Dhiman, V.; Marik, D.; Amrita, ; Shekhawat, R. S.; Swain, A. K.; Dey, A.; Yadav, P.; Pal, A.; Dey, S.; Sadhukhan, A.

We sequenced the drought-response transcriptome of the keystone tree species Prosopis cineraria from the Indian Thar desert to understand the key factors in its drought tolerance mechanism. We identified a network of genes activated in P. cineraria involved in osmotic stress response, phytohormone, calcium, and phosphorelay signal transduction. Of these, up-regulation of 54 APETALA2/Ethylene-Responsive Factor (AP2/ERF) transcription factor genes, validated by real-time PCR, suggests their key role in the drought tolerance of P. cineraria. We conducted a genome-wide study of the AP2/ERF superfamily in P. cineraria, classifying its 232 proteins into 15 clades and analyzing their protein structures, gene structure, and promoter organization. The P. cineraria genome contains more copies of AP2/ERF genes than drought-sensitive plants. Further, we identified sequence polymorphisms in AP2/ERF genes between Arabian and Indian cultivars of P. cineraria. We modeled the DNA-protein complex structures of AP2/ERFs from drought-tolerant and sensitive species using AlphaFold to compare their DNA binding ability. Though the DNA binding domain (DBD) is relatively conserved across species, the unstructured region of these proteins possesses different charge distributions, which might contribute differently to their DNA search and binding. Using all-atom molecular dynamics simulations, we teased out a higher number of specific DBD-DNA hydrogen bonds in P. cineraria, leading to a stronger DNA-binding affinity compared to drought-sensitive Arabidopsis thaliana. These results directly support copy number expansion of AP2/ERF transcription factors and the evolution of their structures for more efficient DNA search and binding as drought adaptation mechanisms in P. cineraria.