Comparative Physiological and Transcriptomic Mechanisms of Defoliation in Cotton in Response to Thidiazuron versus Ethephon

Abstract

Thidiazuron (TDZ) is a widely used chemical defoliant in cotton and can stimulate the production of ethylene in leaves, which is believed to be the key factor in inducing leaf abscission. Ethephon (Eth) can also stimulate ethylene production in leaves, but it is less effective in promoting leaf shedding. In this study, the enzyme-linked immunosorbent assays (ELISA) and RNA-seq were used to determine specific changes at hormonal levels as well as transcriptomic mechanisms induced by TDZ compared with Eth. The TDZ significantly reduced the levels of auxin and cytokinin in cotton leaves, but no considerable changes were observed for Eth. In addition, TDZ specifically increased the levels of brassinosteroids and jasmonic acid in the leaves. A total of 13 764 differentially expressed genes that specifically responded to TDZ were identified by RNA-seq. The analysis of KEGG functional categories suggested that the synthesis, metabolism, and signal transduction of auxin, cytokinin, and brassinosteroid were all involved in the TDZ-induced abscission of cotton leaves. Eight auxin transport genes (GhPIN1-c_D, GhPIN3_D, GhPIN8_A, GhABCB19-b_A, GhABCB19-b_D, GhABCB2-b_D, GhLAX6_A, and GhLAX7_D) specifically responded to TDZ. The pro35S::GhPIN3a::YFP transgenic plants showed lower defoliation than the wild type treated with TDZ, and YFP fluorescence in leaves was almost extinguished after treatment with TDZ rather than Eth. This provides direct evidence that GhPIN3a is involved in the leaf abscission induced by TDZ. We found that 959 transcription factors (TFs) specifically responded to TDZ, and a co-expression network analysis (WGCNA) showed five hub TFs (GhNAC72, GhWRKY51, GhWRKY70, GhWRKY50, and GhHSF24) during chemical defoliation with TDZ. Our work sheds light on the molecular basis of TDZ-induced leaf abscission in cotton.

Keywords: auxin transport; brassinosteroid; cotton; cytokinin; leaf abscission; transcription factors.

Figure 1

Effect of thidiazuron (TDZ) and ethephon (Eth) on cotton leaf defoliation and ethylene production. (A) Representative plants at 3 d and 5 d after treatment with 300 mg/L TDZ, or ethephon with 0.2 (Eth_L), 0.6 (Eth_M), and 1.2 (Eth_H) mg/L. (B) Defoliation rate (%) over time after treatment. (C) Ethylene production of upper (5-6), middle (3-4), and lower (1-2) leaves at 24 h after treatment. Error bars represent the SD of three biological replicates. Homogeneous subgroups with significant differences (p ≤ 0.05) are marked by different lowercase letters.

Figure 2

The content of auxin (IAA, (A)), isopentenyl adenine and isopentenyl adenosine (IP+IPR, (B)), zeatin and zeatin riboside (Z+ZR, (C)), dihydrozeatin and dihydrozeatin riboside (DHZ+DHZR, (D)), brassinosteroid (BR, (E)), abscisic acid (ABA, (F)), jasmonic acid (JA, (G)), and gibberellin (GA₃, (H)) in the fourth leaf (from bottom) of cotton plants at 24 h after treatment with 300 mg/L thidiazuron (TDZ) or 0.6 mg/L ethephon (Eth). Mock: water. Significant differences between TDZ or Eth and Mock were determined by t-test, * indicates p-value < 0.05, ns indicates no significant difference.

Figure 3

Transcriptome features and relationships among all samples. (A) Hierarchical clustering diagram showing the time series distribution of the samples. Cotton plants were treated with 300 mg/L thidiazuron (TDZ) or 0.6 mg/L ethephon (Eth). Mock: water. (B) Principal component analysis (PCA) of 36 libraries taken at 4 time points. (C) The number of up- and downregulated differentially expressed genes in cotton leaf upon TDZ or Eth treatment.

Figure 4

Expression pattern of genes specifically in response to thidiazuron (300 mg/L TDZ) compared with ethephon (0.6 mg/L) (TDZ-SRGs) and functional enrichment analysis over the time course. (A) Heat map of the 13,764 TDZ-SRGs over time using log₂ (fold-change) values of genes. The heat map displays expression patterns of the eight clusters, which are grouped by the start time of each gene’s response to TDZ, with red indicating upregulated expression (C1–C4) and blue indicating downregulated expression (C5–C8). (B) KEGG functional categories enriched in C1–C8 gene clusters. Significant categories (p < 0.05) were displayed.

Figure 5

Genes with specific response to 300 mg/L thidiazuron (TDZ) compared with 0.6 mg/L ethephon (TDZ-SRGs) involved in plant hormone signal transduction. (A) Auxin. (B) Cytokinin. (C) Brassinosteroid.

Figure 6

Genes with specific response to 300 mg/L thidiazuron (TDZ) compared with 0.6 mg/L ethephon (TDZ-SRGs) involved in auxin (A), cytokinin (B) and brassinosteroid (C) synthesis and/or metabolism pathways.

Figure 7

Effect of brassinosteroid (BR) pretreatment (2 h in advance) on thidiazuron (TDZ)-induced cotton leaf abscission. (A) Representative plants at 7 d after treatments. (B) Defoliation rate (%) over time after treatments. Homogeneous subgroups with significant difference (p-value < 0.05) are marked by different lowercase letters.

Figure 8

Expression profiles of genes with specific response to 300 mg/L thidiazuron (TDZ) compared with 0.6 mg/L ethephon (TDZ-SRGs) related to auxin transport. Pearson’s correlation coefficient was used to estimate the agreement (cor) between the RT-qPCR expression levels and RNA-seq data. Red dots indicate FPKM value from RNA-seq. Significant differences between TDZ or Eth and Mock were determined by t-test, * indicates p-value < 0.05.

Figure 9

Effect of thidiazuron (TDZ, 300 mg/L) and ethephon (Eth, 0.6 mg/L) on GhPIN3a::YFP expression and protein localization in vein epidermal cells of the fourth leaf from bottom of cotton plants at six-leaf stage.

Figure 10

The importance of auxin transport in thidiazuron (TDZ)-induced leaf abscission. (A) Representative plants taken at 5 d after treatment with 300 mg/L TDZ, or TDZ plus 100 and 200 μM 1-naphthylphthalamic acid (NPA), respectively. (B) Defoliation rate (%) of cotton leaves over time. Error bars represent the SD of three biological replicates. Homogeneous subgroups with significant difference (p-value < 0.05) are marked by different lowercase letters.

Figure 11

Summary of the top transcription factor families in the genes specifically responsive to 300 mg/L thidiazuron (TDZ) compared with 0.6 mg/L ethephon (TDZ-SRGs). Early: 3, 6, and 12 h after treatment; Late: 24 h after treatment. Red indicates upregulated expression and blue indicates downregulated expression.

Figure 12

Gene co-expression network analysis by WGCNA. (A) Module–sample phenotype association, * indicates p-value < 0.05. (B) Network of Hub transcription factors in green module. Triangles represent transcription factors, circles represent functional genes, red represents hub transcription factors within modules, and blue represents of hub transcription factors target genes.