全 文 ：广 西 植 物 Guihaia 29(3)：393— 399 2009年 5月
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同 等植物花色苷的液泡摄取机制
赵昶灵 ，李孙文2*，张维明1
(1．云南农业大学 农学与生物技术学院，昆明 650201；2．云南农业大学 农科专业基础实验教学中心，昆明 650201)
摘 要：综述了高等植物细胞中花色苷被液泡摄取的机制。花色苷通过细胞质中定位于粗糙内质网细胞质
面的多酶复合体合成后被膜包裹形成囊泡。这些囊泡主要向液泡移动，在移动中相互融合形成更大囊泡，最
终将花色苷带到液泡膜的表面。在大多数情况下，花色苷经过液泡膜上的各种载体被迅速运进液泡。另外两
种较少的是：(1)囊泡直接与液泡融合；(2)液泡膜自主形成大的管状 内陷，使囊泡在内陷处指向液泡内腔“发
芽”。在上述种种可能的具体过程中，花色苷以非修饰或修饰两种形式被摄入液泡。花色苷跨液泡膜运送可
能通过 4种模型实现，即由ATP结合盒型的载体介导、由依赖 pH梯度的载体介导、由24一kD液泡蛋白前体
衍生的蛋白质介导和由多重药物和有毒化合物排出家族的载体介导。据推测，不同植物利用不同的摄取机制
将花色苷积累在液泡中，而多重机制也可能被单个植物种同时使用。
关键词 ：高等植物；花色苷；液泡摄取；机制
中国分类号 ：Q945 文献标识码 ：A 文章编号：1000—3142(2009)03—0393-07
Vacuolar sequestration mechanisms of
anthocyanins in higher plants
ZHAO Chang-Ling ，LI Sun-Wen ，ZHANG Wei—Ming
(1．Colege of Agricultural Sciences and Biotechnology，Yunnan Agricultural University，Kunming 650201，China；2．Teaching
Center of the Basic Experiments of Agricultural Majors，Yunnan Agricultural University，Kunming 650201，China)
Abstract：This review sumed up the mechanisms of anthocyanins being sequestered into vacuole in the cells of higher
plants．After being synthesized by the muhienzyme complexes locating at the cytoplasmic face of the rough endoplas—
mic reticulum in the cytoplasm，anthocyanins are enwrapped by membrane tO form vesicles which migrate mainly to—
ward the vacuole，coalesce each other to form larger vesicles in the migration，and ultimately bring the anthocyanins to
the surface of the tonoplast．In most cases，anthocyanins are expeditiously transported into vacuole by various trans—
porters locating on the tonoplast．Other two minor possibilities are that：(1)the vesicles fuse directly with the vacu—
ole；(2)the tonoplast may independently form a large tubular invagination from which the vesicles“bud of”into the
vacuole lumen．In the concrete courses of above possibilities．anthocyanins are sequestrated into vacuole in two forms．
namely non-modified and modified．The transtonoplast transport of anthocyanins may be accomplished by four rood—
els，namely mediated by ATP—binding cassete(ABC)type transporter，by pH—dependent transporter，by the proteins
derived from the 24一kD vacuolar protein(VP24)precursor and by multidrug and toxic compound extrusion(MATE)
family transporter．It is speculated that diferent plant species utilize diferent sequestration mechanisms tO accumu—
late anthocyanins in vacuole，and multiple mechanisms may be simultaneously used in individual plant species
．
Key words：higher plants；anthocyanins；vacuolar sequestration；mechanisms
Received date：2007—03 19 Accepted date：2008—09—28
Foundation item：Supported by the Provincial Department of Science and Technology of Yunnan Province(2006C0030Q)；the Startup Fund for Doctor of
Yunnan Agricultural University(A2002096)
Biography：ZHAO Chang-1 ing(1969一)I Male-Born in Dujiangyan City of Sichuan Province．Doctor of Science．Associate Professor．working in
Plant physiology，Phyt0chemistry and Plant biochemistry and molecular biology。(E-mail)zhaoplumblossom7@163．com．
Author for correspondence：E—mai1：Sunwen1i68@yahoo．cn
394 广 西 植 物 29卷
Belonging to flavonoids，anthocyanins are the col—
ored end product of the general phenylpropan0id path—
way and consist of anthocyanidins and saccharides(Ho—
lton＆ Cornish，1995；Zhao eta1．，2003)．
Anthocyanins have been found distributing only in
the particular parts of a specific range of plants
(Andersen& Jordheim，2006)．They are commonly
found in angiosperms but are replaced by betacyanins
in all families of Centrospermae except Caryophylaceae
(Harborne 8L Hall，1964；Harbome，1965，1986)．Mo—
reover，they are typically detected in flower and fruit
tissues and in the superficial cells of organs such as
leaves and stems．The anthocyanin-pigmented cells are
typically restricted to the epidermis and hyp odermis
(Harborne，1973；Pecket& Small，1980)．
It has been revealed that anthocyanins are related
not only to plant life but also to human health． In
plant life，anthocyanins provide brilliant pigments in
order to attract insects or animals for pollination and
seed dispersal(Harborne，1976；Gould et a1．，1995；
Grotewold，2006)．Nowadays，anthocyanins are regar—
ded as one of the most important alternatives to a num—
ber of synthetic dyes which have been applied in foods，
cosmetics and medicines，and found to be very harmful
to human health(Mazza＆ Brouillard，1987)．
A series of evidences have been found to support
that the subcellular site of anthocyanins synthesis in
plant cell is the cytoplasm and anthocyanins are syn —
thesized on the cytoplasmic face of the rough endoplas—
mic reticulum(rER)(Hrazdina et a1．，1980；Wagner&
Hrazdina，1 984；Hrazdina & Wagner，1 985；Winkel—
Shirley，1999，2001)．Nevertheless，anthocyanins are
uaually observed not to exist in the cytoplasm(Xu et
a1．，2001)，and normally accumulate in the vacuole
(Harborne，1976；Saunders ＆ Co nn，1978；Wagner，
1979；Hrazdina& Jensen，1992；Go uld et a1．，1995；
Mol et a1．，1998；Kitamura，2006)．Therefore，it is ob—
vious that，after being syn thesized in the cytoplasm of
plant cel，anthocyanins are transported into vacuole．
Up to now，no comprehensive explanation con—
cerning how anthocyanins are transported from their
synthesis site，namely cytoplasm，into vacuole has been
published．Nowadays，anthoeyanins have been one of
the targets of plant metabolic engineering with the ob—
jective of creating new or altering the properties of ex—
isting，colored compounds(Winkel—Shirley，2001)。
Knowing adequately the mechanisms of the vacuolar
sequestration of anthocyanins in the cells of higher
plants is of great significance to biochemi sts and molec—
ular biologists who are interested in realizing the effec—
tive regulations on the biosynthesis and storage of an—
thocyanins．
As a result，this review attempts to sum up the
possible multiple mechanisms of antocyanins being se—
questered into vacuole．
1 From cytoplasm to tonoplast，an—
thocyanins are transported in vesicles
1．1 After being synthesized in the cytoplasm。anthocya—
nins ale el lpped by membrane to form vesicles
It is tempting to speculate that，after anthocyanins
are synthesized by the multienzyme complexes locating
at the cytoplasmic face of the rER，they are enwrapped
by membrane to form vesicles j ust like the tannin vesi—
cles，and are transported within the cytoplasm toward
the tonoplast by means of various vesicle-mediated
processes(Baur＆ Walkinshaw，1 974；Parham ＆
Kaustinen，1977；Pecket& Small，1980；Zobel，1986；
Nozzolillo&Ishikura，1988；Ibrahim，1992；Grotewold，
2001；Grotewold，2004)．These vesicles can be defined
as the pre-vacuolar compartment(PVC)of anthocya—
nins．
However，the formation mechanism of the vesicles
has not been elucidated clearly． The vesiculation
process of anthocyanins probably begins just after the
anthocyanins are synthesized．The anthocyanin-contai—
ning vesicles may be concretely produced via the for—
marion of the double layered structures and the cyto—
plasmi c structures may underlie the forming process
(Facchini，2001)．Initialy，in the cytoplasm，the tiny
vesicles enwrapping anthocyanins are likely to originate
from the rER，and rER is thought to be the initial ac—
cumulation site of the vesicles(Grotewold et a1．，
1998)．
3期 赵昶灵等：高等植物花色苷的液泡摄取机制 395
1．2 The moving behavior
vesicles in the cytoplasm
of the anthocyanin-containing
It is totaly unknown
responsible for the motion
about the impetus which is
of the anthoeyanim contai—
ning vesicles through the cytoplasm．Theoretically，de—
spite the probable help provided by cytoplasmic strue—
tures(Facchini，2001)，it is not possible that the vesi—
cles move in the cytoplasm only by simple diffusion
．
The moving direction of the anthocyanin-contai—
ning vesicles in the cytoplasm is believed mainlv to be
the vacuole．When they mi grate toward the vacuole
，
they coalesce each other to forlTl larger vesicles(Grote—
wold et a1．，1998；Lin et a1． ，2003)．Ultimately。these
vesicles bring anthocyanins to the surfaee of the ton0一
plast(Grotewold eta1．，1998)． Then，three pOSSibilities
are proposed to account for the fate of the anthocya—
nins,(1)they are expeditiously transported into vacuo1e
by various transporters locating on the tonoplast，
which is thought not only to be a joint action 0f trans—
porters and vesicles but also to be the most dominant
possibility in plant cels(Marrs etⅡZ． ，1995；Grotewold
et a1．，1998；Grotewold，2004；Kitamura，2006)；(2)the
anthocyanin_containing vesicles fuse directly with the
vacuole(Scott et a1．，1997；Grotewold et n
． ，1998)：
(3)the tonoplast form，maybe in an autonomous fash—
ion，a large tubular invagination from which the vesi—
cles“bud off”into the vacuole lumen
． The vacuo1ar in—
vagination’s lumen is continuous with the cytoplasm
。
making this inverse budding reaction equiva1ent to mi—
croautophagocytosis(Miller et a1． ，2000)．
It is also observed that not al of the vesicles are
“sported to tonoplast，some stay in the cytoplasm ．
which makes anthocyanins be compartmented within
the cytoplasm and the cytoplasm display special colors
(Nozzolillo et a1．，1988；Calderon PtⅡZ
． ， 1993；Lin P￡
a1．，2003；Goodman et a1
． ，2004)．A case in Doint is
that，in maize(Zea rnays)，the cyanidin-3一glucoside aD
—
pears violet when localized in the vacuole
， but in bz2
corn,anthocyanin remains in the cytoplasm 、 ere it
undergoes oxidation and polymerization reactions
， the
oxMized products appear tan-bronze on the kernels
(Marrs et a1．，1995；Alfenito et a1
． ，1998)．The fact
that the anthocyanins enwrapped in the vesicles are ac-
ylated glycosides appears to be the precondition for the
stay of the vesicles in the cytoplasm(Markham etⅡZ． ，
2001)．
2 Mechanisms of anthocyanins being
sequestrated into vacuole through va—
rious transporters on tonoplast
2．1 Anthocyanins are transported through tonoplast
in two forms
It has been found that the anthocyanins can be
transported through tonoplast in two forms
． One
is non—modified，namely the anthocyanins are di—
rectly transported into vacuole，e
． g．barley antho—
cyanin—g1ucosides(Klein et a1．，1996)． The other is
modified，namely the modification of anthocvanins
is the prerequisite for their effective vacuolar up-
take，and glycosylation or acylation of anthocyanins
appear usually to be the precondition for the vacuo
lar uptake of anthocyanins(Matern Pt“Z
． ， 1 986：
Hopp& Seitz，1987；Wink，1997；Bartholomew
a1．，2002；Springob et a1． ， 2003)．It was further
found that the glucose residue attached to the mo1一
ecules is not sufficient to act as a signal of the vac—
uolar sequestration of anthicyanins(Frangne t n Z
． 。
2002)．However，nothing is known regarding the
mechanisms by which different plant species selects
different anthocyanin forms to finish the vacuo1ar
uptake of the anthocyanins
．
2·2 Possible models of anthocyanins being transpor—
ted through tonoplast
2．2．1 Transport mediated by ATP—binding cas—
sette(ABC)type transporter A growing body of
evidence has demonstrated that glutathione S—
transferase(GST)proteins may be involved in the
vacuolar sequestration of anthocyanins(Marrs et
al，1 995)．However，not all GSTs are responsible
tor the vacuolar sequestration of anthocyanins，e
．
g．in parsley，GST1 appears to act in the early steDs
of a UV light—dependent signal transduction Dath—
way leading to chalcone synthase gene(CHS)ex-
pression(Loyall et a1．，2000)．
Different functions have been described about
396 广 西 植 物 29卷
GST in the anthocyanin transport course mediated
by ABC-type transporter．Previously，GSTs were
thought to form glutathione。conj ugates of anthocy。-
anidin 3一glucosides(Marrs et口Z．，1995)，because
GSTs can catalyze the addition of a glutathione
(GSH)molecule to a heterocyclic organic anion
(Edwards & Dixon，2000)． The corresponding
GSH conj ugate serves the dual purpose of increas—
ing hydrophilicity and marking the molecule for
transport by the multidrug resistance—associated
proteins(MRP)(Ishikawa et a Z．，1997)．It was ev—
er believed that covalent glutathionation is a pre—
requisite for sequestration through a glutathione
pump(GS—X pump)in the tonoplast membrane
(Martinoia et a1．，1993；Li et a1．，1995)．But pres—
ently，GSTs are believed to function as cytoplasmic
“escort”proteins without actually catalyzing GSH
conjugate production in the cytoplasm，because
they may bind anthocyanins through hydrophobic
interactions and the binding reaction occurs near
the tryptophan residues． Afterward，they escort
the bound anthocyanins toward the tonoplast
(Marrs et a1．，1995；M tieller et a1．，2000；M aeller
8L Walbot，2001)．
After recognized and glutathionated by GST，
anthocyanins are transported through tonoplast
and into vacuole by ABC transporter． The gluta—
thione“tag”served as a marker for vacuolar seques—
tration of anthicyanins(M tidier et口Z．，2000)．The
GST—or glutathione—GST—bound anthocyanins are
then taken up into vacuoles through a membrane—
localized specific transporter，namely a M g抖 一ATP—
energized glutathione—specific pump(GS—X pump)
(Martinoia et a1．，1993；Ishikawa et a1．，1997；Ed—
wards et a1．，2000)，which is classified as belonging
to the MRP subfamily and the superfamily of ABC
transporters(Martinoia et a1．，l993；M arrs et a Z．，
1995；Lu et a1．，1997；Borst et a1．，1999；Rea et
nZ．，1998；Rea，1999；Goodman et al，2004；Grote—
wold，2004)． In general，the substrate recognition
of ABC transporters involves not only the gl utathi—
one or glycosyl moieties but also the basic Cl s L ore
of anthocvanin(Klein et a1．，2000)，and the amount
of GSTs binding anthocyanins available in the cell
maybe modulate the activities of GS—X pump，and
the modulation could be via allosteric activation by
intermediates，ph0sphorylation，or protein— trans—
porter interactions(Frangne et a1．，2002)．Finally，
the vacuolar deposition of anthocyanins is accom—
plished by a cotransport mechanism with reduced
GSH ，analogous to the transport of vincristine in
the liver(Loe et a1．，1998；M tidier et aZ．，2000)．
2．2．2 Transport mediated by pH—dependent trans—
porter A specific transporter depending on a pH
gradient across the tonoplast has been supposed for
the transport of anthocyanins acylated with sinapic
acid into vacuole(Hopp& Seitz，1987)．The H 一
electrochemical potential difference is established
by the vacuolar H 一ATPase(V—ATPase)and vacu—
olar H 一pyrophosphatase(Rea Sanders，1 987；
Zhen et a1．，1997；Sze et a1．，1999)． Both pumps
catalyze electrogenic H 一translocation from the cy—
tosol into the vacuole to establish an inside—acid
pH gradient(ApH )and an inside—positive electrical
potential difference(△‘fJ)(Bartholomew et“Z．，
2002)．However，it still remains to be determined
whether anthocyanin transport and vacuolar acidifi—
cation are joined directly(Spelt et a1．，2002)．Mo—
reover，ABC transporter—driven sequestration of an—
thocyanins should theoretically result in much
higher accumulation of the anthocyanins within the
vacuole as compared with the antiport or potential—
driven mechanisms，respectively(Rea＆ Sanders，
1987；Kreuz et a1．，1996；Frangne et a1．，2002)．
2．2．3 Transport mediated by the proteins derived
from the 24一kD vacuolar protein(VP24)precursor
The proteins derived from the VP24 precursor is
believed to mediate the transtonop1ast transport Of
anthocyanins into vacuole because the specific lo—
calization，accumulation of VP24 and the expres—
sion property of VP 24 in plant cells seem to be
closely related with the vacuolar ingestion of an—
thocyanins． In the anthocyanin—containing vacu—
oles，VP24 was found to be localized in anthocyano—
plasts(ACPs)and accumulate as one of the major
vacuolar proteins(Pecket& Small，1 980；Nozue et
3期 赵昶灵等：高等植物花色苷的液泡摄取机制 397
aj．，1995；Nozue et a1．，1997)． 24 expression is
intimately correlated with the accumulation of an~
thocyanins in vacuoles，but no transient increase of
phenylalanine ammonialyase (PAL )or chalcone
synthase(CHS)was determined(Lawton et a1．，
1983；Chappell& Hahlbrock，1984；Xu et a1．，
200 1)．Therefore，it can be thought that the pro～
teins derived from the VP24 precursor is probably
involved in the transport or steady accumulation of
anthocyanins in vacuoles(Xu et a1．，2001)．
2．2．4 Transport mediated by multidrug and toxic corn—
pound extrusion(MATE)family transporter At pres—
ent，MATE family transporter is believed to mediate
the transtonoplast transport of anthocyanins into VaCH—
ole mainly because，in Arabidopsis，ttl2 is proved to
encode a member of the MA TE fami ly transporters
which is involved in the sequestration of flavonoids，
maybe including anthocyanins，into vacuole(Brown et
“Z．，l999；Debeaujon et a1．，2001)．But the universali—
ty of MATE family transporter in the transtonoplast
transfer course of anthocyanins into vacuole stil needs
to be further corroborated．
％ ken together，above four models are virtually
difficult to reconcile．Now，it should be reasonably hy—
pothesized that different plant species make use of dif—
ferent sequestration mechanisms to congregate antho—
cyanins in vacuole，and manifold mechanisms may sire—
uhaneously be used in individual species(Martinoia et
“Z．，2000；Mtidier& Walbot，2001)．
Understanding the molecular mechanisms in—
volved in the transtonoplast transport of anthocyanins
into vacuole stand for a radical，yet weakly illuminated，
problem in botany．Establishing the real pathways in—
volved in the sub—cellular trafficking of anthocyanins is
apparently an essential for the fruitful engineering of
anthocyanin metabolism in higher plants．
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