The Role of Plasma Membrane Aquaporins in
Regulating the Bundle Sheath-Mesophyll
Continuum and Leaf Hydraulics1[C][W][OPEN]
Nir Sade 2,3, Arava Shatil-Cohen 2, Ziv Attia, Christophe Maurel, Yann Boursiac, Gilor Kelly, David Granot,
Adi Yaaran, Stephen Lerner, and Menachem Moshelion*
Institute of Plant Sciences and Genetics in Agriculture, Robert H. Smith Faculty of Agriculture, Food, and
Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel (N.S., A.S.-C., Z.A., G.K., A.Y., S.L.,
M.M.); Biochimie et Physiologie Moléculaire des Plantes, Unité Mixte de Recherche 5004, Centre National de la
Recherche Scientifique/Unité Mixte de Recherche 0386, Institut National de la Recherche Agronomique/
Montpellier SupAgro/Université Montpellier II, F–34060 Montpellier cedex 2, France (C.M., Y.B.); and Institute
of Plant Sciences, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel (G.K., D.G.)
Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and
extravascular, radial hydraulic conductance in leaves (Kleaf), in particular, is still fairly limited. We hypothesized that the AQPs
of the vascular bundle sheath (BS) cells regulate Kleaf. To examine this hypothesis, AQP genes were silenced using artificial
microRNAs that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to
target all five AQP genes from the PLASMA MEMBRANE-INTRINSIC PROTEIN1 (PIP1) subfamily. Our results show that the
constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and
BS osmotic water permeability (Pf), mesophyll conductance of CO2, photosynthesis, Kleaf, transpiration, and shoot biomass. Plants in
which the PIP1 subfamily was silenced only in the BS (SCARECROW:microRNA plants) exhibited decreased mesophyll and BS Pf
and decreased Kleaf but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We
excluded the possibility of SCARECROW promoter activity in the mesophyll. Hence, the fact that SCARECROW:microRNA
mesophyll exhibited reduced Pf, but not reduced mesophyll conductance of CO2, suggests that the BS-mesophyll hydraulic
continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling
leaf water status under normal and limited-water conditions is discussed.
The flow of water through the leaf is one of the most
important, but least understood, components of the
whole-plant hydraulic system (Sack and Holbrook,
2006; Scoffoni et al., 2012). Water is transported axially
from the roots to and through the petiole and veins
and radially from the laminar xylem across the xylem
1
This work was supported by the Israel Science Foundation (grant
no. 1131/12), by the Hohenheim Hebrew University of Jerusalem
Foundation, by the European Molecular Biology Organization
(short-term fellowship no. ASTF 252–2010), and by the Agence Nationale de la Recherche (LeafFlux grant no. ANR–07–BLAN–0206 to
C.M. and Y.B.).
2
These authors contributed equally to the article.
3
Present address: Department of Plant Sciences, University of California, Davis, CA 95616.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Menachem Moshelion ([email protected]).
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www.plantphysiol.org/cgi/doi/10.1104/pp.114.248633
parenchyma and bundle sheath (BS) cells to the mesophyll. Once it exits the xylem vessel, water can flow
through or around mesophyll cells before evaporating
from the cell walls and diffusing out of the stomata
(Esau, 1965). The efficiency of water transport through
the leaf is determined as the hydraulic conductance
(Kleaf [the ratio of the water flow rate to the driving
force of the flow]; for review, see Sack and Holbrook,
2006; Prado and Maurel, 2013), which is the sum of the
vascular-axial and extravascular-radial conductance.
Leaf vascular anatomy (vein density and the distance
between the vein endings and the stomata) has a
strong influence on Kleaf (Sack and Frole, 2006; Brodribb
et al., 2007; Sack and Scoffoni, 2013). Nevertheless, Kleaf
is not constant, and changes in this value reveal highly
dynamic behavior, which is associated with changes in
ambient conditions (i.e. temperature and irradiance)
and abiotic stress (Martre et al., 2002; Sack and Holbrook,
2006; Cochard et al., 2007; Levin et al., 2007).
As the axial hydraulic structure of the mature leaf is
fixed, the assumption is that in nonembolized or undamaged xylem, a substantial part of the dynamic hydraulic regulation is controlled by the radial movement of
water through the parenchymal tissue surrounding the
xylem elements (Sack and Holbrook, 2006). Recent studies
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Sade et al.
have suggested that, in Arabidopsis (Arabidopsis thaliana),
the leaf radial in-flow rate is controlled by the vascular BS,
which is a bottleneck for leaf hydraulic conductance
(Ache et al., 2010). These cells may be involved in regulating the radial transport activity of the xylem system
(Kinsman and Pyke, 1998; Leegood, 2008), partially due
to the extremely low apoplastic flow through the BS
(Shatil-Cohen and Moshelion, 2012).
Recently, new evidence has emerged to support the
hypothesized role of the BS, as well as xylem parenchymal cells, in the regulation of radial Kleaf, possibly via the
regulation of water channels (i.e. aquaporins [AQPs]; for
review, see Moshelion et al., 2014). AQPs are considered
to be key channel proteins for the transport of water,
small and uncharged solutes, and CO2 through plant cell
membranes (Tyerman et al., 2002; Maurel et al., 2008). A
number of previous studies have reported on the regulatory role of AQPs in cellular water transport (Maurel,
1997; Kaldenhoffet al., 2007). Arabidopsis has 35 AQPs
(Johanson et al., 2001), which have been categorized into
four major subfamilies. The PLASMA MEMBRANEINTRINSIC PROTEIN (PIP) subfamily includes 13
members and is divided into two subgroups, AtPIP1
and AtPIP2. The AtPIP1 group includes five proteins
(AtPIP1;1–AtPIP1;5), and the AtPIP2 group includes
eight proteins (AtPIP2;1–AtPIP2;8).
It has been suggested that AQPs play a role in regulating Kleaf (for review, see Sack and Holbrook, 2006).
More recent reports have noted a correlation between
PIP AQP expression and dynamic leaf hydraulics under different irradiance conditions (Cochard et al.,
2007) as well as the role of BS AQPs in the regulation of
Kleaf under stressful conditions (Shatil-Cohen et al.,
2011; Pantin et al., 2013). Recently, it was demonstrated that the activity of the AtPIP2;1 isoform in
veins can account for the responses of rosette hydraulic
conductivity (Kros) under prolonged dark conditions
(Prado et al., 2013). Nevertheless, the identification of
the set of AQPs expressed in vessels and their respective roles in Kleaf is still incomplete, and the direct,
quantitative effect of BS AQPs on leaf hydraulics is not
fully understood.
Many of the studies that have investigated the role
of AQPs in regulating plant hydraulic conductance were
performed using classical reverse-genetic approaches.
For instance, transfer DNA mutant insertion lines lacking the PIP1;2, PIP2;1, or PIP2;6 isoform, which are
abundant in leaf tissue (Alexandersson et al., 2005),
exhibited a 20% decrease in whole-rosette Kros (Postaire
et al., 2010; Prado et al., 2013), and antisense lines targeting PIP AQPs in Arabidopsis and tobacco (Nicotiana
tabacum) exhibited decreased whole-plant hydraulic
conductivity (Martre et al., 2002; Siefritz et al., 2002).
However, these tools are of limited use when it comes to
studying the role of gene-tissue interactions or redundancy caused by a high number of targeted isoforms
(Ossowski et al., 2008).
In an attempt to cope with these challenges, we
employed methodology involving artificial microRNA
(amiRNA), which exploits endogenous microRNA
(miRNA) precursors to generate small RNA sequences
that direct gene silencing in the cell. amiRNA-mediated
gene silencing is a useful method for inducible and partial
gene inactivation as well as the simultaneous, specific
targeting of several related genes (Ossowski et al., 2008).
Moreover, the effect of the construct is autonomous, and
it can be used for studies of tissue-specific expression
(Alvarez et al., 2006).
In this study, we used an amiRNA reverse-genetic
approach to specifically down-regulate multiple PIPs.
We hypothesized that this silencing would lead to
decreased permeability of the cells to water and decreased
Kleaf. We examined this hypothesis using specific downregulation of multiple AQP genes under the control of
both constitutive and BS-specific promoters and tested the
effects of this down-regulation on the flow of water into
the leaf.
RESULTS
Using amiRNA for the Down-Regulation of
PIP1 Homologs
Several studies have reported the involvement of the
PIP subfamily (both PIP1 and PIP2) in the regulation of
hydraulic conductivity (Kaldenhoff et al., 1998; Martre
et al., 2002; Siefritz et al., 2002; Postaire et al., 2010; Prado
et al., 2013). Therefore, we chose to silence the PIP1 subfamily using amiRNA. To that end, we designed a single
sequence of synthetic miRNA containing a consensus sequence of 21 nucleotides targeted at the entire Arabidopsis
PIP1 subfamily (AtPIP1;1–AtPIP1;5; Fig. 1A).
This 21-nucleotide sequence met the criteria for allowable mismatches in number and position as well as
the low free-energy characteristic of endogenous plant
miRNAs (Schwab et al., 2005). It was introduced into the
miR164b backbone (Alvarez et al., 2006) under the control
of the 35S constitutive promoter to generate amiRNApip1,
which was used to transform Arabidopsis plants. Quantitative real-time (q-RT)-PCR was used to identify the
transgenic expression of premiR-PIP1 (Fig. 1B). The plants
that overexpressed 35S:amiRNApip1 (35S:mir1) were
somewhat smaller than the wild-type plants, depending
on the level of 35S:mir1 expression and the physiological
age of the plant (Fig. 1, C–E).
To examine amiRNA activity, we assayed the relative abundance of the full-length PIP1 transcripts using
RNA northern-blot analysis. For all five PIP1s, a significant reduction in RNA level was observed in the
transgenic plants (Supplemental Fig. S1A). In addition,
we detected the presence of amiRNApip1 cleavage
products of three candidate PIP1 genes using 59 RNA
ligase-mediated (RLM)-RACE (Kasschau et al., 2003;
Supplemental Fig. S1B).
Our q-RT-PCR analysis identified a down-regulation
of all PIP1 genes in the leaves from two independent
35S:mir1 lines (35S:mir1-3 and 35S:mir1-8) as compared with the control (Fig. 2). The 35S:mir1-8 line had
2-fold higher expression of the synthetic amiRNApip1
and lower PIP1 transcript levels (70%–98% reduction)
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PIP Aquaporins Regulate Leaf Hydraulics
Figure 1. Physiological and morphological
characteristics of the silenced AtPIP1 plants.
A, Twenty-one base pairs of the consensus sequence
alignment of the amiRNApip1 and the entire
AtPIP1 subfamily (AtPIP1;1–AtPIP1;5). Nucleotides surrounded by squares represent deliberate
mismatches between the amiRNApip1 and the
AtPIP1 gene subfamily. Numbers in parentheses
indicate the position of a mir gene base pair.
B, The mean 6 SE expression level of amiRNApip1
in the transgenic plants (35S:mir1) was characterized using q-RT-PCR. C, At 45 d after planting,
the 35S:mir1-8 plants were smaller than the wildtype (WT) plants. At a younger stage, both lines
were smaller than the control. D, Mean 6 SE rosette dry weight (DW) of 21-d-old plants (black
bar, n = 25; gray bar, n = 17; white bar, n = 28).
This weight difference persisted only for line 35S:
mir1-8. E, Mean 6 SE rosette dry weight of 45-dold plants (black bar, n = 9; gray and white bars,
n = 4). Asterisks indicate significant differences
(P , 0.05) between a genotype and the wild type,
as calculated using Dunnett’s method. [See online
article for color version of this figure.]
as compared with the control and the 35S:mir1-3 line,
in which there was a 52% to 94% reduction in PIP1
expression. Both lines displayed an approximately 50%
reduction in the expression of the PIP2;1 gene, and the
35S:mir1-8 line had 37% less expression of the PIP2;6
gene (Fig. 2). In addition, most of the PIP2 genes were
expressed at lower levels in the 35S:mir1 lines.
Western-blot analysis with specific PIP1 and PIP2
antibodies was used to detect the abundance of PIP1
and PIP2 proteins in the 35S:mir1 plants (Santoni et al.,
2003). Reduced levels of PIP1 proteins were observed
in both 35S:mir1 lines, although (and in accordance with
the observed RNA expression levels) a greater decrease
was observed in the 35S:mir1-8 line (Supplemental Fig. S2).
Figure 2. Relative expression of PIP genes in the
whole leaf. Columns indicate the mean 6 SE expression levels (q-RT-PCR analysis) of all PIP
transcripts in the 35S:mir1 (35S:mir1-3 and 35S:
mir1-8) plants and in the control (wild-type [WT])
plants. The data presented were normalized to the
wild type. Asterisks indicate significant differences (*P , 0.05, **P , 0.01; n = 3–7 independent biological repetitions) between treatments
and the control, as calculated based on the raw
transcript levels using Dunnett’s method. Data
were normalized to ACTIN2.
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The expression of PIP2 proteins was slightly reduced in
the 35S:mir1-8 plants, whereas more significant reduction was observed in the 35S:mir1-3 line (Supplemental
Fig. S2).
Physiological Characteristics of the 35S:mir1 Plants
To compare AQP functioning in the different lines, we
measured the cellular osmotic water permeability coefficient (Pf) of mesophyll and BS protoplasts isolated from
wild-type plants and the two independent 35S:mir1 lines.
Significantly lower Pf values were observed in the 35S:
mir1 mesophyll and BS cells as compared with control
cells (Fig. 3), indicating dominant PIP1 activity in the cells
of wild-type plants.
We examined the whole-rosette Kros of the 35S:mir1
plants using a previously described pressure-chamber
technique (Postaire et al., 2010; Prado et al., 2013). Plants
were transferred to an extended-night (13–21 h of
darkness) environment prior to hydraulic measurements, a condition previously shown to enhance Kros
and its AQP-mediated component (Postaire et al., 2010;
Prado et al., 2013). The 35S:mir1-8 line showed significantly lower Kros than the control, whereas the Kros of
35S:mir1-3 was similar to that of the control (Fig. 4A).
The rosette surfaces of the two lines were similar to that
of the control (Fig. 4B). Analysis of the PIP transcript
levels in the enriched BS tissue (the leaf midveins)
showed that, in both lines, all of the PIP1 genes except
for PIP1:3 were down-regulated. However, the decrease
observed in the 35S:mir1-8 line was more substantial
than that observed in the 35S:mir1-3 line (Fig. 5).
Kleaf was measured using a detached-leaf approach
(Shatil-Cohen et al., 2011) based on the determination
of transpiration rate (E) and leaf water potential (Cleaf;
Figure 4. Constitutive silencing of the AtPIP1 AQP subfamily decreased the hydraulic conductance of 21-d-old 35S:mir1-8 plants.
A, Mean 6 SE Kros for 35S:mir1-8 and 35S:mir1-3 plants (gray bar, n = 14;
white bar, n = 8) and the control (black bar, n = 9). Kros values were
measured under extended-darkness conditions and normalized to rosette surface area. B, Mean 6 SE rosette surface areas (cm2) of two
independent lines expressing amiPIP1, 35S:mir1-8 and 35S:mir1-3
(gray bar, n = 14; white bar, n = 6 cells), and that of the control (black
bar, n = 9). Data were pooled from four independent cultures of plants.
The asterisk indicates a significant difference (P , 0.05) between a
genotype and the wild type (WT), as calculated using Dunnett’s
method.
see “Materials and Methods”), to yield a calculated
Kleaf (ratio of E to Cleaf; Fig. 6). Consistent with the Kros
results and the relative reduction in the transcript
level, the Kleaf of the 35S:mir1-8 line (but not of the
35S:mir1-3 line) was significantly reduced as compared
with the control (Fig. 6A). Nevertheless, examination
of the morphological structure of the vascular systems
of the different lines revealed no differences that could
impact hydraulic conductivity (e.g. abnormal structure
or size or leakage [Supplemental Fig. S3]; for a more
detailed explanation of the relation of these measurements to BS apoplastic activity, see Shatil-Cohen et al.,
2011).
In a parallel experiment, we measured the mesophyll
CO2 conductance and gas-exchange characteristics of
35S:mir1 silenced lines grown in soil. Here again, only
the 35S:mir1-8 line exhibited a significant reduction in
photosynthesis and mesophyll conductance of CO2 (gm)
as compared with the wild-type plants (Table I). This
experiment was performed under saturated light conditions (Flexas et al., 2007). Under these conditions,
lower stomatal conductance (gs) was observed (gs and E
in both 35S:mir1-8 and 35Smir1-3; Table I) as compared
with the wild type.
Physiological Characteristics of the
SCARECROW:microRNA Plants
Figure 3. Constitutive silencing of the AtPIP1 AQP subfamily decreased the Pf of BS and mesophyll cells. Columns indicate the
mean 6 SE Pf for wild-type (WT; black bars), 35S:mir1-8 (gray bars), and
35S:mir1-3 (white bars) BS protoplasts (A; n = 62, n = 15, and n = 24,
respectively) and mesophyll protoplasts (B; n = 17, n = 12, and n = 14,
respectively). Asterisks indicate significant differences (*P , 0.05,
**P , 0.001) between a genotype and the wild type, as calculated
using Dunnett’s method.
As a complementary approach to test the hypothesis
that Kleaf is determined by AQP activity in the BS, we
examined plants in which PIPs are specifically silenced
in the BS cells by the expression of mir1 under the
control of the SCARECROW (SCR) promoter (SCR:
mir1). As described previously, prior to the physiological Kleaf measurements, we followed the expression
patterns of all of the PIP genes in the enriched vein
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PIP Aquaporins Regulate Leaf Hydraulics
Figure 5. Relative expression profile of the PIP
genes in the leaf midveins. Columns indicate the
mean 6 SE PIP transcript levels (q-RT-PCR) in 35S:
mir1-3, 35S:mir1-8, and the control (wild type
[WT]). The data presented were normalized to the
wild type. Asterisks indicate significant differences
(*P , 0.05, **P , 0.01; n = 5 or 6 independent
biological repetitions) between a treatment and the
control based on the raw transcript levels, as calculated using Dunnett’s method. Data were normalized to ACTIN2 levels.
tissue of SCR:mir1 plants and compared them with
those of control plants. Moreover, the morphological
structure of the SCR:mir1 vascular system exhibited no
differences that could impact the hydraulic conductivity
(e.g. abnormal structure or size or leakage; Supplemental
Fig. S3). An analysis of RNA expression revealed downregulation of all of the PIP1 genes (except for AtPIP1:1)
and of four AtPIP2 genes (AtPIP2:5–AtPIP2:8; Fig. 7).
Next, we measured the Pf of the BS cells. The mean
Pf of the SCR:mir1 BS cells was significantly lower than
that of the control cells (Fig. 8A). Surprisingly, the Pf of
the mesophyll cells was also significantly lower in the
SCR:mir1 line (Fig. 8B; see “Discussion”).
Following the molecular and cell Pf analyses of the
SCR:mir1 plants, we measured E and Cleaf in order to
calculate Kleaf (Fig. 9). We found that Kleaf was significantly reduced in this line as compared with the
control (Fig. 9). This significant reduction in Kleaf was
associated with significantly lower Cleaf. Interestingly,
and similar to the 35S:mir1 plants, although a visible
trend was observed, there was no significant reduction
in E (relative to the control) in the detached leaves (Fig. 9).
These results were further confirmed using intact leaves
(Supplemental Fig. S4). Examinations of the mesophyll
CO2 conductance and gas-exchange characteristics of
SCR:mir1 plants grown in soil revealed no reductions in
any of the parameters as compared with the wild type
(Table II), and these plants were slightly larger than the
wild-type plants (Supplemental Fig. S5, A and B). The
fact that these plants did not show any reductions in gs or
E or any increase in their abscisic acid (ABA) biosynthetic
gene transcript levels (9-cis-epoxycarotenoid dioxygenase;
Supplemental Fig. S5C) but did exhibit reduced mesophyll
Pf suggests that hydraulic and biochemical (photosynthesis)
processes proceed separately.
parenchymal tissue-specific PIP2s were recently shown to
control Kros (Prado et al., 2013). Here, we demonstrate
that BS AQPs play a role in the direct control of Kleaf and
also indirectly control mesophyll hydraulic conductance.
amiRNA as a Tool for Down-Regulating AQP Expression
amiRNA silencing is expected to be proportional to
its expression level. That is, if more miRNA molecules
DISCUSSION
AQPs play a major role in mediating the transmembrane
transport of water in plant cells (Maurel et al., 2008). It has
been suggested that the BS acts as a xylem-mesophyll
hydraulic barrier (Heinen et al., 2009; Ache et al., 2010;
Shatil-Cohen and Moshelion, 2012), directing the apoplastic xylem flow into the leaf via the BS transmembrane
pathway, which is controlled by AQPs. Indeed, vascular
Figure 6. Kleaf of the AQP-modified lines. Leaves were harvested from
each of the tested lines, and their petioles were immediately immersed
in AXS. After 1 h, Kleaf was calculated for each individual leaf (A) by
dividing E (B) by Cleaf (C). Asterisks indicate significant differences (P ,
0.05) between a genotype and the wild type (WT), as calculated using
Dunnett’s method. Data are means 6 SE from 14 to 17 replicates from
three independent experiments.
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Sade et al.
Table I. Gas-exchange characteristics of 35S:mir1 and control plants (determined at 1,200 mmol m22 s21, approximately 25˚C, and 400 mmol mol21
CO2, as described by Flexas et al., 2007)
Data are given as means 6 SE (n, number of independent biological repetitions). Asterisks indicate significant differences (P , 0.05) between a
genotype and the wild type, as calculated using Dunnett’s method. AN, Photosynthesis; Ci, substomatal CO2 concentration.
Plant
AN
mmol CO2 m
Wild type (n)
35S:mir1-8 (n)
35S:mir1-3 (n)
Ci
22
21
s
6.9 6 0.31 (33)
5.65 6 0.37 (22)*
6.10 6 0.24 (22)
mmol CO2 mol
gs
21
301.63 6 3.01 (33)
304.95 6 3.37 (22)
296.06 6 3.99 (22)
are present, they will silence more transcripts (Alvarez
et al., 2006). Indeed, the line with the higher level of
amiRNApip1 expression had a lower level of PIP expression (RNA and proteins) and a stronger phenotype (Figs. 1,
2, 4, and 5; Supplemental Fig. S2), confirming the quantitative nature of amiRNA activity.
In contrast to other reverse-genetic approaches, amiRNA
can silence multiple AQP isoforms simultaneously with
minimal risk of off-target effects (Ossowski et al., 2008).
Moreover, amiRNA can be targeted (promoter related)
to a specific tissue, since the miRNA silencing is local
and not systemic (Alvarez et al., 2006).
Monogenic homozygous AQP knockout lines can be
combined to eliminate several isoforms, and this approach can be used to exclude direct side effects on
other related genes. However, there are limitations to
this strategy, including gametophytic or early sporophytic lethality as well as the difficulty of combining
mutant alleles on closely linked (tandem duplicated)
genes. Moreover, targeting more than four AQP isoforms (e.g. the five PIP1 isoforms or eight PIP2 isoforms) using this approach is challenging.
RNA interference (RNAi) or antisense strategies can
be used to silence several isoforms at once. However, the
possibility of side effects, such as the systemic expression of the transformed construct and/or effects on
other unrelated genes, cannot be completely excluded
(Ossowski et al., 2008). For example, in previous studies,
using RNAi to silence AQPs resulted in the unintentional silencing of unrelated genes, yielding an artificial
phenotype (Ma et al., 2004; Schüssler et al., 2008). Since
amiRNA expression results in a single silencing molecule (as opposed to RNAi, which creates a large number
of small interfering RNA molecules), it is possible to
avoid such phenomena in the amiRNA method (Ossowski
et al., 2008). Taking into account sequence homology and
gm
22
mol water m
s
21
0.145 6 0.009 (33)
0.115 6 0.007 (22)*
0.118 6 0.008 (22)*
mol CO2 m
E
22
s
21
0.035 6 0.002 (33)
0.028 6 0.002 (22)*
0.031 6 0.001 (22)
mmol water m22 s21
2.085 6 0.1 (33)
1.716 6 0.09 (22)*
1.755 6 0.095 (22)*
empirical parameters for amiRNA (Schwab et al., 2005),
we constructed an amiRNA sequence that targets all
members of the AtPIP1 subfamily with an acceptable
level of efficiency (Fig. 1A).
Bioinformatic analysis (weigelworld.com) suggested
that some PIP2 AQP genes (AtPIP2;1, AtPIP2;2, AtPIP2;3,
and AtPIP2;7) might serve as possible targets for our
amiRNApip1 (Supplemental Fig. S6). Nevertheless, in
contrast to the steady and constitutive down-regulation
of PIP1 isoforms, PIP2 down-regulation varied between
tissues, conditions, and genotypes (Figs. 2, 5, and 7;
Supplemental Fig. S2), suggesting that PIP2 genes are
most likely not targeted by the amiRNA. This might be
related to the fact that these putative targets have
somewhat higher rates of sequence mismatch (four to
five mismatches; Supplemental Fig. S6). From the physiological point of view, the fact that PIP2 genes were
down-regulated does not interfere with the notion that
AQPs (either PIP1s or others) can determine the membrane Pf and Kleaf.
Whole-Plant Constitutive Silencing of PIP AQPs
The reduction in Pf observed in the constitutively
silenced plants (Fig. 3) was expected, as decreased
mesophyll Pf has been observed previously in AQP
knockout and antisense lines (Martre et al., 2002; Postaire
et al., 2010). The role of PIPs in controlling Kleaf and radial
influx, in particular, is an interesting question. Research
in this area has shown that PIP1 and PIP2 antisense lines
do not exhibit reduced leaf and plant hydraulic conductivity under normal moisture conditions. However,
the lack of functional PIPs results in the significantly
slower recovery of plant hydraulic conductivity following exposure to drought (Martre et al., 2002; Secchi and
Zwieniecki, 2014). Additional studies have described the
Figure 7. Relative expression profile of the PIP
genes in the midveins of SCR:mir1 leaves. Columns indicate the mean 6 SE expression levels
(q-RT-PCR analysis) of all PIP transcript levels in
SCR:mir1 and control leaves. The data presented
were normalized to the control. Asterisks indicate
significant differences between a treatment and
the control based on the raw transcript levels
(Student’s t test, P , 0.05; n = 7–10 independent
biological repetitions). Data were normalized to
ACTIN2 levels.
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PIP Aquaporins Regulate Leaf Hydraulics
Figure 8. Silencing the BS AtPIP1 AQP subfamily decreased the Pf of the
membranes of the BS and the mesophyll cells. Columns indicate the
mean 6 SE Pf of BS protoplasts (A) and mesophyll protoplasts (B) from SCR:
mir1 (gray bars, n = 32 and n = 24, respectively) and control protoplasts
(black bars, n = 25). Asterisks indicate significant differences between a
treatment mean and that of the control (Student’s t test, P , 0.05).
Sade et al., 2014). It has been suggested that AtPIP1;2
may transport CO2 and regulate mesophyll CO2 conductance (Heckwolf et al., 2011; Uehlein et al., 2012) in
addition to its ability to control a cell’s hydraulic conductivity (Postaire et al., 2010). Those observations of
PIP1s affecting both hydraulic conductivity and mesophyll CO2 conductance are not restricted to Arabidopsis;
similar effects have also been observed in tobacco (Nicotiana
tabacum) and poplar (Populus tremuloides; Siefritz et al., 2002;
Flexas et al., 2006; Secchi and Zwieniecki, 2013, 2014).
Indeed, gas-exchange measurements of our 35S:mir1
silenced lines, taken under saturated light, revealed reduced photosynthesis and reduced gm (in 35S:mir1-8), gs,
and E (in both 35S:mir1-8 and 35Smir1-3) in the silenced
plants as compared with the wild type (Table I). Recently,
Atpip1;2 mutants were reported to have lower Kros
(Postaire et al., 2010), less photosynthesis, and lower gm
(Heckwolf et al., 2011). However, in both studies
(Postaire et al., 2010; Heckwolf et al., 2011), the effect
was restricted to one substance (water or CO2), whereas
we have shown that both water and CO2 are affected in
role of Arabidopsis PIP1s and PIP2s in maintaining Kros
under extended-night conditions (Postaire et al., 2010; Prado
et al., 2013). Recently, PIPs in walnut (Juglans nigra) were
shown to control stem hydraulic conductivity (Steppe et al.,
2012).
Our data confirm these findings, with a significant
reduction in Pf as well as Kros and Kleaf in 35S:mir1 plants
(Figs. 3, 4, and 6). The reduction in Kros might be the result
of the reduced Pf in the living cells of the vascular system,
including the BS cells, as demonstrated recently by ShatilCohen et al. (2011), Pantin et al. (2013), and Prado et al.
(2013). Thus, our reverse-genetic approach effectively reduced PIP expression and cellular activity, and as a result,
leaf tissue hydraulic conductivity decreased. Moreover,
using the detached-leaf method, we were able to measure
leaf water efflux (E) and, separately, estimate the ratio
between influx and outflux through Cleaf. These measurements revealed that the reduction in Kleaf was
reflected by a decreased Cleaf and was not due to a decrease in E (i.e. a decrease in water influx but not gs;
Figure 6, B and C). A Cleaf reduction with no significant
change in transpiration was also observed in intact leaves
(from plants grown in soil, as opposed to excised leaves
with submerged petioles and an unlimited water supply;
Supplemental Fig. S7).
These results support the hypothesized role of BS AQPs
in regulating the movement of water into BS cells and Kleaf
(or xylem efflux), as suggested by the hydraulic gs feedback
theory (i.e. down-regulation of AQPs reduces Kleaf, which
reduces Cleaf and serves as a feed-forward signal for stomatal closure; Shatil-Cohen et al., 2011; Pantin et al., 2013).
The Role of PIP1 AQPs in Controlling
Gas-Exchange Parameters
Recent studies have pointed to the ability of PIP1s to
conduct CO2 as well as water (Uehlein et al., 2003, 2008;
Figure 9. Kleaf was lower in the BS-silenced AtPIP1 leaves. Leaves from
the SCR:mir1 and the control lines were harvested and immersed in
AXS. After 1 h, Kleaf was calculated for each individual leaf (A) by
dividing E (B) by the Cleaf (C). Asterisks indicate significant differences
between lines (Student’s t test, P , 0.001). Data are means 6 SE of
values from 18 to 31 replicates from three independent experiments.
Plant Physiol. Vol. 166, 2014
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Sade et al.
Table II. Gas-exchange characteristics of SCR:mir1 and control plants (determined at 1,200 mmol m22 s21, approximately 25˚C, and 400 mmol
mol21 CO2, as described by Flexas et al., 2007)
Data are given as means 6
Plant
Control (n)
SCR:mir1 (n)
SE
(n, number of independent biological repetitions). AN, Photosynthesis; Ci, substomatal CO2 concentration.
AN
Ci
gs
gm
E
mmol CO2 m22 s21
mmol CO2 mol21
mol water m22 s21
mol CO2 m22 s21
mmol water m22 s21
5 6 0.35 (33)
4.69 6 0.21 (38)
317.22 6 2.56 (33)
317.51 6 2.84 (38)
our 35S:mir1 plants (Table I). The fact that SCR:mir1
lines exhibited no reduction in any of the gas-exchange
parameters as compared with the control plants (Table
II), yet did exhibit reduced mesophyll Pf, suggest the
possibility that a BS-mesophyll hydraulic signal controls Kleaf, yet further research is needed to elucidate this
signal.
BS-Mesophyll Hydraulic Feed-Forward Signal
Down-regulation of BS AQPs (SCR:mir1 plants) was
found to be effective at the transcript level (Fig. 7) and
at the functional level, as demonstrated by the lower Pf
of the silenced BS cells of those plants (Fig. 8A). Unexpectedly, the mesophyll Pf of those plants was reduced as well (Fig. 8B), which raises questions about
how it is controlled.
We excluded the possibility of SCR promoter activity in the mesophyll, as this promoter is known to be
expressed specifically in the BS and not at all in the
mesophyll (Wysocka-Diller et al., 2000; Shatil-Cohen
et al., 2011; Sade et al., 2014). In addition, it is not
likely that the silencing agent moves to the mesophyll
because (1) miRNA is known to be cell autonomous
(Alvarez et al., 2006), (2) the relative expression of the
silencing agent was 5 times higher in the midribs than
along the leaf margins (Supplemental Fig. S8), and (3)
unlike the effectiveness of the miRNA agent against
PIP1 members in midvein tissue (Figs. 5 and 7), here
the relative expression of three of the five PIP1s in the
leaf margin area (i.e. enriched mesophyll) was similar
to that of the control (Supplemental Fig. S9). Since the
silencing of the miRNA agent in a cell should not vary
between the different PIPs, we ruled out the possibility
of its presence in the mesophyll cells.
In fact, SCR:mir plants showed mild stress symptoms, in the form of reductions in their Kleaf, Cleaf, and
BS and mesophyll Pf, but they did not exhibit other
stress symptoms of reduced transpiration, gm, or reduced photosynthesis (nor did they exhibit increased
ABA biosynthetic gene transcript levels; Supplemental
Fig. S5). And unlike the 35S:mir1 plants, the SCR:mir
plants were not smaller than the wild-type plants. In
fact, they were a bit larger (Supplemental Fig. S5).
This physiological phenotype suggests that the Pf
and Kleaf are very sensitive and the first to change once
conditions become less than optimal (before any reduction in gs or increase in ABA levels). This raises a
question regarding the hierarchy of the hydraulic signal
0.143 6 0.014 (33)
0.125 6 0.007 (38)
0.022 6 0.002 (33)
0.02 6 0.001 (38)
2.31 6 0.17 (33)
2.1 6 0.1 (38)
cascade controlling leaf water status (for review, see
Chaumont and Tyerman, 2014).
Previous studies addressing this question suggested
the existence of a generalized sensitivity cascade of
a plant’s physiological responses to water stress. Accordingly, the first symptoms to appear at the onset of
stress are turgor loss and the inhibition of cell growth,
followed by gradual increases in ABA levels, the initiation of stomatal closure, and a reduction in the rate
of photosynthesis (for review, see Hsiao and Acevedo,
1974; Taiz and Zeiger, 2010).
Our results in SCR:mir plants suggest that the imposed reduction in Kleaf might serve as a hydraulic
feed-forward signal that regulates the mesophyll Pf
prior to the appearance of secondary and more severe
signals, such as increased ABA levels and reduced gs.
Two more pieces of information support this hydraulic
stress signal cascade hypothesis. First, almost no reduction in the expression levels of the PIP2s was
measured in the SCRmir1 mesophyll. Reduced expression of PIP2s under stress is a well-known symptom of stress (Jang et al., 2004; Alexandersson et al.,
2005; Boursiac et al., 2005). Second, gm, which was
reduced under the constitutive 35s:mir promoter
(probably due to the reduced expression of PIP1 AQPs;
Uehlein et al., 2003, 2008; Flexas et al., 2006; Heckwolf
et al., 2011; Sade et al., 2014), was not affected by the
expression of SCR:mir1 (Tables I and II). The fact that BS
cells are tightly linked with the mesophyll via many
plasmodesmata connections (Ache et al., 2010) serves as
additional evidence supporting the existence of the hypothesized BS-mesophyll hydraulic regulation signal.
These results as well as the literature concerning
the generalized sensitivity hierarchy cascade of plant
physiological characteristics to water stress led us to
the following hypothetical model of action: plants exhibit a rapid decrease in the turgor of their leaf cells in
response to a root-evoked drop in Cw (Christmann
et al., 2013). Under water stress, local changes in
Cw are quickly relayed throughout the plant xylem,
thanks to the cohesion and tension properties of water.
A turgor decrease below the plant’s specific threshold
value in the leaf lamina leads to increased ABA production (Liu et al., 1978; Pierce and Raschke, 1981; Lee
et al., 2006; Tuteja, 2007; Ache et al., 2010), probably at
its synthesis sites in shoot vascular parenchyma tissues
and guard cells (Boursiac et al., 2013). The increase in
the concentration of ABA in the xylem leads to reduced Cleaf and Kleaf in the BS, possibly due to reduced
AQP expression and/or activity (Martre et al., 2002;
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PIP Aquaporins Regulate Leaf Hydraulics
Shatil-Cohen et al., 2011; Pantin et al., 2013; for review,
see Chaumont and Tyerman, 2014). Under these conditions, decreases in AQP expression or activity in the
BS should result in reduced water influx and decreased
Cleaf. Mesophyll Pf also might play a role in this reduction of Kleaf, thus suggesting that the BS-mesophyll
cells form a continuous hydraulic pathway controlling
the leaf hydraulic properties. In this scenario, the BS acts
as a gatekeeper, regulating the rate at which water
flows into the leaf, and the mesophyll tissue controls the
flow of water out of the leaf.
CONCLUSION
For years, the extravascular flow of water through the
plant has been considered to be passively driven by
transpiration and limited by the vascular structure.
While this still holds true for the axial movement of
water through dead xylem vessels, several recent studies
have suggested that radial unloading of water from the
xylem to the leaf is tightly regulated by the membranes
of the cells that surround the xylem. Our results support
the hypothesized role of PIP AQPs in this process. Thus,
Kleaf is regulated via PIP AQPs in BS cells. Moreover, this
control of BS AQP activity and thereby Kleaf can explain
the dynamic Kleaf response to changing ambient conditions, including stress, or via changes in the turgor of the
BS (e.g. in response to light or temperature conditions;
Brodribb and Holbrook, 2006; Sack and Holbrook, 2006;
Shatil Cohen et al., 2011). The fact that BS hydraulics
control the permeability of the mesophyll to water under initial stress conditions suggests the existence of a
tight hydraulic connection between the BS and the mesophyll as well as feed-forward regulation. Further research is needed to elucidate the interaction between the
BS and the mesophyll with respect to their hydraulic
functions and the specific roles of stress and AQP in this
interaction. In addition, it would be interesting to use the
miRNA approach to target the PIP2 subfamily and the
entire PIP family.
behind the 35S promoter (ART7), transferred into the binary pMLBART vector
(Eshed et al.., 2001), and transformed to Arabidopsis (Arabidopsis thaliana) ecotype
Columbia using the floral dip method (Clough and Bent, 1998).
SCR:mir1 Plants
Arabidopsis (ecotype Landsberg) plants were used in the LHG4-10OP twocomponent system, in which the synthetic transcription factor LhG4 was
expressed under the control of the promoter of interest. Transactivation lines
were generated by transcriptional fusion of the SCR promoter (BS-specific
promoter) in front of the chimeric LhG4, and endoplasmic reticulum-GFP was
subcloned behind an operator array in the BJ36 vector (Moore et al., 1998).
(The SCR:LhG4 line and the 10OP:endoplasmic reticulum-GFP line were
generous gifts from Yuval Eshed, Weizmann Institute of Science [Shatil-Cohen
et al., 2011].) Those lines were crossed to generate plants in which GFP was
expressed specifically in the endodermis and BS cells (Shatil-Cohen et al.,
2011). Those plants were further crossed with 10OP:mir1 lines (Columbia
background) in which mir1 was specifically expressed under the control of the
SCR promoter (SCR:mir1 plants). For control plants, a similar cross was made
with wild-type Columbia plants. All Arabidopsis plants were genetically
transformed using the floral dip method (Clough and Bent, 1998).
Protoplast Isolation
Protoplasts were isolated from 30- to 35-d-old plants. The lower leaf epidermis
was peeled off at the leaf center (when extracting the midrib BS cells, the epidermis
right above those cells was peeled off), and the peeled leaves were cut into small
squares and incubated in enzyme solution (3.3% [w/w] of an enzyme mix containing the following enzymes in the given proportions: 0.55 g of cellulase
[Worthington], 0.1 g of pectolyase [Karlan], 0.33 g of polyvinyl pyrrolidone K 30
[Sigma-Aldrich], and 0.33 g of bovine serum albumin [Sigma-Aldrich]) containing
10 mM KCl, 1 mM CaCl2, 540 mM D-sorbitol, and 8 mM MES, pH 5.7. After 20 min
of incubation at 28°C, the leaf tissue was transferred to the same solution, without
the enzymes, and agitated at 100 rpm for 5 min or until all of the protoplasts were
released into the solution. The remaining tissue pieces were removed, and the
remaining solution containing the protoplasts was collected into a 1.5-mL tube
using a cut tip. This protoplast isolation procedure resulted in a very high yield of
protoplasts (20 million protoplasts per gram of leaf tissue). For detailed description (video article) of the protoplast isolation and Pf measurement, please see
Shatil-Cohen et al. (2014).
Measurements of the Protoplast Pf
Pf was measured in single protoplasts based on the initial (recorded) rate of
their increase in volume in response to hypoosmotic challenge (transfer from a
600-mosmol isotonic bath solution to a 500-mosmol hypotonic solution). Pf was
determined using a numerical approach, an offline curve-fitting procedure using
the PFFIT program, as described in detail previously (Moshelion et al., 2002,
2004; Volkovet al., 2007). To identify the BS protoplasts labeled with GFP and the
unlabeled mesophyll cells, we screened the protoplast population as described
by Shatil-Cohen et al. (2011).
MATERIALS AND METHODS
Whole-Rosette Kros
Growth Conditions
Hydrostatic Kros was measured on 21-d-old plants as described by Postaire
et al. (2010) by inserting an excised rosette into a pressure chamber containing
a bathing solution. A flow of liquid water was pressed across the whole rosette
and was monitored as it was released at the hypocotyl section. The flow-versuspressure relationship was used to determine Kros (Supplemental Fig. S10), which
was shown previously to be independent of any stomatal limitation (Postaire
et al., 2010).
All plants were grown in potting mix containing 30% (w/w) vermiculite,
30% (w/w) peat, 20% (w/w) tuff, and 20% (w/w) perlite (Shacham). The plants
were kept in a growth chamber under long-day conditions (14 h of light) at a
controlled temperature of 20°C to 22°C with 70% humidity and light intensity
of 75 to 150 mmol m22 s21. For Kros measurements, plants were grown in
hydroponic solution for 21 d and transferred under extended-night conditions,
prior to the measurements, as described (Postaire et al., 2010).
Plant Material
35S:mir1 Plants
The premiR-PIP1 and synthetic genes were synthesized by DNA 2.0, based on a
premiR164 backbone (Alvarez et al., 2006). We used the Web-based mfold program (http://mfold.rna.albany.edu) to produce premiRNA stem-loop representations (Zuker, 2003). After sequence verification, the premiR-PIP1 was cloned
Measurements of Kleaf
Kleaf was measured using detached leaves (Shatil-Cohen et al., 2011). Fully
expanded leaves were harvested from 32- to 45-d-old Arabidopsis plants in
the dark and immediately immersed (petiole deep) in artificial xylem sap
(AXS; containing 1 mM KH2PO4, 1 mM K2HPO4, 1 mM CaCl2, 0.1 mM MgSO4, 3 mM
KNO3, and 0.1 mM MnSO4 buffered to pH 5.8 with 1 M HCl or KOH; Wilkinson
et al., 1998) while leaf blades were exposed to air. All of the leaves were similar in
size and had similar vascular areas with no noticeable anomalies (Supplemental
Fig. S3).
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E was measured using a Li-Cor 6400 gas-exchange system (LI-COR)
equipped with a 2- 3 3-cm2 aperture standard leaf cuvette (150 mmol m22 s21
light with 400 mmol mol21 CO2 surrounding the leaf [Ca], the leaf temperature
was approximately 24°C, and the vapor pressure deficit was approximately 1.3
kPa). The measuring conditions were set to be similar to the growth chamber
conditions. The measurement of E was followed immediately by the determination of Cleaf using a pressure chamber (ARIMAD-3000; MRC) and a homemade silicon adaptor especially designed for Arabidopsis petioles in order to fit
the O-ring of the pressure chamber. After placing the leaf in the chamber and
tightly closing it, pressure (N2) was gradually applied until the appearance of
water at the cut was observed with the help of a binocular microscope (SZ;
Olympus) under the illumination of a cool light (Intralux 5000; Volpi) directed
toward the petiole. Kleaf, E/Dry weight leaf (Martre et al., 2002; Sack and Holbrook,
2006) was calculated for each individual leaf by dividing the whole-leaf E by the
Cleaf. (In our calculations, Cleaf = ΔCleaf, as the leaf petiole was dipped in AXS at a
water potential of approximately 0.) For the ABA experiment, petioles were left in
AXS supplemented with 10 mM ABA for 1 h before measurement.
Gas-Exchange Measurements
Gas-exchange measurements were assayed using a Li-Cor 6400 portable
gas-exchange system (LI-COR). Analysis was performed on fully expanded
leaves from 32- to 45-d-old plants grown under favorable conditions. All
measurements were conducted between 10 AM and 1 PM.
Photosynthesis was induced under saturating light (1,200 mmol m22 s21)
with 400 mmol mol21 CO2 surrounding the leaf (Ca), as described by Flexas
et al. (2007). In addition, light curves were measured on wild-type plants
(Supplemental Fig. S11). The amount of blue light was set to 10% of the
photosynthetically active photon flux density to optimize stomatal aperture.
Vapor pressure deficit was approximately 1.3 kPa, and the temperature was
approximately 24°C. Chlorophyll fluorescence was measured using an open
gas-exchange system with an integrated fluorescence chamber head (Li-Cor
6400; LI-COR). The actual photochemical efficiency of PSII was determined by
measuring steady-state fluorescence and maximum fluorescence during a
light-saturating pulse of approximately 8,000 mmol m22 s21, as described (Genty
et al., 1989).
Measurements of gm
gm was calculated according to Harley et al. (1992), as described by RibasCarbo et al. (2007), based on coupled gas exchange and chlorophyll fluorescence measurements.
Quantitative Analysis of Gene Expression by
Quantitative PCR
Vascular tissue is found throughout the leaf. Therefore, in order to have a
relatively high number of BS cells in our tissue samples, we excised the
midvein area (Hibberd and Quick, 2002; Brown et al., 2010), which has more
BS cells than mesophyll cells, and used that excised tissue for our analysis.
(Pieces from the edges of the leaves had more mesophyll cells than BS cells.)
Tissue samples were taken from each plant and immediately frozen in liquid
nitrogen.
Total RNA was extracted using Tri-Reagent (Molecular Research Center)
and treated with RNase-free DNase (Fermentas). Complementary DNA
(cDNA) was prepared using the EZ-First Strand cDNA synthesis kit (Biological
Industries) according to the manufacturer’s instructions. Quantitative PCR was
performed in the presence of SYBR Green I (Takara) in a Corbett Research RotorGene 6000 cycler. The reaction was as follows: 30 s at 94°C, followed by 40 cycles
consisting of 10 s at 94°C, 30 s at 60°C, and 20 s at 72°C. A standard curve was
generated for each gene using dilutions of cDNA samples, and data analysis was
performed using Rotor-Gene 6000 series software 1.7. PCR primers used to
amplify specific regions of the genome are listed in Supplemental Table S1. The
specificity of primers was determined by melting-curve analysis; a single, sharp
peak in the melting curve indicates that a single, specific DNA species was
amplified. Arabidopsis ACTIN2 (AT3G18780; Alexandersson et al., 2005) was
used as a reference for the standardization of cDNA amounts.
according to size on a 1% (w/v) denaturing agarose-formaldehyde gel and
transferred to a nylon membrane by capillary blotting. The nucleic acids were
UV light cross linked and prehybridized in DIG Easy Hyb (Roche). Hybridization
with digoxigenin (DIG)-labeled probes and washing were carried out at 42°C (very
stringently) according to the manufacturer’s instructions. The DIG system includes
an anti-DIG antibody that is linked to alkaline phosphatase. Target AQP mRNA
was detected via this enzymatic activity. DIG-labeled probes for specific AQPs
were obtained by using q-RT-PCR primers to amplify the respective sequence of
the AQP.
Western Blotting
About 100 mg of fresh leaf tissue was ground to homogeneity in a 1.5-mL
tube with a piston in a grinding buffer of 500 mM Suc, 10% (v/v) glycerol, 50 mM
NaF, 20 mM EDTA, 20 mM EGTA, 5 mM b-glycerophosphate, 1 mM phenanthroline, 0.6% (w/v) polyvinylpyrrolidone, 10 mM ascorbic acid, 0.5 mg mL21
leupeptin, 1 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, 1 mM
sodium vanadate, and 50 mM Tris-HCl, pH 8. After centrifugation (5 min at
10,000g), the supernatant was subjected to SDS-PAGE on 12% (v/v) acrylamide
gels. After SDS-PAGE, separated proteins were transferred to a polyvinylidene
difluoride membrane (Immobilon, Millipore) according to the manufacturer’s
instructions.
The blot was blocked for 1 h in a modified phosphate-buffered saline
containing 0.1% (v/v) Tween 20 and 1% (w/v) bovine serum albumin (PBSTB)
and then incubated at room temperature (2 h) or at 4°C (overnight) in the
presence of the primary antibodies, anti-PIP1 (AtPIP1;1–AtPIP1;4) and antiPIP2 (AtPIP2;1–AtPIP2;3), diluted 1:5,000 (Santoni et al., 2003). After washing
(2 3 10 min) in PBSTB, the blot was incubated for 1 h with a peroxidaselabeled secondary antibody at 1:20,000 dilution in PBSTB. Secondary antirabbit antibodies were used to detect the anti-PIP1 and anti-PIP2 primary
antibodies. After washing (2 3 10 min) in phosphate-buffered saline, the signal
was observed using a chemiluminescent substrate (Super Signal; Pierce).
RLM-RACE
RACE analysis of cleaved target-gene cleavage sites in the miRNA target
genes was mapped using RLM-RACE, a modified 59 RACE procedure, as
described (Kasschau et al., 2003), using the GeneRacer (Invitrogen) protocol
coupled with nested gene-specific primers 200 to 400 nucleotides downstream
of the predicted miRNA target site. The PCR products were purified and sequenced directly.
Leaf-Clearing Procedure
Leaves were harvested, immersed in 96% (v/v) ethanol, and incubated at
50°C for 3 h. The ethanol was replaced with fresh ethanol three times and then
replaced with lactic acid for an overnight incubation.
Calcofluor White Staining and Imaging
Leaves were cut and immediately immersed (petiole deep) in concentrated
(1 g L21) Calcofluor White, an apoplastic fluorescent marker that stains the
cellulose in cell walls. Twenty-four hours after the staining, fluorescence was
imaged by epifluorescence inverted microscopy (Olympus-IX8 Cell-R; 380-nm
excitation, 475-nm emission) to observe the distribution of the dye in the leaf.
Imaging Leaf Veins and Calculating Their Areas
Following the clearing procedure, leaves were washed with water and put
on microscope slides. Images were taken using NIS-Elements software and a
Leica MZFLIII stereomicroscope on which a Nikon DS-Fi1 digital camera was
mounted. Images were later analyzed to determine leaf vein area using ImageJ
software (http://rsb.info.nih.gov/ij/). A fixed ellipse (6,000-mm length, 3,000-mm
width) was used in all images, and the area of the veins was determined and
calculated relative to the total (ellipse) leaf area.
Supplemental Data
Northern-Blot Analysis
The following materials are available in the online version of this article.
Total RNA was extracted from Arabidopsis leaves using Tri-Reagent
(Molecular Research Center). Ten micrograms of total RNA was separated
Supplemental Figure S1. Northern-blot and RLM-RACE analyses of
AtPIP1 AQP in 35S:mir1 plants as compared with the wild type.
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PIP Aquaporins Regulate Leaf Hydraulics
Supplemental Figure S2. The quantities of PIP1 and PIP2 proteins were
lower in the 35S:mir1 plants.
Supplemental Figure S3. Comparison of the leaf morphology of the different genotypes.
Supplemental Figure S4. E and Cleaf of intact leaves from soil-grown, BSsilenced AtPIP1 plants.
Supplemental Figure S5. Plant morphological characteristics and relative
expression profiles of ABA biosynthetic gene transcript levels (9-cisepoxycarotenoid dioxygenase) in leaves of the SCRmir1 and control
plants.
Supplemental Figure S6. Twenty-one base pairs of the consensus sequence
alignment of amiRNApip1 and some possible targets from the AtPIP2
subfamily.
Supplemental Figure S7. E and Cleaf of intact leaves from the AQP-modified
lines.
Supplemental Figure S8. Relative expression profile of the miRNA
(mir164) in the margins (mesophyll-enriched tissue) and midveins of
SCR:mir1 leaves.
Supplemental Figure S9. Relative expression profile of the PIP genes in the
margins (mesophyll-enriched tissue) of SCR:mir1 leaves.
Supplemental Figure S10. Representative pressure-to-flow relationship
measured in a rosette from an approximately 20-d-old 35S:mir1-3 plant
exposed to prolonged night (11–21 h of darkness).
Supplemental Figure S11. Relationship between net photosynthesis, photosynthesis, and incident light intensity, photon flux density.
Supplemental Table S1. Primers used for q-RT-PCR gene expression analyses.
Received August 13, 2014; accepted September 24, 2014; published September
29, 2014.
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