Abstract
BACKGROUND: The plant hormone cytokinin is a conserved regulator of plant development. LONELY GUY (LOG) proteins are pivotal in cytokinin biosynthesis. However, their origin, evolutionary history, and enzymatic characteristics remain largely uncharacterized. METHODS: To elucidate LOG family evolution history and protein motif composition, we conducted phylogenetic and motif analyses encompassing representative species across the whole green plant lineage. Catalytic activity and structure analysis were conducted to thoroughly characterize the LOG proteins. RESULTS: Our phylogeny showed that LOG proteins could be divided into five groups and revealed three major duplication events giving rise to four distinct groups of vascular LOG proteins. LOG proteins share a conserved structure characterized by a canonical motif arrangement comprising motifs 1, 2, 3, 4, 5, 6, and 7. Two significant changes in LOG motif composition occurred during the transition to land plants: the emergence of motif 3 in charophyte LOG sequences and the subsequent acquisition of motif 8 at the C-terminus of LOG proteins. Enzymatic assays demonstrated that LOG proteins can be classified into two groups based on their enzyme activity. One group act as cytokinin riboside 5'-monophosphate phosphoribohydrolase and primarily convert iPRMP to iP, while the other group act as 5'-ribonucleotide phosphohydrolase, and preferentially produce iPR from the same substrates. TaLOG5-4A1, TaLOG5-4A2, TaLOG5-5B2, and TaLOG5-D1 shared conserved residues in the critical motif and were predicted to have similar protein structures, but displayed distinct enzymatic activities. CONCLUSIONS: Our findings provide a comprehensive overview of LOG protein phylogeny and lay a foundation for further investigations into their functional diversification.