果树学报2026，Vol.43Issue(2)：323-335,13.DOI:10.13925/j.cnki.gsxb.20250209
核桃种皮多酚类物质差异分析
Analysis of differences in phenolic compounds in walnut kernel pellicle
摘要
Abstract
[Objective]This research investigated the variation in tannin and other phenolic compounds in the kernel pellicle of different walnut(Juglans regia L.)cultivars,aiming to elucidate the mecha-nisms underlying their biosynthesis and accumulation as well as to provide a theoretical foundation for targeted improvement of quality traits related to phenolic composition in walnut.[Methods]Three wal-nut cultivars exhibiting distinct tannin levels were selected,including low-tannin Bingta No.1(BT-1),medium-tannin Wen 185(W-185),and high-tannin Zha 343(Z-343).Fruits were collected from five-year or older,vigorously growing trees under consistent water and fertilizer management at the XPCC(Xinjiang Production and Construction Corps)walnut germplasm repository(40°23′N,80°03′E).Sam-pling occurred at eight critical developmental stages:T1(June 12),T2(June 18),T3(June 28),T4(July 5),T5(July 15),T6(July 25),T7(August 9),and T8(August 24).Fresh seed coats were fixed in FAA for paraffin sectioning.Sections were dehydrated through graded ethanol solutions,cleared in xylene,embedded in paraffin,and stained with safranin-fast green.Tannin cells were specifically identified by the purple-red coloration resulting from the safranin-tannin reaction,observed and photographed using optical microscopy.Tannin spatial distribution during development was visualized via imprint staining.Fresh fruit cross-sections were pressed onto a piece of filter paper,which formed purple imprints due to oxidative property of tannins.Total tannin content was determined spectrophotometrically using Folin reagent with gallic acid as standard.Total phenolic content was assayed using the Folin-Ciocalteu meth-od(gallic acid equivalent).Total flavonoid content was determined by the aluminum nitrate colorimet-ric method(rutin equivalent).Phenolic composition was analyzed by HPLC using an Agilent TC-C18(2)column(250 mm×4.6 mm,5 μm)at 35℃.The mobile phase consisted of(A)50%methanol and(B)0.2%aqueous acetic acid,with a gradient elution(A:8%to 40%from 0-40 min,then back to 8%).Flow rate was 0.8 mL·min-1,and injection volume 10 μL.Detection was at 280 nm.Six phenolic acids(shikimic,gallic,caffeic,vanillic,chlorogenic,and p-coumaric)and four flavonoids(catechin,epicate-chin,rutin,and myricetin)were quantified using external standards.[Results]The tannin content in the kernel pellicle of all three cultivars exhibited a pattern of"decline-rise-decline"during fruit develop-ment.Significant differences(P＜0.05)were observed in the timing of key fluctuation nodes and peak accumulation intensity.Peak concentrations were 352.75 mg·g-1,386.84 mg·g-1,and 448.99 mg·g-1for BT-1,W-185 and Z-343.BT-1 accumulated significantly less tannin than W-185 and Z-343 at peak.Im-print staining confirmed tannin deposition was localized specifically within the seed coat,with staining intensity following a"light-dark-light"trend with fruit maturation.Paraffin section revealed that tannin accumulation was primarily associated with specialized tannin cells surrounding the vascular bundles.These cells function as key sites for tannin synthesis,storage,and transport.Cultivars differed markedly in tannin cell abundance and activity.During peak accumulation,W-185 and Z-343 possessed numer-ous,metabolically active,densely packed tannin cells,whereas BT-1 had fewer such cells.This results agreed with the lower tannin synthesis rate and content observed in BT-1.HPLC analysis revealed shi-kimic acid as the predominant phenolic acid and epicatechin as the major flavonoid across cultivars,with distinct accumulation dynamics.BT-1 exhibited significantly lower levels of shikimic acid,gallic acid,p-coumaric acid,and epicatechin compared toW-185/Z-343;Shikimic,vanillic,and chlorogenic acids accumulated continuously,while caffeic acid declined steadily.In Z-343,chlorogenic acid content increased markedly at T5 stage(from 28.32 to 76.29 mg·g-1);rutin accumulated continuously,peaking at 10.54 mg·g-1.In BT-1,a sharp transient increase in rutin was observed at T3 stage.Pearson correla-tion analysis revealed cultivar-specific regulatory networks linking tannins to other phenolics.In BT-1,tannin positively correlated with p-coumaric acid and epicatechin,but negatively correlated with rutin and total flavonoids;in W-185,tannin showed an extremely significant positive correlation with gallic acid(P＜0.01),but negative correlations with caffeic acid and total phenolics;in Z-343,tannin correlat-ed positively only with total phenolics and caffeic acid,but negatively with most phenolic acids.These distinct correlation patterns indicate that variation in partitioning of phenylpropanoid metabolic flux,mediated by differential precursor supply(e.g.,shikimic acid)and the balance of competitive metabo-lites(e.g.,rutin vs tannin precursors),is a critical factor driving divergent tannin accumulation strategies among cultivars,and ultimately influences tannin biosynthetic efficiency.[Conclusion]Tannin accumu-lation in walnut kernel pellicle is co-regulated by cellular structural morphology and phenolic biosynthe-sis pathways.The low tannin phenotype of Bingta No.1(BT-1)results from:(1)a reduced number of tannin cells,limiting synthesis capacity;(2)diminished accumulation of key precursors like shikimic ac-id;and(3)competitive metabolic flux towards rutin synthesis,potentially diverting substrates away from the tannin pathway.In contrast,cultivars Wen 185(W-185)and Zha 343(Z-343)achieve higher tannin accumulation through more abundant tannin cells and enhanced provision of precursors,such as chlorogenic acid in Z-343 and gallic acid in W-185.The identified differentiation in phenolic metabo-lism provides a biochemical basis for targeted regulation of tannin synthesis.As an excellent low-tannin germplasm,Bingta No.1 possesses unique metabolic characteristics valuable for dissecting the molecu-lar mechanisms controlling tannin biosynthesis.Future research should employ multi-omics technolo-gies to identify key regulatory genes within these pathways,enabling precision breeding or biotechno-logical approaches to modulate astringency and improve walnut quality.关键词
核桃种皮/单宁/酚类物质/高效液相色谱Key words
Walnut kernel pellicle/Tannin/Phenolic substances/High performance liquid chromatogra-phy分类
农业科技引用本文复制引用
崔帅帅,马梦琪,丁洋洋,张洪华,虎海防,王红霞,潘志勇,张锐,郭众仲..核桃种皮多酚类物质差异分析[J].果树学报,2026,43(2):323-335,13.基金项目
新疆维吾尔自治区重点研发计划项目(2024B02017) （2024B02017）
南疆重点产业创新发展支撑计划(2022DB022) （2022DB022）
"天山英才"培养计划(2022TSYCCX0120) （2022TSYCCX0120）
塔里木大学研究生科研创新项目(TDGRI202331) （TDGRI202331）
兵团指导性科技计划项目(2024ZD113) （2024ZD113）
塔里木大学校长基金胡杨英才(博士)项目(TDZKBS202419) （博士）
