Physiologia Plantarum 2010 Copyright © Physiologia Plantarum 2010, ISSN 0031-9317

Functional characterization of drought-responsive

aquaporins in Populus balsamifera and Populus simonii ×
balsamifera clones with different drought resistance
strategies
Adriana M. Almeida-Rodrigueza , Janice E.K. Cookeb , Francis Yeha and Janusz J. Zwiazeka∗
a Department of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada T6E 2E3
b Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Correspondence We have characterized poplar aquaporins (AQPs) to investigate their possible

*Corresponding author, functions in differential drought responses of Populus balsamifera and Populus
e-mail: janusz.zwiazek@ualberta.ca
simonii × balsamifera leaves. Plants were exposed to mild and severe levels
Received 23 April 2010; of drought stress and to drought stress recovery treatment, and their responses
revised 20 July 2010 were compared with well-watered controls. Compared with P. balsamifera,
P. simonii × balsamifera used drought avoidance as the main drought
doi:10.1111/j.1399-3054.2010.01405.x resistance strategy, and rapidly reduced stomatal conductance in response to
stress. This strategy is correlated with growth rate reductions. Eleven AQPs
were transcriptionally profiled in leaves from these experiments and five
were functionally characterized for water channel activity. PIP1;3 and PIP2;5
were among the most highly expressed leaf AQPs that were responsive to
drought. Expression of PIP1;3 and five other AQPs increased in response to
drought in the leaves of P. simonii × balsamifera but not in P. balsamifera,
suggesting a possible role of these AQPs in water redistribution in the leaf
tissues. PIP2;5 was upregulated in P. balsamifera, but not in P. simonii
× balsamifera, suggesting that this AQP supports the transpiration-driven
water flow. Functional characterization of five drought-responsive plasma
membrane intrinsic proteins (PIPs) demonstrated that three PIP2 AQPs (PIP2;2,
PIP2;5, PIP2;7) functioned as water transporters in Xenopus laevis oocytes,
while the two PIP1 AQPs (PIP1;2 and PIP1;3) did not, consistent with the
notion that they may be functional only as heterotetramers.

Introduction (drought-tolerant) plants that rapidly reduce leaf water

potential (ψleaf ) in response to drought, isohydric
Water availability is among the most limiting factors for
(drought-avoidant) plants exercise tight control over
plant growth (Bogeat-Triboulot et al. 2007, Touchette
water loss mainly through stomatal movements (Tardieu
et al. 2007). Therefore, considerable research efforts
and Simonneau 1998, McDowell et al. 2008). Since
have focused on understanding drought resistance
isohydric plants rely on stomatal conductance as the
mechanisms in plants. As opposed to anisohydric
main drought resistance mechanism, the water content

Abbreviations – AQP, aquaporin; CNI, close neighbor interchange; LPI, leaf plastochron index; MIPs, major intrinsic proteins;
MP, maximum parsimony; NJ, neighbor joining; NIP, nodulin-like intrinsic protein; PIP, plasma membrane intrinsic protein;
qRT-PCR, quantitative reverse transcription polymerase chain reaction; RH, relative humidity; SIP, small basic intrinsic protein;
SWC, soil water content; TDR, time domain reflectometry; TIP, tonoplast intrinsic protein; XIP, X-intrinsic protein.

Physiol. Plant. 2010

of these plants fluctuates less and they tend to be
more susceptible to xylem cavitation compared with
anisohydric plants (McDowell et al. 2008). Although
hybrid poplars are generally considered to be relatively
isohydric (Tardieu and Simonneau 1998), they widely
vary in their stomatal sensitivity and susceptibility to
xylem cavitation (Awad et al. 2010).
Hydraulic conductivity of plant tissues plays an
important role in drought resistance (Ogasa et al. 2010).
Water transport involves a combination of apoplastic and
cell-to-cell (symplastic and transmembrane) pathways.
Transmembrane movement of water is facilitated by
aquaporins (AQPs) (Maurel et al. 2008), a functional
class of channel proteins that belong to the ancient family
of the major intrinsic proteins (MIPs) (Chaumont et al.
2001, Johanson et al. 2001, Maurel et al. 2008, Siefritz
et al. 2002). This gene family has been highly conserved
during evolution, probably because of the strict structural
requirements of water channels (Shapiguzov 2004). Five
subfamilies of MIPs have been identified in angiosperms
[plasma membrane intrinsic protein (PIPs), tonoplast
intrinsic proteins (TIPs), nodulin-like intrinsic proteins
(NIPs), small basic intrinsic proteins (SIPs) and X-intrinsic
proteins (XIPs); Johanson et al. 2001, Chaumont et al.
2001, Sakurai et al. 2005, Danielson and Johanson
2008]. The latter subfamily has been reported recently
in the moss Physcomitrella patens, Populus trichocarpa
and in a wide variety of dicots, but not in monocots
or Arabidopsis thaliana (Danielson and Johanson
2008, Gupta and Sankararamakrishnan 2009). AQPs
participate in water dynamics as facilitators of water and
small solutes movement across cell membranes (Maurel
et al. 2009). Their differential expression between plant
tissues and in response to abiotic stresses suggests that
they play roles in maintaining water balance in plants
(Luu and Maurel 2005, Parent et al. 2009, Suga et al.
2001, Tyerman et al. 2002). However, the role of AQP s
in leaf water transport under drought conditions remains
unclear (Heinen et al. 2009). As the expression of plant
AQP s is affected by drought (Aharon et al. 2003), it
could be expected that leaf AQP s that are involved in
transpiration-driven water transport are strongly linked
to stomatal movements.
AQP expression in plants subjected to water deficit
stress varies depending on the species, developmen-
tal stage, growth conditions and severity of drought
(Alexandersson et al. 2005, Cocozza et al. 2010, Oono
et al. 2003, Seki et al. 2001, 2002). For example, in
Nicotiana tabacum, drought stress reduced expression
of NtPIP1;1 and NtPIP2;1 while increasing expres-
sion of NtAQP1 (Mahdieh et al. 2008). In A. thaliana
leaves, expression of several PIP genes was downregu-
lated in response to gradual drought stress induced by
withholding water, although AtPIP1;4 and AtPIP2;5 were
upregulated (Alexandersson et al. 2005). Expression of
AQP s in roots and aerial parts of A. thaliana plants
responded differently to mannitol-induced osmotic stress
(Jang et al. 2004) or gradual drought stress (Alexanders-
son et al. 2005). These differential responses of AQP s to
drought stress are likely because of distinct functions that
AQPs may have in different tissues and the functional
differences between various tissues in plants exposed
to stress. In some cases, there can be discrepancies in
AQP expression data reported in different studies (e.g.
Alexandersson et al. 2005, Jang et al. 2004). Variation
in simulating drought stress under laboratory conditions
and the fact that plants may have diverse resistance
strategies under different drought levels (Siemens and
Zwiazek 2003) can confound the interpretation of AQP
expression patterns reported by different studies.
In the present study, we compared a Populus simonii ×
Populus balsamifera hybrid poplar clone, which, similar
to other hybrid poplars, has been characterized as having
high stomatal sensitivity (Stettler et al. 1996, Tardieu and
Simonneau 1998), and a P. balsamifera clone with stom-
ata that exhibits less sensitivity to drought (Pearce et al.
2006). We hypothesized that the expression of leaf AQPs
would respond differently in these two poplar clones
when the plants are exposed to drought conditions in
accordance with their stomatal responses. Therefore, in
the present study, we subjected the plants of these two
clones to mild drought, severe drought and drought
recovery treatments and we compared their physiolog-
ical responses and AQP expression with well-watered
plants.
Materials and methods
Plant material
P. balsamifera (L.) (clone AP1004, gender unknown) and
P. simonii (Carr.) × P. balsamifera (L.) (clone P38P38,
female) shoot cuttings were provided from single trees
by Alberta-Pacific Forest Industries (Boyle AB, Canada).
Ten-centimeter stem segments from 1-year-old trees
were re-hydrated for two days before planting in
45 × 340 cm3 styroblocks (Stuewe and Sons, Tangent,
OR) containing peat moss, vermiculite and Turface cal-
cined clay (Profile Products, Buffalo Grove, IL) (2:1:1/2)
with 2.75 g l−1 of Nutricote Total controlled-release
fertilizer (13–13–13; American Horticultural Supplies,
San Marcos, CA). Rooted cuttings were replanted in
4-l pots with the same soil composition and grown
in the greenhouse under semi-controlled conditions
[22/20◦ C day/night, 18/6 h light/dark, 60% relative
humidity (RH)], with watering six times per week until
runoff and fertilization (2 g l−1 15–30–15) once per
Physiol. Plant. 2010
week. Plants were 130–160-cm tall at the outset of the
drought experiments.
A preliminary experiment was used to empirically
define mild drought stress, severe drought stress, and
stress recovery treatments. Soil apparent permittivity
(Ka ) was measured using a time domain reflectome-
ter (TDR; Tektronix 1502B Cable TDR Cable Tester;
Tektronix, Scarborough, ON, Canada), and volumet-
ric water content (θ) or soil water content (SWC) was
determined based on the equation for organic soils
(Robinson et al. 2003, Toop et al. 1980). Measurements
of stomatal conductance (gs ) were carried out with an
Li-1600 steady-state porometer (LI-COR, Lincoln, NE).
SWC of 20–25% was defined as mild stress, based on
the observation of changes in gs when compared with
well-watered plants. SWC lower than 20% was desig-
nated as severe stress because, at this point, plants were
wilted and they showed low gs . Three days of re-watering
following the point of severe stress was designated as
recovery, because at this point gs was comparable to
that in well-watered plants.
Two duplicate drought experiments were conducted
in this study. One was used for physiological measure-
ments (n = 8) (referred to as the physiological experi-
ment) and one for the gene expression analyses (n = 6)
(referred to as the molecular experiment). In each exper-
iment, trees for each of the two poplar clones were
randomly assigned to the different drought treatments,
i.e. mild drought, severe drought, recovery from severe
drought and well-watered plants (controls). Because of
the diurnal cycle of AQP activity, physiological measure-
ments (physiological experiment) and tissue harvesting
(molecular experiment) were performed between 08:00
and 11:00 h. In the case of the physiological experi-
ment, the plants were measured within two days. For
both experiments, drought treatments were initiated on
different days so that mild drought, severe drought, and
drought recovery treatments ended on the same day.
For logistical reasons, the physiological experiment was
divided into two blocks of four independent replicates
per treatment per block; the treatments for the two
blocks were initiated one day apart so that the plants
from block 1 could be analyzed on one day, and the
plants from block 2 could be analyzed the following
day.
Leaves for analysis were selected according to the leaf
plastochron index (LPI; Larson and Isebrands 1971). For
the physiological experiment, single measurements of
net photosynthesis (Pn ) and gs were measured for LPI 7
on eight plants per treatment with an LI-6400 portable
infrared gas analyzer (LI-COR) with an incorporated
light source. All measurements were taken between
08:00 and 11:00 h. A Scholander pressure chamber
Physiol. Plant. 2010
(PMS Instruments, Corvallis, OR) was used to measure
leaf water potential (ψleaf ) for LPI 7 as described by
Wan and Zwiazek (1999). For both the gas exchange
and pressure chamber measurements, one block of
four replicates per treatment was analyzed per day,
for a total of n = 8. Within each block, the order
in which the plants were measured was randomized.
For the molecular experiment, complete leaves (with
petioles removed) from LPI 7 to LPI 10 were harvested
from each of the six plants and immediately frozen in
liquid nitrogen. Samples were stored at −80◦ C until
processed.
Sequence analysis
P. trichocarpa (Torr. & Gray) AQP gene models were
identified in version 2.0 of the assembled, annotated
genome (http://www.phytozome.net/poplar.php) using
P. trichocarpa AQP sequences reported by Gupta and
Sankararamakrishnan (2009) as BLAST queries (Altschul
et al. 1997) (see Appendix S1). AQP-deduced amino acid
sequences from P. trichocarpa, A. thaliana (L.) Heynh.
(Johanson et al. 2001; www.arabidopsis.org) and repre-
sentative XIPs from Ph. patens (Danielson and Johanson
2008) were aligned using CLUSTALW in MEGA v4 (Tamura
et al. 2007). Phylogenetic trees were constructed using
maximum parsimony (MP) in MEGA v4. A bootstrap
consensus tree of 2000 replicates was obtained, using
close neighbor interchange (CNI) with search level of
3, a random addition of 10 replicates, and a complete
deletion for gaps and missing data. No out group was
included in the analysis.
Quantitative reverse transcription polymerase
chain reaction
Total RNA was extracted using the hexadecyltrimethy-
lammonium bromide (CTAB) extraction protocol of
Chang et al. (1993). Three micrograms of total RNA were
treated with DNase I (New England Biolabs, Ipswich,
MA) and used as template for first strand cDNA synthesis
using oligo(dT)23 VN and Moloney murine leukemia virus
reverse transcriptase, following manufacturer’s instruc-
tions (New England Biolabs).
Gene-specific qRT-PCR primers were designed in the
3 untranslated (UTR)-region using Primer Express v3
(Applied Biosystems, Foster City, CA) (Appendix S2).
Populus AQP genes were selected based on phylo-
genetic proximity to genes associated with drought
responses in other species, or by EST abundance pat-
terns, suggesting relatively high expression in leaves or
in abiotic stress responses (Sterky et al. 2004). Several
potential reference genes were tested for stable levels of
transcript abundance across samples to be compared.
A member of the EF1α gene family was chosen as the
reference gene, as transcript abundance corresponding
to this gene did not show significant differences across
treatments when expressed as 2−Ct (P = 0.514 for the
drought experiment, and P = 0.257 for the tissue com-
parison experiment; Appendix S3).
Transcript abundance was quantified using standard
curves for both target and reference genes. Standard
curves were constructed from amplicons of full-length
or near full-length P. trichocarpa × deltoides cDNAs for
conducting a tissue survey analysis (data not shown)
and for the drought experiment described in this study.
P. trichocarpa × deltoides cDNAs were either obtained
from Université Laval (www.arborea.ca) or, in the
case of PtdSIP1;2 and PtdEF1α, cloned via RT-PCR
with primers described in Appendix S2 (Sambrook and
Russell 2001). Plasmids were purified using GeneJET
Plasmid Miniprep Kit (Fermentas, Burlington, ON,
Canada) according to the manufacturer’s protocol. Each
plasmid (30 ng μl−1 ) was used as template in a PCR
reaction using universal primers. PCR conditions were
as follows: 1 cycle at 94◦ C for 5 min, followed by
40 cycles at 94◦ C for 40 s, 50◦ C for 40 s, and 72◦ C
for 1.5 min, and finishing with an extension step at
72◦ C for 5 min. Amplicons were purified using the
QIAquick PCR purification kit (Qiagen, Mississauga,
ON, Canada), and diluted 1:10 with autoclaved
RNase-free water. Concentration was determined using
a Nanodrop ND-1000 spectrophotometer (Thermo
Scientific, Wilmington, DE). Target and reference genes
were diluted individually to a final concentration of
4 × 109 molecules of DNA. Afterward, a pooled serial
dilution series was made that included all target
genes plus EF1α, comprising 4 × 107 , 4 × 106 , 4 × 104 ,
4 × 103 and 4 × 101 copies of DNA molecules for each
gene.
Real-time PCR was performed on a 7500 Fast Real-
Time PCR system (Applied Biosystems). Three biological
replicates, each with three technical replicates, were
assayed for each sample. The reference gene was
included on each plate. cDNA from a single, pooled
RT reaction representing one sample from the control
(well-watered) plants from the drought experiment was
used as a calibrator to enable plate-to-plate comparison.
PCR was carried out in a volume of 10 μl including a
final concentration of 50 ng cDNA, 1× master mix con-
taining 0.2 mM dNTPs, 0.3 U Platinum Taq Polymerase
(Invitrogen), 0.25× SYBR Green and 0.1× ROX. PCR
conditions were as follows: 1 cycle at 95◦ C for 2 min,
40 cycles at 95◦ C for 15 s, 60◦ C for 1 min, and a disso-
ciation stage including 2 cycles of 95◦ C for 15 s, 60◦ C
for 1 min. Samples were subjected to auto Ct (cycle
threshold) for analysis, and dissociation curves were
verified for each of the genes.
Osmotic water permeability assay
Full-length PtdPIP1;2, PtdPIP1;3, PtdPIP2;2, PtdPIP2;5
and PtdPIP2;7 cDNAs were amplified using the Expand
High Fidelity PCR System (Roche, Indianapolis, IN),
using gene-specific primers incorporating Bgl II sites on
both ends (Appendix S2). Full-length cDNAs were sub-
cloned into the Bgl II site of the pAWPLA (pXLII) vector
[dephosphorylated using calf intestinal alkaline phos-
phatase (Invitrogen)], carrying the 5 - and 3 -UTR regions
of the beta-globin gene from Xenopus laevis. Plasmids
containing the full-length poplar AQPs in the correct
orientation were determined by restriction mapping and
sequencing. Plasmids were linearized downstream of
the 3 -UTR beta-globin sequence using FastDigest SacI
(Fermentas) for PtdPIP1;2 and PtdPIP1;3, NgoMIV (New
England Biolabs) for PtdPIP2;7 and Bpu10I (New Eng-
land Biolabs) for PtdPIP2;2 and PtdPIP2;5. Capped RNA
(cRNA) was synthesized in vitro by T3 RNA polymerase
(mMESSAGE mMACHINE T3 kit, Ambion, Austin, TX)
utilizing the T3 promoter of the vector. DNA was
removed using TURBO DNase, and the cRNA was pre-
cipitated for 2 h at −20◦ C using lithium chloride. cRNA
quality and quantity were determined using the Nan-
odrop and the 2100 Bioanalyser (Agilent, Santa Clara,
CA). cRNA was aliquotted and stored at −80◦ C until use.
X. laevis oocytes were prepared as previously
described (Cao et al. 1992, Daniels et al. 1996). Fifty
nanoliters AQP cRNA (1 mg ml−1 ) or nuclease-free
water were injected to the oocytes using a 10-μl microin-
jector (Drummond Scientific, Broomall, PA). Injected
oocytes were randomly assigned to two separated six-
well cell culture plates, and incubated as described by
Cao et al. (1992). One oocyte was added to one well of
a two-well thick-slide filled with regular Barth’s solution
(200 mosmol), and an initial image of the cell was taken.
Then an oocyte was quickly transferred to the other
well of the slide containing hypotonic Barth’s solution
(Barth’s solution diluted five times with distilled water
for a final concentration of 40 mosmol). Changes in cell
volume were tracked via microscopy images taken at 5-s
intervals for 5 min. Images were analyzed using IMAGEJ
(version 1.38X, Abramoff et al. 2004). The surface area
from the oocyte picture was obtained automatically with
IMAGEJ after a known scale was set. Oocyte surface area
and volume were calculated based on the sphere surface
area and volume geometric formulas correspondingly.
The relative volume (V/Vo ), the initial transmembrane
volume flux (Jv ) and the osmotic water permeability
coefficient (Pf ) were calculated based on Zhang and
Verkman (1991).
Physiol. Plant. 2010
Statistical analysis
Data were analyzed using SAS version 9.1 (SAS Institute,
Cary, NC). Effects of drought on gs , Pn , height and
ψleaf were evaluated by a complete randomized block
design analysis of variance. For every treatment, at
least six replicates were used for each poplar clone.
The experimental design included two blocks, four
treatments, and four replicates per treatment in each
block. The alternative hypothesis of determining the
statistical differences among treatments for each of the
physiological measurements in each of the poplars
was evaluated. Preplanned comparisons were tested
using the least-square means differences statement
with Tukey–Kramer P -values adjustment, and/or with
contrast statement, adjusting the alpha value to 0.03.
Effects of drought on AQP transcript abundance were
evaluated by a randomized factorial analysis of variance.
The alternative hypothesis of determining the statistical
differences among treatments for each of the 11 poplar
AQP genes in each of the poplars was evaluated. Pre-
planned comparisons were tested using the least-square
means differences statement. P -values were adjusted
with Tukey–Kramer or LSD adjusted to a P -value of 0.03.
Effects of PtdPIP s on the osmotic water permeability
coefficient for the oocytes exposed to hypotonic solu-
tion were evaluated by a nested analysis of variance. The
alternative hypothesis of determining the statistical differ-
ences among different oocytes exposed to a hypotonic
solution that were injected individually with different
PtdPIP s. Pairwise comparisons were tested using the
least-square means differences statement. P -values were
adjusted with Tukey–Kramer to a P -value of 0.05.
Results
Effects of drought treatments on Pn , gs , leaf and
height growth
It took two days for P. balsamifera to reduce SWC
to 20–25% (mild drought). In contrast, it took six
days for P. simonii × balsamifera to reach a similar
level. In P. simonii × balsamifera, this mild stress treat-
ment resulted in a reduction of Pn by about 60% and
gs by about 33% compared with well-watered plants
(Fig. 1A,B). In contrast, no significant differences were
observed in Pn and gs between control and mild drought
stress treatment in P. balsamifera. In plants treated with

Fig. 1. Drought responses in two contrasting Populus clones, Populus balsamifera and Populus simonii × P. balsamifera. (A) Photosynthesis (Pn ),
(B) stomatal conductance (gs ) associated with the percentage of SWC, (C) plant height and (D) leaf water potential (ψleaf ). Ten-week-old plants were
well watered (control) or subjected to mild water stress, severe water stress, or stress recovery following severe water stress. Significant differences
among treatments indicated by letters above each bar (P ≤ 0.05, Tukey-Kramer adjustment). Means ± SE are shown (n = 6 minimum).

Physiol. Plant. 2010

severe drought (SWC lower than 20%), both Pn and gs
were reduced but still higher in P. balsamifera compared
with P. simonii × balsamifera (Fig. 1A,B). Following
stress recovery, Pn and gs in both poplars returned
to the levels measured in well-watered control plants
(Fig. 1A,B).
Drought stress treatments did not affect heights of
P. balsamifera plants. However, P. simonii × balsam-
ifera plants were shorter following stress recovery
treatment compared with the control and mild stress
treatments (Fig. 1C). The leaf exhibited trends similar to
Pn and gs in both clones (Fig. 1D). Mild drought did not
significantly affect leaf in P. balsamifera, but decreased
leaf in P. simonii × balsamifera (Fig. 1D). Both poplars
showed a significant reduction of leaf when exposed
to severe drought and returned to near control levels
in re-watered plants (Fig. 1D). Leaves of P. balsamifera
wilted in response to severe drought while only a few
leaves in some P. simonii × balsamifera plants showed
signs of wilting. Similarly, young leaves of P. balsamifera
displayed a relatively high extent of necrosis and leaf
abscission which occurred in only a few P. simonii ×
balsamifera plants (data not shown).
Analysis of the P. trichocarpa AQP family
Fifty-five full-length AQP gene models were identified
in version 2.0 of the P. trichocarpa genome (Appendix
S4), consistent with the findings of Gupta and Sankarara-
makrishnan (2009), who reported on an earlier version
of the P. trichocarpa genome. In agreement with Gupta
and Sankararamakrishnan (2009), phylogenetic analy-
sis (Fig. 2) demonstrated that the P. trichocarpa AQP s
fall into five subfamilies: the PIPs, TIPs, NIPs, SIPs and

Fig. 2. Dendrogram obtained using MP illustrating relationships between members of the AQP family in Populus trichocarpa. P. trichocarpa AQPs
are denoted with black diamonds. The AQP family from Arabidopsis thaliana (AtAQPs) (Johanson et al. 2001) and two XIPs previously reported in
the moss Physcomitrella patens (PpAQPs) (Danielson and Johanson 2008) are included for comparison. Arrows indicate aquaporins selected for gene
expression analysis.

Physiol. Plant. 2010

the recently described XIPs (Danielson and Johanson
2008, Gupta and Sankararamakrishnan 2009). Further
subgroups of proteins could be identified in the TIP
(TIP1, TIP2, TIP3, TIP4, TIP5) and PIP (PIP1 and PIP2)
subfamilies. For consistency, the same gene names are
used in this paper as those proposed by Gupta and
Sankararamakrishnan (2009).
All 55 AQP s were assigned to chromosomes in v2.0
of the P. trichocarpa genome, compared to only 46 in
earlier versions (Gupta and Sankararamakrishnan 2009).
While 15 of 19 linkage groups (chromosomes) exhib-
ited 5 AQP genes or less, linkage group 9 contained
9 AQP s, including 5 of the 6 PtXIP s. Several closely
related gene pairs could be identified based on dendro-
gram location and locations within homologous genome
blocks described by Tuskan et al. (2006), which were
based on the v1.1 genome release: PtPIP1;4/PtPIP1;5,
PtPIP1;3/PtPIP1;2, PtPIP2;4/PtPIP2;3, PtPIP2;1/PtPIP2;2,
PtTIP1;3/PtTIP1;4, PtTIP1;1/PtTIP1;2, PtTIP2;1/PtTIP2;2,
PtNIP3;3/PtNIP3;4, PtNIP3;1/PtNIP3;2, PtSIP1;1/
PtSIP1;2, PtSIP1;3/PtSIP1;4 and PtSIP2;2/PtSIP2;1.
Expression profiling of Populus AQPs
A subset of AQP s was selected for a qRT-PCR survey
of transcript abundance in leaves of P. balsamifera and
P. simonii × balsamifera subjected to drought conditions
identical to the experiments described above. As PIPs
and TIPs have been associated with drought responses
in A. thaliana (Alexandersson et al. 2005, Jang et al.
2004), eight PIP s (PIP1;2, PIP1;3, PIP2;1, PIP2;2, PIP2;3,
PIP2;4, PIP2;5 and PIP2;7 ) and two TIP s (TIP1;6 and
TIP2;1) were chosen for gene expression analysis (Fig. 3).
One SIP (SIP1;2) was also selected for the analysis.
PIP1;2, PIP1;3, PIP2;1, PIP2;2, PIP2;7, TIP1;6 and TIP2;1
showed statistically significant responses to drought
in P. simonii × balsamifera, but not in P. balsamifera.
PIP2;1 and PIP2;2 showed increased transcript abun-
dance in leaves during mild and severe drought and then
reduced transcript abundance following re-watering in
P. simonii × balsamifera. PIP1;2 and PIP2;7 displayed
increased transcript abundance during severe drought,
with the latter also showing a decrease in transcript abun-
dance following re-watering. TIP1;6 exhibited decreased
transcript abundance under severe drought; PIP2;3 and
PIP2;4 showed similar trends, although the difference in
transcript abundance is not statistically significant. Inter-
estingly, only PIP2;5 showed a statistically significant
response to drought in P. balsamifera, exhibiting
increased transcript abundance at mild and severe stress.
In contrast, PIP2;5 showed decreased transcript abun-
dance in P. simonii × balsamifera under mild and severe
stress, although this was not statistically significant.
Physiol. Plant. 2010
Functional analysis of five PIPs
Five PIP s cloned from P. trichocarpa × deltoides were
tested for their ability to transport water using a X. laevis
oocyte swelling assay (Fig. 4). Relative volumes of
oocytes injected with PtdPIP1;2 and PtdPIP1;3 were
similar to those injected with water (controls). In con-
trast, oocytes injected with one of three PtdPIP2s showed
higher relative volumes compared to the control oocytes.
The greatest changes in oocyte volumes were observed
for PtdPIP2;5 followed by PtdPIP2;2 and PtdPIP2;7.
All oocytes injected with any of the PIP2s ruptured
under hypotonic conditions during the 5-min assay.
The osmotic water permeability coefficient (Pf ) was
calculated based on its relationship with the initial trans-
membrane volume flux (Jv ). Pf values for oocytes injected
with cRNAs corresponding to PtdPIP2;5, PtdPIP2;7 and
PtdPIP2;2 were respectively 9-, 3.2- and 5.2-fold higher
than those of control oocytes. For the oocytes injected
with the cRNA corresponding to PtdPIP1;3 and Ptd-
PIP1;2, the Pf values were the same or lower than for the
control (the ratios between the Pf values of the putative
PtdPIP1 and the control were 1 and 0.64, respectively;
Fig. 4).
Discussion
P. balsamifera and P. simonii × balsamifera exhibit
contrasting strategies for coping with drought
P. balsamifera and its descendant P. simonii × bal-
samifera hybrid are phreatophytic poplars (Amlin and
Rood 2003). We have shown that these two poplar
clones have different strategies to resist drought stress.
The responses of P. simonii × balsamifera to drought
can be referred to as drought avoidant, characterized
by rapid stomatal closure. This type of drought strat-
egy has been also previously reported for Populus
euramericana cv. (P. deltoides × nigra) (Tardieu and
Simonneau 1998). It is interesting that despite the dif-
ferences in stomatal conductance, leaf water potentials
were similar in both clones, suggesting possible differ-
ences in osmotic adjustment. This response, combined
with decreased stomatal conductance, could have con-
tributed to less extensive leaf abscission and necrosis in
severely drought-stressed P. simonii × balsamifera com-
pared with P. balsamifera. In contrast, P. balsamifera
maintained relatively high gs and Pn during mild drought.
The capacity of P. balsamifera to maintain relatively
high rates of gas exchange, during sudden drought
events, compared with P. simonii × balsamifera is also
associated with a lower vulnerability to xylem cavita-
tion (Arango-Velezet al., unpublished data), as reported
for other tree species (Maherali et al. 2006), allowing
Fig. 3. Transcript abundance corresponding to 11 AQPs in leaves of Populus balsamifera and Populus simonii × balsamifera subjected to four
different water availability treatments. Transcript abundance for each AQP was quantified by standard curve, and is expressed relative to transcript
abundance corresponding to EF1-α. Mean EF1-α transcript abundance across treatments for each clone was not statistically significantly different, and
thus EF1-α was considered to be constitutively expressed within the context of these experiments (see Appendix S3). n = 3 biological replicates; each
biological replicate was assayed in triplicate. Error bars = SE. Letters above bars designate statistical differences at P = 0.05 in transcript abundance
for a given gene within a clone; significance levels are shown with asterisks (∗ P = 0.03; ∗∗ P < 0.01; ∗∗∗ P < 0.008).

P. balsamifera to maintain a comparable level of carbon
gain. However, while P. balsamifera may be able to
maintain higher photosynthetic activity and growth rates
under short-term mild drought conditions, sluggish stom-
atal responsiveness might compromise its survival under
severe drought (Street et al. 2006).
Although all poplars are relatively susceptible to
cavitation (Cochard et al. 2007a, 2007b), the differences
in stomatal responses of P. balsamifera and P. simonii ×
balsamifera are likely linked to cavitation resistance.
Poplars often cope with drought by reducing their
transpiration area through leaf and branch abscission
(Amlin and Rood 2003). Although in our study, ψleaf , gs
and Pn of severely drought-stressed P. balsamifera and
P. simonii × balsamifera returned to their pre-drought
levels within three days following re-watering, leaf

Physiol. Plant. 2010


Fig. 4. Pf values of oocytes injected with five cRNAs from Populus
trichocarpa × deltoides PIPs or with water (control). These values were
obtained based on the surface area measurements and the relative
volume calculations of a swelling assay in which the injected oocytes
were exposed to hypotonic conditions. Pf values presented are means
± SE of at least nine replicates. Significant differences between the
genes and the control are indicated by lowercase letters (P ≤ 0.05,
Tukey-Kramer adjustment).
senescence and abscission of older leaves still occurred
in plants recovering from drought.
A subset of AQPs are drought responsive in leaves,
and show different responses to drought in
P. balsamifera vs P. simonii × balsamifera
Based on these contrasting stomatal responses, we
hypothesized that AQP s expressed in leaves would show
different responses in P. balsamifera and P. simonii ×
balsamifera when the plants were exposed to drought
conditions. Expression profiling of 11 AQP s representing
3 of the 5 AQP subfamilies revealed that a number of
AQP s were drought responsive, and that some of these
AQP s showed contrasting patterns of responsiveness in
P. balsamifera and P. simonii × balsamifera, supporting
our hypothesis (Fig. 3). The higher level of expression for
several AQP s in control leaves of P. simonii × balsam-
ifera compared with P. balsamifera suggests that, in the
absence of significant differences in leaf hydraulic con-
ductance between the two poplars (data not shown), the
cell-to-cell pathway may play a greater role in the leaf
water transport of P. simonii × balsamifera compared
with P. balsamifera when soil water is not a limiting
factor.
PIP1;3 and PIP2;5 present interesting candidates for
further investigation into potential roles of these AQP s
in regulating cell-to-cell movement of water during
water deficit conditions, because these genes are not
only drought responsive but also show substantially
greater transcript abundance in leaves relative to other
AQP s that were examined (data not shown). When
plants were subjected to mild and severe drought
Physiol. Plant. 2010
stress, expression levels of PIP2;5 increased by almost
fourfold in P. balsamifera and decreased by several-fold
in P. simonii × balsamifera, although the latter was
not significant because of variability in gene expression
across replicates. This supports the notion that PIP2;5
may be involved in the regulation of leaf hydraulic con-
ductance with a possible link to stomatal responses.
The putative ortholog to PIP2;5 was the main leaf AQP
found in P. tremula × tremuloides (Marjanović et al.
2005), bur oak (Quercus macrocarpa; Voicu et al. 2009)
and walnut (Juglans regia; Cochard et al. 2007a, 2007b).
Expression of these putative PIP2;5 orthologs has been
shown to be affected by light conditions in these lat-
ter two species (Cochard et al. 2007a, 2007b; Voicu
et al. 2009), providing additional evidence to support
the hypothesis that PIP2;5 may play a role in leaf water
relations in poplar. We confirmed water channel activity
for PIP2;5. PIP2;5 corresponds to PoptrPIP2.5 in Secchi
et al. (2009), who also demonstrated that this gene prod-
uct functions as a water channel. PIP2s from many other
species function as water channels, including OsPIP2;7
(Oryza sativa; Li et al. 2008), OePIP2;1 (Olea europaea
L.; Secchi et al. 2007), SsAQP2 (Samanea saman; Moshe-
lion et al. 2002), ZmPIP2;5 (Zea mays; Chaumont et al.
2000), ZmPIP2;1, ZmPIP2;4 (Fetter et al. 2004), JrPIP2;1,
JrPIP2;2 (J. regia; Sakr et al. 2003), HvPIP2;1 (Hordeum
vulgaris; Katsuhara et al. 2002), NtPIP2;1 (N. tabacum;
Mahdieh et al. 2008) and PM28A (Spinacea oleracea;
Johansson et al. 1998).
Contrary to PIP2;5, PIP1;3 expression was drought
responsive in P. simonii × balsamifera leaves rather
than P. balsamifera leaves. The role of this AQP in
leaf drought responses is less clear. PIP1;3 is closely
related to PIP1;2, yet shows markedly different pat-
terns and levels of expression, particularly across tissues
(data not shown). This observation supports the notion
that subfunctionalization and/or neofunctionalization
has occurred in the Populus AQP gene family. Sim-
ilar to PIP1;1 in P. trichocarpa (Secchi et al. 2009)
and other plants (Chaumont et al. 2000, Fetter et al.
2004, Mahdieh et al. 2008, Moshelion et al. 2002,
Secchi et al. 2007), PtdPIP1;3 and PtdPIP1;2 had low Pf
when expressed individually in heterologous system of
Xenopus oocytes. PIP1 functionality in cell membranes
may depend on their interactions with PIP2 isoforms.
For example, co-expression of maize ZmPIP1;2 and
ZmPIP2;5 in Xenopus oocytes improved the targeting
of ZmPIP1;2 into the plasma membrane, raising the
Pf values higher than for ZmPIP2;5 expressed alone
(Fetter et al. 2004). In maize mesophyll protoplasts,
when expressed alone, ZmPIP1;2 was retained in the
endoplasmic reticulum and ZmPIP2;1 was found in the
plasma membrane. However, when co-expressed, they
formed hetero-oligomers and ZmPIP1;2 was relocalized
to the plasma membrane (Zelazny et al. 2007). However,
we cannot rule out the possibility that PIP1 isoforms were
not correctly expressed in Xenopus oocytes.
Leaf AQPs and contrasting stomatal behavior in
Populus
It has been suggested that AQP s might play a role in plant
water balance by determining isohydric or anisohydric
behavior in tomato (Solanum lycopersicum; Sade et al.
2009). However, the role of AQPs in leaf water trans-
port and the contribution of the cell-to-cell pathway to
leaf hydraulic conductance remain contentious (Heinen
et al. 2009). Although temperature responses (Sack et al.
2004) and chemical inhibition of leaf hydraulic conduc-
tance (Nardini et al. 2005) suggest that AQPs play a role
in bulk leaf water transport, other studies suggest that
this may not be true for all plants. In Q. macrocarpa,
there was no significant change in expression of AQP
genes in leaves with light-induced high hydraulic con-
ductance compared to leaves with low hydraulic con-
ductance (Voicu et al. 2009). Similarly, in Arabidopsis,
leaf hydraulic conductance was not altered in double-
antisense plants with reduced expression of PIP1 and
PIP2 aquaporins (Martre et al. 2002), while in tobacco,
leaf hydraulic conductance was similar in wild-type and
transgenic plants constitutively overexpressing PIP2;5
and PIP1;4 AQP s (Lee et al. 2009). Therefore, the sig-
nificance of AQP -mediated leaf water transport may
vary between plants and under different environmental
conditions.
In conclusion, we have identified a number of AQP s
that show distinct responses to drought in leaves of
P. simonii × balsamifera and P. balsamifera, two poplar
clones that exhibit contrasting drought response strate-
gies. PIP2;5 and PIP1;3 are the most highly expressed of
these drought-responsive AQP s. The water-transporting
PIP2;5 may be linked to hydraulic and stomatal
responses, while the functions of PIP1;3 are less clear.
The generally higher levels of AQP expression in non-
stressed leaves of P. simonii × balsamifera compared to
P. balsamifera suggest that the cell-to-cell pathway may
constitute a greater role in water transport of leaves for
this drought-avoidant hybrid. Further studies are needed
to delineate the in planta function of these two AQPs in
poplars, particularly with respect to leaf hydraulics.
Acknowledgements – We thank Alfons Weig and François
Chaumont for the pAWPLA (pXLII) vector, François
Chaumont for comments on the manuscript, Leonardo
Galindo-Gonzalez for assistance with sequence analysis,
Walid El Kayal for assistance with cloning, Troy Locke
for assistance with qRT-PCR, and Xing-Zhen Chen and
Jung Woo Yang for assistance with X. laevis oocytes cRNA
injections. This research was funded by the Natural Sciences
and Engineering Research Council of Canada (NSERC).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. AQP sequences from Populus tri-
chocarpa, Arabidopsis thaliana and Physcomitrella
patens that were included in this study.
Appendix S2. List of gene-specific primers used in this
study.
Appendix S3. Expression levels of the reference gene
EF1α, a gene from the elongation factor-1 alpha gene
family.
Appendix S4. AQP gene models from version 2.0 of the
Populus trichocarpa genome.
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material) should be directed to the corresponding author
for the article.