Functional analysis of the CRINKLY4 gene family in Arabidopsis thaliana and Viviparous1 promoter in Zea mays
The cereal endosperm is an excellent system to study cell differentiation and fate specification. The aleurone of maize is one cell layer and requires continuous positional cues to specify and maintain its identity. A genetic hierarchy is implicated in this process. CRINKLY4 ( Cr4), a receptor-like kinase, belongs to cell fate-specifying genes and is required to specify aleurone cell identity. Viviparous1 ( Vp1), a B3 domain-containing transcription factor, functions late in development to regulate the expression of C1 in aleurone cells. It also plays an important regulatory role in late embryogenesis and seed maturation with abscisic acid (ABA).;The Arabidopsis genome encodes ACR4, an ortholog of CR4, and four AtCRRs. ACR4 and AtCRR1 and 2 are mainly expressed in developing young tissues. The kinase domain of ACR4 was shown to be a serine/threonine kinase and had ability to phosphorylate the non-functional kinase domain of AtCRR2. T-DNA insertion mutants of acr4 showed a phenotype restricted to the integument and seed coat, while mutants of atcrrs did not show obvious phenotype and triple mutants of acr4, atcrr1 and 2 had similar phenotype as acr4, suggesting that Arabidopsis might have a redundant function and the hypothetical redundant function is not encoded by AtCRRs.;Vp1 functions in ABA regulated processes. Its expression is regulated by ABA, high salinity and osmoticum. The induction of Vp1 expression by ABA does not require de novo protein synthesis. The sequence analysis of 958 bp of the Vp1 promoter identified a potential ABRC (ABA response complex), which consists a predicted ABRE (ABA response element) and a CE1L (coupling element 1-like) element. Electrophoretic mobility shift assay (EMSA) confirmed that the ABRE and CE1L specifically bound proteins in 20 DAP embryo nuclear protein extracts. ABA or osmoticum induce nuclear protein interaction with the ABRE. The ABRE was also shown to be critical for the induction of Vp1 expression by ABA in a transient gene expression experiment. Sequence analysis and EMSA experiments also identified several other binding sites such as: VPP1, AtMYB2 and ZIP1. This provides a starting point to further study the regulation of Vp1 expression.