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. 2003 Sep;14(9):3565-77.
doi: 10.1091/mbc.e03-02-0109. Epub 2003 Jun 13.

Ectopic expression of atRSZ33 reveals its function in splicing and causes pleiotropic changes in development

Maria Kalyna  1 , Sergiy Lopato, Andrea Barta
Affiliations

Ectopic expression of atRSZ33 reveals its function in splicing and causes pleiotropic changes in development

Maria Kalyna et al. Mol Biol Cell. 2003 Sep.

Abstract

Splicing provides an additional level in the regulation of gene expression and contributes to proteome diversity. Herein, we report the functional characterization of a recently described plant-specific protein, atRSZ33, which has characteristic features of a serine/arginine-rich protein and the ability to interact with other splicing factors, implying that this protein might be involved in constitutive and/or alternative splicing. Overexpression of atRSZ33 leads to alteration of splicing patterns of atSRp30 and atSRp34/SR1, indicating that atRSZ33 is indeed a splicing factor. Moreover, atRSZ33 is a regulator of its own expression, as splicing of its pre-mRNA is changed in transgenic plants. Investigations by promoter-beta-glucuronidase (GUS) fusion and in situ hybridization revealed that atRSZ33 is expressed during embryogenesis and early stages of seedling formation, as well as in flower and root development. Ectopic expression of atRSZ33 caused pleiotropic changes in plant development resulting in increased cell expansion and changed polarization of cell elongation and division. In addition, changes in activity of an auxin-responsive promoter suggest that auxin signaling is disturbed in these transgenic plants.

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Figures

Figure 1.
Figure 1.
Protein and RNA analyses of 35S:atRSZ33 transgenic Arabidopsis plants. (A) Western blot analysis of total protein extracts from control and T3 homozygous 35S:atRSZ33 lines (lanes 1–8) and SR proteins preparation from wild-type plants (lane 9). Lanes 1 and 2, wild-type plants and plants containing pBI121, respectively; 35S: catRSZ33 lines, lanes 3–5; one weaker 35S:gatRSZ33 line, lane 6; and two 35S:gatRSZ33 lines with stronger phenotype, lanes 7 and 8. Asterisks indicate unspecific bands. Arrow indicates position of atRSZ33 protein. (B) RT-PCR analysis of atRSZ33 transcripts in T0 generation. Lane 1, control plants containing pBI121; plants transformed with cDNA (lanes 2 and 3) and with genomic construct (lanes 4 and 5). (C) RT-PCR analysis of atRSZ33 transcripts in T3 generation (top). Lanes 1–8, same plants as indicated in A. Asterisk indicates unspecific band. Schematic representation of atRSZ33 gene structure, its mRNA isoforms detected in transgenic 35S:gatRSZ33 plants, and deduced proteins (bottom). Exons are shown as black boxes, 5′ and 3′-UTRs are gray boxes. 3′alt ss-3′ alternative splice site in the second intron. Bold lines represent included sequences of the second and third introns. Stars indicate premature stop codons. Deduced protein structures are shown as black, RRM; gray, RS-rich region; white, two zinc knuckles; light gray, SP region; and striped boxes, sequences included due to alternative splicing. Positions of primers used for RT-PCR analyses are shown as arrows. Schematic drawings are not to scale.
Figure 2.
Figure 2.
Analysis of transcript patterns of atSRp30, atSRp34/SR1, and AIR3 in plants overexpressing atRSZ33. Primers used for RT-PCR are shown by arrows on the schemes of corresponding genes. Exons are shown as boxes and introns as lines (bold lines are introns included in the mRNAs). 5′- and 3′-UTRs are gray, and coding regions are black. 35S:gatRSZ33n6 and 35S:gatRSZ33n7 are two independent transgenic lines overexpressing atRSZ33. The following control lines were used: transgenic line carrying pBI101, and transgenic line containing atRSZ33 promoter-GUS fusion. RT-PCR of ubiquitin was used to control loading. 1/10, 1/5, x2 and x1 are consecutive dilutions of RNA sample from 35S:gatRSZ33n7 line. Controls for DNA contamination of all RNA preparations were done by omitting reverse transcriptase, and controls for ubiquitin only are shown (–RT).
Figure 3.
Figure 3.
Expression patterns of atRSZ33. Histochemical localization of atRSZ33 promoter-GUS activity (A–N) and in situ hybridization of atRSZ33 transcripts (O–T). (A) Immature seed with embryo at the globular stage of development (arrow). (B) Torpedo stage embryo. (C) Germinating seed. (D–F) Arabidopsis seedling, showing expression in the tip and in the vasculature of cotyledon (D), in the shoot apical meristem (E) and at the site of secondary root formation at the hypocotyl-root junction (F). (G) Expression in the elongation zone of primary root. (H–K) Different stages of the lateral root formation. (L) Young flowers with expression in the tapetum within anthers and in stigma and style. (M) Flower after opening with expression in mature and germinating pollen, in stigma and ovules. (N) Part of immature silique with staining in developing seeds, funiculi and septum. (O) and (P) Shoot apical meristem of 5-d-old seedling. (Q and R) Mature pollen within anther. (S and T) Young silique with developing seeds. (O, Q, and S) Antisense probe. (P, R, and T) Sense probe.
Figure 4.
Figure 4.
Influence of atRSZ33 overexpression on the embryo and seedling development. (A) Wild-type seed with embryo at globular stage. (B) 35S:gatRSZ33 seed, containing twin embryos indicated by arrows 1 and 2. (C and D) Close-up of embryos shown in B. (E and F) Close-up of wild-type and 35S:gatRSZ33 embryos at globular stage, respectively, showing abnormal division in suspensor (arrow in F). (G and H) Wild-type and transgenic embryos at heart stage. (I) Eight-day-old wild-type seedling. (J, M, and N) Eight-day-old transgenic seedlings. (K) Three-day-old transgenic seedling. (J) Twin seedlings, one of them with single cotyledon. (K) Equally developed twin seedlings. (L and M) Seedlings with single cotyledon. Arrows in J, L, and M point out shoot apical meristems. Bars, 50 μm (A–H) and 1 mm (I–N).
Figure 5.
Figure 5.
Ectopic expression of atRSZ33 affects cell size and shape, stomatal development, and formation of ectopic meristems. Plants, containing 35S:gatRSZ33 (E–H, J, and K) were compared with wild-type plants (A–D, and I) of the same age and grown under the same conditions. (A and E) Cotyledons. (B and F) Hypocotyls. (C and G) Root hairs. Arrows in G point out root hairs with abnormal branched shape. (D and H) Trichomes. (I) Stomata in the leaf epidermis of wild-type plants, arrows point out stomata precursors formed due to asymmetric cell divisions, arrows point out areas with multiple misoriented cell divisions. (J) Stomatal development in the leaf epidermis of 35S:gatRSZ33 plants. (K) Formation of stomatal clusters in the leaf epidermis of 35S:gatRSZ33 plants. (L) Ectopic structure (arrow) on the cotyledon of 10-d-old transgenic seedling. Note abnormal shape of the cotyledon surface. (M) Abaxial side of the rosette leaf with ectopic structures formed over midvein and on the edge of the leaf (indicated by arrows). (N) Ectopic meristem developed on the cotyledon surface (arrow points out enlarging trichome precursor). (O) Ectopic meristem formed in the epidermal layer of rosette leaf. Bars, 100 μm (A, B, E, and F), 200 μm (C, D, G, and H) and 25 μm (N and O).
Figure 6.
Figure 6.
Changes in the activity of DR5-GUS promoter construct and of the in vitro regeneration process in 35S:gatRSZ33 plants. (A–C) DR5: GUS activity in the root meristems of primary roots of the control DR5: GUS plants and two homozygous DR5:GUS/35S:gatRSZ33 lines (weak and strong lines), respectively. Arrow in C points out the cell with residual GUS activity. (D and G) DR5:GUS activity in the rosette leaves of control and DR5:GUS/35S:gatRSZ33 plants, respectively. (E) Close-up of rosette leaf of control plant. (H) Close-up of the same area of rosette leaf in the DR5:GUS/35S:gatRSZ33 plant, showing enhanced DR5:GUS activity and additional formation of provascular bands. (F) Close-up of control rosette leaf, including midvein. (I) Close-up showing accumulation of DR5:GUS activity near midvein in the rosette leaf of DR5:GUS/35S:gatRSZ33 plant. GUS activity is visible as blue staining on A–C and as a pink staining in dark field on D–I. (J) Control leaf explant with multiple green clusters of shoot meristems on the shoot-induction medium. (K) 35S:gatRSZ33 leaf explant with roots on the same medium. Bars, 50 μm (A–C), 500 μm (D and G), and 100 μm (E, F, H, and I).
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