南京农业大学学报  2019, Vol. 42 Issue (4): 583-593   PDF    
http://dx.doi.org/10.7685/jnau.201812019
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文章信息

张绍铃, 贾璐婷, 王利斌, 张臻
ZHANG Shaoling, JIA Luting, WANG Libin, ZHANG Zhen
园艺作物果实液泡糖、酸转运与转化研究进展
Recent advance on vacuolar sugar and acid transportation and conversion in horticultural fruit
南京农业大学学报, 2019, 42(4): 583-593
Journal of Nanjing Agricultural University, 2019, 42(4): 583-593.
http://dx.doi.org/10.7685/jnau.201812019

文章历史

收稿日期: 2018-12-13
园艺作物果实液泡糖、酸转运与转化研究进展
张绍铃 , 贾璐婷 , 王利斌 , 张臻     
南京农业大学园艺学院, 江苏 南京 210095
摘要:糖、酸是果实风味的重要组成部分。作为糖、酸等代谢物的主要贮藏场所,液泡在园艺作物果实风味形成过程中起重要作用。液泡中糖、酸组成受膜载体转运特性及液泡内代谢酶催化特性的协同影响。本文总结了园艺作物果实液泡膜糖、酸转运载体类型及其转运特性,探讨了质子泵在液泡糖、酸积累过程中的作用,并对液泡内糖、酸转化的研究进展进行了概述。在此基础之上,整合并构建了园艺作物果实液泡糖、酸转运与转化的初级模型,以期为后续研究提供指导。
关键词液泡   糖、酸转运载体   质子泵   转化   
Recent advance on vacuolar sugar and acid transportation and conversion in horticultural fruit
ZHANG Shaoling , JIA Luting, WANG Libin, ZHANG Zhen    
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
Abstract: Sugar and acid are important contributors to fruit flavor. As the primary storage organelle for sugar and acid, vacuole plays an important role in the formation of flavor in fruit of horticultural crops. The composition of sugar and acid in vacuole is determined by characteristic of transporters on the tonoplast as well as catalytic feature of enzymes inside the vacuole. In this review, information on the type and characteristic of vacuolar sugar and acid transporters in horticultural fruit was summarized firstly; subsequently, the role of proton pump during sugar and acid transportation was discussed; finally, sugar and acid conversion in vacuole was also described. Based on this, the primary model of sugar and acid transportation and conversion in the vacuole from horticultural fruit was integrated and constructed in order to provide clue for further study.
Keywords: vacuole    sugar and acid transporters    proton pumps    conversion   

园艺作物果实种类繁多, 在日常生产生活中占有举足轻重的地位[1]。作为果实风味的重要组成部分, 糖、酸对果实整体感官品质的形成起着至关重要的作用, 也是影响果实经济效益与市场竞争力的关键[2-3]。果实中主要的可溶性糖包括蔗糖、果糖和葡萄糖, 主要的有机酸是苹果酸和柠檬酸[4]。不同糖、酸组分带给人的味觉感受不同[5], 因此糖和酸的种类、含量及配比共同决定着果实风味的形成[2]

叶片光合产物作为果实糖分的主要来源, 以不同形式的转运糖进入果实后, 经相关酶转化, 最终形成蔗糖、果糖和葡萄糖等主要糖分类型[6]。有机酸的合成与转化均在果实内完成[7]。糖、酸各组分分别在果肉细胞胞质和线粒体及乙醛酸循环体中转化形成/合成后, 大部分进入液泡中贮藏[6-7]

液泡作为糖、酸等风味物质的主要贮藏场所[8], 其贮藏功能很大程度上受膜转运特性的影响[7, 9]。此外, 液泡中存在的一些代谢酶可催化物质转化[9], 进而影响液泡中的物质组成。本文结合前人研究, 从液泡膜糖和酸转运载体, 质子泵类型, 液泡中糖、酸转化相关酶, 果实糖、酸积累关系等方面进行总结, 最终构建了园艺作物果实液泡糖、酸转运与转化的初级模型, 旨在为后续研究提供参考。

1 园艺作物果实糖、酸跨液泡膜的转运 1.1 园艺作物果实液泡糖转运蛋白

液泡糖转运蛋白可分为3类:单糖转运蛋白、蔗糖转运蛋白SUC/SUT(sucrose carrier/sucrose transporter)和SWEET(sugars will eventually be exported transporter)转运蛋白[9-10](表 1), 其中前两类转运蛋白属于MFS超家族(major facilitator superfamily), 具有12个跨膜结构域, 而SWEET仅具7个跨膜结构域, 由2个具有3次跨膜α-螺旋的MtN3 motif和1个起连接作用的跨膜α-螺旋组成。

表 1 园艺作物果实液泡糖、酸转运载体 Table 1 Vacuolar sugar and acid transporters of horticultural fruit
类型
Types
物种
Species
编码基因
Encodinggenes
登录号
Accessionnumbers
亚细胞定位
Subcellularlocalization
主要生理功能
Main physiologicalfunction
转运方向
Transportdirection
转运活性
Transportactivity
驱动力
Drivingforce
底物特异性
Substratespecificity
调控因素
Regulatoryfactors
参考文献
References
液泡糖转运载体
Vacuolarsugartransporters
葡萄
Vitis vinifera
VvTMT1 GSVIVT2919001/HQ323282 液泡膜
Tonoplast
葡萄糖转运载体
Glucosetransporter
流入Influx 葡萄糖 > 果糖
Glucose > fructose
[11-12]
白梨
Pyrusbretschneideri
PbTMT4 Pbr032130.1 液泡膜
Tonoplast
单糖转运载体
Monosaccharidetransporter
流入Influx 葡萄糖 > 蔗糖 > 果糖/山梨醇
Glucose > sucrose > fructose/sorbitol
[13]
甜瓜
Cucumismelo
CmTST2 XP_008448165.1 液泡膜
Tonoplast
促进果实糖分积累, 尤其是蔗糖
Promoting sugaraccumulation in fruits, especially for sucrose
可能为流入
Probablyfor influx
[14]
甜橙
Citrussinensis
CsERD6L Cs9g05220 液泡膜
Tonoplast
葡萄糖易化扩散转运体
Facilitated diffusiontransporter for glucose
流出
Efflux
较弱
Weaker
跨膜底物浓度梯度
Transmembrane substrateconcentration gradient
ABA、H2O2及糖饥饿逆境; 可能受转录因子Cs8g12680负调控
ABA, H2O2 and sugar starvation stress; possibly regulated by transcriptionfactor Cs8g12680 negatively
[15]
甜橙
Citrussinensis
CsSUT4 Cs3g16640 液泡膜
Tonoplast
蔗糖/H+同向转运载体
Sucrose/H+symporter
流出
Efflux
pH值为4.0时转运活性最强
With strongest transportactivity at pH value of 4.0
跨膜质子浓度梯度(质子泵)
Transmembrane protonconcentration gradient(proton pump)
蔗糖 > 麦芽糖
Sucrose > maltose
pH、低温、氧化逆境; 麦芽糖; 解偶联剂CCCP; 细胞膜P-ATPase抑制剂
VanadatapH, cold and oxidativestress; maltose; uncouplingagent CCCP; cell-membraneP-ATPase inhibitor Vanadata
[15]

Prunuspersica
PpSUT4 KU198999.1/ppa004620 液泡膜
Tonoplast
蔗糖/H+同向转运载体
Sucrose/H+symporter
流出
Efflux
低于PpSUT1
Lower thanPpSUT1
跨膜质子浓度梯度(质子泵)
Transmembrane protonconcentration gradient(proton pump)
pH [16]
苹果
Malusdomestica
MdSUT4 MDP0000206996 液泡膜
Tonoplast
可能介导液泡蔗糖流出
Possibly mediatingthe efflux ofsucrose fromthe vacuole
可能为流出
Probablyfor efflux
可能受外源糖、ABA、低温调控
Probably regulatedby exogenous sugar, ABA or cold
[17]
番茄
Solanumlycopersicum
SlTDT KC733165 液泡膜
Tonoplast
促进苹果酸积累, 抑制柠檬酸积累
Promoting malateaccumulation, andinhibiting citrateaccumulation
可能介导苹果酸流入, 柠檬酸流出
Possibly mediating the influxof malate into the vacuole; but the efflux of citrateout of the vacuole
[18]
苹果
Malusdomestica
MdtDT1 HM641022 可能定位于液泡膜
Probably locatedon tonoplast
促进苹果酸/柠檬酸积累
Promoting malate/citrate accumulation
可能为流入
Probablyfor influx
[19]
苹果
Malusdomestica
Ma1 MDP0000252114 液泡膜
Tonoplast
苹果酸离子通道
Malate channel
流入
Influx
[20]
葡萄
Vitis vinifera
VvALMT9 GSVIVG01008270001 液泡膜
Tonoplast
苹果酸(酒石酸)离子通道
Malate (tartrate) ion channel
流入
Influx
酒石酸转运活性高于AtALMT9
Conducting tartratebetter thanAtALMT9
膜电位
Membranepotential
[21]
番茄
Solanumlycopersicum
Sl-ALMT9 Solyc06g072910/Solyc06g072920 液泡膜
Tonoplast
促进苹果酸积累
Promoting malateaccumulation
可能为流入
Probablyfor influx
铝离子激活; 转录抑制因子Sl-WRKY42
Activated by Al3+; transcription repressorSl-WRKY42
[22]
脐橙
Citrus sinensis
CsCit1 EF028327 液泡膜
Tonoplast
柠檬酸/H+电中性同向转运体
Citrate/H+electroneutralsymporter
流出
Efflux
跨膜pH梯度
TransmembranepH gradient
Hcitrate2-特异性较高
Higher specificityfor Hcitrate2-
pH梯度
pH gradient
[23]
注: TMT:液泡膜单糖转运蛋白Tonoplast monosaccharide transporter; TST:液泡膜糖转运蛋白Tonoplast sugar transporter; ERD6L:早期干旱响应-6-类似转运子Early response to dehydration-6-like; SUT:蔗糖转运蛋白Sucrose transporter; TDT/tDT:液泡膜二羧酸转运蛋白Tonoplast dicarboxylate transporter; Ma1:一个曾被认为是苹果Ma基因座潜在候选基因的ALMT基因A gene encoding ALMT, previously identified as a potential candidate for the Ma locus in apple; ALMT:铝激活型苹果酸通道蛋白Aluminum-activated malate transporter; Cit:柠檬酸转运蛋白Citrate transporter; ABA:脱落酸Abscisic acid; CCCP:羰基氰化物间氯苯腙Carbonylcyanide- m-chlorophenylhydrazone; P-ATPase:P型腺苷三磷酸酶P-type adenosine triphosphatase.
1.1.1 液泡单糖转运蛋白

单糖转运蛋白分为7个亚家族, 其中4个亚家族的部分成员定位于液泡膜上[24]。液泡膜单糖转运蛋白(tonoplast monosaccharide transporter, TMT)或液泡膜糖转运蛋白(tonoplast sugar transporter, TST)与液泡葡萄糖转运蛋白(vacuolar glucose transporter, VGT)主要负责胞质糖分向液泡的输入[25-26]; 而早期干旱响应-6-类似转运子亚家族(early response to dehydration-6-like, ERD6L)的成员与肌醇转运蛋白(inositol transporter, INT)亚家族的成员INT1(inositol transporter1)分别负责液泡中葡萄糖与肌醇的输出[27-29]

1) TMT:负责葡萄糖由胞质向液泡运输的主要转运体, 介导葡萄糖/H+反向运输[25]。目前已分别从葡萄、柑橘、苹果、梨等果树基因组鉴定出VvTMT1-3CsTMT1-2MdTMT1-5PbTMT1-6等TMT家族成员[12-13, 24, 30]。定位于液泡膜上的VvTMT1与PbTMT4均可恢复己糖转运缺陷酵母菌株EBY.VW4000在低浓度单糖培养基上的生长, 说明两者均具备将胞质单糖输入液泡的功能[11, 13]。VvTMT1与PbTMT4对葡萄糖有较强的偏好性(表 1), 尤其VvTMT1的果糖转运能力较弱, 半乳糖与木糖转运能力更差[11, 13]PbTMT4是调控梨果实可溶性糖分积累的主要PbTMT成员, 其表达量随‘砀山酥梨’果实的发育过程而逐渐增加, 且后期增加更迅速, 基因表达量与糖分(尤其果糖与葡萄糖)含量呈极显著正相关关系[13]。转基因试验也表明, PbTMT4促进番茄果实葡萄糖与果糖积累[13]。虽然尚未验证其他TMT成员的亚细胞定位及糖转运特性, 但大多数成员的基因表达量与果实糖分(尤其是单糖)积累密切相关。VvTMT2在葡萄‘霞多丽’与‘雷司令’果实成熟过程中高表达且显著高于营养器官, 因此相应蛋白可能参与葡萄成熟过程中胞质己糖向液泡的转运[11, 31]。‘嘎啦’苹果果实发育过程中, MdTMT1MdTMT2相对表达量与总糖、还原糖、果糖和蔗糖含量变化呈极显著正相关关系[30]

TMT/TST成员不仅具有单糖转运能力, 部分成员还具备蔗糖转运功能。拟南芥AtTMT1和/或AtTMT2可作为液泡膜蔗糖/H+反向运输载体, 共同参与低温胁迫下蔗糖的积累[25, 32]。甜菜主根中高表达的BvTST2.1则专一介导蔗糖输入液泡, 并耦合H+反向运输[33]。研究表明, 果实中部分TMT家族成员也可能参与蔗糖跨液泡膜运转(表 1)。在‘砀山酥梨’果实发育过程中, PbTMT4表达量与蔗糖含量极显著正相关; 在己糖转运缺陷酵母菌株中异源表达PbTMT4进行转运功能验证, 发现PbTMT4不仅可恢复该菌株在低浓度单糖培养基上的生长能力, 还促进其在低浓度蔗糖培养基上的生长[13]。在甜瓜中鉴定出3个定位于液泡膜上的CmTST, 其中CmTST2在高糖品种‘伊丽莎白’果实发育过程中持续高表达, 且表达量远高于低糖品种‘小麦酥’和‘羊角酥’; 进一步研究发现, CmTST2在草莓与黄瓜中的异源表达也促进果实糖分(尤其蔗糖)的积累[14]

2) VGT:定位于液泡膜且具有活性的VGT成员, 有较高的葡萄糖特异性, 是又一类负责将胞质葡萄糖输入液泡的转运体, 介导葡萄糖/H+反向运输[26]。许海峰等[34]从苹果基因组中发掘出与拟南芥液泡膜AtVGT1同源性较高的MdVGT1, 其编码基因在新疆红肉苹果‘红脆1号’果实发育过程中的表达量与葡萄糖含量呈显著正相关, 并且MdVGT1可能与MdTMT1互作而共同转运葡萄糖进入液泡[34]。此外, 从葡萄、柑橘、梨等果树基因组中也鉴定出相关VGT家族成员[12, 24], 但尚未验证其亚细胞定位及功能特性。

3) ERD6L:该蛋白因最早发现于拟南芥干旱胁迫cDNA文库而得名, 多数成员定位于液泡膜[28-29]。研究发现, 部分液泡膜ERD6L成员可介导液泡中葡萄糖/H+的同向输出[29], 部分则非特异介导糖分的易化扩散[28]。目前关于园艺作物果实液泡膜ERD6L成员的报道还很少。定位于液泡膜的甜橙CsERD6L具备葡萄糖转运活性, 但由于转运速率较低而无法恢复单糖转运缺陷酵母菌株在低浓度单糖培养基上的正常生长, 其所介导的单糖转运属于易化扩散, 自然状态下主要负责液泡葡萄糖的输出[15](表 1)。甜橙果实糖分迅速积累阶段, CsERD6L与液泡酸性转化酶编码基因协同上调表达, 从而导致葡萄糖与果糖维持在低水平而蔗糖迅速积累[15]

4) INT:其成员多属于肌醇/H+同向转运载体, 定位于质膜或液泡膜, 其中液泡膜定位成员负责液泡内肌醇的外排[27]。在凤梨、番茄、葡萄及甜橙基因组中均已挖掘出与拟南芥液泡膜AtINT1同源性较高的INT家族成员[12, 15, 35-36]。其中, 凤梨AcINT1定位于液泡膜, 其在果实中的表达量远低于叶片[36]

1.1.2 液泡蔗糖转运蛋白

SUT4是蔗糖转运蛋白家族中唯一参与蔗糖跨液泡膜转运的亚家族, 主要负责液泡中蔗糖的外排, 此类蛋白多亲和性低而转运能力高, 且大多为H+同向运输载体[35, 37](表 1)。现已从番茄、甜瓜、甜橙、桃、苹果和杏等园艺作物中分别鉴定出SlSUT4 (AF176950)、CmSUT4 (ACJ04700)、CsSUT4 (Cs3g16640)、PpSUT4 (KU198999.1)、MdSUT4 (MDP0000206996)和PaSUT4 (KT223003)等SUT4亚家族基因成员[16-17, 24, 37-38], 并已验证了相应蛋白的液泡膜定位特性(PaSUT4除外)[15, 17, 37]。在蔗糖转运缺陷酵母菌株中异源表达CsSUT 4PpSUT4均可恢复其在单一碳源培养基(低浓度蔗糖)上的生长能力[15-16]。CsSUT4是园艺作物果实液泡中研究较为深入的SUT4亚家族成员。自然状态下CsSUT4主要介导液泡中蔗糖的输出, 但这一转运活性受解偶联剂CCCP(carbonylcyanide-m-chlorophenylhydrazone)与细胞膜P-ATPase(P-type ATPases)抑制剂Vanadata的明显抑制[15]。此外, CsSUT4对蔗糖的转运呈明显的pH值依赖性(pH值为4.0时具有最大转运速率; pH值升高或降低, 转运速率均明显下降)[15]。相对于亲本‘暗柳’甜橙, 较高的液泡pH值导致的CsSUT4低转运活性是‘红暗柳’甜橙蔗糖高积累的重要原因[15]

1.1.3 液泡SWEET蛋白

SWEET成员可顺浓度梯度介导蔗糖或己糖的跨膜运转, 在多数物种中分为4个亚家族:Ⅰ和Ⅱ亚家族倾向于己糖转运, Ⅲ亚家族倾向于蔗糖转运, Ⅳ亚家族则主要控制液泡果糖流通[39]。番茄SlSWEET16/17与拟南芥中具备液泡果糖双向转运功能的AtSWEET16/17同属于Ⅳ亚家族[10, 40], 且预测信息显示其定位于液泡膜的可能性最大[10]。香蕉、葡萄及苹果基因组中的MaSWEET16/17、VvSWEET17a-d及MdSWEET4.1成员也属于Ⅳ亚家族[39, 41-42]

1.2 园艺作物果实液泡酸转运载体

作为水果中重要的有机酸, 苹果酸和柠檬酸分别以二价阴离子(dianion malate, Mal2-)与三价阴离子(trianion citrate, Cit3-)的形式存在于胞质中性或微碱性环境中[7]。Mal2-与Cit3-一旦进入酸性液泡, 便立即被质子化, 以维持其跨膜电化学势差, 从而允许其持续进入液泡[7]

液泡膜上存在的苹果酸转运蛋白与苹果酸离子通道等对于苹果酸和柠檬酸跨液泡膜运转起到重要作用[23, 43-44]。研究表明, 苹果酸至少可以通过1种转运蛋白与3种离子通道来实现跨液泡膜运转[18, 43-44]。然而, 对于柠檬酸而言, 尽管不同物种甚至同一物种中存在不同的柠檬酸转运机制[45], 但至今尚未发现除苹果酸转运蛋白同系物之外其他介导柠檬酸跨液泡膜运输的转运体。

1.2.1 苹果酸转运蛋白

液泡膜二羧酸转运蛋白(tonoplast dicarboxylate transporter, tDT/TDT)是第一类被发现的植物液泡苹果酸载体[46]。现已分别从番茄(SlTDT)、苹果(MdtDT1)、李(tDT-like)及柑橘(CsCit1)等园艺作物中克隆到相应的tDT同源基因[18-19, 23, 47]。SlTDT与CsCit1均定位于液泡膜上, 预测李tDT-like具有类似的定位特点[18-19, 47]。但是, 不同园艺作物tDT的转运特性不同(表 1)。拟南芥AttDT缺失突变体中异源表达MdtDT1对苹果酸与柠檬酸积累的共同促进作用[19], 支持苹果酸与柠檬酸通过相同载体进入液泡的观点[48]。然而, 过表达SlTDT显著提高了番茄果实苹果酸含量, 但降低了柠檬酸含量[18]。抑制番茄果实中SlTDT的表达, 结果则相反[18]。柑橘CsCit1则属于柠檬酸/H+同向转运载体, 主要介导液泡柠檬酸输出, 二价柠檬酸阴离子(HCit2-)是其主要转运底物[23]。柑橘果实发育过程中, CsCit蛋白表达的最高时间点与柠檬酸含量开始下降及pH值开始上升的时间点相吻合[23]

1.2.2 苹果酸通道蛋白

铝激活型苹果酸通道蛋白(aluminum-activated malate transporter, ALMT)家族是普遍存在于植物体内的一类阴离子通道蛋白。其中, ALMT9是最早被报道的液泡膜苹果酸通道蛋白, 可介导苹果酸与延胡索酸跨液泡膜内向电流, 且对苹果酸的特异性高于延胡索酸[49]。定位于液泡膜的番茄Sl-ALMT9与葡萄VvALMT9对果肉细胞有机酸积累起着核心作用[21-22]。GWAS(genome wide association study)与遗传连锁图谱分析均表明, Sl-ALMT9是导致番茄果实苹果酸含量自然变异的重要候选基因, 其启动子区-423 bp至-421 bp处的GTC序列插入/缺失而导致的表达差异是影响苹果酸积累的主要因素。铝离子可激活Sl-ALMT9的表达, 而转录因子Sl-WRKY42则通过与Sl-ALMT9的启动子区的第8个W-box结合而抑制其表达[22]。葡萄中VvALMT9不仅具备苹果酸转运能力, 还可介导酒石酸转运。VvALMT9主要在葡萄的中果皮表达, 酿酒葡萄品种‘Aragonez’中其表达水平随果实成熟而显著增加[21]MdMC1 (HM641023)和MdMC2 (HM641024)是从苹果中发现的AtALMT9的同源基因, 在果实中MdMC1高表达, 且与不同基因型果实苹果酸含量正相关[19]。定位于液泡膜的苹果Ma1(MDP0000252114)与AtALMT9同属于ALMTⅡ亚家族, 具备苹果酸内向通道功能[20]。此外, ALMTⅡ亚家族中的AtALMT6与AtALMT4也能介导跨液泡膜苹果酸内向电流, 且具备液泡苹果酸外向通道的潜能[43-44, 49], 但其同系物在果肉细胞苹果酸积累中的作用尚未报道。

1.3 质子泵在糖、酸跨液泡膜转运过程中的作用

溶质逆浓度梯度积累或释放需要消耗能量。有些化合物进入液泡可通过直接的能量载体, 如:P型ATP酶或ABC转运蛋白(ATP-binding cassette transporter), 然而, 大部分化合物进出液泡依赖电化学梯度[9]。液泡膜上的质子泵可通过水解ATP或焦磷酸盐的富能磷酸键所释放的能量将质子从胞质输送到液泡, 从而降低液泡pH值。这一过程产生的跨液泡膜电化学梯度, 是包括糖转运体、有机酸转运体、无机离子转运体和次生代谢物转运体在内所介导的次级主动运输的重要驱动力[50]。例如, TMT、VGT1、INT1、SUT4和CsCit1等所介导的糖及酸转运均需耦合质子的运输。长期以来研究者认为, 液泡膜质子泵系统由2种不同的质子泵构成, 即V-ATPase(vacuolar H+-ATPase)和V-PPase(vacuolar H+-pyrophosphatase)[9]。V-ATPase结构较为复杂, 是由10多个不同亚基组成的复合物, 而V-PPase仅由单一多肽组成[8]

20世纪末已从果实中分离纯化出V-ATPase和V-PPase蛋白[51-52]。21世纪初, 从不同类型果实(如梨[53]、桃[54]、番茄[55]、苹果[56]及柑橘[57]等)中克隆到V-ATPase亚基编码基因及V-PPase编码基因。研究表明, 抑制‘Micro-Tom’番茄果实中V-ATPase A亚基的表达, 可显著促进蔗糖的积累[55]。V-PPase编码基因MdVHP1的过表达或异源表达显著促进苹果愈伤组织和番茄果实中苹果酸的积累[58]。低酸‘Jalousia’和高酸‘Fantasia’2个桃品种果实发育过程中, 相较于有机酸代谢相关酶编码基因, V-ATPase A亚基编码基因(PRUpe; AtpvA1)和V-PPase编码基因(PRUpe; Vp1PRUpe; Vp2)的表达更符合有机酸的积累模式, 说明液泡质子泵一定程度上决定果实的酸度[54]。‘雁荡雪梨’果实发育后期V-ATPase亚基编码基因(Pp : vVAtp1Pp : vVAtp2)和V-PPase编码基因(Pp : vVpp)的上升表达趋势与‘梗头青’果实中变化相反, 这可能是导致两者柠檬酸含量差异的重要原因[53]。柑橘果实发育过程中V-ATPase亚基编码基因CitVHA-F1CitVHA-F2CitVHA-c4与柠檬酸积累密切相关, 而CitVHA-E1CitVHA-c2CitVHA-dCitVHA-e基因与柠檬酸降解存在更密切的联系[57]。最新研究表明, 苹果果实中MdPP2CH(丝氨酸/苏氨酸蛋白磷酸酶2C家族H亚家族成员, a member belongs to subfamily H of the protein serine/threonine phosphatase 2C family)可通过脱磷酸作用而使3个V-ATPase(MdVHA-A3、MdVHA-B、MdVHA-D2)与1个铝激活型苹果酸转运蛋白(MdALMTⅡ)失活, 进而负调控苹果酸的积累。然而, MdPP2CH的脱磷酸化活性又可被MdSAUR37(生长素早期应答蛋白家族37亚家族成员, a member belongs to subfamily 37 of the small auxin up-regulated RNA family)所抑制[59]

2 园艺作物果实糖、酸在液泡中的转化

叶片光合产物作为果实糖分的主要来源, 可通过蔗糖、山梨醇、棉子糖、水苏糖及甘露醇等形式转运[6]。转运糖进入果实后, 部分直接积累, 而绝大部分会在相应酶作用下转化为果糖和/或葡萄糖以及其他糖类[6, 60-61](表 2)。部分果糖与葡萄糖会进一步分别被果糖激酶和己糖激酶磷酸化, 形成果糖-6-磷酸(fructose-6-phosphate, F6P)和葡萄糖-6-磷酸(glucose-6-phosphate, G6P)。F6P和G6P可在蔗糖磷酸合成酶、蔗糖磷酸磷酸酶以及其他相关酶作用下重新合成蔗糖[60], 也可进入糖酵解途径或戊糖磷酸代谢途径, 亦或参与到其他代谢产物的合成中[61]

表 2 园艺作物果实中转运糖转化第一步相关酶 Table 2 Enzymes involved in the first conversion step of translocation sugars in horticultural fruit
转运糖
Translocation sugars
转化相关酶类
Enzymes involved in conversion
产物
Products
备注
Remarks
蔗糖
Sucrose
细胞壁转化酶
Cell wall invertase中性/碱性转化酶
Neutral/alkaline invertase液泡酸性转化酶
Vacuolar acid invertase
果糖、葡萄糖
Fructose, glucose
蔗糖合成酶
Sucrose synthase
果糖、尿苷二磷酸葡萄糖
Fructose, uridine diphosphoglucose
可逆
Reversible
山梨醇
Sorbitol
NAD依赖型山梨醇脱氢酶
Nicotinamide adenine dinucleotide-sorbitol dehydrogenase
果糖
Fructose
可逆
Reversible
NADP依赖型山梨醇脱氢酶
Nicotinamide adenine dinucleotide phosphate-sorbitol dehydrogenase
葡萄糖
Glucose
可逆
Reversible
山梨醇氧化酶
Sorbitol oxidase
葡萄糖
Glucose
棉子糖Raffinose
水苏糖Stachyose
甘露醇Mannitol
α-半乳糖苷酶与转化酶
α-galactosidase and invertase
甘露醇脱氢酶Mannitol dehydrogenase
半乳糖、葡萄糖、果糖
Galactose, glucose, fructose
果糖Fructose

有机酸的合成与代谢主要由果实自身完成。果肉细胞质中, 来源于糖酵解途径的磷酸烯醇丙酮酸(phosphoenolpyruvate, PEP)的羧化反应属于有机酸合成的第1步, 生成苹果酸和草酰乙酸(oxaloacetate, OAA)。一旦苹果酸和OAA合成, 便可通过TCA循环(tricarboxylic acid cycle)或乙醛酸循环生成柠檬酸与其他二羧酸, 并实现循环转化。此外, 胞质中柠檬酸还可经γ-氨基丁酸支路和ATP-柠檬酸裂解酶分别分解形成琥珀酸和OAA。苹果酸或OAA在胞质中的脱羧反应则可再度合成PEP, 并可能激活糖异生[7]

果实中糖、酸的形成与转化是其积累的前提。但目前所报道的糖和酸转化主要发生于胞质、线粒体及乙醛酸循环体中, 液泡中相关报道较少。

高等植物液泡内存在的酸性转化酶(vacuolar acid invertase, VIN或VI)可将蔗糖水解为葡萄糖和果糖, 这是液泡内单糖的重要来源[62], 也是现有报道中液泡内负责糖分转化的主要酶。野生番茄(Lycopersicon chmielewskii)果实中, VI活性极低, 导致己糖含量极低而蔗糖含量较高; 栽培种番茄(L.esculentum)果实成熟过程中, VI活性逐渐上升, 己糖含量也随之上升[63]。转化反义液泡酸性转化酶编码基因SlTIV1, 可将己糖含量较高的番茄转变成为蔗糖含量较高的番茄[64]。SlVIF(vacuolar invertase inhibitor)则可通过与SlVI互作而抑制SlVI的活性[65]。过表达SlVIF导致番茄果实中蔗糖含量升高而己糖含量降低, 沉默SlVIF则效果相反[65]。此外, 番茄果实成熟调控因子RIN(ripening inhibitor)可分别通过与SlVISlVIF的启动子结合, 促进SlVI的表达, 而抑制SlVIF的表达, 进而影响果实糖分含量[65]。目前在苹果、梨、枇杷、甜橙、桃等果树中均已克隆出液泡酸性转化酶基因[15, 62, 66-67]。在番茄中异源表达枇杷液泡酸性转化酶基因EjVIN, 可促进果实生长, 但果个减小, 蔗糖含量降低[62]。桃果实液泡酸性转化酶基因PpVIN2对于冷藏条件下蔗糖代谢有重要作用, 冷藏‘白凤’和‘玉露’桃果实中蔗糖含量与VIN活性及PpVIN2表达量均极显著正相关[67]。目前尚未发现液泡中有机酸转化的相关报道。

3 总结与展望

结合前期研究结果, 我们构建出园艺作物果实液泡糖、酸转运及转化的初级模型(图 1)。糖分跨液泡膜转运所涉及的载体类型包括:1)单糖转运蛋白:TMT/TST、VGT1、ERD6L与INT1;2)蔗糖转运蛋白:SUT4;3)SWEET蛋白:SWEET16/17。而有机酸跨液泡膜转运所涉及的主要载体类型有:tDT、Cit1与ALMT9/Ⅱ。胞质中果糖/葡萄糖与蔗糖分别在TMT1/VGT1与TMT2.1或其同系物作用下进入液泡, 液泡中葡萄糖与蔗糖又可分别经ERD6L与SUT4或其同系物作用输出到胞质, INT1及其同系物则主要负责液泡中肌醇的外排, 这些转运过程均耦合液泡中质子的输出(除部分ERD6L成员)。SWEET16/17属于糖分异化扩散转运载体, 可依赖糖分跨液泡膜浓度梯度而将果糖、葡萄糖与蔗糖(尤其是果糖)输出或输入液泡。胞质中Mal2-可由ALMT9/Ⅱ与tDT及其同系物介导进入液泡, 后者伴随柠檬酸阴离子输出。胞质中Cit3-也可能经tDT作用输入液泡。Mal2-与Cit3-一旦进入酸性液泡便立即被质子化。Cit1则主要介导液泡中柠檬酸阴离子与H+的电中性输出。V-ATPase和V-PPase是液泡膜上主要质子泵类型, 可为糖、酸跨液泡膜转运提供驱动力。PP2CH可通过脱磷酸化作用负调控V-ATPase与ALMTⅡ的活性, 而SAUR37可抑制PP2CH的脱磷酸化活性进而正调控液泡苹果酸积累。VI是果肉细胞中目前报道的唯一的液泡糖分转化相关酶, 可催化液泡中蔗糖转化为果糖与葡萄糖, 但VI的催化活性可被VIF所抑制。

图 1 园艺作物果实液泡糖、酸转运与转化初级模型[7, 9, 71] Fig. 1 Primary model of vacuolar sugar and acid transportation and conversion in horticultural fruit[7, 9, 71] 1)ATP:三磷酸腺苷Adenosine triphosphate; ADP:二磷酸腺苷Adenosine diphosphate; glc:葡萄糖Glucose; fru:果糖Fructose; suc:蔗糖Sucrose; Mal2-:苹果酸二价阴离子Dianion malate; HMal-:苹果酸单价阴离子Monoanion malate; Cit3-:柠檬酸三价阴离子Trianion citrate; HCit2-:柠檬酸二价阴离子Dianion citrate; H2Cit-:柠檬酸单价阴离子Monoanion citrate; VGT1:液泡葡萄糖转运蛋白1 Vacuolar glucose transporter1;TMT1:液泡膜单糖转运蛋白1 Tonoplast monosaccharide transporter1;TST2.1:液泡膜糖转运蛋白2.1 Tonoplast sugar transporter 2.1;SUT4:蔗糖转运蛋白4 Sucrose transporter 4;ERD6L:早期干旱响应-6-类似转运子Early response to dehydration-6-like; INT1:肌醇转运蛋白1 Inositol transporter1;SWEET16/17:糖外排转运蛋白16/17 Sugars will eventually be exported transporter16/17;tDT:液泡膜二羧酸转运蛋白Tonoplast dicarboxylate transporter; ALMT9/Ⅱ:铝激活型苹果酸通道蛋白9/Ⅱ Aluminum-activated malate transporter 9/Ⅱ; Cit1:柠檬酸转运蛋白1 Citrate transporter1;V-ATPase:液泡H+-腺苷三磷酸酶Vacuolar H+-adenosine triphosphatase; V-PPase:液泡H+-焦磷酸酶Vacuolar H+-Pyrophosphatase; VIK1:丝裂原活化三级激酶类蛋白激酶1 Mitogen-activated triple-kinase-like protein kinase1;VI:液泡酸性转化酶Vacuolar acid invertase; VIF:液泡转化酶抑制子Vacuolar invertase inhibitor; PPi:焦磷酸盐Pyrophosphate; Pi:磷酸盐Phosphate; PP2CH:丝氨酸/苏氨酸蛋白磷酸酶2C家族H亚家族成员Subfamily H of the protein serine/threonine phosphatase 2C family; SAUR37:生长素早期应答蛋白家族37亚家族成员Subfamily 37 of the small auxin up-regulated RNA family.
2)图中红色字体表示该载体蛋白同系物的亚细胞定位及转运特性均已报道; 紫色字体表示该载体蛋白的同系物仅进行亚细胞定位, 未进行明确转运特性研究; 绿色字体表示该载体蛋白同系物尚处于预测阶段; 黑色字体表示其他。
Transporters in red font represent that both subcellular localization and transportation characteristics of their homologs have been reported in horticultural fruit. While those in purple font indicate that in horticultural fruit only subcellular localization of their homologs have been researched, but not for transportation characteristics. Transporters in green font indicate that the information of their homologs in horticultural fruit is merely predictive. Black words represent something else.

上述糖、酸载体/代谢酶中仅少数成员的亚细胞定位及转运/催化特性在园艺作物果实中得到了验证, 有关分子调控机制的报道更是少之又少。因此, 这一模型在所有园艺作物果实液泡糖、酸转运及转化中的适用性, 尚待进一步验证。另外, 液泡糖、酸转运与转化模型有待进一步完善, 如是否存在特异介导山梨醇等跨液泡膜转运的载体, 液泡中是否还存在其他糖、酸转化酶类等。前人研究发现, 苹果种子发育中期的子叶液泡内腔存在山梨醇脱氢酶(sorbitol dehydrogenase, SDH)[68], 叶肉细胞液泡的非透明沉积物中也存在SDH[69], 子叶液泡膜上还存在山梨醇氧化酶(sorbitol oxidase, SOX)[70]。因此, 推测果实液泡中可能也有SDH和SOX以及其他糖、酸代谢酶的存在。随着果实液泡分离技术的不断改进以及液泡多组学数据库的建立, 将有助于我们进一步挖掘参与果实液泡糖、酸转运与转化的关键基因以及核心调控因子, 进而完善果实糖、酸积累的分子调控网络。

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