Exogenous Brassinosteroid Facilitates Xylem Development in Pinus massoniana Seedlings

Abstract

Brassinosteroids (BRs) are known to be essential regulators for wood formation in herbaceous plants and poplar, but their roles in secondary growth and xylem development are still not well-defined, especially in pines. Here, we treated Pinus massoniana seedlings with different concentrations of exogenous BRs, and assayed the effects on plant growth, xylem development, endogenous phytohormone contents and gene expression within stems. Application of exogenous BR resulted in improving development of xylem more than phloem, and promoting xylem development in a dosage-dependent manner in a certain concentration rage. Endogenous hormone determination showed that BR may interact with other phytohormones in regulating xylem development. RNA-seq analysis revealed that some conventional phenylpropanoid biosynthesis- or lignin synthesis-related genes were downregulated, but the lignin content was elevated, suggesting that new lignin synthesis pathways or other cell wall components should be activated by BR treatment in P. massoniana. The results presented here reveal the foundational role of BRs in regulating plant secondary growth, and provide the basis for understanding molecular mechanisms of xylem development in P. massoniana.

Keywords: Pinus massoniana; brassinosteroids; phytohormones; secondary cell wall; secondary xylem; wood formation.

Figure 1

Effects of exogenous BR on stem growth and xylem development after four-month growth at series BR treatment. (A) Quantification of height increase of seedlings. (B) Stem diameter increase at series BR treatment. (C) Normal histology of stem sections. Bar = 500 µm. Xy, xylem. (D) Xylem thickness at series BR treatment. (E) Number of xylem cell layers at series BR treatment. (F) Phloem thickness at series BR treatment. Error bars ± SE (standard error). * and ** indicate significant differences in comparison with control at p < 0.05 and p < 0.01, respectively.

Figure 2

Quantification of the contents of lignin, cellulose and hemicellulose after four-month growth at series of BR treatment. DW means drought weight. Error bars ± SE. * and ** indicate significant differences in comparison with control at p < 0.05 and p < 0.01, respectively.

Figure 3

Endogenous hormone levels of the seedling stems after 1 mg·L⁻¹ BR and H₂O treatment. FW means fresh weight. Error bars ± SE. * indicates significant differences in comparison with control at p < 0.05. The color brightness represents the degree of the content difference, as shown in the color bar. IAA-Val-Me: indole-3-acetyl-L-valine methyl ester; OxIAA: 2-oxindole-3-acetic acid; IAA-Asp: indole-3-acetyl-L-aspartic acid; IAA-Leu: N-(3-Indolylacetyl)-L-leucine; TRA: tryptamine; ICA: indole-3-carboxylic acid; IAA: indole-3-acetic acid; ICAld: indole-3-carboxaldehyde; MEIAA: methyl indole-3-acetate; IPA: 3-indolepropionic acid; GA15: gibberellin A15; GA3: gibberellin A3; 24-epiBL: 24-epibrassinolide; 28-norCS: 28-norcastasterone; CS: castasterone; 28-homoCS: 28-homocastasterone; TY: typhasterol; 6-deoxoCS: 6-deoxocastasterone; BL: brassinolide.

Figure 4

Identification of relative differentially expressed genes (DEGs) after 1 mg·L⁻¹ BR treatment and enrichment analysis of unigenes. (A) Unigene expression abundance of each sample. (B) Identification of DEGs between 1 mg·L⁻¹ BR and H₂O treatment. Red and green dots indicate the up- and down-regulated genes, respectively. (C) Top 20 of GO enrichment analysis of DEGs for cellular component, biological process and molecular function. (D) KEGG enrichment of DEGs. The size of dots represents the gene number, and color of dots represents the FDR value.

Figure 5

Endogenous hormone biosynthesis and signal transduction of auxin, giberellin and brassinosteroid. +u and +p mean ubiquitination and phosphorylation, respectively. Red and green frames indicate significantly upregulated and downregulated, respectively. Orange frame means gene expression was induced, but not significantly.

Figure 6

RT-qPCR validation of selected unigenes involved in xylem development. The blue and orange bars represent the normalized relative expression determined by RNA-seq and RT-qPCR, respectively. Error bars ± SE.