Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice

Abstract

Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our previous studies showed that three major quantitative trait loci (QTL) regulate deepwater-dependent internode elongation in deepwater rice. In this study, we investigated the age-dependent internode elongation in deepwater rice. We also investigated the relationship between deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by physiological and genetic approach using a QTL pyramiding line (NIL-1 + 3 + 12). Deepwater rice did not show internode elongation before the sixth leaf stage under deepwater condition. Additionally, deepwater-dependent internode elongation occurred on the sixth and seventh internodes during the sixth leaf stage. These results indicate that deepwater rice could not start internode elongation until the sixth leaf stage. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the phytohormone contents showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice. Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in deepwater rice. On the contrary, GA feeding mimicked internode elongation under ordinary growth conditions. However, mutations in GA biosynthesis and signal transduction genes blocked deepwater-dependent internode elongation. These data suggested that GA biosynthesis and signal transduction are essential for deepwater-dependent internode elongation in deepwater rice.

Keywords: gibberellin.

Figure 1

Diagram and gross morphology of general cultivated rice, NIL-1 + 3 + 12 and deepwater rice before and after submergence. Diagram of the general cultivated rice T65 (a) and deepwater rice C9285(c) under shallow water (SW) and deepwater (DW) conditions. Arrowhead represents water level. (b) Node and internode components in general cultivated rice. Internode elongation was not induced in general cultivated rice under DW conditions. (d) Node and internode components in deepwater rice. Internode elongation induced in deepwater rice under DW conditions. (e) Gross morphology showing increased plant height before (SW) and after 7 d of DW treatment in T65, NIL-1 + 3 + 12 and C9285. Graphical genotypes are shown below the plant. Green solid rectangle indicates the T65 fragment and red solid rectangle indicates C9285 fragments. Yellow open rectangle shows the base of the internode. (f) Magnified base of internode shown in (e). Longitudinal sections showing only the basal part of the plant composed of the nodes and internodes. Pith cavities (*) are present in NIL-1 + 3 + 12 under DW and C9285 under SW and DW conditions. Scale bars: 5 mm in (f), 5 cm in (e).

Figure 2

Leaf age-dependent internode elongation during submergence in T65, NIL-1 + 3 + 12 and C9285. (a) Diagram represents the longitudinal section of C9285 to show the internode position. (b) Deepwater response of T65 (green rectangle), NIL-1 + 3 + 12 (blue rectangle) and C9285 (red rectangle) was represented based upon comparison of the internode length during the three- to eight-leaf stage (3–8 LS) of the plant under shallow water (SW) and 7 d of deepwater (DW) treatment. X-axis indicates the position of the internode counted from the base to top, as shown in (a). Vertical bars indicate SD of the mean (n = 5–10) of three independent experiments.

Figure 3

Elongation pattern of internode and leaves in C9285 after submergence. (a–c) Diagram of the three- to eight-leaf stage (3–8 LS) of C9285 under shallow water (SW) and deepwater (DW) conditions, showing gross morphology of the plant (a) and anatomical structure of the node and internode (b, c). (a) Gross morphology in the 3–8 LS of C9285 grown under SW conditions. (b, c) Position of internodes and leaves under SW (b) and DW (c) conditions. Only one leaf (marked in green L3-L8) represents the leaf stage. LS; leaf stage. L; leaf.

Figure 4

Gibberellin (GA) feeding and elongation analysis in T65, NIL-1 + 3 + 12 and C9285. (a) Gross morphology of T65, NIL-1 + 3 + 12 and C9285 after treatment in 14 d shallow water (SW) and deepwater (DW), 3 d uniconazole (Uni) and 14 d DW (DW + Uni) treatment and 14 d 10⁻⁵ m of GA₃ treatment (GA). (b, c) Plant height (b) and total internode length (c) in T65, NIL-1 + 3 + 12 and C9285 under SW (green bar), DW (blue bar), DW + Uni (red bar) and GA treatment (yellow bar) (n = 10); 3 independent experiments. Vertical bars indicated SE. Scale bars are 1 m in (a). Asterisks (*) indicate significant differences at P < 0.05 (Student's t-test).

Figure 5

Gross morphology and deepwater response of mutants and mutant pyramiding lines after 7 d of deepwater treatment. (a) Gross morphology of GA biosynthesis (d18-dy), GA signal transduction (gid1-7, gid1-8), DELLA (slr1-dy), and F-box (gid2-2, gid2-5) mutants and mutant pyramiding (MP) lines after 7 d of shallow water (SW) and deepwater (DW) treatments. (b, c) Plant height (b) and total internode length (c) after 7 d of SW and DW conditions were determined (n = 8–10). Vertical bars indicate SE. Asterisks represents significant differences; *P < 0.05 and **P < 0.01 (Student's t-test). ND in (c) means not detected. Scale bar is 1 m in (a).

Figure 6

Measurement of endogenous hormone concentrations under deepwater treatment at 7 LS in T65, NIL-1 + 3 + 12 and C9285. (a) Diagram represents the late step of GA₁ and GA₄ biosynthesis pathway to show enzymes and parallel relationships. (b–g) Endogenous levels of gibberellin (GA₁) (d) and its precursors GA₅₃ (b) and GA₂₀ (c), as well as GA₄ (g) and its precursors GA₁₂ (e) and GA₉ (f), were analysed after 0, 12, 24 and 48 h of deepwater treatment in T65 (green closed circle), NIL-1 + 3 + 12 (blue closed triangle) and C9285 (red closed rectangle) using UPLC-MS/MS (n = 4–7). Vertical bars indicate SD.

Figure 7

Semi-qRT-PCR and qRT-PCR analysis of GA biosynthesis gene expression analysis. (a) mRNA was obtained from the 7 LS of T65 and C9285 after 0, 12 and 24 h of submergence. PCR was performed for 30 and 35 cycles for GA biosynthesis and signal transduction genes and also 30 and 35 cycles for OsActin1. (b) qRT-PCR was performed using the SsoAdvanced SYBR Green Supermix with Step One Plus PCR system. Representation of the relative expression levels of GA20ox2 gene was normalized to the expression level of ubiquitin. The relative value of OsGA20ox2 expression in T65 at 0 h was 1. Vertical bars indicate SD of the mean of four replicates.

Figure 8

Schematic diagram representing three hypotheses of the IM activation mechanism. (a) The image shows a longitudinal section of deepwater rice under shallow water (SW) and deepwater (DW) conditions. (b) Image represents hypothesis 1, where IM formation of deepwater rice started at the sixth internode in the 6 LS. (c) Image represents hypothesis 2, where IM formation initiated upon submergence. (d) Image represents hypothesis 3, where IM formed at every internode but remained dormant until submergence. The old internode disappeared in response to DW. Active (elongation ability) IM is indicated in pink. The dormant or non-elongated (absent elongation ability) IM is indicated in orange.