全 文 ：广 西 植 物 Guihaia 28(3)：395— 401 2008年 5月
Existent states of anthocyanins in vacuole and
ZHAO Chang-Ling，ZHANG Li-Mei，LIU Fu-Cui
(College of Agrlcultural Sciences and Biotechnology，Yunnan Agricultural University，Kunming 650201，China)
Abstract：This review sums up the reasons of anthocyanins being sequestered into vacuole，the existent s~tes of an—
thocyanins in vacuole and the corresponding coloration effects of anthocyanins on plant cells．Transporting of antho—
cyanins from the biosynthesis site，namely the cytoplasm of plant cel，into vacuole is to detoxify the toxicity of antho—
cyanins on the functional molecules of the cel，such as proteins and DNAs．The vacuolar compartmentalization of an—
thocyanins is the prerequisite for anthocyanin function normaly in plant cels
． In a wide rang e of plant species and in
most cases，anthocyanins dissolve completely in vacuole． However，in vacuoles，anthocyanins can also form granules
which can be classified into two categories，namely anthocyanoplast(ACP)and anthocyaninic vacuolar inclusion
(AVI)．ACP is membrane-bounded，its formation is the result of the progressive coalescence of the smaler pigmented
vesicles in vacuole and fuly developed ACP is typicaly spherical and more deeply red-colored than the vacuole．In
vacuole，ACP is high density and insoluble globule highly concentrated wi th anthocyanins
． The emergence of ACP can
provide intense coloration in the vacuole．AVI may be protein matrix and it posseses neither a membrane boundary
nor an internal structure，its formation is the result of the anthocyanins transported into the vacuole bind with a pro—
tein matrix．In vacuole，AVI is irregular and jelly-like in shape．In AVIs，the attachment of anthocyanins to the ma—
trix protein is likely to be via H—bonds to a stericaly restricted site．AVI is suggested to act as vacuolar anthocyanin
“trap”，preferentialy for anthocyanidin 3，5-diglycosides or acylated anthocyanins． The emergence of AVI can en-
hance color intensity and results in the“blueness’of COlOr in the vacuole．
Key words：anthocyanin；existent state；coloration effect；anthocyanoplast；anthocyaninic vacuolar inclusion
CLC Number：Q946．83 Document Code：A Article ID：1000—3142(2008)03—0395—07
Anthocyanins are the coloured end products of the
flavonoid pathway and consist of anthocyanidins and
saccharides(Holton& Comish，1995)．They are typi—
cally found in flower and fruit tissues，and in the super—
ficial cells of organs such as leaves and stems．The an—
thocyanin-pigmented cells are typically restricted to the
epidermis and hypodermis(Pecket& Small，1980)．
The most apparent and important function of an-
thocyanins in plant life is that they act as water-soluble
vacuolar pigments(Harborne，1976；Gould eta1．，1995；
Zhao et a1．，2004a)．Generaly，depending on constitu—
ent substitutions and complexes formed with metalions
and copigm ents，anthocyanins can appear red，blue or
purple colors under the low pH，vacuolar conditions
(Mueler et a1．，2000；Zhao et a1．，2004b)．Almost all
vascular plants possess basic anthocyanins such as pel—
argonidin 3一O-glucoside and cyaniding 3一O-glucoside
responsible for the red to magenta coloration of flowers
and fruits and delphinidin 3一O-glucoside introducing
blue tones to the floral organs of plants(Harborne&
W iliams，2000；Zhao et a1．，2004c，2006)．The colora—
tion of anthocyanins is radically decided by their antho—
cyanidins．Three different classes of anthocyanidins are
responsible for the primary shades of the flower and
Received date：2006—12—10 Accepted date：2007—05—05
Fomdatian ii~n：Supported by the Provincial Dep~tment of Science and Technology of Yunnan(2006C0030Q)；Startup Fund for Doctor of
Yunnan Agricultural University(A2002096)
Biography：ZHAO Chang-Ling(1969一)，male，Born in Dujiangyan City of Sichuan Province，Doctor，Associate professor，working in
Plant physiology，Phytochemistry and Plant biochemistry and molecular biology ．
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396 广 西 植 物 28卷
fruit colors~pelargonidin(orange to brick red)，cyani—
din(pink to red)and delphinidin(purple to blue)(Har—
borne，1976；Zhao et a1．，2004c，2006)．
However，so far，no comprehensive account con—
cerning why anthocyanins accumulate in vacuole and
how their coloration effects are realized has been pub—
lished(Grotewold，2004)． Today，anthocyanins have
been one of the targets of plant metabolic engineering
with the aim of creating new，or altering the properties
of existing colored compounds．Understanding of the
cytological mechanisms of the coloration effects of an-
thocyanins in plants is of great and potential signifi—
cance to plant breeders and molecular biologists who
are interested in creating novel colored flowers or fruits
or enhancing anthocyanin production．
This review attempts to sum up the reasons of an—
tocyanins being sequestered into vacuole，the existent
states of antocyanins in vacuole and the corresponding
coloration efects of anthocyanins on plant cels．
Anthocyanins accumulate in vacuole
1．1 Transporting of anthocyanins from cytoplasm into
vacuole is to detoxify the toxicity of anthocyanins on the
functional molecules of the plant cell
Anthocyanins，including their hydrophobic agly—
cone anthocyanidins，are potentially cytotoxic and geno-
toxic compounds that can oxidize proteins and interca—
late into DNAs(Matile，1984；Matile，1987；Hrazdina，
1992；Ibrahim，1992；Ahmed et a1．，1994；Klein et a1．，
1996；Li et a1．，1997；W ink ，1997；Grotewold et a1．
1998；Klein et a1．，2000；Debeaujon et a1．，2001)，and
the corresponding detoxification process is accom-
plished by the transport of anthocyanins from the syn-
thesis site，namely cytoplasm，into vacuole(Nozue et
aZ．，1993；Coleman et a1．，1997；Klein et aZ．，2000；
Bartholomew et a1．，2002)．Generally，the vacuole has
the potential to detoxify and store not only endogenous
but also foreign，biotic，and abiotic glucosylated sub—
stances(Taiz，1992)．Co mpartmentalization of anthocy—
anins in the vacuole is required both to limit the muta—
genic and oxidative effects of the anthocyanin biosyn—
thetic pathway intermediates and to express the proper
biological function of the anthocyanins(Ahmed et a1．，
1994；Rueff et a1．，1995；Wink，1997)．SO，anthocya—
nins，like a number of other secondary products of
plant metabolism，can not uaually be in the cytoplasm
(Xu et a1．，2001)，and normally accumulate in the、阻c—
uole(Harborne，1976；Saunders& Corm，1978；Wag—
ner，1979；Stafford，1990；Hrazdina 8L Jensen，1992；
Gould et a1．，1995；Mol et a1．，1998)．
It is currently believed that the molecular modifi—
cations of anthocyanins are directly related to the
transport of the anthocyanins from cytoplasm into vac—
uole．Glycosylation or acylation of anthocyanins ap—
pears to be the prerequisite for the vacuolar uptake of
anthocyanins(Matern et a1．，1986；Hopp & Seitz，
1987；Wink，1997；Ba rtholomew et a1．，2002；Springob
et a1．，2003)．It was further found that the glucose
residue attached to the molecules is not SUmcient to act
as a signal of the vacuolar sequestration of anthicyanins
(Frangne et a1．，2002)．
1．2 The vacuolar compartmentalization of anthocyanins
is the precondition of an thocyanin function normally in
plan t cells
In general，vacuoles offer a larger storage space，
which is important for anthocyanins to reach concentra—
tions great enough to function in the protection against
preda tors and pathogens or as UV light sunscreens or
attractants(Klein et a1．，2000；Debeaujon et a1．，2001)．
As a matter of fact，until vacuolar compartmentation of
anthocyanins is completed，they are not stabilized，and
are unable to function in plant ceils，e．g．as pigments
(Winkel-Shirly，2001；Kitamura，2006)．The pigm enta—
tion of anthocyanins is finally realized in vacuole because
it is the acidic environment of vacuole that causes the al—
teration of anthocyanins from colorless to colored pig—
ments(Spelt et a1．，2002；Kitamura，2006)．
2 Existent states of anthocyanins in
vacuole
2．1 Anthoeyanins dissolve totally in vacuole
In a de range of plant species and in most cases。
the water-solubility of anthocyanins ma kes them usually
dissolve uniformly in the vacuolar solution，resulting in
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3期 赵昶灵等：高等植物花色苷在液泡中的存在状态及其着色效应 397
the relatively even color of plant cells(Pecket 8L Small。
1980；Markham et a1．，2000)． Therefore，anthocyanin
pigments become a good marker to reflect the vacuole
size(Wagner et a1．，1978； z& Zeuger，1991)．
2．2 Anthoeyanins form gramfles in vacuole
2．2．1 Discovery of the anthocyanin granules in vacuole
and their diversification of denominations In certain
species，anthocyanins are found to localize in discrete
regions of the vacuole(Pecket 8L Small，1980；Mark—
ham et a1．，2000)，suggesting that anthocyanins can
exist in the form of granules in vacuole．Early in 1 9 1 1，
it appeared to be Politis who ma de the first significant
anatomical observations on anthocyanin-pigm ented
cells．He recognized that an intensely pigm ented struc—
ture was usualy present in some anthocyanin-contai—
ning cells，and he took the lead in terming it“a cyano—
plast”．In 1 926，Lipma a observed a simi lar body in an—
thocyanin-pigm ented cels and na med it“an anthocyan-
ophore”． Since that time，such subcellular structures
have been found in anthocyanin-producing cells of more
than 70 plant species representing at least 33 fami lies
of angiosperm s spanning both dicotyledons and mono—
cotyledons(Pecket{5L Sma l，1980)．
Historically，the anthocyanin granules in vacuole
were denomi nated optionaly and independently．They
have been described as“blue spherules”in the epiderrnal
cells of rose(Rosa rugosa)petal(Yasuda，1 974)，“blue
crystals”in larkspur，Consolida ambigua petals(Asen
et a1．，1975)，“ball—like structures”and“crystals”in
stock，Matthiola incana，petals(Hemleben，1981)，and
red“crystals’’in mung bean(P 口^sP0Z“s radiatus)hy—
pocotyls(Nozzolillo 8L Ishikura，1 988)，“intravacuolar
spherical bodies”in Polygonum cuspidatum seedlings
(Kubo et a1．，1995)．The petals of a number of other
flowers also contain simi lar intensely colored intravacu—
olar bodies(Markham et a1．，2000)．
Despite the tremendous progresses involved in the
worldwide exploition on the anthocyanin granules in vac—
uole，not much is known about the mechanisms by which
anthocyanins form granules in vacuole and give birth to
specific coloration efects on the plant cel1．Nowadays，
from the angle of structural and biochemical diferences，
it should be reasonable to classify the anthocyanin gran—
ules in vacuole into two categories：ACP and ．
2．2．2 Categories of the anthocyanin granules in vacu—
ole and their coloration effects ACP appears to be the
first term proposed by Pecket and Sma ll(1980)to de—
scribe the membrane-bounded anthocyanin granules in
vacuole．It is not the site of anthocyanin biosynthesis
but one of the existent states of anthocyanins in vacu—
ole after anthocyanins are transported into vacuole．
(1)Formation course，existent shape and dynami c
changes：Formation of ACP is the result of the pro-
gressive coalescence of the smaller pigm ented vesicles
in vacuole．First，numerous unassodated sma ll red ves—
icles form in vacuole，while only faint pigmentation is
visible in the vacuole．Then，the red vesicles appear to
be associating，resulting in the gradual decrease of the
vesicle amount．Later，one vesicle becomes substantial—
lY larger than the others and the coloration of the vacu-
ole becomes more obvious．As development proceeds，
the larger red vesicle，na mely the so-caled ACP，in-
creases further in size，and the sma ll vesicles disappear
completely．Normally，only one ACP is present wi thin
the central vacuole．It is thought above process is posi—
tively related wi th the biosyn thesis of anthocyanins
(Pecker 8L Sma l，1980；Nakamura，1993)．In addition，
it is observed that the occurrence of ACP appears to be
inhibited by the presence of absdsic acid(ABA)and 2，
4一D(Nozue et a1．，1993；Kim et a1．，1997)．
After form ation，the existence of ACP in vacuole
is not quiescent but dyn ami c．Fully developed ACP is
typically spherical and more deeply red-colored than
the vacuole，e．g． in the young and relatively short-
lived tissues of red cabbage(Brassica oleracea var．cap—
itata)．When the pigm ented cells approach the comple—
tion of the development，their ACPs tend to degenerate
and become less easily detectable(Pecket 8L Sma l，
1980)，which has something to do with the gradual de—
pigm entation of the mature cells．
(2) Strcutrue and chemi cal property：ACP is
found to be membrane-bounded，e．g．，in the seedling
hypocotyl and leaves of red cabbage(Brassica oleracea
var．capitata)，ACP is bounded by a single tripartite
membrane approximately 10 nnl in thickness(Pecket&
Sma ll，1980)．So，ACP is thought to be one kind of or—
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398 广 西 植 物 28卷
ganelle rather than simply being hydrophobic droplet
(Pecket and Small，1980；Nozzolillo eta1．，1988；Naka-
mura et a1．，1993；Grotewold et a1．，1998；Xu et a1．
2001)．In vacuole，ACP is strongly osmiophilic，high
density and insoluble globule highly concentrated with
anthocyanin(Nozue et a1．，1993)．
(3)Coloration effects：The emergence of ACP can
provide intense coloration in the vacuole．It seems that，
just in the enlarging course of ACP，some anthocyanin
leaks into the vacuole causing it to become progressively
more pigmented(Pecket & Small，1980；Nakarnura，
1993)．The formation and enlargement of resulted
in the intense red coloration of the grape berry(Nakamu—
ra，1989，1994)． At the same time，it is demonstrated
tha t the presence of can not prolong the pigm enta—
tion time of the cell(Pecket& Sma ll。1980)．
厂I appears to be the first term proposed by
Markham et a1．(2000)to describe the non-membrane-
bounded anthocyanin granules in vacuole． 厂I is not the
site of anthocyanin biosynthesis either．Just like ACP。it
is also one of the existent states of anthocyanins in vacu—
ole after anthocyanins are sequestered into vacuole．
(1)Form ation course，existent shape and dynamic
changes：Form ation of AVI is the result of the combi—
nation of anthocyanins wi th proteins in vacuole．After
anthocyanins are transported into the vacuole，they
bind wi th a protein matrix and form AVI(Conn et a1．，
2003)，suggesting that the proteins ma y exist in the
vacuole in advance or they are transported into the vac—
uole just at the time when anthocyanins are being se—
questered．In AⅥ s，the attachment of anthocyanins to
the matrix protein is likely to be via H-bonds to a ster‘-
ically restricted site． At the pH of the vacuole，the
bonding is strong enough to effectively integrate the
anthocyanins and the protein，and the protein acts，in
fact，as an efficacious vacuolar“trap’for anthocyanins
(Markham et a1．，2000)．
After form ation in vacuole。A is irregular and
jelly-like in shape(Markham et a1．，2000)．In addition，
independently of variations in the levels of anthocyanins，
light appears to induce the coalescence of ，Is。resulting
in the spread of anthocyanins from the inclusions into
the vacuolar sap(Irani& Grotewold。2005)．
(2)Strcutrue and chemi cal property．AVI ma y be
protein matrix(Nozue eta1．，1995；Nozue eta1．，1997；
Markham et a1．，2000)and it possesses neither a mem-
brane boundary nor an internal structure(Nozzolillo，
1994；Co rmier，1997；Nozue et a1．，1997)．Therefore，it
appears not to be organelles(Ma rkham et a1．，2000)．
Moreover，AVI ma y be composed of several protein
species(Zhang et a1．，2004)．In the AⅥs of lisianthus
(Eustorna grandiflorum)cells，three proteins were
found to have approxima te molecular weights of
50000，35000 and 34000 Daltons and pIs in the range 4
— 5(Markham et a1．，2000)．
Av1 iS found to be insoluble in most aqueous
buffers and is not solubilized with common detergent
such as SDS。 ton X-100 and Nonidet P40．It is also
only partially soluble in concentrated denaturing solu—
tions such as 6 mol／L urea and 6 mol／L guanidine
hydrochloride．Complete solubilization is only achieved
wi th high concentration of both denaturant and reduc—
tant(Ma rkham et a1．，2000)．
AVI exhibits a high degree of specifcity to the mo-
lecular structures of anthocyanins．A is suggested to
act as vacuolar anthocyanin “traps”，preferentialy for
anthocyanidin 3，5-diglycosides(Ma rkham et a1．，2000)
or acylated anthocyanins(Co nn et a1．，2003；Zhang et
a1．，2004)．In lisianthus(E．grandiflorum)cells，bound
to the肌 ’matrix are four cyanidin and delphinidin
acylated 3，5-diglycosides，and the“trapped”anthocya—
nins are shown to difer from solution anthocyanins only
in tha t they lack a terminal rhamnose on the 3一linked ga—
lactose(Markham et a1．，2000)．
(3)Co loration effects：A I is considered to be the
storage site of anthocyanins(Zhang et a1．，2004)．The
presence of AVI results in intensification in colour and
a significant shift in the absorbance spectra of anthocy—
anins in the cells．
On one ha nd。 厂I can enhance color intensity．In
lisianthus(E．grandiflorum)flowers，the packaging of
anthocyanins into AVI produces marked CO1Our intensifi-
cation by concentrating anthocyanins abo ve Levels that
would be impossible in vacuolar solution(Markham et
a1．，2000)．
On the other hand， 厂I can result in the“blue-
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3期 赵昶灵等：高等植物花色苷在液泡中的存在状态及其着色效应 399
ness”of color．A distinct bluing of color in the A s—
rich petal zone of carnation(Diamhus caryophyllus)is
observed，and the normally pink pelfirgonidin pigments
produce a blue-grey colouration，which is evidenced by
the enhanced absorptance in the longer wavelength
bands at about 625 nm(Markham et a1．，2000)．Flowers
of the“Rhapsody in Blue”rose(Rosa rugosa)cultivfir
show a change in color induced by age，namely from red-
purple to bluish-purple．Ths variation is associated with
a progressive accumulation of anthocyanins into AVI—like
structures，and cyanin is probably“trapped’into AVI at
higher concentrations than would be possible in a vacuo—
lfir solution and in quinonoidal form ，appearing purple-
blue because of additional absorption in the rang e of 580
- 630 um (Gormet，2003)．Theoretically，absorption in
the 580-630 nm region is attributed to the presence of
anthocyanin quinonoidal bases in either neutral or ionized
forms(Hoshino& Goto，1990；Lin et a1．，1992；Brouil-
lard and Dangles，1994；Bfiranac et a1．1996)．The“blu—
ing ”efect could be the result of a concomitant equilibri—
urn shift favouring the quinonoidal base forms of the an—
thocyanin due either to the bo nding or to self association
(Hoshino& Goto，1990)．
As a result，nowadays research advances indicate
that，in vacuole，anthocyanins can dissolve completely or
form granules，affecting the color of plant cels directly
and obviously． However，nothing is known abo ut how
anthocyanins choose different existent modes in the cells
of different plant organs or species．Further studies fire
clearly needed to elucidate the mechanisills by which an—
thocyanins exist in vacuole in different states．Today，the
storage of anthocyanins in vacuole and the corresponding
pigmentation effects fire the important aspect of anthocy-
anin-related studies because they are the cytological
mechanism of the coloration of all anthocyanin-pigmen—
ted cells．It ha s been becomi ng an issue of central im—
portance in understanding how anthoc yanin coloration is
controled in who an d may offer the opportunity to in—
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3期 赵昶灵等：高等植物花色苷在液泡中的存在状态及其着色效应 401
I．Observation on the development of the tannin body in the up-
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等植物花色苷在液泡中的存在状态及其着色效应
赵昶灵，张丽梅，刘福翠
(云南农业大学 农学与生物技术学院 ，昆明 650201)
摘 要：综述了花色苷被摄人液泡的原因、花色苷在液泡中的存在状态及其对植物细胞的着色效应。花色苷在
植物细胞质中合成后转运到液泡里是为了解除其对蛋白质和DNA等细胞功能分子的毒性。花色苷的液泡区隔
化是花色苷在植物细胞 中发挥正常功能的前提。在大多数植物中，花色苷在绝大多数情况下完全溶解在液泡
里。但是，花色苷也能在液泡里形成颗粒 ，这些颗粒可以划分为花色苷体和花色苷液泡包涵体两类 。花色苷体
由膜包裹，其形成是液泡中小的有色囊泡逐渐合并的结果，发育完全的花色苷体为典型的球状、具 比液泡更深的
红色；液泡里的花色苷体具高密度，呈现为含高浓度花色苷的不溶性小球；花色苷体的存在可导致液泡的强烈色
彩。花色苷液泡包涵体可能具备蛋白质基质，既无膜包裹又无内部结构，其形成是转运进液泡的花色苷与蛋白
质基质结合的结果；液泡里的花色苷液泡包涵体形状不规则，象果冻；在花色苷液泡包涵体 中，花色苷可能通过
氢键连接于蛋白质基质的一个有限空间位点；花色苷液泡包涵体被认为是液泡中花色苷的“陷阱”，优先摄取花
色素 3，5一二糖苷或酰化的花色苷；花色苷液泡包涵体的存在可增加液泡色彩的强度并导致“蓝化”。
关键词 ：花色苷 ；存在状态 ；着色效应 ；花色苷体；花色苷液泡包涵体
(上接第 381页 Continue from page 381)
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