Detection of Transcription Factors Related to Axillary Bud Development after Exposure to Cold Conditions in Hexaploid Chrysanthemum morifolium Using Arabidopsis Information

Abstract

Chrysanthemum is one of the most commercially used ornamental flowering plants in the world. As chrysanthemum is self-incompatible, the propagation of identical varieties is carried out through cuttings rather than through seed. Axillary bud development can be controlled by changing the temperature; for instance, axillary bud development in some varieties is suppressed at high temperatures. In this study, we focused on the simultaneous axillary bud growth from multiple lines of chrysanthemum upon changing conditions from low to normal temperature. Transcriptome analysis was conducted on the Chrysanthemum morifolium cultivar 'Jinba' to identify the important genes for axillary bud development seen when moved from low-temperature treatment to normal cultivation temperature. We performed RNA-Seq analysis on plants after cold conditions in two-day time-course experiments. Under these settings, we constructed a transcriptome of 415,923 C. morifolium and extracted 7357 differentially expressed genes. Our understanding of Arabidopsis axillary meristem development and growth showed that at least 101 genes in our dataset were homologous to transcription factors involved in the biological process. In addition, six genes exhibited statistically significant variations in expression throughout conditions. We hypothesized that these genes were involved in the formation of axillary buds in C. morifolium after cold conditions.

Keywords: Chrysanthemum morifolium; RNA-Seq; axillary bud; transcription factor.

Figure 1

The preparation of samples for transcriptome analysis in chrysanthemum. (a) Illustration of plant growth and sampling process for transcriptome analysis. (b) Numbering of sampled nodes. Each node number was added to the leaves. (c) Confirmation of axillary bud development after the transfer from low-temperature condition to 25 °C. In the second node, the formation of axillary buds was detected at 0, 1, and 2 days after the transfer to 25 °C. (d) Axillary buds were visually observed at nodes 1, 2, and 3 at 0, 1, and 2 days after the transfer from 4 °C to 25 °C. (e) Sampled node positions (in red box). After the removal of the petioles, stems bearing axillary-bud-development sites were collected (the chrysanthemum plants in (e) are an example image and not used in this analysis).

Figure 2

Dendrogram of 18 samples by expression patterns of 72,533 genes.

Figure 3

Identification of differentially expressed genes (DEGs) using time-course comparison in nodes 1 and 2. The top and bottom values show upregulated and downregulated genes during the early and late day, respectively. Values in circles represent condition-specific DEGs (specifically expressed in 0, 1, or 2 days). Values on lines represent DEGs from the comparisons between two conditions (0 day vs. 1 day, 0 day vs. 2 day, and 1 day vs. 2 days).

Figure 4

The heatmap of 101 DEGs with FDR < 0.05 among the six conditions based on the gene trimmed mean of M values.

Figure 5

Expression profiles of six chrysanthemum genes: (a) EXE, (b) EXB1, (c) LOB-1, (d) LOB-2, (e) AS1-1, and (f) AS1-2 observed in bud 1 (blue) and bud 2 (orange) over 2 days. Y-axis represents relative expression level against Actin gene. Asterisks indicate statistically significant differences based on the t test (p < 0.05) when comparing the results obtained on days 0 and 1 or days 1 and 2.

Figure 6

The diagram of signal transduction in regulating axillary meristem formation and growth, prepared in reference to that in Arabidopsis. Schematic of signal flow in regulating axillary meristem formation (a) and growth (b). Red letters indicate genes shown in the expression profiles in Figure 5 of this study.