The involvement of wheat F-box protein gene TaFBA1 in the oxidative stress tolerance of plants

Abstract

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants' tolerance to multiple stress conditions.

Fig 1. Expression of TaFBA1 in wheat under MV-induced oxidative stress.

Wheat seedlings with one leaf were subjected to 10-μM MV treatments. Seedlings treated with sterile water were chosen as controls. Seedlings were harvested at different time points for analysis. (A) Expression of TaFBA1 at the mRNA transcript level in shoots, as shown by qPCR; tubulin cDNA was used as a control reference; (B) Expression of TaFBA1 at the protein level in shoots as shown by western blot. After 12.5% SDS-PAGE, protein samples were electro-transferred onto a PVDF membrane and probed with the TaFBA1 antibody produced in our laboratory. The Rubisco large subunit was used as a loading control.

Fig 2. Germination and phenotype of different transgenic lines on MS medium containing different concentrations of MV.

(A) Seed germination; (B) Germination rate. The seeds were allowed to grow for 16 d before the photographs were taken. Each measurement consisted of 50 seeds. Values are averages of three replicates ± SE. (C) Seedling development. (D) Root length. Seedlings grown on MS medium for 7 d were transferred to MS medium containing different concentrations of MV for 7 d before the photographs were taken. Root length was obtained from 30 seedlings in each of three independent experiments.

Fig 3. Methyl viologen-induced oxidative damage of leaves from WT and transgenic lines.

(A) Phenotypes of WT and transgenic (OE-3, 5) seedlings in response to methyl viologen treatments. The corresponding fresh weights are shown in (B). Germinated seedlings with radicle lengths were transferred onto MS medium containing 0 or 10 μM methyl viologen for 7 d. (C) The phenotypic differences in the leaf disks from transgenic vs. WT plants after 24 h of MV treatment. (D) Leaf phenotype of three-month-old WT and transgenic tobacco plants treated with 100 μM MV for 48 h. Chlorophyll content (mg g-1 FW) (E), malondialdehyde (MDA) content (mg g-1 FW) (F) and relative electrical conductivity (%) (G) in leaf disks of transgenic lines vs. WT plants floated on 0, 50 and 100 μM MV solution, respectively, for 48 h under continuous light at 25°C.

Fig 4. Protein carbonylation levels in WT and transgenic lines under oxidative stress.

Protein carbonylation levels were detected with anti-DNP antibodies in WT and transgenic plant leaves under normal conditions or 100-μM MV treatments. Protein gel blot analysis of protein carbonylation following derivatization of protein carbonyls with DNPH in HCl.

Fig 5. ROS accumulation in different transgenic plants and WT under MV-induced oxidative stress.

(A) H₂O₂ accumulation detected by DAB staining; (B) H₂O₂ content_; (C) O₂ ^.- accumulation detected by NBT staining; (D) O₂ ^.- production rate_. Values are averages of three replicates ± SE.

Fig 6. The total activities of the antioxidant enzymes in transgenic plants and WT under MV treatment.

(A) Superoxidase dismutase, SOD; (B) Guaiacol peroxidase, POD; (C) Ascorbate peroxidase, APX; (D) Catalase, CAT. Values are the average of three replicates ± SE. “*” and “**” indicate significant differences between different plants subjected to the same treatment at the P < 0.05 and P < 0.01 levels, respectively.

Fig 7. Expression of antioxidant-related genes in WT and transgenic plants under MV treatment by qPCR.

Transcript levels of these genes in transgenic plants are indicated relative to the level of WT plants taken as 1, referring to the transcript of actin in the same samples. Each column represents the mean ± standard error of five replicates.

Fig 8. Subcellular localization of TaFBA1-GFP fusion protein in onion epidermal cells.

(A) Schematic representation of the 35S::TaFBA1-GFP fusion construct and the 35S::GFP construct. The TaFBA1 coding region was fused upstream of the GFP coding region in the expression vector. (B) The transformed cells of the 35S::TaFBA1-GFP and 35S::GFP constructs were cultured in Murashige-Skoog (MS) medium at 28°C for 2 d and observed under a microscope.