Involvement of G6PD5 in ABA response during seed germination and root growth in Arabidopsis

Abstract

Background: Glucose-6-phosphate dehydrogenase (G6PDH or G6PD) functions in supply of NADPH, which is required for plant defense responses to stresses. However, whether G6PD functions in the abscisic acid (ABA) signaling pathway remains to be elucidated. In this study, we investigated the involvement of the cytosolic G6PD5 in the ABA signaling pathway in Arabidopsis.

Results: We characterized the Arabidopsis single null mutant g6pd5. Phenotypic analysis showed that the mutant is more sensitive to ABA during seed germination and root growth, whereas G6PD5-overexpressing plants are less sensitive to ABA compared to wild type (WT). Furthermore, ABA induces excessive accumulation of reactive oxygen species (ROS) in mutant seeds and seedlings. G6PD5 participates in the reduction of H₂O₂ to H₂O in the ascorbate-glutathione cycle. In addition, we found that G6PD5 suppressed the expression of Abscisic Acid Insensitive 5 (ABI5), the major ABA signaling component in dormancy control. When G6PD5 was overexpressed, the ABA signaling pathway was inactivated. Consistently, G6PD5 negatively modulates ABA-blocked primary root growth in the meristem and elongation zones. Of note, the suppression of root elongation by ABA is triggered by the cell cycle B-type cyclin CYCB1.

Conclusions: This study showed that G6PD5 is involved in the ABA-mediated seed germination and root growth by suppressing ABI5.

Keywords: Abscisic acid; Germination; Glucose-6-phosphate dehydrogenase 5; NADPH oxidases; Reactive oxygen species; Root system architecture.

Fig. 1

Seed germination and root growth of wild type (WT), g6pd5, and g6pd6 mutant in response to ABA. a and b Seeds were germinated on 1/2 MS agar plates with 1 μM ABA. Photographs were taken 4 d after treatment. c 5-day-old seedlings were grown vertically on 1/2 MS agar plates supplemented with 10 μM ABA. Root growth was monitored and analyzed using ImageJ software. d Relative transcript levels of G6PD5 and G6PD6 in wild-type (Col-0) with different concentrations of ABA treatment. The transcript levels were normalized to Actin2. e The activities of G6PD in Arabidopsis WT and mutants exposed to ABA treatment. One-way Duncan’s test was performed, and statistically significant differences are indicated by different lower case letters (P < 0.05). Bar, 1 cm. The experiments were repeated at least three times with similar results, and data from one representative experiment are presented

Fig. 2

Seed germination and root growth of wild type (WT), g6pd5 mutant, and G6PD5-OE lines in response to ABA. a Seeds were germinated on 1/2 MS agar plates with or without ABA. Photographs were taken 4 d after ABA treatment. b Percentage of seed germination with or without ABA treatment for 4 d. c Percentage of seed germination with 1 μM ABA for 3–5 d. d and e 5-day-old seedlings were grown vertically on 1/2 MS agar plates supplemented with indicated concentrations of ABA for 3 d and the length of newly grown roots was measured. Root growth was monitored and analyzed using the ImageJ software

Fig. 3

G6PD5 affects the ROS levels with ABA treatment and the activities and transcript levels of antioxidant enzymes in Arabidopsis seedlings. 5-day-old seedlings were grown vertically on 1/2 MS agar plates supplemented with the 10 μM ABA for 6 h. a Staining of roots with DAB (brown color displaying H₂O₂) after ABA treatment. Bar = 100 μm. b Staining of roots with NBT (blue color displaying O₂^.-) after ABA treatment. Bar = 50 μm. c and d The activities of antioxidant enzymes and transcript levels of antioxidant enzyme responsive genes. The transcript levels were normalized to Actin2. e The seeds and seedlings are incubated with 0.25 μM ASC or 5 μM GSH

Fig. 4

Response of G6PD5 to ABA through NADPH oxidase signaling pathway. a and b Relative transcript levels of NADPH oxidase genes AtrbohD and AtrbohF in Arabidopsis seedlings with or without 20 μM ABA treatment. c Activity of the NADPH oxidase in WT and mutants exposed to ABA treatment. d Relative transcript levels of G6PD5 in WT (Col-0) and NADPH oxidase mutant seeds (atrbohD1, atrbohF1, atrbohD1/F1) with or without 20 μM ABA treatment. The transcript levels were normalized to Actin2. Results are averages ± SE (n = 3). All experiments were repeated at least three times with similar results

Fig. 5

G6PD5 attenuates the expression of several ABA responsive genes. Relative expression levels of ABA-signaling genes ABI3, ABI4, ABI5 in plants. Two-week-old seedlings were incubated in liquid MS medium with or without 10 μM ABA for 12 h. The transcript levels were determined by qRT-PCR analysis. The transcript levels were normalized to Actin2. Results are averages ± SE (n = 3). All experiments were repeated at least three times with similar results

Fig. 6

Gene expression. a Expression of ABA-biosynthesis genes NCED6 and NCED9 and catabolic genes CYP707A3 and CYP707A4 in WT and g6pd5 was analyzed before or after ABA treatment. b The expression of G6PD5 in ABA mutants. Relative transcript levels of G6PD5 in wild-type (Col-0) and ABA mutant seeds (aba2–1, aba2–3, abi4–102) with or without ABA. In all experiments, the expression of Actin2 was used as the control. Three replicates were made for each treatment with similar results. Values are mean ± SE of three different experiments. Means denoted by the same letter do not significantly differ at P < 0.05 according to Duncan’s multiple range test

Fig. 7

G6PD5 knockout on the expression of ABI5 genes in Arabidopsis. a Expression of ABI5::GUS in Col-0/ABI5 and g6pd5/ABI5 seedling roots grown for 7 d then treated with or without 20 μM ABA for 12 h using GUS staining. b Quantification of the GUS activity in Col-0/ABI5 and g6pd5/ABI5 seedlings. The GUS activity of Col-0/ABI5 root was adjusted to 100%. Mean values and SE were calculated from three independent experiments (n = 20). Within each set of experiments, bars with different letters were significantly different at the p < 0.05 level

Fig. 8

G6PD5 regulates root meristem and elongation zone. a and b Root meristems of propidium iodide (PI)-stained images in Arabidopsis WT seedlings. Bars = 100 μm. c Meristem cell number. d Root meristem zone size. e Root elongation zone size. The 5-day-old seedlings were treated with 10 μM ABA for 12 h. Except in a and b, mean values and SE were calculated from three independent experiments (n = 20). Within each set of experiments, bars with different letters were significantly different at the p < 0.05 level

Fig. 9

G6PD5 is involved in cell division in the absence or presence of ABA. a Expression of CYCB1;1::GUS in Col-0/CYCB1;1 and g6pd5/CYCB1;1 seedlings grown for 5 d with or without ABA treatment for 12 h. Scale bars, 200 μm. b Quantification of the GUS activity in CYCB1;1::GUS seedlings. The GUS activity in Col-0/CYCB1;1 roots was adjusted to 100%. c-e Relative transcript levels of CYCB1, PLT1 and PLT2 in WT (Col-0), g6pd5 mutant and OE lines plants with or without ABA treatment. Data are presented as mean values ± SD of three independent experiments. One-way Duncan’s test was performed, and statistically significant differences are indicated by different lower case letters (P < 0.05). The experiments were repeated at least three times with similar results, and data from one representative experiment are presented

Fig. 10

Schematic illustration of a proposed model during Arabidopsis seed germination and root growth. In this model, arrows indicate positive regulation and bars indicate negative regulation. Dotted arrows indicate results from the literature