Synergistic regulation of bZIP53 and dimerizing partners results in abnormal seed phenotype in Arabidopsis: Use of a designed dominant negative protein A-ZIP53

ABSTRACT In Arabidopsis, basic leucine zipper (bZIP) family of transcription factors (TFs) are key proteins to regulate the expression of seed maturation (MAT) genes. bZIPs are functionally redundant and their DNA-binding activity is dependent on dimerization partner. The intervention of loss of function mutation is inadequate to understand and regulate the redundant behavior of TFs and one such example is bZIP53, which is known as a key regulator of seed maturation phenomena. Here, to examine the consequences of hindering the function of bZIP53 and its known and unknown heterodimerizing partners, a transgenic Arabidopsis constitutively expressing a novel dominant negative (DN) protein A-ZIP53 was raised. Transgenic plants demonstrated a delayed growth and retarded seed phenotype. The in vivo inhibition of DNA binding of bZIP53, bZIP10, and bZIP25 to the G-box demonstrated the efficacy of A-ZIP53 protein. In first generation, majority of plants failed to survive beyond four weeks suggesting a pleiotropic nature of bZIP53. Plants expressing A-ZIP53 have small flower, shorter siliques, and small-seeded phenotype. RNA seq analysis of the transgenic lines revealed the reduced expression of target genes of bZIP53 and its heterodimerizing partners. Furthermore, immunoprecipitation followed by mass spectrometry (IP-MS/MS) of transgenic plants helped to identify the additional heterodimerizing partners of the A-ZIP53. The interactions were subsequently confirmed with the transient transfection experiments. Unlike other gene knock out technologies, DN protein can inhibit the function of members of the same group of bZIP TFs.