Background and aims: Carnivorous plants trap and digest insects and similar-sized animals. Many studies have examined enzymes in the digestive fluids of these plants and have gradually unveiled the origins and gene expression of these enzymes. However, only a few attempts have been made at characterization of nucleases. This study aimed to reveal gene expression and the structural, functional and evolutionary characteristics of an S1-type nuclease (DAN1) in the digestive fluid of an Australian sundew, Drosera adelae, whose trap organ shows unique gene expression and related epigenetic regulation.
Methods: Organ-specificity in Dan1 expression was examined using glandular tentacles, laminas, roots and inflorescences, and real-time PCR. The methylation status of the Dan1 promoter in each organ was clarified by bisulphite sequencing. The structural characteristics of DAN1 were studied by a comparison of primary structures of S1-type nucleases of three carnivorous and seven non-carnivorous plants. DAN1 was prepared using a cell-free protein synthesis system. Requirements for metal ions, optimum pH and temperature, and substrate preference were examined using conventional methods.
Key results: Dan1 is exclusively expressed in the glandular tentacles and its promoter is almost completely unmethylated in all organs. This is in contrast to the S-like RNase gene da-I of Dr. adelae, which shows similar organ-specific expression, but is controlled by a promoter that is specifically unmethylated in the glandular tentacles. Comparison of amino acid sequences of S1-type nucleases identifies seven and three positions where amino acid residues are conserved only among the carnivorous plants and only among the non-carnivorous plants, respectively. DAN1 prefers a substrate RNA over DNA in the presence of Zn2+, Mn2+ or Ca2+ at an optimum pH of 4.0.
Conclusions: Uptake of phosphates from prey is suggested to be the main function of DAN1, which is very different from the known functions of S1-type nucleases. Evolution has modified the structure and expression of Dan1 to specifically function in the digestive fluid.
Keywords: Drosera adelae; Carnivorous plant; DNA methylation; S1-type nuclease; S1/P1 nuclease; protein structure; sundew.
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