Biochemical, Genetic and Molecular characterization of TCP3 and TCP4 transcription factors in Arabidopsis thaliana

Abstract

The TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORs (TCP) family of proteins consists of plant-specific, non-canonical basic helix-loop-helix transcription factors that perform diverse developmental processes. Based on sequence similarity within the amino acid residues, TCP proteins are divided into two groups – class I and class II. Arabidopsis genome encodes 24 TCP proteins, 13 belonging to class I and 11 to class II, and a high degree of functional redundancy exists within the members of the same class. Eight class II TCP proteins, known as CINCINNATA-like TCPs (CIN-TCPs) redundantly promote leaf differentiation and maturation. Despite the closest sequence similarity between two CIN-TCPs, TCP3 and TCP4; antagonistic functional differences, primarily with respect to their role in auxin response, has been reported, raising a doubt about whether they have truly redundant function. To test this, we have performed a detailed comparative study of these two proteins using biochemical, genetic, and molecular approaches. We also attempted to identify downstream targets of these proteins by functional genetics approach. The class I TCP proteins bind to the GGNCCCAC sequence element whereas the class II proteins bind to GTGGNCCC consensus element, indicating that members of both the TCP classes bind to distinct but overlapping DNA sequence. In this study, we carried out detailed DNA-binding analysis of these two CIN-TCP proteins - TCP3 and TCP4 and found difference in their DNA-binding properties; TCP3 binds to both class I and class II DNA elements whereas TCP4 binds only to class II sequence. Further analysis suggested that the N-terminal region of TCP3 is responsible for this dual DNA-binding property. Biochemical analysis suggested a divergence in the binding properties of these two redundant members. To study the functional differences, we used various gain of function lines of TCP3 and TCP4 and showed that activation of TCP3 or TCP4 has similar effects on hypocotyl elongation and leaf maturation; both the proteins promote cell elongation in hypocotyl and suppress cell proliferation in leaf primordia. Detailed analysis also suggests similar activity by both the proteins in their temporal effect on leaf primordia maturation. Further, our results suggest that CIN-TCPs are master regulators of compensation in leaves. The transcriptomic comparisons of the differentially regulated genes by TCP3 and TCP4 showed high degree of redundancy between these two proteins in the regulation of the common downstream target genes. Reporter gene analysis demonstrated that both the proteins promote auxin response in leaf primordia. The genetic and molecular studies between these two redundant proteins show convergence in their function. Through functional genetics approach, we identified putative mutants which showed bigger leaves upon TCP4 induction suggesting the probable targets of TCP4 involved in leaf maturation. Further analyses of these putative mutant lines are required to learn the exact effect of the mutation and the mutated genes in leaf growth. In summary, here we show a difference in DNA-binding property of TCP3 and TCP4, yet these two proteins act as true functionally redundant pair in regulating leaf morphogenesis and auxin response.