Cell-surface receptors enable perception of extracellular cytokinins

Abstract

phone 30 +46 90 786 8355 karin.ljung@slu.se 31 32Cytokinins are mobile multifunctional plant hormones with roles in development and 33 stress resilience 1,2 . Although cytokinin receptors are substantially localised to the 34 endoplasmic reticulum [3][4][5] , the cellular sites of cytokinin perception continue to be 35 debated 1,6,7 . Several cytokinin types display bioactivity 8,9 and recently a cell-specific 36 cytokinin gradient was reported in roots 10 . Yet, the importance of spatially heterogeneous 37 cytokinin distribution and the specific cytokinin(s) that account for the different responses 38 remain unclear. Here we show that cytokinin perception by plasma membrane receptors is 39 an effective path for cytokinin response in root cells. Readout from a Two Component 40 Signalling cytokinin-specific reporter (TCSn::GFP; 11 ) is closely matched to intracellular 41 cytokinin content, yet a proportion of bioactive cytokinins are detected in the extracellular 42 fluid. Using cytokinins covalently linked to beads that could not pass the plasma 43 membrane, we demonstrate that strong TCSn activation still occurs and that this response 44 is greatly diminished in cytokinin receptor mutants. Although intracellular receptors play 45 significant roles, we argue for a revision of concepts of cytokinin perception to include the 46 spatial dimensions. In particular, selective ligand-receptor affinities, cellular localisation 47 and tissue distribution of bioactive cytokinins, their receptors, transporters and 48 inactivation enzymes appear all to be components of the signalling regulatory 49 mechanisms. 50 Cytokinins are key hormones regulating cell division and differentiation, root and shoot 51 architecture, senescence and responses to environmental stresses 1 . The active forms are 52 the cytokinin free bases, which comprise a range of N 6 -modified adenine molecules 8,9 , 53 especially trans-zeatin (tZ) and isopentenyl adenine (iP). Homeostatic regulation of active 54 cytokinin pools occurs at the level of biosynthesis, and also through metabolic deactivation 55 by glucosylation, phosphoribosylation or irreversible degradation by cytokinin 56 dehydrogenase (CKX) 9,12 . Cytokinin signalling commences with perception of bioactive 57 molecules by hybrid histidine kinases (HKs) 1,13,14 . Several reports show GFP-fused 58 Arabidopsis HKs (AHKs) mainly localised to the endoplasmic reticulum (ER) membrane 3-5 , 59 yet the originally proposed extracellular site of cytokinin perception at plasma membrane 60 receptors 13,15,16 has never been discounted. Notably, several classes of cytokinin 61 3 transporters facilitate movement of cytokinins in and out of the cell 7 . Cytokinins binding to 62 receptors trigger a phosphorelay cascade, resulting in activation of B-type ARABIDOPSIS 63 RESPONSE REGULATORS (ARR-B) transcription factors 17 . The cytokinin-responsive synthetic 64 promoter fusion TCSn::GFP reflects global ARR-B transcriptional activity 11 and has facilitated 65 in vivo monitoring of cytokinin responses, leading t...

The LONELY GUY gene family: from mosses to wheat, the key to the formation of active cytokinins in plants

“…Cytokinin signal transduction is mediated by a two‐component system (TCS), consisting of histidine protein kinase receptors that sense the input and response regulators (RR) that mediate the output (Wang and Sheen, 2001). Cytokinin free bases are detected by histidine‐kinase receptors (HKs) on the ER (Romanov et al ., 2018) or plasma membrane (Antoniadi et al ., 2020; Kubiasova et al., 2020). Signal transduction is via a phospho relay with phosphate being transferred from the HKs by intermediary histidine phospho transfer proteins (HPs) leading to phosphorylation of type‐B response regulators (type‐B RRs) in the nucleus.…”

IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth

“…Another explanation in our case could be that c Z conversion into t Z occurs via the hydroxylation of N6-isopentenyl side chain or other unknown reaction. In parallel, feeding experiments on Arabidopsis protoplasts and maize cultured cells with labelled c Z indicated to a metabolic route from c Z not only to respective conjugates but also back to its precursor forms (8,29). However, in such feeding experiments on rice seedlings, tobacco cells, oat leaves and Arabidopsis protoplasts no isomerization was observed between t Z- and c Z-ribosides (9,14,86).…”

Auxin signaling: Research advances over the past 30 years

“…Different hormone signals originate at different cellular destinations. For example, brassinosteroid signaling initiates at the PM, ABA signaling initiation occurs at the cytoplasm, and cytokinin signaling initiates at the ER and/or PM (Antoniadi et al, 2020; Kubiasova et al, 2020; Romanov and Schmulling, 2021). Therefore, it is important to systematically analyze the localization, migration, and potential association of auxin receptors to thoroughly understand where auxin signaling is initiated.…”