Effects of cinnamic acid and 4-coumaric acid on shell development and physiological characteristics in walnut

Abstract:【Objective】The germplasm resources of walnut in China are abundant. Walnut varieties with different hard shell thickness, such as thick shell, thin shell, paper shell and exposed-kernel shell, are available in Xinjiang, which provides a good material basis for the study on walnut shell. The thickness of walnut shell is largely affected by the amount of lignin deposited. Cinnamic acid and 4-coumaric acid are products of the lignin metabolic pathway. Therefore, in this paper the effects of different concentrations of cinnamic acid and 4-coumaric acid applied to the Xinlu walnut sample was studied on the development of Endcoarp (hard shell). Through the study on the development of inner pericarp (hard shell) of Xinlu walnut, a unique variety in Xinjiang, the mechanism of kernel exposure and the mechanism of thin shell in Xinjiang were discussed.【Methods】In Wensu County Experimental Forest Farmof Aksu, Xinjiang Uygur Autonomous Region, the inner peel of Xinlu walnut was used as the test material, which was collected from over 15 years old trees at a spacing of 5 m×6 m, with vigorous growth, without pests and diseases, and with consistent water and fertilizer management. In the hardening stage of walnut shell, the walnut tree canopy was sprayed with different concentrations of cinnamic acid and 4-coumadic acid, respectively. The effects of cinnamic acid and 4-coumaric acid on lignin deposition, components of walnut shell (lignin, cellulose and hemicellulose), metabolic enzyme activity (PAL, CAD, C4H and POD) and total phenol and sugar contents during the hardening process of walnut shell were investigated.【Results】Compared with the control, there was a significant difference in the accumulation of lignin in walnut endocarp after treatment with both low and high concentrations of cinnamic acid and 4-coumaric acid. At the later stage of treatment, the endocarp was partially hardened, and there was still a phenomenon of endocarp missing or incompleteness with time. The contents of lignin, cellulose and hemicellulose generally increased with the treatment of cinnamic acid and 4-coumaric acid. The changes of lignin deposition in walnut peel during the shell hardening period were observed by phlorogluphenol staining. The deposition began at the position near the vascular bundle about 64 days after flowering, and the lignin content significantly increased up to 69.69% after 4-coumaric acid treatment (p≤0.05); Cellulose content increased by 33.92% and hemicellulose content increased by 19.6%. After treatment with cinnamic acid, the lignin content increased by 58.98% and hemicellulose content increased by 29.16%. There was a downward trend of PAL enzyme with both cinnamic acid and 4-coumaric acid treatments, and the highest PAL enzyme activity with 4-coumarin treatment was 447.25 U·g^-1 higher than CK at a certain concentration. Low concentration of 4-coumaric acid treatment could promote PAL activity in pre-hardening, while high concentration could inhibit PAL activity in pre-hardening. After treatment with cinnamic acid, PAL activity was significantly higher than CK at 0.2-1.5 h (p≤ 0.05), and the peak value of PAL activity appeared after treatment, which increased by 12.91% compared with CK. PAL activity increased after treatment with cinnamic acid, and 100 mg·L^-1 to 150 mg·L^-1 was better than 50 mg·L^-1 at the early shell-hardening stage. Low concentration of 4-coumaric acid promoted CAD activity, but high concentration of 4-coumaric acid inhibited CAD activity, and a certain concentration of cinnamic acid increased CAD activity by 1.21 times compared with CK. C4H enzyme activity increased first and then decreased, C4H enzyme content decreased rapidly from day 4 after CK treatment, and C4H enzyme began to decrease rapidly 9 days after 4-coumaric acid and cinnamic acid treatments. Under a certain concentration, 4-coumaric acid and cinnamic acid can delay the decrease of C4H content. POD showed a decreasing trend during the hardening process of walnut shell, and POD activity decreased with 4-coumaric acid and cinnamic acid treatment. With the development of walnut pericarp, the total phenol content did not change significantly. After treatment with cinnamic acid, the total phenol content decreased, and reached a peak value of 49.6 mg · g^-1 after 0.2 day of treatment. The content of total sugar in the endocarp was very low, and the content of total sugar was higher than that of CK after 4-coumaric acid treatment for 1.5 days, with the highest increase of 4.82 mg·g-1 . Lignin was positively correlated with cellulose and hemicellulose contents (p≤0.01), indicating that the higher the cellulose content of lignin, the higher the hemicellulose content. The main components of walnut shell (lignin, cellulose and hemicellulose contents) were significantly correlated with the activities of PAL, CAD and POD (p≤0.05), indicating that PAL, CAD and POD are the key components of walnut shell. 【Conclusion】50-150 mg·L^-1 cinnamic acid and 50-150 mg·L^-1 4-coumaric acid promoted the accumulation of lignin in walnut pericarp, but 200 mg·L^-1 4-coumaric acid inhibited lignin synthesis.