Gene knockout study reveals that cytosolic ascorbate peroxidase 2(OsAPX2) plays a critical role in growth and reproduction in rice under drought, salt and cold stresses

Abstract

Plant ascorbate peroxidases (APXs), enzymes catalyzing the dismutation of H2O2 into H2O and O2, play an important role in reactive oxygen species homeostasis in plants. The rice genome has eight OsAPXs, but their physiological functions remain to be determined. In this report, we studied the function of OsAPX2 gene using a T-DNA knockout mutant under the treatment of drought, salt and cold stresses. The Osapx2 knockout mutant was isolated by a genetic screening of a rice T-DNA insertion library under 20% PEG-2000 treatment. Loss of function in OsAPX2 affected the growth and development of rice seedlings, resulting in semi-dwarf seedlings, yellow-green leaves, leaf lesion mimic and seed sterility. OsAPX2 expression was developmental- and spatial-regulated, and was induced by drought, salt, and cold stresses. Osapx2 mutants had lower APX activity and were sensitive to abiotic stresses; overexpression of OsAPX2 increased APX activity and enhanced stress tolerance. H2O2 and MDA levels were high in Osapx2 mutants but low in OsAPX2-OX transgenic lines relative to wild-type plants after stress treatments. Taken together, the cytosolic ascorbate peroxidase OsAPX2 plays an important role in rice growth and development by protecting the seedlings from abiotic stresses through scavenging reactive oxygen species.

Figure 1. The phenotype of the PEG-sensitive T-DNA insertional rice line.

After two days germination, wild-type (WT) (A) and mutant lines (B) were treated with 20% PEG2000. WT and mutant lines were treated with mock (C and D) and 20% PEG2000 (E and F) respectively, for 48 h. Seedlings of treated WT (G) and mutant seedlings (H) were recovered for 4 days. Bar = 1 cm.

Figure 2. The seed phenotype of WT and Osapx2.

A,B: the seed phenotype of WT (odd numbers) and Osapx2 (even numbers), before dehulling (A) and after dehulling (B). C: The thousand kernel weight of WT and Osapx2 seeds.

Figure 3. Molecular analysis of Osapx2 mutants.

A: Structure of OsAPX2 and the location of T-DNA insertion. Black boxes indicate protein-encoding exons. Arrows indicate the locations of PCR primers used. B: Confirmation of co-segregation of the T-DNA insertion with the PEG-sensitive and small seed phenotype in leaves of T₁ heterozygous progeny. Two PEG-sensitive seedlings (Ho; Homo type), PCR-amplified product only from the P1-LB2 primer set (Osapx2/Osapx2); six normal seedlings (He; Hetero type), amplified with both the P1-LB2 and P1–P2 primer sets (Osapx2/OsAPX2); and seven normal seedlings (WT), which amplified only with the P1–P2 primer set (OsAPX2/OsAPX2). C: RT-PCR expression analysis in leaves of wild-type phenotype and PEG-sensitive mutant seedlings. Actin gene is used as control.

Figure 4. Characterization of wild-type, Osapx2, complementation and OsAPX2-OX plants.

A,C: The phenotype of wild-type (WT), Osapx2, complementation (cp) plants and OsAPX2-OX plants at mature stage. B: The leaves of wild-type (WT), Osapx2 (m) and complementation plants (cp) during the vegetative development stage. D: The comparison of the height and the flag leaf angle between wild-type and Osapx2 mutant plants at the mature stage. E: Comparison of anther morphology and pollen viability stained with KI among wild-type (WT), Osapx2 and complementation plants (cp). Bar = 20.0 µm.

Figure 5. The expression of OsAPX2 and seed setting rate in wild-type, Osapx2, complementation and OsAPX2-OX plants.

A: RT-qPCR analysis of OsAPX2 expression in wild-type (WT), Osapx2, complementation plants (cp1 and cp2) and OsAPX2-OX plants. B: Seed setting rates of wild-type (WT), Osapx2, complementation plants (cp1 and cp2) and OsAPX2-OX plants. Values represent means ± SD of three replicates.

Figure 6. Developmentally regulated OsAPX2 expression in rice tissues.

A: RT-qPCR was performed to analysis the expression of OsAPX2 in root, leaf, blade sheath, blade ear, flower (fl), apical meristem and internode (in). B: The expression of OsAPX2 in leaves at different stages. C,D,E,F,G Expression patterns of OsAPX2 revealed by GUS staining. Leaf (D), internode (D), blade ear (F) and the transversely side of internode (G), flower (E). Bar = 200 µm.

Figure 7. OsAPX2 expression response to drought, cold and salt stresses.

A: OsAPX2 expression response to salt stress. B,C: Time course of OsAPX2 expression during drought and cold treatments. Actin was used as an internal control. Data represent means ± SD of three replicates.

Figure 8. The expression of OsAPX1 in Osapx2.

The expression of OsAPX1 was detected in leaves of wild-type and Osapx2 mutant seedlings under normal condition and after stress treatments. Data represent means ± SD of three replicates.

Figure 9. Effect of OsAPX2 expression on drought, salt and cold tolerance in rice.

A: Without OsAPX2, Osapx2 mutant plants showed sensitive to drought, salt and cold treatments. B: OsAPX2-OX transgenic plants showed tolerance to drought, salt and cold treatments.

Figure 10. The effects of stress treatments on wild-type and Osapx2 plants.

A: Relative water content. B: Chlorophyll content. C: H₂O₂ content. D: MDA content. Values represent the mean ± SD of three replicates.

Figure 11. The effects of stress treatments on wild-type and OsAPX2-OX plants.

A: Relative water content. B: Chlorophyll content. C: H₂O₂ content. D: MDA content. Data are mean of three replicates and were compared by one-way analysis of variance and Duncan’s multiple range test. Different letters (a–b) indicate significant differences (P<0.05) between lines.

Figure 12. Spikelet fertility after stress treatments in T₂ OsAPX2-OX transgenic plants and WT plants.

OX1, OX2, OX3 and OX4 are independent OsAPX2-OX lines. A: drought treatment, B: salt treatment, C: cold treatment. Values represent the mean ± SD (N = 10).

Figure 13. APX activity assay of wild-type, Osapx2 and OsAPX2-OX plants.

Five leaf seedlings grown in the half-strengthened MS liquid medium were treated with different treatments. For salt treatment, wild-type, Osapx2 and OsAPX2-OX plants were treated with 150 mM NaCl for 24 h; for cold treatment, plants were transferred to a 10°C incubator for 24 h; for drought treatment, plants were transferred to dry sterile petri dishes or 2 h. Different letters (a–c) indicate significant differences (P<0.05) between lines.

Figure 14. H₂O₂ content assay of wild-type, Osapx2, complementation and OsAPX2-OX plants at different developmental stages.

H₂O₂ content assay was performed in wild-type, Osapx2, independent complementation lines (cp1 and cp2) and independent OsAPX2-OX lines (OX1 and OX2) at different developmental stages. Different letters (a–c) indicate significant differences (P<0.05) between lines.