High‐throughput quantitative assessment of ABA‐responsive elements at single‐nucleotide

The phytohormone abscisic acid (ABA) plays a pivotal role in a variety of developmental processes and adaptive responses to environmental stimuli in plants. Promoter analysis of ABA-regulated genes has led to the identification of ABA-responsive elements (ABREs) for ABA-inducible gene expression. The ABREs contain a conserved ACGT sequence, which is the binding site for basic leucine zipper (bZIP) transcription factors. Sequence variations within ABREs have been shown to affect gene expression by altering bZIP protein binding. ABRE sequences exhibit diversity but are commonly found in the promoter regions of ABA-responsive genes in plant genomes. However, the precise effects of sequence variations on ABRE function are still not well understood. 

Recently, a team from the Zhang lab at Shanghai Jiao Tong University published an article "High-throughput quantitative assessment of ABA-responsive elements at single-nucleotide resolution " in the journal Quantitative Biology. Using synthetic STARR-seq to quantitatively assess the effects of single-nucleotide substitutions on ABRE activity, they found that the nucleotide substitutions in both the ACGT-core and ACGT-flank regions affected transcriptional strength. Alterations in the ACGT-core sequence had a more significant impact on ABRE activity compared to changes in the flanking region. The ACGT-flank variants with high activity exhibited a strong sequence preference in the downstream region, whereas the highly active core variants were diverse in sequence patterns. Furthermore, they identified the preferred nucleotides following the ACGT core and experimentally validated the GGG motif, which could enhance the ABA-responsive activity of ABRE elements.

Figure 1 illustrates the synthetic STARR-seq method used to measure ABRE activity in tobacco leaves. The oligo pool containing degenerate nucleotides (N) within the ABRE element were synthesized and amplified using PCR to add the minimal promoter and the random barcodes. The amplified fragments were cloned into plasmid constructs to drive the expression of a luciferase reporter gene. The STARR-seq libraries were transiently transformed into tobacco leaves, and the transcribed barcodes were counted by targeted RNA sequencing. The promoter strength was quantified by comparing the reporter transcript abundance with that of input plasmid.