Evaluation of adenine derivatives molecules as Cytokinin-like compounds and their involvement in plant metabolism

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(1)BIOS Research Doctorate School in Biological and Molecular Sciences PhD Program: Molecular Biotechnology. Evaluation of adenine derivatives molecules as Cytokinin-like compounds and their involvement in plant metabolism. Student: Dr. Francesca D’Angiolillo. Supervisor: Dr. Laura Pistelli. SSD BIO/04 – Fisiologia Vegetale Department of Agriculture, Food and Environment Plant Via Mariscoglio, 34 - Pisa.

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(3) Table of Contents. 1. Introduction ....................................................................................................... 1 1.1 Plant hormones: Cytokinins.................................................................................... 1 1.2 Roles of cytokinins in plants .................................................................................. 3 1.3 Cytokinins regulate plant adaptation to stress ........................................................ 7 1.4 Cytokinin activity in bioassays ............................................................................... 9 1.5 Cytokinin perception and signal transduction ...................................................... 10 1.6 Cytokinin Receptors Are Sensor Histidine Kinases ............................................. 11 1.7 Proposed model for phosphorelay signal transduction in cytokinin signaling ..... 13 1.8 Arabidopsis response regulators ........................................................................... 14 1.9 Cytokinin Oxidase/Dehydrogenase Irreversibly Degrades Cytokinins ................ 16 1.10 References ............................................................................................................ 17. 2. Aim of the project ........................................................................................... 29. 3. Evaluation of new Adenine derivatives as CK-like compounds ................. 31 3.1 Abstract................................................................................................................. 31 3.2 Introduction .......................................................................................................... 32 3.3 Materials and Methods ......................................................................................... 35 3.4 Results .................................................................................................................. 42 3.5 Discussion............................................................................................................. 54 3.6 Conclusions .......................................................................................................... 56 3.7 References ............................................................................................................ 57. 4. Are the adenine derivatives molecules involved in regulation of stress. metabolism? ..................................................................................................... 62. I.

(4) 4.1 Abstract................................................................................................................. 62 4.2 Introduction .......................................................................................................... 63 4.3 Materials and Methods ......................................................................................... 64 4.4 Results .................................................................................................................. 69 4.5 Discussion............................................................................................................. 76 4.6 Conclusion ............................................................................................................ 78 4.7 References ............................................................................................................ 78. 5. Conclusion and Future Perspectives ............................................................. 83. 6. Acknowledgments ........................................................................................... 85. II.

(5) Figures Figure 1.1:. Structure of natural isoprenoid (ISCK) and aromatic (ARCK) CKs......... 2. Figure 1.2:. Structure of synthetic CKs………………….……………...…………..... 2. Figure 1.3:. Actions of CKs in regulating plant development…………………...….... 6. Figure 1.4:. Strategies for the development of stress-tolerant plants………...…......... 8. Figure 1.5:. Representations of two-component response systems……………......... 11. Figure 1.6:. CK receptors regulate different processes………………….………….... 13. Figure 1.7:. Model for phosphorelay signal transduction in CK signaling…….......... 14. Figure 1.8:. Domain structure of type-A and type-B ARRs…………………….......... 16. Figure 3.1:. Schematic representation of adenine derivatives molecules……….......... 35. Figure 3.2:. Senescence bioassay for CK-like activity………...………….... ............ 39. Figure 3.3:. Typical CKs bioassays ………………………………………………..... 44. Figure 3.4:. Perception of new adenine derivatives by the cytokinin receptors AHK3………………………………...……………………….. 46. Figure 3.5:. Perception of new adenine derivatives by the cytokinin receptors CRE1/AHK4 ……………………………………………….… 47. Figure 3.6:. Live-cell binding assay (AHK3)................................................................ 48. Figure 3.7:. Live-cell binding assay (CRE1/AHK4) …………………………..…..... 49. Figure 3.8:. ARR5::GUS activation activity ………………………………............… 51. Figure 3.9:. ARR5::GUS competition activity………………..………………............ 52. Figure 3.10: Inhibition of AtCKX2 activity……………………...………………….... 53 . Figure 4.1:. Schematic representation of adenine derivatives .......................................65. Figure 4.2:. Senescence bioassay: leaves of Triticum aestivum L. ...............................66. Figure 4.3:. Lipid peroxidation......................................................................................72. Figure 4.4:. Antioxidant enzymatic activities...........................................................74-75. III.

(6) Tables. Table 3.1:. Chemical structures of ten new adenine derivatives molecules...............36. Table 3.2:. Relative CK activity of the adenine derivatives in classical CK bioassays... ………………………………………………………….43. Table 4.1:. Content of chlorophyll (Chl a, Chl b, Chl Tot) and carotenoids in leaves of Triticum aestivum L. ……………………………………….70. Table 4.2:. Content of total polyphenols and DPPH free radical scavenging activity (IC50) in leaves of Triticum aestivum L. …….…....…………….71. IV.

(7) 1 1.1. Introduction Plant hormones: Cytokinins. Plant hormones are defined as naturally occurring substances operating at low concentrations, which are in most cases able to translocate within the plant body and bind to a specific receptor protein. Cytokinins (CKs) belong to one of the most important and well-known classes of plant hormones and discovered over half a century ago they have retained the attention of researchers due to the potential to be used in biotechnology and agriculture CKs play key roles in the regulation of development, morphogenesis and physiological processes, (Riefler et al., 2006) such as cell proliferation, shoot initiation, chloroplast biogenesis, inhibition of leaf senescence, stress responses and programmed cell death (Mok et al.,1982; Carimi et al., 2003; Nishimura et al., 2004). The natural CKs are molecules with a chemical structure based on N6-substitued adenine, and are classified as isoprenoid (ISCK) (Sakakibara, 2006) and aromatic (ARCK) CKs (Mok and Mok, 2001). The isopentenyl pyrophosphate (IPP) is the starting compound for biosynthesis of terpenes and terpenoids and therefore the ISCK (Sakakibara, 2006) (Fig. 1.1). Isoprenoid CKs are the most abundant class and are either isopentenyl (iP)-type cytokinins, having an isopentenyl N6-side chain, or (tZ) zeatin-type cytokinins, having a hydroxylated isopentenyl N6-side chain. The tZ- and iP-type CKs are the major forms in Arabidopsis, whereas substantial amounts of cZ-type CKs are found in maize (Veach et.al, 2003) and rice (Izumi et al., 1988). Aromatic CKs have an aromatic benzyl or hydroxybenzyl group at N6 (e.g. N6benzyladenine (BA), meta-topolin (mT) ortho-topolin (oT), meta-topolin (mT),) and are found only in some plant species (Strnad, 1997) (Fig. 1.1). Moreover, CKs exist in plants not only as free bases but also in the form of nucleosides and nucleotides; the CK bases can be further conjugated to glucose at N3, N7, and N9 of the adenine ring and at the hydroxyl of the side chai (Sakakibara, 2006). Benzyladenine (BAP) and kinetin were initially produced synthetically but were discovered in plants (Barciszewski et al., 2007). Synthetic molecules have been also produced with the cytokinin activity, as phenylurea derivatives (DPU) as N-(2-chloro-4-. 1.

(8) pyridyl)-N-phenylurea (CPPU) or N-phenyl-N’-1,2,3-thidiazol-5-urea (TDZ) that are proved to be positive regulators of cell division and differentiation (Shudo, 1994; Mok and Mok et al., 2001) (Fig. 1.2).. Figure 1.1: Structure of natural isoprenoid (ISCK) and aromatic (ARCK) CKs.. Figure 1.2: Structure of synthetic CKs.. 2.

(9) 1.2. Roles of cytokinins in plants. CKs are involved in many aspects of plant growth and development (Fig. 1.3). Until the appearance of mutants of CK signaling and metabolism, the research was almost devoted to determine the roles of CKs, and focused on the effects of exogenous CKs (Gan and Amasino, 1996). The activity of CKs and the interaction with other hormonal signals, esp. auxin, have been discussed recently in several reviews to explain its role in the plant biology (Werner and Schmülling, 2009; Mazid et al., 2011; Pernisová et al., 2011; Durbak et al., 2012;Vanstraelen and Benková, 2012). CKs influences several light-regulated processes. In fact, CKs mimic photomorphogenesis in etiolated seedlings (Chory et al., 1994) which is mediated by the transcription factors ARR1, ARR10 and ARR12 (Argyros et al., 2008). Moreover, CKs acts as a signal for photosynthetic acclimation to canopy light gradients that induce leaf growth arrest (Carabelli et al., 2007). Exogenous application of CKs stimulates the transition from etioplast to chloroplast in detached leaves and cell cultures, increases the rate of grana and stroma lamella and extends the life span of chloroplasts (Mok, 1994; Synková et al., 2006). Furthermore, CKs influence photosynthesis and related processes (Synková et al., 1999; Cortleven et al., 2012) which is at least partially due to control of gene expression (Rashotte et al., 2003; Brenner et al., 2005). The shoot apical meristem (SAM) is an indeterminate structure that contains a group of pluripotent stem cells essential for the postembryonic formation of aerial organs and tissues. In the SAM the balance between cell proliferation and differentiation is controlled by a local regulatory transcriptional network, by external signals and by CKs. In fact, in Werner et al. (2003), using CK-deficient transgenic Arabidopsis plants, was demonstrated that CKs is a crucial regulator of meristem function. Moreover, also in plants containing mutations in multiple cytokinin receptor or IPT genes was observed the positive role of CKs in the regulation of SAM activity (Nishimura et al., 2004; Miyawaki et al., 2006). CK-deficiency in tobacco caused a reduction in chlorophyll synthesis as well as a strongly reduced sugar and increased starch content in the shoot apex, concomitant with reduced invertase activity, in particular in vacuolar invertase (Werner et al., 2008).. 3.

(10) Other CK actions in the shoot concern the regulation of sink–source relations and leaf senescence. The first demonstration of a role of CK in delaying of leaf senescence was done in 1957 by Richmond and Lang. It is recognized that exogenous application of cytokinin delays degradation of Chl and photosynthetic proteins (Gan and Amasino, 1996) and decreases CO2 assimilation (Rulcova and Pospısilova, 2001). There is evidence that natural endogenous cytokinin levels decrease during dark-induced senescence in Cucurbita cotyledons and natural senescence in maize leaves (He et al., 2005). Genetic analysis of the three cytokinin AHK receptors indicated that AHK3 plays the major role in regulating leaf senescence. This effect of AHK3 on leaf senescence was due to the phosphorylation and activation of ARR2 (Kim et al., 2006), a type-B ARR. Moreover, in Arabidopsis thaliana,, the ahk2 ahk3 double mutant did not display the CK inhibition of dark-induced leaf senescence observed in wild-type detached leaves (Riefler et al., 2006). Moreover, CKs have been shown to be involved in the specification and maintenance of the root vascular meristem and the balance between cell proliferation and differentiation in the vascular system is achieved through a regulatory circuit between CK signaling and AHP6 (Dettmer et al., 2009). On the contrary, in Hejatko et al.,(2009) was reported that the CK control of shoot vascular development depends on the combined action of the AHK2/AHK3 receptors and the Cytokinin Independent 1 (CKI1) His kinase. The best characterized molecular mechanism of CK action is that underlying the role of CKs in root meristem maintenance. In Dello Ioio et al., (2007) was demonstrated, through analysis of the root meristem phenotype of CK signaling, biosynthesis mutants and exogenous CK application, that CKs are acting in restricted regions of the root meristem by controlling cell differentiation rate in the transition zone of vascular tissue. In fact, in certain developmental processes more than one hormone is implicated and, thus, coordination of their overlapping activities is crucial for correct plant development (Ruzickaa, 2009). Adventitious rooting is a postembryonic organogenic process in which new root meristems are induced from a regulated sequence of cell proliferation and differentiation at position where roots do not normally originate. Auxins are the main inducers of adventitious rooting in plants and their role in the process has been widely studied (Pop et al., 2011), whereas CKs, usually considered auxin-antagonists, are described as negative regulators of root meristem activity and of lateral root formation. 4.

(11) (Werner and Schmulling, 2009). CKs control cell differentiation rate during root meristem development by suppressing both auxins signaling and transport, whereas at early stages of embryo development auxins counteract cytokinin signaling to establish the embryonic root stem-cell niche. During the adventitious rooting process it has been reported that TDZ and BAP are the most effective inhibiting molecules (De Klerk et al., 1999). Werner et al. (2003), using cytokinin-deficient transgenic Arabidopsis plants, confirmed that CKs have central, but opposite, regulatory functions both in root and in shoot meristems. However the CKs inhibiting effect is strictly related to the strength of the CK. In fact ‘‘weak’’ cytokinins may even stimulate adventitious rooting in a concentration dependent manner. During the early stages of the adventitious rooting process, CKs and also may stimulate initial cell divisions that are required to achieve adventitious roots; afterwards, they become inhibitory (De Klerk et al., 1999). The antagonistic interaction between CK and auxin seems to also occur in other developmental processes, such as lateral root emergence and leaf initiation (Perilli et al., 2010). New synthetic molecules, DPU derivatives, N,N’-bis-(2,3-methilendioxyphenyl) urea (2,3-MDPU) and N,N’-bis-(3,4-methilendioxyphenyl) urea (3,4-MDPU), have been demonstrated to enhance the adventitious rooting formations, while N-phenyl-N’benzothiazol-6-ylurea (PBU) other physiological processes as somatic embryogenesis (Carra et al., 2006; Ricci and Bertoletti, 2009; Rolli et al. 2012). Somatic embryogenesis is the developmental pathway by which somatic cells develop into structures that resemble zygotic embryos (bipolar and without vascular connection to the parental tissue) through an orderly series of characteristic embryological stages without fusion of gametes (Jimenez, 2001). Somatic cells acquire embryogenic characteristics by means of a complete reorganization of the cellular state, including physiology, metabolism and gene expression. Usually somatic embryogenesis is induced by auxin alone or in combination with CK (Gaj, 2004). Only few data reported the alone involvement of CKs (Carimi et al., 1999) and in particular the only the addition of TDZ can induce the process in some dicots plants. Moreover the synthetic urea derivatives as DPU and PBU are found to induce somatic embryogenesis in Citrus species, exhibiting a higher embryogenic performance compared to other CKs as BAP (Carra et al.; 2006).. 5.

(12) The synthesis of new urea or purine derivatives molecules, as PBU and others, can offer new opportunities for studying the process and the stability of the generated plants (Mik et al., 2011, Ricci and Bartoletti, 2009). In the last years some N9-substituted N6-[(3methylbut-2-en-1yl)amino]purine derivatives (iP derivatives) (Mik et al., 2011) and 6benzylamino-9-tetrahydropyran-2-ylpurine(THPP). and. 9-tetrahydrofuran-2-ylpurine. (THFP) derivatives (Szucova et al., 2009) were tested to clarify the relationship between structure and activity of the new molecules with CK-like activity.. Figure 1.3: Actions of CKs in regulating plant development. (In Curr Opin Plant Biol: Werner and Schmülling, 2009).. 6.

(13) 1.3. Cytokinins regulate plant adaptation to stress. In the past 10 years, was demonstrated that CKs play an important role in the regulation of environmental stress responses (Havlova et al., 2008; Nishiyama et al., 2011) (Fig. 1.4). The content of endogenous CKs depends on the balance between de novo synthesis, the import and export rate, interconversion of distinct forms, transient inactivation by conjugation (mainly glucosylation), and catabolic reactions resulting in a complete loss of biological activity (Sakakibara, 2006). The CK-dependent modulation on stress responses has been studied at various levels and the alteration of endogenous CK levels in reaction to stress suggests that this hormone is involved in stress responses. Moreover, in response to drought, the in planta concentration and transport of trans-zeatin riboside decreases drastically, whereas the ABA levels increase (Davies et al., 2005). In stress condition CKs are known to induce an antioxidant protection mechanism in chloroplasts (Procházková et al., 2008) and alter the transcript levels of many stress-inducible genes (Rashotte et al., 2003; Brenner et al., 2005; Brenner and Schmülling, 2012). In plant during senescence the general antioxidant status of the leaf is diminished and levels of the reactive oxygen species (ROS) are enhanced (Srivalli and Khanna-Chopra, 2004). In stay green maize cv. P3845 the delay of senescence was associated with higher catalase (CAT) and superoxide dismutase (SOD) levels (He et al., 2005). Therefore, leaf senescence is associated with the production of ROS, CKs delay this process and then use of exogenous CK (BAP) in Triticum aestivum L. regulate the oxidative status of the tissue (Zavaleta-Mancera et al., 2007). The negative CK-regulatory function in plants exposed to drought has been demonstrated in genetic studies in which the endogenous CK levels were modified, either by loss of the biosynthesis genes isopentyl transferase(IPT) or by overexpression of cytokinin oxidase (CKX) encoding degradation genes (Werner et al., 2010; Nishiyama et al., 2011). In fact, the application of exogenous CKs to plants can increase stomatal apertures and transpiration in many plants (Pospisilova and Batkova, 2004) and can have a positive influence on photosynthetic activity, indicating an important impact of CKs in the regulation of plant adaptation to environmental stresses (Chernyadev I.I., 2009).. 7.

(14) Even though the specific CKs roles in the individual abiotic stress responses have not been thoroughly identified, it is now becoming clear that modulation of CK levels and CK signal transduction represents a tool to enhance abiotic stress tolerance. In Smigocki. and Owens (1989) was demonstrated that the constitutive IPT expression causes reduced root growth and enhanced sensitivity to water stress; but the stimulation of CK biosynthesis by stress-inducible promoter(s) may diminish stress-induced acceleration of leaf senescence, enhance reactive oxygen species (ROS) scavenging capability and allow the maintenance of photosynthetic activity under stress conditions (Rivero et al., 2007; Xu et al., 2009). This inducible overexpression strategy also enables to maintain the balance between the levels of CKs and other hormones, such as ABA, for normal growth under unstressed conditions. In fact, stress-induced CK synthesis in rice was reported to result in changes in hormone homeostasis and consequently in a stronger sink capacity (Peleg et al., 2011). Inducible or artificially activated promoters driving gene expression in specific stress conditions could be useful for overcoming negative stress effects and for potential applications in agriculture (Peleg and Blumwald, 2010).. Figure 1.4: Strategies for the development of stress-tolerant plants. Transgenic plants that are tolerant to abiotic stresses can be developed with the manipulation of CK metabolism and repression of CK signaling. Stress-associated and senescence-specific overexpression of CK biosynthesis genes can delay plant senescence and render plants stress-tolerant. A reduction in CK levels in the root can enhance root architecture and increase stress tolerance without negative impacts on shoot growth that requires CKs for prosperity. (TRENDS in Plant Sci: Ha et al., 2012).. 8.

(15) 1.4. Cytokinin activity in bioassays. Biological assays are important tests to examine the CKs ability to induce specific physiological processes, in which CKs play a peculiar role. CKs accelerate chloroplast differentiation and stimulate chlorophyll (Chl) production in etiolate cucumber cotyledons (Fletcher et al., 1980; Yu et al., 2006). The amount of Chl produced is proportional to the concentration of CK and is dependent on the age of the cotyledons and on the dark incubation period (Fletche and McCullagh, 1971). Moreover, various aspects of the CK effects on Chl retention have been studied using exogenous applications of CKs on detached leaves, or senescing leaves of intact plants of various species (Varga et al., 1973; Nooden et al., 1997). Another classical bioassay for evaluating the CK role as growth regulators is the betacyanin induction in Amaranthus seedlings. Betacyanins are a class of red and yellow indole-derived pigments found in plants of the Caryophyllales, where they replace anthocyanin pigments (Piatelli, 1981). Moreover CKs have been shown to induce the anthocyanin accumulation in tissue culture and in intact plants. Plant roots are recognized as active sites of CK biosynthesis, metabolism and transport and Arabidopsis thaliana is an excellent plant system for studying the relationship between root development, CKs concentration and chemical structure (Auer, 1996; Higuchi et al., 2004). Many studies have been performed on the evaluation of crucial role of CKs in the rooting and shoot development with particular interest in vitro cultures. Even if the auxins are the well-known growth regulators involved in root formation, the use of in vitro cultures offer the opportunity to investigate the coordination and in some case the overlapping activities of CK and auxins in the phases of rooting process (dedifferentiation, induction and differentiation), each with its specific hormonal requirements (De Klerk et al., 1995; 1999). De Klerk et al. (2001) reported that small amounts of CK, even in the presence of auxin, enhanced adventitious root formation and ‘mung bean’ were used as model plants for the rooting experiments (Ricci et al., 2005; Rolli et al., 2012).. 9.

(16) 1.5. Cytokinin perception and signal transduction. The CK signal transduction pathway is a phosphorelay similar to bacterial two-component response systems (Gao and Stock, 2009; Stock et al., 2000). Many bacterial species contain more than a dozen two-component signaling pathways. For example, the E. coli genome encodes over 60 different two component signaling elements that respond to a diverse array of environmental stimuli (Wuichet et al., 2010). The two components generally consist of a membrane-localized sensor kinase that perceives environmental stimuli and a response regulator that propagates the signal, often by directly regulating transcription of target genes (Fig. 1.5 a). The input domain of the sensor kinase perceives the signal and controls the autophosphorylation of the histidine kinase domain. Active histidine kinas histidine residue in the transmitter domain. This phosphate is then transferred to a conserved aspartate residue in the receiver domain of a cognate response regulator. Many response regulators act as transcription factors and contain DNA-binding output domains in addition to their receiver domains (Stock et al., 2000). Extended versions of the basic two-component system are present in some prokaryotes and predominate in eukaryotic two component signaling (Schaller et al., 2011) (Fig. 1.5 b). Such multi-step phosphor lays typically involve four sequential phosphorylation events that alternate between histidine and aspartate residues, although the number of proteins harboring these phosphorylation sites varies. In eukaryotes the typical multi-step phosphorelay makes use of a “hybrid” kinase that contains both histidine kinase and receiver domains in one protein, a histidine-containing phospho-transfer (HPt) protein, and a separate response regulator (Appleby et al., 1996; Schaller et al., 2011). Genes encoding proteins similar to the bacterial two-component signaling elements are found in the Arabidopsis genome (Mizuno, 2005). These are all found as gene families, and include histidine kinases, histidine-containing phosphotransfer proteins (AHPs), and response regulators (ARRs) (Fig. 1.5 b). Biochemical and genetic analyses support their function in a multi-step phosphorelay. Genetic analysis has indicated that a major role for many of these genes is in cytokinin signal transduction.. 10.

(17) Figure 1.5: Representations of two-component response systems. (a) A basic prokaryotic two-component system with a sensor histidine kinase and a response regulator. H and D represent the conserved phosphoaccepting histidine and aspartate residues involved in phosphorelay signaling. (b) A multistep phosphorelay system involving a hybrid sensor kinase, with input, transmitter and receiver domains, a histidine-containing phosphotransfer protein and a response regulator (In The Arabidopsis Book: Kieber and Schaller, 2014).. 1.6. Cytokinin Receptors Are Sensor Histidine Kinases. The first CK receptor was identified in A. thaliana ten years ago (Inoue et al., 2001; Suzuki et al., 2001). The CK receptor family of Arabidopsis is composed of three histidine kinases: AHK2 (At5g35750), AHK3 (At1g27320), and CRE1/AHK4 (At2g01830) and regulate different processes (Fig. 1.6). These share a similar structure, having transmembrane domains that yield a predicted topology in which there is an extra cytosolic region for signal input and a cytosolic region for signal output. The extra cytosolic portion has a CHASE (cyclases/histidine kinases associated sensor extracellular) domain that functions in cytokinin binding (Anantharaman and Aravind, 2001; Heyl et al., 2007). The cytosolic portion has histidine kinase and C-terminal receiver domains that contain all the highly conserved residues required for enzymatic function. In addition, the receptors contain a diverged second receiver domain sandwiched between the histidine. 11.

(18) kinase and C-terminal receiver domains. This diverged receiver domain lacks some of the highly conserved residues found in other receivers, and in AHK3 and CRE1/AHK4 the putative phosphor accepting aspartate is replaced by a glutamate residue. The ability of the AHK family of cytokinin receptors to specifically perceive bioactive cytokinins has been demonstrated through biochemical and genetic approaches. Genetic, biochemical, and molecular studies supported the role of AHK2, AHK3, and CRE1/AHK4 as cytokinin receptors (Inoue et al., 2001; Suzuki et al., 2001; Ueguchi et al., 2001; Yamada et al., 2001). Different experiments with CRE1/AHK4, AHK2 and AHK3, using both a fission yeast and an E. coli multistep phosphorelay system indicated this receptors were activated in response to CKs (Inoue et al., 2001; Suzuki et al., 2001; Ueguchi et al., 2001; Yamada et al., 2001). Studies of the biochemical properties of the Arabidopsis AHK3 and CRE1/AHK4 CK receptors were performed in E. coli- or yeast-based test systems expressing individual receptors of Arabidopsis (Suzuki et al., 2001; Yamada et al., 2001; Spìchal et al., 2004; Romanov et al., 2005, 2006). A bacterial expression system has been used for a quantitative bioassay in which activation of the CK receptor results in stimulation of a lacZ reporter (Inoue et al., 2001; Spìchal et al., 2004). In addition, direct binding assays with radiolabelled CKs have been performed using bacterial and yeast expression systems (Romanov et al., 2005, 2006; Stolz et al., 2011). Moreover, in planta based evidence for the role of individual receptors in CK binding has been obtained from double mutants of Arabidopsis (Stolz et al., 2011). The receptor genes are expressed in almost all tissues. CRE1/AHK4 showed a higher expression in the root, while AHK2 and, in particular, AHK3 transcripts are more abundant in the shoot (Higuchi et al., 2004). These studies revealed a KD for CKs in the low nano-molar range for both receptors (Yamada et al., 2001; Romanov et al., 2005, 2006). Interestingly, CRE1/ AHK4 showed a high affinity to both principal cytokinins, iP and tZ, and did not recognize other CKs well. In contrast, AHK3 showed a broader spectrum of ligand recognition (Spìchal et al., 2004).. 12.

(19) Figure 1.6: CK receptors regulate different processes. (In Plant Cell: Riefler et al., 2006). 1.7. Proposed model for phosphorelay signal transduction in cytokinin signaling. In Kieber and Schaller, (2014) was proposed a model for phosphorelay signal transduction in CK signaling. As shown in Fig. 1.7 CK binds to the CHASE domains of the AHK2, AHK3 and CRE1/AHK4 CK receptors within the lumen of the ER. Binding of CK activates the transmitter domain, which autophosphorylates on a His (indicated by an H). The phosphate is then transferred an Asp residue (indicated by a D) within the fused receiver domain. The phosphate is then transferred to an AHP protein, which shuttles back and forth between the cytoplasm and the nucleus. In the nucleus, the AHPs transfer the phosphate to type-B ARRs, which then regulate the expression of many target genes, including the type-A ARRs. The type-A ARRs, which are also phosphorylated by the AHPs, in turn feedback to inhibit cytokinin signaling. The pseudo HPt protein AHP6 and nitric oxide (NO) also negatively regulate cytokinin signaling.. 13.

(20) Figure 1.7: Model for phosphorelay signal transduction in CK signaling. (In The Arabidopsis Book: Kieber and Schaller, 2014).. 1.8. Arabidopsis response regulators. In A. thaliana, in response to CK binding, the receptors auto-phosphorylate on a conserved His residue and relay this phosphoryl group to Arabidopsis Response Regulators (ARRs) via an intermediate set of histidine phosphor-transfer (Hpt) proteins called the Arabidopsis Hpt proteins (AHPs) (Suzuki et al., 1998). Similar cytokinin signaling components have been characterized in other plant species (Asakura et al., 2003; Du et al., 2007). The Arabidopsis response regulators fall into four classes based on phylogenetic analysis and domain structure (groups A, B, and C ) and pseudo-response regulators (APRRs) (Schaller et al., 2008). The real transcription factors are the B-type response regulators which contain both the phosphorylated N-terminal receiver domain and a special B-motif including the DNA-binding GARP-domain and the glutamine-rich domain (Sakai et al., 2000; Lohrmann et al., 2001) (Fig 8). Due to the nuclear location signals (NLS) B-type response regulators are localized in the nucleus. The type-B ARRs are 11 elements, and contain C-terminal output domains that have DNA binding and trans-activating activity (Sakai et al., 2000). However, B-type response. 14.

(21) regulators are not identical in terms of CK signal transduction. Type-B ARRs are positive regulators of CK signaling that control the transcription of a subset of CK-regulated targets, including the type-A ARRs (Sakai et al., 2001; Mason et al., 2005; Yokoyama et al., 2007). The ARR1, 10 and 12 genes play the key role; in fact, the triple mutant with knocked out genes is phenotypically similar to the CK receptor triple mutant (Ishida et al., 2008b). The expression of the genes of B-type response regulators is not regulated by CKs (Brenner et al., 2005). It should be mentioned that direct evidence of the interaction between the proteins that are components of the signal transduction circuit and their ability to donate and accept phosphate. As opposed to ARR -B, the genes of A-type response regulators (ARR -A) can be promptly activated by CKs and belong to the primary response genes for these hormones (D’Agostino et al., 2000; Brenner et al., 2005). ARR -A consist of the typical receiver domain and a small C-terminal fragment (Fig. 1.8). The A-type response regulators may accept phosphate from phosphor-transmitters similar to the B-type regulators; however, they cannot induce the typical transcription response. A body of observations allows to conclude that ARR-A act as negative regulators of signal transduction, the conserved aspartate residue being required to implement their inhibitory effect (Hwang et al., 2001). The multiple mutant with respect to the genes of A-type response regulators is characterized by increased sensitivity to CK. It is assumed that the A-type response regulators are capable of suppressing CK signal transduction from the AHP proteins by competing with the B-type regulators for the high energy phosphate. Thus, the participation of ARR–A in the system of cytokinin signal transduction provides negative feedback. Although the structure of C-type response regulators is similar to that of ARR -A, they are not induced by cytokinins and seem not to play a significant role in cytokinin signal transduction (Müller, 2011). The type-C ARRs are more distantly related to type-A and type-B ARR receiver domain sequences. They do not contain the output domain of type-B ARRs and are not transcriptionally regulated by cytokinin, although their overexpression results in reduced sensitivity to cytokinin (Kiba et al., 2003).. 15.

(22) Figure 1.8: Domain structure of type-A and type-B ARRs. Both classes of ARRs contain receiver domains. Type-B ARRs have long C-terminal extensions that include a GARP domain and a glutamine- and proline-rich region. (In The Arabidopsis Book: Kieber and Schaller, 2014).. 1.9. Cytokinin Oxidase/Dehydrogenase Irreversibly Degrades Cytokinins. Cytokinin Oxidase/dehydrogenase (CKX) is the enzyme that degrades the hormone CK and plays an important role in CK regulatory processes. CKX are proteins encoded by a multigene family with a varying number of members (Schmülling et al., 2003). CKX is the only known enzyme that catalyzes irreversible inactivation of CKs and its activity has been reported in many plant species and in a few lower organisms: the moss Funaria hygrometrica (Gerhäuser and Bopp, 1990), the slime mould D. discoideum (Armstrong and Firtel, 1989), and the yeast Saccharomyces cerevisiae (Van Kast and Laten, 1987). CKX irreversibly inactivates CKs by cleaving the bond between the purine ring and the side chain. Its substrates are free CK bases and ribosides with an unsaturated side chain (i.e. iP, iPR, tZ and tZR). Hydroxylated forms (DHZ-type), nucleotides and O-glucosydes cannot be degraded by CKX. In the case of N-glucosides, the data are somewhat contradictory (Mcgaw and Horgan, 1983; Bilyeu et al., 2001) and it is not yet unequivocally clear whether or not cytokinin N-glucosides are degraded by CKX. The products of iP or iPR degraded by CKX are 3-methyl-2-butenal and adenine, or adenine riboside respectively. For years it was assumed that molecular oxygen was essential for CKX activity, but a variety of electron acceptors other than oxygen, especially quinone types such as 2,3dimethoxy-5-methyl-1,4-benzoquinone (Q0), function more efficiently (Galuszka et al., 2001). Therefore, dehydrogenase is a more appropriate description of this enzyme (Galuszka et al., 2001). There is evidence of tissue specific expression of different CKX. 16.

(23) genes (Werner et al., 2006), indicating that degradation of CKs, like their biosynthesis, is a well-regulated process. The availability of A. thaliana plants with a reduced CK status, i.e. plants with a lower CK content or signaling, strongly improves the possibility to explore novel functions of CKs. The knockout of CK receptors is one option to obtain plants with a reduced CK status and mutants lacking one, two or all three receptors have been described (Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006). Alternatively, a reduced CK content can be generated by overexpression of CKX genes, encoding CK dehydrogenases catalyzing CK degradation (Werner et al., 2001, 2003). In fact, Cortleven et al., (2014), used both the CK receptor double mutant ahk2 ahk3 and cytokinin-deficient 35S:CKX4 transgenic plants (overexpressing plants) have observed that reducing levels of endogenous CK increases photo-oxidative stress and reduces photosynthetic activity.. 1.10 References • Anantharaman V. and Aravind L., (2001). The CHASE domain: a predicted ligandbinding module in plant cytokinin receptors and other eukaryotic and bacterial receptors. Trends Biochem Sci 26: 579-582 • Appleby J.L., Parkinson J.S., Bourret R.B., (1996). Signal transduction via the multistep phosphorelay: not necessarily a road less traveled. Cell 86: 845-848 • Argyros R.D., Mathews D.E., Chiang Y.H., Palmer C.M., Thibault D.M., Etheridge N., Argyros D.A., Mason M.G., Kieber J.J., Schaller G.E., (2008). Type B response regulators of Arabidopsis play key roles in cytokinin signaling and plant development. Plant Cell 20: 2102-2113 • Armstrong D.J. and Firtel R.A., (1989). Cytokinin oxidase activity in the cellular slime mold Dictyostelium discoideum. Dev Biol 136: 491-499 • Asakura Y., Hagino T., Ohta Y., Aoki K., Yonekura-Sakakibara K., Deji A., Yamaya T., Sugiyama T., Sakakibara H., (2003). Molecular characterization of His-Asp phosphorelay signaling factors in maize leaves: Implications of the signal divergence by cytokinininducible. Plant Mol Biol 52(2): 331-341. 17.

(24) • Auer C.A., (1996). Cytokinin inhibition of Arabidopsis root growth: An examination of Genotype, Cytokinin Activity and N 6-Benzyladenine Metabolism. J Plant Growth Regul 15: 201-206 • Barciszewski J., Massino F., Clark B.F.C., (2007). Kinetin-A multiactive molecule. Int J Biol Macromol 40: 182–192 • Biagi G., Bianucci A. M., Coi A, Costa B., Fabbrini L., Giorgi I., Livi O., Micco I., Pacchini F., Edoardo Santini E.,. Leonardi M.,. Nofal F.A., LeRoy Salernid O.,. Scartonia V., (2005). 2,9 Disubstituted-N6-(arylcarbamoyl)-8-azaadenines a snew selective A3 adenosine receptor antagonists: Synthesis, biochemical and molecular modelling studies. Bioorg & Med Chem 13: 4679-4693 • Bilyeu K.D., Cole J.L., Laskey J.G., Riekhof W.R., Esparza T.J., Kramer M.D., Morris R.O., (2001). Molecular and Biochemical Characterization of a Cytokinin Oxidase from Maize. Plant Physiol 125: 378-386 • Brenner W. and Schmülling T., (2012). Transcript profiling of cytokinin action in Arabidopsis roots and shoots discovers organ-specific cytokinin responses. BMC Plant Biol 12: 112 • Brenner W.G., Romanov G.A., Kollmer I., Burkle L. and Schmülling, T., (2005). Immediate-early and delayed cytokinin response genes of Arabidopsis thaliana identified by genome-wide expression profiling reveal novel cytokinin-sensitive processes and suggest cytokinin action through transcriptional cascades. Plant J. 44: 314-333 • Carabelli M., Possenti M., Sessa G., Ciolfi A., Sassi M., Morelli G., Ruberti A., (2007). Canopy shade causes a rapid and transient arrest in leaf development through auxin-induced cytokinin oxidase activity. Genes Dev 21: 1863-1868 • Carimi F., De Pasquale F., Crescimanno F.G., (1999). Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus. Plant Cell Rep 18: 935-940 • Carimi F., Zottini M., Formentin E., Terzi M., Lo Schiavo F., (2003). Cytokinins: new apoptotic inducers in plants. Planta 216:413-421 • Carra A., De Pasquale F., Ricci A., Carimi F., (2006). Diphenylurea derivatives induce somatic embryogenesis in Citrus. Plant Cell Tiss Organ Cult 87: 41-48. 18.

(25) • Chernyadev I.I., (2009). The protective action of cytokinins on the photosynthetic machinery and productivity of plants under stress. Appl Biochem Microbiol 45: 351362 • Chory J., Reinecke D., Sim S., Washburn T., Brenner M., (1994). A role for cytokinins in de etiolation in Arabidopsis. Plant Physiol 104: 339-347 • Cortleven A. and Valcke R., (2012). Evaluation of the photosynthetic activity in transgenic tobacco plants with altered endogenous cytokinin content: lessons from cytokinin. Physiol Plant 144: 394-408 • Cortleven A., Nitschke S., Klaumünzer M., AbdElgawad H., Asard H., Grimm B., Schmülling T., (2014). A novel protective function for cytokinin in the light stress response is mediated by the ARABIDOPSIS HISTIDINE KINASE2 and ARABIDOPSIS HISTIDINE KINASE3 receptors. Plant Physiol 164(3): 1470-1483 • D’Agostino I., Deruère J., Kieber J.J., (2000). Characterization of the response of the Arabidopsis ARR gene family to cytokinin. Plant Physiol 124: 1706-1717 • Davies W. J., Kudoyarova G., Hartung W., (2005). Long-distance ABA signaling and its relation to other signaling pathways in the detection of soil drying and the mediation of the plant’s response to drought. J Plant Growth Regul 24(4): 285-295 • De Klerk G.J., Hanecakova J., Jasik J., (2001). The role of cytokinins in rooting of stem slices cut from apple microcuttings. Plant Bio Syst 135: 79-84 • De Klerk G.J., Keppel M., Ter Brugge J., Meekes H., (1995). Timing of the phases in adventitious root formation in apple microcuttings. J Exp Bot 46: 965–972 • De Klerk G.J., Krieken W., De Jong J.C., (1999). The formation of adventitious roots: new concepts, new possibilities. In Vitro Cell Dev Pl 35: 189-199 • Dello Ioio R., Linhares F.S., Scacchi E., Casamitjana-Martinez E., Heidstra R., Costantino P., Sabatini S., (2007). Cytokinins determine Arabidopsis root-meristem size by controlling cell differentiation. Curr Biol 17: 678-682 • Dettmer J., Elo A., Helariutta Y., (2009). Hormone interactions during vascular development. Plant Mol Biol 2009, 69:347-360 • Du L., Jiao F., Chu J., Chen M., Wu P., (2007). The two component signal system in rice (Oryza sativa L.): A genome-wide study of cytokinin signal perception and transduction. Genomics 89: 697-707. 19.

(26) • Durbak A., Yao H., McSteen P., (2012). Hormone signaling in plant development. Curr Opin Plant Biol 15: 92-96 • Fletcher R.A. and McCullagh D., (1971). Cytokinin-in duced chlorophyll for mation in cucumber cotyledons. Planta 101: 88-90 • Fletcher R.A., Kalldumbil V., Steele P., (1980). An improved bioassay for cytokinins using cucumber cotyledons. Plant Physiol 69: 675-677 • Gaj M.D., (2004). Factors influencing somantic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana (L.) Hey Pl Growth Reg 43: 27-47 • Galuszka P., Frebort I., Sebela M., Sauer P., Jacobsen S., Pec P., (2001). Cytokinin oxidase or dehydrogenase?. Eur. J Biochem 268: 450-461 • Gan S. and Amasino R., (1996). Cytokinins in plant senescence: from spray and pray to clone and play. Bioessays 18: 557-565 • Gao R., and Stock A.M., (2009). Biological insights from structures of two-component proteins. Annu Rev Microbiol 63: 133-154 • Gerhäuser D. and Bopp M., (1990). Cytokinin oxidases in mosses. 2. Metabolism of kinetin and benzyladenine in vivo. J Plant Physiol 135: 714-718 • Ha S., Vankova R., Yamaguchi-Shinozaki K.,Shinozaki K. and Tran L.S. P., (2012). Cytokinins: metabolism and function in plant adaptation to environmental stresses. TRENDS Plant Sci17 (3): 172-179 • Havlova, M., Dobrev P.I., Motyka V., Štorchová H., Libus J., Dobrá J., Malbeck J., Gaudinová A., Vanková R., (2008). The role of cytokinins in responses to water deficit in tobacco plants over-expressing trans-zeatin Oglucosyltransferase Cell Environ 31: 341-353 • He P., Osaki M., Takebe M., Shinano T., Wasaki J., (2005). Endogenous hormones and expression of senescence-related genes in different senescent types of maize. J Exp Bot 56: 1117-28 • Hejatko J., Ryu H., Kim G.T., Dobesova R., Choi S., Choi S.M., Soucek P., Horak J., Pekarova B., Palme K., Brzobohatýe B., Hwang I., (2009). The histidine kinases cytokinin-independent1 and Arabidopsis histidine kinase2 and 3 regulate vascular tissue development in Arabidopsis shoots. Plant Cell 21: 2008-2021. 20.

(27) • Heyl A., Wulfetange K., Pils B., Nielsen N., Romanov G.A., Schmülling T., (2007). Evolutionary proteomics identifies amino acids essential for ligand-binding of the cytokinin receptor CHASE domain. BMC Evol Biol 7: 62 • Higuchi M., Pischke M.S., Mahonen A.P., Miyawaki K., Hashimoto Y., Seki M., Kobayashi M., Shinozaki K., Kato T., Tabata S., Helariutta Y., Sussman M.R., Kakimoto T., (2004). In planta functions of the Arabidopsis cytokinin receptor family. Proc Natl Acad Sci USA 101: 8821-8826 • Hwang I. and Sheen J., (2001). Two-component circuitry in Arabidopsis signal transduction. Nature 413: 383-389 • Inoue T., Higuchi M., Hashimoto Y., Seki M., Kobayashi M., Kato T., Tabata S., Shinozaki K., Kakimoto T., (2001). Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409: 1060-1063 • Ishida K., Yamashino T., Yokoyama A., Mizuno T., (2008b). Three Type-B Response Regulators, ARR1, ARR10 and ARR12, play essential but redundant roles in cytokinin signal transduction throughout the life cycle of Arabidopsis thaliana. Plant Cell Physiol 49: 47-57 • Izumi K., Nakagawa S., Kobayashi M., Oshio H., Sakurai A., Takahashi N., (1988). Levels of IAA, cytokinins, ABA and ethylene in rice plants as affected by a gibberellin biosynthesis inhibitor, uniconazole-P. Plant Cell Physiol 29: 97-104 • Jimenez V.M., (2001). Regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. Fisiol Veg 13: 196-223 • Kiba T., Yamada H., Sato S., Kato T., Tabata S., Yamashino T., Mizuno T., (2003). The type-A response regulator, ARR15, acts as a negative regulator in the cytokininmediated signal transduction in Arabidopsis thaliana. Plant Cell Physiol 44: 868-874 • Kieber J.J. and Schaller G.E., (2014). Cytokinins. The Arabidopsis Book 11:e0168. doi:10.1199/tab.0168 • Kim H.J., Ryu H., Hong S.H., Woo H.R., Lim P.O., Lee I.C., Sheen J., Nam,H.G., Hwang I., (2006). Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis. Proc Natl Acad Sci USA 103: 814-819 • Lohrmann J., Sweere U., Zabaleta E., Baurle I., Keitel C., Kozma-Bognar L., Harter K., (2001). The response regulator ARR2: a pollen-specific transcription factor. 21.

(28) involved in the expression of nuclear-encoded mitochondrial complex I genes. Mol. Gen. Genet. 265: 2-13 • Mason M.G., Mathews D.E., Argyros D.A., Maxwell B.B., Kieber J.J., Alonso J.M., Ecker J.R.S., (2005). Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis. Plant Cell 17: 3007-3018 • Mazid M., Khan T.A., Mohammad F., (2011) Cytokinins, a classical multifaceted hormone in plant system. J Stress Physiol Biochem 7: 347-368 • Mcgaw B.A. and Horgan R., (1983).Cytokinin oxidase from Zea mays kernels and Vinca rosea crown-gall tissue. Planta 159: 30-37 • Mik V., Szucuova L., Spìchal L., Plihal O., Nisler J., Zahajska L., Dolezual K., Strnad M., (2011). N9-Substituted N6-[(3-methylbut-2-en-1 yl)amino]purine derivatives and their biological activity in selected cytokinin bioassays. Bioorg & Med Chem19: 72447251 • Miyawaki K., Tarkowski P., Matsumoto-Kitano M., Kato T., Sato S., Tarkowska D., Tabata S., Sandberg G., Kakimoto T., (2006). Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis. Proc Natl Acad Sci U S A 103: 16598-16603 • Mizuno T., (2005). Two-component phosphorelay signal transduction system in plants: from hormone responses to circadian rhythms. Biosci Biotechnol Biochem 69: 22632276 • Mok D.W.S. and Mok M.C., (2001). Cytokinin metabolism and action. Annu. Rev. Plant Physiol Plant Mol Biol 52: 89-118 • Mok M.C., Mok D.WS., Armstrong D.J., Shudo K., Isogai Y., Okamoto T., (1982). Cytokinin. activity. of. N-phenyl-N¢)1,2,3-thiadiazol-5-ylurea. (thidiazuron).. Phytochemistry 21: 1509–1511 • Mok, M.C., (1994). Cytokinins and plant development an overview. In: Mok, D.W.S., Mok, M.C. (Eds.), Cytokinins: Chemistry, Activity and Function. CRC Press, Boca Raton, FL, 155-166 • Müller B., (2011). Generic signal-specific responses: cytokinin and context-dependent cellular responses. J Exp Bot 62: 3273-3288. 22.

(29) • Nishimura C., Ohashi Y., Sato S., Kato T., Tabata S., Ueguchi C., (2004). Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. Plant Cell 16: 1365-1377 • Nishiyama R., Watanabe Y., Fujita Y., Le D.T., Kojima M., Werner T., Vankova R., Yamaguchi-Shinozaki K., Shinozaki K., Kakimoto T., Sakakibara H., Schmülling T., Tran P. L.S., (2011) Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important esponses, and abscisic acid biosynthesis. Plant Cell 23: 2169-2183 • Noodén L.D., Guiamét J.J., John I., (1997). Senescence mechanisms. Physiol. Plantarum 101: 746-753 • Peleg Z. and Blumwald E., (2010). Hormone balance and abiotic stress tolerance in crop plants. Curr. Opin. Plant Biol 14: 290-295 • Peleg Z., Reguera M., Tumimbang E., Walia H., Blumwald E., (2011). Cytokininmediated source/sink modifications improve drought tolerance and increase grain yield in rice under water-stress. Plant Biotechnol J 9: 747-758 • Perilli S., Moubayidin L., Sabatini S., (2010). The molecular basis of cytokinin function. Plant Biol 13: 21-26 • Pernisová M., Kuderová A., Hejátko J., (2011). Cytokinin and auxin interactions in plant development: metabolism, signalling, transport and gene expression. Curr. Protein Pept Sci 12: 137-147 • Piatelli M., (1981). The betalains: structure, biosynthesis, and chemical taxonomy. In EE Conn, ed, The Biochemistry of Plants,. Academic Press, New York, 7: 557-575 • Pop T.I., Pamfil D., Bellini C., (2011). Auxin control in the formation of adventitious roots. Not. Bot. Hort. Agro Bot Cluj 39: 307-316 • Pospisilova J. and Batkova P., (2004). Effects of pre-treatments with abscisic acid and/or benzyladenine on gas exchange of French bean, sugar beet, and maize leaves during water stress and after rehydration. Biol. Plant 48: 395-399 • Procházková D., Haisel D., Wilhelmová N., (2008). Antioxidant protection during ageing and senescence in chloroplasts of tobacco with modulated life span. Cell Biochem Funct 26:582-590. 23.

(30) • Rashotte A.M., Carson S.D.B., To J.P.C., Kieber J.J., (2003). Expression profiling of cytokinin action in Arabidopsis. Plant Physiol 132: 1998-2011 • Ricci A. and Bertoletti C., (2009). Urea derivatives on the move: cytokinin-like activity and adventitious rooting enhancement depend on chemical structure. Plant Biology ISSN 1435-8603 • Ricci A., Carra A., Rolli E., Bertoletti C., Branca C., (2005). The weak cytokinins N,N’-bis-(1-naphthyl)urea and N,N’-bis-(2-naphthyl)urea may enhance rooting in apple and mung bean. Plant Cell Tiss and Org Cult 83: 179-186 • Richmond A.E. and Lang A., (1957). Effect of kinetin on protein content and survival of detached Xanthium leaves. Science, 125: 650-651 • Riefler M., Novák O., Strnad M., 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 • Rivero R.M., Kojima M., Gepstein A., Sakakibara H., Mittler R., Gepstein S. and Blumwald E., (2007). Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl Acad Sci U.S.A. 104: 19631-19636 • Rolli E., Incerti M., Brunoni F., Vicini P., Ricci A., (2012). Structure–activity relationships of N-phenyl-N0-benzothiazol-6-ylurea synthetic derivatives: Cytokininlike activity and adventitious rooting enhancement. Phytochemistry 74: 159-165 • Romanov G.A., Lomin S.N., Schmülling T., (2006). Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay. J Exp Bot 57: 4051-4058 • Romanov G.A., Spíchal L., Lomin S.N., Strnad M., Schmülling T., (2005). A live cell hormone-binding assay on transgenic bacteria expressing a eukaryotic receptor protein. Anal Biochem 347: 129-134 • Rulcova J. and Pospısilova J., (2001). Effect of benzylaminopurine on rehydration of bean plants after water stress. Biol Plant 44:75-81 • Ruzicka K., Simásková M., Duclercq J., Petrásek J., Zazímalová E., Simon S., Friml J., Van Montagu M.C., Benková E., (2009). Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proc. Natl. Acad. Sci. USA 106: 42844289. 24.

(31) • Sakai H., Aoyama T., Oka A., (2000). Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators. Plant J 24: 703-711 • Sakai H., Honma T., Aoyama T., Sato S., Kato T., Tabata S., Oka A., (2001). ARR1, a transcription factor for genes immediately responsive to cytokinins. Science 294: 15191521. • Sakakibara H., (2006). Cytokinins: activity, biosynthesis, and translocation. Annu. Rev. Plant Biol 57: 431-449 • Schaller G.E., Kieber J.J., Shiu S.H., (2008). Two-component signaling elements and histidyl-aspartyl phosphorelays. in The Arabidopsis Book, C. Somerville and E. Meyerowitz, eds (Rockville, MD: American Society of Plant Biologists) • Schaller G.E., Shiu, S.H., Armitage J.P., (2011). Two-component systems and their cooption for eukaryotic signal transduction. Curr Biol 21: R320-R330 • Schmülling T., Werner T., Riefler M., Krupková E., Manns I.B.y., (2003). Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116: 241-252 • Shudo K., (1994). Chemistry of diphenylurea cytokinins. In: Mok DWS, Mok MC (eds) Cytokinins: chemistry, activity and function. CRC Press, Boca Raton, USA, pp. 35-42 • Smigocki A.C., Owens L.D., (1989). Cytokinin-to-auxin ratios and morphology of shoots and tissues transformed by a chimeric isopentenyl transferase gene. Plant Physiol 91: 808-811 • Spíchal L., Rakova N.Y., Riefler M., Mizuno T., Romanov G.A., Strnad M., Schmülling T., (2004). Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol 45: 1299-1305 • Srivalli B. and Khanna-Chopra R., (2004). The developing reproductive ‘sink’ induces oxidative stress to mediate nitrogen mobilization during monocarpic senescence in wheat. Biochem Biophys Res Commun;325:198–202 • Stock A.M., Robinson V.L., Goudreau P.N., (2000). Two-component signal transduction. Annu Rev Biochem 69: 183-215. 25.

(32) • Stolz A., Riefler M., Lomin S.N., Achazi K., Romanov G.A., Schmülling, T., (2011). The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors. Plant J 67: 157-168 • Strnad M., (1997). The aromatic cytokinins. Physiol Plant 101: 674-88 • Suzuki T., Imamura A., Ueguchi C., Mizuno T., (1998). Histidine-containing phosphotransfer (HPt) signal transducers implicated in His-to-Asp phosphorelay in Arabidopsis. Plant Cell Physiol 39: 1258-1268 • Suzuki T., Miwa K., Ishikawa K., Yamada H., Aiba H., Mizuno T., (2001). The Arabidopsis sensor His-kinase, AHK4, can respond to cytokinins. Plant Cell Physiol 42: 107-113 • Synková H., Semorádová Š., Schnablová R., Witters E., Hŭsák M., Valcke R., (2006). Cytokinin-induced activity of antioxidant enzymes in transgenic Pssu-ipt tobacco during plant ontogeny. Biol Plant 50: 31-41 • Synková H., Van Loven K., Pospíšilová J., Valcke R., (1999). Photosynthesis of transgenic Pssu-ipt tobacco. J Plant Physiol 155: 173-182. • Szücová L., Spíchal L., Dolezal K., Zatloukal M., Greplová J., Galuszka P., Kryštof V., Voller J., Popa I., Massino F.J.,. Jørgensen J.E, Strnad M., (2009). Synthesis, characterization. and. biological. activity. of. ring-substituted. 6-benzylamino-9. tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives. Bioorg & Med Chem 17: 1938-1947 • Ueguchi C., Sato S., Kato T., Tabata S., (2001). The AHK4 gene involved in the cytokinin-signaling pathway as a direct receptor molecule in Arabidopsis thaliana. Plant Cell Physiol. 42: 751-755 • Van Kast C.A. and Laten H., (1987) Cytokinin utilization by adenine requiring mutants of the yeast Saccharomyces cerevisiae. Plant Physiol 83: 726-727 • Vanstraelen M. and Benková E., (2012). Hormonal interactions in the regulation of plant development. Annu. Rev. Cell Dev Biol 28: 463-487 • Varga A. and Bruinsma J., (1973). Effects of different cytokinins on the senescence of detached oat leaves. Planta 111: 91-93. 26.

(33) • Veach Y.K., Martin R.C., Mok D.W., Malbeck J., Vankova R., Mok M.C., (2003). OGlucosylation of cis-zeatin in maize. Characterization of genes, enzymes, and endogenous cytokinins. Plant Physiol 131: 1374-80 • Werner T. and Schmulling T., (2009). Cytokinin action in plant development. Curr Opinion Plant Biol 12: 527-538 • Werner T., Holst K., Pors Y., Guivarc’h A., Mustroph A., Chriqui D., Grimm B., Schmulling T., (2008). Cytokinin deficiency causes distinct changes of sink and source parameters in tobacco shoots and roots. J Exp Bot 59: 2659-2672 • Werner T., Kollmer I., Bartrina I., Holst K., Schmulling T., (2006). New Insights into the Biology of Cytokinin Degradation. Plant Biol 8: 371-381 • Werner T., Motyka V., Laucou V., Smets R., Van Onckelen H., Schmülling T., (2003). Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15: 2532-2550 • Werner T., Nehnevajova E., Kollmer I., Novak O., Strnad M., Kramer U. and Schmülling T., (2010). Root-specific reduction of cytokinin causes enhanced root growth, drought tolerance, and leaf mineral enrichment in Arabidopsis and tobacco. Plant Cell 22: 3905-3920 • Wuichet K., Cantwell B.J., Zhulin, I.B., (2010). Evolution and phyletic distribution of two-component signal transduction systems. Curr Opin Microbiol 13: 219-225 • Xu Y., Tian J., Gianfagna T., Huang B., (2009). Effects of SAG12-ipt expression on cytokinin production, growth and senescence of creeping bentgrass (Agrostis stolonifera L.) under heat stress. Plant Growth Regul 57: 281-291 • Yamada H., Suzuki T., Terada K., Takei K., Ishikawa K., Miwa K., Yamashino T., Mizuno T., (2001). The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant Cell Physiol 42: 1017-1023 • Yokoyama A., Yamashino T., Amano Y., Tajima Y., Imamura A. Sakakibara H. and Mizuno T., (2007). Type-B ARR transcription factors, ARR10 and ARR12, are implicated in cytokinin-mediated regulation of protoxylem differentiation in roots of Arabidopsis thaliana. Plant Cell Physiol 48: 84-964. 27.

(34) • Yu X., Li G., Xu D., Dong X., Qi X., Deng Y., (2006). An improvement of cucumber cotyledon greening bioassay for cytokinins. Acta Physiol Plant 28 (1): 9-11 • Zavaleta-Manceraa H.A., Lopez-Delgadob H., Loza-Taverac H., Mora-Herrerab M., Trevilla-Garcıad C., Vargas-Suarezc M., Oughame H., (2007). Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence. Journal of Plant Physiology 164: 1572-1582. 28.

(35) 2. Aim of the project. Natural plant hormones as cytokinins (CKs) are crucial signaling molecules that coordinate multiple aspects of plant growth and development (Mok, 1994). A classical approach to investigate the CK function in plant development was based on analysis of their effects after their exogenous application. The aim of this research was the study at physiological, biochemical and molecular level the effect of new synthesized compounds provided by Department of Pharmacy of University of Pisa (Biagi at al., 2005) and to suggest these as CKs-like molecules after the investigation of their selective activity and properties. These molecules showed interaction with human A3 adenosine receptors, and have been studied at pharmacological and physiological levels to define as potential drugs for the treatment of asthma and inflammatory conditions (Biagi et al., 2005). The structure of this compounds is similar to the Knows CKs, in particular is characterized by the 8-azaadenine nucleus, also present in 6-benzylaminopurine (BAP), and an HNCONH bridge as in diphenylurea derivatives (DPU) (TDZ -N-phenyl-N‟-1,2,3thidiazol-5-urea). During the three-year project, different new adenine derivatives compounds were examined. In the first year of research the attention was focused on testing classical CKs bioassays to evaluate their CK-activity or other involvement in physiological plant development with some molecules. In the second year was evaluated a link between the chemical structure of the new molecules, characterized by different modification on the nucleus at position R, R1 and R2 and their ability to interact in the plant development. Therefore, new molecules adenine derivatives with different substitutions at position R, R1, R2 (Ade1, MM82, 76) were synthesized ex-novo at the Department of Pharmacy of University of Pisa (Biagi et al., 2005; Giorgi et al., 2007). Then most active compounds (73, 76, 77, 68, MM82, LM3, ADE1, 22, 1, 2) were selected and their CK-like activity was evaluated in the different assay: Bacterial CK-receptor Live-cell hormone-binding, Arabidopsis ARR5::GUS reporter gene and inhibition of Arabidopsis CK dehydrogenase 2 (AtCKX2) activity performed in collaboration with. 29.

(36) Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic. In the last year, the involvement of selected molecules was followed during senescence of cutted leaves of Triticum aestivum L. and their effect on the oxidative stress induced by the senescence process was followed by the detection of several parameters. Pigments, polyphenols content, antioxidant activity (by DPPH), the breakdown of cells (as TBARS thiobarbituric acid reactive substances levels) and Enzymatic activities of such scavenger enzymes were detected to evaluate the their activity during stress condition. A correlation between activities, metabolites content and chemical structure should be the key for understand the role of these adenine derivatives compounds in the metabolism of plants.. 30.

(37) 3. Evaluation of new Adenine derivatives as CK-like compounds. Francesca D’Angiolillo1, Irene Giorgi2, Ada Ricci3, Lukáš Spíchal4, Laura Pistelli1 1. Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa, Italy. 2. Department of Pharmacy, University of Pisa, via Bonanno 6, 56126 Pisa, Italy. 3. Dipartimento di Bioscienza, Università di Parma, Parco Area delle Scienze 11/A, 43124, Parma, Italy. 4. Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, Olomouc, CZ78371, Czech Republic. 3.1. Abstract. Cytokinins (CKs) are plant hormones involved in many aspects of plant growth and development, so new synthetic cytokinins have been generated in the last years to increase their effect in plant propagation. New compounds adenine derivatives, providing different substitutions at position R, R1 and R2 were analyzed as CK-like molecules and tested with the classical CKs bioassays. The compounds called 73, 77, LM3, MM82, 1, 2 and 22 showed a low CK-like activity in typical CK bioassays as Amaranthus caudatus betacyanin production, Arabidopsis thaliana root elongation and cucumber cotyledon greening. All the compounds were also tested in the senescence bioassay, and the molecules without the ureidic structures (called 1, 2, 22) showed the best results. The ability of these new molecules to act as CKs was then investigated by the interaction with Arabidopsis CK receptors (CRE1/AHK4 and AHK3) in the heterologous bacterial assay. None of the selected compounds showed any positive response, despite the CK-like activity shown in various bioassays. However some interaction can be observed in ARR5::GUS competition assay for the molecules (MM82, LM3 and 1), together with an interaction with the cytokinin dehydrogenase (CKX).. 31.

(38) Keywords: Cytokinins, adenine derivatives, CK bioassay, Cytokinin receptor, ARR5::GUS, cytokinin dehydrogenase inhibition.. Abbreviations: Cytokinins (CKs), 6-benzylaminopurine (BAP), trans-zeatin (tZ), dimethylsulfoxide (DMSO), Arabidopsis response regulator 5 (ARR5), CRE1/ AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 Arabidopsis histidine kinase 3.. 3.2. Introduction. Cytokinins (CKs) are a well know class of natural plant hormones that coordinate multiple aspects of plant growth: cell division, shoot and root development, leaf expansion and delay of senescence, seedling germination, promotion of chlorophyll synthesis and regulation of interactions with pathogens represent their common function (Mok and Mok, 2001; Carimi et al., 2003; Werner and Schmülling, 2009; Mazid et al., 2011). There are two types of CKs with similar biological activities but they widely differ for the chemical structure. The adenine-type CKs are characterized by either an isoprenoid structure as N62-isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ) and dihydrozeatin (DHZ) or aromatic structure as N6-benzyladenine (BA) and kinetin (KIN). The second type are the synthetic phenyl urea derivatives as N-phenyl-N’-1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ) and N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU; Shudo 1994), than adenine type derivatives (Mok and Mok, 2001). The classical approach to investigate the synthetic CKs function in plant development was based on the effects of the exogenous CK application on plants (reviewed by Mok, 1994). After discovery of 6-benzylaminopurine (BAP) derivatives in plants, the majority of interest has been paid to synthesis and biological activity of fully or partly aromatic CK derivatives. Isoprenoid CK derivatives were often modified with the aim to find a new perspective plant growth regulator and to clarify the relationship between the structure and the activity (Spìchal et al., 2004; Mik et al., 2011). The biological assays as betacyanin induction in Amaranthus seedlings or chlorophyll production in etiolate cucumber cotyledons are important preliminary tests to examine the ability of new compounds to induce CK-like responses (Piatelli, 1981; Fletcher et al., 1982).. 32.

(39) Perception of cytokinins by receptors is the first step in the CK signaling pathway which leads to a biological response triggered by the hormone. The first CK receptor, CRE1/AHK4, was recognized in the model plant Arabidopsis thaliana (Inoue et al., 2001; Suzuki et al., 2001). CKs are perceived by membrane-located sensor histidine kinases belonging to a class of membrane-bound receptor histidine kinases with three members, AHK2, AHK3, and CRE1/AHK4. CK receptors consist of an extra cytosolic cyclase, His kinase-associated sensory extracellular (CHASE) domain flanked by two transmembrane domains at the N terminus, followed toward the C terminus by a His kinase and a response regulator domain in the predicted cytosolic part (Wulfetange et al., 2011). Despite of the model that emphasized the cytokinin signal location at the plasma membrane, these authors showed that the large majority of CK-receptors are localized to the endoplasmic reticulum, suggesting a central role of this compartment in CK-signalling (Wulfetange et al., 2011). AHK3 plays a predominant role in different aspects of shoot development, including the regulation of leaf and shoot growth, chloroplast development, shoot de-etiolation, leaf senescence and chlorophyll retention, while the CRE1/AHK4 receptor is of primary importance in root development (Inoue et al., 2001; Riefler et al., 2006; Heyl et al., 2011). No specific function has been assigned so far to AHK2, although this receptor alone was sufficient to maintain normal plant growth (Higuchi et al., 2004; Riefler et al., 2006). Bacterial test system, where the cytokinin receptors CRE1/AHK4 or AHK3 are functionally expressed in E. coli, was used to evaluate the CK-like activity of new compounds (Suzuki et al. 2001, Yamada et al. 2001). Along the CK signal cascade, the Arabidopsis response regulator ARR4 and ARR5 genes (previously called IBC7 and IBC6; Brandstatter and Kieber, 1998) were identified as cytokinin-regulated gene transcripts (D’Agostino et al., 2000). Their transcript shows a very rapid induction and is specific for CKs, and it is resistant to inhibition of protein synthesis (Brandstatter and Kieber, 1998). In addition to ARR4 and ARR5, the Arabidopsis genome encodes several other response regulator homologs; in fact, ARR gene family is characterized by four classes based on different sequence and domain structure (type A, B, and C ) and pseudoresponse regulators (APRRs) (Schaller et al., 2008).. 33.

(40) The catabolism of CKs is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalysing the irreversible cleavage of N6-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX). CKX is involved in the regulation of endogenous CK contents in plants by the irreversible oxidative cleavage of N6-side chains; seven distinct genes, AtCKX1 to AtCKX7 encode the enzyme in Arabidopsis thaliana (Frebortova et al., 2007). Isolation of the pure protein is difficult because of its extremely low concentration in plant tissues, but the CKX2 gene from A. thaliana has recently been cloned in yeast S. cerevisiae (Werner et al., 2001). This project is based on the evaluation of CK-like activity of new adenine derivatives compounds provided by Department of Pharmacy of University of Pisa (Biagi at al., 2005). These molecules showed interaction with human A3 adenosine receptors, are characterized by a chemical structure similar to DPU derivatives and purine derivatives molecules (Mik et al., 2011). The aim was the investigation at physiological and molecular levels of CKs-like selective activity and properties of these adenine derivatives bioactive compounds. Cytokinin activity of the compounds was determined by classical CK-bioassays. The ability of these compounds to interact with Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 was tested in bacterial receptor and in live-cell binding assays, and in an Arabidopsis ARR5::GUS reporter gene assay. Moreover the new adenine derivatives were also tested in CKX inhibition assay to evaluate their potential interaction with CK metabolism.. 34.

(41) 3.3. Materials and Methods. Characteristics of adenine derivatives molecules Ten new adenine derivatives molecules were previously synthesized the Department of Pharmacy of University of Pisa (Biagi at al., 2005; Giorgi et al., 2007 ), and kindly offered for this project. These molecules exhibited a similar structure to the known CKs and in particular are characterized by the 8-azaadenine nucleus, also present in BAP, and an HNCONH bridge as in diphenylurea derivatives (DPU) as TDZ. The adenine derivatives molecules are also characterized by other different substitutions in R, R1 and R2 position (Fig. 3.1). Ten molecules tested can be divided in two groups characterized by only the presence of the 8-azaadenine nucleus (molecules 1-2-22-Ade1) (Tab. 3.1 B) or by the addition of the HNCONH bridge (molecules 68, 73, 76, 77, MM82 and LM3) (Tab. 3.1 A). The molecules were dissolved in dimethylsulfoxide (DMSO) to prepare a stock solution of 10 mM and stored at -20C°. Various concentrations were used in the experiments (up to 10 μM). The final concentration of DMSO in the assay did not exceed 0,2% in order to avoid the interference of DMSO in the biological activity.. Figure 3.1: Schematic representation of adenine derivatives molecules, different modifications are present on 8-azaadenine nucleus at position R, R1 and R2 (Biagi et al., 2005).. 35.

(42) Table 3.1: Chemical structures of ten new adenine derivatives group A (73, 76, 77, 68, MM82, LM3) and group B (ADE1, 22, 1, 2) synthesized at the Department of Pharmacy of University of Pisa (Biagi et al., 2005).. 36.