Expression characteristics of apoplastic ROS-related-enzyme genes at different development stages of Kuerlexiangli pear

Abstract: 【Objective】Kuerlexiangli pear (Pyrus sinkiangensis Yü) is a cultivar selected from a hybrid pear species of Xinjiang Autonomous Region in China. Kuerlexiangli pear is distinctive for its aroma, rich juicy flesh and crispy texture. However, recently the formation of rough skin fruits has been one of the major factors that adversely affect fruit quality, and increasing stone cell content is the typical phe- notype of rough skin fruits. Stones cells are dead ones derived from parenchyma cells and mainly com- posed of cellulose, hemicellulose and lignin. The formation of stone cells is closely related to reactive oxygen species (ROS) in plasma membrane, especially for apoplastic H2O2. Apoplastic H2O2 serving as an oxidant, plays important roles in oxidative coupling of lignin monomers, and cross-linking between cell wall polymers by diferulic linkages. In addition, H2O2 can also promote the process of programmed cell death (PCD). Since the polymerization of lignin is carried out on the cell wall, in this experiment, we tried to explore the possible relationship between the formation of stone cells and the apoplastic ROS production in Kuerlexiangli pear by analyzing the expression patterns of genes encoding, respiratory burst oxidase homologue D (RBOHD), superoxide dismutase (SOD), lipoxygenase(LOX), germin- like protein (GLP) with oxalate oxidase activity and polyamine oxidase (PAO) that were related to apo- plasic ROS production, so as to provide a scientific basis for regulating the content of stone cells in 'Ko- rla Fragrant Pear' fruit and improving fruit quality.【Methods】15 years old Kuerlexiangli pear trees were used as the plant materials to study the possible roles of apoplastic ROS during the formation of stone cells. Stone cell staining and relative expression levels of apoplastic ROS generating genes were then performed on fruit samples that were collected at five different developmental stages (10, 30, 60, 80 and 100 days after flowering, DAF), representing the prime stage (10-30 DAF), late (60 DAF) and sta- tionary stage (80-100 DAF) of stone cell differentiation, respectively. Stone cell staining and lignin content determination were also performed with the fruit pulp and pericarp of rough skin and normal fruits.【Results】The number of stone cells increased first and reached the highest level at 30 DAF, and at this period stone cell content also reached the highest level (19.64%). The stone cell content de- creased rapidly thereafter, with the growth of surrounding parenchyma cells and the trend starting to level off 80 DAF, and reached the lowest level of 3.7% 100 DAF with a 5.3-fold decrease. Meanwhile, the numbers and diameters of stone cells in the pulp and skin of rough skin fruits were significantly higher than those of normal fruits, and lignin contents in the pulp and skin of rough skin fruits were 1.12-2.7-fold higher than those of normal fruits. Meanwhile, qPCR results showed that expression trends of Germin-like protein 5-1 (GLP5-1) with OXO activity, RBOHD, SOD [Cu-Zn] 2-like, LOX ,LOX5 , PAO1 and PAO 5-like genes were also highly expressed at the early stages of fruit develop- ment, the expression levels of these enzyme genes decreased gradually with the development of fruit, and tended to be low and stable at later stages of fruit development (80-100 DAF). However, there were some differences in the occurrence of peak expression value among these genes, LOX and LOX5peaked at 10 DAF, GLP5-1 peaked at 60 DAF, and the rest peaked at 30 DAF, indicating that all of ap- oplastic ROS related genes showed peak expression during the prime stage of stone cell differentiation, the expression levels of these genes decreased rapidly at the stationary stage of stone cell differentia- tion, and expression levels of these genes ranged from 5 to 157 among the prime and stationary stages of stone cell differentiation. The result was consistent with the changes of stone cells in pear fruit, indi- cating that genes may participate in the differentiation of stone cells.【Conclusion】The increased con- tent of stone cells is one of the major factors that lead to the formation of rough skin in Kuerlexiangli pear. Apoplastic ROS, especially apoplastic H2O2, plays important roles in oxidative coupling of lignin monomers, cross-linking between cell wall polymers and the process of PCD. The time from 10 to 60 DAF was the key period for stone cells formation in Kuerlexiangli pear. Similarly, all of genes related to apoplastic ROS production were abundantly expressed at the early stage of fruit development, and expression levels gradually decreased along with fruit-development, indicating that apoplastic ROS accu- mulation basically overlapped with the period of stone cells formation. These results suggest that the ap- oplast may be the main source of ROS that participates in the process of stone cell differentiation.