High planting density induces the expression of GA3-oxidase in leaves and GA mediated stem elongation in bioenergy sorghum

Abstract

The stems of bioenergy sorghum hybrids at harvest are > 4 m long, contain > 40 internodes and account for ~ 80% of harvested biomass. In this study, bioenergy sorghum hybrids were grown at four planting densities (~ 20,000 to 132,000 plants/ha) under field conditions for 60 days to investigate the impact shading has on stem growth and biomass accumulation. Increased planting density induced a > 2-fold increase in sorghum internode length and a ~ 22% decrease in stem diameter, a typical shade avoidance response. Shade-induced internode elongation was due to an increase in cell length and number of cells spanning the length of internodes. SbGA3ox2 (Sobic.003G045900), a gene encoding the last step in GA biosynthesis, was expressed ~ 20-fold higher in leaf collar tissue of developing phytomers in plants grown at high vs. low density. Application of GA3 to bioenergy sorghum increased plant height, stem internode length, cell length and the number of cells spanning internodes. Prior research showed that sorghum plants lacking phytochrome B, a key photoreceptor involved in shade signaling, accumulated more GA1 and displayed shade avoidance phenotypes. These results are consistent with the hypothesis that increasing planting density induces expression of GA3-oxidase in leaf collar tissue, increasing synthesis of GA that stimulates internode elongation.

Figure 1

Plant spacing alters sorghum stem growth and morphology. (a) Photograph of stems of bioenergy sorghum plants grown for 60 days in the field at 1 m or 0.15 m spacing, (b) average height of plants grown at 1 m and 0.15 m spacing. Average length (c) and diameter (d) of elongated internodes of plants grown at 1 m or 0.15 m spacing. Asterisks indicate two-tailed P value; ****P < 0.0001, ***P = 0.0007, **P = 0.0019, by Welch’s t test (n = 5). Error bars: SEM.

Figure 2

Plant spacing alters sorghum stem internode growth during development. Average internode lengths (cm) (a) and diameters (mm) (b) of 9 phytomers of bioenergy sorghum grown at 1 m and 0.15 m spacing for 60 days (DAE) in the field. Phytomer 12 is located at the base of the stem. Asterisks indicate two-tail Welch’s t test, ****P < 0.0001, ***P < 0.001, **P < 0.005, *P < 0.05, by one-way ANOVA (n = 5). Error bars: SEM.

Figure 3

Plant spacing affects the number and length of cells in sorghum internodes. (a) The average number of cells spanning fully elongated internodes of phytomer 7 and (b) average length of cells in phytomer 7 of plants grown at 1 m and 0.15 m spacing. Asterisks indicate two-tailed P value, ****P < 0.0001, by Welch’s t test [n = 96 (1 m), n = 84 (0.15 m)]. Error bars: SEM.

Figure 4

GA3 alters sorghum internode length, cell length and number. (a) Photograph of sorghum stems from control plants 14 days after removal of the leaf sheath (LS) from phytomer 7 (− LS) and plants treated with GA3 after LS removal (− LS, 1% GA3) (right). (b) Average length of internodes of control (blue line), and plants treated with 1% GA3 (green line), 1% PAC (red line) or both compounds (yellow line). Site of LS removal and GA3/PAC application is marked (solid inverted triangle). (c) Micrographs of longitudinal sections from the middle section of fully elongated internode from phytomer 7 (solid star) stained for cellulose. (d) Average length of cells and (e) average number of cells spanning the length of internode 7 following 14 days of treatment. One-way ANOVA, followed by Tukey comparison test, indicated significant differences between control, + PAC and + GA conditions with P < 0.0001 (n = 291). Different letters indicate significance. Error bars: SEM.

Figure 5

Relative expression of SbGA3ox2 in sorghum grown at 0.15 m (a) and 1 m (b) spacing. Tissues were collected from the shoot apex (Apex), mid leaf blade (LB center), base of the leaf blade (LB base), tissue between the leaf blade and leaf sheath (LB:LS collar), mid leaf sheath (LS center), base of the leaf sheath (LS base), leaf sheath collar (LS collar), stem nodal plexus, internode, and pulvinus of phytomers (Phy) 3–8. Relative expression is shown in bar graphs (leaf blade = green, leaf sheath = blue, stem = brown). Expression values are the average of three biological replicates. Error bars: SEM. Photograph and diagram of the development of sorghum phytomer tissues is shown in Supplementary Figure S4.

Figure 6

GA3ox2 is differentially expressed at high vs. low plant spacing. (a) Differential expression of SbGA3ox2 in the leaf blade (LB) and leaf blade/leaf sheath collar tissue (LB:LS collar) of phytomers 3–8 of plants grown at 0.15 m spacing (high density) and 1 m spacing (low density). (b) Differential expression of SbGA3ox2 in the stem apex, nodal plexus, internode and pulvinus of plants grown at 0.15 m and 1 m spacing. Expression values are the average of three biological replicates. Differences in expression were analyzed by one-way ANOVA followed by Tukey comparison test. ****P < 0.0001, **P < 0.005. Error bars: SEM.