Transcription profiling of 5 to 7 days old Arabidopsis thaliana seedlings of wild type (Col-0) and the cytokinin deficient transgenic line 35S:CKX1
ABSTRACT: 5 to 7 days old Arabidopsis thaliana seedlings of wildtype (Col-0) and the cytokinin deficient transgenic line 35S:CKX1 (Werner et al. Plant Cell 15, 2532) at growth stage 1.00 (Boyes scale) grown in liquid medium were induced witn 5 µM 6-Benzylaminopurine for different periods of time in order to find cytokinin-related changes in gene expression.
Project description:The synthetic cytokinins kinetin and 6-benzylaminopurine exhibit equilibrium-type binding to purified chinese-cabbage leaf ribosomes. At 23mum and 4 degrees C one molecule of kinetin and 1.34 molecules of 6-benzylaminopurine are bound per ribosome. Adenine and adenine derivatives that are inactive as cytokinins showed much less affinity for ribosomes. Pretreatment of ribosomes with 0.5m-ammonium chloride or Triton X-100 did not decrease the extent of cytokinin binding. Binding appeared to be to the 83S ribosome species. A positive correlation between the extent of binding and the biological effect of various cytokinin analogues was demonstrated. These results are discussed in terms of cytokinin control of growth processes at the ribosomal level.
Project description:A BAP (benzylaminopurine, cytokinin) solution (10-7 M) was applied to root tips of Medicago truncatula (Jemalong A17 genotype) for 1h. Microarrays were used to compare the transcriptome of these plants (4 replicates) with the transcriptome of control plants, not exposed to BAP (4 replicates)
Project description:In Arabidopsis thaliana, cytokinin responsive B-type ARR transcription factors and HD-ZIP III transcription factors such as REVOLUTA (REV), act cooperatively as master regulators of shoot regeneration. To identify the downstream targets of ARR-HD-ZIP III transcriptional complex, we used an inducible line of REV, 35S::FLAG-GR-rREV, in which FLAG-tagged miR165/6-non-targetable form of REV (rREV)-GR fusion protein was expressed from 35S promoter. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. We treated 35S::FLAG-GR-rREV seedlings with 6-benzylaminopurine (6-BA, a cytokinin), dexamethasone (DEX), or 6-BA+DEX for 2 hours. Total RNAs were extracted and subjected to Agilent Arabidopsis Gene Expression Microarray analyses. The differentially expressed genes (>1.5-fold, p<0.05) were identified. 10-day-old 35S::FLAG-GR-rREV plants were treated with 6-benzylaminopurine (6-BA), dexamethasone (DEX), or 6-BA+DEX for 2 hours. DEX treatment induced activation of REV by translocation of FLAG-GR-rREV fusion protein from cytoplasm to the nucleus. Total RNA was extracted with RNeasy Mini Kit and hybridized to Agilent Arabidopsis Gene Expression Microarray. Differentially expressed genes were defined by a 1.5-fold expression difference with a P value<0.05. Biological replicates were performed.
Project description:Columbia (Col) seeds were sown on half-strength Murashige and Skoog (MS) medium, supplemented with 1% sucrose and 0.8% agar and grown vertically in culture room conditions. The 5-d-old homogenous seedlings were washed five times with sterile water and lastly with liquid half strength MS medium without sugar to remove residual exogenous sugar. In order to deplete internal sugars seedlings were grown in sugar free liquid half strength MS medium for 24 h in dark. Thereafter, the seedlings were treated with half-strength MS medium containing 0% G, 0% G + 1 uM BAP, 3% G, and 3% G + 1 uM BAP for 3 h in dark. RNA was extracted and microarray analysis was performed. Please note: G stands for glucose and BAP stands for 6-Benzylaminopurine (cytokinin)
Project description:UNLABELLED:Endophytic bacteria are harmless in most plant species; and known to boost the growth and development of the host plants probably by secreting growth hormones. The isolation, identification and screening of endophytic bacteria for the plant growth regulators like cytokinin are needed to get the leads for their applications in agriculture sector. We describe the isolation and identification of the bacterial endophytes from the leaves of Sambung Nyawa [Gynura procumbens (Lour.) Merr.] and their screening for cytokinin-like compounds. We isolated three endophytic bacteria from the leaves of G. procumbens collected from the forest research institute of Malaysia (FRIM). They were further identified using amplified 16S rRNA gene sequence based method of bacterial identification. The ethyl acetate extracts of the isolates-broth were analyzed using cucumber cotyledon greening bioassay (CCGB) to determine the presence of cytokinin-like compounds. Consequently, the bacterial putative endophytes were identified as Psuedomonas resinovorans, Paenibacillus polymaxa, and Acenitobacter calcoaceticus. Broth-extracts from two (Psuedomonas resinovorans and Paenibacillus polymaxa) of the three putative bacterial endophytes show the positive results in their screening for cytokinin-like compounds using CCGB. Thus, we hypothesize that the bacterial putative endophytes of G. procumbens that produce cytokinin-like compounds might have a role in the growth and development of G. procumbens. ABBREVIATIONS:CCGB - Cucumber cotyledon greening bioassay, rDNA - Ribosomal DNA, K12, BAP - 6-Benzylaminopurine, Db1, MSA - Multiple sequence alignment. 8081.
Project description:Heterotrimeric GTP binding proteins (G proteins) and cytokinin play important roles in regulating plant growth and development. However, little is known about the mechanism by which they coordinate the regulation of grain size in rice. We functionally characterized one gene, RGG1, encoding a type-A G? subunit. Strong GUS staining was detected in young panicles and spikelets, suggesting a role for this gene in modulating panicle-related trait development. Overexpression of RGG1 in Nipponbare (NIP) and Wuyunjing 30 (WYJ30) significantly decreased plant height, panicle length and grain length by regulating cell division. However, rgg1 mutants generated by the CRISPR/Cas9 system exhibited no obvious phenotypic differences, which may be due to the extremely low expression level of this gene in vivo. The transcriptomes of young panicles of NIP, the NIP-rgg1-2 mutant and the NIP-OE2 overexpression line were sequenced, and the results showed that many differentially expressed genes (DEGs) were associated with the cytokinin biosynthetic pathway. We confirmed this result by measuring the endogenous cytokinin levels and found that cytokinin content was lower in the overexpression lines. Additionally, increased expression of RGG1 decreased sensitivity to low concentrations of 6-benzylaminopurine (6-BA). Our results reveal a novel G protein-cytokinin module controlling grain size in rice and will be beneficial for understanding the mechanisms by which G proteins regulate grain size and plant development.
Project description:Many metabolic processes in plants are regulated by phosphorylation of proteins by kinases, but little is known of the roles that specific protein kinase play in the various signal transduction pathways or the mechanisms by which these kinases themselves are regulated. We report here the isolation of a gene, wpk4, encoding a putative protein kinase from wheat that appears to belong to the SNF1 kinase subfamily and that shows increased transcript levels in response to multiple stimuli: light, nutrient deprivation, and cytokinin application. Although wpk4 mRNA is undetectable in etiolated seedlings, it rapidly accumulates within 1 hr of illumination. General nutrient deprivation also increases wpk4 mRNA levels, but only under light conditions. In addition, of the various phytohormones tested, cytokinin (N6-benzylaminopurine) specifically increases wpk4 mRNA levels regardless of the light conditions, whereas in the presence of a cytokinin antagonist the level of wpk4 mRNA is not increased by either light or nutrient deprivation. These results suggest that the light and nutrient signals that induce wpk4 mRNA accumulation may be mediated through cytokinins and provide a strong basis for examining the coordinated regulation of protein phosphorylation by light, cytokinins, and nutritional cues in a single transduction pathway.
Project description:BACKGROUND: When applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays . Those showing comparable activities with N(6)-benzylaminopurine were further tested in planta. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants. CONCLUSIONS/SIGNIFICANCE: Tetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected hormonal effects in plant propagation techniques in the future.