Effects of canopy exposure and layers on lignin formation and related enzyme activities in endocarp of walnut

Abstract：【Objective】Walnut (Juglans regia L.) is one of the most important traditional economic forest trees in China. It is cultivated in 1 046 counties and cities across the country, and its cultivated area has increased by 10% per year in recent years. Walnut nuts are composed of hard shells and seed kernels. The seed kernels are rich in nutrients, and have the functions of brain- enhancing, nourishing blood, warming the lungs and invigorating the intestines, moistening and drying phlegm, etc., which are deeply loved by people. In the past times, the lignified hard shells were mostly abandoned and attention was rarely received. As an important part of walnut nuts, hard shell plays an important role in nut growth, development, maturation, rinsing, transportation and storage, and activated carbon produced from hard shells can effectively absorb mercury, acid dye, phenol, etc. in industrial wastewater. Therefore, it is of great significance to study the formation mechanism and influencing factors of walnut nut hard shell. In recent years, with the continuous expansion of walnut growing area, the early-bearing walnuts with short growth cycle, high yield and good all-in-one have been vigorously promoted. However,in the actual production, the early- bearing walnut orchard has a common problem that affects the appearance quality of walnut nuts, such as“white face”and“Lu Ren”(Exposed kernel). In order to improve this phenomenon, accelerate the development of the walnut industry and improve the economic value of the walnut market, the growth and development of endocarp and the main factors affecting its growth and development were studied to explore the dynamic changes of the effects of light intensities on the activity of lignin and related enzymes in endocarp during the development of walnut fruit.【Methods】‘Xinxin No. 2’cultivar with a planting space of 3 m × 6 m was used as a material in the walnut seedling demonstration garden of Xinjiang Academy of Forestry in Wensu county, Akesu region. Six sample trees with relatively consistent vigor were chosen and nuts were collected from the same part of each tree and mixed, and the mixed nut sample was divided into three parts as three replicates. During sampling, the sample tree was divided into different parts. Fruit samples were collected from the lower layer (less than 2.5 m from the base of the trunk), the middle layer (2.51 to 5 m from the base of the trunk), and the upper layer (more than 5m from the base of the trunk). On the horizontal distribution, fruit samples were picked in the middle of the tree (within 1.5 m from the central trunk), south and north (more than 1.5 m from the central trunk); from June 5, 2019 to July 26, 2019, at 7 d intervals. Sampling was performed once for a total of 7 times. After each picking, the fruits were classified and bagged according to different parts, and brought back to the laboratory for storage at -40 ℃ in a lowtemperature refrigerator for the determination of various indicators. To observe the continuous deposition process of endodermal lignin, the content of lignin and the activities of related enzymes (PAL, POD, PPO) were determined. According to the changes of lignin deposition, the trend of lignin content and the change of related enzyme activities were analyzed in relation to the different positions of the canopy. Effects of light intensities on endodermal lignin deposition, lignin content, and changes in related enzyme activities were studied, and correlation analysis was performed.【Results】Lignin deposition began to accumulate from both ends of the endocarp to the middle, and the color changed from cherry red to purplish red, and the thickness and hardness of the endocarp increased with the increase of walnut fruit. The lignin content continued to increase with the development of walnut fruits. On different exposures and in different canopy layers, the order of the lignin content was as follows: south & upper layer＞south & middle layer＞ south & lower layer＞ north & upper layer＞ north & middle layer＞ north & lower layer＞ middle & upper layer＞ middle & middle layer＞ middle & lower layer. On different exposures and in different canopy layers, the order of PAL and POD enzyme activity was as follows: south & upper layer＞ south & middle layer＞ south & lower layer＞ north & upper layer＞ north & middle layer＞ north & lower layer＞ middle & upper layer＞ middle & middle layer＞ lower & middle layer; The order of PPO enzyme activity was: north & lower layer＞ north & middle layer＞ north & upper layer＞ middle & lower layer＞ middle & middle layer＞ middle & upper layer＞ south & lower layer＞ south & middle layer＞ south & upper layer. Correlation analysis showed that lignin was significantly positively correlated with POD enzyme activity (p < 0.01, r = 0.808**), and negatively correlated with PPO enzyme activity.【Conclusion】The south-exposed and upper canopy layer received the highest light intensity, and the nuts had the highest lignin content, the strongest PAL and POD enzyme activities and the lowest PPO enzyme activity. The lignin content was extremely significantly positively related to the POD enzyme activity (p < 0.01, r = 0.808**), indicating that light intensity induced the lignin accumulation and enhanced the related enzyme activities in the endocarp of walnut fruits.