Plant aquaporins: Roles in plant physiology☆
Guowei Li, Véronique Santoni, Christophe Maurel⁎
Biochimie et Physiologie Moléculaire des Plantes, UMR 5004 CNRS/UMR 0386 INRA/Montpellier SupAgro/Université Montpellier 2, F-34060 Montpellier Cedex 2, France
a b s t r a c t a r t i c l e i n f o
Received 20 August 2013
Received in revised form 28 October 2013
Accepted 4 November 2013
Available online 15 November 2013
Background: Aquaporins are membrane channels that facilitate the transport of water and small neutral
molecules across biological membranes of most living organisms.
Scope of review: Here, we present comprehensive insights made on plant aquaporins in recent years, pointing to
their molecular and physiological specificities with respect to animal or microbial counterparts.
Major conclusions: In plants, aquaporins occur as multiple isoforms reflecting a high diversity of cellular
localizations and various physiological substrates in addition to water. Of particular relevance for plants is the
transport by aquaporins of dissolved gases such as carbon dioxide or metalloids such as boric or silicic acid.
The mechanisms that determine the gating and subcellular localization of plant aquaporins are extensively
studied. They allow aquaporin regulation in response to multiple environmental and hormonal stimuli. Thus,
aquaporins play key roles in hydraulic regulation and nutrient transport in roots and leaves. They contribute to
several plant growth and developmental processes such as seed germination or emergence of lateral roots.
General significance: Plants with genetically altered aquaporin functions are now tested for their ability to
improve plant resistance to stresses. This article is part of a Special Issue entitled Aquaporins.
© 2013 Elsevier B.V. All rights reserved.
Terrestrial plants establish a continuum between the soil and the
atmosphere and contribute to water transfer between these two
entities. In transpiring plants, the ascent of water is mediated through
xylem vessels, capillaries made from dead cells. For a long time, water
diffusion across the lipid phase of membranes was thought to be
sufficient to support water exchanges in living plant cells and tissues
. In the very early 1990s, the existence of water channels in plants
had not been clearly hypothesized, even though some aquaporins
had been molecularly characterized due to their high abundance or
remarkable expression properties. Thus, the functional characterization
of plant aquaporins shortly after the pioneering work of Preston et al.
 on human AQP1 opened unprecedented perspectives in the field of
plantwaterrelations.Todate,thefunction andregulation ofaquaporins
is quite extensively integrated to explain the remarkable hydraulic
properties of plants. However, additional surprises have come with
the identification of other aquaporin substrates than water, some of
them such as boron, silicon or carbon dioxide (CO2) being of great
physiological significance. Thus, the term “aquaporin” has been used
in a broad sense and now refers to all plant Major Intrinsic Proteins
(MIPs), whether or not their main role is in water transport.
In the present review, we present the comprehensive insights
made on plant aquaporins in recent years, pointing to their molecular
and physiological specificities with respect to animal or microbial
counterparts. We discuss the diversity of plant aquaporin isoforms,
of their substrates and cellular localizations. We emphasize their
physiological functions with respect to whole plant hydraulics, plant
development, nutrient acquisition, and plant responses to various
2. The family of plant aquaporins and their substrates
2.1. High diversity of isoforms
Aquaporins belong to the large class of MIPs, with first member
(Nodulin-26, GmNOD26) identified in plants (soybean) as early as
1987 . The water transport activity of plant aquaporins was first
was thereafter described in numerous herbaceous or ligneous, wild or
cultivated plant species. Aquaporins of higher plants exhibit a high
diversity with 35, 36, 33 isoforms in Arabidopsis, maize and rice,
respectively [5–7]. Plant aquaporin homologs can be classified
according to their sequence into up to seven subfamilies [8,9],
which may also correspond to distinct sub-cellular localizations. The
so-called Plasma membrane Intrinsic Proteins (PIPs), which localize to
the plasma membrane mostly, can be further divided into PIP1 and
PIP2 subclasses. The Tonoplast Intrinsic Proteins (TIPs) are targeted to
the vacuolar membrane (tonoplast). Although GmNOD26 is exclusively
expressed in the peribacteroid membrane of nitrogen-fixing symbiotic
Biochimica et Biophysica Acta 1840 (2014) 1574–1582
☆ This article is part of a Special Issue entitled Aquaporins.
⁎ Corresponding author at: Biochimie et Physiologie Moléculaire des Plantes, Bât. 7,
Campus INRA/SupAgro, 2 place Viala, F-34060 Montpellier Cedex 2, France. Tel.: +33
499 612011; fax: +33 467 525737.
E-mail address: email@example.com (C. Maurel).
0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
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nodules of legume roots, the NOD26-like Intrinsic Proteins (NIPs),
which form the third subfamily, are also found in non-legume plant
species, where they localize to the plasma membrane [10,11] or the
endoplasmic reticulum (ER) . The Small basic Intrinsic Proteins
(SIPs) group comprises only a few isoforms (3 and 2 in Arabidopsis
and rice, respectively). The uncategorized X Intrinsic Proteins (XIPs)
were recently discovered in protozoa, fungi and plants and are of
as yet unknown functions [13–15]. This subfamily is absent from
plants such as Arabidopsis, maize, rice. Two additional subfamilies, the
GlpF-like Intrinsic Proteins (GIPs) and the Hybrid Intrinsic Proteins
(HIPs) are present exclusively in moss, not in vascular plants [8,9].
Whereasthefirstfoursubfamilies (PIPs,TIPs,NIPs and SIPs)are present
in all terrestrial plants, from non-vascular plants to vascular plants, the
PIPs are the only ones that are shared between algae and higher plants.
Thus, the PIPs could represent the ancestor aquaporins that have been
conserved throughout evolution of terrestrial plants. By contrast, the
GIPs and HIPs may have been lost during this process.
2.2. High diversity of sub-cellular localization
With respect to their animal counterparts, plant aquaporins show a
broader array of sub-cellular localizations, in relation with the high
degree of compartmentalization of plant cells. Aquaporins have been
localized in nearly all of plant cell sub-cellular compartments, including
plasma membrane, tonoplast, ER, Golgi apparatus and chloroplast.
Localization of aquaporins in the chloroplast, which quasi exclusively
relies on proteomic studies, is still debated . Interestingly, some
aquaporins exhibit multiple sub-cellular localizations. For instance,
NtAQP1, a tobacco PIP1 homolog, was observed in both the plasma
membrane and chloroplast inner envelope membrane of tobacco leaf
cells . Seed-specific TIPs of Arabidopsis (AtTIP3;1, AtTIP3;2), which
predominantly sit in the protein storage vacuoles, are also transiently
expressed at the plasma membrane during the early stages of seed
germination and maturation . In these two cases, however, it will
be important that these observations can be extended to other plant
species. Several mechanisms that determine the trafficking of newly
synthesized PIPs or TIPs to their destination membranes have recently
been discovered. In PIP2s, diacidic motifs and C-terminal phosphoryla-
tion were found to favor export from the ER [19–21]. In contrast,
homologs of the PIP1 sub-class showed trafficking defects, unless they
were co-expressed with PIP2s and formed heterotetramers [21–23].
These molecular interactions seem to be necessary for PIP1s to reach
the plasma membrane (see 4.3). In addition, a role for Soluble NSF
Attachment Protein REceptors (SNAREs) in PIPtraffickingto theplasma
membrane was recently uncovered [24,25]. However, the mechanisms
that determine the multiple localizations of some aquaporins are as
2.3. High diversity of substrates
Functional expression of plant aquaporins in heterologous systems
such as Xenopus oocytes or yeast cells revealed a great diversity of
substrates. They include water and the related molecule H2O2, solutes
transported by animal and bacterial homologs (urea, glycerol),
metalloid species [boric acid: B(OH)3; silicic acid: Si(OH)4, arsenious
acid: As(OH)3], lactic acid, or dissolved gas molecules (CO2, ammonia:
NH3) [26,27]. With respect to this large array of substrates, plant
aquaporins can exhibit complex yet specific selectivity profiles. For
instance, AtTIP1;1 facilitates the transport of H2O, H2O2and urea
[4,28,29] whereas OsNIP2;1 from rice functions as a transporter
of H2O, methylated arsenic species, silicic acid and antimonite
[10,30–32]. Atomic structures of microbial, animal, and plant homologs
have shown that highly conserved structural features can confer on
aquaporins their transport selectivity for water and/or solutes .
Accordingly, homology modeling approaches based on the aromatic/
arginine selectivity filter have been developed to predict plant
aquaporin selectivity [34,35]. However, further investigations are still
required. For instance, mammalian AQP1 and AQP4 facilitate the
transport of nitric oxide (NO) [36,37]. NO plays a role in plant signaling
and may well be transported by plant aquaporins.
3. Aquaporins and water transport in plant roots and leaves
3.1. Root hydraulic conductivity (Lpr)
radial and axial paths. Axial water transport is mediated by xylem
vessels, which do not present significant membrane barriers. The radial
path allows water transport from the soil to the vessels and involves
three concurrent pathways: apoplastic (across cell walls), symplastic
(through plasmodesmata and cytoplasmic continuities) or transcellular
(across membranes) . The latter pathway is contributed in part
by aquaporins but is difficult to distinguish experimentally from
the symplastic pathway. They together form the cell-to-cell pathway.
Many aquaporins are known to be highly expressed in roots
[7,39–41], supporting a role of aquaporins in root water transport.
Mercury ions (Hg2+), which act as common aquaporin blockers by
binding to Cys residues within or in the vicinity of the pore , were
first used in tomato roots  and later on in various other species
[44,45] to show that aquaporins can contribute to N70% of Lpr. This
figure was confirmed using other types of aquaporin inhibitors
(azide, weak acids) which showed a very similar inhibition profile as
mercury among natural accessions of Arabidopsis . First genetic
evidence for the contribution of a specific aquaporin to overall Lprwas
reported by Javot et al. . These authors showed that Arabidopsis
AtPIP2;2 is highly expressed in several root cell types including
endodermis, and that, by comparison to wild-type plants, the Lprof
corresponding knock-out mutants (pip2;2) was reduced by 14%. More
recently, Sutka et al.  reported that the transcript abundance of
several PIPs (AtPIP1;1, AtPIP1;2, AtPIP1;4, AtPIP2;1, AtPIP2;3, AtPIP2;4
and AtPIP2;5) in Arabidopsis roots is positively correlated with Lpr, in
good agreement with published genetic data. For instance, the Lprof
pip1;2 mutants and pip2;1 pip2;2 double mutants was decreased by
20% and 40%, respectively, compared to that of wild type [47,48].
3.2. Leaf hydraulics
Aquaporins are also highly expressed in plant leaves, where they
contribute to the hydraulic conductance of inner tissues [49–51]. First
evidence for aquaporin function was obtained in sunflower leaves by
inhibition experiments using HgCl2. More recently, combined
physiologicalandgenetic approacheshaveindicatedthat,in Arabidopsis
at least, thefunction of PIP aquaporins in leaf veins (xylem parenchyma
and bundle sheath) critically determines leaf hydraulics [47,53,54].
However, the contribution of aquaporins to leaf hydraulic conductance
(~25%) was much less than that in the roots (N70%) [45,47,55]. This
indicates that vessels represent an important hydraulic limitation in
leaves. The interplay between vascular and extra-vascular (mediated by
aquaporins mostly) transport of water will deserve more physiological
studies in the future.
3.3. Regulation of root and leaf hydraulics by aquaporins
Plants have the remarkable ability to sense various signals from the
surrounding environment and accordingly adjust their water transport
properties. For instance, many abiotic stresses imposed by soil, such as
salinity, oxygen deprivation or nutrient starvation, markedly reduce
Lprin various plant species . Irradiance and the stress hormone
abscisic acid (ABA) both act as potent regulators of stomata-mediated
transpiration and also regulate aquaporin-dependent leaf hydraulic
conductance. Recent studies in Arabidopsis have established the
involvement in light-dependent leaf hydraulic conductance of a single
G. Li et al. / Biochimica et Biophysica Acta 1840 (2014) 1574–1582
aquaporin isoform (AtPIP2;1) expressed in veins . Moreover, root
hydraulic conductance was found to be positively correlated with the
size of the shoots among Arabidopsis natural accessions , and recent
cording to the transpiration demand from shoots . Thus, complex
aquaporin regulation mechanisms are at work in both roots and shoots.
The followingsection addresses themolecular and cellular mechanisms
4. Modes of aquaporin regulation
4.1. Cotranslational and posttranslational modifications
Application over thelast10 years of well-developed proteomicsand
mass spectrometry techniques have led to a comprehensive description
of cotranslational and posttranslational modifications of plant aquapo-
rins in their native membranes [54,59–61]. Combined with in vivo and
in vitro labeling studies, these approaches have indicated that PIP1s
and PIP2s from various species can be phosphorylated at multiple
sites on the N-terminal or C-terminal tail, respectively. In addition,
various environmental conditions such as drought, salinity or oxidative
stresses induce quantitative changes in PIP, TIP or NIP phosphorylation
[20,59,61–63]. However, knowledge of the protein kinases and protein
phosphatases determining reversible aquaporin phosphorylation is
still scarce [64,65]. Whereas aquaporin phosphorylation is very
common, glycosylation was identified in only a few homologs such as
soybean GmNOD26 and a TIP from Mesembryanthemum crystallinum
(ice plant) [66,67]. First evidence for methylation in aquaporins,
and even plant membrane proteins, was obtained with Arabidopsis
AtPIP2;1, Lys3 and Glu6 of which were di- and monomethylated,
respectively . Altogether, these studies suggest that intricate
co- and post-translational regulation mechanisms regulate plant
aquaporins (Fig. 1). However, we are far from having a comprehensive
view of how the numerous environmental or hormonal signals that
intervene during plant growth and development target, in time and
space,themultipleaquaporin isoformsexpressed throughouttheplant.
water channel pore, can be regulated by multiple effectors, such as
phosphorylation, protons (H+), and divalent cations. An activating
role for phosphorylation was first proposed for pea PvTIP3;1,
GmNod26 and spinach SoPIP2;1, based on functional expression in
oocytes of wild-type and phosphorylation mutant forms, together
with alterations of oocyte protein phosphatase and/or kinase activities
[59,63,69]. Water transport assays in purified plasma membranes
from Arabidopsis and sugar beet (Beta vulgaris) also indicated that H+
and divalent cations, with calcium (Ca2+) being the most efficient, can
regulate the activity of PIPs [70,71]. These effects were further
established after reconstitution of purified AtPIP2;1 in proteoliposomes
. Furthermore, the sidedness and molecular bases of H+-dependent
gating were dissected using Xenopus oocyte expression and the central
role as pH sensor of a His residue, perfectly conserved in the second
cytosolic loop (loop D) of PIPs, was established  (Fig. 1).
 were crucial in determining a conserved molecular mechanism for
PIPgating. Inbrief, the model showed howa hydrophobic (Leu) residue
of loop D can protrude in the cytosolic pore vestibule, to prevent any
passage of water. The conformation of loop D itself is determined by
ionic and H-bond interactions between residues of loops D (His193)
and B (Ser115) and the N-terminal tail (Asp28, Glu31). Divalent cations
binding to Asp28 and Glu31 or protonation of loop D at His193
can tighten this interaction, locking the water channel in a closed
state. By contrast, phosphorylation of Ser115 in loop B or Ser274 in
the C-terminus favors the unfolding of loop D to open the water
4.3. Cellular trafficking
Regulated trafficking also plays a key role in plant aquaporin
expression and regulation. PIP1s have been described since long as
having no or a weak water transport activity when individually
expressed in Xenopus oocytes. This can be explained by a failure to
traffic properly to the oocyte plasma membrane , this defect being
overcome after co-expression with PIP2 homologs. Direct evidence for
a physical interaction between PIP1s and PIP2s was obtained in oocytes
or plants by affinity copurification, coimmunopurification and fluo-
rescence resonance energy transfer (FRET) methods [22,23]. Thus,
formation of heterotetramers comprising various PIP1 and PIP2
combinations may be crucial for proper localization of PIPs at the
surface of plant cells.
Stimulus-induced subcellular trafficking of plant aquaporins was
first characterized in ice plant McTIP1;2. This aquaporin was found to
be redistributed from tonoplast to intracellular vesicles during osmotic
stress by a glycosylation-dependent mechanism . Regulated
trafficking of PIPs and TIPs was also proposed to play an important
role during root response to salt and oxidative stresses [24,25].
Following treatment of Arabidopsis roots with 100 mM NaCl for 4 h
or with 2 mM H2O2for 15 min, Lprwas inhibited by N70% and an
intracellular relocalization of several highly expressed plasma
membrane and tonoplast aquaporins was observed . Further
studies showed that the salt-induced relocalization of PIPs is mediated
by reactive oxygen species (ROS)-activated cell signaling cascades. In
addition, the role of AtPIP2;1 phosphorylation at Ser283 in directing
the protein to a specific endosomal (prevacuolar) compartment was
demonstrated [20,73]. Advanced fluorescence microscopic approaches
have recently brought deeper insights into the effects of salt on the
membrane dynamics of PIPs in root epidermal cells. Variable-angle
IIIIIV IVI VI
Fig. 1. Multiple post-translational regulations of PIP aquaporins in Arabidopsis. The figure
shows a schematic representation of a PIP aquaporin with its 6 transmembrane domains
(I to VI), five connecting loops (A–E), and N- and C-terminal tails bathing in the cytosol.
Covalent modifications indicated in green, black and blue concern AtPIP1;1, AtPIP2;1
and AtPIP2;6, respectively. The modification profiles of Arabidopsis PIPs were
mostly determined by mass spectrometry: in AtPIP2;1, the initiating Met residue
is co-translationally cleaved (cross) whereas in AtPIP1;1 this residue is N-α-acetylated
(Acet). Di-methylation (Met Met) of Lys3 (K3), mono-methylation (Met) of Glu6 (E6),
and phosphorylation (P) of Ser280 (S280) and Ser283 (S283) have been established
experimentally. By contrast, the phosphorylation of loop B at Ser119 (S119) was
inferred from studies on spinach SoPIP2;1 [33,63]. According to PhosPhAt database
(http://phosphat.mpimp-golm.mpg.de/), the N-terminal tail of AtPIP2;6 is phosphorylat-
ed at Thr3 (T3) and/or Thr7 (T7) as well as at Ser11 (S11) and/or Ser13 (S13). Because
ormodified form,a great variety of modifiedforms can beanticipatedfor PIPs.Inaddition
to covalent modifications, PIPs are post-translationally regulated (gated) by calcium
(Ca2+) and protons (H+) [55,70,71]. Their sites of action (binding) are shown.
G. Li et al. / Biochimica et Biophysica Acta 1840 (2014) 1574–1582
evanescent wave microscopy indicated that the diffusion rate of a
PIP2;1-GFP fusion at the cell surface was increased by 2-fold by salt
stress, whereas fluorescence correlation spectroscopy revealed that
PIP2;1-GFP density in the plasma membrane was decreased by 46%
. Moreover, a novel Fluorescence Recovery After Photobleaching
(FRAP) approach indicated that salt stress enhances PIP cycling
between the cell surface and endosomes, by acting on both endocytosis
diffusion and cycling of PIPs are still undetermined.
5. Aquaporins and plant nutrient acquisition
5.1. Mineral nutrient uptake
Boron is an essential element for plant growth, which, however, can
be toxic when present at high concentrations. A role of aquaporins in
boric acid transport was first proposed by Dordas et al. . These
authors showed that plasma membrane vesicles purified from squash
roots had boric acid permeability, which was 6-fold higher than that
of microsomal vesicles, and was partly inhibited by HgCl2. AtNIP5;1
represents the first aquaporin shown to play a significant contribution
to boron uptake in plants. Its gene is strikingly induced in response to
boron deficiency and boric acid transport activity of the protein was
demonstrated after oocyte expression or using nip5;1 knock-out plants
leaf nodes and floral anthers, respectively [78,79], were also identified
as boric acid channels. Takano et al.  proposed a cooperative boron
transport mechanism whereby AtNIP5;1 absorbs boric acid [B(OH)3]
from the soil, whereas AtBOR1 serves a secondary active efflux
transporter of borate [B(OH)4
accordance with this model, Arabidopsis plants were conferred with
enhanced tolerance to low boron concentrations by functional
activation of both AtNIP5;1 and AtBOR1. Conversely, tolerance of a
barley cultivar to high boron toxicity was associated to low expression
of a NIP homolog .
Silicon is another major mineral component for certain plants
including cereals (it can account for 10% of shoot dry weight in rice),
where it enhances resistance to abiotic and biotic stresses [82,83]. A
forward genetic approach in rice identified Lsi1 (OsNIP2;1) as the first
silicon transport protein of plants . OsNIP2;1 and its maize homolog
(ZmNIP2;1) are both localized on the distal sides of exodermal and
endodermal root cells. OsNIP2;1 functions as an influx channel for
silicic acid and works in concert with the efflux transporter Lsi2, to
facilitate silicon uptake from the soil into the root stele and vascular
tissues [10,84]. Another silicic acid-transporting NIP homolog (Lsi6 or
OsNIP2;2) exhibits a polar localization in xylem transfer cells of rice
nodes and may play a role in xylem unloading of silicon to enhance
transfer into panicles . In Arabidopsis, silicon also plays a role in
resistance to fungal pathogen . However, phylogenetic or structural
analyses did not reveal any clear functional homologs of cereal silicic
acid channels (OsNIP2;1, ZmNIP2;1, ZmNIP2;2) among Arabidopsis
NIPs [35,87]. Thus, it will be interesting to investigate whether NIPs
can similarly function as silicic acid transporters in dicot plants.
−] , to load boron into the xylem. In
5.2. Ammonia transport
fertilizer for crops. Whereas NH4
plants since long, NH3was initially suggested to cross membrane by
free diffusion . Yet, several TIP2 homologs of Arabidopsis and
wheat were found to have a remarkable permeability to NH3, and may
therefore participate in NH3 compartmentalization in vacuoles
[88–90]. This idea remains to be assessed at the whole plant level and,
for instance, overexpression of AtTIP2;1 orAtTIP2;3 in Arabidopsis failed
to enhance whole plant NH4
+/NH3) is an important nitrogen
+transporters have been identified in
The symbiotic interaction of plants with soil microorganisms may
provide an interesting context to understand the role of aquaporins in
significant changes in aquaporin expression in host plants [91–93] and
specific aquaporins of legumes, such as GmNOD26, are expressed in
the peribacteroid membrane of N2-fixing root nodules . Secondly,
recent work has demonstrated that GmNOD26 is a NH3-transporting
aquaporin that binds cytosolic glutamine synthase to create a
metabolite funnel and possibly enhance ammonia assimilation
efficiency [94,95]. Thirdly, some of fungal aquaporins expressed in
ectomycorrhiza showed a high NH3permeability , and could
contribute to NH3export from the fungal cytoplasm into the plant
apoplasm. Interestingly, a high-affinity NH4
be specifically expressed in arbuscular mycorrhizal (AM) roots of Lotus
japonicus . The interplay between these two types of NH4
transport proteins may provide a basis to explain the fungus-based
nitrogen nutrition of plants in symbiotic roots.
+/NH3 acquisition. Firstly, mycorrhizal symbiosis causes
+transporter was found to
Several lines of evidence suggest that aquaporins may contribute to
CO2diffusion within leaf tissues, to favor its transfer from the atmo-
sphere to the sites of photosynthetic carboxylation in the mesophyll
cell chloroplasts. The first evidence was that CO2-dependent photosyn-
thesis and deduced mesophyll conductance to CO2in Vicia faba and
Phaseolus vulgaris (French bean) leaves were reversibly inhibited by a
HgCl2treatment . In addition, NtAQP1 and AtPIP1;2 were shown
to facilitate CO2transmembrane transport after heterologous expres-
sion in Xenopus oocytes or yeast cells [99,100]. Genetic alteration of
their function in tobacco or Arabidopsis plants revealed a positive
correlation between their expression and mesophyll conductance to
CO2[99,101]. There have been concerns, however, that with respect to
cell walls and carbonic anhydrases, the contribution of membranes to
mesophyll conductance to CO2may not be predominant . We
also note that both NtAQP1 and AtPIP1;2 significantly contribute to
whole plant water transport [47,103]. This suggests a fine interplay
between CO2and H2O transport in inner leaf tissues. Also, it cannot be
excluded that hydraulic alterations in plant aquaporin mutants exerts
indirect effects on mesophyll conductance to CO2.
6. Aquaporins and plant development
Seeds play a crucial role in the reproductive cycle and dissemination
of higher plants.Most seeds are highly desiccated organs,and extensive
and well-defined water exchanges are associated with seed maturation
and germination, the latter process including seed imbibition and
subsequent embryo growth . A fine regulation of aquaporin
expression during these processes has been described in many species,
including ice plant and Brassica napus [105,106]. In particular, TIP3s of
all plant species examined show seed-specific expression and their
abundance markedly decreased during germination [107–110]. TIP3
expression may accompany the massive deposition of storage proteins,
oligosaccharides and phytins in protein storage vacuoles during late
seed development . In Arabidopsis and pea, mercury derivatives
reduced the rate of seed germination and seed imbibition, respectively
[110,112], suggesting a role of aquaporins in these processes. To date,
clear genetic evidence for a role of aquaporins in seed germination has
only been provided in rice using transgenic plants with loss- and gain-
of-function of OsPIP1;3. Gene expression studies further indicated that
this aquaporin may mediate the effects of NO on seed germination
A strong link between aquaporin expression, cell expansion and
plant growth has emerged in recent years. For instance, the expression
pattern of the AtTIP1;1 promoter in Arabidopsis is correlated with
cell enlargement in roots, hypocotyls, leaves and flower stems ,
and AtTIP1;1 expression was induced by the growth-promoting
G. Li et al. / Biochimica et Biophysica Acta 1840 (2014) 1574–1582
the exchange of water and solutes across the tonoplast, during the for-
mation of the large central vacuoles of mature cells. Numerous studies
using transgenic plants also point to a positive role of aquaporins in
plant growth. For instance, over-expression of Arabidopsis AtPIP1;2 in
tobacco and of Panax ginseng PgTIP1 in Arabidopsis significantly in-
creased plant growth [116,117]. Whereas these results may suggest
that growth was hydraulically limited in these materials, we cannot ex-
clude that altered aquaporin expression resulted in stomatal deregula-
tion or enhanced mesophyll conductance to CO2, thereby promoting
carbon fixation and plant growth. In line with the former hypothesis,
the transgenic tobacco plants overexpressing AtPIP1;2 showed en-
hanced leaf dehydration under drought stress conditions .
A recent work on the role of PIPs during lateral root emergence 
provides a more complete dissection of the role of aquaporins in plant
tissue growth. The hormone auxin, which orchestrates root growth
and development, was found to dramatically down-regulate the tran-
providing a fine control of aquaporin expression at the sites of lateral
root emergence. The hormone also inhibited water transport at the
cell and whole root levels. Aquaporin mutant analysis allowed demon-
strating the role of several aquaporin isoforms in facilitating root
emergence. A mathematical model was elaborated showing how aqua-
porin regulation favors water influx into the root primordium, which
thereby forces its way through the surrounding layers of cells in
the main root . Thus, plant roots appear to use auxin to regulate
aquaporins and therefore fine-tune water flow to speed up lateral root
7. Aquaporins and response to abiotic stresses
7.1. Plants under multiple environmental stresses
Maintaining their water balance under adverse conditions is a
formidable challenge for land plants. Under dry air, windy and/or high
temperature conditions, for instance, the evaporative demand is mark-
edly increased. Depending on plant species and physiological contexts
(e.g. availability or deficit of water in the soil), transpiration can either
be restricted through stomatal closure or maintained to favor CO2up-
take and lower leaf temperature. In the latter case, the hydraulics of
roots and leaves should not be non-limiting to prevent plant dehydra-
ly adapt to a variable environment and various kinds of abiotic stresses.
Thus, onemajor objective of currentresearch is to identify thesignaling
mechanisms that govern the regulation of aquaporin expression and
activity in plants under stresses. Several types of signaling molecules
involved are shown in Fig. 2 and discussed below.
7.2. Plant hormones
Plant hormones are important signaling molecules that play
numerous vital roles in controlling plant hydraulics and growth under
both favorable and stressful conditions. The drought-induced hormone
ABA not only induces stomatal closure, but also regulates plant aquapo-
rin function in the whole plant. Treatment of plants with exogenous
ABA [118–121], and the characterization of ABA-deficient and
overproducing plants [122,123] have revealed positive effects of ABA
on Lpr. By contrast, ABA reduced the leaf hydraulic conductance in
Arabidopsis, by down-regulating aquaporins in bundle sheath cells
, with consistent reducing effects on phosphorylation of several
PIP2s in Arabidopsis plantlets . Hose et al.  were among the
first to report that auxin (IAA) reduces the hydraulic conductivity of
root cortical cells. Recent studies in Arabidopsis indicated that IAA acts
through an Auxin Response Factor 7 (ARF7)-dependent path to inhibit
the expression of most PIPs at both transcriptional and translational
levels . ARF7 was previously identified as one of the major
transcription factors involved in auxin-regulated hypocotyls growth
and lateral root development. Salicylic acid, a hormone induced by
pathogen attack and abiotic stresses, acts similar to salt and down-
regulates PIP aquaporins and Lprby a ROS-mediated mechanism
[73,126]. Other growth-promoting hormones such as GA3 and
brassinolides also regulate aquaporin expression [127,128], but by as
yet unknown mechanisms.
7.3. Ca2+and pH
Cytosolic Ca2+and pH, which function as potent regulators of plant
aquaporins, are crucial signaling intermediates in plant responses to
stresses and hormones. For instance, soil flooding results in an oxygen
deprivation in roots, which traps cells in a deep cytosolic acidosis, and
thereby inhibits Lprbyproton-dependentgatingof PIPs. Theeffects
of Ca2+seem to be more diverse and contribute to multiple signaling
salt stress in maize and melon [130,131]. Whereas structure–function
analyses have elucidated the mode of Ca2+and proton-dependent gat-
ing of PIPs [33,55,70], the numerous Ca2+-dependent protein kinases
that exist in plant certainly provide additional regulation mechanisms
that need to be elucidated.
Root hydraulic conductivity
Cell water permeability
Fig. 2. Signaling diagram of root hydraulic conductivity (Lpr) regulation under various
stress conditions. Lpris repressed by various stresses (e.g. flooding, salt, drought, cold or
pathogen attack), which effects are mediated by phytohormones (ABA, SA) [56,126] or
signaling intermediates (cytosolic pH, Ca2+and H2O2) [55,70,71,73]. These signals can
alter the modification status (for instance, phosphorylation) and/or the sub-cellular
dynamics (endocytosis and intracellular sorting) of PIP aquaporins. These mechanisms
determine the gating, storage and recycling, or degradation of PIPs, and ultimately root
cell hydraulic conductivity. Recently, it was reported that auxin (IAA) down-regulates
Lpr, through a pathway involving the auxin response factor-7 (ARF7) . For the sake
of the indicated stresses or hormones is not shown.
G. Li et al. / Biochimica et Biophysica Acta 1840 (2014) 1574–1582
This ROS is a by-product of plant metabolism which can be toxic
at high (mM) concentrations. At lower concentrations, it can serve as
a signal molecule during plant response to various stresses, including
aquaporins,H2O2is a potentinhibitor of aquaporins and root water
transport. For instance, exogenous H2O2reduced cell and/or root
water conductivity in cucumber, maize, and Arabidopsis [73,132,133].
Whereas H2O2was proposed to gate algal aquaporins by a direct
oxidation mechanism , this compound was not able to
inhibit Arabidopsis aquaporins after expression in Xenopus oocytes. In
Arabidopsis roots, H2O2rather acts through signaling pathways involv-
ing a Ca2+influx, phosphorylation events, and leading to relocalization
of PIPs in intracellular vesicles . However, this response seems to
vary according to species and for instance, no effect of exogenous
H2O2on Lprwas observed in maize or figleaf gourd (Cucurbita ficifolia)
[132,135] whereas a low exogenous H2O2concentration increased Lpr
in French bean .
7.5. Aquaporins and plant resistance to abiotic stresses
As discussed above, exposure of plants to abiotic constraints as
diverse as soil water deficit or dry air, heat or cold stress, ionic stress
or nutrient deprivation, or changes in irradiance challenge the plant
water status and trigger highly specific hydraulic responses [27,56].
Molecular analyses on regulation of the whole aquaporin family in
these contexts have often revealed complex transcriptional and post-
translational response patterns, with sometimes opposite profiles
between isoforms. Genetic approaches have been developed, as a
complement of these expression studies. However, they also led to
somewhat contrasting results, depending on the plant species or stress
conditions investigated. For instance, antisense inhibition of PIP1s in
transgenic tobacco plants reduced Lprand leaf water potential, and
of PIP1s and/or PIP2s using a similar antisense approach in Arabidopsis
did not modify leaf water potential and hydraulic conductivity in plants
under normal or water deficit conditions. After rewatering, however,
the recovery of leaf water potential and plant hydraulic conductivity
was significantly delayed in antisense as compared to wild-type plants
. These data indicate that PIPs can play an important role during
the early phase of water stress, by acting on root water transport
(a transient ABA-mediated increase in Lprcan be observed before a
longer term inhibition), or during recovery from water stress,
by favoring water mobilization in dehydrated leaves.
Overexpression of aquaporin genes has become a widely used
strategy to understand and possibly engineer plant water relations
under stress. Numerous studies have shown that enhancing aquaporin
expression can confer on transgenic plants either a higher resistance
[116,139–143], or a higher sensitivity [117,144,145] to stresses.
Remarkably, negative effects on stress resistance were rather seen
when an aquaporin of interest was over-expressed in a heterologous
plant species [117,144,145]. We speculate that the foreign
aquaporin may not be properly recognized by the endogenous stress
response machinery. Among the few success stories, we may cite the
case of OsPIP1;3, which is induced by water-deficit in a drought-
resistant rice cultivar. Its expression in a lowland drought-sensitive
rice cultivar, using a stress-induced promoter, significantly enhanced
plant water stress resistance . Spectacular results were also
obtained with SlTIP2;2, a stress-induced aquaporin of tomato . Its
over-expression in the same species dramatically altered plant water
relations, enhancing transpiration and modifying leaf water potential
maintenanceunder drought. Nevertheless,the transgenehad beneficial
effects on plant growth and fruit yield under both control and water
Despite these punctual successes, aquaporin manipulation will
not be sufficient for developing optimal stress-resistant genotypes.
Whereas beneficial effects may be observed in certain conditions, it
will be difficult to optimize plant growth in a wide range of climatic
scenarios. In addition, targeting other genes and functions that may be
needed to avoid or repair the damage caused by the stresses has been
proposed [132,147]. The most relevant genes are those involved in
antioxidant metabolism, or encoding heat-shock proteins and stress-
responsive transcription factors.
During the last twenty years, tremendous progresses have been
achieved in understanding the structure and function of plant aquapo-
rins. The realization that plant aquaporins transport water but also
many other physiological substrates has contributed to the great
expansion of this research field. As a result, specific aquaporin isoforms
were identified for theircontribution to physiological anddevelopmen-
tal processes as diverse as seed germination, regulation of leaf and
root hydraulics, lateral root emergence but also carbon fixation or
nutrient absorption. Yet, we are still far from a fully integrated view.
Aquaporins show a particularly high molecular diversity in plants and
the function of many isoforms, even in the most studied model species
(Arabidopsis, rice), is as yet undetermined. In addition, each aquaporin
often contributes, in concert with other isoforms, to several physiologi-
cal functions. Thus, thorough cell-specific expression analyses of the
whole plant aquaporin family, in plants under optimal or stress condi-
tions, are still needed. Reverse genetic analyses of aquaporins in plants
require sharp phenotyping procedures and careful examination of
possible genetic redundancies. However, they provide a necessary
functional dimension to these integrative studies.
govern aquaporin regulation will also require more research efforts. At
present, we know that numerous co- and post-translational modifica-
tions, and molecular interactions involving distinct aquaporin isoforms
and regulatory proteins act, together with signaling intermediates
such as cytosolic H+and Ca2+, to regulate aquaporin gating and sub-
cellulartrafficking.However,thesemechanisms often needtobeplaced
in their genuine cellular context. The upstream signaling events and
their cross-talks during plant cell response to hormone or environmen-
tal stimulations are also largely unknown.
Another important avenue for future studies is the role of aquapo-
rins in plant growth. Understanding how aquaporins can hydraulically
control tissue expansion, as recently discovered in lateral roots, will
deserve a specific attention. Also, many reports indicate that some
ic plants to adverse environmental conditions, whereas others have
opposite effects. Further analyses of the mechanisms behind may
improve our knowledge on the role of aquaporins in plant stress
This work was supported in part by the Agence Nationale de la
Recherche (PhosphoStim, ANR-08-GENM-013). G. L. acknowledges a
post-doctoral fellowship from the Agropolis Fondation (Montpellier,
France). We apologize to all colleagues whose work was not cited due
to space limitation.
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