2.1 Comparison of nuclear morphological changes in endosperm between weedy rice and cultivated rice
The process of PCD is often accompanied by degeneration of cell nuclei. The DAPI is a highly sensitive and specific DNA fluorescent dye, which has excellent fluorescence staining effect on the cell nucleus and chromosome. The endosperm cell nuclei of weedy rice and cultivated rice were in the coenocyte stage or cellularization stage, and the endosperm nucleus was small and regular spherical at 3 days post anthesis (DPA). Starch accumulated continuously in endosperm cells, and the nucleus of starch endosperm was extruded, gradually deformed and disintegrated at 5 to 9 DPA (Fig. 1).
Morphological and statistical results of starch endosperm cell nuclei of weedy rice and cultivated rice (normal nuclei, deformed nuclei and degraded nuclei) at 3, 5, 7 and 9 DPA were shown in Fig. 1 and Fig. 2. After DAPI staining, the nuclei of endosperm of weedy rice and cultivated rice in Taizhou were 100% normal nuclei at 3 DPA. The percentage of normal nuclei of TZWR was 3% lower than that of TZCR, and the percentage of deformed nuclei and degraded nuclei of TZWR were 2% and 1% higher than that of TZCR at 5 DPA, respectively. The percentage of normal nuclei and deformed nuclei of TZWR was 25%-48% lower than that of TZCR, and the percentage of degraded nuclei of TZWR were 74% and 63% higher than that of TZCR at 7 and 9 DPA, respectively (Figs. 1A1-A4, Figs. 1B1-B4; Fig. 2A).
After DAPI staining, the percentage of normal nuclei of YZWR was 44% and 15% lower than that of YZCR at 3 and 5 DPA, respectively, and the percentage of deformed nuclei and degraded nuclei of YZWR were 2%-34% higher than that of YZCR at 3 and 5 DPA. There were no normal nuclei in the endosperm cells of YZWR and YZCR, and the percentage of deformed nuclei of YZWR was 83% and 3% lower than that of YZCR at 7 and 9 DPA, respectively. The percentage of degraded nuclei of YZWR were 83% and 3% higher than that of YZCR at 7 and 9 DPA, respectively (Figs. 1C1-C4, Figs. 1D1-D4; Fig. 2B).
The endosperm cells of MMWR and MMCR were normal nuclei at 3 DPA. From 5 DPA to 9 DPA, the percentage of normal nuclei and deformed nuclei of MMWR were 5%-49% lower than that of MMCR, and the percentage of degraded nuclei of MMWR was 14%-59% higher than that of MMCR (Figs. 1E1-E4, Figs. 1F1-F4; Fig. 2C). The percentage of deformed nuclei and degraded nuclei were 18% and 2% at 3 DPA in DDWR, respectively. However, the percentage of normal nuclei was 100% in DDCR at 3 DPA. From 5 DPA to 9 DPA, the percentage of normal nuclei and deformed nuclei of DDWR were 1%-47% lower than that of DDCR, and the percentage of degraded nuclei of DDWR were10-70% higher than that of DDCR (Figs. 1G1-G4, Figs. 1H1-H4; Fig. 2D). Generally speaking, the PCD process of endosperm cell nuclei of weedy rice was faster than that of associated cultivated rice (Figs. 1, 2).
2.2 Comparison of endosperm cell viability between weedy rice and cultivated rice
Viability staining provides a means to follow the pattern and progression of cell death during endosperm development. Evans blue dye only stains cells which are no longer capable of excluding the dye, indicating a loss of membrane integrity and consequently viability. The embryos of weedy rice and cultivated rice could not be dyed blue by Evans blue, which means that the embryos were always active during endosperm development. The endosperm of weedy rice and cultivated rice was gradually dyed blue by Evans blue with the development of endosperm, that is, endosperm cells gradually lost membrane permeability and became dead cells(Fig. 3). All starch endosperm cells of TZWR were completely stained dark blue by Evans blue at 13 DPA, while those of TZCR were at 21 DPA. The starch endosperm cells of YZWR and MMWR were completely stained dark blue by Evans blue at 13 DPA, and the starch endosperm cells of YZCR and MMCR were completely stained dark blue by Evans blue at 15 DPA. Endosperm cells of DDWR were completely stained dark blue by Evans blue 4 days earlier than that of DDCR (Fig. 3). In all, the whole starch endosperm of weedy rice was completely dyed dark blue by Evans blue 2–8 days earlier than that of associated cultivated rice, that is, endosperm cells of weedy rice lost membrane permeability and became dead cells 2–8 days earlier than associated cultivated rice (Fig. 3).
The embryo of weedy rice and cultivated rice can be dyed red by TTC, which means that the embryo has strong viability during endosperm development. The endosperm of weedy rice and cultivated rice could not be dyed red by TTC with the development of endosperm, which indicated that endosperm cells gradually lost viability (Fig. 4). The endosperm cells of DDWR could not be dyed red at 9 DPA by TTC, while the endosperm cells of weedy rice in other three places could not be dyed red by TTC at 15 DPA. However, the endosperm cells of four cultivated rice varieties could not be dyed red by TTC at 18 DPA (Fig. 4).
2.4 Comparison of anti-oxidative enzymes system between weedy rice and cultivated rice
The anti-oxidative enzymes activity decreased gradually both in weedy rice and cultivated rice, and the CAT activity of weedy rice was significantly lower than that of associated cultivated rice (Fig. 5). The CAT activity levels of TZWR was 10.39–82.95 U/g lower than that of TZCR at 3–25 DPA, while similar at 30 DPA (Fig. 5A). The CAT activity of YZWR was 37.72–53.81 U/g lower than that of YZCR at 3–15 DPA, but there was no significant difference between YZWR and YZCR at 20–30 DAP (Fig. 5B). The CAT activity of MMWR was significantly lower than that of MMCR at 3–15 DPA, but there was no significant difference between MMWR and MMCR at 20–30 DPA (Fig. 5C). At 3 and 5 DPA, there was no significant difference between the CAT activity of DDWR and DDCR, and the CAT activity of DDWR was 23.62–42.20 U/g lower than that of DDCR at 10–30 DPA.
The change trend of SOD activity of weedy rice was similar to that of associated cultivated rice, and there was no significant difference between MMWR and MMCR. The decline rate of SOD activity of weedy rice in the other three areas was faster than that of associated cultivated rice (Fig. 6). The SOD activity of TZWR was the highest at 3 DPA, which was 3.45 U/mg higher than that of TZCR. The SOD activity of TZCR was the highest at 5 DPA, which were increased continuously after the anthesis, reached a maximum, and declined thereafter. The SOD activity of TZCR was 0.73–1.52 U/mg higher than that of TZWR at 10–20 DPA (Fig. 6A). The SOD activity of weedy rice and cultivated rice in Yangzhou showed a downward trend, but the SOD activity of YZWR was 0.99–1.96 U/mg significantly lower than that of YZCR at 3, 25 and 30 DPA (Fig. 6C). SOD activity of weedy rice and cultivated rice was higher from Dandong at 3 to 10 DPA, and there was no significant difference between them. The SOD activity decreased at 15 to 30 DPA, but the decline rate of weedy rice was faster, which was significantly lower than that of cultivated rice by 1.25–2.50 U/mg (Fig. 6D).
The POD activity of weedy rice in Taizhou showed a downward trend, the highest at 3 DPA, and was 184.53 U/g higher than that of TZCR, the POD activity of TZCR reached the maximum at 15 DPA, and declined thereafter. The POD activity of TZCR was 357.52-559.19 U/g higher than that of TZWR at 15–30 DPA (Fig. 7A). The POD activity of YZWR and YZCR showed a downward trend, but the decline rate of YZWR was slower. The POD activity of YZWR was 103.25 U/g lower than that of YZCR at 5 DPA, and was significantly higher than that of cultivated rice by 218.56 U/g at 20 DPA (Fig. 7B). The POD activity of MMWR reached the highest at 5 DPA, which was 219.77 U/g higher than that of MMCR, while the POD activity of MMCR reached the highest at 3 DPA, the POD activity of MMCR was 154.35 U/g higher than that of MMWR at 20 DPA (Fig. 7C). There was no significant difference in POD activity between weedy rice and cultivated rice in Dandong (Fig. 7D).