Exogenous GA₃ Application Enhances Xylem Development and Induces the Expression of Secondary Wall Biosynthesis Related Genes in Betula platyphylla

Abstract

Gibberellin (GA) is a key signal molecule inducing differentiation of tracheary elements, fibers, and xylogenesis. However the molecular mechanisms underlying the effect of GA on xylem elongation and secondary wall development in tree species remain to be determined. In this study, Betula platyphylla (birch) seeds were treated with 300 ppm GA₃ and/or 300 ppm paclobutrazol (PAC), seed germination was recorded, and transverse sections of hypocotyls were stained with toluidine blue; the two-month-old seedlings were treated with 50 μM GA₃ and/or 50 μM PAC, transverse sections of seedling stems were stained using phloroglucinol-HCl, and secondary wall biosynthesis related genes expression was analyzed by real-time quantitative PCR. Results indicated that germination percentage, energy and time of seeds, hypocotyl height and seedling fresh weight were enhanced by GA₃, and reduced by PAC; the xylem development was wider in GA₃-treated plants than in the control; the expression of NAC and MYB transcription factors, CESA, PAL, and GA oxidase was up-regulated during GA₃ treatment, suggesting their role in GA₃-induced xylem development in the birch. Our results suggest that GA₃ induces the expression of secondary wall biosynthesis related genes to trigger xylogenesis in the birch plants.

Keywords: Betula platyphylla SUK; GA3; secondary wall biosynthesis related genes; seed germination; xylem development.

Figure 1

The germination of birch seeds treated with GA₃ and/or paclobutrazol (PAC). (A) Germination percentage (%) of birch seeds treated with GA₃ and/or PAC; (B) Germination energy (%) of birch seeds treated with GA₃ and/or PAC; (C) Germination time (day) of birch seeds treated with GA₃ and/or PAC; (D) Growth of birch seedlings; (E) Hypocotyl height of birch seedling; and (F) Fresh weight of birch seedlings. Lower case letter indicates p < 0.05.

Figure 1

The germination of birch seeds treated with GA₃ and/or paclobutrazol (PAC). (A) Germination percentage (%) of birch seeds treated with GA₃ and/or PAC; (B) Germination energy (%) of birch seeds treated with GA₃ and/or PAC; (C) Germination time (day) of birch seeds treated with GA₃ and/or PAC; (D) Growth of birch seedlings; (E) Hypocotyl height of birch seedling; and (F) Fresh weight of birch seedlings. Lower case letter indicates p < 0.05.

Figure 2

Transverse sections of 15-day-old birch seedlings germinating under GA₃ and/or PAC. The hypocotyl base was sectioned 15 days after germination. (A) Toluidine blue staining analysis of the differentiation of xylem. xy, xylem cell. Bars = 50 μm; (B) The diameter of the hypocotyl (μm); and (C) The number of xylem cell. Lower case letter indicates p < 0.05.

Figure 3

Phenotypic changes of 2-month-old birch seedlings under GA₃ and/or PAC.

Figure 4

Transverse sections of 2-month-old birch seedlings and the ratio of xylem area to total area under GA₃ and/or PAC treatments. (A–D) GA and/or PAC treatment for 3 days; (E–H) GA and/or PAC treatment for 7 days; (I–L) GA and/or PAC treatment for 14 days; and (M–P) GA and/or PAC treatment for 21 days. (A,E,I,M) GA treatment; (B,F,J,N) GA + PAC treatment; (C,G,K,O) PAC treatment; and (D,H,L,P) water treatment (control). Phloroglucinol–HCl was used to stain lignin to highlight xylem vessels and fibers. Bars = 1 mm; (Q) The ratio of xylem area to total area in birch plants under GA₃, GA₃ + PAC, PAC and control for 3, 7, 14 or 21 days. Error bars were obtained from multiple replicates of the real-time PCR. Lower case letter indicates p < 0.05.

Figure 4

Transverse sections of 2-month-old birch seedlings and the ratio of xylem area to total area under GA₃ and/or PAC treatments. (A–D) GA and/or PAC treatment for 3 days; (E–H) GA and/or PAC treatment for 7 days; (I–L) GA and/or PAC treatment for 14 days; and (M–P) GA and/or PAC treatment for 21 days. (A,E,I,M) GA treatment; (B,F,J,N) GA + PAC treatment; (C,G,K,O) PAC treatment; and (D,H,L,P) water treatment (control). Phloroglucinol–HCl was used to stain lignin to highlight xylem vessels and fibers. Bars = 1 mm; (Q) The ratio of xylem area to total area in birch plants under GA₃, GA₃ + PAC, PAC and control for 3, 7, 14 or 21 days. Error bars were obtained from multiple replicates of the real-time PCR. Lower case letter indicates p < 0.05.

Figure 5

Expression analysis of the genes in birch plants under GA₃ and/or PAC. These genes include NAC, MYB, PAL, CESA, and GA20ox. Error bars were obtained from multiple replicates of the real-time PCR. Lower case letter indicates p < 0.05.

Figure 5

Expression analysis of the genes in birch plants under GA₃ and/or PAC. These genes include NAC, MYB, PAL, CESA, and GA20ox. Error bars were obtained from multiple replicates of the real-time PCR. Lower case letter indicates p < 0.05.