CHARACTERIZATION OF AP-4 COMPLEX PROTEIN IN GUAR AND OTHER PLANTS

Abstract

AP-4 complex plays an important role in intracellular protein transport carried out by vesicular trafficking mechanism. The first comprehensive study in plants on the characterization of genes encoding AP-4 complex has been presented here. These genes were characterized for introns, CDS, GC content, CpG islands, conserved motifs, cis regulatory elements (CREs) and SNPs. Two conserved motifs, one in the mu subunit for binding to the sorting signal of the cargo protein and the other in the beta subunit for binding to the accessory protein, were observed in each studied AP-4 complex. CREs that regulate stress response, cellular development and hormonal regulation were found in the promoters of AP-4 complex genes. Out of about 1376 SNPs observed in these genes, 31 were found in the functional binding pockets of proteins of Zea mays and soybean. Out of these 31 SNPs 6 were showing damaging effects. Transcriptome analysis of Arabidopsis thaliana, Z. mays and Glycine max showed that the genes coding for four subunits of AP-4 complex express in various tissues, however, the expression was significantly more in seed and other reproductive tissues. The RNA-Seq analysis of genes coding for ARFs, Epsins and VSR1 accessory proteins in all studied tissues of A. thaliana and Z. mays showed expression in all studied tissues, however, their expression like AP-4 complex genes was high in seed. The expression of AP-4 complex genes in different tissues of guar, Z. mays and G. max while ARFs, epsin, and VSR1 in studied tissues of Z. mays was further confirmed by real time PCR analysis. Among the 15 studied genes, genes coding for AP4E, AP4B, AP4M, AP4S, ARFA1A, ARFA1B, ARFA1E, ARFA1F, epsin2 and VSR1 were found to co-express in anther while AP4E, AP4B, AP4M, AP4S, ARFA1A, ARFA1B, ARFA1E, ARFA1F, ARFB1B, epsin 1 and epsin 2 proteins were found to co-express in the seed tissue of A. thaliana. In Z. mays, genes coding for AP4E, AP4B, AP4M, AP4S, ARFA1A, ARFA1B, ARFA1C, ARFA1E, ARFA1F, ARFB1B, ARF3, epsin 1 and VSR1 proteins were observed to co-express in the anther tissue whereas whereas the genes encoding AP4E, AP4B, AP4M, AP4S, ARFA1A, ARFA1B, ARFA1C, ARFA1E, ARFA1F, ARFB1B, ARF3, epsin 1, epsin 2 and VSR1 proteins were the co-expressing genes in seed. The protein- protein interaction studies of the AP-4 complex and its accessory proteins generated a protein network containing 39 interacting combinations. Molecular docking revealed that Ile 49 residue of ARFA1A is critical for interaction with epsilon subunit in maize. These results indicated a role of the AP-4 complex and its accessory proteins in the development and functioning of various tissues, especially reproductive tissues.