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 and CK Top row – the plant on the right is silenced in a CK receptor and deficient in root nodule formation. Bottom row: The figure on the left shows a rice variety with slightly increased CK levels leading to increased seed production. In the middle is the classic experiment showing the effects of CK, auxin or both on differentiation of tobacco leaf discs. On the right the AtCKX1 overexpression plant has an increased turnover of CK leading to increased root growth.

Structure of major cytokinins Cytokinins are N6-substituted adenine-related compounds. Trans-zeatin and isopentenyl-adenine are the most active and abundant CKs adenine trans-zeatin (tZ) Isopentenyl-adenine (iP)

Agrobacterium tumefaciens induces hormone-based tumors Plant Cell Nucleus DNA Agrobacterium tumefaciens SEM courtesy of Martha Hawes, University of Arizona; grown gall by C-M Agrobacterium tumefaciens carries tumor-inducing (Ti) plasmids. A subset of the plasmid DNA called transfer-DNA (T-DNA) is mobilized into the plant nucleus Agrobacterium tumefaciens on the surface of a plant cell 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

T-DNA includes genes for biosynthesis of auxin and cytokinin Ti plasmid normal tumor Auxin and CK produced No tmr gene No tms gene rooty tumor shooty tumor Auxin produced CK produced Normal T-DNA These studies showed that the Agrobacterium tmr gene encodes a cytokinin biosynthetic enzyme

The tmr gene encodes isopentenyl-transferase, a key enzyme in CK synthesis iPRMP isopentenyladenine (iP) riboside phosphate Isopentenyl-transferase (IPT) DMAPP – dimethylallyl-diphosphate The bacterial IPT gene uses AMP exclusively as a substrate whereas plant IPT genes prefer ADP or ATP AMP Haberer, G. and Kieber, J.J. (2002) Cytokinins. New insights into a classic phytohormone. Plant Physiol. 128: 354-362.

IPT overexpression causes reduced apical dominance, reduced root growth and delayed leaf senescence Medford, J.I., et al. (1989) Alterations of endogenous cytokinins in plants using a chimeric isopentenyl transferase gene Plant Cell1: 403-413. Wild type IPT overexpression Elevated CK promotes shoot growth and restricts root growth Elevated CK promotes shoot bud outgrowth

Cytokinins can be inactivated by conjugation or degradation 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. A ACTIVE FORM CKX genes are CK-induced Irreversible Degradation LOG Cytokinin oxidase (CKX) Reversible Conjugation Glucosylation site O-glycosylation or O-acetylation Adenylation Adenine Phosphoribosyl Transferase 1 (APT1) The CKX genes are important regulators of active cytokinin levels

Cytokinin signaling takes place through a phosphorelay system Type-C ARR ARR22 ARR24 Adapted from Schaller, G.E., Kieber, J.J., and Shiu, S (2008) Two-component signaling elements and histidyl-aspartyl phosphorelays. The Arabidopsis Book: ASPB. 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

Arabidopsis has three CK receptors, AHK2, 3 and 4 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. Cytokinin receptor (AHK2) Cytokinin receptor (AHK3) AHK4 was identified as a wooden-leg mutant (wol) and a cytokinin response mutant (cre1) WT wol Wild-type cre1 The wol root is truncated due to impaired differentiation in central cylinder. The cre1 mutants do not produce shoots in tissue culture

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.

Receptors relay phosphoryl groups to His phosphotransfer proteins Type-C ARR ARR22 ARR24 Adapted from Schaller, G.E., Kieber, J.J., and Shiu, S (2008) Two-component signaling elements and histidyl-aspartyl phosphorelays. The Arabidopsis Book: ASPB. 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

There are three types of response regulators D E Arabidopsis: 10 Type-A ARRs Arabidopsis: 2 Type-C ARRs Arabidopsis: 11 Type-B ARRs Negative regulators Pseudoresponse regulators (PRRs) are related proteins but not involved in CK signaling DNA-binding domain Receiver domain Type-B ARRs are transcriptional activators with C-terminal DNA- binding domains PRRs usually lack the conserved Asp residue in the receiver domain

ARR1, a type-B ARR, is a positive regulator of CK signaling Overexpression of ARR1 makes tissues more sensitive to CK ARR1 overexpression Wild-type arr1 loss-of-function Loss-of-function arr1 mutants are less sensitive to CK The concentration of CK needed to produce green tissues is a good measure 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.

Type A ARRs are negative regulators of CK signaling LUC CK-inducible ARR6 promoter D Expression of type-A ARRs interferes with CK-induced transcription in protoplasts Type-A ARR 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,

Type-C ARRs may remove phosphoryl groups from the system Phospho-relay system Histidine kinase activity adds phosphoryl groups to system Type-A ARRs Type-B ARRs – CK-induced transcription Type-C ARRs remove phosphoryl groups from the system via histidine phosphatase activity Putative competition between type-A and type-B ARRs ? Type-B ARRs are activated by phosphorylation. The total input of phosphoryl groups is derived from histidine kinase activity. The phosphoryl groups can be removed from the system by phosphatase activity of both the type-C ARRs, and as will be seen later, the AHK4 receptor when it is not bound to CK. The phosphorylation state of type-B ARRs is also dictated by the phosphorelay system moving in the reverse direction.

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 Synthesis CK Transport Perception (receptor) TF activation/ inactivation Target genes Biological Functions Catabolism Conjugation Cytokinin’s roles in whole-plant processes

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.

Cytokinin and auxin regulate organogenesis in tissue culture + CK + auxin and CK Tobacco leaf discs are placed into sterile culture dishes on medium containing various hormones TIME Images courtesy of Richard Amasino.

CKs contribute to nutrient uptake and allocation NO3- SO42- PO43- CO2 Sink Source Root systems take up mineral nutrients such as nitrate, sulfate and phosphate Shoot systems are a source of sugars and primary metabolites that are distributed to nutrient sinks including flowers and fruits, roots, and young leaves Elevated CK levels increase expression of photosynthetic enzymes and delay leaf senescence

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.

Cytokinins delay leaf senescence 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.

CKs can negatively affect stress tolerance Three-week-old plants exposed to drought stress for13 or 14 days and then re-watered for three days C Wild type ahp2,3,5 Loss-of-function mutants affecting CK synthesis (atipt) or signaling (ahp) are drought tolerant Drought tolerance is conferred in part by an increase in membrane stability CK also reduces the plant’s sensitivity to ABA, so it acts through ABA-dependent and independent pathways Stress tolerance CK ABA Stress 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.

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.

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

Ongoing investigations Synthesis CK Transport Perception (receptor) TF activation/ inactivation Target genes Biological Functions Catabolism Conjugation Why is localized CK synthesis sometimes critical and sometimes not? What signals are carried by xylem-borne tZ versus phloem-borne iP? How do catabolism and conjugation contribute to in vivo functions? What are the target genes, and what do they do? Are signals from the three receptors integrated or kept separate? How do all these pieces fit together to make a functioning plant???? How do the type-A and type-C ARRs work? What is the relationship with CRFs? 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.