Cytokinin Oxidase/Cytokinin Dehydrogenase Assay: Optimized Procedures and Applications

doi:10.1006/abio.2002.5670

Analytical Biochemistry

Volume 306, Issue 1, 1 July 2002, Pages 1-7

https://doi.org/10.1006/abio.2002.5670 Get rights and content

Abstract

Spectrophotometric methods for determining the activity of cytokinin oxidase/cytokinin dehydrogenase (EC 1.5.99.12) were developed and optimized. A sensitive end-point method based on a combination of the electron acceptor 2,6-dichlorophenolindophenol and Schiff base formation of the reaction product with 4-aminophenol under acidic conditions can be applied to crude cell and tissue extracts. The assay was also adapted for other substrates than N⁶-(2-isopentenyl)adenine, such as zeatin and the aromatic cytokinins, although an enzyme which degrades the latter compounds has not yet been identified. The second novel method is an initial rate method based on the coupled redox reaction of phenazine methosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide resulting in the formation of a formazan dye. This method can be used for kinetic studies with purified enzyme and is entirely substrate independent.

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This study investigated if and how cytokinins would mediate the effect of post-anthesis soil-drying on source-to-sink remobilization of nitrogen (N) in rice. Field experiments were conducted with normal (NN) and high amount of N (HN) applications. Two irrigation regimes, well-watered (WW) and moderate soil-drying (MD), were imposed from 8 days after full heading until maturity. The results showed that N use efficiency (NUE) including internal N use efficiency, N partial factor productivity and N harvest index, was substantially increased in MD relative to in WW. The MD promoted the reallocation and transfer of N, free amino acids (FAAs) and trans-zeatin (Z) + trans-zeatin riboside (ZR) from the source organs (stems and leaves) to the sink organ (panicles). Activities of protein degradation enzymes including endopeptidase (EP) and aminopeptidase (AP) in stems and leaves, and expression levels of FAAs transporter genes in panicles were substantially increased in MD than in WW, and were closely associated with Z + ZR content there. Application of kinetin to stems and leaves significantly decreased the activities of EP and AP, resulting in more N retention in stems and leaves and lower N harvest index. Application of kinetin to panicles significantly increased the expression levels of FAAs transporter genes in panicles, leading to less N retention in stems and leaves and higher N harvest index. Such facilitation in source-to-sink remobilization of N induced by the MD was greater at HN than at NN. Collectively, a post-anthesis MD can enhance source-to-sink remobilization of N and synergistically increase grain yield and NUE via redistributing cytokinins (Z + ZR) in rice.
OsCKX2 regulates phosphate deficiency tolerance by modulating cytokinin in rice
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Citation Excerpt :
The hydroponic nutrient solution was changed every 2 days throughout the treatment. The measurement of CKX activity was performed according to the methods described previously [25,26]. Seedling samples of OE, KO, and WT plants under NP and LP conditions were homogenized and prepared as a slurry by mixing chilled Tris/HCl buffer (0.2 M, pH 8.0), containing 1 mM phenylmethylsulphonyl fluoride (PMSF) and 0.3% Triton X-100.
Cytokinin oxidase/dehydrogenases (CKXs) are key enzymes that degrade cytokinins (CTKs) and play an essential role in plant growth and development. The present study analyzed the phenotypic and physiological characteristics of OsCKX2 overexpressing (OE) and knockout (KO) rice plants after exposure to phosphate (Pi) deficiency and the transcriptome and metabolome to investigate the function of OsCKX2 in response to Pi deficiency. OsCKX2 KO plants demonstrated higher endogenous CTK levels than wild-type (WT) under Pi deficiency. Further analysis indicated more robust tolerance of OsCKX2 KO plants to Pi deficiency, which exhibited higher phosphorus concentration, larger shoot biomass, and lesser leaf yellowing under Pi deficiency; whereas the opposite was observed for OsCKX2 OE plants. Transcriptome and metabolome analyses revealed that overexpression of OsCKX2 downregulated the transcriptional levels of genes related to Pi transporters, membrane lipid metabolism, and glycolysis, and reduced the consumption of metabolites in membrane lipid metabolism and glycolysis. On the contrary, knockout of OsCKX2 upregulated the expression of Pi transporters, and increased the consumption of metabolites in membrane lipid metabolism and glycolysis. These results indicated that OsCKX2 impacted Pi uptake, recycling, and plant growth via Pi transporters, phospholipid hydrolysis, and glycolysis under Pi deficiency. Overall, OsCKX2 negatively regulated Pi deficiency tolerance by modulating CTKs in rice.
Blue light suppression alters cytokinin homeostasis in wheat leaves senescing under shading stress
2018, Plant Physiology and Biochemistry
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Cell debris was removed by centrifugation at maximum speed for 20 min at 4 °C. The supernatant was used for the enzymatic activity determination by the end-point assay with 4-aminophenol (Frébort et al., 2002). The reaction mixture of 0.6 mL final volume contained 100 μL of the protein extract, 200 μM of iP as substrate and 0.5 mM of 2,6-dichlorophenolindophenol (DCPIP) as electron donor, in 200 mM McIlvaine buffer (citric acid/disodium hydrogen phosphate, pH = 6.8).
Blue light (BL) suppression accelerates the senescence rate of wheat (Triticum aestivum L.) leaves exposed to shading. In order to study whether this effect involves the alteration of different cytokinin (CK) metabolites, CK-degradation, as well as the expression profile of genes responsible of CK-perception, -inactivation, -reactivation and/or -turnover, leaf segments of 30 day-old plants were placed in boxes containing bi-distilled water and covered with blue (B) or green (G) light filters, which supplied a similar irradiance but differed in the percentage of BL transmitted (G << B). A neutral (N) filter was used as control. When appropriate, different CK metabolites or an inhibitor of CK-degradation were added in order to alter the endogenous CK levels. A rapid decrement of trans-zeatin (tZ) and cis-zeatin (cZ) content was observed after leaf excision, which progressed at a higher rate in treatment G than in the control and B treatments. Senescence progression correlated with an accumulation of glycosylated forms (particularly cZ-derivatives), and an increment of CK-degradation, both of which were slowed in the presence of BL. On the contrary, CK-reactivation (analyzed through TaGLU1-3 expression) was delayed in the absence of BL. When different CK were exogenously supplied, tZ was the only natural free base capable to emulate the senescence-retarding effect of BL. Even though the signaling components involved in the regulation of senescence rate and CK-homeostasis by BL remain elusive, our data suggest that changes in the expression profile and/or functioning of the transcription factor HY5 might play an important role.
Maize cytokinin dehydrogenase isozymes are localized predominantly to the vacuoles
2016, Plant Physiology and Biochemistry
Citation Excerpt :
Protein concentration in clarified extract and concentrated media was quantified using the Bradford method (Bradford, 1976). CKX activity in cell lysates and in culture media was measured by the end point method (Frébort et al., 2002). Aliquots of cell lysate (1–50 μL) or concentrated media (2–100 μL) were incubated at 37 °C for 2–16 h in a reaction mixture (total volume of 0.6 mL) consisting of 250 mM McIlvaine buffer (pH 7.5), 0.125 mM dichlorophenolindophenol, and 250 μM substrate iP.
The maize genome encompasses 13 genes encoding for cytokinin dehydrogenase isozymes (CKXs). These enzymes are responsible for irreversible degradation of cytokinin plant hormones and thus, contribute regulating their levels. Here, we focus on the unique aspect of CKXs: their diverse subcellular distribution, important in regulating cytokinin homeostasis. Maize CKXs were tagged with green fluorescent protein (GFP) and transiently expressed in maize protoplasts. Most of the isoforms, namely ZmCKX1, ZmCKX2, ZmCKX4a, ZmCKX5, ZmCKX6, ZmCKX8, ZmCKX9, and ZmCKX12, were associated with endoplasmic reticulum (ER) several hours after transformation. GFP-fused CKXs were observed to accumulate in putative prevacuolar compartments. To gain more information about the spatiotemporal localization of the above isoforms, we prepared stable expression lines of all ZmCKX–GFP fusions in Arabidopsis thaliana Ler suspension culture. All the ER-associated isoforms except ZmCKX1 and ZmCKX9 were found to be targeted primarily to vacuoles, suggesting that ER-localization is a transition point in the intracellular secretory pathway and vacuoles serve as these isoforms' final destination. ZmCKX9 showed an ER-like localization pattern similar to those observed in the transient maize assay. Apoplastic localization of ZmCKX1 was further confirmed and ZmCKX10 showed cytosolic/nuclear localization due to the absence of the signal peptide sequence as previously reported. Additionally, we prepared GFP-fused N-terminal signal deletion mutants of ZmCKX2 and ZmCKX9 and clearly demonstrated that the localization pattern of these mutant forms was cytosolic/nuclear. This study provides the first complex model for spatiotemporal localization of the key enzymes of the cytokinin degradation/catabolism in monocotyledonous plants.
Transgenic barley overexpressing a cytokinin dehydrogenase gene shows greater tolerance to drought stress
2016, New Biotechnology
Citation Excerpt :
The blast results were then filtered to accept only those results having >80% identity, and 20,108 genes of the reference genome were definitely assigned to the Zea mays genes. The CKX activity was determined spectrophotometrically [30] using 0.5 mM 2,3-dimetoxy-5-methyl-1,4-benzoquinone as an electron acceptor and 0.25 mM substrate iPR in McIlvaine buffer (pH 5.5). All measurements were performed in three biological replicates.
Together with auxins, cytokinins are the main plant hormones involved in many different physiological processes. Given this knowledge, cytokinin levels can be manipulated by genetic modification in order to improve agronomic parameters of cereals in relation to, for example, morphology, yield, and tolerance to various stresses. The barley (Hordeum vulgare) cultivar Golden Promise was transformed using the cytokinin dehydrogenase 1 gene from Arabidopsis thaliana (AtCKX1) under the control of mild root-specific β-glucosidase promoter from maize. Increased cytokinin degradation activity was observed positively to affect the number and length of lateral roots. The impact on morphology depended upon the recombinant protein's subcellular compartmentation. While assumed cytosolic and vacuolar targeting of AtCKX1 had negligible effect on shoot growth, secretion of AtCKX1 protein to the apoplast had a negative effect on development of the aerial part and yield. Upon the application of severe drought stress, all transgenic genotypes maintained higher water content and showed better growth and yield parameters during revitalization. Higher tolerance to drought stress was most caused by altered root morphology resulting in better dehydration avoidance.
Cytokinin Oxidase/Dehydrogenase as an Important Target for Increasing Plant Productivity
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Regular ArticleCytokinin Oxidase/Cytokinin Dehydrogenase Assay: Optimized Procedures and Applications

Abstract

Biochem. Biophys. Res. Commun.

Anal. Biochem.

J. Plant Physiol.

J. Plant Physiol.

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Anal. Biochem.

Plant Sci.

J. Biol. Chem.

Cytokinin oxidase: Biochemical features and physiological significance

Physiol. Plant.

Cytokinin oxidase or dehydrogenase? Mechanism of the cytokinin degradation in plants

Eur. J. Biochem.

Molecular and biochemical characterization of a cytokinin oxidase from maize

Plant Physiol.

Cytokinin metabolism and action

Annu. Rev. Plant Physiol. Plant Mol. Biol.

Degradation of cytokinins by cytokinin oxidases in plants

Plant Growth Reg.

Cytokinin oxidase from Zea mays: Purification, cDNA cloning and expression in moss protoplasts

Plant J.

Regular Article
Cytokinin Oxidase/Cytokinin Dehydrogenase Assay: Optimized Procedures and Applications