Cyclanilide Induces Lateral Bud Outgrowth by Modulating Cytokinin Biosynthesis and Signalling Pathways in Apple Identified via Transcriptome Analysis

Abstract

Cyclanilide (CYC), a plant growth regulator, is a potent shoot branching agent in apple. However, its mechanism remains unclear. The current study revealed that CYC treatment resulted in massive reprogramming of the axillary bud transcriptome, implicating several hormones in the response. We observed a marked increase (approximately 2-fold) in the level of zeatin riboside and a significant decrease (approximately 2-fold) in the level of abscisic acid (ABA). Zeatin metabolism gene cytokinin (CTK) oxidase 1 (CKX 1) was down-regulated at 168 h after CYC treatment compared with the control. Weighted gene co-expression network analysis of differentially expressed genes demonstrated the turquoise module clusters exhibited the highest positive correlation with zeatin riboside (r = 0.92) and the highest negative correlation with ABA (r = -0.8). A total of 37 genes were significantly enriched in the plant hormone signal transduction pathway in the turquoise module. Among them, the expressions of CTK receptor genes WOODEN LEG and the CTK type-A response regulators genes ARR3 and ARR9 were up-regulated. ABA signal response genes protein phosphatase 2C genes ABI2 and ABI5 were down-regulated in lateral buds after CYC treatment at 168 h. In addition, exogenous application of 6-benzylaminopurine (6-BA, a synthetic type of CTK) and CYC enhanced the inducing effect of CYC, whereas exogenous application of lovastatin (a synthetic type of inhibitor of CTK biosynthesis) or ABA and CYC weakened the promoting effect of CYC. These results collectively revealed that the stimulation of bud growth by CYC might involve CTK biosynthesis and signalling, including genes CKX1 and ARR3/9, which provided a direction for further study of the branching promoting mechanism of CYC.

Keywords: apple; branching; cyclanilide; cytokinin; transcriptome.

Figure 1

Apple bud phenotype and bud length of axillary buds subjected to cyclanilide application in 1-year apple seedlings. Scale bar = 2.0 cm. (a) treatment method; (b) bud phenotype and length. Cyc: cyclanilide. Data represent the mean ± SD (n = 15). Asterisk indicates significant differences based on the Tukey test (p < 0.05).

Figure 2

Overview of the BRC1 expression level using qRT-PCR. (a) The BRC1 expression level among the different maturity state axillary buds at 0 h with A-1, A-2, and A-3 materials (b) The BRC1 expression level after cyclanilide (Cyc) treatment at 24 h and 168 h with A-1 material. Significant differences analysis based on the Tukey test in (a) and t-test in (b) (* p < 0.05). Data represent the mean ± SD (n = 3).

Figure 3

Hormone content in axillary buds at 6–168 h after application of cyclanilide. (a) IAA (indole-3-acetic acid) content (b) ZR (zeatin riboside) content (c) ABA (abscisic acid) content (d) GA₃ (Gibberellin 3) content (e) JA-me (methyl jasmonate) content. Cyc: cyclanilide. Data represent the mean ± SD (n = 3). Asterisk indicates significant differences based on a t-test (p < 0.05). Double asterisks indicate extremely significant differences based on the t-test (p < 0.01).

Figure 4

Principle component analysis and differentially expressed genes count. (a) Principle component analysis of controls and treatment samples at 0, 24 and 168 h. (b) the numbers of differentially expressed genes from all sample comparisons at different time points after treatment.

Figure 5

Pathway analysis based on Kyoto encyclopaedia of genes and genomes (KEGG) analysis. (a) The KEGG dot map of differentially expressed genes at 24 h after treatment. (b) The KEGG dot map of differentially expressed genes at 168 h after treatment.

Figure 6

Module-trait associations. Each row corresponds to a module and each column to a trait. Each cell contains the corresponding correlation and p-value.

Figure 7

The heatmap of 37 genes involved in the plant hormone signal transduction pathway.

Figure 8

The phenotype and bud length of different pharmacologic treatments. (a) The phenotype of different pharmacologic treatments, Scale bar = 5.0 cm. 6-BA (6-benzylaminopurine, a kind of cytokinin). Lov, lovastatin (a synthetic inhibitor of CTK biosynthesis). ABA (abscisic acid). CYC (cyclanilide). (b) The bud length of different pharmacologic treatments. Data represent the mean ± SD (n = 12). Bars with different letters are significantly different at α = 0.05.