Identification and Functional Characterization of cis-Regulatory Elements of Key Photorespiratory Genes in Response to Short-Term Abiotic Stress Conditions.

The cis-regulatory elements (CREs) are the short stretches of noncoding DNA upstream of a gene, which play a critical role in fine-tuning gene expression. Photorespiration is a multi-organellar, energy-expensive biochemical process that remains intricately linked to photosynthesis and is conserved in plants. Recently, much focus has been devoted in generating plants with engineered alternative photorespiratory bypasses to enhance photosynthetic efficiency without compromising the beneficial aspect of photorespiration. Varied constitutive or inducible promoters for generating transgenic plants harboring multiple transgenes have been introduced over years; however, most of them suffer from unintended effects. Consequently, a demand for synthetic tunable promoters based on canonical CRE signatures derived from native genes is on the rise. Here, in this chapter, we have provided a detailed method for in silico identification and characterization of CREs associated with photorespiration. In addition to the detailed protocol, we have presented an example of a typical result and explained the significance of the result. Specifically, the method covers how to identify and generate tunable synthetic promoters based on native CREs using three key photorespiratory genes from Arabidopsis and two web-based tools, namely, PlantPAN3.0 and AthaMap. Finally, we have also furnished a protocol on how to test the efficacies of the synthetic promoters harboring predicted CREs using transient tobacco expression coupled with luciferase-based promoter assay in response to ambient conditions and under short-term abiotic stress conditions.