Archives of Biochemistry and Biophysics 539 (2013) 102–109

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Archives of Biochemistry and Biophysics

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Review

Chromoplast biogenesis and carotenoid accumulation

Li Li ⇑, Hui Yuan
Robert W. Holley Center for Agriculture and Health, USDA-ARS, Cornell University, Ithaca, NY 14853, USA
Department of Plant Breeding and Genetics, Cornell University, Ithaca, NY 14853, USA

a r t i c l e i n f o a b s t r a c t

Article history: Chromoplasts are special organelles that possess superior ability to synthesize and store massive
Available online 12 July 2013 amounts of carotenoids. They are responsible for the distinctive colors found in fruits, flowers, and roots.
Chromoplasts exhibit various morphologies and are derived from either pre-existing chloroplasts or
Keywords: other non-photosynthetic plastids such as proplastids, leucoplasts or amyloplasts. While little is known
Chromoplast biogenesis about the molecular mechanisms underlying chromoplast biogenesis, research progress along with pro-
Carotenoid accumulation teomics study of chromoplast proteomes signifies various processes and factors important for chromo-
Metabolic sink
plast differentiation and development. Chromoplasts act as a metabolic sink that enables great
biosynthesis and high storage capacity of carotenoids. The formation of chromoplasts enhances caroten-
oid metabolic sink strength and controls carotenoid accumulation in plants. The objective of this review
is to provide an integrated view on our understanding of chromoplast biogenesis and carotenoid accumu-
lation in plants.
Published by Elsevier Inc.

Introduction Chloroplasts as photosynthetic plastids are the most conspicuous

Plastids are major organelles ubiquitously found in plant cells.
Based on color, structure, and metabolites accumulated, plastids
can be categorized into different types, such as proplastids, etiop-
lasts, chloroplasts, chromoplasts, leucoplasts, elaioplasts and amy-
loplasts [1]. Proplastids are the progenitors of other types of
plastids. They are colorless with limited internal membrane vesi-
cles and found in the meristematic cells of shoots, roots, and young
fruits. Etioplasts contain prolamellar bodies and found in flowering
plants grown in dark. Chloroplasts are green, photosynthetic
organelles with distinctive internal thylakoid discs and occur in
leaves and other green tissues. Chromoplasts contain large quan-
tity of carotenoids and are associated with the red, orange, and yel-
low colors in flowers, fruits and roots. Leucoplasts are a general
term for colorless plastids that include elaioplasts and amyloplasts.
Elaioplasts fill with oil in oil-accumulating storage organs, and
amyloplasts contain starch granules in roots and storage tissues
[2].
These different plastids fulfill various distinctive and essential
functions for plant growth and development, and serve as the main
sites for photosynthesis and/or many important primary and sec-
ondary metabolisms [2,3]. All plastids are enclosed by double
envelope membranes and retain a semi-autonomous character.
⇑ Corresponding author at: Robert W. Holley Center for Agriculture and Health,
USDA-ARS, Department of Plant Breeding and Genetics, Cornell University, Ithaca,
NY 14853, USA. Fax: +1 607 2551132.
E-mail address: ll37@cornell.edu (L. Li).
plastid type and have been subjected to extensive studies. Among
non-photosynthetic plastids, chromoplasts have received the most
attention due to the accumulation of pigments that are important
for nutritional and sensory quality of agricultural products [4].
Chromoplasts like all other plastids are descendants of prokary-
otes [5]. Chromoplasts are characterized by massive accumulation
of carotenoid pigments. The vivid red, orange and yellow colors of
many flowers, fruits, and vegetables owe their hue to the high lev-
els of carotenoid accumulation in chromoplasts. Chromoplasts de-
velop unique mechanisms to synthesize and deposit large amounts
of carotenoids. Recent research has demonstrated that regulation
of chromoplast biogenesis plays a crucial role in controlling carot-
enoid content by enabling great biosynthesis and high storage
capacity [6–8]. This review focuses on the recent progress toward
our understanding of various processes of chromoplast develop-
ment and carotenoid accumulation in plants.
Carotenoid accumulation, a net result of biosynthesis,
degradation and sequestration
Significant progress has been made in our understanding of
carotenoid metabolism in plants [6–15]. Carotenoid composition
and relative abundance in green leaf tissues of plants are remark-
ably conserved for optimal function of photosynthesis, with lutein,
b-carotene, violaxanthin, and neoxanthin as dominant carotenoids
occurring in a decreased order [16]. In contrast, carotenoid content
and identity in non-green tissues of plants vary largely from negli-
gible in white tissues to high quantity in colored organs that

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http://dx.doi.org/10.1016/j.abb.2013.07.002
 L. Li, H. Yuan / Archives of Biochemistry and Biophysics 539 (2013) 102–109 103

accumulate specific carotenoids such as lycopene in watermelon

and tomato fruits, a-/b-carotene in carrot and sweet potato, and lu-
tein in marigold flower. Carotenoids can be synthesized in nearly
all kind of plastids except proplastids [17], but they accumulate
in large quantity in chloroplasts of green tissues and in chromop-
lasts of many fruits, roots, and flowers. The amount of carotenoids
accumulated is determined by the rate of biosynthesis and degra-
dation, as well as by the capacity of stable storage of synthesized
products in a sink structure (Fig. 1). Thus, factors that associate
with these processes all regulate final carotenoid levels.
Carotenoid biosynthetic catalytic activity is a critical factor in
determining carotenoid level. Phytoene synthase (PSY)1 is gener-
ally accepted as the rate-limiting step in the carotenoid biosynthetic
pathway. Its expression is tightly coordinated and controlled by
source and sink metabolites [7]. While a single copy of PSY exists
in Arabidopsis, multiple PSYs regulate carotenoid metabolic flux in
a tissue-specific manner in many other plants [18,19]. Because of
the central role of PSY in directing metabolic flux into the carotenoid
biosynthetic pathway, alternation of PSY expression exerts profound
effects on carotenoid accumulation, as demonstrated in many trans-
genic studies [20–25]. A recent study shows that PSY gene expres-
sion and subsequently carotenoid accumulation is negatively
regulated by phytochrome-interacting factors in Arabidopsis during
de-etiolation [26]. In addition, by examining the epistasis in tomato
color mutants, PSY1 gene expression is suggested to be controlled by
cis-carotenoids in tomato fruits [27]. PSY isozymes exhibit differen-
tial plastid localization, which could link to its activity in promoting
carotenoid biosynthesis [28]. While PSY is the major determinant for
carotenoid content in plants, other carotenoid biosynthetic enzymes
alter carotenoids accumulated [13]. Lycopene e-cyclase (LCY - e)
plays a key role in controlling metabolic flux from one branch to an-
other branch within the carotenoid biosynthetic pathway. LCY - e is Fig. 1. Carotenoid accumulation is determined by the rate of biosynthesis, the rate
of turnover, and stable storage in a sink structure. Phytoene synthase (PSY) is a
found to be a major regulator in determination of the b-carotene/a-
major determinant of carotenoid content, whereas lycopene-e-cyclase (LYC-e)
carotene ratio in maize [29,30]. It underlies a major b-carotene quan- regulates flux between the branches of the biosynthetic pathway and b-carotene
titative trait locus in maize kernel [31]. Suppression of LCY - e in hydroxylase (BCH) controls b-carotene level. Or represents the only known gene
Arabidopsis and potato alters the ratio of b-carotenoids to lutein that governs chromoplast differentiation.
[32,33], which further confirms a key role of LCY - e as a determi-
nant of flux between the branches. b-carotene hydroxylase (BCH)
catalyzes the conversion of b-carotene into xanthophylls. Recently, mature leaves of Arabidopsis shows continuous carotenoid turn-
a study reveals that Hydroxylase3 locus contributes to b-carotene over and the turnover rates are influenced by the carotenoid spe-
level in maize [34]. Down-regulation of BCH expression enhances cies [48].
b-carotene content in potato [35]. It is likely that BCH represents an- Carotenoids are synthesized in the membranes of nearly all
other control point for a,b-carotene level in crops. types of plastids except proplastids [15,17]. The capacity of seques-
The rate of carotenoid degradation or turnover inversely deter- tration and stable storage of the synthesized products in a plastid
mines carotenoid content as the case of accumulation of many sink also contributes to the final levels of carotenoid accumulation
other metabolites in living cells. While random cleavage of carote- [6–8]. Various types of plastids including etioplasts, amyloplasts,
noids caused by photooxidation or peroxidase/lipoxygenase co- leucoplasts, chloroplasts, and chromoplasts exist in plant tissues.
oxidation occurs, a family of carotenoid cleavage dioxygenases These plastids have different capacities in synthesizing and seques-
(CCDs) catabolizes specific enzymatic turnover of carotenoids into tering carotenoids. Etioplasts are found in dark-grown plants and
apocarotenoids [9,36–38]. CCDs help maintain carotenoid homeo- defined by the presence of paracrystalline prolamellar bodies that
stasis in plants and some members of the CCD family also control eventually become thylakoids of chloroplasts upon illumination
the synthesis of phytohormones ABA and strigolactone [39–42]. [49]. Etioplasts have low biosynthetic catalytic activity and storage
Expression of CCD1 and CCD4 has been found to negatively corre- capacity of plastids [26], thus, they contain extremely low levels of
late with carotenoid accumulation in a number of plant species carotenoids. Amyloplasts or leucoplasts are lack of carotenoid
such as in chrysanthemum flowers [43,44], in potato [45,46], and sequestering structures within these plastids [50], which may
in strawberry [47]. A recent 14CO2 pulse-chase labeling study in make the synthesized carotenoids more vulnerable for degrada-
tion. Many important staple crops such as wheat, rice, barley,
1
maize, potato, and cassava have low to mediate levels of carote-
Abbreviations used: ABA, abscisic acid; accD, acetyl-CoA carboxylase, beta subunit;
noids synthesized and accumulated in the membranes of amylop-
BCH, b-carotene hydroxylase; CCD, carotenoid cleavage dioxygenase; CCS, capsan-
thin/capsorubin synthase; CHRC, chromoplast-specific carotenoid-associated protein; lasts or leucoplasts in edible seeds or roots [17]. In chloroplasts,
HDS, hydroxymethylbutenyl diphosphate synthase; Hsp, heat shock protein; HYD, b- carotenoids constitute photosynthetic complexes in thylakoid
carotene hydroxylase; LCY-e, lycopene e-cyclase; LTR, long terminal repeat; MEP, membranes [7,8]. The formation of thylakoid membranes pro-
mevalonate pathway; NCED, 9-cis-epoxycarotenoid dioxygenase; OPP, oxidative motes sequestration and storage of the synthesized carotenoids
pentose phosphate; PDS, phytoene desaturase; Pftf, plastid fusion translocation
factor; PSY, phytoene synthase; QTL, quantitative trait locus; ROS, reactive oxygen
for high level of accumulation in chloroplasts. Chromoplasts accu-
species; sHSPs, small heat shock proteins; SnRK, Snf1-related protein kinase; ZDS, f- mulate massive amounts of carotenoids and possess a superior
carotene desaturase. storage capacity to deposit carotenoids in carotenoid-lipoprotein
104 L. Li, H. Yuan / Archives of Biochemistry and Biophysics 539 (2013) 102–109
sequestering structures and/or plastoglobules [4]. These structures
enhance the sink strength of chromoplasts, leading to the massive
accumulation of carotenoids found in many flowers, fruits and
vegetables.
Chromoplasts as a metabolic sink
Chromoplasts synthesize and sequester massive amounts of
carotenoids by generating carotenoid–lipoprotein substructures
and/or accumulating plastoglobules inside the plastids [4,6,51]. A
large number of studies show that biosynthesis of components of
the carotenoid sequestering structures is well associated with
carotenoid accumulation [50,52–58]. It has been shown in some
instances that the proliferation of carotenoid sequestering struc-
tures is more strongly associated with increases in the carotenoid
accumulation than the changes in carotenogenic gene expression
or enzyme abundance [53,59,60]. Additionally, plastoglobules as
lipoprotein particles enlarge considerably and sometimes become
the dominant features within chromoplasts during the transition
from chloroplasts to chromoplasts [61]. Carotenoids in lipid body
plastoglobules are highly stable. Thus, it is not surprising to find
that high level of carotenoid enrichment in the oil droplets is
achieved [20,53]. Plastoglobules have been suggested to be in-
volved in chromoplast biogenesis [62]. In addition to their well-
established function of carotenoid sequestration and storage [62],
plastoglobules are found to have the capacity of synthesizing
carotenoids [63]. These carotenoid sequestering substructures
serve two roles for massive amounts of carotenoid accumulation
in chromoplasts. One role is to facilitate the sequestration of newly
synthesized carotenoids for stable storage and the other one is to
avoid overloading of end products at the site of carotenoid biosyn-
thesis for continuous biosynthesis [53,55].
Chromoplasts serve as a metabolic sink for carotenoid accumu-
lation. Indeed, biogenesis of chromoplasts to enhance sink strength
has been shown to exert a strong effect on carotenoid accumula-
tion [50,60]. Increase of chromoplast compartment size and num-
ber contributes to enhanced carotenoid levels. For example, high
carotenoid content in the tomato high pigment mutants as well as
in the tomato flacca and sitiens mutants are found to be linked to
an increased plastid number and/or compartment size, which en-
ables greater biosynthesis and higher storage capacity [64–66]. A
recent study reveals that the carotenoid associated protein CHRC
is up-regulated in these high pigment mutants and the increased
CHRC level regulates carotenoid sequestration and storage for the
enhanced carotenoid accumulation in these tomato fruits [57].
Chromoplast morphology
Carotenoids are sequestered and stored in carotenoid–lipopro-
tein substructures and/or plastoglobules within chromoplasts.
Based on the variation of these storage substructures, chromop-
lasts are classified into five main categories as globular, crystalline,
membranous, fibrillar, and reticulo-tubular type [4,5]. More than
one type of carotenoid sequestering substructures are often found
within a chromoplast, making the categorization of chromoplasts
difficult, and often chromoplasts from the same plant species fall
into different types. Globular chromoplasts are characterized with
accumulation of plastoglobules. A number of fruits and vegetables,
such as yellow and orange pepper, citrus, squash, mango, and yel-
low papaya contain chromoplasts abundant with plastoglobules
[67–69]. Crystalline chromoplasts are normally associated with
hyperaccumulation of b-carotene and lycopene as crystal struc-
tures. Typical examples include chromoplasts found in carrot root,
red papaya, tomato [67,69,70] and watermelon [71]. Membranous
chromoplasts contain extended concentric membranes with low
amounts of plastoglobules. Chromoplasts from the daffodil flowers
represent the best known membranous chromoplasts, where the
plastid inner envelope membrane proliferates, producing a concen-
trically stacked array of massive membranes [72]. By analog, chro-
moplasts from orange curd cauliflower are classified as
membranous chromoplasts although they can also be categorized
as crystalline type [73]. Fibrilla chromoplasts are characterized
by the presence of lipoprotein fibrils with extensive bundled
microfibrillar structures. Red pepper is found to accumulate 95%
of carotenoids in the specific fibrils in chromoplasts [74]. Reticu-
lo-tubular chromoplasts contain twisted fibril network filling plas-
tid stroma. Mango chromoplasts are assigned to be reticulotubular
type in addition to globular type [68]. The highly heterogeneous
nature of chromoplasts from different plant species may contribute
to the various profiles of carotenoid accumulation found in fruits,
vegetable, and roots.
Path of chromoplast differentiation
Plastid types are generally interconvertable [49]. Chromoplasts
can be differentiated from a number of paths (Fig. 2). Chromoplasts
are best known to be derived from fully developed chloroplasts as
seen during green fruit ripening in tomato and pepper. Chromop-
lasts also originate from non-green plastids as shown during fruit
ripening and root development from white tissues to carotenoid
enriched tissues in melon, watermelon, papaya, mango, carrot
and sweet potato. In these cases, chromoplasts are either differen-
tiated directly from proplastids, or arise from leucoplasts or amy-
loplasts (Fig. 2).
Chromoplast differentiation from chloroplasts is characterized
by complete degradation of chlorophyll and disappearance of thy-
lakoid structures of chloroplasts, accompanied with remodeling of
internal membrane systems and dramatic accumulation of carote-
noids in the newly formed carotenoid sequestering substructures
within chromoplasts [4,61]. The transition of chloroplasts into
chromoplasts during fruit ripening represents one of the most vis-
ible events. Chromoplasts are believed to arise from pre-existing
chloroplasts during fruit ripening. A recent spectral confocal
microscopy analysis of carotenoids and chlorophylls in isolated
plastids from tomato fruits shows intermediate plastids containing
both carotenoids and chlorophylls at the breaker stage, providing
direct evidence of chromoplast biogenesis from pre-existing chlo-
roplasts [75]. While chromoplast differentiation from chloroplasts
Fig. 2. Path of chromoplast biogenesis from other plastids. Chromoplasts are best
known to be derived from fully developed chloroplasts during fruit ripening from
green tissues. Chromoplasts also evolve from non-green plastids (proplastids,
leucoplasts or amyloplasts) during fruit ripening and root development from white
tissues.
 L. Li, H. Yuan / Archives of Biochemistry and Biophysics 539 (2013) 102–109
in tomato and pepper fruits is regarded as terminal, the fully differ-
entiated chromoplasts in citrus fruit pericarps and pumpkins can
redifferentiate back into chloroplasts [49]. Sucrose and nitrogen
as well as phytohormones are found to be associated with the plas-
tid transition in citrus fruits [76].
Chromoplasts can also be differentiated directly from proplast-
ids in plants. Proplastids are undifferentiated plastids and present
in high abundance in meristematic tissues. All plastids in plants are
ultimately derived from proplastids. Cauliflower orange curd mu-
tant accumulates high levels of carotenoids in shoot apical meris-
tems as well as in curd inflorescence meristems, yielding a
striking orange shoot and curd phenotype [77]. Microscopy studies
of the meristematic tissues from contrasting genotypes reveal that
while cells from wild type shoot apices contain multiple proplast-
ids with simple interior substructures, cells from the mutant plant
contain large chromoplasts as the only type of plastids within
these pigmented cells [73,77], supporting chromoplast differentia-
tion directly from proplastids.
In many cases, chromoplasts evolve from leucoplasts existed in
young, non-photosynthetic tissues during fruit and root develop-
ment. In papaya fruits, leucoplasts are found to be among the dom-
inant plastids in unripe papaya [69]. Chromoplasts are believed to
emerge directly from these plastids as no intermediate plastids
such as amyloplasts or chloroplasts are observed during fruit rip-
ening in papaya [69]. Similarly, chromoplasts are considered to dif-
ferentiate from leucoplasts in carrot root during root development
[78] and to be derived from amyloplasts as the disappearance of
amyloplasts is concomitant with carotenoid accumulation in chro-
moplasts [70].
Chromoplasts are also shown to originate from amyloplasts in
saffron red stigma of young floral buds [79] and in tobacco floral
nectaries [80]. Saffron chromoplasts in red stigma evolve from
amyloplasts since amyloplasts are the only plastids existing in
the colorless basal portion of style, and the formation of transition
forms of plastids as amylo-chromoplasts are observed [79]. In
developing tobacco floral nectaries as they change to orange color,
chromoplasts are directly converted from amyloplasts, and the
conversion process is accompanied with breakdown of starch and
production of nectar sugars [80].
Genes initiating chromoplast differentiation
Plastid biogenesis is crucial for plant survival. Considerable pro-
gress has been made toward understanding various aspects of
chloroplast biogenesis and development [3,81–84]. Many plastidic
and nuclear factors required for chloroplast biogenesis and devel-
opment are identified [3,81–84]. However, not much is known
about those factors that govern chromoplast biogenesis.
The Or gene originally isolated from cauliflower orange curd
mutant represents the only known gene that acts as a bona fide
molecular switch to trigger the differentiation of non-colored plas-
tids into chromoplasts [60,85]. The Or gene is isolated via posi-
tional cloning [86,87]. The mutation results from an insertion of
a copia-like LTR retrotransposon in the mutant allele, which posi-
tively controls b-carotene accumulation in the apical shoot and
curd tissues [60]. Or encodes a plastid associated protein contain-
ing a DnaJ cysteine-rich zinc finger domain and is highly conserved
among divergent plant species [60]. Since the OR protein lacks the
typical J-domain that defines DnaJ-like molecular chaperones, OR
represents a novel protein with a cellular function in triggering
chromoplast differentiation [60,73,77]. As discussed above, micro-
scopic analysis of plastids reveals that the Or mutated gene con-
verts numerous colorless proplastids or leucoplasts in each cell
from wild type shoot and curd tissues into one or two large chro-
moplasts that constitute the entire plastidome in those affected
105
cells [60,73,77]. The fact that expression of the Or transgene in
both white cauliflower and potato tubers induces chromoplast for-
mation [50,60] further confirms its crucial role in triggering chro-
moplast biogenesis.
Recent studies show that a melon Or homologous gene is
responsible for b-carotene synthesis in melon fruit. The Or homol-
ogous gene is aligned with a major QTL controlling b-carotene con-
tent in melon [88]. Analysis of the genetic variation of Or in a wide
melon germplasm collection reveals a complete co-segregation be-
tween the Or allele and fruit color (Tadmor et al., unpublished
data). Functional characterization of the melon Or homolog in
transgenic plants confirms its role in controlling the inheritance
of orange fruit in melon. Moreover, a recent microscopic examina-
tion of orange melon mesocarp tissue reveals that each cell also
contains a large chromoplast, consistent with the case shown in
cauliflower orange mutant (Chayut et al., unpublished data), which
further supports the role of Or in conferring chromoplast
biogenesis.
Metabolic processes important for chromoplast biogenesis
Carotenoid metabolism
The prominent role of chromoplasts is associated with the syn-
thesis and accumulation of carotenoid pigments. Thus, carotenoid
metabolism is crucial for chromoplast biogenesis and represents
the most well studied metabolic process in chromoplasts [4,89].
Carotenoids are derived from MEP pathway and massively accu-
mulated or sequestrated as carotenoid lipoprotein substructures
within chromoplasts [6]. Carotenoids are also actively metabolized
to produce diverse aroma and flavor compounds in fruits [90].
Expression of carotenoid biosynthetic genes has been subjected
to extensive studies in many chromoplast-containing tissues.
Transcriptional regulation prevails in chromoplasts during fruit
ripening from green fruits in tomato and pepper [89]. The accumu-
lation of lycopene in tomato and capsanthin/capsorubin in pepper
is paralleled with a coordinated up-expression of upstream genes
and down-regulation of downstream genes of the accumulated
metabolites during fruit ripening [91]. In contrast, transcriptional
regulation is more ambiguous in chromoplasts derived from non-
colored plastids in white tissues. Accumulation of carotenoid
metabolites is not found to be associated with increased expres-
sion of upstream genes during fruit ripening in watermelon and
root maturation in carrot [92–94], and in orange curd cauliflower
[77]. The non-correlation between expression of carotenoid bio-
synthetic genes and specific carotenoids accumulated suggests a
complicated regulation of carotenoid accumulation in plants.
Carotenoid metabolic enzymes are present at low abundance
and normally escape detection [28]. Limited information is avail-
able for regulation of carotenogenic enzymes at protein levels. Re-
cent advances in proteomics technology enable detection of a
relatively large number of core enzymes related to carotenoid
metabolism in chromoplast proteomes. Analysis of chromoplasts
from carotenoid enriched tissues of various fruits and vegetables
repeatedly identifies a number of early carotenoid pathway en-
zyme proteins prior to lycopene biosynthesis, i.e. HDS, PSY, PDS,
and ZDS, as well as carotenoid degradation enzymes, i.e. CCD and
NCED [95]. In contrast, downstream enzyme proteins in the carot-
enoid biosynthetic pathway are not generally detected [95–97].
The detection of these early pathway enzymes for lycopene biosyn-
thesis in chromoplast proteomes implies a relative abundance of
early pathway proteins, which may be important for metabolic flux
into carotenoid biosynthetic pathway. Interestingly, while caroten-
oid metabolic enzyme proteins are generally present in low
quantity in chromoplast proteomes, the specific enzyme for
106 L. Li, H. Yuan / Archives of Biochemistry and Biophysics 539 (2013) 102–109
capsanthin/capsorubin biosynthesis in red pepper, capsanthin/cap-
sorubin synthase (CCS), represents the most abundant protein in
pepper chromoplast proteome [95,98]. The high abundant CCS
along with abundance of the other carotenoid metabolic enzymes
and fibrillins indicates that pepper chromoplasts are characterizing
with predominant carotenoid biosynthesis and sequestration [95].
Noticeably, while enhanced carotenogenic biosynthetic activity
stimulates carotenoid biosynthesis, carotenoid synthetic enzymes
by themselves may not be responsible for chromoplast differentia-
tion. Similarly, high levels of carotenoid accumulation do not trig-
ger chromoplast biogenesis. For example, overexpression of PSY in
many plant species dramatically enhances carotenoid accumula-
tion, but appears to exhibit no effect on chromoplast biogenesis
although functional overexpression of PSY drives sequestration of
overproduced carotenoids into crystal structures like the substruc-
tures found in chromoplasts [20,21,24].
Lipid metabolism
Chromoplast biogenesis and development are linked with
membrane proliferation. Remodeling of the internal membrane
system to develop carotenoid-lipoprotein sequestration substruc-
tures as well as plastoglobules represents one of the most promi-
nent changes during chromoplast differentiation [4]. Plastid
envelope membrane budding or fusion allows chromoplast sub-
structure formation and acquires new biosynthetic capacity [5]. In-
deed, membranes are found to be newly synthesized from vesicles
derived from the inner plastid membranes during chromoplast
biogenesis in tomato fruits [56]. Membranes are also observed to
proliferate strongly in the course of chromoplast differentiation
in daffodil flower [99]. Massive carotenoid lipoprotein sheet struc-
ture formation is closely associated with carotenoid accumulation
in chromoplasts [55]. A plastid fusion and/or translocation factor
(Pftf) from pepper fruits is known to be involved in chromoplast
membrane biogenesis [100]. An altered expression of Pftf homolog
is also observed during chromoplast differentiation in cauliflower
orange curd mutant [60] and during fruit ripening in tomato [101].
Active fatty acid biosynthesis and lipid metabolism are critical
for membrane proliferation. Lipids function as structural compo-
nents of membranes, carotenoid-lipoprotein sequestration sub-
structures, and plastoglobules in chromoplasts. A large number
of proteins involved in lipid metabolism, including those key en-
zyme proteins for the synthesis of fatty acids, sulfolipids, and gly-
colipids are identified in chromoplast proteomes from various crop
species [95,96]. Transcriptomics and translatomics analyses iden-
tify accD, a plastid-encoded gene involved in fatty acid biosynthe-
sis, whose expression is required to fulfill the high demand for
membrane lipid synthesis during chloroplasts to chromoplast tran-
sition in tomato fruits [102]. A rapid increase in plastid specific po-
lar lipids along with carotenoid accumulation is observed during
chromoplast formation in daffodil flower [103]. A recent metabolic
study using various radiolabeled precursors shows that most of the
[14C]-pyruvate is converted into lipophilic compounds in tomato
fruit chromoplasts, supporting that lipid biosynthesis is a very effi-
cient process in chromoplasts [104], as observed in early studies
[105].
Carbohydrate metabolism
Carotenoid accumulation and chromoplast differentiation are
concomitant with the accumulation of sugars in fruits and roots
during maturation. Indeed, a number of previous studies provide
evidence showing that sugars play important roles in stimulating
chromoplast biogenesis and carotenoid accumulation [76,80,106].
Citrus fruit epicarps undergo color break to convert chloroplasts
into chromoplasts during ripening and this process is reversible
as seen during degreening and regreening [106]. Both in vivo and
in vitro studies show that chromoplast differentiation in citrus
fruit epicarps is stimulated by sucrose [76]. High sucrose supple-
mentation promotes the conversion of chloroplasts into chromop-
lasts and depletion of sucrose reverses the process. The rate of
color break is positively correlated with sucrose content [76]. As
defoliation blocks endogenous sucrose build up, defoliation results
in inhibiting carotenoid accumulation and preventing color break
[76]. Sucrose limitation also delays lycopene and phytoene accu-
mulation in tomato fruit pericarp discs and the modulation by su-
crose is found to occur via regulating PSY1 gene expression and
hexose accumulation [107]. A positive correlation between in-
creased levels of sucrose and reducing sugars with carotenoid
accumulation and chromoplast formation has been observed in
other studies [55,80]. During tobacco nectary development, chro-
moplast differentiation from amyloplasts is associated with starch
degradation and production of nectar sugars [80]. Similarly, during
tomato fruit ripening, the chromoplast differentiation and caroten-
oid accumulation are correlated with the declining of plastid starch
content and progressive conversion into reducing sugars [108–
110]. Increased levels of sucrose and reducing sugars are detected
in the Or transgenic potato tubers that contain enhanced caroten-
oid accumulation in chromoplasts [55].
Sugars, as hexose phosphates, are actively metabolized via gly-
colysis and the oxidative pentose phosphate (OPP) pathway to pro-
vide precursors, ATP, and reductants for the biosynthesis of
carotenoids and many other metabolites within chromoplasts
[89]. Recent proteomics analysis of chromoplast proteomes from
a number of crop species identifies nearly all the enzymes of gly-
colysis from glucose to pyruvate [95,96]. Several glycolytic en-
zymes including aldolase, triosephosphate isomerase,
phosphoglycerate kinase, enolase, pyruvate kinase, and glyceralde-
hyde-3-phosphate dehydrogenase are persistently found in chro-
moplasts from most of the examined plant species [95].
Molecular analysis of a chromoplast-specific glyceraldehyde-3-
phosphate dehydrogenase in red pepper shows that the enzyme
is expressed mainly in fruits and roots, and plays a specific role
in glycolytic energy production of nongreen plastids [111]. Glu-
cose-6-phosphate dehydrogenase is a main enzyme in the OPP
pathway and identified in chromoplast proteomes from a number
of crop species [95,96]. Glucose-6-phosphate dehydrogenase is
found to be highly active in tomato chromoplasts, showing an ac-
tive OPP metabolism [112]. Chromoplasts isolated from sweet pep-
per and buttercup also have been found to have OPP pathway
activity [110,113].
In addition, sugars may act as signaling molecules in the sugar
signaling pathways for chromoplast biogenesis and development.
Sugars have been shown to act as signaling molecules for chloro-
plast biogenesis [114]. In the transgenic Or potato tubers, the en-
hanced carotenoid accumulation in chromoplasts is associated
with an increased abundance of a Snf1-related protein kinase
(SnRK) [55], which is a global regulator of carbon metabolism in
plants [115].
Chaperones and reactive oxygen species in chromoplast
biogenesis
Chaperone proteins
Molecular chaperones are a group of heat shock proteins (Hsp)
that participate in protein folding or unfolding, protein assembly or
disassembly, and translocation into organelles [116,117]. Chaper-
one proteins are known to play an important role in plastid biogen-
esis [84,118]. Hsp90 and Hsp70 as the most abundant chaperones
and cytosolic factors recruit precursor proteins to the plastid
 L. Li, H. Yuan / Archives of Biochemistry and Biophysics 539 (2013) 102–109
translocation machinery, and work along with Hsp93, Cpn60, and
other chaperones to facilitate the preprotein import into plastids,
mature protein folding and assembly, and intraorganellar localiza-
tion [118]. Accordingly, some of these chaperone proteins are con-
sistently detected in chromoplast proteomes from various crops
[95]. Hsp70 forms a complex with phytoene desaturase in daffodil
chromoplast to actively regulate phytoene desaturase enzyme
activity [59].
Small heat shock proteins (sHSPs) constitute a diverse group of
proteins that have been suggested to have unique physiological
functions in plants [116,117]. A sHSP is recently shown to function
as a cofactor in facilitating preprotein import during plastid differ-
entiation and development [119]. A chloroplast sHSPs, HSP21,
along with a number of other sHSPs, is dramatically induced in
developing fruits during the transition from chloroplasts into chro-
moplasts in tomato [120]. Overexpression of HSP21 in transgenic
tomato promotes carotenoid accumulation early, providing direct
evidence for the role of this protein in color changes and the con-
version of chloroplasts into chromoplast during fruit maturation
[121]. sHSP21 is suggested to be necessary for obtaining maximal
carotenoid accumulation through promoting the activities of carot-
enoid biosynthetic enzymes [121]. A number of studies also show
that the expression of sHSPs is closely correlated with chromoplast
differentiation [55,122,123].
Reactive oxygen species (ROS)
An intrinsic feature during fruit ripening is the increased pro-
duction of ROS. Recent studies reveal that ROS are central players
in the complex signaling network of cells, and acquire dynamic
and specific roles in signaling [124]. Indeed, an early study shows
that the plastid generated ROS act as a novel class of secondary
massagers and induce chromoplast specific carotenoid gene
expression during chromoplast differentiation [125]. ROS are also
believed to act as retrograde signaling molecules from plastids to
the nucleus [126]. They are suggested to play a vital role in coordi-
nately activating numerous morphological and biochemical
changes, leading to the ultimate transition into chromoplasts
[125].
The ROS homeostasis is maintained by redox system enzymes.
Interestingly, many key enzyme proteins involved in maintaining
cell redox status, such as ascorbate peroxidase, glutathione reduc-
tase, glutathione peroxidase, and superoxide dismutase, are not
only identified in chromoplast proteomes, but also present in high
abundance in chromoplasts [95–97]. Increase in redox protein lev-
els has been shown to represent one of the main features during
the transition from chloroplast into chromoplast in tomato fruits
[120]. Similarly, redox enzymes are found to be up-regulated and
play a critical role during chloroplast to chromoplast transition in
pepper fruits [127]. The increased redox enzyme activities indicate
that the rate of ROS generation is stimulated during chromoplast
biogenesis. The higher redox activity also protects plastid compo-
nents against oxidation and allows the plastids to acclimate vari-
ous metabolic processes during chromoplast development [4].
Recently, redox is identified to be involved in the control of plastid
protein import for chloroplast biogenesis [128].
Energy synthesis and import for chromoplast biogenesis
Chromoplasts as heterotrophic plastids are a site of pronounced
anabolic reactions that rely on the supply of energy along with car-
bon sources for essential metabolic activities [2,4,129]. Thus, active
energy production and import are required for chromoplast bio-
genesis. As energy in the form of ATP is either generated within
the plastids or provided by importing ATP from cytosol through
107
the plastidial adenine nucleotide translocators, it is expected that
both ATP synthase, the enzyme that catalyzes the synthesis of
ATP, and adenine nucleotide translocators, the transports of ATP
or ADP across membranes, affect chromoplast development. A re-
cent proteomics study reveals that ATP synthase and adenine
nucleotide translocator represent two of the most highly abundant
proteins in chromoplast proteomes of various crops [95], support-
ing an important role of energy production and transport in chro-
moplast development.
ATP synthase converts the energy of protons moving down the
electrochemical gradient into ATP synthesis and is responsible for
the bulk production of cellular ATP. Although ATP synthesis mainly
occurs in mitochondria and chloroplasts within plant cells, it has
been demonstrated to take place in chromoplasts too. ATP syn-
thase from daffodil chromoplasts is shown to be able to synthesize
ATP by chromorespiration linked with the NAD(P)H-dependent
respiratory activity [130]. Similarly, a recent study demonstrates
that the isolated tomato chromoplasts are also able to synthesize
ATP de novo using NADPH as an electron donor and this process in-
volves a plastidial ATP synthase harboring an atypical c-subunit
that is induced during chromoplast differentiation [131]. Using
radiolabeled precursors to feed purified tomato chromoplasts, the
isolated chromoplasts are also shown to be able to actively sustain
anabolic reactions in absence of exogenous supply of ATP [104],
further supporting that chromoplasts contain endogenous func-
tional ATP synthesis system to produce ATP. Moreover, elevated
ATP energy production components are found to be associated
with chromoplast differentiation during the chloroplast to chro-
moplast transition in tomato fruits [120].
Adenine nucleotide translocator is also known as ADP, ATP
translocator and facilitates ADP and ATP transport across orga-
nelle membranes. In heterotrophic plastids, adenine nucleotide
translocator preferentially mediates ATP import into the plastids
to influence the metabolic sink strength [2,129]. As a result,
alteration of adenine nucleotide translocator affects metabolism
within plastids. For example, inhibition of plastid adenine nucle-
otide translocator activity is found to cause substantial reduc-
tion of lipid content in leucoplasts of developing Arabidopsis
seeds [132]. Suppression of potato plastid adenine nucleotide
translocator also leads to decreased starch accumulation in amy-
loplasts of potato tubers [133]. Furthermore, overexpression of
plastid adenine nucleotide translocator along with a carbon
translocator results in increased starch content and yield of po-
tato tubers [134]. Although currently there is no experimental
evidence supporting the role of plastid adenine nucleotide trans-
locator in the control of carotenoid biosynthesis, the presence of
active translocators is implied to be needed for massive mem-
brane proliferation during chromoplast development in daffodil
flower [130].
Conclusions
Chromoplast biogenesis is a complex process and under control
of developmental and environmental signals. Many factors and
processes influence and control chromoplast differentiation and
development. Apart from those discussed here, others may also ex-
ert profound effects on chromoplast biogenesis. For example, plas-
tid proteome sizes are estimated ranging from 2000 to 3500
proteins and over 95% of plastid proteins are synthesized as cyto-
solic preproteins [135]. Plastid biogenesis depends on the import
of these nuclear-encoded proteins [136]. Emerging evidence from
studies of chloroplasts supports the critical role of protein translo-
cation machinery for plastid biogenesis [137,138]. The plastid
translocation machinery likely plays a critical role for chromoplast
biogenesis too.
108 L. Li, H. Yuan / Archives of Biochemistry and Biophysics 539 (2013) 102–109
Current evidence clearly shows that regulation of chromoplast
biogenesis plays a crucial role in controlling carotenoid content
in plants. This is due to that chromoplasts provide a site not only
for active carotenoid biosynthesis, but for stable storage of the syn-
thesized products. The unique characteristics of chromoplasts en-
hance metabolic sink strength, providing a potential for massive
carotenoid synthesis and stable accumulation. Many important
staple crops such as wheat, rice, barley, maize, potato, and cassava
synthesize and store carotenoids in amyloplasts in the edible or-
gans and retention of these carotenoids has been a major concern
during post-harvest storage. Induction of chromoplast formation to
effectively sequester carotenoids within chromoplasts in the edible
organs may facilitate carotenoid synthesis and stable storage in
these major staple crops.
Acknowledgments
The authors are grateful to many colleagues and collaborators
for their contribution to this research. We thank Dr. Yaakov Tad-
mor, Dr. Joseph Burger, and Mr. Noam Chayut for sharing their
unpublished data and Ms. Tara Fish for carefully reading the man-
uscript. This work was partially supported by grant No. US-4423-
11 of BARD, the United States-Israel Binational Agricultural Re-
search and Development Fund, and by the USDA-ARS base fund.
USDA is an equal opportunity provider and employer.
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