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Cytokinins Cytokinins (CKs): •promote cell division in the shoot •delay leaf senescence •regulate nutrient allocation •promote root nodule development •contribute to environmental signaling and pathogen responses •regulate auxin action and distribution + CK + auxin + auxin and CK © 2013 American Society of Plant Biologists Structure of major cytokinins adenine Cytokinins are N6substituted adeninerelated compounds. Trans-zeatin and isopentenyl-adenine are the most active and abundant CKs trans-zeatin (tZ) Isopentenyladenine (iP) © 2013 American Society of Plant Biologists Agrobacterium tumefaciens induces hormone-based tumors Agrobacterium tumefaciens is a natural plant pathogen. It causes crown gall disease and tumorlike growths by inducing the plant to produce auxin and cytokinin Plant Cell Nucleus DNA Agrobacterium tumefaciens Agrobacterium tumefaciens on the surface of a plant cell Agrobacterium tumefaciens carries tumor-inducing (Ti) plasmids. A subset of the plasmid DNA called transfer-DNA (T-DNA) is mobilized into the plant nucleus SEM courtesy of Martha Hawes, University of Arizona; grown gall by C-M © 2013 American Society of Plant Biologists T-DNA includes genes for biosynthesis of auxin and cytokinin Normal T-DNA Ti plasmid Auxin and CK produced normal tumor Auxin produced These studies showed that the Agrobacterium tmr gene encodes a cytokinin biosynthetic enzyme rooty tumor No tmr gene CK produced shooty tumor No tms gene © 2013 American Society of Plant Biologists The tmr gene encodes isopentenyltransferase, a key enzyme in CK synthesis Isopentenyltransferase (IPT) AMP DMAPP – dimethylallyldiphosphate iPRMP isopentenyladenine (iP) riboside phosphate The bacterial IPT gene uses AMP exclusively as a substrate whereas plant IPT genes prefer ADP or ATP Haberer, G. and Kieber, J.J. (2002) Cytokinins. New insights into a classic phytohormone. Plant Physiol. 128: 354-362. © 2013 American Society of Plant Biologists IPT overexpression causes reduced apical dominance, reduced root growth and delayed leaf senescence Elevated CK promotes shoot bud outgrowth Elevated CK promotes shoot growth and restricts root growth Wild type IPT overexpression Medford, J.I., et al. (1989) Alterations of endogenous cytokinins in plants using a chimeric isopentenyl transferase gene Plant Cell1: 403-413. © 2013 American Society of Plant Biologists Cytokinins can be inactivated by conjugation or degradation The CKX genes are important regulators of active cytokinin levels Adenine Phosphoribosyl A Transferase 1 (APT1) O-glycosylation or O-acetylation Glucosylation site Irreversible Degradation Cytokinin oxidase (CKX) Reversible Conjugation Adenylation LOG ACTIVE FORM CKX genes are CK-induced Kieber, J.J. (2002) Cytokinins: March 27, 2002. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists. doi: 10.1199/tab.0063 See also Bajguz, A. and Piotrowska, A. (2009) Conjugates of auxin and cytokinin. Phytochemistry 70: 957–969. © 2013 American Society of Plant Biologists Cytokinin signaling takes place through a phosphorelay system Type-C ARR ARR22 ARR24 CK binding to the membrane-bound receptor histidine kinases leads them to autophosphorylate The receptor HKs transfer the phosphoryl group to histidine phosphotransfer proteins (HPTs) The HPTs transfer the phosphoryl group to response regulators Adapted from Schaller, G.E., Kieber, J.J., and Shiu, S (2008) Two-component signaling elements and histidyl-aspartyl phosphorelays. The Arabidopsis Book: ASPB. © 2013 American Society of Plant Biologists Arabidopsis has three CK receptors, AHK2, 3 and 4 Cytokinin receptor (AHK2) Cytokinin receptor (AHK3) wol WT AHK4 was identified as a wooden-leg mutant (wol) and a cytokinin response mutant (cre1) The wol root is truncated due to impaired differentiation in central cylinder. The cre1 mutants do not produce shoots in tissue culture Wild-type cre1 Reproduced with permission from Scheres, B. et al. (1995) Mutations affecting the radial organisation of the Arabidopsis root display specific defects throughout the embryonic axis. Development 121: 53-62. Reprinted by permission from Macmillan Publishers Ltd.: Nature. Inoue, T., et al. (2001). Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409: 1060-1063. © 2013 American Society of Plant Biologists Downstream of the receptors: HPTs and RRs The receptors pass the phosphoryl groups to a histidine phosphotransfer protein (HPT or AHP*) which passes it to a response regulator (RR or ARR*). Type-B ARRs are transcription factors, whereas Type- A ARRs are inhibitors of CK signaling *AHP and ARR refer to the Arabidopsis proteins Reproduced with permission from El-Showk, S., Ruonala, R. and Helariutta, Y. (2013) Crossing paths: cytokinin signalling and crosstalk. Development 140: 1373–1383. © 2013 American Society of Plant Biologists Receptors relay phosphoryl groups to His phosphotransfer proteins Type-C ARR ARR22 ARR24 Three CK receptors Five histidine phosphotransfer proteins (HPTs) - In Arabidopsis known as AHPs 23 response regulators (RRs) of three types – In Arabidopsis known as ARRs Adapted from Schaller, G.E., Kieber, J.J., and Shiu, S (2008) Two-component signaling elements and histidyl-aspartyl phosphorelays. The Arabidopsis Book: ASPB. © 2013 American Society of Plant Biologists There are three types of response regulators Receiver domain D Arabidopsis: 10 Type-A ARRs D Arabidopsis: 2 Type-C ARRs D Arabidopsis: 11 Type-B ARRs DNA-binding domain E Negative regulators Type-B ARRs are transcriptional activators with C-terminal DNA- binding domains Pseudoresponse regulators (PRRs) are related proteins but not involved in CK signaling PRRs usually lack the conserved Asp residue in the receiver domain © 2013 American Society of Plant Biologists ARR1, a type-B ARR, is a positive regulator of CK signaling ARR1 overexpression Overexpression of ARR1 makes tissues more sensitive to CK Wild-type arr1 loss-of-function The concentration of CK needed to produce green tissues is a good measure of CK sensitivity Loss-of-function arr1 mutants are less sensitive to CK From Sakai, H., Honma, T., Aoyama, T., Sato, S., Kato, T., Tabata, S., and Oka, A. (2001). ARR1, a transcription factor for genes immediately responsive to cytokinins. Science 294: 1519-1521; reprinted with permission from AAAS. © 2013 American Society of Plant Biologists Type A ARRs are negative regulators of CK signaling Type-A ARR D CK-inducible ARR6 promoter Expression of type-A ARRs interferes with CK-induced transcription in protoplasts LUC Reprinted by permission from Macmillan Publishers Ltd.: Nature . Hwang, I., and Sheen, J. (2001). Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413: 383-389, copyright 2001, © 2013 American Society of Plant Biologists Type-C ARRs may remove phosphoryl groups from the system Histidine kinase activity adds phosphoryl groups to system Phosphorelay system Type-A ARRs Putative competition between type-A and type-B ARRs ? Type-C ARRs remove phosphoryl groups from the system via histidine phosphatase activity Type-B ARRs – CKinduced transcription © 2013 American Society of Plant Biologists CK action in whole-plant processes CKs regulate •Root vascular tissue development •Shoot and root developmental patterning •Nutrient uptake and allocation •Leaf senescence •Many other processes Catabolism Synthesis CK Cytokinin’s roles in wholeplant processes Conjugation Transport Perception (receptor) TF activation/ inactivation Target genes Biological Functions © 2013 American Society of Plant Biologists Cytokinins contribute to developmental patterning and meristem functions CK promotes cell division and stem cell fate at the shoot apical meristem CK inhibits root meristem size and cell division and promotes cell differentiation at the root apical meristem Reprinted by permission from Macmillan Publishers, Ltd: NATURE Wolters, H., and Jürgens, G. (2009). Survival of the flexible: Hormonal growth control and adaptation in plant development. Nat. Rev. Genet. 10: 305–317. Copyright 2009. © 2013 American Society of Plant Biologists Cytokinin and auxin regulate organogenesis in tissue culture + CK + auxin Tobacco leaf discs are placed into sterile culture dishes on medium containing various hormones + auxin and CK TIME Images courtesy of Richard Amasino. © 2013 American Society of Plant Biologists CKs contribute to nutrient uptake and allocation Sink Source CO2 Sink Shoot systems are a source of sugars and primary metabolites that are distributed to nutrient sinks including flowers and fruits, roots, and young leaves Source Root systems take up mineral nutrients such as nitrate, sulfate and phosphate Sink NO3SO42PO43- Elevated CK levels increase expression of photosynthetic enzymes and delay leaf senescence © 2013 American Society of Plant Biologists CKs contribute to nutrient uptake and allocation Elevated levels of nitrate or phosphate increase the rate of CK synthesis, which ultimately decreases root growth rate. In turn, elevated CK represses nutrient uptake Model showing the role of CK and other hormones on nitrogen acquisition Reprinted from Kiba, T., Kudo, T., Kojima, M. and Sakakibara, H. (2011). Hormonal control of nitrogen acquisition: roles of auxin, abscisic acid, and cytokinin. J. Exp. Bot. 62: 1399-1409 by permission from Oxford University Press. © 2013 American Society of Plant Biologists Cytokinins delay leaf senescence SAG:IPT Control Plants that express IPT under the regulation of a senescence-induced promoter (SAG) have significantly delayed leaf senescence SAG:IPT Control From Gan, S., and Amasino, R.M. (1995) Inhibition of leaf senescence by autoregulated production of cytokinin Science 270: 1986-1988. Reprinted with permission from AAAS. © 2013 American Society of Plant Biologists CKs can negatively affect stress tolerance Loss-of-function mutants affecting CK synthesis (atipt) or signaling (ahp) are drought tolerant ahp2,3,5 C CK also reduces the plant’s sensitivity to ABA, so it acts through ABA-dependent and independent pathways Stress Wild type Three-week-old plants exposed to drought stress for13 or 14 days and then re-watered for three days Drought tolerance is conferred in part by an increase in membrane stability CK ABA Stress tolerance Nishiyama, R., et al., (2011) Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis. Plant Cell. 23 : 2169-2183. Nishiyama, R., Watanabe, Y., Leyva-Gonzalez, M.A., Ha, C.V., Fujita, Y., Tanaka, M., Seki, M., Yamaguchi-Shinozaki, K., Shinozaki, K., Herrera-Estrella, L., Tran, L.S. (2013) Arabidopsis AHP2, AHP3, and AHP5 histidine phosphotransfer proteins function as redundant negative regulators of drought stress response. Proc. Natl. Acad. Sci. USA. 2013 110: 4840–4845. © 2013 American Society of Plant Biologists CKmediated processes There are many other processes mediated by CK. Identifying the specific genes that contribute to each of these will help us to understand the myriad roles that CK plays in coordinating plant growth Reprinted from Werner, T., and Schmülling, T. (2009). Cytokinin action in plant development. Current Opinion in Plant Biology 12: 527-538, with permission from Elsevier copyright 2009. © 2013 American Society of Plant Biologists Cytokinin action - summary CKs have diverse roles – from regulating vascular differentiation and meristem function to regulation of nutrient allocation and leaf senescence We are beginning to correlate specific genes with specific functions but there are still many unresolved questions CKs provide many unexploited opportunities for improving agricultural yields through increased stress tolerance and seed yields © 2013 American Society of Plant Biologists Ongoing investigations How do catabolism and conjugation contribute to in vivo functions? Catabolism Synthesis What signals are carried by xylem-borne tZ versus phloemborne iP? Conjugation CK Why is localized CK synthesis sometimes critical and sometimes not? Transport Perception (receptor) Are signals from the three receptors integrated or kept separate? What are the target genes, and what do they do? TF activation/ inactivation Target genes Biological Functions How do the type-A and type-C ARRs work? What is the relationship with CRFs? How do all these pieces fit together to make a functioning plant???? Adapted from Kieffer, M., Neve, J., and Kepinski, S. (2010). Defining auxin response contexts in plant development. Current Opinion in Plant Biology 13: 12-20. © 2013 American Society of Plant Biologists