Multistep phosphorelay (MSP) signaling integrates hormonal and environmental signals to control both plant development and adaptive responses. Type-A RESPONSE REGULATOR (RRA) genes, the downstream members of the MSP cascade and cytokinin primary response genes, are thought to mediate primarily the negative feedback regulation of (cytokinin-induced) MSP signaling. However, transcriptional data also suggest the involvement of RRA genes in stress-related responses. By employing evolutionary conservation with the well-characterized Arabidopsis thaliana RRA genes, we identified five and 38 novel putative RRA genes in Brassica oleracea and Brassica napus, respectively. Our phylogenetic analysis suggests the existence of gene-specific selective pressure, maintaining the homologs of ARR3, ARR6, and ARR16 as singletons during the evolution of Brassicaceae. We categorized RRA genes based on the kinetics of their cytokinin-mediated up-regulation and observed both similarities and specificities in this type of response across Brassicaceae species. Using bioinformatic analysis and experimental data demonstrating the cytokinin and abiotic stress responsiveness of the A. thaliana-derived TCSv2 reporter, we unveil the mechanistic conservation of cytokinin- and stress-mediated up-regulation of RRA genes in B. rapa and B. napus. Notably, we identify partial cytokinin dependency of cold stress-induced RRA transcription, thus further demonstrating the role of cytokinin signaling in crop adaptive responses.

• Klíčová slova
• Arabidopsis thaliana, Brassica napus, Brassica oleracea, Brassica rapa, cytokinins, multistep phosphorelay, osmotic stress, salinity, two-component signaling, type-A response regulator,
• MeSH
• Arabidopsis genetika   fyziologie   metabolismus   MeSH
• Brassica napus genetika   fyziologie   metabolismus   MeSH
• Brassica * genetika   fyziologie   metabolismus   MeSH
• cytokininy * metabolismus   MeSH
• fylogeneze MeSH
• fyziologický stres * MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné proteiny * genetika   metabolismus   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokininy * MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny * MeSH

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormones act over short and long distances to control plant responses to environmental cues. CEP and cytokinin pathway mutants share phenotypes, however, it is not known if these pathways intersect. We show that CEP and cytokinin signalling converge on CEP DOWNSTREAM (CEPD) glutaredoxins to inhibit primary root growth. CEP inhibition of root growth was impaired in mutants defective in trans-zeatin (tZ)-type cytokinin biosynthesis, transport, perception, and output. Concordantly, mutants affected in CEP RECEPTOR 1 showed reduced root growth inhibition in response to tZ, and altered levels of tZ-type cytokinins. Grafting and organ-specific hormone treatments showed that tZ-mediated root growth inhibition involved CEPD activity in roots. By contrast, root growth inhibition by CEP depended on shoot CEPD function. The results demonstrate that CEP and cytokinin pathways intersect, and utilise signalling circuits in separate organs involving common glutaredoxin genes to coordinate root growth.

• MeSH
• Arabidopsis * metabolismus   MeSH
• cytokininy * metabolismus   MeSH
• glutaredoxiny genetika   metabolismus   MeSH
• kořeny rostlin metabolismus   MeSH
• peptidy metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• výhonky rostlin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy * MeSH
• glutaredoxiny MeSH
• peptidy MeSH

Brassinosteroids (BRs) are group of plant steroidal hormones that modulate developmental processes and also have pivotal role in stress management. Biosynthesis of BRs takes place through established early C-6 and late C-6 oxidation pathways and the C-22 hydroxylation pathway triggered by activation of the DWF4 gene that acts on multiple intermediates. BRs are recognized at the cell surface by the receptor kinases, BRI1 and BAK1, which relay signals to the nucleus through a phosphorylation cascade involving phosphorylation of BSU1 protein and proteasomal degradation of BIN2 proteins. Inactivation of BIN2 allows BES1/BZR1 to enter the nucleus and regulate the expression of target genes. In the whole cascade of signal recognition, transduction and regulation of target genes, BRs crosstalk with other phytohormones that play significant roles. In the current era, plants are continuously exposed to abiotic stresses and heavy metal stress is one of the major stresses. The present study reveals the mechanism of these events from biosynthesis, transport and crosstalk through receptor kinases and transcriptional networks under heavy metal stress.

• Klíčová slova
• BR biosynthetic pathway, BR signaling, heavy metal, hormone crosstalk, stress, transcription,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The epidermal cell layer of plants has important functions in regulating plant growth and development. We have studied the impact of an altered epidermal cytokinin metabolism on Arabidopsis shoot development. Increased epidermal cytokinin synthesis or breakdown was achieved through expression of the cytokinin synthesis gene LOG4 and the cytokinin-degrading CKX1 gene, respectively, under the control of the epidermis-specific AtML1 promoter. During vegetative growth, increased epidermal cytokinin production caused an increased size of the shoot apical meristem and promoted earlier flowering. Leaves became larger and the shoots showed an earlier juvenile-to-adult transition. An increased cytokinin breakdown had the opposite effect on these phenotypic traits indicating that epidermal cytokinin metabolism can be a factor regulating these aspects of shoot development. The phenotypic consequences of abbreviated cytokinin signaling in the epidermis achieved through expression of the ARR1-SRDX repressor were generally milder or even absent indicating that the epidermal cytokinin acts, at least in part, cell non-autonomously. Enhanced epidermal cytokinin synthesis delayed cell differentiation during leaf development leading to an increased cell proliferation and leaf growth. Genetic analysis showed that this cytokinin activity was mediated mainly by the AHK3 receptor and the transcription factor ARR1. We also demonstrate that epidermal cytokinin promotes leaf growth in a largely cell-autonomous fashion. Increased cytokinin synthesis in the outer layer of reproductive tissues and in the placenta enhanced ovule formation by the placenta and caused the formation of larger siliques. This led to a higher number of seeds in larger pods resulting in an increased seed yield per plant. Collectively, the results provide evidence that the cytokinin metabolism in the epidermis is a relevant parameter determining vegetative and reproductive plant growth and development.

• Klíčová slova
• Arabidopsis, cytokinin, developmental transitions, epidermis, seed yield, shoot growth,
• Publikační typ
• časopisecké články MeSH

Cytokinins (CKs) are known to regulate the biogenesis of chloroplasts under changing environmental conditions and at different stages of plant ontogenesis. However, the underlying mechanisms are still poorly understood. Apparently, the mechanisms can be duplicated in several ways, including the influence of nuclear genes that determine the expression of plastome through the two-component CK regulatory circuit. In this study, we evaluated the role of cytokinins and CK signaling pathway on the expression of nuclear genes for plastid RNA polymerase-associated proteins (PAPs). Cytokinin induced the expression of all twelve Arabidopsis thalianaPAP genes irrespective of their functions via canonical CK signaling pathway but this regulation might be indirect taking into consideration their different functions and versatile structure of promoter regions. The disruption of PAP genes contributed to the abolishment of positive CK effect on the accumulation of the chloroplast gene transcripts and transcripts of the nuclear genes for plastid transcription machinery as can be judged from the analysis of pap1 and pap6 mutants. However, the CK regulatory circuit in the mutants remained practically unperturbed. Knock-out of PAP genes resulted in cytokinin overproduction as a consequence of the strong up-regulation of the genes for CK synthesis.

• Klíčová slova
• Arabidopsis thaliana, PEP-associated proteins, chloroplast biogenesis, cytokinin, plastid transcription machinery,
• MeSH
• Arabidopsis účinky léků   genetika   MeSH
• buněčné jádro účinky léků   genetika   MeSH
• chloroplasty účinky léků   genetika   MeSH
• cytokininy farmakologie   MeSH
• fotosyntéza účinky léků   MeSH
• genetická transkripce účinky léků   MeSH
• geny chloroplastů * MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace genetika   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• zeatin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• messenger RNA MeSH
• zeatin MeSH

To elucidate the effect of light intensity on the cold response (5°C; 7 days) in Arabidopsis thaliana, we compared the following parameters under standard light (150 μmol m-2 s-1), low light (20 μmol m-2 s-1), and dark conditions: membrane damage, photosynthetic parameters, cytokinin oxidase/dehydrogenase (CKX) activity, phytohormone levels, and transcription of selected stress- and hormone-related genes and proteome. The impact of cytokinins (CKs), hormones directly interacting with the light signaling pathway, on cold responses was evaluated using transformants overexpressing CK biosynthetic gene isopentenyl transferase (DEX:IPT) or CK degradation gene HvCKX2 (DEX:CKX) under a dexamethasone-inducible promoter. In wild-type plants, cold treatment under light conditions caused down-regulation of CKs (in shoots) and auxin, while abscisic acid (ABA), jasmonates, and salicylic acid (SA) were up-regulated, especially under low light. Cold treatment in the dark strongly suppressed all phytohormones, except ABA. DEX:IPT plants showed enhanced stress tolerance associated with elevated CK and SA levels in shoots and auxin in apices. Contrarily, DEX:CKX plants had weaker stress tolerance accompanied by lowered levels of CKs and auxins. Nevertheless, cold substantially diminished the impact from the inserted genes. Cold stress in dark minimized differences among the genotypes. Cold treatments in light strongly up-regulated stress marker genes RD29A, especially in roots, and CBF1-3 in shoots. Under control conditions, their levels were higher in DEX:CKX plants, but after 7-day stress, DEX:IPT plants exhibited the highest transcription. Transcription of genes related to CK metabolism and signaling showed a tendency to re-establish, at least partially, CK homeostasis in both transformants. Up-regulation of strigolactone-related genes in apices and leaves indicated their role in suppressing shoot growth. The analysis of leaf proteome revealed over 20,000 peptides, representing 3,800 proteins and 2,212 protein families (data available via ProteomeXchange, identifier PXD020480). Cold stress induced proteins involved in ABA and jasmonate metabolism, antioxidant enzymes, and enzymes of flavonoid and glucosinolate biosynthesis. DEX:IPT plants up-regulated phospholipase D and MAP-kinase 4. Cold stress response at the proteome level was similar in all genotypes under optimal light intensity, differing significantly under low light. The data characterized the decisive effect of light-CK cross-talk in the regulation of cold stress responses.

• Klíčová slova
• acclimation, cold stress, cytokinin, cytokinin oxidase/dehydrogenase, isopentenyl transferase, karrikin, light intensity, phytohormone,
• Publikační typ
• časopisecké články MeSH

Five poplar CHASE-containing histidine kinase receptors bind cytokinins and display kinase activities. Both endogenous isoprenoid and aromatic cytokinins bind to the receptors in live cell assays. Cytokinins are phytohormones that play key roles in various developmental processes in plants. The poplar species Populus × canadensis, cv. Robusta, is the first organism found to contain aromatic cytokinins. Here, we report the functional characterization of five CHASE-containing histidine kinases from P. × canadensis: PcHK2, PcHK3a, PcHK3b, PcHK4a and PcHK4b. A qPCR analysis revealed high transcript levels of all PcHKs other than PcHK4b across multiple poplar organs. The ligand specificity was determined using a live cell Escherichia coli assay and we provide evidence based on UHPLC-MS/MS data that ribosides can be true ligands. PcHK2 exhibited higher sensitivity to iP-type cytokinins than the other receptors, while PcHK3a and PcHK3b bound these cytokinins much more weakly, because they possess two isoleucine residues that clash with the cytokinin base and destabilize its binding. All receptors display kinase activity but their activation ratios in the presence/absence of cytokinin differ significantly. PcHK4a displays over 400-fold higher kinase activity in the presence of cytokinin, suggesting involvement in strong responses to changes in cytokinin levels. trans-Zeatin was both the most abundant cytokinin in poplar and that with the highest variation in abundance, which is consistent with its strong binding to all five HKs and activation of cytokinin signaling via A-type response regulators. The aromatic cytokinins' biological significance remains unclear, their levels vary diurnally, seasonally, and annually. PcHK3 and PcHK4 display the strongest binding at pH 7.5 and 5.5, respectively, in line with their putative membrane localization in the endoplasmic reticulum and plasma membrane.

• Klíčová slova
• Aromatic cytokinin, Histidine kinase, Hormone, Isoprenoid cytokinin, Poplar, Topolin,
• MeSH
• cytokininy metabolismus   MeSH
• histidinkinasa metabolismus   MeSH
• Populus metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• terpeny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokininy MeSH
• histidinkinasa MeSH
• terpeny MeSH

Isoprenoid and aromatic cytokinins occur in poplar as free compounds and constituents of tRNA, poplar isopentenyltransferases are involved in the production of isoprenoid cytokinins, while biosynthesis of their aromatic counterparts remains unsolved. Cytokinins are phytohormones with a fundamental role in the regulation of plant growth and development. They occur naturally either as isoprenoid or aromatic derivatives, but the latter are quite rare and less studied. Here, the spatial expression of all nine isopentenyl transferase genes of Populus × canadensis cv. Robusta (PcIPTs) as analyzed by RT-qPCR revealed a tissue preference and strong differences in expression levels for the different adenylate and tRNA PcIPTs. Together with their phylogeny, this result suggests a functional diversification for the different PcIPT proteins. Additionally, the majority of PcIPT genes were cloned and expressed in Arabidopsis thaliana under an inducible promoter. The cytokinin levels measured in the Arabidopsis-overexpressing lines as well as their phenotype indicate that the studied adenylate and tRNA PcIPT proteins are functional in vivo and thus will contribute to the cytokinin pool in poplar. We screened the cytokinin content in leaves of 12 Populus species by ultra-high performance-tandem mass spectrometry (UHPLC-MS/MS) and discovered that the capacity to produce not only isoprenoid, but also aromatic cytokinins is widespread amongst the Populus accessions studied. Important for future studies is that the levels of aromatic cytokinins transiently increase after daybreak and are much higher in older plants.

• Klíčová slova
• Cytokinin, Expression, Isopentenyltransferase, Poplar, Topolin, tRNA,
• MeSH
• alkyltransferasy a aryltransferasy genetika   metabolismus   MeSH
• Arabidopsis genetika   metabolismus   MeSH
• cytokininy biosyntéza   MeSH
• fylogeneze MeSH
• geneticky modifikované rostliny MeSH
• listy rostlin genetika   metabolismus   MeSH
• Populus genetika   metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylate isopentenyltransferase MeSH Prohlížeč
• alkyltransferasy a aryltransferasy MeSH
• cytokininy MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny 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

BACKGROUND: Cytokinin is a negative regulator of root growth, and a reduction of the cytokinin content or signalling causes the formation a larger root system in model plants, improves their growth under drought and nutrient limitation and causes increased accumulation of elements in the shoot. Roots are an important but understudied target of plant breeding. Here we have therefore explored whether root enhancement by lowering the cytokinin content can also be achieved in oilseed rape (Brassica napus L.) plants. RESULTS: Transgenic plants overexpressing the CKX2 gene of Arabidopsis thaliana encoding a cytokinin-degrading cytokinin oxidase/dehydrogenase showed higher CKX activity and a strongly reduced cytokinin content. Cytokinin deficiency led to the formation of a larger root system under different growth conditions, which was mainly due to an increased number of lateral and adventitious roots. In contrast, shoot growth was comparable to wild type, which caused an enhanced root-to-shoot ratio. Transgenic plants accumulated in their leaves higher concentrations of macro- and microelements including P, Ca, Mg, S, Zn, Cu, Mo and Mn. They formed more chlorophyll under Mg- and S-deficiency and accumulated a larger amount of Cd and Zn from contaminated medium and soil. CONCLUSIONS: These findings demonstrate the usefulness of ectopic CKX gene expression to achieve root enhancement in oilseed rape and underpin the functional relevance of a larger root system. Furthermore, the lack of major developmental consequences on shoot growth in cytokinin-deficient oilseed rape indicates species-specific differences of CKX gene and/or cytokinin action.

• Klíčová slova
• Brassica napus, Cytokinin, Cytokinin oxidase/dehydrogenase, Leaf mineral enrichment, Phytoremediation, Plant growth, Root enhancement,
• MeSH
• biodegradace * MeSH
• Brassica napus genetika   růst a vývoj   metabolismus   MeSH
• chlorofyl analýza   metabolismus   MeSH
• cytokininy genetika   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• kadmium analýza   metabolismus   MeSH
• kořeny rostlin růst a vývoj   metabolismus   MeSH
• listy rostlin chemie   metabolismus   MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• půda chemie   MeSH
• zinek analýza   metabolismus   MeSH
• živiny analýza   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl MeSH
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• kadmium MeSH
• oxidoreduktasy MeSH
• půda MeSH
• zinek MeSH