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Advances in systematic computational biology and rapid elucidation of synergistic interplay between cis and trans factors governing transcriptional control have facilitated functional annotation of gene networks. The generation of data through deconstructive, reconstructive and database assisted promoter studies, and its integration to principles of synthetic engineering has started an era of designer promoters. Exploration of natural promoter architecture and the concept of cis engineering have not only enabled fine tuning of single or multiple transgene expression in response to perturbations in the chemical, physiological and environmental stimuli but also provided researchers with a unique answer to various problems in crop improvement in the form of bidirectional promoters.

BackgroundTranscription factor binding is regulated by several interactions, primarily involving cis-element binding. These binding sites maintain specificity by means of their sequence, and other additional factors such as inter-motif distance and spacer specificity. The ACGT core sequence has been established as a functionally important cis-element which frequently regulates gene expression in synergy with other cis-elements. In this study, we used two monocotyledonous – Oryza sativa and Sorghum bicolor, and two dicotyledonous species – Arabidopsis thaliana and Glycine max to analyze the conservation of co-occurring ACGT core elements in plant promoters with respect to spacer distance between them. Using data generated from Arabidopsis thaliana and Oryza sativa, we also identified conserved regions across all spacers and possible conditions regulating gene promoters with multiple ACGT cis-elements.ResultsOur data indicated specific predominant spacer lengths between co-occurring ACGT elements, but these lengths were not universally conserved across all species under analysis. However, the frequency distribution indicated local regions of high correlation among monocots and dicots. Sequence specificity data clearly revealed a preference for G at the first and C at the terminal position of a spacer sequence, suggesting that the G-box motif is the most prevalent for the ACGT class of promoters. Using gene expression databases, we also observed trends suggesting that co-occurring ACGT elements are responsible for gene regulation in response to exogenous stress. Conservation in patterns of ACGT (N) ACGT among orthologous genes also indicated the possibility that emergence of functional significance across species was a result of parallel evolution of these cis-elements.ConclusionsAlthough the importance of ACGT elements has been acknowledged for several plant species, ours is the first study that attempts to compare their occurrence across four species and analyze conservation among them. The apparent preference for particular spacer distances suggest that these motifs might be implicated in important physiological functions which are yet to be identified. Variations in correlation patterns among monocots and dicots might arise out of differences in transcriptional regulation in the two classes. In accordance with literature, we established the involvement of co-occurring ACGT elements in stress responses and showed how this regulation differs with variation in the ACGT (N) ACGT motif. We believe that our study will be an essential resource in determining optimum spacer length and spacer sequence between ACGT elements for promoter design in future.

Gene expression is mediated through interaction between cis regulatory elements and its cognate transcription factors. Cis regulatory elements are defined as non-coding DNA sequences that provide the binding sites for transcription factors and are clustered in the upstream region of genes. ACGT cis regulatory element is one of the important cis regulatory elements found to be involved in diverse biological processes like auxin response, salicylic acid (SA) response, UV light response, ABA response and jasmonic acid (JA) response. We identified through in silico analysis that the upstream region of protein phosphatase 2C (PP2C) gene has a distinct genetic architecture of ACGT elements. In the present study, the activation of the full length promoter and its deletion constructs like 900 base pair, 500 base pair, 400 base pair and NRM (Nathji Rajesh Mehrotra) were examined by stable transformation in Arabidopsis thaliana using β-glucuronidase as the reporter gene. Evaluation of deletion constructs of PP2C-like promoter was carried out in the presence of phytohormones like abscisic acid (ABA), SA and JA. Our result indicated that the full length and 900 base pair promoter-reporter constructs of PP2C-like promoter was induced in response to ABA but not to methyl jasmonate and SA.

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Transcription control of gene expression depends on a variety of interactions mediated by the core promoter region, sequence specific DNA-binding proteins, and their cognate promoter elements. The prominent group of cis acting elements in plants contains an ACGT core. The cis element with this core has been shown to be involved in abscisic acid, salicylic acid, and light response. In this study, genome-wide comparison of the frequency of occurrence of two ACGT elements without any spacers as well as those separated by spacers of different length was carried out. In the first step, the frequency of occurrence of the cis element sequences across the whole genome was determined by using BLAST tool. In another approach the spacer sequence was randomized before making the query. As expected, the sequence ACGTACGT had maximum occurrence in Arabidopsis thaliana genome. As we increased the spacer length, one nucleotide at a time, the probability of its occurrence in genome decreased. This trend continued until an unexpectedly sharp rise in frequency of (ACGT)N25(ACGT). The observation of higher probability of bigger size motif suggests its directed evolution in Arabidopsis thaliana genome.

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