Exogenous application of brassinosteroids regulates tobacco leaf size and expansion via modulation of endogenous hormones content and gene expression

Abstract

Brassinosteroids (BR) play diverse roles in the regulation of plant growth and development. BR promotes plant growth by triggering cell division and expansion. However, the effect of exogenous BR application on the leaf size and expansion of tobacco is unknown. Tobacco seedlings are treated with different concentrations of exogenous 2,4-epibrassinolide (EBL) [control (CK, 0 mol L^-1), T1 (0.5 × 10^-7 mol L^-1), and T2 (0.5 × 10^-4 mol L^-1)]. The results show that T1 has 17.29% and T2 has 25.99% more leaf area than control. The epidermal cell area is increased by 24.40% and 17.13% while the number of epidermal cells is 7.06% and 21.06% higher in T1 and T2, respectively, relative to control. So the exogenous EBL application improves the leaf area by increasing cell numbers and cell area. The endogenous BR (7.5 times and 68.4 times), auxin (IAA) (4.03% and 25.29%), and gibberellin (GA₃) contents (84.42% and 91.76%) are higher in T1 and T2, respectively, in comparison with control. Additionally, NtBRI1, NtBIN2, and NtBES1 are upregulated showing that the brassinosteroid signaling pathway is activated. Furthermore, the expression of the key biosynthesis-related genes of BR (NtDWF4), IAA (NtYUCCA6), and GA₃ (NtGA3ox-2) are all upregulated under EBL application. Finally, the exogenous EBL application also upregulated the expression of cell growth-related genes (NtCYCD3;1, NtARGOS, NtGRF5, NtGRF8, and NtXTH). The results reveal that the EBL application increases the leaf size and expansion by promoting the cell expansion and division through higher BR, IAA, and GA₃ contents along with the upregulation of cell growth-related genes. The results of the study provide a scientific basis for the effect of EBL on tobacco leaf growth at morphological, anatomical, biochemical, and molecular levels.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-021-00971-x.

Keywords: Brassinosteroids; Cell division and expansion; Gene expression; Hormones content; Leaf area and expansion; Tobacco leaf growth.

Fig. 1

Setup of the experiment. This schematic flow shows a brief introduction to the entire experiment from start to end. 2,4-epibrassinolide (EBL) was exogenously applied to the leaves of tobacco seedlings with different concentrations. CK symbolizes control (spraying of seedlings with ddH₂O); T1 represents seedlings sprayed with EBL of 0.5 × 10⁻⁷ mol L⁻¹ concentration; T2 denotes seedlings sprayed with EBL of 0.5 × 10⁻⁴ mol L⁻¹ concentration; DAS shows days after sowing

Fig. 2

Effect of exogenous application of 2,4-epibrassinolide (EBL) on leaf growth and morphology. Growth performance under various concentrations of EBL along with a control (A and B). Leaf length, leaf width, and leaf area after EBL treatments (C, D and E, respectively). CK represents control seedlings sprayed with ddH₂O, T1 represents seedlings sprayed with EBL of 0.5 × 10⁻⁷ mol L⁻¹ concentration, and T2 represents seedlings sprayed with EBL of 0.5 × 10⁻⁴ mol L⁻¹ concentration. Values represent the mean ± standard deviation (SD). Different letters indicate significant differences (LSD test; p < 0.05)

Fig. 3

Leaf structure changes in response to exogenous application of 2,4-epibrassinolide. Representative scanning electron microscopic images of the fifth leaf of control and EBL-treated seedlings (A, B and C). Epidermal cell area (D) and epidermal cell numbers (calculated in 4.9152 mm²) (E) in response to exogenous application of EBL. CK represents control seedlings sprayed with ddH₂O, T1 represents seedlings sprayed with EBL of 0.5 × 10⁻⁷ mol L⁻¹ concentration, and T2 represents seedlings sprayed with EBL of 0.5 × 10⁻⁴ mol L⁻¹ concentration. Values represent the mean ± SD. Different letters indicate significant differences (LSD test; p < 0.05)

Fig. 4

The endogenous hormones content and its biosynthesis-related genes expression in response to EBL spraying. Brassinosteroids (BR) concentration (A), auxin (IAA) concentration (B), and gibberellin (GA₃) concentration (C) under various EBL concentrations along with a control. NtDWF4 (BR biosynthesis gene) (D), NtYUCCA6 (IAA biosynthesis gene) (E), NtGA3-ox2 (GA₃ biosynthesis gene) (F), and NtGA2-ox3 (GA₃ inhibitor gene) (G) differentially expressed under various EBL treatments. Values represent mean ± SD. Different letters indicate significant differences (LSD test; p < 0.05). CK represents control seedlings sprayed with ddH₂O, T1 represents seedlings sprayed with EBL of 0.5 × 10⁻⁷ mol L⁻¹ concentration, and T2 represents seedlings sprayed with EBL of 0.5 × 10⁻⁴ mol L⁻¹ concentration

Fig. 5

Differential regulation of BR signaling pathway-related genes under the exogenous EBL application. Expression levels of the corresponding genes-related to the BR signaling pathway are NtBRI1 (A), NtBIN2 (B), and NtBES1 (C). Values represent mean ± SD. Different letters indicate significant differences (LSD test; p < 0.05). CK represents control seedlings sprayed with ddH₂O, T1 represents seedlings sprayed with EBL of 0.5 × 10⁻⁷ mol L⁻¹ concentration, and T2 represents seedlings sprayed with EBL of 0.5 × 10⁻⁴ mol L⁻¹ concentration

Fig. 6

EBL application significantly regulates the expression of cell growth-related genes. The differential expression of cell growth-related genes; NtCYCD3;1 (A), NtXTH (B), NtARGOS (C), NtGRF5 (D), NtGRF8 (E), and NtEXP13 (F) are presented relative to the control samples. Different letters above vertical bars indicate significant differences using the LSD test at p < 0.05. CK represents control seedlings sprayed with ddH₂O, T1 represents seedlings sprayed with EBL of 0.5 × 10⁻⁷ mol L⁻¹ concentration, and T2 represents seedlings sprayed with EBL of 0.5 × 10⁻⁴ mol L⁻¹ concentration

Fig. 7

Promotion effect of the exogenous 2,4-epibrassinolide application on tobacco leaf growth and expansion. This schematic model shows the proposed mechanism of the involvement of EBL in growth and expansion from a morphological level to cellular and molecular levels