Bioinformatics analysis of TaPCNA
The structure domain of PCNA was conservative, and the topological structure of N-terminal and C-terminal was similar upon analysis of the protein structure via online website. Three PCNA molecules formed a loop closed to DNA double helix. In the presence of limitation on carried gene fragments of the virus vector, the PCNA was divided into two fragments based on both ends of N-terminal and C-terminal in this research, which were respectively named PCNA-1 (400bp) and PCNA-2 (402bp).
Agrobacterium mediated BSMV VIGS in N.benthamiana and Wheat
Figure 2a showed the 7-15 days N.benthamiana leaves after inoculated with BSMV VIGS mediated Agrobacterium. Among the three treatments inoculated with virus vectors, BSMV:00 showed virus symptoms. BSMV:TaPCNA-1 and BSMV:TaPCNA-2 showed different degrees of leaf edge curling, small leaf area and plant dwarfing. Considering the evolutionary conservatism of PCNA, it is similar to the gene-silencing phenotype reported by Gareth Bruce et al.[20], indicating that N.benthamiana was successfully infected with the virus. We inferred that the phenotype changed since the PCNA is a key factor on meristem growth. After BSMV::TaPCNA infected the wheat, the overall growth of the plant was weak and dwindled that observed at 13-15 days (Fig. 2b and 2c). Also, wheat leaves showed some signs of virus infection.
The growth rate of the gene silenced plants was statistically calculated (Fig. 3). The 5-day old wheat seedlings were inoculated, and the calculation began on the 10th day and lasted until the 27th day. BSMV:TaPCNA-1 and BSMV:TaPCNA-2 groups showed obvious growth stagnation on the 15th day during the growth period (at 10 days after inoculation). While the growth rates of WT and BSMV:00 was slow during plant growth to the later stage. And the growth rate dropped slowly with the extension of the growth cycle. In order to prove the plants growth stagnation is a result of gene silencing, the length of mesophyll cells was recorded and caculated (Fig. 4). It was found that the cell length of BSMV:TaPCNA-1 and BSMV:TaPCNA-2 groups were significantly shorter than WT and BSMV:00. And the WT and BSMV:00 was not significantly different.
The expression of TaPCNA after VIGS and exposed to enhanced UV-B radiation
The phenotype of wheat processed with VIGS maintained for 15-25 days. However, the plants can recover at the 30th day after inoculations. Therefore, total RNA was extracted from 22 days wheat plants after gene silencing , and semi-quantitative RT-PCR was performed to determine the efficiency of VIGS. The expression of TaPCNA in BSMV:TaPCNA-1 and BSMV:TaPCNA-2 groups decreased significantly (Fig. 5), indicating that the BSMV::TaPCNA interfered the expression of TaPCNA. To test the response of TaPCNA to enhanced UV-B radiation, we extracted total RNA from seedlings after irradiated with enhanced UV-B radiation. The RT-PCR showed the expression level increased in leaves, whilethe expression reduced in root (Fig. 5). We can infer that the reduced expression of TaPCNA in root is a result of compenstroy function under enhanced UV-B radiation. Thus the germination could get more energy and restore the normal growth.
Changement of physiochemical and metabolisms of Wheat after VIGS and treated with enhanced UV-B radiation
Peroxidase (POD) was associated with respiration, photosynthesis and auxin oxidation of plants. The activity of POD was measured on the 7th day after TaPCNA silencing (Fig. 6). The results showed the activity of POD in BSM::00 group was not significantly different from WT, while the activity of POD in BSMV::TaPCNA-1 and BSMV::TaPCNA-2 were decreased obviously compared with WT and BSMV::00. The activity of POD on the 7th day was much higher than that on the 1st day after virus infection. These results indicated that silenced TaPCNA affected plant growth and caused tremendous accumulation of peroxides affecting the overall growth of the plant. Catalase (CAT) can remove the excess accumulated H2O2 and indirectly reflect the stress resistance and metabolic intensity of plants (Fig. 6). The activity of CAT in BSMV::TaPCNA-1 and BSMV::TaPCNA-2 was lower when compared with WT and BSMV::00. The concentration of Malondialdehyde (MDA) is a reflection of the degree of lipid peroxidation in plant cell membrane (Fig. 6). The MDA content in BSMV::TaPCNA-1 and BSMV::TaPCNA-2 content increased when compared with WT and BSMV::00. Besides, MDA was proven a substance product of membrane lipid under the action of reactive oxygen species (ROS), indicating that the membrane was damaged after the silencing of TaPCNA. Superoxides dismutase (SOD) was functioned as remove free oxygen radicals in cells. As shown in figure 6, the activity of SOD of BSMV::TaPCNA-1 and BSMV::TaPCNA-2 were decreased compared with WT and BSMV::00. After treated with enhanced UV-B radation, the activity of POD and CAT were decreased more and SOD activity and MDA content increased more in each treatment group. Moreover, it was found WT+B and BSMV::TaPCNA-1+B and BSMV::TaPCNA-2+B showed the same trend. Soluble sugar and soluble protein are useful parameters on reflect the metabolic process of plants. As shown in figure 6, the content of soluble sugar and soluble protein in BSMV::TaPCNA-1 and BSMV::TaPCNA-2 decreased a lot compared with WT and BSMV::00. These indicated that the metabolism of plants were slowed down. It also indirectly proves that silence of TaPCNA affected plant growth. Especially, after being exposed to enhanced UV-B radation the reduction was exacerbated.
Apoptosis in wheat leaves after VIGS and exposed to enhanced UV-B radiation
The DNA of wheat plants in TaPCNA silencing was extracted. As shown in figure 7, the gel electrophoresis showed the complete DNA bands of WT and BSMV::00, while the DNA of BSMV::TaPCNA-1 and BSMV:TaPCNA-2 were dispersed as the DNA laddering. Both WT+B and BSMV::00+B groups showed regular low-molecular weight fragments, and similar fragmentation and dispersion with BSMV::TaPCNA-1+B and BSMV::TaPCNA-2+B, presenting that the DNA damage and apoptosis in wheat cells had happend.
To confirm the apoptosis in wheat cells after TaPCNA silenced, the PI staining was carried out on the VIGS treated leaves. According to figure 8, the dead cells showed obvious contour. The view of WT and BSMV::00 was dark heavy while BSMV::TaPCNA-1 and BSMV::TaPCNA-2 showed more shiny colored parts than WT and BSMV::00. After magnified, normal cells showed clear notch red. The apoptosis cells nucleus could be stained in red. This indicated the inhibition of wheat growth caused by TaPCNA silencing induced apoptosis. PI staining was also carried out for each treatment group after UV-B radiation. As shown in figure 8, after treated with enhanced UV-B radiation all groups could be observed and with different degrees of red colored parts. While BSMV::TaPCNA-1+B and BSMV::TaPCNA-2+B showed more colored parts than WT+B and BSMV:00+B.