A key ABA catabolic gene, OsABA8ox3, is involved in drought stress resistance in rice

Abstract

Expressions of ABA biosynthesis genes and catabolism genes are generally co-regulated in plant development and responses to environmental stress. Up-regulation of OsNCED3 gene, a key gene in ABA biosynthesis, has been suggested as a way to enhance plant drought resistance but little is known for the role of ABA catabolic genes during drought stress. In this study, we found that OsABA8ox3 was the most highly expressed gene of the OsABA8ox family in rice leaves. Expression of OsABA8ox3 was promptly induced by rehydration after PEG-mimic dehydration, a tendency opposite to the changes of ABA level. We therefore constructed rice OsABA8ox3 silencing (RNA interference, RNAi) and overexpression plants. There were no obvious phenotype differences between the transgenic seedlings and wild type under normal condition. However, OsABA8ox3 RNAi lines showed significant improvement in drought stress tolerance while the overexpression seedlings were hypersensitive to drought stress when compared with wild type in terms of plant survival rates after 10 days of unwatering. Enzyme activity analysis indicated that OsABA8ox3 RNAi plants had higher superoxide dismutase (SOD) and catalase (CAT) activities and less malondialdehyde (MDA) content than those of wild type when the plants were exposed to dehydration treatment, indicating a better anti-oxidative stress capability and less membrane damage. DNA microarray and real-time PCR analysis under dehydration treatment revealed that expressions of a group of stress/drought-related genes, i.e. LEA genes, were enhanced with higher transcript levels in OsABA8ox3 RNAi transgenic seedlings. We therefore conclude that that OsABA8ox3 gene plays an important role in controlling ABA level and drought stress resistance in rice.

Figure 1. ABA accumulation and expression analysis of the OsABA8ox genes.

(A) Expression patterns of OsABA8ox genes in different tissues of rice. (B) ABA accumulation and (C) OsABA8ox genes expression in rice leaves during PEG-mimic dehydration treatment and rehydration. Four-leaf stage seedlings were treated with 20% PEG for 4 h, and then transferred to nutrient solution for rehydration. Error bars are standard deviations based on three replicates.

Figure 2. Stress tolerance assays of wild type plants and the transgenic rice.

(A) Relative expressions of OsABA8ox3 gene in RNAi and overexpression lines; (B) Performance of WT control and OsABA8ox3 RNAi-9, RNAi -27 and overexpression lines after 7 d soil drought stress and 4 d recovery. (C) Survival rate of rice seedlings after drought stress and rewatering. Error bars are standard deviations based on three replicates (n > 40). ** Indicates significant difference from wild-type at P = 0.01.

Figure 3. Physiological changes in the OsABA8ox3-RNAi and -overexpression transgenic plants.

(A) ABA contents, (B) MDA contents and (C) antioxidant enzyme activities in the WT and OsABA8ox3-RNAi and -overexpression transgenic seedlings under normal condition and PEG treatment. Four-leaf stage seedlings were treated with 20% PEG for 2 h, then the samples were quickly frozen with liquid nitrogen. Error bars are standard deviations based on three replicates. ** Indicates significant difference from wild-type at P = 0.01.

Figure 4. Relative expression levels of 9 stress-related genes in the WT and OsABA8ox3 RNAi transgenic plants under normal and PEG-treated conditions detected by qRT-PCR.

Four-leaf stage seedlings were treated with 20% PEG for 2 h, and then the samples were quickly frozen with liquid nitrogen for RNA extraction. The genes include dehydration-responsive element-binding protein (Os06g03670), zinc finger protein (Os05g10670), late embryogenesis abundant proteins (Os05g46480, Os01g50910 and Os04g49980), dehydrin family proteins (Os11g26760 and Os01g50700) and heat shock protein (Os03g16920 and Os02g54140).

Figure 5. ABA sensitivity of the OsABA8ox3 transgenic seeds during germination and post-germination growth.

(A) Seed germination, (B) Post-germination root growth and (C) Root length of WT and OsABA8ox3-RNAi and -overexpression transgenic plants in response to exogenous ABA. Seeds treated with water and ABA (1μM and 5μM) were imbibed at 28°C to facilitate germination and post-germination growth. Error bars are standard deviations based on three replicates. ** Indicates significant difference from wild-type at P = 0.01.