Study on the relationship between the stone cell formation and apoptosis during the fruit development of ’Kuerlexiangli’ pear

【Objective】‘Kuerlexiangli’pear(Pyrus sinkiangensis Yu) is a native species of the Xinjiang Autonomous Region in China. At present, high stone cell content, which leads to rough skin, is one of the most important factors decreasing fruit quality. Stone cells are a type of sclerenchyma cell formed by the secondary thickening of cell walls followed by the deposition of lignin on the primary walls of parenchyma cells. While we understand a great deal of the genes involved in lignin synthesis and deposition, we know surprisingly little concerning the transition from the primary to the secondary cell wall synthesis. Secondary walls are formed between the plasma membrane and the primary walls in specialized cells after the cessation of cell expansion. They are mainly composed of cellulose, hemicelluloses and lignin, the proportions of which may vary in different cell types and plant species. Therefore, the development of stone cells cannot be fully explained by the deposition of lignin in the secondary cell walls. All the biosynthesis genes for cellulose, hemicelluloses and lignin need to be turned on as well as the genes responsible for the supply of nucleotide sugars, phenylpropanoid pathway precursors, methyl and acetyl donors and many other secondary wall biosynthetic pathway precursors need to be up-regulated to construct secondary walls. After their biosynthesis, lignin monomers must be transported to the cell wall where they are oxidized for polymerization, reactive oxygen species(ROS) plays a key role in this process. Meanwhile, ROS is the main trigger of programmed cell death(PCD). PCD,also called apoptosis, is an active behavior, genetically controlled either by so-called death receptors or intrinsic signals that direct the cell to eliminate itself during development and in response to pathogens or environmental insults. Therefore, it is speculated that the formation of stone cells is essentially the process of apoptosis. Some parenchymal cells in pear fruit stop growing and differentiate early at cell division stage. ROS, as signal molecules, activate apoptosis in some fruit cells and lignin deposits in the secondary cell walls of apoptotic cells to form stone cells. In order to reveal this scientific problem and theoretical inference, in this study, we explored the possible relationship between the formation of stone cells and apoptosis during the fruit development of‘Kuerlexiangli’pear. Knowledge gained through this study could be helpful for understanding the molecular mechanisms of stone cell formation in pear and could be further used to develop strategies to reduce or eliminate the stone cell formation.【Methods】To obtain a better insight into the stone cell formation process in fruit tissues, 15-year-old‘Kuerlexiangli’pear trees were used as test material to analyze the variation of stone cells by microscopic observation and content determination. Meanwhile, three different methods of apoptosis detection, namely Hoechst staining, DAPI(4’, 6-diamidino-2-phenylindole), and TUNEL(terminal dexynucleotidyl transferase dUTP nick-end labeling), were used to observe the chromatin condensation and DNA fragmentation in fruit cells during the nine different developmental stages. ROS content, as the main apoptotic stress factor, in the pulp of‘Kuerlexiangli’pear was also examined during fruit development.【Results】The content of stone cells increased first and then decreased during the fruit development. The value reached the peak of 20.22% at day 50 after flowering and decreased sharply thereafter. The content of stone cells reached its lowest level of 3.7% at 120 days after flowering. Microscopic observation results also showed that most of the stone cells were still in the initial stage of loosely aggregation at 10-20 d after flowering. Although some of stone cells had formed cluster at this period, they didn’t reach the maximum volume. The stone cells began to cluster at 30 days after flowering, forming a stone cell cluster.The numbers and size of the stone cells tended to be stable 50 d after flowering. Changes in ROS content were consistent with those in stone cells,increasing first and decreasing later. The staining test showed that apoptosis did occur in‘Kuerlexiangli’pear fruit during fruit development. TUNEL, DAPI and Hoechst staining indicated that the early stage of fruit development(10 d to 40 d after flowering)was the key stage of apoptosis in the pulp cells. Fluorescence signals of cell apoptosis were very weak or not detectable at the late stage of fruit development. From day 10 to day 50 was the key stage of stone cell formation in‘Kuerlexiangli’pear pulp.【Conclusion】Stone cell formation mainly occurs during the early stage of fruit development, and apoptosis occurs along with the development of stone cells. The apoptosis period basically overlaps with the period of stone cell formation and ROS accumulation.