A 9 bp cis-element in the promoters of class I small heat shock protein genes on chromosome 3 in rice mediates L-azetidine-2-carboxylic acid and heat shock responses

Abstract

In rice, the class I small heat shock protein (sHSP-CI) genes were found to be selectively induced by L-azetidine-2-carboxylic acid (AZC) on chromosome 3 but not chromosome 1. Here it is shown that a novel cis-responsive element contributed to the differential regulation. By serial deletion and computational analysis, a 9 bp putative AZC-responsive element (AZRE), GTCCTGGAC, located between nucleotides -186 and -178 relative to the transcription initiation site of Oshsp17.3 was revealed. Deletion of this putative AZRE from the promoter abolished its ability to be induced by AZC. Moreover, electrophoretic mobility shift assay (EMSA) revealed that the AZRE interacted specifically with nuclear proteins from AZC-treated rice seedlings. Two AZRE-protein complexes were detected by EMSA, one of which could be competed out by a canonical heat shock element (HSE). Deletion of the AZRE also affected the HS response. Furthermore, transient co-expression of the heat shock factor OsHsfA4b with the AZRE in the promoter of Oshsp17.3 was effective. The requirement for the putative AZRE for AZC and HS responses in transgenic Arabidopsis was also shown. Thus, AZRE represents an alternative form of heat HSE, and its interaction with canonical HSEs through heat shock factors may be required to respond to HS and AZC.

Fig. 1.

Localization of the L-azetidine-2-carboxylic acid response element (AZRE) by 5′ deletion analysis of the Oshsp17.3 promoter. (A) Nucleotide sequence of the intergenic region between Oshsp17.3 and Oshsp18.0. The transcription start site is in bold. The direction of transcription is shown by an arrow with a vertical line. The putative TATA box is indicated by a thin solid underline. The putative heat shock elements (HSEs) are underlined in bold with dots to designate matches to the core consensus (GAA/TTC) of the canonical HSE. The putative CAAT/CCAAT box is indicated by bold italic letters. The downward-pointing arrowhead shows the position used to generate the promoter–GUS-deleted construct in the Oshsp17.3 direction, and the upward-pointing arrowhead shows the same construct in the Oshsp18.0 direction. The putative AZRE is indicated by a dashed line. (B) Schematic representation of the 5′ deletion and internal deletion derivatives of the Oshsp17.3 promoter fused to the GUS reporter gene shown on the left. The number indicates the distance from the transcription start site of Oshsp17.3. The TATA box is shown as a vertical black rectangle. The putative perfect HSE is represented by a grey ellipse, and the putative imperfect HSE is represented by a hollow ellipse. The putative 9 bp AZRE is indicated by a hatched box. The thin black angled lines in the p567ΔAZRE and p310ΔAZRE constructs represent the truncation of the 9 bp AZRE in the Oshsp17.3 promoter. The bombarded coleoptiles were incubated at 28 °C in the dark for at least 6 h and then incubated with 5 mM AZC in phosphate buffer for 4 h. Then, the samples were incubated in fresh phosphate buffer at 28 °C in the dark for at least 12 h. The relative GUS activity was determined as shown in the right-hand panel. The fold induction relative to the control (28 °C) is indicated to the right of the scale bar. Each experiment was repeated at least three times. Relative GUS activites with ±SE are from at least 12 independent bombardments. Constructs with <2-fold induction by AZC and their P-values are shown at the right of the scale bar. All P-values are >0.05, which suggests no significant difference in induction.

Fig. 2.

Position and nucleotide sequence alignment of the four putative AZREs in AZC-inducible sHSP-CI gene promoters. The numbers indicate the number of nucleotides relative to the translation start codon (ATG). The putative AZREs are in bold uppercase letters. The putative HSEs in the promoters of Oshsp17.9A and Oshsp17.7 are underlined.

Fig. 3.

Effect of the 9 bp AZRE deletion on response to HS. The bombarded coleoptiles were incubated at 28 °C in the dark for at least 6 h and then at 41 °C for 2 h. Then, the samples were incubated in fresh incubation buffer at 28 °C in the dark for at least 12 h. The fold induction relative to the control (28 °C) is indicated next to the bars. Each experiment was repeated at least three times. Mean ±SE GUS activites are from at least 12 independent bombardments. The P-values for p270, p567(–)ΔAZRE, and p265 are shown. p270 and p567(–)ΔAZRE show a significant difference from the control with HS treatment (P ≤0.05), and p265 shows no significant difference (P=0.47).

Fig. 4.

Effect of a protein synthesis inhibitor on accumulation of Oshsp17.3 transcript induced by AZC and HS. Before 5 mM AZC or HS (41°C) treatment, 3-d-old rice seedlings were treated or not with cycloheximide (CHX) (2 μg ml⁻¹) for 30 min at 28 C. A 16 ng aliquot of DNase I-treated total RNA was used for RT-PCR. The RT-PCR products of Oshsp17.3 are shown by ethidium bromide staining, and the RT-PCR product of the 18S rRNA was used as an internal PCR control.

Fig. 5.

Electrophoretic mobility shift assay results for the AZRE. (A) Sequence-specific interaction of nuclear proteins with the AZRE. The 23 bp AZRE containing a fragment between nucleotides –190 and –168 relative to the transcription start site of Oshsp17.3 was radiolabelled, and 1 ng of the probe was incubated with 10 μg of the nuclear proteins prepared from rice etiolated seedlings under AZC treatment for 6 h. The positions of the DNA–protein complexes are shown as C1 and C2 by arrows. The free probe is indicated with an asterisk (*). (B) Effect of CHX on the interaction of nuclear proteins with the AZRE. Three-day-old rice seedlings were treated with CHX (2 μg ml⁻¹) and with (left) or without (right) AZC for 6 h. (C) Cross-competition analysis of the HSE. The 23 bp HSE-containing fragment located at –87 to –65 relative to the transcription start site of Oshsp17.3 was used as a competitor. (D) Stress-dependent interaction of nuclear proteins with the AZRE. Nuclear extracts were prepared from seedlings treated with AZC, As, or HS for 2 h and by 28 °C [control (C)] or Cd for 6 h.

Fig. 6.

Effect of the 9 bp AZRE on activation of HSFs on the Oshsp17.3 promoter. (A) Schemes of gene constructs. Arrowheads indicate the orientation of each gene. (B) The heat-inducible reporter construct was co-bombarded with the Ubi-Empty vector (–) or the effector construct (+) at a 1:1 ratio. The bombarded coleoptiles were incubated in fresh shaking buffer at 28 °C in the dark for at least 12 h, and then their relative GUS activity was determined. Each experiment was repeated at least three times. Mean ±SE relative GUS activites are from at least 12 independent bombardments. The P-values of p567+pHsfA4b and p567+pHsfB4b are shown on the right. (This figure is available in colour at JXB online.)

Fig. 7.

Effect of AZRE truncation in transgenic Arabidopsis. A GUS reporter gene was fused to the full-length Oshsp17.3 promoter (Oshsp17.3Pro::GUS) and the AZRE-truncated Oshsp17.3 promoter (Oshsp17.3ProΔAZRE::GUS). (A) Seedlings from independent transgenic lines (four lines for each contruct) underwent AZC treatment for 4 h at 23 °C or HS treatment (39 °C) for 150 min and then 18 h of no treatment at 23 °C. Untreated seedlings were used as the control (23 °C). (B) The relative GUS activity of seedlings. The magnitude of induction relative to the control is indicated at the top of the bars. Mean GUS activites ±SE are from at least three independent experiments. For Oshsp17.3ProΔAZRE::GUS, the asterisks indicate no significant difference from control in induction with HS or AZC treatment (P ≥0.7). The P-values for transgenic plants #11 of Oshsp17.3Pro::GUS and #2 and #4 of Oshsp17.3ProΔAZRE::GUS are shown at the top of the scale bar. (This figure is available in colour at JXB online.)