Cytokinins PDF

Cytokinins www.plantcell.org/cgi/doi/10.1105/tpc.110.tt0610 © 2013 American Society of Plant Biologists  Cytokinins Cytokinins (CKs): promote cell division in the shoot delay leaf senescence regulate nutrient allocation promot...

Cytokinins www.plantcell.org/cgi/doi/10.1105/tpc.110.tt0610 © 2013 American Society of Plant Biologists  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  The discovery of cytokinin Cytokinin was Control With kinetin discovered through efforts to identify compounds that increase the growth of plant cells in culture In the 1950s, Folke Skoog’s research group identified a synthetic cytokinin, kinetin Amasino, R. (2005) 1955: Kinetin Arrives. The 50th Anniversary of a newplant hormone. Plant Physiol. 138: 1177 – 1184. © 2013 American Society of Plant Biologists  Cytokinin and auxin regulate organogenesis in tissue culture + CK + auxin + auxin and CK Tobacco leaf discs are placed into sterile culture dishes on medium containing various TIME hormones Images courtesy of Richard Amasino. © 2013 American Society of Plant Biologists  Skoog’s group showed that CK promotes shoot growth in culture Increasing [Auxin] Increasing [Cytokinin] Roots They also recognized Roots that auxin and CK act antagonistically, and that the ratio between the hormones is critical Shoots for their effects Shoots Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissue cultured in vitro. Symp Soc Exp Biol XI: 118–131; Figure from Amasino, R. (2005) 1955: Kinetin Arrives. The 50th Anniversary of a newplant hormone. Plant Physiol. 138: 1177 – 1184. © 2013 American Society of Plant Biologists  Cytokinins delay leaf senescence Senescence is dramatically reduced in kinetin-treated leaves or leaves of plants producing higher levels of cytokinins Plants overproducing Control plants cytokinin Smart, C.M., Scofield, S.R., Bevan, M.W., and Dyer, T.A. (1991). Delayed leaf senescence in tobacco plants transformed with tmr, a gene for cytokinin production in Agrobacterium. Plant Cell 3: 647-656. © 2013 American Society of Plant Biologists  Lecture outline Cytokinin Cytokinin perception Cytokinin’s roles in whole- homeostasis and signaling plant processes Catabolism Conjugation Synthesis CK Transport Perception TF activation/ Target Biological (receptor) inactivation genes Functions 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  Cytokinin homeostasis Cytokinin homeostasis Catabolism Conjugation Synthesis CK Transport Perception TF activation/ Target Biological (receptor) inactivation genes Functions 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  Structure of major cytokinins Cytokinins are N6- substituted adenine- related compounds. adenine Trans-zeatin and isopentenyl-adenine trans-zeatin (tZ) are the most active and abundant CKs Isopentenyl- adenine (iP) © 2013 American Society of Plant Biologists The plant pathogen Agrobacterium tumefaciens induces hormone- based tumors Agrobacterium tumefaciens is a natural plant pathogen. It causes crown gall disease and tumor-like growths by inducing the plant to produce auxin and cytokinin Agrobacterium tumefaciens on the surface of a plant cell SEM courtesy of Martha Hawes, University of Arizona; grown gall by C-M © 2013 American Society of Plant Biologists  Agrobacterium tumefaciens transfers T-DNA to plant genomes Plant Cell Nucleus Agrobacterium tumefaciens Agrobacterium tumefaciens carries tumor-inducing (Ti) plasmids. A subset of the plasmid DNA called Agrobacterium tumefaciens on the surface of a plant cell 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 Normal tumor plasmid Auxin synthesis Cytokinin synthesis © 2013 American Society of Plant Biologists  T-DNA includes genes for biosynthesis of auxin and cytokinin Auxin and CK Normal T-DNA produced Ti plasmid normal tumor Auxin produced These studies showed rooty tumor that the Agrobacterium No tmr gene tmr gene encodes a CK produced cytokinin biosynthetic shooty tumor enzyme No tms gene © 2013 American Society of Plant Biologists  The tmr gene encodes isopentenyl- transferase, a key enzyme in CK synthesis Isopentenyl- transferase (IPT) AMP DMAPP – iPRMP isopentenyladenine (iP) dimethylallyl- riboside phosphate diphosphate 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  The bacterial IPT gene was used to isolate plant IPT genes AtIPT2 (and AtIPT9) encode tRNA- IPT enzymes that use tRNA as a substrate Arabidopsis and rice encode 7 and 8 IPTs respectively Takei, K., Sakakibara, H., and Sugiyama, T. (2001) Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana. J. Biol. Chem. 276: 26405-26410. © 2013 American Society of Plant Biologists  trans-zeatin synthesis Isopentenyl- CYP735A transferase (IPT) (trans- hydroxylation) ADP/ + iPDP/ tZDP/ tZTP iPTP transZeatin (tZ) ATP riboside phosphate iP tZ The iP precursors iPDP or iPTP can be trans-hydroxylated by CYP735A to produce tZDP or tZTP, precursors of tZ Therefore, CYP735A activity contributes to the relative abundance of iP vs tZ 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  Production of active CKs Isopentenyl- CYP735A transferase (IPT) (trans- hydroxylation) ADP/ + iPDP/ tZDP/ ATP iPTP IP and tZ are tZTP produced by dephosphorylation and deribosylation tZ ACTIVE trans-zeatin FORMS iP Isopentenyl- adenine 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  Production of active CKs iPDP/ tZDP/ iPTP LONELY tZTP This can occur via one GUY or two enzymatic steps; LONELY GUY encode an enzyme that produces active CKs in a single step tZ ACTIVE trans-zeatin FORMS iP Isopentenyl- adenine 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 Altering expression of CK synthetic enzymes affects plant growth… Isopentenyl- transferase (IPT) CYP735A LONELY GUY tZ ACTIVE trans-zeatin FORMS iP Isopentenyl- adenine 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  ipt mutants have reduced shoot growth and enhanced root elongation growth WT A quadruple ipt loss-of- function mutant reduces CK levels to less than 20% of those in wild-type plants atipt1;3;5;7 These CK deficient mutants have dramatically reduced shoot apical meristems and shoot growth and enhanced root growth WT atipt1; WT atipt1; 3;5;7 3;5;7 Miyawaki, K., et al. (2006). Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis. Proc. Natl. Acad. Sci. USA 103: 16598-16603. © 2013 American Society of Plant Biologists  CYP735A expression (and tZ production) is tightly regulated CYP735A genes are root- specific, induced by cytokinins and repressed by auxin or ABA iPDP/ tZDP/ CYP735A iPTP tZTP iP tZ Takei, K., Yamaya, T., and Sakakibara, H. (2004). Arabidopsis CYP735A1 and CYP735A2 encode cytokinin hydroxylases that catalyze the biosynthesis of trans-zeatin. J. Biol. Chem. 279: 41866-41872. © 2013 American Society of Plant Biologists Loss-of-function log mutants fail to maintain the shoot meristem LONELY GUY (LOG) mRNA is specifically localized in shoot meristem tips. Loss-of-function log mutants show reduced shoot branching and abnormal flowers, presumably caused by reduced levels of active CK Reprinted by permission from Macmillan Publishers Ltd.: Nature. Kurakawa, T., et al. (2007). Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445: 652-655, copyright 2007. © 2013 American Society of Plant Biologists LONELY GUY genes contribute to CK production in Arabidopsis As in rice, loss-of-function log mutants in Arabidopsis have smaller inflorescences and fewer flowers and seed pods WT 35S::LOG4 LOG overexpression phenotypes are more subtle than IPT- overexpression; shown here is a delay in leaf senescence WT log3 log4 log7 Kuroha, T., et al. (2009) Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in Arabidopsis.Plant Cell 21: 3152–3169. © 2013 American Society of Plant Biologists  Cytokinin can be inactivated by conjugation or degradation The CKX genes are important regulators of active cytokinin levels Adenine O-glycosylation Phosphoribosyl or O-acetylation A Transferase 1 LOG Irreversible (APT1) Degradation Cytokinin Reversible oxidase Conjugation (CKX) CKX genes are Adenylation ACTIVE CK-induced Glucosylation site FORM 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 CK biosynthesis and degradation is compartmentalized within cells Apoplast Plastid The cellular and Nucleus Vacuole subcellular AtIPT1 AtCKX2 AtIPT3 distribution of AtCKX4 AtIPT5 AtCKX1 synthetic and AtIPT8 degradation AtCKX5 AtCKX3 Cytosol enzymes AtCKX6 Mitochondria AtIPT4 ensures that endogenous AtIPT7 AtCKX7 cytokinin levels can be precisely regulated Adapted from Frébort, I., Kowalska, M., Hluska, T., Frébortová, J., and Galuszka, P. (2011). Evolution of cytokinin biosynthesis and degradation. J. Exp.Bot. 62: 2431–2452. © 2013 American Society of Plant Biologists CKX overexpression enhances root growth and decreases shoot growth Notice that these growth effects are very similar to those caused by the loss-of-function ipt mutations! Werner, T., Motyka, V., Strnad, M., and Schmülling, T. (2001). Regulation of plant growth by cytokinin. Proc. Natl. Acad. Sci. USA 98: 10487-10492, Copyright 2001 National Academy of Sciences, USA. © 2013 American Society of Plant Biologists  Increasing [CK] through ckx knock- outs leads to increased seed yields Elevated levels of CK correlates with increased meristem size and increased numbers of seeds per plant, suggesting a non-transgenic approach to increasing crop yields Batrina, I., Otto, E., Strnad, M., Werner, T., and Schmülling, T. 2011. Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana. Plant Cell 23: 69-80. © 2013 American Society of Plant Biologists Natural variation in CKX expression affects grain production in rice The rice variety Koshihikari (Ko) has elevated expression of OsCKX2, lower CK levels, and reduced grain production as compared to the closely related near- inbred-line (NIL) Rice plants that accumulate more CK can produce more grain per plant because of changes in inflorescence architecture Ashikari, M. et al. (2005) Cytokinin oxidase regulates rice grain production. Science 309: 741 – 745, with permission from AAAS; © 2013 American Society of Plant Biologists CK acts as a paracrine and a long- distance signal Induction of A localized induction IPT at node of IPT promotes lateral bud outgrowth only at the induced node, indicating that the synthesized CK acts locally only (like a paracrine signal) The log phenotype also demonstrates a requirement for localized CK synthesis TIME From Faiss, M., Zalubìlová, J., Strnad, M., and Schmülling, T. (1997). Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants. Plant J. 12: 401-415. © 2013 American Society of Plant Biologists Two non-specific cytokinin transport proteins have been identified PUP preferentially moves the free base, and ENT moves the riboside, but neither is specific for CKs and their biological functions are not well characterized Purine Equilibrative permease Nucleoside (PUP) Transporter (ENT) Kudo, T., Kiba, T., and Sakakibara, H. (2010) Metabolism and long-distance translocation of cytokinins. J. Integrative Plant Biol. 52: 53-60. © 2013 American Society of Plant Biologists  CKs translocate through the xylem and phloem Different CKs are produced and transported differently throughout the plant. These different forms may convey different information The phloem sap contains iP and tZ Xylem Phloem The xylem sap mostly contains tZ and tZ In the roots riboside which is synthesized at high levels in CYP735A iPRP tZRP the roots by CYP735A Lomin, S.N., Krivosheev, D.M., Steklov, M.Y., Osolodkin, D.I. and Romanov, G.A. (2012) Receptor properties and features of cytokinin signaling. Acta Naturae 3: 31–45. © 2013 American Society of Plant Biologists  Wild-type and ipt1;3;5;7 mutant grafts reveal CK translocation The ipt1;3;5;7 mutants (m) make very narrow stems with little secondary growth. This phenotype is rescued by grafting with wild-type (w) shoots or wild-type roots, demonstrating long-distance translocation of CKs These studies also demonstrate that CKs have a critical role in vascular cambium activity Matsumoto-Kitano, M., et al. (2008). Cytokinins are central regulators of cambial activity. Proc. Natl. Acad. Sci. USA 105: 20027-20031. Copyright (2008) National Academy of Sciences, USA. © 2013 American Society of Plant Biologists  CK homeostasis - summary CKs are synthesized by IPTs, CYP735As and LOGs CKs can be reversibly and irreversibly inactivated Increasing CK levels: enhance shoot growth decrease apical dominance delay leaf senescence decrease root growth CKs can act as paracrine or long-distance signals CKs can be translocated through xylem and phloem © 2013 American Society of Plant Biologists  Cytokinin perception and signaling Cytokinin perception and signaling Catabolism Conjugation Synthesis CK Transport Perception TF activation/ Target Biological (receptor) inactivation genes Functions © 2013 American Society of Plant Biologists  CK signaling is mediated by histidine-kinase receptors CK receptors CK receptors all have a CHASE Cytokinin receptor (AHK2) domain that binds CK Cytokinin receptor (AHK3) Related HKs Related proteins include ethylene receptors and HKs without CHASE domains All these proteins are hybrid- histidine kinase proteins related to Histidine bacterial two-component signaling Kinase domain systems 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  Higher plants have ≥ 3 CK receptors with slightly different properties CK binding domain Basal plants Higher plants The receptors apparently diversified early in the CHASE stands for “cyclase / evolution of histidine kinase associated higher plants sensory extracellular” Pils, B. and Heyl, A. (2009). Unraveling the evolution of cytokinin signaling. Plant Physiol. 151: 782-791; Reprinted from Heyl, A., Riefler, M., Romanov, G.A. and Schmülling, T. (2011) Properties, functions and evolution of cytokinin receptors. Eur. J. Cell Biol. (2011), doi:10.1016/j.ejcb.2011.02.009 with permission from Elsevier. © 2013 American Society of Plant Biologists Cytokinin signaling is mediated by a two-component-like system Input Transmitter Receiver Output domain domain domain domain H D Histidine Kinase Response Regulator A two-component system is a short signaling pathway that moves information from an input to an output. In bacteria it usually consists of two proteins, a histidine kinase (HK) and a response regulator (RR) © 2013 American Society of Plant Biologists Cytokinin signaling is mediated by a two-component-like system ATP ADP P H D Perception at the input domain activates the histidine kinase domain. In bacterial systems the input domain is involved in environmental sensing © 2013 American Society of Plant Biologists Cytokinin signaling is mediated by a two-component-like system ATP ADP P H D P H D The phosphoryl group is relayed to an aspartate (D) on the receiver domain of the response regulator © 2013 American Society of Plant Biologists Hybrid histidine kinases participate in multistep signaling H Typical bacterial HK H D H D Histidine-containing Response phosphotransfer protein regulator (HPT) (In Arabidopsis the HPTs are referred to as AHPs) © 2013 American Society of Plant Biologists The Arabidopsis cytokinin response pathway Type-C ARR ARR22 ARR24 Three CK receptors Five histidine 23 response phosphotransfer regulators (RRs) of proteins (HPTs) - In three types Arabidopsis known – In Arabidopsis as AHPs 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 Arabidopsis has three CK receptors, AHK2, 3 and 4 Cytokinin receptor (AHK2) Cytokinin receptor (AHK3) AHK4 was identified as a wooden-leg mutant wol (wol) and a cytokinin response mutant (cre1) The wol root is Wild-type cre1 truncated due to impaired differentiation in central cylinder. The cre1 mutants do not produce shoots in WT tissue culture 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  CK receptor function can be assayed in a protoplast system +AHK CK-inducible ARR6 promoter LUC Protoplasts carrying a CK-induced promoter fused to a reporter gene (LUC) can reveal enhanced CK responses © 2013 American Society of Plant Biologists  Arabidopsis CK receptors have partially redundant activities in vivo CK response was determined by CK- induced greening and inhibition of root growth. Both responses are abolished in the triple mutant. Double mutant phenotypes are generally more severe than single mutant phenotypes Higuchi, M., et al. (2004). In planta functions of the Arabidopsis cytokinin receptor family. Proc. Natl. Acad. Sci. USA 101: 8821-8826.Copyright (2004) National Academy of Sciences, USA. © 2013 American Society of Plant Biologists  CK receptors have distinct roles in CK responses Fertility, seed size Chlorophyll retention Primary root Leaf cell formation Germination CK-induced elongation Root branching Cytochrome metabolism photomorphogenesis Root response to Cold-stress slginalng exogenous CK In vitro shoot regeneration Riefler, M., Novak, O., Strnad, M., and Schmülling, T. (2006). Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. Plant Cell 18: 40-54. © 2013 American Society of Plant Biologists Downstream of the receptors: HPs 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 23 response phosphotransfer regulators (RRs) of proteins (HPTs) - In three types Arabidopsis known – In Arabidopsis as AHPs 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 HPTs shuttle between the cytoplasm and nucleus cytoplasm nucleus P P H D H H D Receptor His Response regulator phosphotransfer protein © 2013 American Society of Plant Biologists HPTs / AHPs are necessary but act somewhat redundantly ahp2 ahp2 ahp2 ahp3 ahp3 WT ahp3 ahp5 ahp5 ahp5 Loss-of-function of one or two AHPs has no apparent effect, suggesting that they act somewhat redundantly in transducing CK signaling. A triple mutant has a short root characteristic of many mutants deficient in CK signaling Hutchison, C.E., et al. (2006). The Arabidopsis Histidine Phosphotransfer Proteins Are Redundant Positive Regulators of Cytokinin Signaling. Plant Cell 18: 3073-3087. © 2013 American Society of Plant Biologists  Response regulators are positive or negative regulators of CK signaling Type-C ARR ARR22 ARR24 Response regulators (RRs) aka ARRs in Arabidopsis) Type-B ARRs are positive regulators, type-A and type-C are negative 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  There are three types of response Receiver regulators domain D Arabidopsis: 10 Type-A ARRs Negative regulators D Arabidopsis: 2 Type-C ARRs D Arabidopsis: 11 Type-B ARRs DNA-binding Type-B ARRs are transcriptional activators domain with C-terminal DNA- binding domains Pseudoresponse regulators (PRRs) are related E 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 Wild-type arr1 loss-of-function The concentration of CK Loss-of-function arr1 Overexpression of needed to produce green mutants are less ARR1 makes tissues tissues is a good measure sensitive to CK more sensitive to CK of CK sensitivity 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-B response regulators are partially redundant Single loss-of- function arr mutants are still sensitive to CK Double and triple mutants are +CK impaired in CK responses Mason, M.G., Mathews, D.E., Argyros, D.A., Maxwell, B.B., Kieber, J.J., Alonso, J.M., Ecker, J.R., and Schaller, G.E. (2005). Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis. Plant Cell 17: 3007-3018. © 2013 American Society of Plant Biologists  Type-B response regulators bind to a DNA cytokinin response motif Type-B ARR -A/GGAT(T/C)- T-rich enhancer -AAGAT(T/C)TT- Type-B response regulators In the ARR6 promoter, other type-B ARRs bind -(A/G)GAT(T/C)- bind to a cytokinin response to an extended CRM, and a 27 bp enhancer motif (CRM) region is necessary for full gene activation by cytokinin Reprinted from Ramireddy, E., Brenner, W.G., Pfeifer, A., Heyl, A. and Schmülling, T. (2013) In Planta analysis of a cis-regulatory cytokinin response motif in Arabidopsis and identification of a novel enhancer sequence. Plant Cell Physiol. 54: 1079–1092 by permission of Oxford University Press; See also Hosoda, K., Imamura, A., Katoh, E., Hatta, T., Tachiki, M., Yamada, H., Mizuno, T. and Yamazaki, T. (2002) Molecular structure of the GARP family of plant myb-related DNA binding motifs of the Arabidopsis response regulators. Plant Cell 14: 2015–2029. © 2013 American Society of Plant Biologists  Transcriptional responses can be visualized by a reporter construct LUC/GFP The TCS cytokinin reporter consists CaMV 35S min A(A/G)GAT(C/T)TT promoter of a concatamer of 24 repeats of Cytokinin responses in different Arabidopsis tissues the consensus B- type-ARR cytokinin response motif and a minimal 35S promoter to drive luciferase or pavement shoot apical lateral root GFP reporter cells and meristem primordium expression. root stomata meristem Zürcher, E., Tavor-Deslex, D., Lituiev, D., Enkerli, K., Tarr, P.T. and Müller, B. (2013) A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta. Plant Physiol 161: 1066–1075. © 2013 American Society of Plant Biologists Type-A ARRs are rapidly induced by cytokinins Minutes after CK application ARR5pro::GUS 0 µM BA 0.5 µM BA 5 µM BA Control - -tubulin D'Agostino, I.B., Deruere, J., and Kieber, J.J. (2000). Characterization of the response of the Arabidopsis response regulator gene family to cytokinin. Plant Physiol. 124: 1706-1717. © 2013 American Society of Plant Biologists  Overexpression of a type-A ARR interferes with shoot initiation in vitro Control Overexpression of rice type-A RR6 Hirose, N., Makita, N., Kojima, M., Kamada-Nobusada, T., and Sakakibara, H. (2007). Overexpression of a type-A response regulator alters rice morphology and cytokinin metabolism. Plant Cell Physiol. 48: 523-539. © 2013 American Society of Plant Biologists  What are the downstream effects of the phosphorelay signaling system? What happens downstream of the phosphorelay system? H D H D Type-B ARRs are transcriptional activators. Their targets include: Type-A ARRs CKXs Other transcription factors The Aux/IAA auxin repressor SHY2 © 2013 American Society of Plant Biologists Perception and signalling: Summary CKs are perceived by hybrid histidine kinases CK binding initiates a phosphorelay that ultimately activates type-B ARR (Response Regulator) transcription factors CK signaling is negatively regulated by type-A and type-C ARRs CK signaling is also mediated by CRFs (Cytokinin Response Factors) © 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 Cytokinin’s roles in whole- Many other processes plant processes Catabolism Conjugation Synthesis CK Transport Perception TF activation/ Target Biological (receptor) inactivation genes 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  Formation and maintenance of the shoot meristem requires cytokinin stm1 mutant Arabidopsis expressing IPT from stm1 mutant STM promoter STM IPT CK The shootmeristemless1 mutant (stm1) fails to initiate a shoot apical meristem. This mutant can be rescued by CK application or IPT gene expression at the SAM. STM is a transcription factor that induces expression of an IPT gene Reprinted from Yanai, O., et al. (2005). Arabidopsis KNOXI Proteins activate cytokinin biosynthesis. Curr Biol. 15: 1566-1571, with permission from Elsevier. © 2013 American Society of Plant Biologists  CK regulates meristem size and phyllotaxy by interaction with auxin Wild Type: aberrant phyllotaxy alternate (abph1) mutant: leaves opposite leaves The ABPH1 gene encodes a type-A response regulator that inhibits meristem size in wild type maize. The abph1 mutant has an enlarged meristem that disrupts auxin signaling andleads to incorrect leaf positioning. Lee, B.-H., Johnston, R., Yang, Y., Gallavotti, A., Kojima, M., Travençolo, B.A.N., Cast L. da F., Sakakibara, H. and Jackson, D. (2009) Studies of aberrant phyllotaxy1 mutants of maize indicate complex interactions between auxin and cytokinin signaling in the shoot apical meristem. Plant Physiol. 150: 205-216; Giulini A, Wang J, Jackson D. (2004) Control of phyllotaxy by the cytokinin-inducible response regulator homologue ABPHYL1. Nature 430: 1031–1034. © 2013 American Society of Plant Biologists  CKs regulate leaf shape complexity Tomato Leaf 6 Leaflet Leaf 6 Leaflet Leaf 6 Leaflet Increased CK expression in the Decreased CK leaf margins leads to increased expression in the leaf leaf complexity margins leads to decreased leaf complexity The control of leaf shape is dependent on Knotted1-like homeobox (KNOX1) gene function, which positively regulates CK biosynthesis. CKs can partially substitute for KNOX1 function. Shani, E., Ben-Gera, H., Shleizer-Burko, S., Burko, Y., Weiss, D., Ori, N. (2010) Cytokinin regulates compound leaf development in tomato. Plant Cell 22: 3206–3217. © 2013 American Society of Plant Biologists  Cytokinin stimulates lateral bud outgrowth together with auxin Cytokinins can directly stimulate lateral bud outgrowth, but auxins repress outgrowth by downregulating cytokinin synthesis at the node. If plants are decapitated, newly-syntehsized cytokinins at the node are transported into the bud and cause its outgrowth Reprinted by permission from Macmillan Publishers Ltd from Damagalska, M. and Leyser, O. (2011) Signal integration in the control of shoot branching. Nature Reviews: Molecular Cell Biology 12: 211–221. © 2013 American Society of Plant Biologists  Cytokinin and auxin interact to regulate lateral root outgrowth Cytokinin, via the CRE1 receptor, negatively controls lateral root initiation by repressing PIN1 expression in the pericycle cells flanking the lateral root founder cells (light blue), causing an auxin response maxima (dark blue) in the lateral root founder cells. Reprinted from Perilli, S., Moubayidin, L. and Sabatini, S. (2010) The molecular basis of cytokinin function. Curr. Opin. Plant. Biol. 13: 21-26, with permission from Elsevier. Cytokinin interacts with several other hormones besides auxin Reproduced with permission from El-Showk, S., Ruonala, R. and Helariutta, Y. (2013) Crossing paths: cytokinin signalling and crosstalk. Development 140: 1373–1383.  CKs contribute to nutrient uptake and allocation Sink Shoot systems are a source of sugars and Source primary metabolites that are distributed to CO2 nutrient sinks including Sink flowers and fruits, roots, and young leaves Source Elevated CK levels Root systems take increase expression of up mineral nutrients Sink photosynthetic enzymes such as nitrate, and delay leaf NO3- senescence sulfate and SO42- phosphate PO43- © 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  Regulation of leaf senescence by CKs 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 CK also reduces the plant’s sensitivity to Loss-of-function ABA, so it acts through mutants affecting CK ABA-dependent and synthesis (atipt) or independent pathways signaling (ahp) are drought tolerant Stress ahp2,3,5 Wild type C Drought CK ABA tolerance is conferred in part by an increase in Stress Three-week-old plants exposed to membrane tolerance drought stress for13 or 14 days and then re-watered for three days stability 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  However, by delaying senescence, CKs increase drought tolerance Drought-induced IPT expression This suggests that an inducible confers drought-tolerance system to boost CK levels prior to leaf senescence can Wild-type Elevated CK contribute to drought tolerance Rivero, R. M. et al. (2007) Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc. Natl. Acad. Sci. USA 104: 19631-19636 Copyright National Academy of Sciences; Ha, S., Vankova, R., Yamaguchi.Shiozaki, K., Shiozaki, K. and Tran, L.S. (2012) Cytokinins: metabolism and function in plant adaptation to environmental stresses. Trends Plant Sci. 17: 172–179. © 2013 American Society of Plant Biologists  CKs contribute to plant defenses… CK enhances Arabidopsis 1 μM CK ARR2, a type-B ARR, immunity to the interacts with TGA3, a bacterial pathogen salicylic acid-responsive Pseudomonas transcription factor, to syringae induce PR1 and other defense-response genes CK receptor mutants (ahk2 ahk3) are more susceptible ASA Leaves pretreated with cytokinins are more resistant to Pseudomonas syringae infection Reprinted from Choi, J., Huh, S.U., Kojima, M., Sakakibara, H., Paek, K.-H., and Hwang, I. (2010). The cytokinin-activated transcription factor ARR2 promotes plant immunity via TGA3/NPR1-dependent salicylic acid signaling in Arabidopsis. Devel. Cell 19: 284-295 with permission from Elsevier. Großkinsky, D.K., et al and and Roitsch, T. (2011). Cytokinins mediate resistance against Pseudomonas syringae in tobacco through increased antimicrobial phytoalexin synthesis independent of salicylic acid signaling. Plant Physiol. 157: 815-830. © 2013 American Society of Plant Biologists  CKs contribute to plant defenses, but can be exploited by pathogens Phytopathogenic bacteria can modify plant development through production of cytokinins, or cytokinins and auxin, to produce leafy galls or crown galls Leafy gall produced by the The gall tissue bacterium Rhodococcus fascians provides a good substrate for the bacteria Crown gall produced by Agrobacterium tumefaciens Stes, E., Vandeputte, O.M., El Jaziri, M., Holsters, M. and Vereecke, D. (2011). A successful bacterial coup d'état: How Rhodococcus fascians redirects plant development. Annu. Rev. Phytopathology. 49: 69-86. See also Giron, D., Frago, E., Glevarec, G., Pieterse, C.M.J. and Dicke, M. (2013). Cytokinins as key regulators in plant–microbe–insect interactions: connecting plant growth and defence. Functional Ecology. 27: 599-609. Image credit Royal Horticultural Society, Edward L. Barnard, Florida Department of Agriculture and Consumer Services, Bugwood.org © 2013 American Society of Plant Biologists  CKs contribute to plant defenses, but can be exploited by herbivores Leaf-mining insects can maintain nutrient-rich “green islands” through CK accumulation – the CKs may be produced by the insects’ symbiotic bacteria! The development of insect-induced galls involves cytokinins that may be produced by the insect larvae they house Giron, D., Kaiser, W., Imbault, N. and Casas, J. (2007). Cytokinin-mediated leaf manipulation by a leafminer caterpillar. Biol.Lett. 3: 340-343. Kaiser, W., Huguet, E., Casas, J., Commin, C. and Giron, D. (2010). Plant green-island phenotype induced by leaf-miners is mediated by bacterial symbionts. Proc. Roy. Soc. B. 277: 2311-2319. Mapes, C.C. and Davies, P.J. (2001). Cytokinins in the ball gall of Solidago altissima and in the gall forming larvae of Eurosta solidaginis. New Phytologist. 151: 203-212. See also Stirk, W.A. and Staden, J. (2010). Flow of cytokinins through the environment. Plant Growth Regulation. 62: 101-116. Photo credit: SriMesh © 2013 American Society of Plant Biologists  CK- mediated 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 What signals are carried by in vivo functions? xylem-borne tZ versus phloem- What are the target borne iP? genes, and what do Catabolism Conjugation they do? Synthesis CK Transport Perception TF activation/ Target Biological (receptor) inactivation genes Functions Why is localized Are signals from How do the type-A How do all these CK synthesis and type-C ARRs sometimes the three receptors work? What is the pieces fit together to critical and integrated or kept relationship with make a functioning separate? CRFs? plant???? sometimes not? 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