Light-mediated regulation defines a minimal promoter region of TOP2

Abstract

Light signaling has been demonstrated to be an important factor for plant growth and development; however, its role in the regulation of DNA replication and cell cycle has just started to be unraveled. In this work, we have demonstrated that the TOP2 promoter of Pisum sativum (pea) is activated by a broad spectrum of light including far-red light (FR), red light (RL) and blue light (BL). Deletion analyses of the TOP2 promoter in transformed plants, Arabidopsis thaliana and Nicotiana tobaccum (tobacco), define a minimal promoter region that is induced by RL, FR and BL, and is essential and sufficient for light-mediated activation. The minimal promoter of TOP2 follows the phytochrome- mediated low-fluence response similar to complex light regulated promoters. DNA-protein interaction studies reveal the presence of a DNA binding activity specific to a 106 bp region of the minimal promoter that is crucial for light-mediated activation. These results altogether indicate a direct involvement of light signaling in the regulation of expression of TOP2, one of the components of the DNA replication/cell cycle machinery.

Figure 1

Light-regulated expression of TOP2 gene in pea. (A) Seedlings were grown for 6 days in either constant dark (D) or in constant white light (WL) for RNA gel blot analysis. Total RNA (25µg) was loaded in each lane; 18S rRNA (18S) was shown as a loading control. A representative autorad from three independent experiments was shown. (B) Five-day-old dark grown seedlings were transferred to red light (RL), far-red light (FR) or blue light (BL) for 6 or 24 h for transcript analysis. Total RNA (25µg) was loaded in each lane and 18S rRNA (18S) was used as a loading control. A representative autorad from four independent experiments was shown. (C) Seedlings were grown for 7 days in constant dark (D), or grown for 7, 14 or 21 days in constant white light (WL) for RNA gel blot analysis. Total RNA (25µg) was loaded in each lane; 18S rRNA (18S) was shown as a loading control. A representative autorad from three independent experiments was shown. (D) Quantification of the data in (A) by Fluor-S-MultiImager (Bio-Rad) (E) Quantification of the data in (B) by Fluor-S-MultiImager (Bio-Rad) (F) Quantification of the data in (C) by Fluor-S-MultiImager (Bio-Rad).

Figure 2

Minimal promoter region of TOP2 that is activated by light in transgenic Arabidopsis. (A) Schematics of deletion constructs of TOP2 promoter fused to GUS reporter. The arrow indicates the transcriptional start site, and the numbers indicate the length of each undeleted or deleted constructs from the transcriptional start site. (B) Three independent lines (UD1, UD2 and UD3) containing UD-TOP2-GUS transgene were used to determine the GUS activity. Six-day-old constant dark (D) or constant white light (WL) grown seedlings were used for GUS activity measurement. The error bars indicate standard deviation from at least three independent experiments; the experiment was repeated three times. (C) Three independent lines (D1–1, D1–2 and D1–3) containing D1-TOP2-GUS transgene were used to determine the GUS activity. For experimental detail see legend to (B). (D) Three independent lines (D2–1, D2–2 and D2–3) containing D2-TOP2-GUS transgene were used to determine the GUS activity. For experimental detail see legend to (B). (E) Four-day-old dark grown seedlings were transferred to WL for 12 (12h), 24 (24h), 48 h (48h) or kept in the dark for another 48 h (0h) and GUS activities were measured. D1 indicates D1-TOP2-GUS transgene and D2 indicates D2-TOP2-GUS transgene. The error bars indicate standard deviation from at least three independent experiments; the experiment was repeated four times.

Figure 3

Activation of TOP2 minimal promoter by various wavelengths of light. Four-day-old dark grown seedlings were transferred to red light (RL), far-red light (FR), blue light (BL) and white light (WL) for 48 h and GUS activities were measured. Six-day-old dark grown seedlings were used as dark (D) control. The level of activation at various wavelengths of light of (A) UD-TOP2-GUS transgene, (B) D1-TOP2-GUS transgene and (C) D2-TOP2-GUS transgene.

Figure 4

RL pulse-mediated induction and its cancellation by FR light of dark grown seedlings containing UD-TOP2-GUS, D1-TOP2-GUS or CAB1-GUS transgenes. Five-day-old dark grown seedlings (D) were exposed for 2 min to RL or followed by 10 min exposure of FR light (RL+FR). After the light treatments the seedlings were kept in dark for an optimum period of 20 h before the seedlings were harvested for GUS activity measurements. (A) UD-TOP2-GUS transgene. (B) D1-TOP2-GUS transgene. (C) CAB1-GUS transgene.

Figure 5

Tissue specific expression of UD-TOP2-GUS, D1-TOP2-GUS or D2-TOP2-GUS transgenes in Arabidopsis or tobacco light grown seedlings. (a–c) Six-day-old Arabidopsis seedlings containing UD-TOP2-GUS, D1-TOP2-GUS or D2-TOP2-GUS transgenes, respectively. (d–f) Tobacco seedlings containing UD-TOP2-GUS, D1-TOP2-GUS or D2-TOP2-GUS transgenes, respectively, were used for transverse section of the stem and GUS staining. (g) The tobacco seedling used for serial transverse section and GUS staining. (h–l) Tobacco seedling as shown in (g) was used for serial transverse section of the stem and staining. The arrows indicate the leaf traces.

Figure 6

Identification of TOP2 minimal promoter-specific DNA-binding activity. (A) DNA sequences of TOP2 minimal promoter region from –468 to –262 bp (D1–D3), and the multiple cloning site (MCS) of pBluescript from 653 to 759 bp. The arrow indicates the start of D2–D3 promoter fragment. The cis-acting elements are underlined: two I boxes and GA box by thin line and AT1 box by thick line. (B) Electrophoretic mobility shift (gel shift) analysis using the whole cell extracts of 6-day-old light grown pea seedlings and 207 bp D1-D3 DNA as probe. No protein extract was added in lane 1. Four micrograms of extract were added in lane 2, 8 µg of whole cell extract were added in lanes 3–6. The amount of competitors added in lanes 4, 5 and 6 was 50 ng D1–D3, 100 ng D1–D3 and 100 ng MCS, respectively. The increasing concentration of D1–D3 unlabeled DNA is shown by triangles. Plus and minus signs indicate the presence and absence, respectively, of whole cell extracts (Pro. Ext.) or competitors (Comp.). (C) Electrophoretic mobility shift (gel shift) analysis using the whole cell extracts of 6-day-old light grown pea seedlings and 207 bp D1–D3 DNA as probe. No whole cell extract was added in lanes 1. Four micrograms of whole cell extract were added in lane 2, and 8 µg in lanes 3–7. The amount of competitors added in lanes 4–7 was 50 ng D2–D3, 100 ng D2–D3, 50 ng D1–D2 and 100 ng D1–D2, respectively. The increasing concentration of D1–D2 or D2–D3 unlabeled DNA is shown by triangles. Plus and minus signs indicate the presence and absence, respectively, of whole cell extracts (Pro. Ext.) or competitors (Comp.).

Figure 7

DNase I foot printing analysis of 207 bp D1–D3 fragment (top strand) of TOP2 promoter. Lane 1 shows the A+G Maxam and Gilbert sequencing ladder. Lane 2 is the control lane (cont.) without any protein. Lanes 3 and 4 show the ladder caused by DNase I cleavage with 10 and 20 µg of whole cell extract (Pro. Ext.), respectively. The triangle indicates the increasing concentrations of whole cell extracts. The hypersensitive nucleotides are marked as open circles and protected nucleotides as stars.

Figure 8

Electrophoretic mobility shift (gel shift) analysis using the whole cell extracts of 6-day-old dark or light grown pea seedlings and D1–D2 DNA as probe. (A) The DNA sequence of AT1&I box and its mutated version, AT1&I (m). (B) No protein extract was added in lane 1. Eight micrograms of protein extract were added in lane 2 from dark grown seedlings. Eight micrograms of protein extract from light grown seedlings were added in lanes 3–7. The amount of AT1&I box DNA fragment added in lanes 4 and 5 was 50 and 100 ng, respectively. The amount of AT1&I (m) box DNA fragment added in lanes 6 and 7 was 50 and 100 ng, respectively. The increasing concentration of unlabelled competitor DNA is shown by triangles. Plus and minus signs indicate the presence and absence, respectively, of whole cell extracts (Pro. Ext.) or competitors (Comp.).