Distribution of actin filaments in different transgenic lines
Since we focused on the response of actin filaments to environmental factors in Arabidopsis thaliana roots, we tried to select a transgenic line that could be used to better observe actin filaments in roots from the fABD2-GFP and Lifeact-Venus transgenic lines. The 7 DAG root tip, mature root regions, mesophyll cells, and leaf epidermal cells of these two lines were observed, as shown in Fig. 1. For the fABD2-GFP line, weak green signals appeared in different tissues of the seedling (Fig. 1a–e), but the strongest signal was in the leaf epidermis (Fig. 1e). For the Lifeact-Venus line, the green signal was much stronger in the root region than in any other parts of the seedling, and we could not detect a fluorescence signal in mesophyll or epidermal cells (Fig. 1n, o). Therefore, considering the subject matter of this study, the Lifeact-Venus transgenic line was chosen as the experimental line.
UV-B inhibits hypocotyl elongation and root growth of Arabidopsis seedlings
Hypocotyl elongation was inhibited after UV-B exposure in Lifeact-Veuns and uvr8 seedlings, as shown in Fig. 2a–d. The reduction in the length of the hypocotyl in the UV-B-treated group was ~ 63.3% in the control group and 20.9% in the uvr8 group (Fig. 2o). UV-B exposure also inhibited root growth, as shown in Fig. 2m. Compared with that of the untreated plants, root length was reduced by ~ 68.2% in the control group and 33.8% in the uvr8 group. H2DCF (Fig. 2e–h) staining showed that the H2O2 concentration in the root increased sharply after UV-B treatment in the Lifeact-Venus line but not in the uvr8 line. After calculating the H2DCF fluorescence intensity (Fig. 2n), it was found that UV-B treatment resulted in a 30.1% increase in H2O2 content in the Lifeact-Venus line. Confocal microscopy observations revealed that the dynamics of actin filaments were changed, and the shape converted from a bundle to a much thinner arrangement in the less elongated hypocotyl in the Lifeact-Venus line after exposure to UV-B (Fig. 2i, k). The uvr8 line also showed reorganization of actin filaments in the shortened hypocotyl under UV-B exposure (Fig. 2j, l). The orientation of the actin filament bundles changed from parallel to the hypocotyl axis to perpendicular to the elongation of the hypocotyl axis under UV-B exposure. The skewness of actin filaments increased substantially under UV-B in both the Lifeact-Venus line and uvr8 lines (Fig. 2p). These results indicated that the inhibition of hypocotyl elongation by UV-B exposure may be due to changes in the distribution of actin filaments. This change was independent of ROS since the ROS burst was not detected in the hypocotyl.
Exogenous H2O2 inhibited root growth in Arabidopsis
Previous results showed that UV-B caused an increase in ROS in Arabidopsis roots, especially an increase in H2O2 content (Fig. 2e–h). Therefore, we wanted to determine the effect of exogenous H2O2 on root growth in Arabidopsis. As shown in Fig. 3a, an increase in H2O2 concentration inhibited the root growth of Arabidopsis seedlings. Moreover, this inhibitory effect was positively correlated with the H2O2 concentration. The 7 DAG and 21 DAG seedlings showed the same trend. The H2O2 scavenger DMTU partly recovered root growth at lower H2O2 concentrations (Fig. 3b).
Effects of exogenous H2O2 on the dynamics and distribution of actin filaments in Arabidopsis roots
As shown in Fig. 4, panels A0-F6, 7 DAG Arabidopsis seedlings (Lifeact-Venus and uvr8) were treated with different concentrations of exogenous H2O2 and UV-B, and the distribution of actin filaments in the roots was observed by confocal microscopy. The morphology of the root tip in response to different H2O2 treatments was similar to that observed in the absence of UV-B exposure (Fig. 4A0–A4), and the fluorescence signal at the root tip was stronger than that in other regions of the root. The green signal was significantly weakened by treatment with 1 mM exogenous H2O2 (Fig. 4A3). The morphology of the actin filaments in different groups was much clearer after enlargement, as shown in Fig. 4B0–B4. In the control and 0.05 mM exogenous H2O2 treatment groups, the apical actin filaments were obviously filamentous (Fig. 4B0, B1). Under the 0.5 mM exogenous H2O2 treatment, the actin filaments disaggregated into bright puncta (Fig. 4B2). In the 1 mM H2O2 treatment, almost all the actin filaments disaggregated into puncta (Fig. 4B3). After treatment with the highest concentration of 2 mM exogenous H2O2, the actin filaments in the root tip changed into a thick bundle-like structure (Fig. 4B4). This suggested that the cell ductility is greatly restricted under treatment with high concentrations of H2O2. In Fig. 4B0–B4, the actin filaments first depolymerized and then aggregated into a thicker bundle-like structure with increasing H2O2 concentrations. This may be related to the resistance of plants to stresses induced by external H2O2. After UV-B treatment, actin filaments in the root tips underwent depolymerization (Fig. 4A5, B5), similar to the results obtained after exogenous 1 mM H2O2 treatment in the Lifeact-Venus line. In contrast, actin filaments in root apical cells remained filamentous under UV-B exposure in the uvr8 line (Fig. 4A6, B6). This result indicates that the UV-B-induced change in actin filament morphology in root tip cells is dependent on H2O2 generation.
The distribution of actin filaments in the elongation zone under different treatments was also observed and analyzed (Fig. C0–D6). Only treatment with 1 mM exogenous H2O2 showed lower fluorescence intensity in the elongation zone under low magnification (Fig. 4C3). The enlarged results showed that the arrangement of actin filaments in the elongation zone of the control group was diffuse, and the arrangement direction was transverse (Fig. 4C0, D0). After treatment with 0.05 mM H2O2, the arrangement of the actin filaments was similar to that of the control group (Fig. 4C1, D1). When the exogenous H2O2 concentration was increased to 0.5 mM, the intracellular actin filaments changed from filaments to puncta (Fig. 4C2, D2). When the concentration reached 1 mM, the number of puncta was the highest (Fig. 4C3, D3), and the presence of filamentous actin was not observed. Under 2 mM H2O2 treatment, the actin filaments were a thick bundle (Fig. 4C4, D4), and the direction was parallel to the longitudinal axis of the cells (Fig. 4D4). The changes in the distribution of actin filaments in the elongation zone were similar to the changes in the apical area. After UV-B treatment, the actin filaments in cells in the root elongation zone showed both short filamentous structures and more punctate structures in the Lifeact-Venus line (Fig. 4C5, D5). In the uvr8 mutant, the distribution of microfilaments in the cells in the elongation zone (Fig. 4C6, D6) was similar to that of the control without any treatment (Fig. 4C0, D0).
Finally, we observed and analyzed the response of exogenous H2O2 to actin filaments in the mature region of the root. The actin filaments in the mature region of the control group (Fig. 4E0) were filamentous, and the 0.05 mM exogenous H2O2 treatment group (Fig. 4E1) was not significantly different from the control. In the 0.5 mM H2O2 treatment group (Fig. 4E2, F2), a large number of actin filaments were distributed as green puncta. While the fluorescence intensity of the actin filaments in the 1 mM H2O2-treated group was much weaker, only partially punctate actin was visible (Fig. 4E3). This result also implies that the mature region is more sensitive to ROS accumulation. After treatment with a high concentration of H2O2, green fluorescence could be detected in several cells in the short mature region, and the actin filaments gathered in a thick bundle (Fig. 4E3, F4). UV-B treatment resulted in complete degradation of actin filaments in the cells in the root maturation zone, with no filamentous or punctate structures present in the Lifeact-Venus line (Fig. 4E5, F5). In contrast, cells in the root maturation zone of the uvr8 mutant still showed more microfilament bundles (Fig. 4E6, F6).
We analyzed the integrated density after different treatments and in different root regions, as shown in Fig. 4g. The results showed that the integrated density in the Lifeact-Venus line continued to decrease with increasing exogenous H2O2 concentrations. The integrated density also greatly diminished after exposure to UV-B in the Lifeact-venus line. In contrast, the overall integrated density of the uvr8 mutant roots under UV-B exposure remained high. The skewness results (Fig. 4h) showed that 1 mM exogenous H2O2 increased skewness in the maturation zone, which showed the same tendency as uvr8. This means that the maturation zone could be a region of signal transduction in the regulation of actin filament dynamics.