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Petr Galuszka Hana Popelková Tomáš Werner Jitka Frébortová Hana Pospíšilová Václav Mik Ireen Köllmer Thomas Schmülling Ivo Frébort 《Journal of Plant Growth Regulation》2007,26(3):255-267
Transgenic tobacco plants overexpressing single Arabidopsis thaliana cytokinin dehydrogenase (CKX, EC 1.5.99.12) genes AtCKX1, AtCKX2, AtCKX3, AtCKX4, AtCKX5, AtCKX6, and AtCKX7 under the control of a constitutive 35S promoter were tested for CKX-enzymatic activity with varying pH, electron acceptors,
and substrates. This comparative analysis showed that out of these, only AtCKX2 and AtCKX4 were highly active enzymes in reaction
with isoprenoid cytokinins (N
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-(2-isopentenyl)adenine (iP), zeatin (Z)) and their ribosides using the artificial electron acceptors 2,6-dichlorophenol indophenol
(DCPIP) or 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0). Turnover rates of these cytokinins by four other AtCKX isoforms (AtCKX1, AtCKX3, AtCKX5, and AtCKX7) were substantially
lower, whereas activity of AtCKX6 was almost undetectable. The isoenzymes AtCKX1 and AtCKX7 showed significant preference
for cytokinin glycosides, especially N
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-(2-isopentenyl)adenine 9-glucoside, under weakly acidic conditions. All enzymes preferentially cleave isoprenoid cytokinins
in the presence of an electron acceptor, but aromatic cytokinins are not resistant and are degraded with lower reaction rates
as well. Cytokinin nucleotides, considered as resistant to CKX attack until now, were found to be potent substrates for some
of the CKX isoforms. Substrate specificity of AtCKXs is discussed in this study with respect to the structure of the CKX active
site. Further biochemical characterization of the AtCKX1, AtCKX2, AtCKX4 and AtCKX7 enzymes showed pH-dependent activity profiles. 相似文献
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The expression of cytokinin oxidase/dehydrogenase (CKX EC: 1.5.99.12) is subject to fine regulation and it provides a rapid
turnover of cytokinins, which serves as a signal for triggering developmental events during plant growth. The activity of
this enzyme is believed to be responsible for the changes in cytokinin pool under adverse environmental conditions. CKX gene-specific
assay to measure the expression in response to different stress treatments in the tissues of Pisum sativum plants was developed. Pea CKX genes were amplified and sequenced using primers designed from the sequences of Medicago truncatula CKX genes. Expression of two P. sativum CKX genes was assessed using relative-quantification in real time two-step RT-PCR, in leaves and roots of drought-, glufosinate-
and atrazine-treated cv. Manuela pea plants. Varied CKX responses support the existence of complicated regulating mechanism
of cytokinin oxidase/ dehydrogenase gene expression. 相似文献
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Ethylene and gibberellins have a synergistic stimulatory effect on hypocotyl elongation of light-grown Arabidopsis thaliana (L.) Heynh. seedlings. A screen for mutants with decreased response to these hormones led to the isolation of a novel allele
(amp1-7) of the ALTERED MERISTEM PROGRAM (AMP) 1 locus. The amp1-7 allele contains a missense mutation causing a phenotype, which is weaker than that of the amp1-1 mutant that carries a nonsense mutation. The mutant phenotype prompted the hypothesis that AMP1 is involved in ethylene and
GA signalling pathways or in a parallel pathway-controlling cell and hypocotyl elongation and cellular organization. Amp1 mutants contain higher zeatin concentrations causing enlargement of the apical meristem, which was confirmed by cytokinin
application to wild type seedlings. Light grown amp1 seedlings have shorter hypocotyls than wild type; however, application of cytokinins promotes hypocotyl elongation of both
Col-0 and amp1. We suggest that in amp1 mutants either zeatin overproduction or its action is strictly localized.
Nelson J. M. Saibo and Wim H. Vriezen contributed equally to this work. 相似文献
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Cytokinin dehydrogenase (CKX) is responsible for regulating the endogenous cytokinin content by oxidative removal of the side
chain and seven distinct genes, AtCKX1 to AtCKX7, code for the enzyme in Arabidopsis thaliana. The recombinant enzyme AtCKX2 was produced in Saccharomyces cerevisiae after expressing the corresponding gene from a plasmid (pDR197) or following chromosomal integration, under either the constitutive
promoter PMA1 or the inducible promoter GAL1. The recombinant protein was purified from yeast culture media using a sequence of chromatographic steps. The purified enzyme
had a molecular mass of 61 kDa and a typical flavoprotein spectrum. The specific activity of the enzyme was 87.8 μkat g−1, with isopentenyladenine as a substrate and 2,3-dimethoxy-5-methyl-p-benzoquinone as an electron acceptor. The pH optimum lay between 7.0 and 8.0, depending on the electron acceptor used. AtCKX2
reacts both with isoprenoid and aromatic cytokinins, the activity with isoprenoid cytokinins being two to three orders of
magnitude higher. AtCKX2 prefers p-quinones and the synthetic dye 2,6-dichlorophenol indophenol as electron acceptors, although low reactivity with oxygen can
also be observed. This study presents the first purification and characterization of the enzyme from Arabidopsis thaliana. 相似文献
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A. E. Valdés P. Galuszka B. Fernández M. L. Centeno I. Frébort 《Biologia Plantarum》2007,51(1):193-197
In the present study cytokinin dehydrogenase (CKX) activity was for the first time found in a conifer species, Pinus sylvestris. The activities were correlated with the endogenous cytokinin contents. Several enzyme substrates and two different electron
acceptors were used to search for the enzyme activity in the extract from seeds, seedlings and plantlets. The highest specific
activity was found in one-year-old plantlets with isopentenyladenine as the substrate and 2,6-dichlorophenolindophenol as
the electron acceptor, at pH 8. An enhancement in the CKX specific activity corresponded to increasing contents of cytokinins,
mainly isopentenyladenine and isopentenyladenosine, indicating that the enzyme activity is affected by the endogenous supply
of cytokinins. CKX affinity for the ribosylated form of isopentenyladenine was dependent on the developmental stage, being
higher in seeds than in seedlings, and not detectable in plantlets. The results are indicative of the presence of different
isoenzymes throughout the development. 相似文献
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Marek Klemš Zdeňka Slámová Václav Motyka Jiří Malbeck Alena Trávníčková Ivana Macháčková Josef Holík Stanislav Procházka 《Plant Growth Regulation》2011,65(3):427-437
The uptake and metabolism of trans-zeatin and/or dihydrozeatin, in correlation with cytokinin oxidase/dehydrogenase (CKX) and β-glucosidase activity, were studied
in leaf segments derived from wild-type (WT) and transgenic (T) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) during in vitro induction of shoot organogenesis. T explants harbored the maize gene Zm-p60.1β-glucosidase. Higher levels of shoot regeneration were observed on T explants in the early stages of cultivation. In WT explants, the
content of cytokinin (CK)-O- and N-glucosides increased. In T explants, a higher content of Z-9-riboside and Z-9-riboside-5′-monophosphate and higher CKX activity
during the early stage of cultures were found. A positive correlation was obtained for bioactive CK content and the organogenic
response in T explants. Our results indicate a connection between the organogenic capacity of tobacco explants, metabolism
of endogenous CKs and uptake of exogenous CKs from the cultivation medium. 相似文献
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Elitsa Blagoeva Petre I. Dobrev Jiří Malbeck Václav Motyka Alena Gaudinová Radomíra Vaňková 《Plant Growth Regulation》2004,44(1):15-23
The role of cytokinin N-glucosylation and degradation by cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) in response to application of exogenous auxins (2,4-dichlorophenoxyacetic acid [2,4-D] and -naphthaleneacetic acid [NAA]) and cytokinins (N
6-benzyladenine [BA] and trans-zeatin [Z]) was investigated in de-rooted seedlings of Raphanus sativus L. cv. Rampouch. Both auxins applied for 24 h at 1 and 10 M concentration increased N-glucosylation of exogenously applied [3H]dihydrozeatin (DHZ) by up to 20%. The level of endogenous 7N-glucosides (of Z, isopentenyladenine [iP] and DHZ) was increased by 2,4-D and NAA at 10 M concentration by 28 and 23%, respectively, the level of Z being decreased by 90 and 59%, respectively. 2,4-D and NAA suppressed CKX activity ca. by half. Exogenous cytokinins Z and BA applied at 1 and 10 M concentration stimulated 7N-glucosylation of [3H]DHZ (by up to 40%). BA both at 1 and 10 M, increased the level of endogenous Z by up to 35% and that of 7N-glucosides by up to 27%. BA application also strongly stimulated CKX activity (by up to 180%). Feeding with 1 and 10 M Z resulted in ca. 100-fold and 2000-fold increase of Z level, respectively. The main metabolite, Z7G, was increased ca. 6-fold and 60-fold, respectively. Levels of Z 9-glucoside (Z9G), trans-zeatin riboside (ZR) and Z O-glucoside (ZOG) were elevated to lesser extent. As compared to BA, Z had only negligible effect on CKX activity. Adenine (1–500 M) was preferentially 7N-glucosylated inhibiting competitively 7N-glucosylation of [3H]DHZ. At high concentrations (100–500 M) it increased endogenous levels of active cytokinins, especially of Z, however, it had no effect on CKX activity. Cytokinin N-glucosylation proved to be involved in down-regulation of active cytokinins in response to auxin and in the re-establishment of cytokinin homeostasis following application of exogenous cytokinins. 相似文献
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Zuping Cao Zhiwei Jia Yongjun Liu Meng Wang Jinfeng Zhao Jun Zheng Guoying Wang 《Molecular biology reports》2010,37(2):1089-1097
A small HSP gene, ZmsHSP, was isolated from Zea mays. Sequence analysis revealed that the open reading frame of ZmsHSP was 477 bp and that it encodes a protein composed of 159 amino acid residues with a calculated molecular mass of 18.17 kD
and a predicated isoelectric point (pI) of 5.63. ZmsHSP contains a CS domain (p23-like domain) and shares similarity with
the HSP90 co-chaperone p23. The expression level of ZmsHSP was different among various tissues with the highest expression in leaves and the lowest in silks. Results also showed that
the expression of ZmsHSP in maize was significantly up-regulated by dehydration. Transgenic Arabidopsis plants overexpressing ZmsHSP under the control of the CaMV 35S promoter had lower endogenous cytokinin content and showed more sensitivity to cytokinin
during the germination and early seedling stage than wild-type plants, suggesting that ZmsHSP might has a function in cytokinin response in Zea mays. 相似文献
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Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N6-benzyladenine (BA) treatment in the presence of Ag+. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag+ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by -naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis. 相似文献
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Li Y Yang L Pathak M Li D He X Weng Y 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2011,123(6):973-983
The compact (dwarf) plant architecture is an important trait in cucumber (Cucumis sativus L.) breeding that has the potential to be used in once-over mechanical harvest of cucumber production. Compact growth habit
is controlled by a simply inherited recessive gene cp. With 150 F2:3 families derived from two inbred cucumber lines, PI 308915 (compact vining) and PI 249561 (regular vining), we conducted
genome-wide molecular mapping with microsatellite (simple sequence repeat, SSR) markers. A framework genetic map was constructed
consisting of 187 SSR loci in seven linkage groups (chromosomes) covering 527.5 cM. Linkage analysis placed cp at the distal half of the long arm of cucumber Chromosome 4. Molecular markers cosegregating with the cp locus were identified through whole genome scaffold-based chromosome walking. Fine genetic mapping with 1,269 F2 plants delimited the cp locus to a 220 kb genomic DNA region. Annotation and function prediction of genes in this region identified a homolog of
the cytokinin oxidase (CKX) gene, which may be a potential candidate of compact gene. Alignment of the CKX gene homologs from both parental lines revealed a 3-bp deletion in the first exon of PI 308915,
which can serve as a marker for marker-assisted selection of the compact phenotype. This work also provides a solid foundation
for map-based cloning of the compact gene and understanding the molecular mechanisms of the dwarfing in cucumber. 相似文献
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Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse
that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m−2) containing BA at 100, 200, or 400 mg L−1 or 25, 50, or 100 mg L−1 each of BA and gibberellins A4 + A7 (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L−1 had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant
than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates
tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L−1 at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants
received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones
when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at
29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least
partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature. 相似文献
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Piestrzyńska-Kajtoch A Gurgul A Polak MP Smołucha G Zmudziński JF Rejduch B 《Molecular biology reports》2012,39(3):2575-2583
Scrapie, a fatal transmissible spongiform encephalopathy (TSE) occurs in two phenotypes: classical and atypical. Many authors
point out that the polymorphism of three codons (136, 154, 171) of the PRNP (PrP gene) is associated with a sheep susceptibility to classical scrapie. Until now, only one PRNP gene variant coding phenylalanine at codon 141 has been found to be associated with atypical scrapie. Another recently identified
and interesting candidate gene for scrapie susceptibility in sheep is an SPRN gene coding for Shadoo protein (Sho). Sho is a highly interspecies conserved protein and an insertion/deletion (indel) found
in a sheep Sho gene was associated with classical scrapie occurrence. Here we determined the polymorphism of PRNP and SPRN genes in nine atypical scrapie cases (six in native born sheep and three in imported sheep) and compared these results with
a control group of healthy animals comprising six corresponding Polish sheep breeds. In atypical scrapie cases five PRNP diplotypes were identified: A136R154Q171/ARQ, AHQ/ARQ, ARR/ARQ, ARR/AHQ and AHQ/AHQ. The ARR/AHQ diplotype was found only in imported sheep. A previously unobserved
SNP in PRNP (E224K) was also found in both atypical scrapie and in a few control animals. In the ORF of the SPRN gene, six SNPs and one indel were identified. None of these variations was exclusive for scrapie animals and they were probably,
naturally occurring polymorphisms. Special attention was given to the 6-bp indel SPRN polymorphism which was previously associated with classical scrapie occurrence. 相似文献