, Volume 249, Issue 5, pp 1449–1463 | Cite as

Effects of exogenous 24-epibrassinolide and brassinazole on negative gravitropism and tension wood formation in hybrid poplar (Populus deltoids × Populus nigra)

  • Junlan Gao
  • Min Yu
  • Shiliu Zhu
  • Liang Zhou
  • Shengquan LiuEmail author
Original Article


Main conclusion

Exogenous 24-epibrassinolide (BL) and brassinazole (BRZ) have regulatory roles in G-fiber cell wall development and secondary xylem cell wall carbohydrate biosynthesis during tension wood formation in hybrid poplar.

Brassinosteroids (BRs) play important roles in regulating gravitropism and vasculature development. Here, we report the effect of brassinosteroids on negative gravitropism and G-fiber cell wall development of the stem in woody angiosperms. We applied exogenous 24-epibrassinolide (BL) or its biosynthesis inhibitor brassinazole (BRZ) to slanted hybrid poplar trees (Populus deltoids × Populus nigra) and measured the morphology of gravitropic stems, anatomy and chemistry of secondary cell wall. We furthermore analyzed the expression levels of auxin transport and cellulose biosynthetic genes after 24-epibrassinolide (BL) or brassinazole (BRZ) application. The BL-treated seedlings showed no negative gravitropism bending, whereas application of BRZ dramatically enhanced negative gravitropic bending. BL treatment stimulated secondary xylem fiber elongation and G-fiber formation on the upper side of stems but delayed G-fiber maturation. BRZ inhibited xylem fiber elongation but induced the production of more mature G-fibers on the upper side of stems. Wood chemistry analyses and immunolocalization demonstrated that BL and BRZ treatments increased the cellulose content and modified the deposition of cell wall carbohydrates including arabinose, galactose and rhamnose in the secondary xylem. The expression of cellulose biosynthetic genes, especially those related to cellulose microfibril deposition (PtFLA12 and PtCOBL4) was significantly upregulated in BL- and BRZ-treated TW stems compared with control stems. The significant differences of G-fibers development and negative gravitropism bending between 24-epibrassinolide (BL) and brassinazole (BRZ) application suggest that brassinosteroids are important for secondary xylem development during tension wood formation. Our findings provide potential insights into the mechanism by which BRs regulate G-fiber cell wall development to accomplish negative gravitropism in TW formation.


Brassinosteroids Gelatinous layer Cellulose biosynthesis PtFLA12 PtCOBL4 Immunofluorescence Cell wall carbohydrates 



This work was supported by the National Key Research and Development Program of China (No. 2017YFD0600201), the China Postdoctoral Science Foundation (No. 2016M601995), and the Anhui Province Postdoctoral Science Foundation (No. 2017B165). We are very grateful to Mr K. Liu for suggestions in identification of tension wood experimental design and manuscript writing. We would also like to thank Robbie Lewis for his assistance with language editing.

Supplementary material

425_2018_3074_MOESM1_ESM.tif (4.8 mb)
Supplementary Fig. S1 Transverse stem sections stained with DAPI showing the distribution of cellulose. (a) Blue fluorescence is observed when sections are treated with the DAPI staining. (b) Scan mode shows the scanning image of the section. Bar: 500 μm (TIFF 4907 kb)
425_2018_3074_MOESM2_ESM.tif (3.2 mb)
Supplementary Fig. S2 Negative LM5 immunolabeling controls of the inclined stem cross-sections from trees subjected to 1 week BL or BRZ stimulation. No signal is present when the primary LM5 antibodies are omitted and sections are treated with the 488 and 633 Alexa secondary antibodies. Blue fluorescence is observed when sections are treated with the DAPI staining. Scan mode shows the scanning image of the section. Bar: 50 μm (TIFF 3268 kb)
425_2018_3074_MOESM3_ESM.tif (1.1 mb)
Supplementary Fig. S3 Negative LM10 immunolabeling controls of the inclined stem cross-sections from trees subjected to 1 week BL or BRZ stimulation. No signal is present when the primary LM10 antibodies are omitted and sections are treated with the 488 and 633 Alexa secondary antibodies. Blue fluorescence is observed when sections are treated with the DAPI staining. Scan mode shows the scanning image of the section. Bar: 50 μm (TIFF 1165 kb)
425_2018_3074_MOESM4_ESM.tif (1.2 mb)
Supplementary Fig. S4 Negative LM21 immunolabeling controls of the inclined stem cross-sections from trees subjected to 1 week BL or BRZ stimulation. No signal is present when the primary LM21 antibodies are omitted and sections are treated with the 488 and 633 Alexa secondary antibodies. Blue fluorescence is observed when sections are treated with the DAPI staining. Scan mode shows the scanning image of the section. Bar: 50 μm (TIFF 1201 kb)
425_2018_3074_MOESM5_ESM.tif (1.1 mb)
Supplementary Fig. S5 Negative 2F4 immunolabeling controls of the inclined stem cross-sections from trees subjected to 1 week BL or BRZ stimulation. No signal is present when the primary 2F4 antibodies are omitted and sections are treated with the 488 and 633 Alexa secondary antibodies. Blue fluorescence is observed when sections are treated with the DAPI staining. Scan mode shows the scanning image of the section. Bar: 50 μm (TIFF 1159 kb)
425_2018_3074_MOESM6_ESM.tif (37.8 mb)
Supplementary Fig. S6 Immunolocalizations with LM5, LM10, LM21 and 2F4 antibody (in red) and cellulose (DAPI in light blue) in transverse sections of BL or BRZ-treated stems after 1 week of OW. (a) BRZ-treated stems with LM5 labeling. (b) Mock control-treated stems with LM5 labeling. (c) BL-treated stem with LM5 labeling. (d) BRZ-treated stems with LM10 labeling. (e) Mock control-treated stems with LM10 labeling. (f) BL-treated stem with LM10 labeling. (g) BRZ-treated stems with LM21 labeling. (h) Mock control-treated stems with LM21 labeling. (i) BL-treated stem with LM21 labeling. (j) BRZ-treated stems with 2F4 labeling. (k) Mock control-treated stems with 2F4 labeling. (l) BL-treated stem with 2F4 labeling. Box indicates the areas shown in detail in a–l. cz, cambial zone; pf, phloem fiber; xy, xylem; v, xylem vessel; OW, opposite wood. Arrowhead indicates the LM5, LM10, LM21 and 2F4 labeling in these fibers. Bars: a-l = 100 μm; box in a-l = 20 μm (TIFF 38,738 kb)
425_2018_3074_MOESM7_ESM.docx (20 kb)
Supplementary Table S1 Oligonucleotide sequences used in the semi-quantitative RT-PCR analysis (DOCX 19 kb)
425_2018_3074_MOESM8_ESM.xlsx (23 kb)
Supplementary Table S2 The original data of xylary fibers length (XLSX 22 kb)
425_2018_3074_MOESM9_ESM.xlsx (18 kb)
Supplementary Table S3 The original data of xylary fibers width (XLSX 18 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Forestry and Landscape ArchitectureAnhui Agricultural UniversityHefeiPeople’s Republic of China

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