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1 222 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 沈丽红等 / 一氧化氮信号在脱落酸诱导丹参酚酸类成分积累中的作用 February 25, 2016, 32(2): Chin J Biotech, All rights reserved 组织工程与细胞培养沈丽红 1, 任加惠 1, 金雯芳 1, 王瑞杰 1, 倪春红 1, 童梦姣 1, 梁宗锁 1,2, 杨东风 ,2,,,.., 2016, 32(2): Shen LH, Ren JH, Jin WF, et al. Role of NO signal in ABA-induced phenolic acids accumulation in Salvia miltiorrhiza hairy roots. Chin J Biotech, 2016, 32(2): 摘要 : 为探讨一氧化氮 (Nitric oxide,no) 信号在脱落酸 (Abscisic acid,aba) 诱导丹参酚酸类成分积累中的作用, 采用不同浓度一氧化氮外源供体硝普钠 (Sodium nitroprusside,snp) 处理丹参毛状根,6 d 后采收, 测定酚酸类成分含量 ;ABA 联合一氧化氮清除剂 (2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl- 3-oxide,c-PTIO) 或一氧化氮合酶抑制剂 (N G -nitro-l-arginine methyl ester,l-name) 对丹参毛状根进行处理, 测定酚酸类成分含量和关键基因表达量结果表明,100 μmol/l SNP 对丹参毛状根中迷迭香酸与丹酚酸 B 积累的诱导效果最显著, 迷迭香酸和丹酚酸 B 含量分别增加了 3 倍和 4 倍 ABA 处理能显著促进 PAL (Phenylalanine ammonia lyase) TAT (Tyrosine aminotransferase) 和 RAS (Rosmarinic acid synthase) 基因的表达, 促进丹参毛状根中酚酸类成分的积累, 而联合 c-ptio 或 L-NAME 共同处理后,3 种关键基因表达下调, 并显著抑制了酚酸类成分的积累研究证明 NO 和 ABA 均能够促进丹酚酸类成分的积累,NO 信号可能介导了 ABA 对丹酚酸生物合成的诱导作用 : 丹参, 一氧化氮, 脱落酸, 酚酸 Received: June 12, 2015; Accepted: September 22, 2015 Supported by: Natural Science Foundation of Zhejiang Province (No. LQ13C020003), National Natural Science Foundation of China (Nos , ). Corresponding authors: Zongsuo Liang. Tel/ Fax: ; liangzs@ms.iswc.ac.cn Dongfeng Yang. Tel/Fax: ; ydf807@sina.com (No. LQ13C020003) (Nos , )

红 1, 童梦姣 1, 梁宗锁 1,2, 杨东风 1 310018 2 310018 1,2,,,.., 2016, 32(2): 222 230. Shen LH, Ren JH, Jin WF, et al.

2 沈丽红等 / 一氧化氮信号在脱落酸诱导丹参酚酸类成分积累中的作用 223 Role of NO signal in ABA-induced phenolic acids accumulation in Salvia miltiorrhiza hairy roots Lihong Shen 1, Jiahui Ren 1, Wenfang Jin 1, Ruijie Wang 1, Chunhong Ni 1, Mengjiao Tong 1, Zongsuo Liang 1,2, and Dongfeng Yang 1,2 1 School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou , Zhejiang, China 2 Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Hangzhou , Zhejiang, China Abstract: To investigate roles of nitric oxide (NO) signal in accumulations of phenolic acids in abscisic acid (ABA)-induced Salvia miltiorrhiza hairy roots, S. miltiorrhiza hairy roots were treated with different concentrations of sodium nitroprusside (SNP)-an exogenous NO donor, for 6 days, and contents of phenolic acids in the hairy roots are determined. Then with treatment of ABA and NO scavenger (2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide, c-ptio) or NO synthase inhibitor (N G -nitro-l-arginine methyl ester, L-NAME), contents of phenolic acids and expression levels of three key genes involved in phenolic acids biosynthesis were detected. Phenolic acids production in S. miltiorrhiza hairy roots was most significantly improved by 100 μmol/l SNP. Contents of RA and salvianolic acid B increased by 3 and 4 folds. ABA significantly improved transcript levels of PAL (phenylalanine ammonia lyase), TAT (tyrosine aminotransferase) and RAS (rosmarinic acid synthase), and increased phenolic acids accumulations. However, with treatments of ABA+c-PTIO or ABA+L-NAME, accumulations of phenolic acids and expression levels of the three key genes were significantly inhibited. Both NO and ABA can increase accumulations of phenolic acids in S. miltiorrhiza hairy roots. NO signal probably mediates the ABA-induced phenolic acids production. Keywords: Salvia miltiorrhiza, nitric oxide, abscisic acid, phenolic acids Salvia miltiorrhiza Bunge ( B ) ( Ⅰ ⅡA ) [1] B B [2-3] [4] (PAL) (TAT) (HPPR) 4-4- A 4- (RAS) P450 [5] PAL TAT RAS B [6] (Abscisic acid ABA) (Nitric oxide NO) [7] NO ABA cjb@im.ac.cn

Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, Hangzhou 310018, Zhejiang, China Abstract: To investigate roles of nitric oxide (NO) signal in accumulations of phenolic

3 224 ISSN CN /Q Chin J Biotech February 25, 2016 Vol.32 No.2 ABA [8] NO ABA (Sodium nitroprusside SNP) NO c-ptio (2-(4-carboxy- 2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 -oxide) NO [9] L-NAME (N G -nitro-larginine methyl ester) NO [10-11] SNP c-ptio NO L-NAME NO NO ABA 1 材料与方法 1.1 诱导子与抑制剂的制备 (ABA) (Wolsen ) (SNP) L-NAME (Sigma ) c-ptio (Amresco ) 0.22 µm 1.2 毛状根培养及处理 ( ATCC15834 ) 30 g/l 6,7-V 0.2 g 50 ml 110 r/min μmol/l 18 d c-ptio (100 μmol/l) L-NAME (200 μmol/l) ABA (50 μmol/l) 18 d 酚酸类有效成分的提取与 HPLC 定量分析 0.45 mm 0.10 g 2 ml 45 min r/min 15 min 0.45 µm B B 10 µl B B Y=1.3E+7X+3.0E+5 R 2 = Y=3.3E+7X 2.4E+5 R 2 = Waters 1525 Waters 2996 Waters SunFire C18 (250 mm 4.6 mm 5 µm) Empower ml/min µl 270 nm t = 0 min 40% t = 5 min 60% t = 20 min 60% t = 23 min 80% t = 25 min 100% 1.4 RNA 分离 cdna 合成及荧光实时定量分析 24 d RNA Trizol PrimeScript RT reagent Kit (TaKaRa Tokyo ) cdna RT-PCR

NO L-NAME NO NO ABA 1 材料与方法 1.1 诱导子与抑制剂的制备 (ABA) (Wolsen ) (SNP) L-NAME (Sigma ) c-ptio (Amresco ) 0.22 µm 1.2 毛状根培养及处理 ( ATCC15834 ) 30 g/l 6,7-V 0.

4 沈丽红等 / 一氧化氮信号在脱落酸诱导丹参酚酸类成分积累中的作用 225 Brilliant Ⅱ SYBR Green QPCR Master Mix (Agilent Santa Clara ) 10 µl 5.2 µl Brilliant Ⅱ SYBR Green QPCR Master Mix, 0.4 µl 10 μmol/l 0.4 µl 10 μmol/l 1.0 µl cdna 3.0 µl s s min S PAL TAT RAS 18S C t = 2 - Ct C t =(C t C t ) (C t C t ) DPS 5% (a b c ) 2 结果与分析 2.1 硝普钠对丹参毛状根生长的影响 1 50 μmol/l 100 μmol/l (200 μmol/l) (25 μmol/l) 图 1 SNP ABA c-ptio 和 L-NAME 对丹参毛状根生长状况的影响 Fig. 1 Effects of SNP and ABA, c-ptio, L-NAME on the growth of S. miltiorrhiza hairy roots. Different letters (such as a, b, c) indicate significant difference at P 0.05 using Duncan s multiple-range test cjb@im.ac.cn

0 µl 95 30 s 95 15 s 58 30 min 40 3 18S PAL TAT RAS 18S C t = 2 - Ct C t =(C t C t ) (C t C t ) DPS 5% (a b c ) 2 结果与分析 2.

5 226 ISSN CN /Q Chin J Biotech February 25, 2016 Vol.32 No.2 14% 16% ABA g 14% ABA c-ptio L-NAME g g 11% 21% c-ptio L-NAME 2.2 硝普钠对丹酚酸类成分积累的影响 B 2 50 μmol/l 200 μmol/l B μmol/l B mg/g 3 B mg/g 4 NO 2.3 c-ptio 和 L-NAME 对 ABA 诱导丹酚酸类成分积累的影响 3 ABA B 7.5 mg/g 20.5 mg/g 45% 40% c-ptio L-NAME ABA ABA ABA+c-PTIO B 5.3 mg/g 16.6 mg/g ABA 31% 27% ABA+L-NAME 4.6 mg/g 14.2 mg/g ABA 40% 38% c-ptio L-NAME ABA NO ABA 图 2 SNP 对丹参毛状根中酚酸类成分积累的影响 Fig. 2 Effects of SNP on the accumulation of phenolic acids in S. miltiorrhiza hairy roots. Different letters (such as a, b, c) indicate significant difference at P 0.05 using Duncan s multiple-range test. 图 3 ABA 及 c-ptio L-NAME 对丹参毛状根中酚酸积累的影响 Fig. 3 Effects of ABA, c-ptio and L-NAME on the accumulation of phenolic acids in S. miltiorrhiza hairy roots. Different letters (such as a, b, c) indicate significant difference at P 0.05 using Duncan s multiple-range test.

5 mg/g 45% 40% c-ptio L-NAME ABA ABA ABA+c-PTIO B 5.3 mg/g 16.6 mg/g ABA 31% 27% ABA+L-NAME 4.6 mg/g 14.2 mg/g ABA 40% 38% c-ptio L-NAME ABA NO ABA 图 2 SNP 对丹参毛状根中酚酸类成分积累的影响 Fig.

6 沈丽红等 / 一氧化氮信号在脱落酸诱导丹参酚酸类成分积累中的作用 c-ptio 和 L-NAME 对丹酚酸合成关键基因表达的影响 (PAL) (TAT) (RAS) 3 ABA ABA PAL TAT RAS 4 ABA PAL TAT RAS c-ptio L-NAME 3 ABA c-ptio ABA PAL TAT RAS 97% 48% 79% ABA L-NAME 72% 78% 63% NO c-ptio NO L-NAME ABA 图 4 c-ptio 和 L-NAME 对丹参毛状根中丹酚酸合成关键基因表达量的影响 Fig. 4 Effects of c-ptio and L-NAME on the key gene expressions in S. miltiorrhiza hairy roots. Different letters (such as a, b, c) indicate significant difference at P 0.05 using Duncan s multiple-range test. NO ABA 3 讨论 [12] [13] [14] (Nitric oxide NO) (Abscisic acid ABA) NO [15] NO [16] NO NO [17] [18] NO [19] ABA [20] ABA [21-23] ABA [24] NO [7] ABA NO ABA SNP 100 μmol/l SNP cjb@im.ac.cn

63% NO c-ptio NO L-NAME ABA 图 4 c-ptio 和 L-NAME 对丹参毛状根中丹酚酸合成关键基因表达量的影响 Fig. 4 Effects of c-ptio and L-NAME on the key gene expressions in S. miltiorrhiza hairy roots.

7 228 ISSN CN /Q Chin J Biotech February 25, 2016 Vol.32 No.2 NO ABA ABA NO NO ABA NO ABA [25] ABA NO NO ABA [26-27] ABA NO [28] ABA NO NO (c-ptio) NO (L-NAME) ABA NO ABA [29] ABA B (PAL) (TAT) (RAS) NO ABA c-ptio L-NAME ABA c-ptio L-NAME ABA PAL TAT RAS ABA NO c-ptio L-NAME ABA NO [29] NO ABA ABA NO REFERENCES [1] Yang YX, Li Y, Zhou TS. Impacts of drying methods on the main chemical ingredients and antioxidant activities in cultured hairy roots of Salvia miltiorrhiza. Anhui Med Pharm J, 2014, 18(12): (in Chinese).,,.., 2014, 18(12): [2] Yuan Y, Wu Q, Shi JS, et al. Advance in studies on hepatoprotective effect of Salvia miltiorrhiza and its main components. China J Chin Mat Med, 2015, 40(4): (in Chinese).,,,.., 2015, 40(4): [3] Lin C, Liu ZG, Qian X, et al. Research progress of salvianolic acid B in cardiovascular diseases. Chin Pharmacol Bull, 2015, 31(4): (in Chinese).,,,. B., 2015, 31(4): [4] Huang YX, Huang RG, Zheng XZ. Progress in the research on the pharmacological actions of rosmarinic acid. Strait Pharmaceut J, 2010, 22(5): (in Chinese).,,.., 2010, 22(5): [5] Di P. Exploring the biosynthetic pathway of phenolic acids and functional study of the involved genes in Salvia miltiorrhiza bunge[d]. Shanghai: Department of Pharmacy of the Long March Hospital Affiliated to Second Military Medical University, 2012: (in Chinese).. [D]. :, 2012: [6] Feng K. The study on establishment of Salvia miltiorrhiza bunge hairy root culture system and elicitor s effect[d]. Yangling: Northwest A&F University, 2010: 1 8 (in Chinese)..

NO c-ptio L-NAME ABA NO [29] NO ABA ABA NO REFERENCES [1] Yang YX, Li Y, Zhou TS.

8 沈丽红等 / 一氧化氮信号在脱落酸诱导丹参酚酸类成分积累中的作用 229 [D]. :, 2010: 1 8. [7] Liang ZS, Yang DF, Liang X, et al. Roles of reactive oxygen species in methyl jasmonate and nitric oxide-induced tanshinone production in Salvia miltiorrhiza hairy roots. Plant Cell Rep, 2012, 31(5): [8] Cui BM, Liang ZS, Liu Y, et al. Effects of ABA and its biosynthetic inhibitor fluridone on accumulation of penolic acids and activity of PAL and TAT in hairy root of Salvia miltiorrhiza. China J Chin Mat Med, 2012, 37(6): (in Chinese).,,,. ABA., 2012, 37(6): [9] Wang SH, Zhang Q, Li ZP, et al. Effect of nitric oxide on the production of extracellular polysaccharide and intracellular polysaccharide of Ganoderma lucidum. J Anhui Sci Technol Univ, 2013, 27(3): (in Chinese).,,,.., 2013, 27(3): [10] Zhang AY, Zhang J, Zhang JH, et al. Nitric oxide mediates brassinosteroid-induced ABA biosynthesis involved in oxidative stress tolerance in maize leaves. Plant Cell Physiol, 2011, 52(1): [11] Omar NM, Marshall JM. Age-related changes in carotid vascular responses to adenosine and nitric oxide in the rat: in vitro and in vivo studies. J Appl Physiol, 2010, 109(2): [12] Chao MX, Ye BP. Influence of environmental abiotic factors on plant secondary metabolite biosynthesis. Pharmaceut Biotechnol, 2013, 20(4): (in Chinese).,.., 2013, 20(4): [13] Guo YL, Zhang PY, Guo MR, et al. Secondary metabolites and plant defence against pathogenic disease. Plant Physiol J, 2012, 48(5): (in Chinese).,,,.., 2012, 48(5): [14] Wang YM, Li J, Zhang LY, et al. Research overview on accumulation of medicinal plant secondary metabolites. Central South Pharm, 2012, 10(2): (in Chinese).,,,.., 2012, 10(2): [15] Oz MT, Eyidogan F, Yucel M, et al. Functional role of nitric oxide under abiotic stress conditions//khan MN, Mobin M, Mohammad F, et al. Eds. Nitric Oxide Action in Abiotic Stress Responses in Plants. Berlin Heidelberg: Springer, 2015: [16] Zhou WH, Shi SL, Kou JT. Effects of exogenous nitric oxide on the growth and nitrogen metabolism of alfalfa seedlings under salt stress. Chin J Appl Ecol, 2012, 23(11): (in Chinese).,,. NO., 2012, 23(11): [17] El-Beltagi HS, Ahmed OK, Hegazy AE. Molecular role of nitric oxide in secondary products production in Ginkgo biloba cell suspension culture. Not Bot Horti Agrobo, 2015, 43(1): [18] Liu JF, Wang MY, Tang YJ, et al. Effects of salicylic acid and nitric oxide on the growth and accumulation of secondary metabolites in Curcuma longa. Acta Horticult Sin, 2015, 42(4): (in Chinese).,,,.., 2015, 42(4): [19] Wang JW, Zheng LP, Tan RX. Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in Taxus yunnanensis suspension cells. Appl Microbiol Biotechnol, 2007, 75(5): [20] Yoshida T, Mogami J, Yamaguchi-Shinozaki K. ABA-dependent and ABA-independent signaling in cjb@im.ac.cn

[8] Cui BM, Liang ZS, Liu Y, et al. Effects of ABA and its biosynthetic inhibitor fluridone on accumulation of penolic acids and activity of PAL and TAT in hairy root of Salvia miltiorrhiza.

9 230 ISSN CN /Q Chin J Biotech February 25, 2016 Vol.32 No.2 response to osmotic stress in plants. Curr Opin Plant Biol, 2014, 21: [21] Gagné S, Cluzet S, Mérillon JM, et al. ABA initiates anthocyanin production in grape cell cultures. J Plant Growth Regul, 2011, 30(1): [22] Chang K, Chen M, Zeng L, et al. Abscisic acid enhanced ajmalicine biosynthesis in hairy roots of Rauvolfia verticillata by upregulating expression of the MEP pathway genes. Russian J Plant Physiol, 2014, 61(1): [23] Kai GY, Teng XJ, Cui LJ, et al. Effect of three plant hormone elicitors on the camptothecin accumulation and gene transcript profiling in Camptotheca acuminata seedlings. Int J Sci, 2014, 3: [24] Yang DF, Ma PD, Liang X, et al. PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots. Physiol Plant, 2012, 146(2): [25] Song XG, Yan MH, Zhu YP, et al. Effects of nitric oxide in ABA- and darkness-induced stomatal closure of V. faba. J Anhui Agri Sci, 2011, 39(2): (in Chinese).,,,.., 2011, 39(2): [26] León J, Castillo MC, Coego A, et al. Diverse functional interactions between nitric oxide and abscisic acid in plant development and responses to stress. J Exp Bot, 2014, 65(4): [27] Wang PC, Du YY, Hou YJ, et al. Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1. Proc Natl Acad Sci USA, 2015, 112(2): [28] Chen Q, Chen ZW, Lu L, et al. Interaction between abscisic acid and nitric oxide in PB90-induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures. Biotechnol Progr, 2013, 29(4): [29] Du XH, Zhang CL, Guo WL, et al. Nitric oxide plays a central role in water stress-induced tanshinone production in Salvia miltiorrhiza hairy roots. Molecules, 2015, 20(5): ( 本文责编陈宏宇 )

Abscisic acid enhanced ajmalicine biosynthesis in hairy roots of Rauvolfia verticillata by upregulating expression of the MEP pathway genes. Russian J Plant Physiol, 2014, 61(1): 136 140.

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填表说明 1. 本表所填数据截至时间为 2011 年 9 月底 2. 本表请用 A4 纸双面打印, 加盖学校公章后上报 3. 表内所填数据请学校认真核实, 确保准确无误 4. 建设完成情况对照填表说明 1. 本表所填数据截至时间为 2011 年 9 月底 2. 本表请用 A4 纸双面打印, 加盖学校公章后上报 3. 表内所填数据请学校认真核实, 确保准确无误 4. 建设完成情况对照表等相关表格栏高不够请自行增加 5. 表格中填写的相关量