Molecular Horticulture

Jiang et al. Molecular Horticulture(2021) 1:4
https://doi.org/10.1186/s43897-021-00008-7

REVIEW

Open AccessMultiple functions of the vacuole in plant
growth and fruit quality
Yu-Tong Jiang1, Lu-Han Yang1, Ali Ferjani2 and Wen-Hui Lin1*

Abstract
Vacuoles are organelles in plant cells that play pivotal roles in growth and developmental regulation. The mainfunctions of vacuoles include maintaining cell acidity and turgor pressure, regulating the storage and transport ofsubstances, controlling the transport and localization of key proteins through the endocytic and lysosomal-vacuolar
transport pathways, and responding to biotic and abiotic stresses. Further, proteins localized either in the tonoplast(vacuolar membrane) or inside the vacuole lumen are critical for fruit quality. In this review, we summarize anddiscuss some of the emerging functions and regulatory mechanisms associated with plant vacuoles, including
vacuole biogenesis, vacuole functions in plant growth and development, fruit quality, and plant-microbe
interaction, as well as some innovative research technology that has driven advances in the field. Together, thefunctions of plant vacuoles are important for plant growth and fruit quality. The investigation of vacuole functions
in plants is of great scientific significance and has potential applications in agriculture.
Keywords: Vacuole, Biogenesis, Plant growth and development, Protein trafficking, Fruit quality

Background
The vacuoles of plant cells are multifunctional organelles
that display strong plasticity during plant growth and development. Lytic vacuoles (LVs) function as reservoirs
for ions and metabolites (e.g., pigments, acids, and toxic
substances), and are crucial for general cell homeostasis(Andreev, 2001; Marty, 1999). Vacuoles also play key
roles in cellular responses to abiotic and biotic stresses(e.g., microbial invasion) (Miransari, 2014; Nguyen et al.,2015; Swarbreck et al., 2019). In plant vegetative organs,vacuoles act in combination with the cell wall to establish and maintain turgor, the driving force underlying
hydraulic stiffness and cell growth (Marty, 1999; Zhang
et al., 2014). In seeds and specialized storage tissues,vacuoles serve as storage sites for proteins and soluble
carbohydrates. Vacuoles are also reported to modulate
stomatal activity (Gao et al., 2010), and to control the* Correspondence: whlin@sjtu.edu.cn
1
Joint International Research Laboratory of Metabolic & Developmental
Sciences, School of Life Sciences & Biotechnology, Joint Center for Single
Cell Biology, Shanghai Jiao Tong University, Shanghai 200240, China
Full list of author information is available at the end of the article

localization and transport of key proteins via vacuolar
trafficking (Marty, 1999; Offringa & Huang, 2013;
Reinhardt et al., 2016; Saini et al., 2017). Thus, vacuoles
have several physical and metabolic functions that are
essential for plant life.
Vacuole functions are tightly connected with vacuolar
proteins, many of which are embedded in the lipid
monolayer vacuolar membrane, referred to as the tonoplast. The tonoplast is an important physical barrier that
separates the acidic vacuolar lumen compartment fromthe cytoplasm. Tonoplast-specialized proton pumps,
channel proteins, ion transporters, and enzymes locatedin the tonoplast are essential for the normal function of
the vacuole.
The biogenesis and function of plant vacuoles havebeen topics of interest for decades. Advances in live imaging technology have resulted in constant updates tothe field of vacuole-related research. For example, recent
studies have shed light on the role of the vacuole inplant embryo development and patterning, throughregulating cell division in the embryo (Jiang et al., 2019;© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
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Jiang et al. Molecular Horticulture(2021) 1:4

Kimata et al., 2019). This review summarizes recent
advances in research on vacuole biogenesis, technicalmethods, and the functions of the vacuole in plant
growth and fruit quality.
Popular technology in current vacuole research

Our understanding of the regulatory mechanisms underlying vacuole-mediated control of plant growth andpattern formation remains fragmentary. As the study of
vacuoles usually requires focus on deep subcellular levelprocesses, most observations of vacuoles have been performed using seedling roots, which have the advantagesof lacking chloroplasts and thick cell walls. Direct observation of vacuoles in vivo is the best way to disentangle
their functions in different tissues, organs, and developmental stages; however, it remains technically challenging to study vacuoles in particular tissues and/ororgans, such as ovules and embryos, because they are
deeply embedded. One approach to solve this problem isto overexpress Arabidopsis LEAFY COTYLEDON2(LEC2), a gene encoding a key factor in embryo development and somatic embryogenesis. Overexpression of
LEC2 triggers the development of embryos in plantleaves, which allows for a relatively clear view of vacuole
morphology (Feeney et al., 2013). Although this systemcannot perfectly mimic embryonic vacuolar functions, it
has greatly facilitated vacuolar marker signal capture.
Technological improvements have also allowed formore detailed investigations of plant vacuoles. First,
technology used for vacuole extraction has matured.
Vacuoles from different plants can be extracted andenriched independently, which is convenient for further
experiments such as proteomics analysis (Robert et al.,2007). Further, the indirect observation of vacuoles andrelated proteins in plant cells has improved. Laser confocal
scanning microscopy (LCSM), which was originally developed to allow live imaging, is often combined with one ormore fluorescent trackable markers, such as VAMP7,
VHA-a3, or 2S1; dyes including the pH-sensitive agents,
BCECF-AM [3′-O-acetyl-2′,7′-bis (carboxyethyl)-4]; neutral red; time-based dyes (FM-64[N-(N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl)
pyridinium dibromide)]; or propidium iodide (Tejos et al.,1789). With continuous improvements in microscope
hardware and image processing software, spatial Z-axis
and three-dimensional (3D) reconstruction on the T-axis
have become rapid and convenient (Cui et al., 2019; Viotti
et al., 2013). In addition, LCSM-based live imaging is a
powerful tool to monitor the effect of acute pharmacological treatments on signal intensity in living systems.
High background noise or poor definition can occur in
LCSM when the microscope resolution is less than 1 μm,
or when the signal is weak or non-specific. Multiple-layerscans of non-staining fluorescence in living cells can cause

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fluorescence quenching, resulting in unsatisfactory reconstructed 3D images (Viotti et al., 2013).
Sectioning technique is another important factor for
vacuole observation. Although it is possible to obtain
sections as thin as 50 nm, or even 1 nm, ultrathin sectioning is time consuming, technically difficult, and challenging to apply to large-scale imaging of living samples,in which co-localized signals cannot be distinguished.
Currently, 3D tomography, combined with field emission
scanning electron microscopy, is frequently used to build
3D structures, where sections are combined into thehighest accuracy steric model of tissue cells. This approach can solve the problem of low-resolution LCSM
(Kalinowska et al., 2015; Kolb et al., 2015; Scheuring
et al., 2015).
With the rapid development of fluorescence microscopy, technologies involving single-molecule fluorescence imaging in living cells have gradually been applied
to research into plant membrane systems and key proteins; relevant approaches include variable-angle total internal reflection fluorescence microscopy (VA-TIRFM)
and fluorescence correlation spectroscopy technologies,
among others (Lv et al., 2017; Tsuganezawa et al., 2013;
Wang et al., 2015a). VA-TIRFM has high resolution, canbe used to track the movement rate, lateral displacement, and movement trajectory of membrane proteins,and is most commonly used to study tonoplast proteins(Lv et al., 2017; Wang et al., 2015a). In summary, continuous technological developments provide new perspectives for vacuole study.
Biogenesis of different vacuole types

Vacuoles can be divided into two types, according totheir main function: LVs and protein storage vacuoles(PSVs) (Marty, 1999; Jiang et al., 2000). LVs are specialized compartments found in almost all vegetative tissues.
They are involved in substance transportation, storage,
and degradation, similar to the roles of lysosomes in animal cells.
PSVs mainly occur from the late embryonic developmental stage to the seed germination stage, and function
to store proteins and important minerals during seedfilling (Feeney et al., 2018; Zheng & Staehelin, 2011).
The above-mentioned authordemonstrated that LVs and
PSVs can be mutually transformed during different biological processes (Feeney et al., 2018; Zheng & Staehelin,2011). The process of vacuole biogenesis has long been
an attractive topic for a broad researcher community.
Vacuole initiation has been one of the most controversial issues in plant biology research over the past half
century. Evolutionary studies suggest that the important
tonoplast proton pump, vacuole H+-ATPase, is derived
from the P-type ATPase (H+-ATPase of the plasma
membrane (PM)) of archaea. In addition, evolutionary

Jiang et al. Molecular Horticulture(2021) 1:4

analyses indicate that the plant tonoplast has a wide
range of origins, and that the proteins positioned on it
show strong homology with those in plant cell membrane
systems (Axelsen & Palmgren, 1998; Vasanthakumar &Rubinstein, 2020). Studies of vacuole initiation are usuallyobservational, and mainly conducted using LCSM technology, sectioning, and other approaches combined withthose technologies.
(1) Lytic vacuole biogenesis
As mentioned above, vacuoles are the largest
membrane-bound organelles and have essential roles inplant growth and development, yet several importantquestions about the biogenesis and dynamics of LVs remain unanswered. The LV is the main type of vacuoleand found in most plant organs and plays an importantrole in maintaining homeostasis within plant cells. There
are two hypotheses regarding the biogenesis of LVs: oneis that they originate from the endoplasmic reticulum(ER) (Viotti et al., 2013), and the other that they originate from the Golgi apparatus; these two pathways havebeen well-described by Cui et al. (Cui et al., 2019).
The ER initiation hypothesis is based on observations
from the VHA-a3 (a subunit of the tonoplast proton
pump V-ATPase) marker line. This hypothesis wastested by specifically blocking various steps of the vacuolar transport pathway and tracking the VHA-a3 signal
and vacuole morphology. In that study, the initiation of
LVs, including transportation of the tonoplast proton
pump and important lipids, appeared to be independentof key proteins in the vacuolar transport system, Rab5
and Rab7. Moreover, the initiation of LVs did not occur
in the region containing the Golgi apparatus. It was
postulated that the precursors of LVs form in an area
enriched with sterols, directly shed from the ER. At thevery beginning, vacuole precursors were empty, subsequently gradually expanding to accommodate highly
acidic fluid. This process appears to be related to autophagosomes; however, there is no experimental evidence
for the involvement of a typical autophagy process in theinitiation of LVs (Viotti et al., 2013). Other studies suggested that VHA-a3 transport depends on the traffickingof the small G protein, Rab5, but is independent of regulation by Rab7, with the VHA-a3 protein finally reaching
the tonoplast via the trans-Golgi/early endocytosis(TGN/EE) pathway, which is part of the typical vacuolar
transport pathway (Feng et al., 2017; Uemura & Ueda, 2014).
Another hypothesis is that LVs in plant cells are independent and separate from each other. Cui et al. (Cuiet al., 2019) found no experimental evidence for a clear
connection between the vacuole and other membrane
systems; using 3D reconstruction techniques, based oncontinuous ultra-thin slices, they found that LVs could

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be initiated from multivesicular bodies (MVBs). Further,
they observed that internal small vesicles fused together
following induction by the SNARE protein, and the bodyof fused vesicles gradually enlarged, eventually forming
an LV (Cui et al., 2019).
(2) Biogenesis of protein storage vacuoles
The PSV is a storage organelle specifically formed
during plant seed development that plays a key role instoring nutrients from the seed development stage to thegermination stage. The initiation of PSVs varies among
species. LVs have been reported to transform into PSVs
and vice versa; however, the mechanism underlying thisprocess remains unclear.
Pea (Pisum sativum) PSVs form de novo, while thoseof Arabidopsis thaliana form via functional reprogramming of LVs (Feeney et al., 2018; Robinson et al., 1995).
Observations of Arabidopsis embryos from the late torpedo stage showed that the LVs in embryo cells gradually transformed into PSVs. After seed germination,
PSVs rapidly transformed back into LVs (Feeney et al.,2018). It is unlikely that such transformations are governed by the same mechanism in all plants. For example,
during the germination of tobacco (Nicotiana tabacum)seeds, PSVs in root cells were converted into LVs in two
different ways: de novo biogenesis and functional reprogramming genesis. Both types of genesis were observedin epidermal, exodermal, endothelial, and vascular cells(Feeney et al., 2018).
Vacuole-related trafficking influences the transportationand localization of key proteins

Plant cells have complex inner membrane systems, including ER, Golgi, TGN, EE, vacuoles, and so on. The
trafficking of intracellular proteins begins with cargosorting and the formation of transport vesicles. This
process is mediated by SAR/ARF GTPases, coat protein
complexes (COPI and COPII), and clathrin (Zhang
et al., 2014; Uemura & Ueda, 2014; Fan et al., 2015;
Takehiko & Takashi, 2017). After vesicles detach fromthe donor membrane, effectors/tethers interacting withthe protein-specific RAB GTPases or GTPases are transported to the target membrane and fuse with the target
membrane to unload the protein. Most important
physiological activities in plant cells, including the precise localization of key proteins in the cell, depend on
membrane system transport pathways (Zhang et al.,2014; Uemura & Ueda, 2014; Fan et al., 2015; Takehiko& Takashi, 2017). There are three main types of membrane system transport pathway: the secretory pathway,
the endocytic pathway, and the lysosomal-vacuolar
transport pathway (Zhang et al., 2014; Uemura & Ueda,
2014; Fan et al., 2015; Takehiko & Takashi, 2017).

Jiang et al. Molecular Horticulture(2021) 1:4

Vacuoles have key roles in the endocytic and lysosomalvacuolar transport pathways, and LV and PSV haveunique regulatory pathways for the transport of different
proteins in these processes (Bottanelli et al., 2011; Ebine
et al., 2014; Kang & Hwang, 2014).
The distribution of most membrane-localized proteinsin the PM in plants does not exhibit polarity, while afew proteins with polar localization are of great significance during plant development. The polar localizationof these proteins in PM depends on various molecularmechanisms. For example, proteins such as PEN3 and
NIP5;1 rely on the extracapsular subunit, EXO84b, toorient in the abaxial-lateral direction in the PM (Maoet al., 2016). Heterogeneous cell growth depends on thegeometric edge-directed transport of proteins within thecell, which requires activation of the RAB11/RABA
group member, RABA5c/ARA4 (Kirchhelle et al., 2016).
Among proteins with polar localization, the auxin polartransporter, PIN1, has been well-studied in recent years.
This protein is synthesized in the rough ER, then passes
through the TGN/EE and reaches the PM via the endomembrane system. Several studies have shed light on the
dynamics of PIN1, and how it is controlled. Initially,
PIN1 is evenly distributed in the PM with no polarity. It
is then shed from the PM and recovered by the TGN/EE
through clathrin-mediated endocytosis. Some PIN1 relocalizes to the PM through a recycling process, is distributed in a polar manner, and functions as a polartransporter of auxin. Remaining PIN1 is transported to
the TGN/EE via endocytosis and then moved to thevacuole for degradation. This PIN1 protein vacuolar
transport pathway is regulated by auxin concentration
and ubiquitination level (Gälweiler et al., 1998; Friml,
2003; Kleine-Vehn & Friml, 2008; Steinmann & Grebe,
1999; Wiśniewska et al., 2006). Intracellular auxin levels
that are too high or too low induce PIN proteins toenter the vacuolar degradation pathway following endocytosis. Mono-ubiquitination induces PIN endocytosis;
however, poly-ubiquitination of a lysine residue of thehydrophilic ring induces their degradation within thevacuole after endocytosis and transport (Offringa &Huang, 2013; Saini et al., 2017; Dhonukshe et al., 2015;
Huang et al., 2010; Kim & Bassham, 2011; Kleine-Vehn
et al., 2009; Leitner et al., 2012). When PINs aredegraded through the vacuolar degradation pathway,
cellular microtubules disaggregate via interactions withthe associated proteins CLIP-ASSOCIATED PROTEIN(CLASP) and SORTING NEXIN (SNX). This process
triggers movement of PIN from the TGN/EE to thevacuole, and the endosome sorting transport complex(ESCRT) then transfers PINs to endosomes for subsequent degradation in the vacuole via recognition ofubiquitination sites (Offringa & Huang, 2013; Saini et al.,2017; Dhonukshe et al., 2015; Huang et al., 2010; Kim &Page 4 of 13

Bassham, 2011; Kleine-Vehn et al., 2009; Leitner et al.,2012). In this way, the vacuolar degradation pathway
plays a key role in maintaining PIN levels and auxin
homeostasis through regulating PIN metabolism.
In mutants lacking vacuolar proton pumps, the leveland distribution of auxin and PIN1 proteins are dramatically affected during embryo and seedling development,
and this is tightly connected with abnormal number,size, and shape of vacuoles. PIN1 in the mutant background is insensitive to Brefeldin A treatment, suggesting that PIN1 vesicular trafficking may be defective inthe vap3 background, resulting in abnormal PIN1 polar
localization and auxin distribution (Fig. 1) (Jiang et al.,2019).
Vacuole functions in plant growth and fruit quality(1) Basic storage function of vacuoles
As the largest organelle in mature plant cells, thevacuole exhibits complex and diverse functions. First, asa closed compartment, vacuoles can store free aminoacids, sugars, and ions. They can also transport key molecules through specific channel proteins on the tonoplast. In addition, tonoplast aquaporins participate inlong-distance water transport, and enhance resistance toabiotic stresses, such as drought and flooding (Srivastava
et al., 2014). Stomata are the ultimate gas exchange gatein plants, and their morphology changes depending on
vacuole water content (Reinhardt et al., 2016; Chrispeels& Daniels, 1997; Footitt et al., 2019).
Secondary metabolism and secondary metabolites are
typical characteristics of plants and some microorganisms, and are the result of adaptation to the external environment during evolution. Most secondary metabolites
are produced in the cytoplasm. Since some metabolites
are toxic to even the plant itself, they are preferentiallystored inside vacuoles, where they are isolated fromother cellular compartments. Vacuolar-related secondary
metabolic processes are widely involved in plant growthand development. The vacuole undergoes regularchanges in growth and morphology during the production and secretion of colored nectar, which contains
secondary metabolites, such as alkaloids, terpenes, andcyclic olefin ether glycosides (Davies et al., 2005; Fahn,2010). This process helps to attract pollinators for crosspollination of plants (Davies et al., 2005; Leshem et al.,2007). During the pollination process, correct guidanceof the pollen tube to the micropyle also depends on
growth and movement of the vacuole in the correct
direction (Ju & Kessler, 2020).
(2) Vacuole-related cell growth and patternformation

Jiang et al. Molecular Horticulture(2021) 1:4

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Fig. 1 Relationship between tonoplast proton pumps and vesicular trafficking of PIN1 protein. The polar localization of PIN1 protein depends onthe vacuolar transport system. The basal-side transport of non-phosphorylated PIN1 protein depends on GNOM, and phosphorylated PIN1 proteinmoves to the apical region of the membrane. In mutants lacking tonoplast proton pumps, transport of PIN1 to the basal side is inhibited,demonstrating that the tonoplast proton pump affects the vesicular trafficking of PIN1, thereby influencing the polar transport of auxin

The effect of the vacuole on cell growth under theaction of auxin is a research hotspot that has been wellsummarized by Kaiser and Scheuring (Kaiser &cheuring, 2020). The acid-growth theory proposes that
auxin activates the PM H+-ATPase, leading to acidification of the apoplast and the cell wall. This activates pHresponsive non-enzymatic proteins, ultimately resulting
in xyloglucan sliding, which triggers cell wall loosening(Cosgrove, 2000; McQueen-Mason et al., 1992). Subsequently, cell elongation is achieved by vacuole swellingthrough water uptake and deposition of new cell wallmaterial (McQueen-Mason et al., 1992; Barbez et al.,2017). Hence, the process of cell elongation relies on thefine tuning of auxin signaling and precise changes invacuole morphology (Barbez et al., 2017).
Vacuole distribution has an essential role in embryonicdevelopment and pattern formation. Studies on Arabidopsis

embryo development revealed the dynamics of the large
vacuole in the basal part of the mature egg cell. The volumeof the large vacuole immediately decreases after the fertilized egg shrinks (Jensen, 1968; Mayer et al., 1993; Suzuki
et al., 1992), leading to loss of polarity of the zygote. Subsequently, the zygote nucleus moves to one end via the actionof F-actin. The zygote continuously grows and the polar
distribution of the vacuole is re-established in the basal partof the zygote. After the first unequal division of the zygote,a small apical cell and a large basal cell form. At this point,
there are several small vacuoles in the dense cytoplasm of
the apical cell and a large vacuole in the basal cell (Kimata
et al., 2019; Kimata et al., 2016). Very recent work has
shown that the morphology and distribution of vacuoles
are critical for cell division and pattern formation of theembryo in the early stage of development. Mutants lacking
vacuolar proton pumps (namely V-ATPase and V-PPase)Jiang et al. Molecular Horticulture(2021) 1:4

showed severe disruption of vacuole morphology and distribution in early embryos (Jiang et al., 2019). Compared withwild-type embryos, mutants formed bigger vacuoles in apical cells and smaller vacuoles in basal cells, leading to an
aberrant pattern of embryonic cell division (Fig. 2).
(3) Contribution of vacuoles to fruit quality
Vacuoles are closely related to plant gametophyte development and fertilization. The position of the vacuole
plays a key role in the development of crop sperm cells.
In rice, OsGCD1 (GAMETE CELLS DEFECTIVE1) dysfunction changes the dynamics of the central vacuole.
This leads to incorrect positioning of the male gametophyte, which ultimately affects pollen development anddisrupts pollen germination (Huang et al., 2018). The
vacuolar invertase, GhVIN1, in cotton (Gossypium hirsutum) plays a key role in the timing of pollen release andthe normal accumulation of nutrients, such as starch, inthe female gametophyte (Wang & Ruan, 2016). GhVIN1
mediates hexose signal transduction and regulates the

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early differentiation of cotton fibers from the ovule epidermis and their subsequent elongation (Fig. 3) (Wang& Ruan, 2016).
Vacuoles play a key role in seed development. The
endosperm and aleurone layer are tissues unique to theseeds of cereal crops (Fath et al., 2000). The endosperm
is mainly responsible for storing nutrients, such as proteins and lipids. The aleurone layer wraps around theendosperm tissue of cereal seeds, but is morphologicallyand biochemically distinct from it. As the only viabletissue after seed maturation, the aleurone layer is
responsible for secreting key enzymes. An increase invacuolization is followed by programmed cell death(PCD), which releases nutrients and enzymes to promote
seed germination (Fath et al., 2000; Fath et al., 2010;
Pennell & Lamb, 1997). The vacuole is an extremely important organelle in this process. During seed germination, polymers are rapidly hydrolyzed in the PSV lumen
by pre-existing enzymes. Gradual fusion of LVs releases
key minerals and amino acids to fuel seed germination.
This transformation process is promoted by gibberellin

Fig. 2 Influence of vacuoles on cell division and morphology of plant embryos and seedlings. After fertilization, the zygote shrinks and loses
polarity, and then re-establishes polarity under the action of F-actin and vacuoles. The morphology and distribution of vacuoles in embryo cells
and suspensor cells are very important for polarity establishment, embryo patterning, and cotyledon initiation

Jiang et al. Molecular Horticulture(2021) 1:4

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Fig. 3 Diverse roles of vacuoles in influencing crop quality. The vacuole is important in many aspects of crop growth, including seedgermination, vegetative growth, flowering, and fruit development

and inhibited by abscisic acid (ABA) (Fath et al., 2000;
Pennell & Lamb, 1997). The nutrients released from vacuoles are used by the embryo and trigger the PCD
process in rice aleurone layer cells. Tonoplast intrinsic
proteins in barley (Hordeum vulgare) help to prevent theaggregation of small PSVs in aleurone cells (Lee et al.,2015). In barley, ABA was found to induce HvTIP3; 1transcription and prevent PSV fusion (Lee et al., 2015).
There are two main types of vacuole fusion during the
PCD of aleurone cells in rice. The first type is whenmembranes of multiple small vacuoles fuse to generatelarge vacuoles. The second type is when large vacuoles
engulf small ones, which then rupture inside the large
vacuoles and release their contents (Zheng et al., 2017).

Jiang et al. Molecular Horticulture(2021) 1:4

In this process, vacuolar processing enzyme (VPE)
promotes tonoplast fusion and accelerates PCD. RiceOsVPE3 is also involved in the regulation of leaf widthand guard cell length (Fig. 3) (Lu et al., 2016).
The taste and quality of fruits are important issues inhorticulture research. Vacuoles are the main storage
compartments for flavor-related substances, such as
sugars and acids (Shiratake & Martinoia, 2007). Vacuolar
invertase (VIN or VI) in the vacuole can hydrolyze sucrose into glucose and fructose (Wang et al., 2015b).
Both tonoplastic transporters and some hexose metabolic enzymes in the vacuole lumen, can catalyze theconversion of certain substances. Sugar transferases at
the tonoplast can be classified as monosaccharide transporters, sucrose transporters SUC/SUT (Sucrose Carrier/Sucrose Transporter), or SWEET (Sugars Will Eventually be Exported Transporters) transporters (Fig. 3)(Feng et al., 2015; Martinoia et al., 2012). The malatetransporter and malate ion channels at the tonoplast
help to move malic acid and citric acid across the tonoplast. During this process, the tonoplast proton pumps
transport hydrogen ions to generate the primary electromotive force; this activity is closely related to fruit flavor.
Inhibition of the V-ATPase A subunit in ‘Micro-Tom’tomato fruit results in significant accumulation of
sucrose in the fruit (Amemiya et al., 2005), while theoverexpression or heterologous expression of MdVHP1(encoding V-PPase) can significantly promote the accumulation of malic acid in apple callus and tomato fruit(Yao et al., 2011). Grapevine (Vitis vinifera L.) is a major
cultivated fruit crop worldwide. The processes involvedin the induction of grape berry ripening have been intensively investigated, with particular focus on the vacuole,since it occupies more than 99% of the total intracellularvolume in grape berry (Storey, 1987). The hydrolytic activities of V-PPase and V-ATPase increase throughout
development, but especially during ripening, and thisprocess is controlled at both the transcriptional and protein levels (Terrier et al., 2001). The vacuolar acid invertase, PbrAc-Inv1, which is located in the tonoplast of“Fengshui” pears, participates in sucrose hydrolysis andaffects the sugar composition and taste of pear fruits.
PbrII5 is located in the vacuole lumen and inhibits theactivity of PbrAc-Inv1 by combining with it to form an
inactive complex and inhibiting the activity of vacuolaracid invertase, thereby reducing sucrose hydrolysis(Ma et al., 2020).
Involvement of vacuoles in plant stress responses

Horticultural crops are a major source of food, feed, andfuel, and their yields and qualities are related to theirability to cope with fluctuations in the environment.
Stress is a major factor that affects crop productivity.
Higher plants are often exposed to biotic stress

Page 8 of 13(pathogen invasion) and/or abiotic stress (e.g., salt stress,
temperature stress). In this regard, vacuoles are key organelles in maintaining ion homeostasis and stabilizing
the intracellular environment. Thus, vacuoles help plants
to cope with environmental fluctuations, particularlywater scarcity (Lobell et al., 2014).
(1) Vacuole functions in response to abiotic stress
Transporters at the tonoplast and proteins in the vacuole lumen are vital for tolerance to abiotic stress. The
two types of tonoplastic proton pumps, V-ATPase and
V-PPase, pump protons from the cytoplasm into thevacuole and maintain relative pH stability in the vacuole,cytoplasm, and other organelles (Ferjani, 2011; Kriegel
et al., 2015). V-ATPase and V-PPase have closely related
functions, with respect to stress tolerance. Overexpression of V-PPase leads to an enhanced electrochemical
gradient across the tonoplast, increased transport andaccumulation of toxic ions in the vacuole, and enhanced
salt stress tolerance in transgenic tobacco (Li et al.,2017) and creeping bentgrass (Agrostis stolonifera L.) (Liet al., 2010); enhanced drought and salt stress tolerancein Arabidopsis (Gamboa et al., 2013); and greaterdrought tolerance in maize (Wang et al., 2016). VATPase is important for the development of mung bean
(Vigna radiata) under cold stress (Kuo et al., 1999;
Shahram et al., 2018).
Other membrane transporters, such as the tonoplast
Na+/H+ antiporter, NHX (Na+/H+ exchanger), use thetransmembrane electrochemical potential gradient generated by V-ATPase and V-PPase to sequester toxic Na+
in the vacuole, thereby reducing its harmful effects oncells. In addition to tonoplastic transporters, proteins in
the vacuole lumen are critical for plant stress resistance(Heven & Salil, 2018; Yokoi et al., 2002). For example,
MPK6, a member of the mitogen-activated protein kinase family, up-regulates transcription of VPE (encoding
vacuolar processing enzyme), and plays an important rolein heat shock-induced PCD (Li et al., 2012; Ye et al., 2013).
(2) Vacuole functions in response to biotic stressVacuoles are also important in resistance against microbial infection. Plant-microbe interactions are complex, and include parasitic, antagonistic, and mutually
beneficial symbiotic relationships, which have been described in detail previously (Dickman & Fluhr, 2013).
During these processes, the vacuole is an important mediator of microbial infection. Bacteria and pathogens
must stay inside the vacuole of eukaryotes to be isolated
from the cytosolic phagocytic system and lysosomes. For
example, the pathogen, Herbaspirillum rubrisubalbicans,negatively affects rice plant growth by suppressing V-

Jiang et al. Molecular Horticulture(2021) 1:4

ATPase activity and increasing ethylene content (Fig. 4)(Valdameri et al., 2017). Salmonella establishes a Salmonella-containing vacuole (SCV) through membrane
remodeling, actin rearrangement, microtubule movement, and adjustment of the autophagy system (Fig. 4)(Steele-Mortimer, 2008). In this way, it is protected fromhost defenses and can control various processes after entering the cell. Salmonella can invade Arabidopsis using
the same infection strategies with which it infects
humans and its broad host range (Huang et al., 2016;
Schikora et al., 2011). This finding greatly impacted thestudy of vacuole function. Although this research is limited to date, we have included a summary in the model
diagram, denoted by a dotted line (Fig. 4). Rhizobium coexist with legume cells in the form of bacteroids andhelp plants to fix nitrogen. After bacteroid invasion, theoriginal central vacuole in the plant cell shrinks to makespace for the resident bacteroid. Transporters in the
tonoplast also provide nutrients for the bacteroid(Gibson et al., 2008; Jones et al., 2007). For example,
the sugar-phosphate/anion anti-porter, GmG3PT3,
which is located in the tonoplast, participates in inorganic phosphate transport from vacuole to cytoplasm
and affects the distribution of phosphorus in nodules(Chen et al., 2019; Li et al., 2018).

Page 9 of 13

Plant cells restrict the spread of pathogens via thehypersensitive response, which involves PCD (Wu &Jackson, 2018). During this process, enzymes with caspase activity alter vacuole morphology and tonoplast
structure (Hara-Nishimura & Hatsugai, 2011). Destructive PCD occurs due to tonoplast collapse, which releases vacuolar hydrolases into the cytoplasm, resulting
in rapid and direct cell death. This process of cell destruction can effectively eliminate viruses that proliferate
in the cytoplasm. In non-destructive PCD, the fusion of
the vacuole and PM is triggered in a proteasomedependent manner. This results in the discharge of vacuolar
defense proteins into the extracellular space where bacteria
are located (Fig. 4) (Hara-Nishimura & Hatsugai, 2011).
Concluding remarks

The vacuole is a specific and extremely important organelle in plant cells. Vacuole initiation is related to the evolutionary history of species. Some fungi, bacteria, andprotists have vacuoles or analogous organelles. Changes
in the morphology, distribution, and function of thevacuole during cell proliferation and budding can provide crucial clues about evolution. The vacuole stores
nutrients in cells, and its contents determine the color ofcells and tissues and the turgor pressure of the cell.

Fig. 4 Roles of the vacuole in plant responses to biotic stresses. Plant vacuoles have important roles in resistance to microbial invasion. They mediate
two types of programmed cell death (PCD) to eliminate microorganisms from plant cells. Some microorganisms can avoid these elimination strategies
and change the morphology and function of plant cells. This model includes information about the PCD process and three types of bacteria

Jiang et al. Molecular Horticulture(2021) 1:4

Tonoplast proteins are involved in intracellular ion
transport, pH regulation, and vacuole transport
pathways.
Studies on vacuole initiation have focused on the
dynamics of the vacuole cavity and the transport and relocalization of vacuole-related proteins. How vacuoles
originate remains a matter of debate, hence there is anurgent need to obtain experimental evidence supportingor opposing the various hypotheses that have been proposed. The hypothesis of direct initiation from the ER
proposes that proteins on the tonoplast reach thevacuole from the ER, without passing through proteintransport pathways (the Golgi apparatus) (Uemura &Ueda, 2014; Lupanga et al., 2020). Are there other quality control systems for such proteins? Are they produced
in a functionally mature form? When do the contents ofthe vacuole become acidic? These questions warrant further exploration. The hypothesis that the vacuole is derived from the Golgi proposes that an acidic state exists
from the beginning, and that the transport pathway of
tonoplast proteins differs from that proposed in the ER
hypothesis. Vacuoles may form in multiple ways. For
example, observations have suggested that vacuoles
originate from MVBs and separate from one another;
however, studies on tubular vacuoles suggest that not all
vacuoles develop from MVBs. Vacuole initiation may
differ depending on plant cell functions. Therefore, there
may be other, as-yet unidentified, pathways involved invacuole initiation. The storage function of plant vacuoles
is the basis of plant secondary metabolism, while the distribution of vacuoles also affects plant growth, development, and pattern formation.
Plant products have great impact on human life, andthe fruit quality of edible plants is closely related to nutrition intake. Most proteins important for transport andconversion of sugar and acid in fruits are located in the
tonoplast and vacuole lumen. The function and activityof these proteins are major determinants of fruit taste
and nutrition; however, there has been limited researchon the function and regulatory mechanisms of those
proteins in different fruits to date. With technologicalimprovements in vacuole extraction methods and the establishment of a vacuole multi-omics database, the key
proteins and core regulatory factors underlying thetransportation and conversion of sugars and acids infruit vacuoles will be further explored, and are expected
to reveal the regulatory mechanisms underlying theaccumulation of sugar and acid in fruit.
Plants tolerate various stresses (including abiotic andbiotic stress) in different environments by changing their
metabolic processes, which can reduce quality and decrease yield. Vacuoles are also crucial in plant resistanceto abiotic stress or bacterial invasion. Although a varietyof bacteria and fungi can infect plants, only a few (partial

Page 10 of 13

rhizobia, nitrogen-fixing bacteria, etc.) can actually enter
plant cells and form symbiotic relationships with plants(otherwise, they cause disease). During this process, thecentral vacuole provides growth space for the microbe,
while transporters in the tonoplast provide the necessary
ions; however, vacuole functions in the interactions between plants and microorganisms remain unclear. How
plant endophytes survive in different organs and plants
requires further study. Hence, vacuoles play importantroles in multiple physiological processes and the investigation of vacuole functions in plants is of great scientificsignificance and has potential applications in agriculture.
Abbreviations
3D: Three-dimensional; ABA: Abscisic acid; ER: Endoplasmic reticulum;
LCSM: Laser confocal scanning microscopy; LVs: Lytic vacuoles;
MVBs: multivesicular bodies; PCD: Programmed cell death; PM: Plasma
membrane; PSVs: Protein storage vacuoles; TGN/EE: Trans-golgi/earlyendocytosis; VA-TIRFM: Variable-angle total internal reflection fluorescencemicroscopy; VPE: Vacuolar processing enzyme
Acknowledgements
We thank Dr. Ren-Jie Tang for helping us to collect literatures. We apologize
to researchers whose work could not be included in this manuscript due tospace constraints.
Authors’ contributions
W-H.L., Y-T J. and A. F. wrote and edited the manuscript, Y-T. J. drew the pictures, L.-H. Y. helped to collected and organized the literatures. All authors
read and accepted the final manuscript. The author(s) read and approved
the final manuscript.
Funding
This work is supported by National Natural Science Foundation of China(Grant No. 32070342), the Project MDS-JF-2020-8 supported from SJTUJiRLMDS Joint Research Fund, the Agri-X Interdisciplinary Fund of Shanghai
Jiao Tong University (20200204), the bio-X Interdisciplinary Fund of Shanghai
Jiao Tong University (20CX-04), and Shanghai Jiao Tong University Scientificand Technological Innovation Funds (19X160020009).

Declarations
Competing interests
The authors declare that they have no competing interests in this section.
Author details
1
Joint International Research Laboratory of Metabolic & Developmental
Sciences, School of Life Sciences & Biotechnology, Joint Center for Single
Cell Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
2
Department of Biology, Tokyo Gakugei University, Koganei-shi 184-8501,
Japan.
Received: 9 February 2021 Accepted: 9 April 2021

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