Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers.

Centre of the Region Haná for Biotechnological and Agricultural Research Central Laboratories and Research Support Faculty of Science Palacky University and Department of Genetic Resources for Vegetables Medicinal and Special Plants Crop Research Institute CZ 78371 Olomouc Czech Republic

Centre of the Region Haná for Biotechnological and Agricultural Research Department of Metabolomics Institute of Experimental Botany CZ 78371 Olomouc Czech Republic

Department of Plant and Microbial Biology University of Zurich Zurich Basel Plant Science Center 8008 Zurich Switzerland

Department of Plant Sciences Weizmann Institute of Science Rehovot 76100 Israel

Institute of Plant Sciences and Genetics in Agriculture Hebrew University of Jerusalem Rehovot 76100 Israel

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