Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds

Abstract

Exposure of imbibed seeds to low temperature (typically 4 degrees C) is widely used to break seed dormancy and to improve the frequency of germination. However, the mechanism by which temperature accelerates germination is largely unknown. Using DNA microarray and gas chromatography-mass spectrometry analyses, we found that a subset of gibberellin (GA) biosynthesis genes were upregulated in response to low temperature, resulting in an increase in the level of bioactive GAs and transcript abundance of GA-inducible genes in imbibed Arabidopsis thaliana seeds. Using a loss-of-function mutant, the cold-inducible GA biosynthesis gene, AtGA3ox1, was shown to play an essential role in mediating the effect of low temperature. Besides temperature, AtGA3ox1 also is positively regulated by active phytochrome and negatively regulated by GA activity. We show that both red light and GA deficiency act in addition to low temperature to elevate the level of AtGA3ox1 transcript, indicating that multiple signals are integrated by the AtGA3ox1 gene to control seed germination. When induced by low temperature, AtGA3ox1 mRNA was detectable by in situ RNA hybridization in an additional set of cell types relative to that in red light-induced seeds. Our results illustrate that the GA biosynthesis and response pathways are activated during seed imbibition at low temperature and suggest that the cellular distribution of bioactive GAs may be altered under different light and temperature conditions.

Figure 1.

The Major GA Biosynthesis Pathway in A. thaliana. Dotted lines indicate the steps that are catalyzed by GA 20-oxidase. CPS, ent-copalyl diphosphate synthase; ent-CDP, ent-copalyl diphosphate; GGDP, geranylgeranyl diphosphate; KAO, ent-kaurenoic acid oxidase; KO, ent-kaurene oxidase; KS, ent-kaurene synthase.

Figure 2.

Expression of GA Biosynthesis Genes at 4 and 22°C in Dark-Imbibed Seeds. (A) Diagram showing light and temperature treatments. Wild-type seeds were imbibed in the dark and then irradiated with an FR light pulse (hatched box). The seeds then were incubated in the dark at 22 or 4°C until harvested (see below). The triangle indicates the start time of imbibition. (B) Transcript accumulation of GA biosynthesis genes as determined by QRT-PCR. Wild-type seeds were imbibed as shown in (A) and harvested 48 h after FR light pulse. Values are means with standard errors from three measurements. Values for AtGA3ox1 and AtGA3ox2 are multiplied by 10 for clarity. (C) Time-course changes in transcript accumulation of AtGA3ox1 and AtGA3ox2 genes as determined by QRT-PCR. Wild-type seeds were imbibed as shown in (A). Start of imbibition was set as 0 h. Experiments were repeated twice with similar results. Data from one of the replicates are shown.

Figure 3.

Endogenous GA Levels at 4 and 22°C in Dark-Imbibed Seeds. As illustrated in Figure 2A, wild-type seeds were imbibed in the dark, irradiated with an FR light pulse, and then incubated in the dark at 22 or 4°C for 96 h until harvested. Data are means with standard errors from three measurements using independent seed batches unless otherwise indicated. The asterisks indicate means from two measurements (with similar results). Note that the y axis scale for 13-nonhydroxylated GAs (top; GA_{12,15,24,9,4,34,51}) and that for 13-hydroxylated GAs (bottom; GA_{53,44,19,20,1,8,29}) are different for clarity. gdw, grams dry weight; n.d., detectable but could not be quantified because of comigration of impurities.

Figure 4.

Expression of GA-Inducible Genes at 4 and 22°C in Dark-Imbibed Seeds. (A) GA responsiveness of genes used in this study. The ga1-3 mutant seeds were imbibed for 2 d at 4°C in the dark and then incubated at 22°C for 1 d under continuous white light as described by Ogawa et al. (2003). The seeds then were exposed to varying concentrations of GA₄ for 6 h. At-EXP1 and At-EXP2 encode expansins (http://www.bio.psu.edu/expansins/arabidopsis.htm). AtCP1 is a gene for putative Cys proteinase. At-XTH5 and At-XTH31 code xyloglucan endotransglycosylase/hydrolase (Rose et al., 2002). Asterisk, the transcript level at 50 μM GA₄ was set as 100. (B) Transcript levels of GA-upregulated genes at 4 and 22°C. Wild-type seeds were imbibed at 4 or 22°C under the light conditions depicted in Figure 2A. The transcript abundance was determined 48 h after the start of imbibition. Values are means with standard errors from three replicates. (C) Effect of the ga4-2 mutation on transcript levels of GA-upregulated genes at 4 and 22°C. Wild-type and ga4-2 mutant seeds were imbibed at 4 or 22°C under the light conditions depicted in Figure 2A. The transcript abundance was determined 48, 72, and 96 h after the start of imbibition. Experiments were repeated twice with similar results. Data from one of the replicates are shown.

Figure 5.

Effects of Cold Treatment on Germination of Wild-Type and ga4-2 Mutant Seeds. (A) Diagram showing temperature and light conditions. Wild-type and ga4-2 mutant seeds were imbibed in the dark, irradiated with an FR light pulse (hatched box), and then incubated in the dark at 4°C for 48 h before being placed at 22°C for 72 h in the dark to allow germination (+ cold). As a control, the incubation at 4°C for 48 h was omitted (− cold). Seeds also were imbibed at 22°C under continuous white light. The triangle indicates the start time of imbibition. (B) Germination rates under conditions described in (A). Asterisk, no germination (0%).

Figure 6.

Effects of R light and GA deficiency on AtGA3ox1 Transcript Accumulation at 4°C. Schematic diagram is shown at the top of each panel for light and temperature conditions. Graphs below the diagram show relative AtGA3ox1 mRNA levels. Values are means with standard errors from three replicates. (A) Effect of an R light pulse during cold treatment. Wild-type seeds were imbibed in the dark, irradiated with an FR light pulse (hatched box), and then incubated in the dark at 4°C for 96 h. During the dark incubation at 4°C, the seeds were treated with a brief R light pulse (dotted box) 3 or 6 h before being harvested. (B) Effect of the ga1-3 mutation at 4°C. Wild-type and ga1-3 mutant seeds were imbibed in the dark, irradiated with an FR light pulse, and then incubated in the dark at 4°C for 96 h.

Figure 7.

In Situ Hybridization Analysis of AtGA3ox1 mRNA. (A) Schematic drawing of a longitudinal section. ale, aleurone; cot, cotyledon; HY, hypocotyl; RA, radicle; SA, shoot apical meristem; SC, seed coat. (B) Schematic drawing of a transverse section. ale, aleurone; cor, cortex; cot, cotyldon; end, endodermis; epi, epidermis; PV, provasculature; SC, seed coat. (C), (D), and (E) Longitudinal (left) and transverse (center) sections of imbibed seeds were hybridized with digoxigenin-labeled antisense AtGA3ox1 RNA probe. Magnified views (right) highlight the aleurone layer (the area surrounded by a box in (C) was magnified). Wild-type seeds were incubated at 22°C in (C) or 4°C in (D) for 96 h after FR light pulse as depicted in Figure 2A. All incubations were performed at 22°C in (E) as described by Yamaguchi et al. (1998). Wild-type seeds were incubated at 22°C for 24 h after FR light pulse and then harvested 4 h after the R light pulse. Bars = 50 μm.

Figure 8.

Summary of the Regulation of AtGA3ox1 and AtGA3ox2 during Seed Germination. Thin arrows indicate positive regulation. Feedback inhibition is shown by the T bar. Conversion of GA₉ into bioactive GA₄ is indicated by the thick arrow. Regulation by phytochrome and by GA activity has been reported previously (Chiang et al., 1995; Yamaguchi et al., 1998).