Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.

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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...

ABSTRACT:Effective reproductive management is a crucial concern in the dairy industry and missed oestrus and late recognition of unsuccessful insemination can lead to substantial financial loss. Progesterone levels change in a predictable manner with progress of the oestrus cycle and with the onset of pregnancy. We report here the development of a simple ELISA test for the measurement of progesterone levels in bovine milk. The method is based on the use of polyclonal antibodies raised against 11α-hydroxyprogesterone-hemisuccinate and competition for antibody binding sites between free progesterone and horseradish peroxidase-labelled progesterone. Binding is quantified by means of the colour reaction between horseradish peroxidase and 3,3' ,5,5'-tetramethylbenzidine. The method has proved to be a cost effective, sensitive and easy to perform alternative to currently available methods of oestrus and pregnancy detection. The practical feasibility of the method has been proven under dairy farm conditions.