNA
(Romac and Keene, 1995).
Quantitative analysis of nuclear and cytoplasmic RNA revealed an increase of
the amount of both intronless and unspliced nuclear messages due to U1-70K
overexpression. Interestingly, overexpression of the carboxy-terminal part of
U1-70K alone showed a similar effect. These results suggest that excess of a
splicing component can cause an imbalance in splicing factors and disruption
at the very early stages of spliceosome formation. A yeast strain lacking the
U1-70K can grow at low temperature, suggesting that U1-70K is
dispensable in yeast (Hilleren et al.,
1995). However, it is not known if U1-70K is dispensable in
multicellular eukaryotes in which both basic and alternative splicing play an
important role in growth and development.
In this study, we addressed this question by expressing the U1-70K
antisense transcript in a tissue-specific manner in petals and stamens to
determine if it is essential for the development of these organs. Because
U1-70K is coded by a single gene in Arabidopsis
(Golovkin and Reddy, 1996;
Arabidopsis Genome Initiative,
2000) it is an ideal candidate to block the expression using an
antisense approach. Several reports have shown that inhibition of expression
of endogenous genes in plants can be achieved at a reasonable frequency by
overexpressing the antisense transcript corresponding to the endogenous gene
(Kuipers et al., 1997). Our
antisense approach to block U1-70K in specific organs (petal and stamens)
resulted in suppression of development of these organs, suggesting that in
plants, and most likely in other multicellular organisms, U1-70K is essential.
Several lines of evidence presented here indicate that the observed phenotype
of flowers is due to expression of U1-70K antisense transcript and
not due to disruption of the AP3 or PI gene. First, Southern
analysis with these genes verified that they are intact in transgenic plants
(Fig. 5A). Second, the severity
of the phenotype was correlated with the extent of expression of
U1-70K antisense transcript in petals and stamens
(Fig. 4B). Third, unlike in
mutants of AP3 or PI where petals and stamens are
transformed to sepals and carpels, respectively, flowers of transgenic plants
expressing U1-70K antisence RNA had either rudimentary or partially
developed petals and stamens (Fig.
3A). Fourth, several independent transgenic lines showed similar
phenotype. Finally, we did not observe this phenotype in hundreds of
transgenic plants that are transformed with the same promoter fused to a GUS
reporter gene. Taken together, our results clearly show that the targeted
expression of U1-70K antisense transcript in petals and stamens
effectively suppresses their development. Based on the role of U1-70K, it is
likely that low or no expression of U1-70K adversely affects the splicing of
pre-mRNAs, thereby altering the levels of several proteins.
Expression of antisense genes in transgenic plants has been used
successfully in plants to block expression of the corresponding genes
(Chuang and Meyerowitz, 2000).
The reductions in endogenous mRNA, protein, and/or enzyme activity level could
vary greatly in different transgenic lines
(Kuipers et al., 1997;
Chuang and Meyerowitz, 2000).
Here, we observed the expected phenotype only in 11% of transgenic lines.
Furthermore, although the lines with abnormal flowers were all male sterile,
the extent to which petals and stamens developed varied. In some lines,
flowers showed very rudimentary petals and stamens, whereas in others, these
organs were partially developed. The observed variation in phenotype is due to
the level of expression of antisense transcript because high levels of
antisense transcript correlated with the severe phenotype. The reason for
strong level of expression in some plants but not in others could be due to
the number of copies of the introduced gene, the influence of adjacent plant
genomic DNA sequences, and/or chromosomal structure at the integration site
(Peach and Velten, 1991).
Variability in expression level of introduced gene and its phenotype is a
general feature of antisense lines
(Kuipers et al., 1997;
Chuang and Meyerowitz, 2000).
Similar variations in promoter activity were observed with other promoters
also (Peach and Velten, 1991).
As shown in Figure 1, with the
AP3-GUS construct there is significant difference in the strength and
specificity of the expression in petals and stamens. The appearance of
carpel-like structures rarely in some flowers could be caused by splicing
alterations due to partial suppression of U1-70K, which may have
resulted in the unbalanced expression of homeotic flower development genes
(Jack et al., 1992;
Goto and Meyerowitz, 1994).
The presence of very rudimentary petals and stamens suggests that either the
expression of U1-70K is not completely blocked by antisense RNA or the U1-70K
protein present in the precursor cells of stamens and petals, before
activation of the AP3 promoter, may be sufficient for the development of these
rudimentary organs. The latter possibility is most likely if the U1-70K
protein is stable with a long half-life. Despite the fact that the depletion
of U1snRNP in in vitro splicing extracts derived from animal cells can be
complemented with an excess amount of a single SR protein or mixture of SR
proteins (Crispino et al.,
1994; Tarn and Steitz,
1994), our in vivo studies show that U1-70k is essential in plants
and likely in other multicellular organisms.
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MATERIALS AND METHODS
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Preparation of U1-70K Antisense Construct
We used pCGN1547-based pLB215 plasmid
(Jack et al., 1994) to prepare
U1-70K antisense construct. The bacterial reporter gene uidA
(GUS) in pLB215, which is between the APETALA3 (AP3)
promoter and NOS3 terminator, was replaced with the long cDNA of
U1-70K in antisense orientation. First, a fragment containing both
the AP3 promoter and GUS reporter was removed from pLB215 plasmid by
partial XbaI/BamHI digestion and placed into
SpeI/XbaI of pBluescript (KS) plasmid. The GUS gene was then
excised from this construct by BsmI/SpeI digestion. The
resulting plasmid contained the AP3 promoter and a small (50-bp) GUS fragment
with a unique BamHI site. The 750-bp AP3 promoter from this construct
was released by XbaI/BamHI digestion and subcloned back into
the same XbaI/BamHI pLB215 vector from the first step. The
resulting plasmid contained AP3 promoter and NOS3 terminator with a convenient
BamHI site in between. The Arabidopsis U1-70K cDNA from the
large transcript (Golovkin and Reddy,
1996) was obtained by ApaI/SmaI digestion. The
fragment was blunted with T4 polymerase and inserted into the unique
BamHI site (blunted by Klenow) to generate the pAP3-U1-70K
antisense construct.
Generation of Transgenic Plants
A vacuum infiltration procedure was used to generate Arabidopsis transgenic
plants essentially as described by Bechtold et al.
(1993). Plasmid constructs
(AP3-GUS or AP3-U1-70K) were introduced into
Agrobacterium tumefaciens strain ASE by electroporation using a Cell
Porator (Invitrogen, Carlsbad, CA), and the transformants were selected on
Luria-Bertani medium plates with antibiotics (kanamycin, chloramphenicol, and
gentamycin). Three- to four-week-old Arabidopsis (Columbia) plants were
infiltrated and grown to maturity. Seeds were collected, dried for at least 10
d, sterilized, and stratified at 4°C in a 0.1% (w/v) agarose solution for
3 d. Seeds were then spread on selection plates (Murashige and Skoog medium
containing 1% [w/v] Suc, 50 µg mL–1 kanamycin,
and 500 µg mL–1 timentin) and grown under
continuous light. Plants that grew on these plates were transferred to fresh
plates and finally to soil. About 100 transgenic plants were obtained and
analyzed. Using cross-pollination with pollen from wild-type Arabidopsis
(Columbia) flowers, we were able to maintain the mutant phenotype of these
plants for several generations. Flowers from transgenic plants carrying the
GUS reporter driven by the AP3 promoter were stained for GUS
activity.
Scanning Electron Microphotography
Young inflorescences were incubated in 2% (w/v) glutaraldehyde in 0.025
M sodium phosphate (pH 7.0) at 4°C. Samples were transferred to
1% (w/v) osmium tetroxide in 0.025 M sodium phosphate buffer (pH
7.0) for 4 to 6 h at room temperature and in 0.05 M sodium
cocodylate buffer (pH 7.0) for 12 h. Dehydration was performed through a
standard ethanol battery (50%, 70%, 80%, 90%, and twice at 100% [v/v]) and
then dried in liquid CO2. Individual flowers were removed from the
inflorescence and mounted on a scanning electron microphotography table.
Flower buds were carefully opened using glass needles and then coated with
gold (4:1 [w/v]) and photographed at an accelerating voltage of 20 kV.
DNA and RNA Gel-Blot Analyses
DNA gel-blot analysis of individual transgenic plants was performed as
described earlier (Golovkin and Reddy,
1996). Genomic DNA was digested with BamHI, which leaves
the U1-70K intact. Ten micrograms of genomic DNA was separated in a 0.8% (w/v)
Tris-acetate EDTA agarose gel, blotted onto a Hybond N+ (Amersham,
Buckinghamshire, UK) nylon filter, and probed with a 1.1-kb fragment of
32P-labeled U1-70K cDNA. Hybridization was carried out for
3 h at 65°C. Total RNA from flowers and flower buds was isolated using
"Trizol" (Life Technologies/Gibco-BRL). RNA (50 µg) was
resolved in a formaldehyde-containing agarose gel and transferred onto a
Hybond N+ (Amersham) nylon filter. The 910-bp PCR product of an
"alternative intron" from the Arabidopsis U1-70K gene was
used to detect both the U1-70K antisense transcript and the
endogenous U1-70K large transcript.
To exclude the possibility that flower developmental genes are disrupted by
T-DNA integration, we used the Arabidopsis AP3 gene and the PI
cDNA probes to hybridize with the same blots of BamHI-digested
genomic DNA.
RT-PCR Analysis for AP3 Gene Expression
One microgram of total RNA from wild type and transgenic plants was treated
with RNAse-free DNAse and used to synthesize first strand cDNA. Primers
5'-CGATTATCATGTTCTCTAGCTCC-3' and
3'-TTGTCTACTAGTCCATAGTGAGG-5' specific to the first and seventh
exon of AP3 gene were used to amplify a approximately 400-bp fragment
of AP3 transcript. The PCR reactions were performed using the "Expand
Long template" PCR system (Boehringer Mannheim/Roche, Basel) in a final
volume of 50 µL. The first six cycles of PCR consisted of 92°C for 1
min, 58°C for 30 s, and 68°C for 3 min, and the next 29 cycles
consisted of 92°C for 1 min, 60°C for30 s, and 68°C for 3 min with
a 10-s gradient increase in extension time. The amplified products were
separated in a 1% (w/v) agarose gel and blotted onto a nylon membrane. A
7.5-kb BamHI fragment of the AP3 gene from pD275 plasmid was
used as a probe to detect the amplified 400-bp product. The amount of first
strand cDNA in each reaction was verified using the primers (forward,
5'-GTC TGATAGAGATCTCACGT-3'; and reverse,
5'–AATCGGCAACAACCACAG GC-3') corresponding to a
constitutively expressed cyclophilin gene
(Golovkin and Reddy,
1999).
|
ACKNOWLEDGMENTS
|
|---|
We thank Dr. Thomas Jack for providing the AP3 gene and A.
tumefaciens ASE strain and Dr. Day for her critically reading the
manuscript.
Received March 6, 2003;
returned for revision April 8, 2003;
accepted April 24, 2003.
|
FOOTNOTES
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|---|
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.103.023192.
1 This work was supported by the Department of Energy, Division of Energy
Biosciences (grant no. DE–FG03–01ER15199 to A.S.N.R.). 
*
Corresponding author, e-mail
reddy{at}colostate.edu;
fax 970–491–0649.
|
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