Rice dwarf mutant d1, which is defective in the alpha subunit of the heterotrimeric G protein, affects gibberellin signal transduction

Abstract

Previously, we reported that the rice dwarf mutant, d1, is defective in the alpha subunit of the heterotrimeric G protein (Galpha). In the present study, gibberellin (GA) signaling in d1 and the role of the Galpha protein in the GA-signaling pathway were investigated. Compared with the wild type, GA induction of alpha-amylase activity in aleurone cells of d1 was greatly reduced. Relative to the wild type, the GA(3)-treated aleurone layer of d1 had lower expression of Ramy1A, which encodes alpha-amylase, and OsGAMYB, which encodes a GA-inducible transcriptional factor, and no increase in expression of Ca(2 +)-ATPase. However, in the presence of high GA concentrations, alpha-amylase induction occurred even in d1. The GA sensitivity of second leaf sheath elongation in d1 was similar to that of the wild type in terms of dose responsiveness, but the response of internode elongation to GA was much lower in d1. Furthermore, Os20ox expression was up-regulated, and the GA content was elevated in the stunted internodes of d1. All these results suggest that d1 affects a part of the GA-signaling pathway, namely the induction of alpha-amylase in the aleurone layer and internode elongation. In addition, a double mutant between d1 and another GA-signaling mutant, slr, revealed that SLR is epistatic to the D1, supporting that the Galpha protein is involved in GA signaling. However, the data also provide evidence for the presence of an alternative GA-signaling pathway that does not involve the Galpha protein. It is proposed that GA signaling via the Galpha protein may be more sensitive than that of the alternative pathway, as indicated by the low GA responsiveness of this Galpha-independent pathway.

Figure 1

(A) Phenotypes of d1 (Center), wild-type (Left), and d18 (Right) plants at 6 weeks. (B) Comparison of internode lengths of d1 (Center), wild-type (Left), and d18 (Right). Data are average internode lengths of 10 plants.

Figure 2

(A) GA induction of α-amylase activity in d1 and wild type. Embryoless half seeds were incubated for 4 days at 30°C in culture medium containing various concentrations of GA₃ and then assayed for α-amylase activity as described in Materials and Methods. (B) RNA gel blot analyses of GA-inducible genes in aleurone cells. Fifty embryoless half seeds were incubated at 30°C for 0, 6, 12, 24, 36, and 48 h in culture medium containing 10⁻⁷ M GA₃. Total RNA was extracted and analyzed by RNA gel blotting. Ten micrograms of total RNA for detection of GAMYB and Ca^{2 +}-ATPase and 2 μg for detection of Ramy1 was loaded onto each lane. The gel was stained with ethidium bromide (EtBr).

Figure 3

Elongation of the second leaf sheath in response to GA treatment in d1 (triangle) and the wild plant (circle) pretreated with (▵, ○) and without uniconazol (▴, ●). LS_max/LS₀ is the ratio of the second leaf sheath length with GA₃ treatment at 10⁻⁴ M (LS_max) to the length without GA₃ treatment (LS₀). n = 10.

Figure 4

(A) Internode elongation in response to GA treatment in d1 and the wild type. (B) Expression of Os20ox in the first to third internodes of d1 and first to fifth internodes of wild-type plants. Total RNA was extracted from each internode and analyzed by RNA gel blotting. Ten micrograms of total RNA was loaded onto each lane, and the gel was stained with ethidium bromide (EtBr).

Figure 5

Epistatic analysis of d1 and slr mutants. (A) A plate assay of α-amylase induction. Embryoless half seeds of the wild type (Left), d1, slr, and d1/slr (right) were sterilized, washed, and incubated on starch plates without GA₃ for 4 days at 30°C. (B) Two-week-old seedlings of the wild type (W), d1, slr, and d1/slr.

Figure 6

Diagram summarizing the two proposed GA-signaling pathways in rice. (Model A) Gα works in high sensitive GA receptor system. There may be an alternative GA receptor system for low sensitivity to GA that does not involve Gα. High sensitive α-amylase induction and internode elongation by GA may be mediated mainly through the high sensitive reception system (red), whereas leaf sheath elongation may be through the low sensitive reception system (blue). (Model B) Gα works in a pathway that regulates the GA signaling. α-Amylase induction and internode elongation may be regulated mainly by this pathway to result in high sensitivities to GA (red), whereas leaf sheath elongation may be not regulated by this pathway to result in low sensitivity to GA (blue). The SLR protein works at the downstream site of these two pathways.