SPINDLY inhibits class I TCP proteolysis to promote sensitivity to cytokinin

Abstract

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.O-linked GlcNAc (O-GlcNAc) modification of Ser and Thr residues by the nucleocytoplasmic O-GlcNAc transferases (OGTs) regulates the posttranslational fate and function of target proteins (Hart et al., 2007;Butkinaree et al., 2010). In mammalian cells, O-GlcNAcylation affects protein localization, phosphorylation, and stability and plays a role in signal transduction, transcription, and proteasomal degradation (Roos and Hanover,

Cytokinin activity increases stomatal density and transpiration rate in tomato

“…We examined transgenic tomato expressing the GUS reporter gene under the regulation of the synthetic CK-induced promoter TCS v2. TCSv2 harbors the concatemerized type-B RR-binding motifs (Steiner et al , 2016). Guard cells of the transgenic plants exhibited strong GUS activity following BA treatment (Supplementary Fig.…”

“…The sequence of the synthetic promoter Two-Component Signaling Sensor ( TCS ) version 2 (Steiner et al , 2016) was ligated to the β-galactosidase (GUS) gene from Escherichia coli to generate a TCSv2:GUS fusion in pART27. This construct was introduced into Agrobacterium tumefaciens and then transferred to M82 cotyledons using the transformation and regeneration methods described by McCormick (1991).…”

Coordination of auxin-triggered leaf initiation by tomato LEAFLESS

“…1G). To determine whether the SAMs of the pin-like shoots retained their function, we monitored the expression of the two-component sensor (TCS) that marks cytokinin signaling (10). In WT, this reporter is expressed in the SAM proper, is excluded from the outermost two cell layers, and is weakly expressed in young leaf primordia (Fig.…”

CLAUSA is a MYB Transcription Factor that Promotes Leaf Differentiation by Attenuating Cytokinin Signaling

“…To further examine whether CK dynamics could be altered by CLAU, we compared the expression of a CK activity reporter TCS fused to VENUS (Zürcher et al, 2013;Steiner et al, 2016) between wild-type tomato plants and the clau mutant ( Figure 6). We first confirmed that TCS is CK responsive in tomato as was reported in Arabidopsis (Supplemental Figure 4).…”

“…Tomato (M82, sp) plants expressing pFIL>>IPT and pFIL>>CKX were generated by the LhG4 transactivation system (Moore et al, 1998) and described previously (Shani et al, 2010). Tomato plants overexpressing TCSv2:3XVENUS were obtained from the Muller, Eshed, and Weiss labs (Steiner et al, 2016;Zürcher et al, 2013), confirmed to be CK responsive in tomato, and backcrossed into a clau background.…”