Using plant growth regulators to alter cytokinin homeostasis with the aim of enhancing endogenous cytokinin levels has been proposed as a strategy to increase yields in wheat and barley. The plant growth regulators INCYDE and CPPU inhibit the cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (CKX), while TD-K inhibits the process of senescence. We report that the application of these plant growth regulators in wheat and barley field trials failed to enhance yields, or change the components of yields. Analyses of the endogenous cytokinin content showed a high concentration of trans-zeatin (tZ) in both wheat and barley grains at four days after anthesis, and statistically significant, but probably biologically insignificant, increases in cisZ-O-glucoside, along with small decreases in cZ riboside (cZR), dihydro Z (DHZ), and DHZR and DHZOG cytokinins, following INCYDE application to barley at anthesis. We discuss possible reasons for the lack of efficacy of the three plant growth regulators under field conditions and comment on future approaches to manipulating yield in the light of the strong homeostatic mechanisms controlling endogenous cytokinin levels.

• Klíčová slova
• CKX, CPPU, INCYDE, IPT, TD-K, barley, cytokinin, cytokinin oxidase/dehydrogenase, isopentenyl transferase, thidiazuron, wheat, yield,
• Publikační typ
• časopisecké články MeSH

Modifying the cytokinin content in plants is a means of improving plant productivity. Here, we report the development and biological activity of compound TD-K (1-(furan-2-ylmethyl)-3-(1,2,3-thiadiazol-5-yl)urea)which is related to thidiazuron. TD-K-which exhibited extremely high antisenescence activity in the wheat leaf bioassay-and INCYDE (2-chloro-6-(3-methoxyphenyl)aminopurine)-a plant growth regulator reported to inhibit cytokinin oxidase/dehydrogenase (CKX), an enzyme involved in the degradation of the plant hormone cytokinin-were selected for investigation of their effects on the model plant Rapid Cycling Brassica rapa (RCBr). We monitored the expression of BrCKX and isopentenyl transferase (BrIPT), which codes for the key cytokinin biosynthesis enzyme, in developing leaves following INCYDE and TD-K application. Growth room experiments revealed that INCYDE increased RCBr seed yield per plant, but only when applied multiple times and when grown in 5 mM KNO3. Expression in control leaves showed transient, high levels of expression of BrCKX and BrIPT at true leaf appearance. Following INCYDE application, there was a rapid and strong upregulation of BrCKX3, and a transient downregulation of BrIPT1 and BrIPT3. Interestingly, the upregulation of BrCKX3 persisted in a milder form throughout the course of the experiment (16 days). TD-K also upregulated BrCKX3. However, in contrast to INCYDE, this effect disappeared after two days. These results suggest that both compounds (CKX inhibitor and cytokinin TD-K) influenced cytokinin homeostasis in RCBr leaves, but with different mechanisms.

• Klíčová slova
• CKX, INCYDE, IPT, TD-K, cytokinin, cytokinin oxidase/dehydrogenase, isopentenyl transferase, thidiazuron,
• Publikační typ
• časopisecké články MeSH

The arbuscular mycorrhizal (AM) symbiosis between terrestrial plants and AM fungi is regulated by plant hormones. For most of these, a role has been clearly assigned in this mutualistic interaction; however, there are still contradictory reports for cytokinin (CK). Here, pea plants, the wild type (WT) cv. Sparkle and its mutant E151 (Pssym15), were inoculated with the AM fungus Rhizophagus irregularis. E151 has previously been characterized as possessing high CK levels in non-mycorrhizal (myc-) roots and exhibiting high number of fungal structures in mycorrhizal (myc+) roots. Myc- and myc+ plants were treated 7, 9, and 11 days after inoculation (DAI) with synthetic compounds known to alter CK status. WT plants were treated with a synthetic CK [6-benzylaminopurine (BAP)] or the CK degradation inhibitor INCYDE, whereas E151 plants were treated with the CK receptor antagonist PI-55. At 13 DAI, plant CK content was analyzed by mass spectrometry. The effects of the synthetic compounds on AM colonization were assessed at 28 (WT) or 35 (E151) DAI via a modified magnified intersections method. The only noticeable difference seen between myc- and myc+ plants in terms of CK content was in the levels of nucleotides (NTs). Whereas WT plants responded to fungi by lowering their NT levels, E151 plants did not. Since NTs are thought to be converted into active CK forms, this result suggests that active CKs were synthesized more effectively in WT than in E151. In general, myc+ and myc- WT plants responded similarly to INCYDE by lowering significantly their NT levels and increasing slightly their active CK levels; these responses were less obvious in BAP-treated WT plants. In contrast, the response of E151 plants to PI-55 depended on the plant mycorrhizal status. Whereas treated myc- plants exhibited high NT and low active CK levels, treated myc+ plants displayed low levels of both NTs and active CKs. Moreover, treated WT plants were more colonized than treated E151 plants. We concluded that CKs have a stimulatory role in AM colonization because increased active CK levels were paralleled with increased AM colonization while decreased CK levels corresponded to reduced AM colonization.

• Klíčová slova
• AM symbiosis, INCYDE, PI-55, Pisum sativum L., Rhizophagus irregularis, cytokinin, legume, plant hormone,
• Publikační typ
• časopisecké články MeSH

Cytokinins modulate a number of important developmental processes, including the last phase of leaf development, known as senescence, which is associated with chlorophyll breakdown, photosynthetic apparatus disintegration and oxidative damage. There is ample evidence that cytokinins can slow down all these senescence-accompanying changes. Here, we review relationships between the various mechanisms of action of these regulatory molecules. We highlight their connection to photosynthesis, the pivotal process that generates assimilates, however may also lead to oxidative damage. Thus, we also focus on cytokinin induction of protective responses against oxidative damage. Activation of antioxidative enzymes in senescing tissues is described as well as changes in the levels of naturally occurring antioxidative compounds, such as phenolic acids and flavonoids, in plant explants. The main goal of this review is to show how the biological activities of cytokinins may be related to their chemical structure. New links between molecular aspects of natural cytokinins and their synthetic derivatives with antisenescent properties are described. Structural motifs in cytokinin molecules that may explain why these molecules play such a significant regulatory role are outlined.

• Klíčová slova
• antioxidant, antioxidant enzymes, antisenescent, cytokinin, derivative, genes, photosynthesis, plant defence, structure and activity relationship,
• MeSH
• antioxidancia chemie   metabolismus   MeSH
• cytokininy chemie   metabolismus   MeSH
• flavonoidy analýza   MeSH
• fotosyntéza MeSH
• listy rostlin chemie   růst a vývoj   fyziologie   MeSH
• molekulární struktura MeSH
• rostliny chemie   MeSH
• vývoj rostlin MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antioxidancia MeSH
• cytokininy MeSH
• flavonoidy MeSH

Two new TDZ derivatives (HETDZ and 3FMTDZ) are very potent inhibitors of CKX and are promising candidates for in vivo studies. Cytokinin hormones regulate a wide range of essential processes in plants. Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, TDZ), formerly registered as a cotton defoliant, is a well known inhibitor of cytokinin oxidase/dehydrogenase (CKX), an enzyme catalyzing the degradation of cytokinins. TDZ thus increases the lifetime of cytokinins and their effects in plants. We used in silico modeling to design, synthesize and characterize twenty new TDZ derivatives with improved inhibitory properties. Two compounds, namely 1-[1,2,3]thiadiazol-5-yl-3-(3-trifluoromethoxy-phenyl)urea (3FMTDZ) and 1-[2-(2-hydroxyethyl)phenyl]-3-(1,2,3-thiadiazol-5-yl)urea (HETDZ), displayed up to 15-fold lower IC 50 values compared with TDZ for AtCKX2 from Arabidopsis thaliana and ZmCKX1 and ZmCKX4a from Zea mays. Binding modes of 3FMTDZ and HETDZ were analyzed by X-ray crystallography. Crystal structure complexes, solved at 2.0 Å resolution, revealed that HETDZ and 3FMTDZ bound differently in the active site of ZmCKX4a: the thiadiazolyl ring of 3FMTDZ was positioned over the isoalloxazine ring of FAD, whereas that of HETDZ had the opposite orientation, pointing toward the entrance of the active site. The compounds were further tested for cytokinin activity in several cytokinin bioassays. We suggest that the combination of simple synthesis, lowered cytokinin activity, and enhanced inhibitory effects on CKX isoforms, makes 3FMTDZ and HETDZ suitable candidates for in vivo studies.

• Klíčová slova
• Crystal structure, Cytokinin, Cytokinin oxidase/dehydrogenase, Molecular docking, Organic synthesis, Thidiazuron,
• MeSH
• cytokininy metabolismus   MeSH
• fenylmočovinové sloučeniny chemie   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• oxidoreduktasy antagonisté a inhibitory   MeSH
• thiadiazoly chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• fenylmočovinové sloučeniny MeSH
• inhibitory enzymů MeSH
• oxidoreduktasy MeSH
• thiadiazoly MeSH
• thidiazuron MeSH Prohlížeč