采后BTH处理对苹果果实苯丙烷代谢和病程相关蛋白积累的增强作用

1 材料与方法

1.1 材料与仪器

国光苹果于2013年9月采自甘肃省国营条山农场,单果包装入纸箱后当天运抵甘肃农业大学采后生物学实验室,常温((22±2)℃,RH 50%～60%)下贮藏待用;BTH由Novatis公司提供(有效浓度50%)。

UV-2450型分光光度计日本岛津;H-1850R型离心机长沙湘仪离心机仪器有限公司;WYX-A微型旋涡混合器上海跃进医疗器械厂。

1.2 实验方法

1.2.1 BTH处理大小均匀,无机械损伤和病虫害的果实分别用100mg/L BTH溶液和清水浸泡(对照)10min(内含0.05% Tween 20),取出晾干后入包装箱,于常温条件下((22±2)℃,RH 55%～60%)贮藏待用,每处理用果实30个,重复3次。

1.2.2 取样参照范存斐等方法[16]。BTH和清水处理后的果实分别于0、2、4、6、8d取皮下1～5mm 处果肉组织3g,铝箔纸包裹,用液氮速冻后在-80℃超低温冰箱中保存待用。

1.2.3 苯丙氨酸解氨酶(PAL)活性测定参照Liu等方法[8]。酶反应体系包括:2mL 0.2mol/L硼酸缓冲液(pH8.8),300μL酶粗提液和1mL L-苯丙氨酸(0.02mol/L)。24℃下反应2min 后测其OD290。空白以缓冲液代替酶粗提液。以U/g FW(鲜重)表示酶活性,每分钟内OD290变化0.01为1个活性单位(U)。

1.2.4 4-香豆素-辅酶A连接酶(4 CL)活性测定参照范存斐等方法[16]。以每分钟吸光值变化0.01为1个活性单位(U),4 CL活性以U/g FW表示。

1.2.5 总酚和类黄酮含量的测定参照范存斐等方法[16]。总酚含量以OD280/g FW表示,类黄酮含量表示为OD325/g FW。

1.2.6 木质素含量的测定参照范存斐等方法[16]。木质素含量以OD280/g FW表示。

1.2.7 过氧化物酶(POD)活性的测定参照葛永红等方法[17]并修改。酶反应体系包括1.8mL愈创木酚(0.05mol/L),30μL酶粗提液和100μL,2% H2O2,然后于470nm波长下测定吸光度的变化,以每分钟内OD470变化0. 01为1个活性单位(U),POD活性以U/g FW表示。

1.2.8 β-1,3-葡聚糖酶(GLU)活性的测定参照Ge等方法[18]并修改。酶反应体系包括0.8mL,1mg/mL昆布多糖,0.2mL酶液,2mL铜试剂,2mL砷钼酸试剂,反应后在660nm处测定吸光度,对照标准曲线求出样品液中的葡萄糖量。在最适反应条件下(37℃)下每秒钟每升酶液催化底物昆布多糖产生1μmol的葡萄糖为一个酶活力单位(U)。

1.2.9 几丁质酶(CHT)活性的测定参照葛永红等方法[17]。以每分钟生成1μmol N-乙酰氨基葡萄糖所需的酶量为一个酶活力单位(U)。

1. 3 数据处理

全部实验数据用Microsoft Excel 2003 处理并作图,用SPSS16.0统计软件进行LSD统计分析,图中以标准误差(±SE)表示。

2 结果与分析

2.1 BTH处理对苹果果实PAL、4CL活性的影响

PAL 是苯丙烷代谢途径的一个限速酶,其活性高低与酚类、黄酮类和木质素的积累密切相关。随着贮藏时间的延长,苹果果实PAL先升高后降低,但BTH处理显著提高了果实PAL活性,在贮藏第4d达到最大,高出对照71.4%,第8d BTH处理果实PAL活性又略有升高,而对照则呈下降趋势(图1A)。4CL活性的变化趋势基本和PAL一致,BTH处理也显著提高了苹果果实4CL活性,在贮藏第6d活性达到最大,高出对照32.2%(图1B)。

图1 采后BTH处理对苹果果实PAL(A) 和4CL(B)活性的影响Fig.1 Effect of postharvest BTH dipping treatment on PAL(A)and 4CL(B)activities in apple fruit

2.2 BTH处理对苹果果实总酚和类黄酮含量的影响

总酚和类黄酮是苯丙烷代谢的产物,其含量高低与果实预防病原菌的侵入有关。处理和对照苹果果实总酚含量在贮藏期间总体呈先升高后下降趋势,但BTH处理显著提高了果实总酚含量,并且在贮藏第4d出现高峰,高出对照25.9%(图2A)。贮藏期间果实类黄酮含量先升高后降低,BTH处理果实类黄酮含量显著高于对照果实,如在贮藏第4d时类黄酮含量高出对照27.2%(图2B)。

图2 采后BTH处理对苹果果实总酚(A) 和类黄酮(B)含量的影响Fig.2 Effect of postharvest BTH dipping treatment on the content of total phenolic compounds(A) and flavonoids(B)in apple fruit

2.3 BTH处理对苹果果实木质素含量的影响

木质素是苯丙烷代谢途径的产物,果实中木质素的积累有利于增强细胞壁厚度,从而提高对病原物的抵抗力。苹果果实在贮藏期间,木质素含量总体呈先升高后下降趋势,对照果实中木质素含量一直维持在一个较低的水平,但BTH处理果实中木质素含量显著高于对照,并且在贮藏第6d达到最大,高出对照35.4%(图3)。

图3 采后BTH处理对苹果果实木质素含量的影响Fig.3 Effect of postharvest BTH dipping treatment on lignin content in apple fruit

2.4 BTH处理对苹果果实POD活性的影响

POD广泛存在于植物中,在果实体内主要参与活性氧的清除、木质素的合成等过程。在整个贮藏期间对照和BTH处理果实POD活性都呈先上升后下降的趋势,但是BTH处理果实POD活性显著高于对照,如在贮藏第4、6d分别高出对照49.8%和55.8%(图4)。

图4 采后BTH处理后苹果果实POD活性的变化Fig.4 Changes in the activity of POD in apple fruit after BTH dipping treatment

2.5 BTH处理对苹果果实GLU和CHT活性的影响

GLU和CHT是两种主要的病程相关蛋白,主要用来降解病原物细胞壁,从而导致病原物死亡。贮藏期间对照果实GLU活性总体维持在一定水平,而BTH处理显著提高果实中GLU活性,在贮藏第6d出现活性高峰,高出对照63.7%(图5A)。CHT活性总体呈先升高后降低趋势,但BTH处理果实中CHT活性显著高于对照果实,在贮藏第6d达到最大,高出对照30.3%(图5B)。

图5 采后BTH处理后苹果果实GLU(A) 和CHT(B)活性的变化Fig.5 Changes in the activity of GLU(A)and CHT(B) in apple fruit after BTH dipping treatment

3 讨论

苯丙烷代谢是植物和果蔬中次生代谢产物合成的主要途径,在抗病中具有重要的作用[19]。PAL和4CL是该途径中的关键酶[25],PAL能够调控植物和果实中酚类、黄酮类、生物碱、花青素、木质素等抗菌物质的合成,木质素的合成有利于强化细胞壁,增强对病原物侵染的抵抗力[20],而4CL主要调控苯丙烷代谢主途径向分支途径的转折[21]。本实验表明,BTH能显著诱导苹果果实中PAL和4CL活性的升高,同时促进了果实中总酚、类黄酮和木质素的积累。桃[8]、梨[9],厚皮甜瓜[22]、草莓[23]等果实的研究中也得到了类似的结果。本实验还发现BTH处理苹果果实贮藏后期PAL活性有所升高,这与我们在甜瓜果实上的研究结果类似[22],可能是因为前期PAL和4CL活性的升高促进了黄酮类、生物碱和花青素的积累,而木质素的合成过程比较复杂,需要在POD、过氧化氢及漆酶的共同作用下合成,因此后期PAL的升高主要是调控木质素的合成。还有研究发现采前BTH处理也能诱导甜瓜果实中苯丙烷代谢产物的积累,从而降低潜伏侵染的发生率[28]。酚类物质在多酚氧化酶(PPO)的作用下氧化成对病原菌产生高毒性的醌类物质,直接抑制侵染寄主的病原物生长[24]。此外,酚类物质还可以作为木质素合成的前体物质,促进细胞壁中木质素的积累,从而增厚细胞壁提高对病原物抵抗力[25-26]。类黄酮具有直接的抑菌活性,可抑制真菌孢子萌发、芽管伸长和菌丝生长[27]。由此表明,采后BTH处理可明显激活苹果果实体内酚类和类黄酮合成途径,以增加果实对病原物侵染的抵抗能力。

病程相关蛋白(Pathogenesis-related proteins,PRs)是植物受到病原菌侵染或非生物胁迫条件下产生的小分子蛋白质,在抗病过程中发挥着重要作用[21]。目前已经发现的PRs有17种,根据其结构和功能的不同表示为PR-1至PR-17,其中PR-2和PR-3,8,11分别具有GLU和CHT活性,可以直接分解真菌细胞壁主要成分为葡聚糖和几丁质,破坏其结构[21]。本研究结果发现,BTH处理可显著提高GLU和CHT活性。已有研究报道,采前或采后BTH处理能够诱导甜瓜[28]、桃[8,29]、芒果[30]、梨[31]果实中GLU和CHT的活性,从而提高果实的抗病性。PR9具有POD活性,参与果实体内木质素的合成、细胞壁结构蛋白的交联以及活性氧的清除等[15,21]。本研究结果表明,BTH处理诱导了POD活性的提高,促进了木质素含量的积累,提高了果实的抗病性。这与在甜瓜[17]、砂糖橘[32]、芒果[33]上的研究结果相一致。此外,BTH处理还能诱导芒果和番木瓜果实的过氧化物酶基因(POD)表达[34-35]。有关BTH处理后苹果果实中苯丙烷代谢和病程相关蛋白基因表达有待进一步分析研究。

4 结论

采后100mg/L BTH处理显著提高了苹果果实PAL和4CL活性,并且促进了苯丙烷代谢产物总酚、类黄酮和木质素含量的提高。BTH处理还促进了苹果果实POD,GLU和CHT活性的提高。由此表明,采后BTH处理可通过激活苹果果实苯丙烷代谢途径增强组织结构,促进病程相关蛋白的产生,从而提高了果实抵抗病原物侵染的能力。

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Activation of postharvest BTH treatment on phenylpropanoid pathwayand pathogenesis-related proteins accumulation in apple fruit

GE Yong-hong1,LI Can-ying1,ZHU Dan-shi1,DONG Bo-yu2,LAN Lin2

(1.College of Chemistry,Chemical Engineering and Food Safety,Jinzhou 121013,China;2.College of Food Science and Engineering,Gansu Agricultural University,Lanzhou 730070,China)

The effect of benzothiadiazole(BTH)dipping on phenylpropanoid pathway and accumulation of pathogenesis-related proteins in apple fruit was investigated in this paper. Apple fruit(cv. Guoguang)were dipped in 100mg/L BTH for 10min to study the effect of BTH on key enzymes activities,metabolites accumulation in phenylpropanoid pathway and production of pathogenesis-related proteins(PRs). The results indicated that BTH treatment significantly increased the activities of phenylalanine ammonia-lyase(PAL)and 4-coumaric acid-coenzyme A ligase(4CL),and the content of total phenolic compounds,flavonoids and lignin. The activities of β-1,3-glucanase(GLU),chitinase(CHT)and peroxidase(POD)were also induced by BTH treatment. These results suggest that postharvest BTH treatment induced disease resistance in apple fruit by activating phenylpropanoid pathway and promoted the accumulation of PRs.

fruit;benzothiadiazole(BTH);phenylpropanoid pathway;pathogenesis-related proteins;induced resistance

2014-06-03

葛永红(1979-)，男，博士，副教授，研究方向：果蔬贮藏与保鲜。

国家自然科学基金(31160405)；渤海大学博士启动基金(bsqd201405)；辽宁省食品安全重点实验室项目(LNSAKF2013021)。

TS255.1

1002-0306(2015)05-0306-05

10.13386/j.issn1002-0306.2015.05.056