Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals

Abstract

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.

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.

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).

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.

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.

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.