cytokinin oxidase/dehydrogenase (CKX) Dotaz Zobrazit nápovědu
KEY MESSAGE: 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.
Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.
- MeSH
- Arabidopsis enzymologie genetika MeSH
- cytokininy metabolismus MeSH
- exprese genu MeSH
- geneticky modifikované rostliny chemie genetika fyziologie MeSH
- období sucha MeSH
- oxidoreduktasy genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- tabák chemie genetika fyziologie MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Degradation of the plant hormone cytokinin is controlled by cytokinin oxidase/dehydrogenase (CKX) enzymes. The molecular and cellular behavior of these proteins is still largely unknown. In this study, we show that CKX1 is a type II single-pass membrane protein that localizes predominantly to the endoplasmic reticulum (ER) in Arabidopsis (Arabidopsis thaliana). This indicates that this CKX isoform is a bona fide ER protein directly controlling the cytokinin, which triggers the signaling from the ER. By using various approaches, we demonstrate that CKX1 forms homodimers and homooligomers in vivo. The amino-terminal part of CKX1 was necessary and sufficient for the protein oligomerization as well as for targeting and retention in the ER. Moreover, we show that protein-protein interaction is largely facilitated by transmembrane helices and depends on a functional GxxxG-like interaction motif. Importantly, mutations rendering CKX1 monomeric interfere with its steady-state localization in the ER and cause a loss of the CKX1 biological activity by increasing its ER-associated degradation. Therefore, our study provides evidence that oligomerization is a crucial parameter regulating CKX1 biological activity and the cytokinin concentration in the ER. The work also lends strong support for the cytokinin signaling from the ER and for the functional relevance of the cytokinin pool in this compartment.
- MeSH
- Arabidopsis metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- membránové proteiny chemie metabolismus MeSH
- multimerizace proteinu * MeSH
- oxidoreduktasy chemie metabolismus MeSH
- proteinové domény MeSH
- proteiny - lokalizační signály MeSH
- proteiny huseníčku chemie metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Zinc (Zn) is an essential element in human nutrition. The concentration of Zn in cereals, which is a staple food in developing countries, is often too low thus contributing to Zn malnutrition in nearly two billion people worldwide. We have reported recently that transgenic barley plants expressing a cytokinin-degrading CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene in their roots form a larger root system and accumulate a higher concentration of Zn in their grains when grown under greenhouse conditions. Here, we have tested this trait under field conditions. Four independent pEPP:CKX lines accumulated an up to 30% higher Zn concentration in their grains as compared to the untransformed control suggesting that this is a stable trait. The increased Zn concentration exceeded the limit set by the HarvestPlus program for wheat. We, therefore, propose that root enhancement achieved by increased degradation of cytokinin in roots can be a sustainable strategy to combat malnutrition caused by Zn deficiency.
- MeSH
- cytokininy metabolismus MeSH
- geneticky modifikované rostliny genetika metabolismus MeSH
- ječmen (rod) genetika metabolismus MeSH
- jedlá semena genetika metabolismus MeSH
- kořeny rostlin genetika metabolismus MeSH
- oxidoreduktasy genetika metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- zinek metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hydroxamic acid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-one (DIMBOA) was isolated from maize phloem sap as a compound enhancing the degradation of isopentenyl adenine by maize cytokinin dehydrogenase (CKX), after oxidative conversion by either laccase or peroxidase. Laccase and peroxidase catalyze oxidative cleavage of DIMBOA to 4-nitrosoresorcinol-1-monomethyl ether (coniferron), which serves as a weak electron acceptor of CKX. The oxidation of DIMBOA and coniferron generates transitional free radicals that are used by CKX as effective electron acceptors. The function of free radicals in the CKX-catalyzed reaction was also verified with a stable free radical of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid. Application of exogenous cytokinin to maize seedlings resulted in an enhanced benzoxazinoid content in maize phloem sap. The results indicate a new function for DIMBOA in the metabolism of the cytokinin group of plant hormones.
- MeSH
- benzoxaziny chemie metabolismus MeSH
- biokatalýza MeSH
- cytokininy metabolismus MeSH
- floém enzymologie MeSH
- kukuřice setá enzymologie MeSH
- lakasa metabolismus MeSH
- molekulární struktura MeSH
- oxidace-redukce MeSH
- oxidoreduktasy metabolismus MeSH
- peroxidasa metabolismus MeSH
- volné radikály metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- 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
The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N(6)-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q(0)). In slightly acidic conditions (pH 5.0), N(6)-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N(6)-(2-isopentenyl)adenine 9-riboside-5'-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N(6)-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate.
- MeSH
- Arabidopsis enzymologie genetika metabolismus MeSH
- cytokininy metabolismus MeSH
- elektroforéza kapilární MeSH
- Escherichia coli enzymologie genetika MeSH
- geneticky modifikované rostliny enzymologie genetika MeSH
- oxidoreduktasy genetika metabolismus MeSH
- Pichia enzymologie genetika MeSH
- rekombinantní proteiny metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- tabák enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.
- MeSH
- Agrobacterium tumefaciens MeSH
- cytokininy biosyntéza genetika MeSH
- exprese genu * MeSH
- fertilita genetika MeSH
- geneticky modifikované rostliny enzymologie genetika MeSH
- ječmen (rod) enzymologie genetika MeSH
- kořeny rostlin embryologie genetika MeSH
- oxidoreduktasy biosyntéza genetika MeSH
- rostlinné proteiny biosyntéza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The formation of glycoconjugates depends on nucleotide sugars, which serve as donor substrates for glycosyltransferases in the lumen of Golgi vesicles and the endoplasmic reticulum (ER). Import of nucleotide sugars from the cytosol is an important prerequisite for these reactions and is mediated by nucleotide sugar transporters. Here, we report the identification of REPRESSOR OF CYTOKININ DEFICIENCY 1 (ROCK1, At5g65000) as an ER-localized facilitator of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) transport in Arabidopsis thaliana. Mutant alleles of ROCK1 suppress phenotypes inferred by a reduced concentration of the plant hormone cytokinin. This suppression is caused by the loss of activity of cytokinin-degrading enzymes, cytokinin oxidases/dehydrogenases (CKXs). Cytokinin plays an essential role in regulating shoot apical meristem (SAM) activity and shoot architecture. We show that rock1 enhances SAM activity and organ formation rate, demonstrating an important role of ROCK1 in regulating the cytokinin signal in the meristematic cells through modulating activity of CKX proteins. Intriguingly, genetic and molecular analysis indicated that N-glycosylation of CKX1 was not affected by the lack of ROCK1-mediated supply of UDP-GlcNAc. In contrast, we show that CKX1 stability is regulated in a proteasome-dependent manner and that ROCK1 regulates the CKX1 level. The increased unfolded protein response in rock1 plants and suppression of phenotypes caused by the defective brassinosteroid receptor bri1-9 strongly suggest that the ROCK1 activity is an important part of the ER quality control system, which determines the fate of aberrant proteins in the secretory pathway.
- MeSH
- Arabidopsis metabolismus ultrastruktura MeSH
- biologický transport MeSH
- cytokininy metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- fenotyp MeSH
- meristém metabolismus ultrastruktura MeSH
- proteiny huseníčku metabolismus MeSH
- transportní proteiny metabolismus MeSH
- uridindifosfát-N-acetylgalaktosamin metabolismus MeSH
- uridindifosfát-N-acetylglukosamin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis↔trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.
- MeSH
- Arabidopsis enzymologie růst a vývoj MeSH
- biokatalýza MeSH
- biologický transport MeSH
- biotest MeSH
- časové faktory MeSH
- fylogeneze MeSH
- isomerie MeSH
- kultivované buňky MeSH
- listy rostlin metabolismus MeSH
- oves metabolismus MeSH
- oxidoreduktasy metabolismus MeSH
- protein - isoformy MeSH
- rostliny enzymologie metabolismus MeSH
- semena rostlinná metabolismus MeSH
- signální transdukce MeSH
- substrátová specifita MeSH
- tabák cytologie enzymologie MeSH
- tritium metabolismus MeSH
- zeatin genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
