Effects of pre-flowering and flowering irrigation on reactive oxygen species metabolism and programmed cell death in the calyx tubes of Kuerlexiangli pear

Abstract: 【Objective】The objective of this study is to disclose the effect of pre-flowering and flowering irrigation on sepal abscission of Kuerlexiangli pear.【Methods】This experiment was conducted by measuring the calyx abscission rate, soil water content, water potential in fruiting spurs, reactive oxygen species (ROS) content and antioxidant enzymes activity of detached calyx, so as to verify the cell death in detached calyx by microscopic observation, DNA ladder detection, DAPI staining and TUNEL labeling. The programmed cell death (PCD) characteristics of calyx abscission were also demonstrated by microscopic observation, DNA ladder assay, DAPI staining and TUNEL labeling.【Results】There was no significant difference between pre-flowering and flowering irrigation treatments in terms of calyx abscission rate, and non-irrigation (control) promoted calyx abscission during flowering although the highest calyx shedding rate was 56.57%. The soil water content of non-irrigation (control) was low-er than the irrigation treatments at different phenological stages and exhabited a continuous decreasing trend. By comparing the spur water potential changes between pre-flowering or flowering stage irrigation treatments and control 10 days after those, the spur water potential decreased by (-0.31 MPa), (-0.33 MPa) and (-0.20 MPa), respectively, among which the spur water potential of non-irrigation (control) decreased the least. With the increase of drought stress, the changes of reactive oxygen contents and antioxidant enzyme activity of persistent calyx tubes with pre- flowering and flowering irrigation treatments and control were not significant at bloom and 10 days after bloom, while those of shedding calyx at bloom and 10 days after bloom were significant. Among them, the ·OH scavenging rate, O2 - · production rate and H2O2 content in the non- irrigated (control) calyx tubes were significantly higher by 46.24%, 36.12% and 53.84%, respectively, compared with the bloom stage, while SOD, POD and CAT activities decreased significantly by 45.57%, 69.76% and 62.47%, showing the most significant changes. By observing the microstructure of paraffin sections of calyx tubes, no significant saffron coloration was found in the cells of the persistent calyx isolates during flowering and 10 days after flowering; the percentage of saffron coloration in the cells of the desiccated isolates during flowering was 48%, and that 10 days after flowering was as high as 85%, indicating that the continued lignification of the desiccated isolates increased with drought stress and promoted sepal abscission. The total DNA in the persistent calyx cells showed a complete band during flowering and 10 days after flowering, indicating that no programmed cell death occurred during this period. The degradation of cellular DNA in this isolated region was observed at this time, and the DNA electrophoresis pattern of the calyx at 10 days after flowering was“smear-like”with the disappearance of the ladder band, probably due to the degradation of intracellular DNA into lower DNA fragments as the programmed cell death deepened. This is probably due to the fact that as the programmed cell death deepens, the DNA inside the cell is degraded into smaller fragments, which are absorbed and used by the cell as part of the cell wall. No changes were observed in the TUNEL labeling and DAPI staining of the isolated cells from the persistent calyx during and 10 days after flowering, and no dead cells were found; during the desiccation of the calyx at flowering, a few nuclei showed positive TUNEL labeling, indicating that the PCD process had started in the isolated cells and occurred first in the differentiated mature isolated cells; during the desiccation of the calyx 10 days after flowering, fewer nuclei were seen with TUNEL labeling, and the occasional nuclei that were visible were brownish in color, indicating that the nuclei had been absorbed and used or were severely atrophied at this stage. At 10th day after anthesis, the nuclei of the DAPI stained isolated cells showed nuclear condensation and deepened staining, and the nuclear chromatin was gathered in a crescent shape on one side of the nuclear membrane, indicating that the cells had begun with apoptosis; 10 days after anthesis, the nuclei of the DAPI stained isolated cells broke into round vesicles of different sizes and were surrounded by the cell membrane, indicating that the cells had entered apoptosis.【Conclusion】The combination of paraffin sectioning, DNA ladder detection, DAPI staining and TUNEL labeling showed that with the increase of drought stress, the calyx tube ROS contents increased, leading to the inability of antioxidant enzymes to scavenge, thus affecting the development of the calyx tube free zone, affecting the PCD process and promoting the sepal abscission of Kuerlexiangli pear. This study showed that the effect of irrigation on sepal shedding of Kuerlexiangli pear was not significant during pre- flowering and flowering stage, while the effect of continuous drought stress on sepal shedding was mostly obvious.