Dynamic observation of split-pit nectarines and corresponding changes in lignin deposition-related enzyme activity

【Objective】The split- pit phenomenon impairs the appearance and commercial value of peach fruit, resulting in significant economic losses. This research investigated the relationship between pit splitting and physiological changes during endocarp development in nectarines, to provide a reference for preventing and controlling the disorder.【Methods】This research used a comparative physiological approach with two nectarine cultivars: the split-pit susceptible cultivar Zijinhong 3 and the split-pit resistant cultivar Zijinhong 1, which served as the control. The experimental design covered the full endocarp development period, from fruit set through to maturation. The dynamic process of lignin deposition in the endocarp was carefully tracked using histological staining techniques, with phloroglucinolHCl staining as the primary method, which specifically detects lignin. Concurrently, we conducted a comprehensive analysis of the physicochemical changes associated with endocarp development. Furthermore, we systematically measured the activities of key enzymes involved in the lignin biosynthesis pathway-including phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarateCoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), peroxidase (POD), and polyphenol oxidase (PPO)-across different developmental stages. To quantitatively assess the relationship between enzyme activities and lignin accumulation, grey relational analysis (GRA) was applied, which would provide a mathematical evaluation of the regulatory influence of these enzymes on pit-splitting susceptibility.【Results】The investigation revealed distinct patterns of endocarp development and lignification between the two cultivars. In Zijinhong 3, lignin accumulation commenced 36 days after full bloom (36 DAFB), displaying a basipetal deposition pattern extending from the stem end toward the stylar end. Lignification was largely completed by 54 DAFB, with pit splitting typically observed at 58 DAFB. By contrast, Zijinhong 1 initiated lignin deposition at the same developmental stage (36 DAFB) but completed the lignification process later, around 58 DAFB, which coincides with the occurrence of splitting in susceptible fruits. Throughout the developmental period, lignin content increased progressively in both cultivars. However, split-pit fruit exhibited a significantly lower deposition rate during the mid-development stage, resulting in a consistently lower final lignin content compared with normal endocarps. GRA identified PAL, C4H, and POD as the enzymes most strongly associated with lignin content in nectarine endocarp (GRA＞0.7), highlighting their pivotal roles in the lignification process. Detailed enzyme activity profiling revealed cultivar- specific and splitting- related patterns: PAL regulates the initial commitment to the phenylpropanoid pathway, predominantly influencing early deposition rates. C4H decreased to a minimum before cell splitting, but recovered in coincidence with the splitting event. CAD showed a contrasting trend during the critical splitting period: it increased in split- pit fruits while decreasing in normal fruits. Both cultivars exhibited 4CL activity peaks at 58 DAFB, with similar fluctuation patterns around this peak, though their absolute activity levels differed. POD activity first increased, then decreased before splitting, and subsequently remained stable with minor fluctuations. PPO displayed a distinct cultivar- specific regulatory pattern. Furthermore, hemicellulose content varied significantly between cultivars, suggesting that hemicellulose may play an important role in maintaining the integrity of cell wall assembly. Crucially, at 58 DAFB-the pivotal splitting period-the activities of C4H, 4CL, POD, and PPO in split-pit fruits of Zijinhong 3 were significantly lower than those in normal fruits, indicating a coordinated dysregulation of multiple enzymes in the lignin biosynthesis pathway of susceptible fruits. GRA revealed a strong association (GRA＞0.7) between C4H, POD, and PPO activities and lignin content in both normal and split- pit fruits of Zijinhong 3. For Zijinhong 1, the enzymes with strong associations (GRA＞0.7) were PAL, C4H, 4CL, and PPO in normal fruits, while this changed to PAL, 4CL, CAD, and PPO in split-pit fruits.【Conclusion】The occurrence of pit splitting in oil peaches is closely related to lignin deposition, and the severity of pit splitting varies among different varieties. In the variety Zijinhong 3, which exhibits severe pit splitting, the activities of C4H, POD, and PPO enzymes in the inner pericarp are lower than in non-split fruits. This leads to insufficient lignin accumulation and, consequently, the occurrence of pit splitting.