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. 2016 Dec 8;16(1):260.
doi: 10.1186/s12870-016-0932-z.

Characterization of the Cytokinin-Responsive Transcriptome in Rice

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Free PMC article

Characterization of the Cytokinin-Responsive Transcriptome in Rice

Tracy Raines et al. BMC Plant Biol. .
Free PMC article

Abstract

Background: Cytokinin activates transcriptional cascades important for development and the responses to biotic and abiotic stresses. Most of what is known regarding cytokinin-regulated gene expression comes from studies of the dicotyledonous plant Arabidopsis thaliana. To expand the understanding of the cytokinin-regulated transcriptome, we employed RNA-Seq to analyze gene expression in response to cytokinin in roots and shoots of the monocotyledonous plant rice.

Results: We identified over 4,600 and approximately 2,400 genes differentially expressed in response to cytokinin in roots and shoots respectively. There were some similarities in the sets of cytokinin-regulated genes identified in rice and Arabidopsis, including an up-regulation of genes that act to reduce cytokinin function. Consistent with this, we found that the preferred DNA-binding motif of a rice type-B response regulator is similar to those from Arabidopsis. Analysis of the genes regulated by cytokinin in rice revealed a large number of transcription factors, receptor-like kinases, and genes involved in protein degradation, as well as genes involved in development and the response to biotic stress. Consistent with the over-representation of genes involved in biotic stress, there is a substantial overlap in the genes regulated by cytokinin and those differentially expressed in response to pathogen infection, suggesting that cytokinin plays an integral role in the transcriptional response to pathogens in rice, including the induction of a large number of WRKY transcription factors.

Conclusions: These results begin to unravel the complex gene regulation after cytokinin perception in a crop of agricultural importance and provide insight into the processes and responses modulated by cytokinin in monocots.

Keywords: Arabidopsis; Cytokinin; Plant hormone; Rice; Transcriptomics.

Figures

Fig. 1
Fig. 1
Gene expression in BA-treated and mock-treated rice seedlings. a RNA-Seq coverage graphs from chromosome 1 showing mock-treated rice root and shoots. Graphs in the different tracks use the same y-axis scale, and peaks represent regions of high expression. The region in view includes the following genes: 1) LOC_Os01g72170DE; 2) LOC_Os01g72190; 3) LOC_Os01g72200; 4) LOC_Os01g72205 DE; 5) LOC_Os01g72210 DE; 6) LOC_Os01g72220; 7) LOC_Os01g72230; 8) LOC_Os01g72240; 9) LOC_Os01g72250; 10) LOC_Os01g72260DE; 11) LOC_Os01g72270DE; 12) LOC_Os01g72280DE; 13) LOC_Os01g72290DE; where (DE) indicates genes that are differentially expressed between roots and shoots. b Expression in BA-treated samples in log2 of RPKM compared to mock-treated samples in roots (left) and shoots (right). c Venn diagram showing the number of genes up or down-regulated in roots and shoots. d Plot showing log2 fold change in roots versus shoots for genes that were differentially regulated by BA treatment in both tissues. Data points in the upper right and lower left quadrants represent genes that were changed in the same direction in both shoots and roots. Blue data points indicate type-A RRs
Fig. 2
Fig. 2
Comparison of gene expression changes detected by NanoStrings and RNA-Seq. Expression of cytokinin-responsive genes in roots (a) and shoots (b) was assayed using RNA-Seq and Nanostrings on treated samples from two separate experiments. The log2 fold change between BA-treated and mock-treated samples was calculated from three biological replicates. Linear regression lines are shown in red. R2 was 0.89 and 0.74 for the root and shoot, respectively
Fig. 3
Fig. 3
OsRR22 binding sites in differentially expressed genes in rice roots and shoots. a Position weight matrix representations of the top-scoring DNA-binding motifs for OsRR22 as determined by the protein binding microarray. b-c Percentage of cytokinin-regulated genes in roots that contained one or more of the top 25 8-mer sequences that showed significant binding for OsRR22 (see methods). Percentages indicated with vertical dashed lines were (b) 27.8% of up-regulated genes, (c) 23.7% of down-regulated genes. Histograms show the distribution of percentages obtained from 1,000 random samples of equivalent size
Fig. 4
Fig. 4
Mapman categorization of genes differentially regulated by cytokinin. MapMan representations of overview of regulation (a), cellular response (b) and metabolism (c) of genes identified as differentially regulated in response to cytokinin in rice or roots. Differentially expressed genes are shown as color-coded squares. Blue indicates down-regulated genes and red indicates up-regulated genes with the relative level of log2 fold change indicated by the scale shown
Fig. 5
Fig. 5
MapMan categories enriched in cytokinin-responsive genes in rice tissues. Representation of the MapMan categories enriched in the number of cytokinin-regulated genes in rice roots (left) and shoots (right). The numbers (above each bar indicate the number of cytokinin-regulated genes per category. Arrow length and direction and bar shading indicate how many differential expressed genes were up-regulated (red bar, arrows pointing up) or down-regulated (blue bar, arrows pointing down). Categories are ordered by significance (FDR) with the most significant terms on the left. Only categories with FDR 0.001 or smaller are shown
Fig. 6
Fig. 6
Similarity between biotic stress-regulated and cytokinin-regulated gene. expression in rice. Overlap of the differentially expressed genes in response to benzyladenine (BA) in roots and (a) benzothiadiazole (BTH) [76], (b) Xanthomonas oryzae pv. oryzicola (Xoc) [77] or (c) Magnaporthe oryzae (Mor) [78]. Previously reported biotic stress data are from microarray experiments on whole seedlings. Only genes that could be assayed by a single, non-cross hybridizing probe on the array are shown
Fig. 7
Fig. 7
Cytokinin regulation of WRKY transcription factors in rice. Log2 fold change in the level of the WRKY transcription factors that are differentially expressed in rice roots (blue bars) or shoots (green bars) in response to cytokinin treatment. WRKY1 (indicated in red) is regulated in both tissues

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