LES CYTOKININES. 1962: la molécule active du lait de coco a les caractéristiques d une base nucléique, l adénosine (base purique)

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1 dia1 LES CYTOKININES Les cytokinines (kutos, cellule; kinein, mouvoir, au sens de séparer) sont nécessaires à la division de cellules 1941: Blakeslee essaie de créer des hybrides entre différentes espèces de Datura, les embryons immatures meurent. L utilisation de lait de coco (un albumen liquide) permet de les faire développer in vitro 1948: le lait de coco, l extrait d albumen de maïs activent la prolifération plus que l auxine et permettent la culture de tissus ne répondant pas à l auxine 1962: la molécule active du lait de coco a les caractéristiques d une base nucléique, l adénosine (base purique) Indépendamment Skoog et Miller 1957, montrent que les produits de dégradation de l ADN stimulent la prolifération. Isolement Zéatine d albumen de maïs (1963), ribosyl zéatine de lait de coco La carotte et les hybrides N.Glauca/N.Langsdorffii ont de fort taux de cytokinines endogènes

2 dia Synthétique À partir de l autoclave de l ADN 1ère découverte synthétique

3 dia3 trans-zeatin synthesis Isopentenyltransferase (IPT) CYP735A (transhydroxylation) ADP/ ATP Adénosine 5 (di/tri) Phosphate + Hémiterpène diphosphate ipdp/ iptp Isopentenyl Adénosine (di/tri) phosphate tzdp/ tztp transzeatin (tz) riboside ip (di/tri) phosphate Isopentenyl Adénine (di/tri) phosphate The ip precursors ipdp or iptp can be trans-hydroxylated by CYP735A to produce tzdp or tztp, precursors of tz 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:

4 dia4 Production of active CKs Isopentenyltransferase (IPT) CYP735A (transhydroxylation) ADP/ ATP + ipdp/ iptp IP and tz are produced by dephosphorylation and deribosylation tzdp/ tztp ip Isopentenyladenine ACTIVE FORMS tz trans-zeatin Haberer, G. and Kieber, J.J. (2002) Cytokinins. New insights into a classic phytohormone. Plant Physiol. 128:

5 dia5 Production of active CKs This can occur via one or two enzymatic steps; LONELY GUY encode an enzyme that produces active CKs in a single step ipdp/ iptp ip Isopentenyladenine LONELY GUY ACTIVE FORMS tzdp/ tztp tz trans-zeatin Haberer, G. and Kieber, J.J. (2002) Cytokinins. New insights into a classic phytohormone. Plant Physiol. 128:

6 dia6 Cytokinin can be inactivated by conjugation or degradation Reversible Conjugation Irreversible Degradation Cytokinin CKX genes are CK-induced oxidase (CKX) ACTIVE FORM La kinétine est beaucoup plus résistante à la dégradation par la cytokinine oxydase que les cytokinines naturelles (utilisée en culture in vitro) Quand traitement au diphénylurée, mime un traitement aux cytokinines car les cytokinines oxydases sont inhibées Kieber JJ (2002) Cytokinins: March 27, The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists. doi: /tab.0063

7 dia7 Conjugaison des cytokinines Glycosylation sur le groupe hydroxyle de la chaîne latérale, O-glycosylation * Présents naturellement dans la plante En grande quantité dans la graine de riz en développement O-glycosylation sert à inactiver les cytokinines mais réversible, donc rôle de stockage des cytokinines, libérées après action de glycosidases

8 dia8 N-Glycosylation en position 3, 7 et 9 * Présents naturellement dans la plante N-Glycosylation, inactivation irréversible des cytokinines car probablement résistant au clivage enzymatique

9 dia9 Conjugaison avec l alanine inactivation irréversible des cytokinines car probablement résistant au clivage enzymatique Lieux de synthèse chez les plantes Principalement les racines, serait transportée par le xylème dans les parties aériennes mais aussi synthèse dans les cellules du méristème apical et les graines en développement

10 dia10 Rôles des cytokinines dans la division cellulaire Certaines bactéries possèdent des gènes codant des enzymes de la voie de biosynthèse des cytokinines Smith et Townsend montrent en 1907 que la galle du collet ou «crown-gall» est causée par une bactérie Agrobacterium tumefaciens, «cancer des plantes» ADN-T, gène catalysant la surproduction de cytokinines et d auxines Gène ipt = isopentényl transférase

11 dia11 Synthèse de cytokinines dans les tumeurs produites par Agrobacterium CYTOKININE

12 dia12 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:

13 dia13 Les cytokinines favorisent la division cellulaire en conjugaison avec l auxine Diminution de la phosphorylation Cyclin- Dependent Kinases /accumulation D-type cyclin (CYCD et CYCB) Mitose: noyau et cellule se divisent Gap2 ou G2 Gap1 ou G1 Activités cellulaires Synthèse: réplication ADN

14 dia14 Rôle des cytokinines dans l organogenèse Le rapport auxines / cytokinines détermine le devenir des tissus en culture En concentrations égales->division de cellules indifférenciées Auxine -> formation de racines Cytokinines -> bourgeons IBA: indole-3-butyric acid

15 dia15 Rôle des cytokinines dans la sénescence et la dominance apicale Tabac transgénique contenant une construction 35SCaMV-ipt d agrobacterium tumefaciens Contenu important en Z, [9R]Z et [9R-5 P]Z Retard dans la sénescence des feuilles Perte de la dominance apical

16 dia16 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:

17 dia17 Rôle des cytokinines dans la sénescence et la dominance apicale

18 dia18 Rôle des cytokinines dans la sénescence et la dominance apicale Tabac transgénique contenant une construction avec promoteur 35SCaMV modifié inductible par la tétracycline 35STET-ipt constitutif Activateur transcriptionnel Tet Tet CaMV CaMV ipt tétracycline Application locale de tétracycline

19 dia19 Rôle des cytokinines dans le développement -Tet Similaire à WT + Tet Application très tôt dans le développement Similaire WT Augmentation de l activation [Tet]

20 dia20 ipt mutants have reduced shoot growth and enhanced root elongation growth A quadruple ipt lossof-function mutant reduces CK levels to less than 20% of those in wild-type plants WT atipt1; 3;5;7 WT atipt1; 3;5;7 These CK deficient mutants have dramatically reduced shoot apical meristems and shoot growth and enhanced root growth Miyawaki, K., et al. (2006). Roles of Arabidopsis ATP/ADP isopentenyltransferases and trna isopentenyltransferases in cytokinin biosynthesis. Proc. Natl. Acad. Sci. USA 103:

21 dia21 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 seeds WT log3 log4 log7 WT 35S::LOG4 LOG overexpression phenotypes are more subtle than IPToverexpression; shown here is a delay in leaf senescence 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:

22 TRANSPORT DES CYTOKININES

23 dia22 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 permease (PUP) Equilibrative Nucleoside Transporter (ENT) Kudo, T., Kiba, T., and Sakakibara, H. (2010) Metabolism and long-distance translocation of cytokinins. J. Integrative Plant Biol. 52:

24 dia23 CKs translocate through the xylem and phloem The phloem sap contains ip and tz Different CKs are produced and transported differently throughout the plant. These different forms may convey different information In the roots CYP735A iprp tzrp Phloem Xylem The xylem sap mostly contains tz and tz riboside which is synthesized at high levels in the roots by CYP735A

25 dia24 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

26 The Arabidopsis cytokinin response pathway

27 dia25 Cytokinin perception and signaling Cytokinin perception and signaling Catabolism Conjugation Synthesis CK Transport Perception (receptor) TF activation/ inactivation Target genes Biological Functions

28 dia26 CK signaling is mediated by histidine-kinase receptors Type-C ARR ARR22 ARR24 Three CK receptors Five HPts (AHPs) Histidine-containing phosphotransfer protein (HPt) 23 response regulators (ARRs) (In Arabidopsis the HPts are referred to as AHPs) Adapted from Schaller, G.E., Kieber, J.J., and Shiu, S (2008) Two-component signaling elements and histidyl-aspartyl phosphorelays. The Arabidopsis Book: ASPB.

29 dia27 CK receptors have distinct roles in CK responses Fertility, seed size Germination Cytochrome metabolism Cold-stress signalling Chlorophyll retention CK-induced photomorphogenesis Primary root elongation Root response to exogenous CK In vitro shoot regeneration Leaf cell formation Root branching 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:

30 dia28 Cytokinin signaling is mediated by a two-component-like system Input domain Transmitter domain Receiver domain Output 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)

31 dia29 Cytokinin signaling is mediated by a two-component-like system ATP H P ADP D Perception at the input domain activates the histidine kinase domain. In bacterial systems the input domain is involved in environmental sensing

32 dia30 Cytokinin signaling is mediated by a two-component-like system ATP H P ADP D H P D The phosphoryl group is relayed to an aspartate (D) on the receiver domain of the response regulator

33 dia31 Cytokinin signaling is mediated by a two-component-like system ATP H P ADP D H P D this step involves a transfer of a phosphoryl group rather than another kinase step involving ATP. A twocomponent phosphorelay system is different from a protein kinase cascade

34 dia32 Downstream of the Receptors: AHPs and ARRs The receptor passes the phosphoryl group to an AHP which passes it to an ARR. Type-B ARRs are transcription factors To, J.P.C., et al. (2007) Cytokinin regulates type-a Arabidopsis response regulator activity and protein stability via two-component phosphorelay. Plant Cell 19:

35 dia33 AHPs shuttle between the cytoplasm and nucleus cytoplasm nucleus H D H H P P D

36 dia34 AHPs are necessary but act somewhat redundantly WT ahp2 ahp3 ahp2 ahp5 ahp3 ahp5 ahp2 ahp3 ahp5 Loss-of-function of one or two AHPs has no apparent effect, suggesting that they act somewhat redundantly in transducing CK signalling. A triple mutant has a short root characteristic of many mutants deficient in CK signalling. Hutchison, C.E., et al. (2006). The Arabidopsis Histidine Phosphotransfer Proteins Are Redundant Positive Regulators of Cytokinin Signaling. Plant Cell 18:

37 dia35 The ARRs are positive or negative regulators of CK signalling Type-C ARR ARR22 ARR24 CK receptors AHPs Response regulators (ARRs) 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.

38 dia36 How do type-a ARRs interfere with CK signaling? There are two nonmutually exclusive possibilities: Type-A ARRs might compete with type-b ARRs for phosphoryl groups Type-A ARRs might have other phosphorylationspecific functions To, J.P.C., et al. (2007) Cytokinin regulates type-a Arabidopsis response regulator activity and protein stability via two-component phosphorelay. Plant Cell 19:

39 dia37 CRF activity depends on AHKs and AHPs but not ARRs. CRFs:(cytokinin-response factors) are another family of CK-induced transcription factors CRFs act in parallel with ARRs Mutations in the ARR genes do not affect CRF localization, suggesting it acts in parallel with, not downstream of, the ARRs. Rashotte, A.M., Mason, M.G., Hutchison, C.E., Ferreira, F.J., Schaller, G.E., and Kieber, J.J. (2006). A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway. Proc. Natl. Acad. Sci. 103: , Copyright 2006 National Academy of Sciences, USA.

40 dia38 Perception and signaling - summary CKs are perceived by hybrid histidine kinases CK binding initiates a phosphorelay that ultimately activates type-b ARR transcription factors CK signalling is negatively regulated by type-a and type-c ARRs CK signalling is also mediated by CRFs

41 dia39 CK action in whole-plant processes CKs regulate Root vascular tissue development Shoot and root apical meristem functions Nutrient uptake and allocation Leaf senescence Many other processes Cytokinin s roles in wholeplant processes Catabolism Conjugation Synthesis CK Transport Perception (receptor) TF activation/ inactivation Target genes Biological Functions

42 dia40 Cell differentiation Auxin and cytokinin work together to regulate root meristem function Auxin transport In the root meristem, cytokinin promotes cell differentiation, whereas auxin sustains root meristem activity by promoting cell division. Auxin transport and response Cytokinin (Cell differentiation) Cytokinin biosynthesis Cell division Auxin (Cell division) cytokinin causes auxin redistribution, prompting cell differentiation Adapted from Dello Ioio, R., et al. (2008). A genetic framework for the control of cell division and differentiation in the root meristem. Science 322:

43 dia41 Rôles des cytokinines

44 dia42 CK-mediated processes There are many other processes mediated by CK. Identifying the specific genes that contribute to each of these will help 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: , with permission from Elsevier copyright 2009.

45 dia43 Role of cytokinin in the regulation of root gravitropism. Aloni R, Langhans M, Aloni E, Ullrich CI. Planta Nov;220(1): Epub 2004 Sep 10 Department of Plant Sciences, Tel Aviv University, 69978, Tel Aviv, Israel, The models explaining root gravitropism propose that the growth response of plants to gravity is regulated by asymmetric distribution of auxin (indole-3-acetic acid, IAA). Since cytokinin has a negative regulatory role in root growth, we suspected that it might function as an inhibitor of tropic root elongation during gravity response. Therefore, we examined the free-bioactive-cytokinin-dependent ARR5::GUS expression pattern in root tips of transformants of Arabidopsis thaliana (L.) Heynh., visualized high cytokinin concentrations in the root cap with specific monoclonal antibodies, and complemented the analyses by external application of cytokinin. Our findings show that mainly the statocytes of the cap produce cytokinin, which may contribute to the regulation of root gravitropism. The homogenous symmetric expression of the cytokinin-responsive promoter in vertical root caps rapidly changed within less than 30 min of gravistimulation into an asymmetrical activation pattern, visualized as a lateral, distinctly stained, concentrated spot on the new lower root side of the cap cells. This asymmetric cytokinin distribution obviously caused initiation of a downward curvature near the root apex during the early rapid phase of gravity response, by inhibiting elongation at the lower side and promoting growth at the upper side of the distal elongation zone closely behind the root cap. Exogenous cytokinin applied to vertical roots induced root bending towards the application site, confirming the suspected inhibitory effect of cytokinin in root gravitropism. Our results suggest that the early root graviresponse is controlled by cytokinin. We conclude that both cytokinin and auxin are key hormones that regulate root gravitropism. A novel regulatory pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4-mediated cytokinin-dependent regulation. Maruyama-Nakashita A, Nakamura Y, Yamaya T, Takahashi H. Plant J Jun;38(5): RIKEN Plant Science Center, Suehiro-cho, Tsurumi-ku, Yokohama , Japan. Cytokinin is an adenine derivative plant hormone that generally regulates plant cell division and differentiation in conjunction with auxin. We report that a major cue for the negative regulation of sulfur acquisition is executed by cytokinin response 1 (CRE1)/wooden leg (WOL)/Arabidopsis histidine kinase 4 (AHK4) cytokinin receptor in Arabidopsis root. We constructed a green fluorescent protein (GFP) reporter system that generally displays the expression of the high-affinity sulfate transporter SULTR1;2 in Arabidopsis roots. GFP under the control of SULTR1;2 promoter showed typical sulfur responses that correlate with the changes in SULTR1;2 mrna levels; accumulation of GFP was induced by sulfur limitation (-S), but was repressed in the presence of reduced sulfur compounds. Among the plant hormones tested, cytokinin significantly downregulated the expression of SULTR1;2. SULTR1;1 conducting sulfate uptake in sultr1;2 mutant was similarly downregulated by cytokinin. Downregulation of SULTR1;1 and SULTR1;2 by cytokinin correlated with the decrease in sulfate uptake activities in roots. The effect of cytokinin on sulfate uptake was moderated in the cre1-1 mutant, providing genetic evidence for involvement of CRE1/WOL/AHK4 in the negative regulation of high-affinity sulfate transporters. These data demonstrated the physiological importance of the cytokinin-dependent regulatory pathway in acquisition of sulfate in roots. Our results suggested that two different modes of regulation, represented as the -S induction and the cytokinindependent repression of sulfate transporters, independently control the uptake of sulfate in Arabidopsis roots.

46 dia44 The phenotype of Arabidopsis thaliana det1 mutants suggests a role for cytokinins in greening. Chory J, Aguilar N, Peto CA. Plant Biology Laboratory, Salk Institute for Biological Studies, San Diego, CA When grown in the absence of light, the det1 mutants of Arabidopsis thaliana (L.) Heynh. develop characteristics of light-grown plants as determined by morphological, cellular, and molecular criteria. Further, in light-grown plants, mutations in the DET1 gene affect cell-typespecific expression of light-regulated genes and the chloroplast developmental program. Here we show that the addition of exogenously added cytokinins (either 2-isopentenyl adenine, kinetin, or benzyladenine) to the growth medium of dark-germinated wild-type seedlings results in seedlings that resemble det1 mutants, instead of having the normal etiolated morphology. Like det1 mutants, these dark-grown seedlings now contain chloroplasts and have high levels of expression of genes that are normally 'light'-regulated. These results suggest an important role for cytokinins during greening of Arabidopsis, and may implicate abnormal cytokinin levels or an increased sensitivity to cytokinins as explanations for some of the observed phenotypes of det1 mutants stokomorphogenèse + cytokinine avec chloroplaste avec chloroplaste photomorphogenèse Fig. 18.4