Ethylene-signaling mutants have altered Glc response during germination. Germination kinetics was determined in Col-0 or ethylene-signaling mutants ctr1-1, etr1-1, or ein2. Plant material was prepared as described in Figure 1, except that no sugar (Murashige and Skoog) or high concentration of Glc (indicated in the figure) was added to the Murashige and Skoog plate. Germination kinetics was determined by measuring the time of radicle emergence after transfer to constant light and 24°C.

Proposed models for Glc-, ABA-, and Glc/ABA-signaling pathways in controlling seed germination and seedling development. ABA2 and ABA3 affect the plant response to Glc during both germination and seedling development due to their effects on cellular ABA concentration. Both ABI4 and CTR1 are involved in plant response to high levels of exogenous Glc during seedling development but not during seed germination. In contrast, ABI1, ABI4, and CTR1 are involved in plant response to exogenous ABA during seed germination but not during seedling development (except ABI1). HXK appears to be a positive regulator for Glc-induced rescue of seed germination inhibited by ABA. However, plants do not develop further unless either ABI1 or ABI4 is defective.

Sugar sensing is affected by developmental program, because previously known sugar-insensitive mutants abi4 and gin2 are sensitive to a high level of Glc during germination. Plant material was prepared as described in Figure 1, except that no sugar (Murashige and Skoog), or Glc (concentration indicated in the figure) or 1 μmABA was added to the Murashige and Skoog plates. Germination kinetics was determined by measuring the time of radicle emergence after transfer cultures to constant light and 24°C. A, Compared with the background ecotype Col-0, whereas ABA biosynthetic mutants aba2-1 and aba3-1 are less sensitive to Glc, abi4 is less sensitive to ABA. B, HXK AtHXK1 is not involved in sensing either Glc or ABA during seed germination. Compared with the background ecotype, there is no apparent difference in germination kinetics in either HXK (AtHXK1) loss-of-function mutant gin2 (in Ler background) or AtHXK1 overexpresser 35SAtHXK1 (in BE background).

Osmotic effect does not mimic the inhibitory effect of Glc on seed germination. Plant material was prepared as described in Figure 1, except that no sugar (Murashige and Skoog) or various concentrations of either mannitol or Glc (indicated in the legend) were added to the Murashige and Skoog plates. Germination kinetics was determined by measuring the time of radicle emergence after transfer cultures to constant light, 24°C. A, Compared with mannitol, Glc is more effective in delaying seed germination. B, ABA biosynthetic mutants aba2-1 and aba3-1 and ABA perception mutant abi4 are less sensitive to 333 mm mannitol than the background ecotype Col-0. C, HXK does not appear to be involved in osmotic sensing. Compared with the background ecotype, there is no apparent difference in germination kinetics in response to mannitol in either HXK (AtHXK1) loss-of-function mutant gin2 (in Ler background) or AtHXK1 overexpresser 35SAtHXK1 (in BE background).

Arabidopsis can sense a low level of exogenous Glc during seed germination. Arabidopsis seeds were surface-sterilized and water-imbibed in the dark for 3 d at 4°C. Seeds were transferred to 1× Murashige and Skoog plates containing B5 vitamins, 0.05% (w/v) MES (pH 5.7), and 0.7% (w/v) phytagar (Invitrogen) without sugar (Murashige and Skoog) or with Glc (concentration indicated in the figure). The plant material was incubated in the dark at 4°C for 3 d to break dormancy and was then transferred to light at 24°C. Germination kinetics was determined by measuring the time of radicle emergence after transfer to constant light and 24°C. A, ABA biosynthetic mutants aba2-1 and aba3-1 and ABA perception mutant abi4 are less sensitive to Glc than their background ecotype Col-0. B, Compared with the background ecotype, there were no apparent changes in Glc sensitivity in either HXK (AtHXK1) loss-of-function mutant gin2 (in Ler background) or AtHXK1 overexpresser 35SAtHXK1 (in BE background).

Glc partially relieves ABA-induced inhibition of seed germination but not subsequent seedling development. A, Compared with the background ecotype Col-0, Glc rescue of ABA-induced inhibition of seed germination is not altered in either ABA biosynthetic mutants aba2-1 and aba3-1 or ABA perception mutant abi4. However, HXK seems to be a positive regulator for this rescue, because faster germination kinetics was observed in AtHXK1 overexpresser 35SAtHXK1 (in BE background). Plant material was prepared as described in Figure 1, except that 167 mm Glc and 1 μm ABA were added to the Murashige and Skoog plates. Germination kinetics was determined by measuring the time of radicle emergence after transfer cultures to constant light and 24°C. B, Although Glc can rescue the inhibition of seed germination induced by ABA, subsequent seedling development only occurred in ABA-insensitive mutants (abi1 and abi4). Seedlings of aba2-1 (Col-0 background), abi4 (Col-0 background), and abi1 (Ler background) were grown for 21 d in constant white light (90 μE m^–2 s^–1) at 25°C. Plant material was prepared as described in Figure 1, except that either 333 mm Glc, 1 μm ABA, or a combination of 167 mm Glc and 1 μm ABA was added to the Murashige and Skoog plates.

Developmental program-dependent induction of ABA2 and ABI4 by Glc. A, Imbibition exerts an opposing effect on the expression of ABA2 and ABI4. Col-0 seeds were surface disinfected and immediately collected (Dry seed), imbibed for 3 d in water at 4°C and collected, or imbibed at 4°C water for 3 d and incubated in 4°C Glc-free 1× Murashige and Skoog medium for another 3 d and then collected. ABA2 expression was determined by RT-PCR following the manufacturer's protocol (Panvera, Madison, WI), and ABI4 was determined by RNA gel-blot analysis. Five micrograms of RNA was loaded in each lane. RT-PCR of ubiquitin expression and ethidium bromide staining of rRNA bands are shown for quality and loading controls, respectively. B, RNA gel-blot analysis of ABA2 expression during germination. Col-0 seeds were surface disinfected, imbibed 3 d in water at 4°C and another 3 d at 4°C in Glc-free 1× Murashige and Skoog liquid medium containing B5 vitamins and MES (pH 5.7) without sugar (control) or with Glc and/or ABA (concentration indicated in the figure). After transferring cultures to constant light at 24°C, samples were collected 2, 4, or 6 d after incubation (DAI), and total RNA was isolated. Five micrograms of RNA was loaded in each lane. Ethidium bromide staining of rRNA bands for 2-DAI samples is shown for loading control. RNA quality and loading are similar for 4- and 6-DAI samples. C, RNA gel-blot analysis of ABI4 expression during germination. Plant material was prepared identically to the procedure in Figure 6B, except that samples were collected 4 h after incubation (HAI) in addition to 2, 4, and 6 DAI. Five micrograms of RNA was loaded in each lane. Ethidium bromide staining of rRNA bands for 4-HAI and 2-DAI samples is shown for loading control. RNA quality and loading are similar for 4- and 6-DAI samples. D, RNA gel-blot analysis of ABI1 expression during germination. Plant material was prepared identically to the procedure in Figure 6B, except that samples were collected 4 and 6 DAI. Five micrograms of RNA was loaded in each lane. Ethidium bromide staining of rRNA bands for 4-DAI samples is shown for loading control. RNA quality and loading are similar for the 6-DAI samples.