Volume 7, Issue 4, Pages 626-641 (April 2014) Distinct Role of Core Promoter Architecture in Regulation of Light-Mediated Responses in Plant Genes  Srivastava Rakesh , Rai Krishan Mohan , Srivastava Meenal , Kumar Verandra , Pandey Bindu , Singh Sudhir P. , Bag Sumit K. , Singh Brahma Deo , Tuli Rakesh , Sawant Samir V.   Molecular Plant  Volume 7, Issue 4, Pages 626-641 (April 2014) DOI: 10.1093/mp/sst146 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Selection of Different Types of Light-Regulated Promoters. (A) Heat map showing gene expression profiles in different light conditions for four selected light-regulated promoters using the dChip software. The color scale (representing log signal values) is shown at the bottom; AL: 5-min UV-A light pulse followed by 4 h of darkness; AS: 5-min UV-A light pulse followed by 40 min of darkness; BL: 4-h continuous blue light; BS: 45-min continuous blue light; DL: 4-h continuous darkness; DS: 45-min continuous darkness; FL: 4-h continuous far-red light; FS: 45-min continuous far-red light; PL: 1-min red light pulse followed by 4 h of darkness; PS: 1-min red light pulse followed by 44 min of darkness; RL: 4-h continuous red light; RS: 45-min continuous red light; UL: 5-min UV-A/B light pulse followed by 4 h of darkness; US: 5-min UV-A/B light pulse followed by 40 min of darkness; WL: 4-h continuous white light; WS: 45-min continuous white light. (B) Gene expression patterns of different selected light-regulated promoters in Col-0 ecotype seedlings grown for 6 d in the complete dark (24 h) and white light (16-h light/8-h dark). (C) Real-time PCR for effect of different qualities of light in TATA-box-containing and TATA-less Inr-containing genes. L (white light), R (red), FR (far-red), B (blue), and D (dark). x-axis: monochromatic light. RNA was extracted from Col-0 wild-type seedlings that were grown in the dark for 5 d and then exposed to different quality (R, FR, and B) of light for 4 h. (D) Time-course analysis of light-regulated promoters. RNA was extracted from Col-0 wild-type seedlings that were grown in the dark for 6 d and then exposed to continuous white light (120 μmol m–2 s–1) for 0, 2, and 4 h. The values above the bars indicate the SE (standard error) from the means of two independent experiments. The asterisks denote statistically significant differences (P < 0.05). TC, TATA-box-containing; IC, TATA-less Inr-containing; LA, Light-Activated; LR, Light-Repressed. Molecular Plant 2014 7, 626-641DOI: (10.1093/mp/sst146) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Effect of Mutation on Core Promoter Sequences in TATA-Box and TATA-Less Inr-Containing Light-Regulated Promoters. Gus activities in 12-day-old light-grown (white bars) or dark-grown (black bars) transgenic seedlings containing light-regulated promoters. (A) Effect of mutation on the TATA-box-containing light-activated promoter (TC-LAP). (B) Effect of mutation on the TATA-box-containing light-repressed promoter (TC-LRP). (C) Effect of mutation on TATA-less Inr-containing light-activated promoter (IC-LAP). (D) Effect of mutation on TATA-less Inr-containing light-repressed promoter (IC-LRP). The values above the bars indicate the SE of the means of three independent experiments. ‘n’ indicates the total number of independent T1 tobacco transgenic lines that were analyzed. The asterisks denote statistically significant differences (P < 0.05) between plants expressing the native light and dark transgenic lines and all other mutant constructs. TC, TATA-box-containing; IC, TATA-less Inr-containing; LAP, Light-Activated Promoter; LRP, Light-Repressed Promoter; superscript ‘m’, mutated element; superscript ‘r’, restored element. Molecular Plant 2014 7, 626-641DOI: (10.1093/mp/sst146) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Effect of Mutation in the TATA-Box and Inr Region on Nucleosome Formation in the Core Promoter Region. (A) Nucleosome occupancy in the core promoter region of native light-activated and -repressed promoters. Nuclei were isolated from tobacco T1 transgenic seedlings. MNase-treated mononucleosome were isolated, purified, and used as a template for qRT–PCR for nucleosome occupancy at the core promoter region of light-regulated promoters. (B) Effect of the mutation in the core promoter region of mutated and swapped versions (TATA-box/Inr swapped) of light-regulated promoters. The values above the bars indicate the SE from the means of two independent experiments. TC, TATA-box-containing; IC, TATA-less Inr-containing; LA, Light-Activated; LR, Light-Repressed. Molecular Plant 2014 7, 626-641DOI: (10.1093/mp/sst146) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Effect of Single Nucleotide Polymorphisms in TATA-Box-Containing and TATA-Less Promoters in Different Arabidopsis Ecotypes. The qRT–PCR was performed using cDNA prepared from RNA, which was extracted from Col-0 and different ecotypes from seedlings, flower, siliques, or leaves. (A) TATA-box-containing promoters. (B) TATA-less promoters. The values above the bars indicate the SE from the means of two independent experiments. The asterisks denote statistically significant differences (p < 0.05). Molecular Plant 2014 7, 626-641DOI: (10.1093/mp/sst146) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Spatial Distribution of Motifs of HY5 Light-Activated Promoter Obtained from MEME Database. Frequency of motif in the 5′ UTR to –250-bp upstream sequences of HY5 light-activated promoter. (A) Motifs from 49 TATA-box-containing light-activated promoters. (B) Motifs from 151 TATA-less light-activated promoters. Molecular Plant 2014 7, 626-641DOI: (10.1093/mp/sst146) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Effect of Interchange of Core Promoter in TATA-Box and TATA-Less Inr-Containing Light-Regulated Promoters. (A) Schematic representation of construct formation of core promoter sequences of TATA-less Inr-containing light-activated promoter (IC-LAP) with activator region (proximal promoter) of TATA-box-containing light-activated promoter (TC-LAP). (B) Schematic representation of construct formation of core promoter sequences of TATA-box-containing light-activated promoter (TC-LAP) with activator region (proximal promoter) of TATA-less Inr-containing light-activated promoter (IC-LAP). (C) Effect of swapping of core promoter sequences in TC-LAPacti-IC-LAPcp with comparison of TATA-box-containing light-activated promoter (TC-LAP TATA+Inr–). (D) Effect of swapping of core promoter sequences in IC-LAPacti-TC-LAPcp with comparison of TATA-less Inr-containing light-activated promoter (IC-LAP TATA–Inr+). Each reading is an average of four- to six-leaf disc bombardments over three independent experiments. The values above the bars indicate the SE of the means of three independent experiments. The asterisks denote statistically significant differences (P < 0.05) between plants expressing in the light and dark conditions. TC, TATA-box-containing; IC, TATA-less Inr-containing; LAP, Light-Activated Promoter; acti, activator region (proximal promoter); cp, core promoter sequences. Molecular Plant 2014 7, 626-641DOI: (10.1093/mp/sst146) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Model for Light-Mediated Gene Expression from Core Promoter Elements. Selective activation models of the core promoter elements of light-regulated promoter. (A) The assemblies of transcription factors and complex that identify the promoter lead to the activation of gene expression in plants. In the natural promoter of TATA/Inr type, the light signal mediated by upstream activators operates on TATA+/Inr− or TATA−/Inr+ to activate transcription initiation. Transcription initiation starts with cooperative interaction between upstream sequences, transcription factor, and PIC complex, which, in turn, removes the repressive nucleosome structure over core promoter elements. (B) When the core promoter region contains mutated TATA/Inr, transcription initiation is stopped. (C) In TATA/Inr swap or vice versa, transcription initiation is not established due to a lack of interaction between downstream of the TSS, PIC along with TATA/Inr in promoter. (D) Interchanging the core promoter sequences either with proximal promoter of TATA/Inr light-activated promoter re-established the transcription. LRE, light-responsive element; TC, TATA-box-containing; IC, TATA-less Inr-containing; LAP, Light-Activated Promoter; acti, activator region (proximal promoter); cp, core promoter sequences. Molecular Plant 2014 7, 626-641DOI: (10.1093/mp/sst146) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions