浙江农业学报, 2024, 36(4): 978-996 DOI: 10.3969/j.issn.1004-1524.20230660

综述

高等植物花青素生物合成、调控、生物活性及其检测的研究进展

牛钰,1, 李晶1, 王俊文1, 李瑞瑞1, 田强1, 武玥,1,*, 郁继华,1,2,*

1.甘肃农业大学 园艺学院,甘肃 兰州 730070

2.省部共建干旱生境作物学重点实验室,甘肃 兰州 730070

Research progress of anthocyanin biosynthesis, regulation, bioactivity and detection in higher plants

NIU Yu,1, LI Jing1, WANG Junwen1, LI Ruirui1, TIAN Qiang1, WU Yue,1,*, YU Jihua,1,2,*

1. College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China

2. State Key Laboratory of Aridland Crop Science, Lanzhou 730070, China

通讯作者: 郁继华,E-mail:yujihua@gsau.edu.cn*武玥,E-mail:wuy@gsau.edu.cn;

责任编辑: 张韵

收稿日期: 2023-05-18  

基金资助: 甘肃省高等学校创新基金项目(2021B-124)
“双一流”科研重点项目(GSSYLXM-02)
甘肃省拔尖领军人才培养计划(GSBJLJ-2021-14)

Received: 2023-05-18  

作者简介 About authors

牛钰(1999—),女,甘肃卓尼人,藏族,硕士研究生,研究方向为设施蔬菜栽培生理与生长调控研究。E-mail:2262832454@qq.com

摘要

花青素是广泛存在于植物体中的一种重要次级代谢物,是影响植物呈色的关键物质,其生物合成具有一定的组织表达特异性,并能够受到内源和外源因素的调控,包括转录因子、植物生长调节剂以及环境条件。文章综述了近年来植物花青素的研究进展和现状,对花青素的生物合成、调控网络、影响因素、生物活性和检测策略进行了系统地阐述,并对存在的问题和未来研究方向进行了探讨。

关键词: 花青素; 生物合成途径; 转录因子; 调控机理; 生物活性; 检测方法

Abstract

Anthocyanin is an important secondary metabolite in higher plants, and is a key substance affects the color of plants. The biosynthesis of anthocyanin has specificity in certain tissue and can be regulated by endogenous and exogenous factors, including transcription factors, plant growth regulators and environmental conditions. In this paper, the research progress and advance of anthocyanin in recent years were reviewed, including the biosynthesis pathway, regulatory network, influenced factors, bioactivity and detection strategies of anthocyanins. Moreover, the existing problems and future research directions were discussed.

Keywords: anthocyanin; biosynthetic pathway; transcription factor; regulative mechanism; bioactivity; detection method

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本文引用格式

牛钰, 李晶, 王俊文, 李瑞瑞, 田强, 武玥, 郁继华. 高等植物花青素生物合成、调控、生物活性及其检测的研究进展[J]. 浙江农业学报, 2024, 36(4): 978-996 DOI:10.3969/j.issn.1004-1524.20230660

NIU Yu, LI Jing, WANG Junwen, LI Ruirui, TIAN Qiang, WU Yue, YU Jihua. Research progress of anthocyanin biosynthesis, regulation, bioactivity and detection in higher plants[J]. Acta Agriculturae Zhejiangensis, 2024, 36(4): 978-996 DOI:10.3969/j.issn.1004-1524.20230660

近年来,随着生活水平的提高,消费者在选购瓜果蔬菜产品过程中越来越注重产品的外观品质。果蔬产品的色泽不仅是反映其成熟度的关键因素,同时也是影响其外观品质的核心因素[1-3]。果蔬菜品的商业价值存在较强的地域性,不同的地区消费者的喜好不同,因此,根据不同的市场需求来改良果蔬的外观性质,尤其是果皮颜色,对提高果蔬的商业价值具有重要意义。

植物器官的各种颜色通常是由特定的类黄酮、类胡萝卜素和生物碱的种类和积累量的差异引起的[4-6]。花青素是最重要的类黄酮化合物之一,普遍存在于许多植物和水果中,对植物器官的色素沉着起着至关重要的作用[4,7]。例如,花青素显著影响紫红色菊花花瓣、深紫色茄子果实和粉红色百合花朵[8-10]。一品红红叶和葡萄红黑莓的颜色均与花青素的积累有显著的相关性[11-12]。先前的研究表明,类黄酮生物合成途径中基因表达水平的变化导致植物器官中花青素大量积累,从而导致颜色多态性[13]。由于菠萝成熟果实中AcHOX21和AcMYB12相对表达水平下调,花青素含量减少,以及内源茉莉酸、赤霉素酸和生长素水平的波动,导致菠萝果皮因花青素介导的变色而变色。NsMYB1基因促进西伯利亚白刺黑色果实花青素的积累[14]DbMYB2的瞬时过表达显著促进烟叶花青素积累[15]。紫色杜鹃花花朵中可以检测到各种各样的花青素,而白色花瓣中则没有检测到花青素。此外,还发现花瓣颜色较深的原因是其胚和种皮中花青素大量积累[16]。这些研究结果一致表明,颜色的增强与花青素水平的提高密切相关。阳光可以促进植物花青素的吸收,尤其是在苹果和葡萄的果皮中,缺乏光线则会产生相反的效果[17-18]。花青素相对含量的高低可以明显地反映植物的生长生理状态,还可以显著提高植物非生物胁迫抗性[19-20]。此外,花青素作为一种食品添加剂,具有多种生物活性,包括抗氧化性[21]、抗炎症性[22]、抗突变性和抗肿瘤性[23-24],并在全国得到了广泛的应用。医学家利用花青素的强抗氧化性治疗人体癌症,发现有显著的效果[25]

目前,已在自然界鉴定出6 000多种花青素,其中92%由天竺葵色素(Pg)、矢车菊素(Cy)、锦葵色素(Mv)、飞燕草色素(DP)、芍药色素(Pn)、牵牛花色素(Pt)等色素及其衍生物组成(表1)。除了一系列酶和转录因子调控花青素合成以外,外界因素也发挥重要作用。木糖(C5H10O5)、阿拉伯糖(C5H10O5)、葡萄糖(C6H12O6)、鼠李糖(C6H12O5)等与花青素结合,通过糖基化、酰基化、甲基化等过程进一步进行修饰,在植物液泡中以糖苷的形式存在[26]。不同修饰方式及修饰程度对糖苷的种类和稳定性的影响均不相同,对糖苷种类的区分主要在于糖苷C骨架结构中的羟基数目、甲基化与酰基化的位置和种类数目(图1)。

表1   六种常见的花青素及其取代官能团位置

Table 1  Six kinds of anthocyanins and their substituted chemical group

花青素种类
Anthocyanins species
R1碳位官能团
R1 carbon funtional group
R2碳位官能团
R2 carbon funtional group
物种
Plant species
参考文献
References
天竺葵色素
Pelargonidin (Pe)
HH桑葚Morus alba L.
玫瑰Rosa spp.
[27-28]
矢车菊色素Cyanidin (Cy)OHH蓝莓Vaccinium spp.[29]
飞燕草色素
Delphindin (DP)
OHOH黑枸杞Lycium ruthenicum Murr.
蓝莓Vaccinium spp.
[30-31]
芍药花色
Peonidin (Pn)
OMeH玫瑰Rosa rugosa
黑胡萝卜Daucus carota L.
[32]
[28]
牵牛花色素
Petunidin (Pt)
OMeOH马铃薯Solanum tuberosum
黑枸杞Lycium ruthenicum Murr.
[33-34]
锦葵色素
Malvidin (Mv)
OMeOMe葡萄Vitis vinifera L.
越橘Vaccinium corymbosum
[35-36]

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图1

图1   花青素的结构

Fig.1   Structure of anthocyanins


在近10年的研究中,研究者对花青素的生物学功能进行了广泛的研究,对花青素的研究重点转向外源刺激信号对花青素生物合成的影响及其调控机制,如光照、温度、糖和外源调节物质(5-氨基乙酰丙酸ALA 、脱落酸ABA、乙烯Eth、褪黑素MT)等,但对花青素全面概述的报道较少。因此,本文就近年来对花青素的生物合成、外界影响因素、相关基因表达、转录因子调控机制及其生物活性和检测方法进行了系统的阐述,为今后花青素研究提供理论支持。

1 花青素生物合成途径及其关键调控基因

由苯丙氨酸到花色苷的转化过程即生成花青素的过程(图2)。苯丙氨酸依次经苯丙氨酸裂解酶(PAL)、肉桂酸羟化酶(C4H)、香豆酰COA连接酶(4CL)、查尔酮合成酶(CHS)催化生成查尔酮(chalcone)。查尔酮经查尔酮异构酶(CHI)催化生成柚皮素(naringenin),再经黄烷酮3-羟化酶(F3H)作用转化生成香橙素(dihydrokaempferol),又名二氢黄酮醇。香橙素之后分为两条支路。其一,依次经二氢黄酮醇-4-还酶(DFR)和花青素合成酶(ANS)催化生成天竺葵素(pelargonidin);其二,依次经过类黄酮3'-羟化酶(F3'H)、二氢黄酮醇-4-还酶和花青素合成酶催化生成矢车菊素(cyanidin)。之后,经过糖基化、丙二酰化等过程后形成稳定的花青素[37-39]

图2

图2   花青素的生物合成

Fig.2   Biosynthesis of anthocyanins


多种因素共同调控着花青素的生物合成[40]。在结构基因中,合成通路上游PALCHSCHIF3H等酶基因起到了重要调控作用,并且这些酶基因的表达参与果实发育进程[17,41-42]。而在合成通路下游中发现DFR、ANS以及其他酰基化酶、糖基化酶等酶基因则主要调控果实果皮的颜色,且这些基因的高效表达及其酶活性与果皮颜色呈正相关关系[43-45]

1.1 苯丙氨酸裂解酶基因PAL

PAL是花青素合成初始反应的第一个关键限速酶,也是植物次生代谢的一个重要调控点,几乎存在于所有植物中[46]。对草莓喷施PAL抑制剂 L-α-氨基β苯基丙酸有效抑制了花青素的合成[47]。分别在未成熟和成熟苹果表皮上喷施乙烯,发现未成熟苹果中PAL活性与花青素积累水平增加,而成熟的苹果PAL活性与花青素积累水平无显著变化,说明乙烯通过激发PAL酶的活性,促进了苹果表皮花青素的积累[48]。对萝卜幼苗采用UV-B照射的处理,24 h后萝卜幼苗下胚轴花青素的积累水平迅速增加,发现萝卜幼苗花青素的积累与PAL活性的激活呈显著正相关[49]

1.2 查尔酮合成酶基因CHS

4-香豆酸和丙二酰CoA经过CHS催化生成查尔酮,为花青素后续合成和代谢构建基础碳架结构[50]。据报道,查尔酮合成酶是CHS超基因家族的核心酶,自Mehdy等[51]在1987年首次在豌豆中克隆并分离出CHS基因后,目前,已从20多个植物和细菌中识别并克隆出了查尔酮超基因家族成员的基因序列[52]。在甘薯中,已克隆得到CHS编码基因IbCHS1,经生物信息学分析发现IbCHS1具有典型的查尔酮合成酶的结构特征,表达分析结果发现,其主要在甘薯果肉中表达,且表达量与甘薯中花青素含量呈显著正相关[53]。由于CHS在花青素生物合成的上游代谢过程中发挥作用,研究发现,抑制查尔酮合成酶基因的表达时,培育出的花卉花色较淡。Aida等[54]的研究发现,在夏堇中导入CHS的反义基因后,其花色变浅;而Fukusaki等[55]利用RNAi技术沉默了夏堇CHS基因后,得到了花朵颜色为白色和灰白色的新品种。

1.3 查尔酮异构酶基因CHI

CHI在花青素合成过程中也起着重要的催化作用,主要在查尔酮转化生成黄烷酮的过程中起催化作用。目前,已在番茄(Lycopersicon esculentum Mill.)[41]、洋葱(Allium cepa)[56]、大豆[Glycine max (Linn.) Merr.][57]、菜豆(Phaseolus vulgaris L.)[58]等植物中分离克隆出了CHI 基因,其表达对花青素以及类黄酮的积累有重要作用。将矮牵牛CHI基因转入番茄中过度表达,转基因番茄果皮中类黄酮化合物含量增加78倍[41]。Morita等[59]在日本牵牛花中鉴定出了一种花青素合成增强基因EFP,发现敲除EFP基因使牵牛花(Petunia hybrida)和斗牛花(Torenia hybrida)中花青素的含量显著降低,表现为花色褪去。

有学者在研究过程中发现, CHI主要存在于葡萄果实的表皮细胞的细胞质、细胞核及叶绿体中,而在葡萄果实的其他组织中,CHI主要存在于细胞质、细胞壁和细胞核中,该基因存在表达特异性[60]

1.4 黄烷酮-3-羟化酶基因F3H

黄烷酮-3-羟化酶属于一种加氧型酶,是调控花青素合成的重要酶基因,发挥作用时要依赖于Fe2+、氧等辅助因子。F3H是类黄酮等多个代谢合成途径中关键限速酶,黄烷酮经F3H催化转化为苯丙氨酸下游代谢物的直接前体物质——二氢黄酮醇。据1995年Charrier等[61]报道了F3H基因序列以来,目前已在猕猴桃(Actinidia)[62]、玉米(Zea mays L.)[63]、菊花(Dendranthema morifolium)[64]、桔梗(Platycodon grandiflorus)[65]中克隆出了他们对应的F3H基因。在反义抑制黄烷酮-3-羟化酶的转基因苹果植株中,表现为阻断花青素以及类黄酮物质的合成[66]。Jiang等[67]构建含有编码F3H基因的RNAi基因沉默载体后,将含有RNAi基因沉默载体的农杆菌导入授粉14 d的草莓10 d后,经检测与野生型相比F3H的相对表达量下调了70%,且花青素含量大幅度降低。

1.5 二氢黄酮醇-4-还原酶基因DFR

无色的花色苷是二氢黄酮醇通过二氢黄酮醇-4-还原酶催化生成[68]。有研究表明,DFR基因与CHI基因相似,存在表达特异性。如凤兰(Cymbidium dayanum Rchb. F.)中,DFR基因主要在幼叶中表达,而在花器官中几乎检测不到[69]。通过克隆百合(Lilium Asiatic)DFR酶编码基因LhDFR并分析其时空表达,发现在白色鳞茎以及未着色的叶和茎中,LhDFR基因均未表达,而在花药、花丝,以及雌蕊中大量表达[70]DFR的表达是一个复杂的调控网,调控方式需要进一步去探究。

1.6 花青苷合成酶基因ANS

花青苷合成酶是花青素生物合成过程中最后一步反应的关键酶,其作用主要将无色的花色素氧化生成有色但易分解的花色素[71]。对花青苷合成酶的研究发现其表达具有一定的品种或组织特异性。Li等[72]测定了紫色和白色品种的桑果中ANS编码基因的表达量,发现ANS基因只在紫色桑果果肉中高表达。在菠菜中,ANS在大多数器官和组织中均未表达,且只在种子中检测到了ANS的活性[73]。利用RT-PCR技术从桃果皮中分离出了ANS编码基因PpANS,对其在桃植株中的表达进行了研究,结果发现,PpANS在桃花、果皮,以及果肉中高表达,其中以果皮中的表达量最高,而在桃植株的根、茎、叶中均未表达[74]。这些研究结果表明,花青素的合成与积累与ANS的表达有直接关系。

2 影响花青素合成及积累的调控因素

2.1 转录因子

在花青素合成过程中,调控基因在转录水平上同样起到了决定性的作用。结构基因经转录因子调控以识别特异性结合的区域,影响花青素的生物合成[75]。转录因子MYB、bHLH和WD40主要调控花青素的合成。MYBbHLHWD40基因在作用的过程中能够形成二元复合物或三元复合物,以此正向或负向调控花青素的合成[76]

2.1.1 MYB

在高等植物中,MYB是极其重要的一类转录因子,参与了植物多种发育过程以及植物抗逆性的形成。1987年Paz-Ares等[77]在玉米中分离并鉴定出了ZmC1,这是植物中被鉴定的第一个 MYB家族转录因子。后来,随着技术手段的成熟,相继在许多植物中鉴定出了MYB转录因子。目前,已在大豆中鉴定出了244种R2R3型MYBs,其中与两种非生物应激调节剂ATmyb44和ATmyb77密切相关的R2R3型MYBs(GmMYB81)被证明在大豆组织和胚胎中差异表达,且在大豆逆境条件下的表达显著上调[78]。MYB转录因子家族各成员能够调控植物组织着色,在花青素合成的过程中发挥了重要的作用(表2)。许多MYB型转录因子已经被证明参与了花青素生物合成的正向调控。在山竹(Garcinia mangostana L.)果皮中,研究者克隆得到GmMYB1,GmMYB7,GmMYB10和GmUFGT,发现GmMYB10和GmUFGT在山竹果实成熟和着色过程中高表达,促进了山竹花青素的积累[79]。在苹果中,MdMYB3的过表达促进了CHSCHIUFGTFLS等结构基因上调,且发现在红色果皮中MdMYB3的表达高于黄色果皮苹果;另外,转MdMYB3的烟草植株比野生型的花颜色更深[80]。Li等[81]在荔枝中(Litchi chinensis Sonn.)克隆并鉴定了一种新型R2R3-MYBS LcMYB5转录因子,发现在荔枝中的表达与其果皮花青素的积累无相关性关系,而在烟草和矮牵牛中过表达,能够通过直接激活花青素合成关键基因的表达促进其积累。另一方面,部分MYB转录因子也能够负向调控花青素的生物合成。在葡萄中发现了一个R2R3-MYB转录因子VvMYBC2L2能够负向调控花青素的生物合成。Zhu等[82]发现VvMYBC2L2作为一种核蛋白,在葡萄果实发育过程中在果皮细胞中高表达,而果实到达转色期时表达降低,另外,花青素生物合成关键基因VvDFR1和VvDLOX1与VvMYBC2L2的表达模式相一致。Colanero等[83]在番茄突变体atv的候选基因中发现一种R3-MYB的突变基因,其过表达时,通过负调控内源MBW复合物来介导花青素合成,且可以直接和MBW中bHLH转录因子结合,形成底物竞争关系干扰番茄花青素的合成和积累。

表2   部分花青素合成途径相关转录因子

Table 2  Transcription factors related to anthocyanin synthesis pathway

类别
Category
转录因子
Transcription factors
物种
Plant species
调节基因
Regulate genes
参考文献
Reference
MYBPmMYBa1梅花Armeniaca mume Sieb.DFR, ANS[95]
EsAN2淫羊藿Epimedium brevicornu Maxim.CHIS, CHI, ANS[96]
CmMYB6菊花Dendranthema morifolium Tzvel.DFR[97]
GmMYB10山竹Garcinia mangostana L.DFR, UFGT[79]
TaMyb1D烟草Nicotiana tabacum L.PAL, CHS, CHI, F3H, DFR, FLS[98]
OsPL水稻Oryza sativa L.PAL, CHS, ANS[99]
AtMYBL2拟南芥Arabidopsis thalianaAtTT8, DFR[100]
TaPL1小麦Triticum aestivumPAL, CHS, CHI, F3H, DFR[37]
VvMYBA1/6/7葡萄Vitis vinifera L.UFGT, 3AT[101]
DcMYB6黑胡萝卜Daucus carota L.CHS, DFR[102]
PpMYB10.1Prunus persicaDFR, UFGT[103]
PpMYBPA1Prunus persicaLAR1[103]
bHLHPsbHLH1牡丹Paeonia suffruticosaAndr.DFR, ANS[104]
PubHLH2石榴Punica granatum L.DFR, ANS[105]
AtGL3/TT8拟南芥Arabidopsis thalianaCHS, CHI, F3H, F3'H, DFR, ANS, GT[106]
LeAH番茄Lycopersicon esculentum Mill.F3'5'H, DFR, ANS, 3GT, GST[86]
MdMYC2苹果Malus×domesticaDFR, UF3GT, F3H, CHS[107]
MdbHLH3苹果Malus×domesticaDFR, UFGT[108]
AcbHLH42猕猴桃Actinidia chinensis PlanchF3GT1, ANS[87]
SmTT8茄子Solanum melongena L.CHI, F3H, DFR, 3GT, 5GT[109]
WD40MdTTG1苹果Malus×domesticaPAL, CHI, CHS[76]
PhAN11矮牵牛Petunia hybrida Vilm.DFR[90]
Pu TTG1石榴Punica granatum L.DFR, LDOX[105]
GhTTG1/TTG3棉花Gossypium spp.DFR[110]
StAN11马铃薯Solanum tuberosumDFR[111]

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2.1.2 bHLH

bHLH也是植物中重要的一类转录因子,其结构存在两个高度保守且功能不同的功能区,一个是C端的螺旋-环-螺旋(bHLH),另一个为N端的碱性区域。目前,大部分关于bHLH的研究都关注于其对植物抗性的效果以及参与信号转导的作用,对花青素的生物合成影响也是其中一个重要的部分。Shan等[84]通过克隆小苍兰(Freesia hybrida)WD40基因家族的调节蛋白FhTTG1并分析其功能,结果表明FhTTG1与bHLH 两个非等位基因相互作用显著激活调控花青素合成相关的启动子,进而促进花青素或原花青素的积累。在拟南芥种皮中,编码原花青素的酶基因TT2、TT8、TTG1与MBW复合体互作,bHLH调控TTG1发挥作用[85]。在缺失bHLH 蛋白的番茄中,对应bHLH 蛋白位点的ahin FMTT271基因研究发现,ahin FMTT271的过表达上调了花青素生物合成关键酶基因,提高了花青素在幼苗下胚轴、叶片和果皮中的积累水平[86]。Wang等[87]发现猕猴桃AcMYB123和AcbHLH42的组合作用,激活了编码花青素合成AcANS和AcF3GT1的启动子,且通过对烟草和拟南芥瞬时表达分析表明,AcMYB123和AcbHLH42的共同表达是花青素生物合成的必要条件。bHLH对花青素生物合成的调控是一个复杂的网络,但有趣的是,并不是所有植物的花青素合成都需要bHLH的参与,早在1995年Solano等[88]在对矮牵牛的研究中发现,MYB3可以激活CHS的表达,但在作用的过程中不需要bHLH的协作。

2.1.3 WD40

转录因子在花青素合成过程中主要促进MYB-bHLH-WD40蛋白间的复合物的相互作用,从而为花青素的合成提供一个相对稳定的环境条件[89]。目前,有研究者已经从不同物种中分离并鉴定出WD40,另外,发现在多个物种中WD40基因家族中的AN11、TTG1、PAC1、PFWD等蛋白参与并调控了花青素合成整个过程的关键结构酶基因。De Vetten等[90]分离了第一个植物体中的WD40蛋白——矮牵牛AN11,研究发现在矮牵牛an11突变体中,DFR的表达量下调,另外在矮牵牛花瓣中,AN2过表达后,AN11恢复,上调了花青素结构基因的表达,促进了花青素的积累。在其后的研究中,在拟南芥中克隆并鉴定出了的矮牵牛AN11同源基因TTG1基因,两者具有高度的相似性[91]。在蓝莓中,VcMYBL1、VcbHLH1、VcWDL2的表达与花青素的积累和颜色的变化呈正相关[92]。Meng等[93]发现紫花苜蓿WHITE PETAL1(WP1)基因和MtTT8基因通过和MtWD40-1相互作用激活了类胡萝卜素生物合成基因MtLYCeMtLYCb,进而促进了类胡萝卜素和花青素的合成。Kang等[94]发现紫番茄中,果皮花青素的产生受MYB、bHLH和WD40转录因子的共同调节。

2.2 外源调节物质

2.2.1 5-氨基乙酰丙酸(ALA)

外源ALA通过诱导花青素的积累,从而影响植物果实果皮花青素的含量。对葡萄喷施ALA,发现100 mg·L-1的ALA显著增加了果皮花青素的含量同时也显著增加了PAL的活性,与对照水平相比,分别增加了194.71%、82.35%[112]。对苹果设置不同浓度梯度的ALA进行喷施,发现100 mg·L-1 ALA对苹果果皮花青素的积累量的影响最为显著,且在处理25 d后,与对照水平相比,苹果花青素合成仍表现出促进效应[113]。ALA对花青素的促进积累的效应还表现在对花青素生物合成关键酶基因的调控中。在桃表皮涂抹ALA,研究结果发现ALA不仅促进了桃果皮花青素含量,还显著提高了UFGT、DFR、LDOX 和 CHS等酶的活性,4个基因的表达量高峰也随之提前[114],在转录水平上,显著促进了桃果皮中MYB10和WD40基因的表达量,而BHLH3蛋白的相对表达量无显著影响,这些酶基因在转录水平与花青素的积累水平相一致[74]。对喷施ALA的离体葡萄果皮进行半定量RT-PCR,检测结果表明ALA能有效促进花青素合成过程中结构基因(PAL、CHS、CHI、DFR、UFGT)以及调控因子(MYB、bHLH3、WD40)的表达。而对于ALA对花青素合成的调控机制,可能是由于在果皮喷施ALA后,ALA的向下代谢成卟啉化合物来实现对花青素的调控,但具体的调控机制有待进一步研究。

2.2.2 脱落酸(ABA)

ABA作为一种植物激素参与了植物的多种生理过程,如种子休眠、气孔关闭以及光合作用等。此外, ABA还参与了跃变果实的成熟[115],而对非跃变果实的成熟作用不显著[116]。近年来,研究证明ABA不仅参与果实成熟,也参与调控果皮花青素的积累。Shen等[117]用ABA的生物合成抑制剂去二氢愈创木酸(NDGA)和1 mmol·L-1 ABA处理樱桃,发现NDGA抑制了花青素的积累,ABA处理樱桃2 h后,检测到PacMYBA的表达量达到了高峰,另外,构建一种烟草病毒(TRV)沉默樱桃ABA合成过程中的编码关键酶基因PacNCED1,发现樱桃果实内源ABA的含量降低,以及花青素合成关键酶活性及其基因表达的下调,抑制了花青素的积累。对荔枝果实分别进行ABA和光照处理,研究结果表明两种处理均能促进LcMYB1基因的表达,且在LcMYB1启动子区鉴定了与光响应性和脱落酸响应性相关的顺式元件,数据显示,在花青素合成的结构基因中只有LcUFGTLcMYB1高度相关[72]。目前,对于ABA调控花青素合成的机理还有待进一步研究。

2.2.3 乙烯(Eth)

植物的成熟和衰老过程都有乙烯的参与以及调控。在乙烯信号转导的过程中,EIN3和EIL1两个蛋白发挥着重要的转录调控作用。有研究表明敲除拟南芥中ein3和eil1基因后,双突变体拟南芥的种子和叶片均表现为紫色,与单突变体ein3-1和野生型拟南芥相比较,其花青素含量显著增加[118]。同时,在野生型拟南芥体内乙烯抑制糖信号诱导的花青素的合成过程中,发现糖信号被抑制的同时抑制了其相关转录因子MYBL2的表达,与此同时,一类影响花青素合成的负调控因子R3-MYB表达量提高,从而抑制了花青素的积累[119]。El-Kereamy等[120]在葡萄表皮喷施乙烯释放物2-CEPA,24 h后葡萄内源乙烯含量与对照相比较增加了6倍,与此同时,2-CEPA还上调了花青素合成关键酶CHI和F3H的转录水平。乙烯在参与果实成熟过程中调控了果实花青素的合成,但果实中乙烯是直接作用于关键基因还是间接刺激转录因子来调控花青素的积累,具体的调控机制是一项亟待探究的课题。

2.2.4 褪黑素(MT)

褪黑素作为色氨酸的一种衍生物类激素,近年来在农业生产中得到了广泛的运用[121]。有学者发现,在寒冷条件下,野生型拟南芥与缺少褪黑素生物合成酶基因的拟南芥突变体snat的叶片的颜色更深,这一发现揭示了褪黑素能正向促进花青素的积累[122]。将草莓果实分别在50 mol·L-1和100 mol·L-1的褪黑素浸泡30 min后,测定出其花青素合成相关调控基因表达量上调,果实花青素积累增多,说明外源褪黑素可以正向调控苯丙氨酸代谢过程[123]。在Wei等[124]对海棠的研究中表明,外源喷施褪黑素可以上调相关合成酶基因和转录因子的表达量,进而提高花青素的含量。有趣的是,有研究发现,充足的ATP和PEP可以有效增加甘薯和双孢菇PAL的活性,且可以增加花青素的积累量[125-126]。有研究者推测,外源褪黑素可能促进了TCA循环中柠檬酸合成酶活性并且上调了柠檬酸合成相关基因的表达,为花青素合成提供更为丰富的合成底物,以此促进花青素的生物合成,但具体的调控机制仍为一项研究薄弱点。

2.2.5 蔗糖和葡萄糖

糖作为一种信号转导物质已在多个领域研究和应用。对植物的生长和发育过程也起重要作用[127]。糖参与了植物的多种代谢过程,调控花青素生物合成过程中的大部分结构基因和调控的表达,如PAL、CHS、DFR、UFHT等[128]。Li等[81]将草莓用50 mmol·L-1的蔗糖溶液处理后,发现草莓果实果肉中花青素含量增加;Wang等[87]发现,在光照条件下拟南芥经蔗糖溶液处理后,能显著诱导拟南芥花青素的积累。另外,植物内源蔗糖转运载体也能够参与花青素合成和积累途径。利用农杆菌转化法将在苹果中克隆的蔗糖转运载体基因MdSUT2转化到拟南芥中,发现MdSUT2的过表达会显著增加拟南芥花青素含量,而抑制MdSUT2的表达则会降低花青素含量,说明蔗糖转运蛋白参与并调控花青素的生物合成[129]。在苹果中,MdSnRK1.1与茉莉酸信号途径中的抑制因子MdJAZ18相互作用,磷酸化后促进了26S蛋白酶介导的降解,调控了花青素生物合成相关基因的表达,并促进了花青素的积累[130]MdJAZ18、MdMYB1、MdMYB9和MdMYB11均能与MdbHLH3相互作用,MdbHLH3不仅能上调MdJAZ18、MdMYB9和MdMYB11的表达,并且激活了花青素生物合成基因MdDFRMdUFGTMdANSMdANRMdLAR和调控基因MdMYB1的表达,促进了原花青素和花青素的积累[17,131]。葡萄糖是植物光合作用的主要产物,同时也是参与细胞内众多生理生化过程的重要能源物质和碳源,而作为葡萄糖信号转导物质,苹果己糖激酶MdHXK1也参与了葡萄糖的感应和花青素的调控,其在调控的过程也是通过激活MBW复合蛋白和靶基因的表达来完成。

2.3 环境条件

2.3.1 光照

光照是非生物环境因子中影响花青素生物合成的关键因子,光照时长对植物色泽和花青素的积累都有重要的影响。Lotkowska等[132]发现苹果延长光照时间5 h后,上调了花青素合成转录因子MYB112的表达,并诱导了苹果花青素的合成和积累。在茄子中分离出2个蓝光受体基因SmCRY1、SmCRY2和花青素调节阴性基因SmCOP1和阳性基因SmHY5,发现SmCRY1、SmCRY2和阳性基因SmHY5在光照条件下的表达上调,而阴性基因SmCOP1的表达下调[133]。另一方面,不同植物品种花青素合成积累对光照的响应也不同。利用RT-PCR技术分析FGM(非光敏型)和禾线(光敏型)两个光敏性不同品种茄子在套袋处理后果皮花青素合成酶基因和转录因子的表达情况,发现FGM与不套袋相比较,SmMYB1的表达量显著增加,光形态建成因子SmCOP1的表达量显著减少,而禾线与对照相比较,SmMYB1 和SmCOP1的表达情况则截然相反[134]。同时 Guan等[135]以葡萄为试验材料,也得到了类似的结果,在光照条件下,Gamay葡萄的果皮和果肉中花青素含量显著多于Gamay Fre’aux。

2.3.2 温度

对13个无性系葡萄品种分别进行24 ℃/14 ℃、28 ℃/18 ℃的处理,结果28 ℃下葡萄果皮花青素的积累量大于24 ℃下花青素的积累量[136]。对桃分别于16 ℃和12 ℃下贮藏后,发现在16 ℃下桃果皮存在花青素积累,而12 ℃或以下贮藏时花青素不积累,且16 ℃下桃果皮花青素生物合成酶基因PpPAL1/2、PpC4HPp4CLPpF3HppF3'H、PpDFRPpANS转录水平增加,促进了花青素的积累[137]。Liu等[138]发现低温和低pH条件下有利于维持蓝莓总花色苷的最大稳定性。通过对生长在高温环境(30 ℃/15 ℃)下的草莓进行研究,发现其果皮和果肉中花青素浓度与对照(20 ℃/15 ℃)相比较均呈下降趋势,且转录因子FaMYB10以及草莓花青素合成结构基因FaDFRFaANSFaUFGT的表达均显著下调[139]。Ryu等[140]通过下调和上调夜间温度来探讨夜间生长温度对苹果果皮色泽以及花青素积累的影响,结果发现降低夜温促进了苹果花青素生物合成基因MdCHSMdF3HMdDFRMdANSMdUFGT的表达,增加了花青素的积累,而上调夜温表现出相反的结果。

3 花青素生物活性

化合物的生物活性高度依赖于它们与环境的相互作用[141]。芸薹属蔬菜的生物活性与多种植物化学物质的活性有关,包括维生素C和E、类胡萝卜素、酚类化合物和硫代葡萄糖[142]。在红卷心菜中发现花青素是最丰富的酚类化合物[143],通过降低胆固醇含量有效保护心脏和肝脏。

3.1 抗氧化活性

人体代谢过程中产生多种自由基,而过多的自由基可导致脂质、蛋白质、DNA、RNA和糖的氧化[144-145]。Teow等[21]采用ORAC、DPPH和ABTS研究了19种甘薯白色、浅黄色、黄色、橙色和紫色果肉的自由基清除能力,结果表明紫色甘薯的抗氧化能力最强。这主要是因为花青素可以诱导各种氧化产物含量下降,防止氧化应激引起的胁迫损伤。Wiczkowski等[146]探究了红甘蓝花青素的抗氧化活性,并报道了所有酰化的花青素苷比非酰化的花青素苷具有更强的抗氧化能力,此外,二酰基化的花青素比单酰基化的花青素具有更高的抗氧化能力。Wang等[147]在对红萝卜花青素抗氧化性的研究中也得出类似结论。Fang等[148]报道富红甘蓝花青素提取物可降低H2O2诱导的人体肝癌(HepG2)细胞氧化应激,改善细胞凋亡和细胞活力。Kou等[149]在之前对蓝莓多酚提取物抗氧化性能分析的基础上,对粗提取物和纯化的花青素和多酚的抗氧化性能进行了探究,结果发现蓝莓花青素纯提取物具有较高的抗氧化活性。

3.2 抗诱变活性

花青素和多酚类化合物对肿瘤的特殊抑制作用是近年来植物提取物治疗或辅助治疗癌症的研究热点。Zhao等[24]的研究表明,小鼠体内植入S180肿瘤细胞后体重降低,然而紫甘薯花青素能够通过提高谷胱甘肽过氧化物酶和超氧化物歧化酶水平,增强抗氧化能力,丙二醛水平降低,从而抑制小鼠体内肿瘤生长;另一项关于紫甘薯的研究表明,花青素通过诱导细胞凋亡抑制膀胱癌BIU87细胞的增殖,且具有剂量依赖性[150]。此外,花青素对小鼠肿瘤生长的抑制率与花青素浓度成正比。槲皮素可抑制子宫肿瘤细胞的增殖,且其治疗效果优于其他活性物质。从葡萄中提取的花青素对乳腺癌MCF-7细胞的抗肿瘤作用优于纯化的花青素[151],这与Yang等[152]的研究结果一致。另外,在一定程度上,活性物质对体外肿瘤增殖的抑制能力与抗氧化活性并不呈正相关[152]

3.3 其他生物活性

除上述功能活性外,高等植物花青素还具有保护肝脏、神经系统、抗炎、降血糖、改善肠道微生态等功能。

来自医学研究的试验数据表明,含有花青素的提取物或纯花青素对保护心脏具有巨大的潜力。Brader等[153]研究了富含花青素的蔓越莓和黑加仑浆果的饮食(每天5 g含有172 mg花青素的浆果粉)对糖尿病脂肪大鼠的脂质特征和其他生物指标的影响。实验进行56 d后的结果显示,胆固醇水平降低,有效保护动物体肝脏。Al-Dosari等[154]发现,高剂量的红白菜提取物可以改善大鼠因胆固醇积累导致的肝组织的坏死、炎症和纤维化。Arjinajarn等[155]研究发现富含矢车菊素-3-O-葡萄糖苷和芍药苷-3-O-葡萄糖苷(分别为13.24和5.33 mg·g-1粗提物)的米糠提取物可有效预防庆大霉素引起的肝脏中毒。此外,口服富含花青素的越橘浆果提取物(100 mg·kg-1,连续7 d)可抑制心理应激诱导的小鼠大脑氧化应激和多巴胺异常[156]。从黑大豆中提取的花青素也被证明可以逆转成年小鼠中d-半乳糖或脂聚糖诱导的氧化应激、神经炎症和神经变性[157-159]。另外,在研究过程中还发现花青素处理后,恢复了线粒体电子传递链复合物Ⅰ和Ⅱ的活性[160]。Matsui等[161]先给予8周龄雄性小鼠2 g·kg-1 BW的麦芽糖,然后灌胃注入100 mg·kg-1 BW的二酰化紫甘薯花青素,结果显示30 min后小鼠血糖浓度明显下降16.5%,二酰化花青素通过抑制α-葡萄糖苷酶活性降低血糖水平。Zhang等[162]通过观察紫甘薯花青素处理后不同时间点细菌数量和短链脂肪酸浓度的变化,分析其对肠道菌群的影响。结果发现,花青素能明显增加双歧杆菌和乳酸杆菌/肠球菌的种群数量和浓度。因此,摄入富含花青素的紫甘薯有利于肠道微生态和宿主健康。

在人体水平上,Zhang等[163]报道,从黑加仑中提取花青素(每天320 mg)用于临床治疗84 d后,可改善非酒精性脂肪肝患者的肝损伤。在另一项研究中,测试了含有高多酚和花青素含量的果汁(40%红葡萄汁、20%黑莓汁、15%酸樱桃汁、15%黑加仑汁和10%接骨木果汁)对血液透析患者的预防作用,结果表明,DNA氧化损伤、蛋白质和脂质过氧化水平显著降低,还原型谷胱甘肽水平升高[164]。血液透析患者经常饮用浓缩红葡萄汁可通过降低中性粒细胞NADPH氧化酶活性以及炎症标志物水平降低进而缓解病症[165]

4 花青素含量测定方法

花青素作为植物色素中研究的重点领域,其诸多测定技术已被广泛报道(表3)。

表3   花青素测定方法

Table 3  Analytical method of anthocyanin

物种
Species
测定样品/部位
Measuring sample/parts
方法
Method
参考文献
Reference
葡萄Vitis vinifera L.果肉Pulp纤维素薄层色谱-密度分析法Thin-layer chromatography[166]
葡萄Vitis vinifera L.果皮Pericarp高效液相色谱法High-performance liquid chromatography[167]
越橘Vaccinium dunalianum叶片Leaf紫外分光光度计法The UV spectrophotometry[168]
蓝莓Vaccinium spp.果实Fruit酶辅助提取Enzyme-assisted extraction[169]
黑胡萝卜Daucus carota L.根RootpH示差法The pH-differential spectrophotometry[170]
葡萄Vitis vinifera L.果皮Pericarp反向高效液相色谱法[171]
Reversed phase high-performance liquid chromatography
苹果Malus×domestica果实Fruit对-二甲基氨基肉桂醛法The P-dimethylamino-cinnamaldehyde method[172]
覆盆子Rubus idaeus L.果实Fruit超高效液相色谱-光电二极管阵列检测器-四极杆飞行时间质谱法[173]
UPLC-PDA-Q/TOF-MS
巴西莓冻干巴西莓粉液相色谱-质谱联用测定法LS-MS[174]
Euterpe oleracea Mart.Freeze-dried acaie powder
蔓越莓Oxycoccos果肉Pulp香草醛法Vanillin method[175]
蓝莓Vaccinium spp.果肉Pulp电化学发光法Electrochemiluminescence[176]
甘草根Root超高效液相色谱-二极管阵列检测器串联质谱法MS/MS- UPLC-PDA[177]
Glycyrrhiza uralensis Fisch.
山楂果肉Pulp定量核磁共振法Quantitative NMR[178]
Crataegus pinnatifida Bge.
马铃薯Solanum tuberosum块茎Tuber超高效液相色谱-二极管阵列检测器法UPLC-PDA[179]
黑稻Oryza sativa L.种子Seeding欧姆加热辅助萃取Ohmic heating assisted extraction[180]
马齿苋Portulaca oleracea L.叶片Leaf超高效液相色谱-电喷雾串联三重四级杆质谱法UPLC-ESI-MS/MS[181]

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紫外分光光度计法是测定花青素含量简便且成本较低的办法[168]。目前,高效液相色谱法是分离、提取。在高效液相色谱法测定植物花青素含量的基础上,超高效液相色谱-二极管阵列检测器串联质谱定性分析法(MS/MS-UPLC-PDA)、超高效液相-四级杆飞行时间质谱联用法(UPLC-PDA-Q/TOF-MS)以及超高液相色谱-电喷雾二级质谱联用(UPLC-ESI-MS/MS)等方法被证明具有更精确、重复性更好的特点[173,179,181]。有研究发现,植物花青素糖苷结构会随着pH值的变化而变化,而其他会造成测定干扰的物质的特征光谱并不会在环境条件的影响下改变,因此,在样品中含有干扰物时,要测定花青素总量选用pH值示差法是比较合适的测定方法[170,182]。此外,也有利用花青素化学结构而添加对-二甲基氨基肉桂醛或香草醛等使其官能团发生化学反应,以此形成稳定的花青素缩合产物,然后用成本较低、简便的分光光度计法测定花青素物质的量浓度[172,175]。为了快速检测大量样品中的特征物质,用定量核磁共振法测定花青素种类和含量,具有分析速度快,精确度高的特点[178,183]。另外,纤维素薄层色谱-密度分析法也被常用于分离和定性分析花青素的测定中,对其测定过程中杂质以及干扰物质的鉴定具有重要的作用[166,184]

5 展望

目前,已经有学者在多个物种中研究花青素的合成过程,研究关注点也从结构基因转向调控基因越来越多的转录因子在多种植物中被鉴定出来,然而,在植物不同组织中许多调控花青素合成的转录因子的表达及功能还未被鉴定,调控花青素合成的转录因子microRNA、MYB、BHLH、WD40以及MBW复合体对基因表达的调控机制也还未揭示,未来应加强对MYB、BHLH、WD40以及MBW复合体等调控机制的研究,借助基因组测序技术、转基因技术、蛋白质组学技术等技术去鉴定植物中的各类转录因子,进一步明确植物各类结构基因与转录因子间相互作用的表达机制以及转录因子的作用模式,为植物生长发育以及花青素的生物合成及其调控提供相应的理论依据。

外界因素对植物的生长调节以及花青素的生物合成是一个复杂的网络,许多外界因素都能影响花青素的生物合成及积累量的变化,但这些因素对花青素合成的调控机制依然缺乏更深入的研究,目前,该方向的研究仅涉及到对结构基因表达的调控,但对转录调控因子的研究更是极度欠缺。因此,今后应深入对外界因素引起的花青素生物合成调控转录因子表达的研究,结合前人研究结果并综合分析外界因素对花青生物合成的调控和作用机制,进一步阐明花青素生物合成作用机制。

目前,检测花青素种类和含量主要以高效液相色谱法为主,通常在测定的过程中会联合其他的仪器一起测定,其测定结果具有重复性好、精确度高的特点,但同时也存在耗时长、成本高的问题。因此,开发简便快捷、精确度高且成本低的检测仪器和其他测定方法是研究者们需要关注和解决的问题之一。未来应通过加强转基因技术以及定点突变等技术,实现定向调控植物颜色,从而提高观赏植物的观赏价值、提高果蔬产品的营养品质。

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