Distinct roles of the first introns on the expression of Arabidopsis profilin gene family members

Abstract

Profilin is a small actin-binding protein that regulates cellular dynamics of the actin cytoskeleton. In Arabidopsis (Arabidopsis thaliana), five profilins were identified. The vegetative class profilins, PRF1, PRF2, and PRF3, are expressed in vegetative organs. The reproductive class profilins, PRF4 and PRF5, are mainly expressed in pollen. In this study, we examined the role of the first intron in the expression of the Arabidopsis profilin gene family using transgenic plants and a transient expression system. In transgenic plants, we examined PRF2 and PRF5, which represent vegetative and reproductive profilins. The expression of the PRF2 promoter fused with the beta-glucuronidase (GUS) gene was observed in the vascular bundles, but transgenic plants carrying the PRF2 promoter-GUS with its first intron showed constitutive expression throughout the vegetative tissues. However, the first intron of PRF5 had little effect on the reporter gene expression pattern. Transgenic plants containing PRF5 promoter-GUS fusion with or without its first intron showed reproductive tissue-specific expression. To further investigate the different roles of the first two introns on gene expression, the first introns were exchanged between PRF2 and PRF5. The first intron of PRF5 had no apparent effect on the expression pattern of the PRF2 promoter. But, unlike the intron of PRF5, the first intron of PRF2 greatly affected the reproductive tissue-specific expression of the PRF5 promoter, confirming a different role for these introns. The results of a transient expression assay indicated that the first intron of PRF1 and PRF2 enhances gene expression, whereas PRF4 and PRF5 do not. These results suggest that the first introns of profilin genes are functionally distinctive and the first introns are required for the strong and constitutive gene expression of PRF1 and PRF2 in vegetative tissues.

Figure 1.

Histochemical analysis of GUS expression in transgenic Arabidopsis containing pPRF2-5′ and pPRF2e-p2i constructs. A, Schematic representations of PRF2 promoter-GUS fusion constructs. The box with PRF2 pr is the promoter region of PRF2; the box with e is the first exon of PRF2; the black box with GUS is the GUS coding region; the line with p2i is the first intron of PRF2; ATG is the start codon. B to G, From pPRF2-5′; H to M, from pPRF2e-p2i; B and H, 3-d-old seedlings; C and I, roots of 3-d-old seedlings; D and J, 15-d-old plants; E and K, rosette leaves; F and L, flowers from 5-week-old plants; G and M, young siliques. GUS staining was performed for 12 h. Scale bars, 1 mm (B, D, E, H, J, and K), 0.2 mm (C, G, I, and M), and 0.5 mm (F and L).

Figure 2.

RT-PCR analysis of transcript expression of transgenic Arabidopsis containing pPRF2-5′ and pPRF2e-p2i constructs in various tissues. One microgram of total RNA from each organ was used for RT. TUBULIN2 (TUB2) amplified with TUB-F (5′-CTCAAGAGGTTCTCAGCAGTA-3′) and TUB-R (5′-CTCAAGAGGTTCTCAGCAGTT-3′) primers was used as a control. After electrophoresis of PCR products, it was transferred to the nylon membrane and hybridized with ³²P-labeled GUS or TUB2 probes.

Figure 3.

Western-blot analysis and determination of GUS activity in transgenic Arabidopsis containing pPRF2-5′ and pPRF2e-p2i. A, Total soluble protein (20 μg) from mature leaves was loaded per lane and analyzed using monoclonal GUS antibody. B, GUS activity is expressed as pmol 4-methyl-umbelliferone min⁻¹ mg⁻¹ protein. Values are means ± sd (n = 3).

Figure 4.

Histochemical analysis of GUS expression in transgenic Arabidopsis containing pPRF5e and pPRF5e-p5i constructs. A, Schematic representations of PRF5 promoter-GUS fusion constructs. The gray box with PRF5 pr is the promoter region of PRF5; the gray box with e is the first exon of PRF5; the black box with GUS is the GUS coding region; the line with p5i is the first intron of PRF5; ATG is the start codon. B to D, From pPRF5e; E to G, from pPRF5e-p5i; B and E, 3-d-old seedlings; C and F, 15-d-old plants; D and G, flowers. Scale bars, 0.5 mm (B, D, E, and G), 1 mm (C and F), and 0.1 mm (anthers in small box).

Figure 5.

Histochemical analysis of GUS expression in transgenic Arabidopsis containing pPRF2e-p5i and pPRF5e-p2i. A, Schematic representation of promoter-GUS fusion constructs. The white box with PRF2 pr is the promoter region of PRF2; the white box with e is the first exon of PRF2; the line with p2i is the first intron of PRF2; the gray box with PRF5 pr is the promoter region of PRF5; the gray box with e is the first exon of PRF5; the line with p5i is the first intron of PRF5; the black box with GUS is the GUS coding region; ATG is the start codon. B to D, From pPRF2e-p5i; E to G, from pPRF5e-p2i; B and E, 3-d-old seedlings; C and F, 15-d-old plants; D and G, flowers. Scale bars, 0.5 mm (B, D, E, and G), 1 mm (C and F), and 0.1 mm (anthers in small box).

Figure 6.

GUS staining patterns of transgenic Arabidopsis containing pPRF2-5′ and pPRF2e-p2i constructs at various incubation time points. Seedlings grown on Murashige and Skoog medium for 3 d were incubated for 1, 4, 8, and 12 h, respectively, and the reaction was stopped and the tissues cleared by incubation with 100% ethanol.

Figure 7.

Transient expression analysis of various PRF2 and PRF5 promoter-GUS fusion constructs. A, Schematic representation of construction for transient expression. GP at the end of the construct name indicates a vector for transient expression. The white box with m is a 35S minimal promoter; the black box with GUS is the GUS ORF; the thin line with p2i is the first intron of PRF2; the white box with PRF2 pr is the PRF2 promoter region; the white box with e is the first exon of PRF2; ATG is the start codon; ORF is the ORF of PRF2 cDNA; the white boxes with e2 and e3 are the second and third exons of PRF2; the line with p2i-2 is the second intron of PRF2; the gray box with PRF5 pr is the PRF5 promoter region; the gray boxes with e2 and e3 are the second and third exons of PRF5; the lines with p5i and p5i-2 are the first and second introns of PRF5. Arrows indicate the orientation of intron. The numbers in intron deletion constructs indicate the deletion points from the 5′ end of the intron. B, Relative normalized GUS activity in percentage. The full name of each construct is the addition of each insert name into the brackets of the vector name. For example, p[p2iF-]35SmGP is the pp2iF-35SmGP construct. Each transfection was performed at least three times. GUS activity was normalized with cotransfected luciferase activity and relative activity of pPRF2eGP was set to 100%. Bars are means ± sd.

Figure 8.

Effect of the first intron on the expression of the Arabidopsis profilin gene family. A, Schematic representation of construction for transient expression. GP at the end of the construct name indicates a vector for transient expression. The white box is vegetative genes; the gray box is reproductive genes; the white or gray box with e is the first exon of each gene; the black box with GUS is GUS ORF; the thin lines with p1i, p2i, p3i, p4i, and p5i are the first introns of PRF1, PRF2, PRF3, PRF4 and PRF5, respectively; the white or gray box with gene name pr is the promoter region of each gene; ATG is the start codon. B, Relative normalized GUS activity in percentage. A plus (+) indicates the presence of an intron within the construct; a minus (−) indicates no intron. Each transfection was performed at least three times. GUS activity was normalized with cotransfected luciferase activity and relative activity of pPRF2eGP was set to 100%. Bars are means ± sd.