Cytosolic Glucose-6-Phosphate Dehydrogenase Is Involved in Seed Germination and Root Growth Under Salinity in Arabidopsis

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH or G6PD) is the key regulatory enzyme in the oxidative pentose phosphate pathway (OPPP). The cytosolic isoforms including G6PD5 and G6PD6 account for the major part of the G6PD total activity in plant cells. Here, we characterized the Arabidopsis single null mutant g6pd5 and g6pd6 and double mutant g6pd5/6. Compared to wild type, the mutant seeds showed a reduced germination rate and root elongation under salt stress. The seeds and seedlings lacking G6PD5 and G6PD6 accumulate more reactive oxygen species (ROS) than the wild type under salt stress. Cytosolic G6PD (cy-G6PD) affected the expression of NADPH oxidases and the G6PD enzymatic activities in the mutant atrbohD/F, in which the NADPH oxidases genes are disrupted by T-DNA insertion and generation of ROS is inhibited, were lower than that in the wild type. The NADPH level in mutants was decreased under salt stress. In addition, we found that G6PD5 and G6PD6 affected the activities and transcript levels of various antioxidant enzymes in response to salt stress, especially the ascorbate peroxidase and glutathione reductase. Exogenous application of ascorbate acid and glutathione rescued the seed and root phenotype of g6pd5/6 under salt stress. Interestingly, the cytosolic G6PD negatively modulated the NaCl-blocked primary root growth under salt stress in the root meristem and elongation zone.

Keywords: NADPH oxidases; NaCl; germination; glucose-6-phosphate dehydrogenase; reactive oxygen species; root system architecture.

FIGURE 1

The analysis of cy-G6PD in Arabidopsis seedlings with or without salt treatment. (A,B) The qRT-PCR analysis of G6PD5 (WT and 5OE#9) and G6PD6 (WT and 6OE#21) expression in Arabidopsis different organs. (C) Relative transcript levels of G6PD5 and G6PD6 in wild-type (Col-0) seedlings with the 150 mM NaCl treatment. Uppercase letters represent the error analysis of G6PD6, and lowercase letters represent the error analysis of G6PD5. (D,E) The activities of G6PD or cy-G6PD in Arabidopsis WT and mutants exposed to salt treatment. (F) Western blot analysis of G6PD expression in Arabidopsis. In this experiment, 150 mM NaCl was used for treatment. The Coomassie Brilliant Blue-stained gel was present to show that an equal amount of proteins was loaded in all lanes. Data are mean ± SE of three independent experiments, bars with different letters are significantly different at the level of P < 0.05. The experiment was repeated three with similar results.

FIGURE 2

The qRT-PCR analysis of G6PDs expression in WT and cy-G6PD mutants. The transcript levels were normalized to Actin2 gene expression. Results are averages ± SE (n = 3), bars with different letters are significantly different at the level of P < 0.05. All experiments were repeated at least three times with similar results.

FIGURE 3

Seed germination and root growth of WT, g6pd5 mutant, g6pd6 mutant, g6pd5/6 mutant, G6PD5-OE, and G6PD6-OE Arabidopsis in response to NaCl stress. Seeds were germinated on ½ MS agar plates with or without various concentrations of NaCl. (A) Photographs were taken 3 days in terms of radical emergence after NaCl treatment. (B) Percentage of seed germination in WT, g6pd5 mutant, g6pd6 mutant, g6pd5/6 mutant, G6PD5-OE, and G6PD6-OE with or without different NaCl treatment. (C,D) 5-day-old seedlings were grown vertically on ½ MS agar plates supplemented with the indicated concentrations of NaCl for 3 days. Root growth was monitored and analyzed using ImageJ software. Data are reported as the average value of three replicates using >50 seeds for each genotype. 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.

FIGURE 4

The g6pd5, g6pd6, and g6pd5/6 mutant affect the ROS levels under salt stress. 1-day-old seeds and 5-day-old seedlings were grown vertically on ½ MS agar plates supplemented with the 150 mM NaCl for 12 h. (A) The levels of H₂O₂ were measured using the H₂DCF-DA fluorochrome dyes in Arabidopsis seeds. Bar, 200 μm. (B) The levels of H₂O₂ were measured using the H₂DCF-DA fluorochrome dyes in Arabidopsis roots. Bar, 200 μm. (C,D) Quantification of the fluorescence in Arabidopsis seeds and roots under NaCl treatment. (E) 10-day-old seedlings were grown vertically on ½ MS agar plates supplemented with the 150 mM NaCl for 12 h. Data are mean ± SE of three independent experiments, bars with different letters are significantly different at the level of P < 0.05. The experiment was repeated three with similar results.

FIGURE 5

The response of G6PD5 and G6PD6 to salt stress through NADPH oxidases signaling pathway. (A,B) Relative transcript levels of NADPH oxidases AtrbohD and AtrbohF genes in Arabidopsis seedlings with or without 150 mM NaCl treatment. (C) The activities of NADPH oxidase in Arabidopsis WT and mutants exposed to salt treatment. (D,E) Relative transcript levels of G6PD5 and G6PD6 in WT and NADPH oxidases mutant seeds (atrbohD1, atrbohF1, and atrbohD1/F1) exposed to salt treatment. The transcript levels were normalized to Actin2 gene expression. (F) The activities of G6PD in Arabidopsis WT and mutants exposed to salt treatment. Results are averages ± SE (n = 3), bars with different letters are significantly different at the level of P < 0.05. All experiments were repeated at least three times with similar results.

FIGURE 6

G6PD5 and G6PD6 affects NADPH content in Arabidopsis under salt stress. 10-day-old seedlings were grown vertically on ½ MS agar plates supplemented with the 150 mM NaCl for 12 h. Results are averages ± SE (n = 3), bars with different letters are significantly different at the level of P < 0.05. All experiments were repeated at least three times with similar results.

FIGURE 7

G6PD5 and G6PD6 regulate root meristem and elongation zone. (A) Root meristems of propidium iodide (PI)-stained images in Arabidopsis WT seedlings. The meristem zone was marked with white arrows in (A). Bars = 100 μm. (B) Root meristem cell number, meristem zone size, elongation cell number, and elogation zone size in Arabidopsis WT seedlings. The 5-day-old seedlings were treated with 100 mM NaCl for 12 h. 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 0.05 level.

FIGURE 8

Schematic illustration of a proposed model for the link between G6PD5, G6PD6, ROS, and APX-GR in Arabidopsis seed germination and root growth. In this model, arrows indicate positive regulation, bars indicate negative regulation. Salt stress induces cy-G6PD, which subsequently maintain the intracellular NADPH homeostasis, and involved in regulating key enzymes (APX and GR) in ASC-GSH cycle. The APX and GR inhibit the level of H₂O₂ in cells through GSH content. cy-G6PD is involved in H₂O₂ accumulation through applying NADPH to PM NADPH oxidase. The enhanced cy-G6PD thus control germination of Arabidopsis seeds and growth of Arabidopsis primary roots.