Regulation of gibberellin 20-oxidase and gibberellin 3beta-hydroxylase transcript accumulation during De-etiolation of pea seedlings

Abstract

Gibberellin (GA) 20-oxidase (GA 20-ox) and GA 3beta-hydroxylase (GA 3beta-hy) are enzymes that catalyze the late steps in the formation of active GAs, and are potential control points in the regulation of GA biosynthesis by light. We have investigated the photoregulation of the GA 20-ox and GA 3beta-hy transcript levels in pea (Pisum sativum L.). The GA 20-ox transcript level was higher in light-grown seedlings than in etiolated seedlings, whereas GA 3beta-hy mRNA accumulation was higher in etiolated seedlings. However, transfer of etiolated seedlings to light led to a 5-fold increase in the expression of both transcripts 4 h after transfer. GA 20-ox mRNA accumulation is regulated by both phytochromes A and B. Transfer to light also resulted in a 6-fold decrease in GA(1) levels within 2 h. These results suggest that the light-induced drop in GA(1) level is not achieved through regulation of GA 20-ox and GA 3beta-hy mRNA accumulation. The application of exogenous GA(1) to apical buds of etiolated seedlings prior to light treatments inhibited the light-induced accumulation of both GA 20-ox and GA 3beta-hy mRNA, suggesting that negative feedback regulation is an important mechanism in the regulation of GA 20-ox and GA 3beta-hy mRNA accumulation during de-etiolation of pea seedlings.

Figure 1

Simplified 13-hydroxylation pathway of GA biosynthesis in vegetative tissues of pea. The steps affected by mutations at the LS, LH, NA, LE, and SLN loci are indicated. GGDP, Geranylgeranyldiphosphate; CDP, copalyl diphosphate.

Figure 2

GA 20-ox and GA 3β-hy transcript accumulation in light- and dark-grown seedlings. Seedlings were grown in constant light (L) or darkness (D) for 6 d. Buds (Bd) and stems (St) were harvested from dark-grown seedlings. Apices (Ap), first expanded nodes (Lf), and stems were harvested from light-grown seedlings. Total RNA was prepared and 5 (CAB) or 30 (GA 20-ox and GA 3-βhy) μg per lane was used for RNA-blot analysis. Blots were hybridized with probes for chlorophyll a/b-binding protein (CAB) or GA 20-oxidase (GA 20-ox) and GA 3β-hydroxylase (GA 3β-hy).

Figure 3

GA 20-ox and GA 3β-hy transcript accumulation during de-etiolation. Seedlings were grown in constant darkness for 6 d before transfer to constant light (L). Control seedlings were maintained in constant darkness (D). A, Buds and stems were harvested at 0, 2, 4, and 24 h after transfer to light and RNA-blot analysis was performed as described in Figure 2. As a loading control, blots were rehybridized with an oligo to the 18S rRNA (18S rRNA). B, RNA-blot analysis of buds were harvested 0, 2, 4, and 24 h after transfer to light, and hybridization signals were quantitated with a phosphor imager. Each data set was normalized to the maximum signal. The experiment was repeated at least three times, and bars indicate the se of the mean. ○, Constant light; ●, constant darkness.

Figure 4

Phytochrome mediation of GA 20-ox and GA 3β-hy transcript accumulation in wild-type and phytochrome-deficient mutants. Seedlings were grown in continuous darkness (D) for 6 d. After irradiation with 10 min of red light (R), 10 min of red light followed by 10 min of far-red light (R+FR), or 10 min of far-red light (FR), seedlings were returned to the dark. Buds were harvested at 5 h after light treatment. Control seedlings were maintained in constant darkness. RNA-blot analysis was performed as described in Figure 2. A, RNA-blot analysis of cv Alaska seedlings. B, Quantitation of hybridization signal from RNA-blot analysis for the GA 20-ox transcript. Genotypes used were cv Torsdag (WT), fun1-1 (phyA- deficient), and lv-5 (phyB-deficient). Each data set was normalized to the maximum signal (red-light induction of the wild type, cv Torsdag). Experiments were repeated five times for cv Torsdag, three times for fun1-1 and lv-5. Error bars indicate the se of the mean. Black bars, Continuous darkness; white bars, red light; shaded bars, red plus far-red light; hatched bars, far-red light.

Figure 5

Endogenous GAs in pea epicotyls during de-etiolation. 13-Hydrodroxylated GAs were determined in 6-d-old etiolated seedlings at 0, 2, 4, and 24 h after transfer to light (panels on the right side) and in etiolated seedlings maintained in darkness (panels on the left side) for the same period of time. Top panels show the levels of the inactivated GAs GA₈ (▾), and GA₂₉ (▿). Bottom panels show the levels of precursors of GA₁, GA₄₄ (▵), GA₅₃ (○), GA₁₉ (▴), and GA₂₀ (●), and GA₁ (▪). Three independent experiments were performed. Error bars are the se of the mean.

Figure 6

Effect of GA₁ application on light regulation of GA 20-ox and GA 3β-hy transcript accumulation. A, Effect of 30 min of exposure to dim-green safelight (G) on transcript accumulation in apical buds. B, Exogenous application of GA₁ on apical buds of 6-d-old etiolated seedlings 30 min prior transfer to light. Light treatment and tissue harvestings were identical to Figure 3A. C, Two micrograms of GA₁ was applied to the apical buds of 6-d-old etiolated seedlings 30 min prior to the red light pulse. Light treatments were identical to Figure 5. RNA-blot analysis was performed as described in Figure 2. D, Darkness; L, light; R, red light.