Abstract Water movement across root tissues occursby parallel apoplastic, symplastic, and transcellular

pathways that the plant can control to a certain extent.

Because water channels or aquaporins (AQPs) play an

important role in regulating water flow, studies on

AQP mRNA and protein expression in different root

tissues are essential. Here, we quantified and localized

the expression of Zea mays plasma membrane AQPs

(ZmPIPs) in primary root tip using in situ and quan-

titative RT-PCR and immunodetection approaches.

All ZmPIP genes except ZmPIP2;7 were expressed in

primary roots. Expression was found to be dependent

on the developmental stage of the root, with, in gen-

eral, an increase in expression towards the elongation

and mature zones. Two genes, ZmPIP1;5 and

ZmPIP2;5, showed the greatest increase in expression

(up to 11- and 17-fold, respectively) in the mature

zone, where they accounted for 50% of the total ex-

pressed ZmPIPs. The immunocytochemical localiza-

tion of ZmPIP2;1 and ZmPIP2;5 in the exodermis and

endodermis indicated that they are involved in root

radial water movement. In addition, we detected a

polar localization of ZmPIP2;5 to the external pericli-

nal side of epidermal cells in root apices, suggesting an

important role in water uptake from the root surface.

Finally, protoplast swelling assays showed that root

cells display a variable, but globally low, osmotic water

permeability coefficient (Pf < 10 lm/s). However, thepresence of a population of cells with a higher Pf (up to

26 lm/s) in mature zone of the root might be corre-lated with the increased expression of several ZmPIP

genes.

Keywords Aquaporin Æ PIP Æ mRNA and proteinexpression Æ Root water movement

Abbreviations

ABA abscisic acid

AQP aquaporin

Lpr root hydraulic conductivity

Pf osmotic water permeability coefficient

RT-PCR reverse transcription-PCR

ZmPIP Zea mays plasma membrane intrinsic

protein

Introduction

To move from the soil into the root vascular tissues,

water must flow radially across a series of concentric

cell layers, including the epidermis, the exodermis

(when present), one or several layers of cortex cells,

the endodermis, the pericycle, the xylem parenchyma

cells, and, finally, the vessels. This anatomical structure

of roots results in a complex pattern of water flow.

After reaching the vascular bundles, water moves

C. Hachez Æ M. Moshelion Æ E. Zelazny Æ D. Cavez ÆF. Chaumont (&)Unité de Biochimie physiologique, Institut des Sciences dela Vie, Université catholique de Louvain, Croix du Sud 5-15B-1348 Louvain-la-Neuve, Belgiume-mail: chaumont@fysa.ucl.ac.be

M. MoshelionThe Robert H. Smith Institute of Plant Sciences andGenetics in Agriculture, Faculty of Agricultural, Food &Environmental Quality Science, The Hebrew UniversityRehovot 76100, Israel

Plant Mol Biol (2006) 62:305–323

DOI 10.1007/s11103-006-9022-1

123

Localization and quantification of plasma membrane aquaporinexpression in maize primary root: a clue to understanding theirrole as cellular plumbers

Charles Hachez Æ Menachem Moshelion ÆEnric Zelazny Æ Damien Cavez Æ François Chaumont

Received: 7 March 2006 / Accepted: 22 May 2006 / Published online: 15 July 2006� Springer Science+Business Media B.V. 2006

through the entire plant by the long path (root-stem-

leaf), which is characterized by a low resistance to

water movement. The radial transfer through the root

can be explained by the ‘‘composite transport model’’,

which includes the contributions of three different

pathways (Steudle 2000). The first is the apoplastic

pathway, referring to the flow around protoplasts. The

second is the symplastic pathway from cell to cell via

plasmodesmata. The third is called the transcellular

pathway, in which water and solutes have to cross cell

membranes. The transcellular pathway is important for

water, but of minor importance for solutes. The sym-

plastic and transcellular pathways cannot be experi-

mentally separated and are considered as a cell-to-cell

path. This model including a combination of three

different parallel pathways best describes the radial

flow of water along the apoplast with its barriers.

Depending on the conditions, the relative contribution

of these pathways to overall uptake or hydraulic con-

ductivity may change substantially. Thus, the radial,

rather than the axial, component of water transport

limits water uptake by roots (Steudle 2001).

Different zones corresponding to different matura-

tion levels can be distinguished in the primary maize

root. The first region, at the tip of the root, is the cap,

which is made up of cells originating from the calyp-

trogen. Above this is the apical meristem region,

responsible for the production of new root cells. This

region shows a high mitotic activity, except in the qui-

escent center, which lacks such activity (Ishikawa and

Evans 1995). The region of cell division extends for a

few millimeters behind the apical meristem and it is here

that cells start to undergo elongation and enlargement

(Ishikawa and Evans 1995). In the elongation zone, cells

enlarge and develop as specific cell types according to

their position in the root. Cells that leave the meriste-

matic zone elongate rapidly and their volume can in-

crease up to 40-fold during their development (Moore

and Smith 1990). Frensch et al. (1996) reported that,

during development, the root tissues in the division and

differentiation zones become less permeable for trans-

membrane water and solute movement.

Roots must take up water and necessary solutes from

the soil into the stele, while avoiding the influx of

unnecessary solutes. Since roots manage to fulfill these

contradictory requirements, the internal tissues

responsible for these selectivity functions must be pre-

cisely regulated in a manner depending on growth

conditions (Karahara et al. 2004). The development of

Casparian strips by the hypodermis and endodermis,

which cut off apoplastic water and solute flow, occurs

along the root axis. While the suberization of the

endodermis surrounding the stele occurs before the

maturation of the protoxylem and the development of

root hairs (Raven et al. 1992), formation of the exo-

dermis, defined as suberized hypodermis, is highly

variable and highly dependent on growth conditions.

As an example, hydroponically grown maize seedlings

usually do not develop an exodermis, whereas aerop-

onically grown ones develop a suberized layer approx-

imately 0.5–1 cm after the region in which

differentiation of the endodermis occurs (Enstone and

Peterson 2005; Hose et al. 2001). These observations

reflect a mean of protection against excessive water loss

due to aeration (Hose et al. 2001). The distance from

the root tip to the lowest position of the exodermal

Casparian strip decreases in maize roots under osmotic

stress (Perumalla 1986). However, the mechanism

responsible for Casparian strip development closer to

the root tip remains to be fully characterized (Karahara

et al. 2004). In addition, the apoplastic barriers may not

be completely impermeable to water and solutes, as

bypasses have been demonstrated for both the exo-

dermis and endodermis (Schraut et al. 2004).

For a given root size and anatomy, plants also have

the ability to alter their root hydraulic conductivity (Lpr)

in less than 3 days (Javot and Maurel 2002). This fine

and rapid regulation is assumed to be under control by

internal and external stimuli, such as the day/night cycle,

nutrient deficiency, water shortage, soil salinity, water

demand of the transpiring shoot, anoxia, temperature,

heavy metals, and abscissic acid (ABA) (Lopez et al.

2003; Steudle 2000). Depending on the species and

conditions, water conductivity can change rapidly by up

to three orders of magnitude (Steudle 2001). These

modifications are due to changes in cell membrane

permeability triggered by the expression and specific

regulation of aquaporins (AQPs), which facilitate the

movement of water or other small solutes through the

membrane. These proteins are ubiquitous in plants and

have been detected in high amounts in regions of high

transcellular water transport, such as the endodermis

(Otto and Kaldenhoff 2000; Schaffner 1998).

AQPs are seen as ‘‘cellular plumbers’’ regulating the

movement of water across membranes. A significant

proportion of root water transport (20–80%) is under

rapid metabolic control involving AQPs (Maurel and

Chrispeels 2001). When present and active, they can

increase the cell membrane osmotic water permeability

coefficient (Pf) by up to 20-fold. AQPs are abundantly

expressed in roots, where they mediate soil water up-

take (Alexandersson et al. 2005; Boursiac et al. 2005;

Jang et al. 2004; Javot and Maurel 2002). The contri-

bution of AQPs to root water transport can be assessed

by the use of AQP inhibitors, such as mercurial

compounds. For instance, mercurials reduce the Lpr by

306 Plant Mol Biol (2006) 62:305–323

123

32–90% in Buckhorn cholla cactus, tomato, wheat,

paprika pepper, melon, sugar beet, and barley (Amo-

deo et al. 1999; Carvajal et al. 2000; 1996; 1999; Maggio

and Joly 1995; Martre et al. 2001; Tazawa et al. 1997).

Hukin et al. (2002) also showed a rapid 4-fold decrease

in maize root elongation rate (from 1.83 mm h–1 to

under 0.4 mm h–1) after mercurial treatment, suggest-

ing a central role of AQPs in this process.

Thirteen plasma membrane AQPs (PIPs), split into

two groups (PIP1 and PIP2), are expressed in maize

(Chaumont et al. 2001). Functional assays in Xenopus

oocytes indicated that all ZmPIP2s tested so far

(ZmPIP2;1, ZmPIP2;4, ZmPIP2;5) have a high water

channel activity whereas ZmPIP1s (ZmPIP1;1;

ZmPIP1;2 and ZmPIP1;5) have no or low activity

(Chaumont et al. 2000; Fetter et al. 2004; Gaspar

et al. 2003). However, when co-expressed, ZmPIP1;2

and ZmPIP2 isoforms physically interact to enhance

both ZmPIP1;2 targeting to the plasma membrane

and membrane osmotic water permeability (Fetter

et al. 2004). Transcripts of all ZmPIP genes except

ZmPIP2;7 have been detected in the primary maize

root (Aroca et al. 2005; Zhu et al. 2005). Moreover,

the expression of several ZmPIPs is regulated by

chilling and salt stress, ABA and diurnal cycle

(Aroca et al. 2005; Lopez et al. 2003; Zhu et al. 2005).

These findings indicate that specific AQP isoform

can be regulated differently and it is therefore

essential to have a comprehensive analysis of the

expression pattern of each AQP gene in maize root

tissues or cells.

To obtain a better understanding of PIP function in

the primary root, we quantified and localized AQP

mRNA and protein expression using in situ and

quantitative RT-PCR approaches, Western blots, and

immunocytochemistry. We found that most ZmPIP

genes were expressed in the roots and their expression

was developmentally regulated. The high expression of

ZmPIP2;1 and ZmPIP2;5 in the exodermis and endo-

dermis indicates an essential role of these channels in

facilitating water movement through these critical cell

layers surrounded by Casparian strip.

Materials and methods

Plant material

Zea mays (B73 line) seeds were disinfected using

50% sodium hypochloride, rinsed with distilled

water, placed overnight in oxygenated 1 mM CaCl2solution, and incubated in water for an additional

24 h at 24�C. Seedlings were then grown aeroponi-

cally on an 8 h-dark (18�C)/16 h-light (24�C) regimein a system exposing the roots to a humid atmo-

sphere (de Dorlodot et al. 2005). Humidification of

the atmosphere was achieved through pulverization

of nutrient solution (half-strength Hoagland medium,

Sigma, Steinheim, Germany). The pulverization rate

was set to create a mist by projection of small

nutrient medium droplets (1 mm) into the air for

20 s periods separated by 40 s rest periods. Using

these conditions, the relative humidity of the atmo-

sphere was maintained close to 100%. The age of the

seedlings was calculated from the time they were

placed in the aeroponic system. For the experiments,

we chose 7- to 8-day-old roots harvested 3.5 h after

the start of illumination (200 lmol photons m–2 s–1),which corresponds roughly to the peak of ZmPIP

mRNA production (Lopez et al. 2003). These growth

conditions were used in all experiments except the Pfexperiment, in which the system described by Fetter

(Fetter et al. 2004) was used.

In situ reverse transcriptase-mediated PCR

Root segments were fixed, embedded in Paraplast Plus

(Sherwood Medical, St. Louis, MO, USA), sectioned

into 10 lm slices, dewaxed, and hydrated as previouslydescribed (Fetter et al. 2004). Slide pre-treatment was

performed as described by Fetter et al. (2004), except

that 2% pectinase (Calbiochem, Darmstadt, Germany)

was used and no proteinase K digestion was carried

out. RT-PCR and amplicon localization were per-

formed as described previously (Fetter et al. 2004).

Signals were recorded 16 h after the beginning of the

staining procedure using an epifluorescent Leica DMR

microscope (Wetzlar, Germany).

Detection of apoplastic barriers

Detection of apoplastic barriers was performed on

sections hand-cut from fresh root tissue which were

stained for 1 h with 0.1% berberine hemisulfate and

for 45 min with 0.5% toluidine blue O (w/v) (Brundrett

et al. 1988), mounted in 0.1% FeCl3 in 50% glycerol,

and immediately examined under a microscope (exci-

tation at 340–380 nm, emission at 425 nm).

Quantitative RT-PCR

Total RNA was extracted from fresh root sections

using an RNeasy� plant extraction minikit (Qiagen,Maryland, USA). DNAse I digestion was performed

on the column during RNA extraction according to the

manufacturer’s recommendations.

Plant Mol Biol (2006) 62:305–323 307

123

For cDNA synthesis, 3 lg of total RNA and 1 lMoligo dT(18) in a volume of 12.5 ll were incubated at70�C for 10 min and chilled on ice, then 12.5 ll of acocktail containing 500 lM dNTP Mix (Roche,Mannheim, Germany), 20 units of RNase inhibitor

(Roche, Mannheim, Germany), and 200 units of Mo-

loney murine leukemia virus reverse transcriptase

(Promega, Madison, USA) was added and the mixture

incubated for 60 min at 42�C, followed by 5 min at85�C. cDNA was purified using a DNA purification kit(Roche) and eluted in 50 ll of water at 60�C.

Real-time PCR analysis was performed using 1 ll of1:10 diluted cDNA samples in 25 ll of reaction med-ium containing 300 nM gene-specific primers (Table 3

in Supplementary Data) and 12.5 ll of SYBR Green 1PCR Mix (Eurogentec, Liège, Belgium) in an Applied

Biosystems PE-7000 (Darmstadt, Germany). For each

RNA sample, a reaction without reverse transcription

was performed to control for contamination by geno-

mic DNA. PCR using intron-flanking primers was also

used to check for genomic DNA contamination. In the

calibration step, a control experiment without cDNA

was performed to test for primer–dimer formation,

primers forming dimers being excluded. The PCR

program was as follows: 2 min at 50�C, 10 min at 95�Cfor DNA polymerase activation, and 40 cycles of 15 s

at 95�C and 60 s at 60�C. Data collection was per-formed at 60�C. The melting curve analysis programconsisted of 10 s at 95�C, 30 s at 60�C, and heating to90�C at a rate of 0.2�C s–1, data being continuouslycollected. The data were analyzed by the 2–DDCt

method (Pfaffl 2001). The calibration step of the

experiment checked for equivalent PCR efficiency of

the different genes (to allow comparison and normal-

ization). Standard curves (log of cDNA dilution vs. Ct)

using serial 10-fold dilution of cDNA were built for

each pair of selected primers, a 100% PCR efficiency

corresponding to a slope of –3.3 (Marino et al. 2003).

Practically, only pairs of primers yielding a slope of –

3.3 ± 0.1 were selected. The specificity of the amplifi-

cation (checked by dissociation curve analysis and gel

electrophoresis) and the use of appropriate control

genes were also assessed. Normalization of the results

was achieved using the glyceraldehyde phosphate

dehydrogenase (GAPDH) (gi:22237) or alpha tubulin

(gi:450292) gene as internal control, both yielding the

same results. Results obtained on the different root

zones were normalized using three maize internal

control genes, alpha tubulin, plasma membrane H+-

ATPase (gi:758354), and polyubiquitin (gi:248338), the

expression of which was shown to be stable in the

investigated tissues. This method smoothes normali-

zation errors due to the small variation of expression of

a single control gene (Vandesompele et al. 2002). The

elongation factor 1-alpha (EF1-a) gene (gi:2282583)was used as a control for meristematic activity.

Root cell protoplast isolation

Portions of the root tip at known positions from the

root apex were excised, chopped into small fragments,

and placed in a multiplate containing 500 ll of300 mOsm isotonic buffer (10 mM KCl, 1 mM CaCl2,

8 mM MES-KOH, pH 5.7) containing a cocktail of 2%

cellulase (Worthington, Lakewood, USA), 1% Drisel-

lase (Fluka, Buchs, Switzerland), 0.1% BSA (Sigma,

St. Louis, MO, USA), 0.1% Pectolyase (Karlan, Santa

Rosa, USA), and 1.5% Macerozyme (Yakult Phar-

maceutical Ind. Co., Tokyo, Japan) to degrade the cell

wall. Digestion was carried out for 1.5 h at 30�C in thedark on a rotating table (90 rpm), then the protoplasts

were filtered on a 100 lm mesh and centrifuged at 90gfor 1 min. To remove the enzymes, 80% of the super-

natant was removed and replaced by the same amount

of isotonic solution, then the sample was recentrifuged;

this step was repeated a further twice. The protoplasts

were kept at room temperature and analyzed as rapidly

as possible as described in Moshelion et al. (2004).

GST-ZmPIP-H expression

Genetic constructs were prepared using a modified

version of pGex-KG plasmid (pGex-KG’) (Guan and

Dixon 1991), in which a sequence encoding six Histi-

dine residues was introduced between the HindIII and

SmaI restriction sites. The DNA sequences encoding

the cytoplasmic amino-terminal ends of ZmPIP iso-

forms were amplified by PCR using specific primers,

incorporating EcoRI and HindIII sites at the

ends (Table 4 in Supplementary data) and subcloned

into EcoRI and HindIII sites of pGex-KG’. These

constructs were checked by sequence analysis. The

encoded fusion proteins consist in glutathione

S-transferase in frame with the amino-terminal part of

each ZmPIP and a His tag (GST-ZmPIP-H).

Twelve milliliters of overnight precultures of indi-

vidual transformed E. coli cells were used to inoculate

1 liter Luria-Bertani broth medium containing 100 lg/ml of ampicillin. The cells were cultured at 37�C untilmid-log phase (3–4 h, or OD600 = 0.6) before induction

of GST-ZmPIP-H expression with 1 mM isopropyl b-D-thiogalactopyranoside. After 3 h incubation at 37�Cthe cells were harvested by centrifugation at 5000g,

resuspended in 10 ml PBS containing 100 lg/ml lyso-zyme and incubated for 15 min on ice. The cells were

308 Plant Mol Biol (2006) 62:305–323

123

then sonicated 4 · 30 s and the lysate centrifuged for15 min at 20800g. GST-ZmPIP-H proteins were

extracted from the soluble phase by nickel affinity

chromatography according to the manufacturer’s rec-

ommendations (Qiagen, Maryland, USA).

Subcellular fractionation

To prepare the microsomal fraction, root sections,

collected 3.5 h after light onset, were ground in 800 llof a mixture of 250 mM sorbitol, 50 mM Tris–HCl (pH

8), 2 mM EDTA, and protease inhibitors [1 mM

phenylmethylsulfonyl fluoride, 1 lg/ml each of leu-peptin, aprotinin, antipain, chymostatin, and pepstatin

(Sigma, St. Louis, MO, USA)]. All subsequent steps

were performed at 4�C. The homogenate was centri-fuged at 2,700g for 5 min, and the resulting supernatant

centrifuged at 10,000g for 10 min. The second super-

natant was then centrifuged at 100,000g for 30 min and

the resulting pellet (microsomal fraction) resuspended

in 30–60 ll of suspension buffer (5 mM KH2PO4,330 mM saccharose, 3 mM KCl, pH 7.8) and sonicated

twice for 4 s.

Protein analysis

Fifteen micrograms of crude microsomal membranes

or 0.5 lg of purified GST-ZmPIP-H fusion proteinwere solubilized for 15 min at 60�C in solubilizationbuffer (27 mM Tris/HCl, 0.7% SDS, 3.3% glycerol,

0.0016% bromophenol blue, 1% DTT) and the pro-

teins separated by SDS-PAGE on a 12% polyacryl-

amide gel. After electrophoresis, the gel was incubated

for 15 min in semi-dry buffer (48 mM Tris, 39 mM

glycine, 20% methanol, 0.0375% SDS) before semi-dry

transfer to a polyvinylidene fluoride (PVDF; Millipore)

membrane was performed for 30 min at 22 V, the

membrane being previously incubated for 1 min in

pure methanol and 5 min in semi-dry buffer.

Western blot analysis was performed on the PVDF

membrane using antisera raised against the amino-

terminal peptides of ZmPIP1;2, ZmPIP2;1, ZmPIP2;5,

and ZmPIP2;6 (ZmPIP1;2, AQGAADDKDYKEP;

ZmPIP2;1, KDDVIESGAGGEFAAKD; ZmPIP2;5,

AKDIEAAAAHEGKD; ZmPIP2;6, KEVDVSTLEA

GGVRDRD). The peptides were coupled to KLH

using an Imject Maleimide Activated mcKLH Kit

(Pierce, Rockford, USA) and the resulting conjugates

used to immunize rabbits. The anti-RsPIP1 antiserum

was raised against radish PIP1 isoforms (Suga

et al. 2001). The anti-PMA antiserum was raised

against plasma membrane H+-ATPases (Morsomme

et al. 1996). The dilutions used were 1/500 for the anti-

ZmPIP1;2 and anti-ZmPIP2;5 antisera, 1/750 for

anti-His (Qiagen), 1/1000 for the anti-ZmPIP2;6 and

anti-RsPIP1 antisera, 1/5000 for the anti-ZmPIP2;1/2;2

antiserum, and 1/10000 for the anti-PMA antiserum in

TBS (0.02 M Tris, 0.136 M NaCl). Stripping of the

membrane was achieved by incubation in 0.5 M NaOH

for 5 min and 3 washes in TBS-Tween 20 0.1%.

In situ immunolocalization

Hand-cut sections of freshly harvested roots were fixed

in 0.8% paraformaldehyde in PBS (68 mM NaCl,

1.34 mM KCl, 5.070 mM Na2HPO4, 0.85 mM

KH2PO4, pH 7.4) for 1 h at room temperature under

light vacuum, followed by 5 washes with PBS. Perme-

abilization of the samples was achieved by dipping the

sections for 30 min in 0.25% Triton X-100 in PBS. The

sections were incubated for 1 h at 37�C in PBS con-taining 5% BSA (blocking solution), then for 1 h at

37�C with anti-PIP antiserum diluted 1/100 in blockingsolution. After 3 · 20 min washes in blocking solution,the slides were incubated for 1 h in the dark at 37�Cwith fluorescein-coupled goat anti-rabbit IgG anti-

bodies (Molecular Probes, Eugen, Oregon, USA;

dilution 1/100 in blocking solution). To quench auto-

fluorescence, the sections were incubated for 2 min in

PBS containing 0.1% toluidine blue. The slides were

then rinsed for 4 · 5 min in PBS and analyzed underUV (excitation: 460–500 nm, emission: 510–530 nm).

The controls included immunodetection using pre-im-

mune serum (primary antibody specificity), detection

without incubation with primary antibody (secondary

antibody specificity), and untreated slides (false posi-

tive signals due to autofluorescence).

Results

Expression of ZmPIP mRNAs in aeroponically

grown maize roots

The level of expression of ZmPIP genes was first

determined in entire aeroponically grown primary roots

using real time PCR on RNA extracted from the first

10 cm of the root (from the apex). To allow comparison

between different ZmPIP amplicons in terms of rela-

tive abundance, we made sure that the PCR efficiency

was very similar (see Materials and methods). The roots

were sampled 3.5 h after light onset. The time when the

roots are collected is of major importance, as some

maize PIPs display a diurnal expression pattern, with a

Plant Mol Biol (2006) 62:305–323 309

123

peak of expression at 2–4 h after the beginning of the

light period (Lopez et al. 2003).

All ZmPIP genes except ZmPIP2;7 were expressed,

although to different extents (Fig. 1). ZmPIP1;1,

ZmPIP1;5, ZmPIP2;1, and ZmPIP2;5, were the most

highly expressed and, together, accounted for more

than 75% of all the expressed ZmPIPs, ZmPIP2;5

alone accounting for 30%. All other genes were ex-

pressed at a much lower level, each accounting for

between 0 and 6% of total ZmPIP expression. Abso-

lute quantification of the expression of the ZmPIP2;4

and ZmPIP2;6 genes was performed by constructing a

calibration curve using serial 10-fold dilutions of plas-

mids bearing ZmPIP2;4 or ZmPIP2;6 cDNA for which

the initial concentration was known. Assuming an

equal efficiency of amplification as checked during real

time PCR experiment set up (see Materials and

methods) and knowing the relative expression of the

different ZmPIPs (Fig. 1), we were able to estimate

ZmPIP expression in terms of transcript copy number

(Table 1 in Supplementary Material). The four

most highly expressed genes, ZmPIP1;1, ZmPIP1;5,

ZmPIP2;1, and ZmPIP2;5, were present at 2500–7000

copies par nanogram of total RNA. ZmPIP1;2,

ZmPIP1;4, ZmPIP2;4, and ZmPIP2;6 represented an

intermediate group with copy numbers in the range

of 850–1500 copies per nanogram of total RNA.

Finally, ZmPIP1;3, ZmPIP1;6, ZmPIP2;2, and

ZmPIP2;3 showed low expression, below 500 copies

per nanogram of total RNA. These ZmPIP mRNA

copy numbers are in the range of those obtained in

Arabidopsis roots for AtPIP genes (500–7,000 copies

per nanogram of total RNA) (Jang et al. 2004).

Development of apoplastic barriers

It is generally believed that AQPs play a role in elon-

gating tissues. As the root tissues mature, the cells be-

come more symplastically isolated and apoplastic

barriers develop in two regions, the endodermis and the

exodermis. The localization of both cell layers was

examined to determine whether ZmPIP gene expres-

sion correlated with regions in which symplastic water

movement is hindered, i.e. regions requiring a high

membrane water permeability. We analyzed transverse

sections of the primary root from aeroponically grown

plants. Lignin and suberin deposits were examined at

different positions from the root tip up to 10 cm from the

cap (Fig. 2 and data no shown). Suberization of the

endodermis was detected 3 cm from the root tip, the

suberin and lignin deposits being located in the radial

cell walls of the endodermis. The suberized hypodermis,

defined as the exodermis, was detected 0.7–1 cm above

the first suberized Casparian strip of the endodermis at

3.7–4 cm from the root tip (Fig. 2). These data agree

with previous reports concerning suberin deposits in

maize roots (Enstone and Peterson 2005; Hose et al.

2001; Karahara et al. 2004).

Expression of ZmPIPs according to root

developmental stage

To investigate ZmPIP gene expression along the lon-

gitudinal axis of the developing root in aeroponic

conditions and how it relates to the development of

apoplastic barriers, we extracted total RNA from dif-

ferent segments of the root, each corresponding to a

tissue at a different developmental stage. For practical

reasons, we studied segments of at least 5 mm along

the root axis. By visual inspection and measurement of

cell length, we identified six distinct root zones. The

first, between 0 and 5 mm, consisted of the cap, apical

meristem, and distal elongation zone (Ishikawa and

Evans 1995) and corresponded to a region of fast cell

division and elongation. The second, between 5 and

10 mm, corresponded roughly to the elongation zone.

The third, between 10 and 20 mm, was the zone in

which the process of cell elongation/differentiation

stopped and root hair started to appear. The fourth,

between 30 and 40 mm, was in the root hair region,

where water absorption is reported to reach a maxi-

mum (Boyer 1985), the endodermis being suberized,

while the exodermis is still forming. The fifth, between

50 and 60 mm, corresponded to what we call ‘‘mature’’

primary root tissue in this report because both apo-

plastic barriers are present (Fig. 2). Finally, the sixth,

0

1

2

3

4

5

6

ZmPI

P1;1

ZmPI

P1;6

ZmPI

P2;1

ZmPI

P1;2

ZmPI

P1;3

ZmPI

P1;4

ZmPI

P1;5

ZmPI

P2;2

ZmPI

P2;3

ZmPI

P2;4

ZmPI

P2;5

ZmPI

P2;6

ZmPI

P2;7

GAP

DH

Arb

itrar

y un

its

Fig. 1 Levels of ZmPIP transcripts in the primary maize root.cDNA was synthesized from total RNA extracted from primarymaize roots and real-time PCR was performed as described inthe Materials and methods. The relative expression level ofZmPIPs was assessed using glyceraldehyde phosphate dehydro-genase (GAPDH) as internal standard. The mean and standarddeviation for three independent experiments are shown

310 Plant Mol Biol (2006) 62:305–323

123

between 100 and 110 mm, was the zone in which lateral

roots start to protrude from the root and corresponded

to the zone with ‘‘secondary’’ mitotic activity.

Interestingly, with the exceptions of ZmPIP2;7,

which was not expressed, and ZmPIP2;6, which

showed a fairly stable level of expression throughout

the root segments, expression of the ZmPIP genes

was developmentally regulated (Fig. 3 and Table 2 in

Supplementary data). ZmPIP expression was low (or

nonexistent in the case of ZmPIP1;6) in the first and

second zones and gradually increased to a plateau in

either zone 3 or zone 4, corresponding to mature

primary root tissues. ZmPIP1;5 and ZmPIP2;5, two of

the most highly expressed genes, both showed the

same dramatic change in their expression pattern

(Fig. 3). Assuming an equal efficiency of PCR

amplification (see Materials and methods), we com-

pared the relative contribution of each ZmPIP within

one segment. ZmPIP1;5 and ZmPIP2;5 expression

was low in the first segment (less than 6% of the total

expressed ZmPIPs), then gradually increased to

about 20% (ZmPIP1;5) or 30% (ZmPIP2;5) of the

expressed ZmPIPs in mature primary root tissue,

accounting for 50% of the expressed ZmPIPs

(Table 1). ZmPIP1;1 accounted for 27% of the ex-

pressed ZmPIPs in the first root segment, but its

contribution gradually decreased to 9% in the fourth

segment, where root hairs appear, and was about 12%

in the mature zones. ZmPIP2;1 showed the same

pattern of a decrease from 28 to 14% (Table 1).

These decreases in relative contribution were not

attributable to a decrease in absolute ZmPIP1;1 and

ZmPIP2;1 expression levels, which actually increased,

but rather to a huge increase in ZmPIP1;5 and

ZmPIP2;5 expression. On average, all the other genes

each accounted for less than 5% of ZmPIP expression

in the different root segments, with the exczeption of

ZmPIP2;6, which averaged 8% (Table 1). These data

show that ZmPIP1;5 and ZmPIP2;5 are major

isoforms in the mature root.

The EF1-a (elongation factor) gene was chosen as amarker for meristematic activity. Its expression peaked

in the meristematic zone, then decreased to a constant

and much lower value in the subsequent segments, again

increasing (to a lower level than in zone 1) in segment 6,

in which secondary root meristems were activated

4 cm

3 cm

(A) (B) (C)

Suberized hypodermis

(D)

(E)

(F)

Suberized endodermis

D

E

F

Fig. 2 Development ofapoplastic barriers in primaryroots cultivated in aeroponicconditions. A, Schematic viewof apoplastic barrierdevelopment in maize rootsgrown in aeroponicconditions. B–F, Transversesections of the roots at 4 (B–D), 3 (E), and 1 (F) cm fromthe root tip stained withberberine hemisulfate andtoluidine blue as described inthe Materials and methods.No suberized cell layer wasdetected in the zone 0–3 cmfrom the root tip (F). At 3 cm,suberization of theendodermis was detected (E,white arrow). At 4 cm, both asuberized endodermis andexodermis were detected (D,white arrows). B and C,Magnifications of theendodermis and exodermis,respectively, at 4 cm from theroot tip. Bars = 25 lm in Band C and 100 lm in D–F

Plant Mol Biol (2006) 62:305–323 311

123

(Fig. 3). Although it underwent the same normalization,

the expression pattern of EF1a displayed an oppositetrend to that of the PIP genes, clearly indicating that the

observed developmental PIP expression results were

not due to an artifact of the method.

Expression of ZmPIP genes in the cap, meristem,

and elongation zones

In situ RT-mediated PCR is a powerful tool for

localizing specific gene expression and is claimed to

be more sensitive than RNA in situ hybridization

because it includes an amplification step (Nuovo

1996). As appropriate controls, the full process was

checked for efficiency and specificity by carrying out

an in vitro RT-PCR on total root RNA extract using

the same conditions (primers, PCR cycles, stock

solutions etc.) as for the in situ reactions. The RT-

PCR products obtained for all ZmPIP genes were

checked on a 2% agarose gel. The presence of a

single band of the expected size was considered as

resulting from a specific amplification. To distinguish

between a specific signal and background noise, some

sections did not undergo the RT step and were con-

sidered as negative controls; as a result, since genomic

DNA was destroyed by the DNAse treatment and

mRNA was not transcribed into cDNA, no signal was

observed (Fig. 4E, H and J). A true positive signal for

in situ RT-PCR consists of labeling of both the nu-

cleus and cytoplasm (Nuovo 1996) (Fig. 4K), while a

false positive signal generated by incomplete genomic

DNA digestion results in only nuclear labeling

(Fig. 4L). A positive signal shows the presence of the

targeted mRNA, but its intensity cannot be linked to

0

10

20

30

40

50

60

70

800-5 mm

5-10 mm

10-20 mm

30-40 mm

50-60 mm

100-110 mm

ZmPI

P1;1

ZmPI

P1;6

ZmPI

P2;1

ZmPI

P1;2

ZmPI

P1;3

ZmPI

P1;4

ZmPI

P1;5

ZmPI

P2;2

ZmPI

P2;3

ZmPI

P2;4

ZmPI

P2;5

ZmPI

P2;6

EF1-

Aα-

tubu

linUb

iqui

tin

PMA

Arb

itrar

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its

Fig. 3 Levels of ZmPIP transcripts in different segments of theprimary root. cDNA was synthesized from total RNA extractedfrom six different segments of the primary maize root and real-time PCR performed as in Fig. 1. The geometric mean of theexpression level of three control genes (a-tubulin, ubiquitin, andH+-ATPase) was used to standardize the results. The distance ofthe different segments from the root tip were 0–5 mm (cap,

meristem, start of the elongation zone), 5–10 mm (elongationzone), 10–20 mm (end of the elongation zone, start of the roothair zone), 30–40 mm (root hair zone), 50–60 mm (matureprimary root tissue), and 100–110 mm appearance of secondaryroots). The mean and standard deviation for three independentexperiments are shown

Table 1 Expression ofZmPIPs as a percentage ofthe total ZmPIP expression ineach investigated root zone.N.D. stands for ‘‘NotDetected’’

Percentage contribution to ZmPIP expression in different root segments

0–5 mm 5–10 mm 10–20 mm 30–40 mm 50–60 mm 100–110 mm

ZmPIP1;1 26.9 ± 5 25.6 ± 2.8 19.2 ± 3.7 9.3 ± 3.0 11.7 ± 4.7 12.9 ± 4.1ZmPIP1;2 2.1 ± 0.5 6.4 ± 1.9 6.2 ± 1.4 3.5 ± 1.8 2.2 ± 0.4 2.3 ± 0.7ZmPIP1;3 2.7 ± 0.9 3.6 ± 0.6 3.8 ± 1.1 6.0 ± 2.6 4.2 ± 1.2 5.1 ± 1.3ZmPIP1;4 1.1 ± 0.3 1.8 ± 0.8 2.3 ± 1.4 2.6 ± 0.6 2.4 ± 0.3 2.5 ± 0.6ZmPIP1;5 5.8 ± 1.4 12.3 ± 1.8 14.6 ± 3.0 19.9 ± 2.4 21.3 ± 4.1 22.6 ± 3.2ZmPIP1;6 N.D. N.D. 0.2 ± 0.1 1.1 ± 0.1 1.0 ± 0.8 1.1 ± 0.5ZmPIP2;1 27.6 ± 12.6 18.0 ± 13.9 18.3 ± 11.9 19.3 ± 4.7 12.1 ± 5.7 14.1 ± 8.0ZmPIP2;2 8.3 ± 2.5 5.9 ± 1.6 4.9 ± 1.9 5.4 ± 1.3 3.9 ± 0.4 3.3 ± 0.5ZmPIP2;3 1.6 ± 0.5 3.8 ± 0.6 2.6 ± 1.6 2.8 ± 0.8 3.9 ± 0.8 3.0 ± 1.3ZmPIP2;4 0.5 ± 0.3 1.6 ± 0.3 1.5 ± 0.3 1.3 ± 0.1 1.4 ± 0.7 1.4 ± 0.4ZmPIP2;5 5.0 ± 1.4 15.4 ± 2.1 20.0 ± 4.2 22.0 ± 0.5 29.2 ± 3.3 27.2 ± 2.3ZmPIP2;6 18.3 ± 5 5.7 ± 4.5 6.3 ± 2.6 6.7 ± 0.8 6.7 ± 1.2 4.5 ± 0.6

312 Plant Mol Biol (2006) 62:305–323

123

mRNA levels. Thus, this approach can be seen as a

complementary to quantitative RT-PCR, which yields

quantitative results, but no information about the

location of transcript expression.

With the exception of the ZmPIP1;6 and ZmPIP2;7

transcripts, which were not detected, all ZmPIP

mRNAs were detected in most cell types in the meri-

stem, elongation, and mature zone (Fig. 4A–F and data

not shown). The cap constituted an exception to the

rule. Indeed, with the exception of ZmPIP2;6 and

ZmPIP2;4 which were found in the root cap (Fig. 4D

and F), the ZmPIP isoforms yielded a very low signal

not significantly different from background noise

(Fig. 4A–C and E). However, the nuclei of the cap

cells were strongly labeled in the positive controls (no

DNAse treatment), suggesting that our method is able

to stain the cap tissue. Transverse sections showed a

clear ZmPIP1;1 signal in the epidermis and exodermis

(Fig. 4G) and in that the ZmPIP2;4 signal was stronger

in the stele than the cortex (Fig. 4I)

Fig. 4 Localization ofZmPIP mRNAs revealed byin situ RT-PCR. A–F, 10 lmthick longitudinal sectionsthrough the maize root tipshowing the presence ofZmPIP transcripts. AZmPIP2;1, B ZmPIP1;2, CZmPIP1;1, D ZmPIP2;4, Enegative control (no RT), FZmPIP2;6. G–H: Localizationof ZmPIP1;1 mRNA in theepidermis and exodermis (G)and negative control (no RT)(H), I–J: Transverse sectionsat 5 cm from the root tipshowing the localization ofZmPIP2;4 mRNA in all thetissues, with a stronger signalin the stele (I) or the negativecontrol (No RT) (J). K,Typical signal for in situ RT-PCR showing that both thecytoplasm and the nuclei werelabeled for ZmPIP1;1transcripts. L, Positive controlin which no DNAse and noRT treatments wereperformed, leading to a strongnuclear signal (ZmPIP2;2gene). Bars = 200 lm in A–F,I, J and 20 lm in G, H, K, L

Plant Mol Biol (2006) 62:305–323 313

123

The ubiquitous appearance of AQP mRNAs might

seem puzzling, but is not abnormal considering the

fairly high level of ZmPIP gene expression observed

by quantitative RT-PCR. Moreover, the PCR step

renders the method ultrasensitive for the detection of

low transcript copy numbers, a few copies of mRNA

being theoretically enough to obtain a signal at the

single cell level (Nuovo 1996).

ZmPIP protein levels in roots

Expression at gene level is not always reflected at the

protein level. Therefore, ZmPIP protein expression in

maize primary root was investigated using Western

blots. ZmPIPs were detected using antibodies raised

against amino-terminal peptides derived from

ZmPIP2;1, ZmPIP2;5, ZmPIP2;6, ZmPIP1;2, and rad-

ish PIP1s (Suga et al. 2001) (Fig. 5A–E). To test the

specificity of the antibodies, fusion proteins consisting

in the glutathione-S-transferase, amino-terminal end of

each ZmPIP (21–56 amino acid residues) and six His

tag were expressed in Escherichia coli, purified on a

nickel column, separated on SDS-PAGE and immu-

nodetected using each serum (Fig. 5F). The anti-

ZmPIP2;1 antibodies recognized ZmPIP2;1 and

ZmPIP2;2, as expected on the basis of amino acid se-

quence comparison (Fig. 5A), whereas the antisera

against ZmPIP2;5, ZmPIP2;6 and ZmPIP1;2 were iso-

form-specific. The anti-radish RsPIP1 antiserum

recognized all maize PIP1s but a higher signal was

obtained for ZmPIP1;6.

Crude membrane proteins from the different root

segments were extracted and separated on SDS-PAGE,

then ZmPIPs were immunodetected. Anti-PMA anti-

body recognizing plasma membrane H+-ATPases was

used as a loading control (Morsomme et al. 1996). All

the immunoblot analyses of the different ZmPIPs re-

vealed two major bands at approximately 28 and 56 kD,

corresponding to monomeric and dimeric forms

(Fig. 6). Interestingly, the variations in protein levels in

the different root segments correlated quite well with

the variation in transcript amount. ZmPIP2;1, encoded

by one of the most highly expressed ZmPIP genes in

the root, showed low expression in the first segment

(cap, meristem, and elongation zone), but its expression

gradually increased to peak in the fourth segment

(Fig. 6). The same expression pattern was observed at

the RNA level (Fig. 3). In each of the two major bands,

Fig. 5 Antibody specificity.A–E, Alignments of peptidesused to raise antisera againstthe N-terminal region ofZmPIP2 or ZmPIP1 proteins.The peptide synthesized tomake the antiserum is shownat the top. Identical aminoacid residues are shown inbold. The numbers indicatethe peptide position withinthe protein. F. Purified GST-ZmPIP-H fusion proteins(0.5 lg) were separated bySDS-PAGE, transferred toPVDF membrane andimmunostained usingantibodies against His-tag,ZmPIP2;1, ZmPIP2;5,ZmPIP2;6, ZmPIP1;2, orRsPIP1 as described in‘‘Materials and methods’’

314 Plant Mol Biol (2006) 62:305–323

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this antibody detected two bands with very close

molecular masses. The upper band could correspond to

the ZmPIP2;2 isoform, which has a slightly higher

molecular mass and isoelectric point (30,259 Da, 8.15)

than ZmPIP2;1 (30,214 Da, 7.81) and was less ex-

pressed at the RNA level (Figs. 3 and 6), or to a post-

transcriptional modification of ZmPIP2;1. ZmPIP2;5

was barely detected in the two first root segments but

clearly observed in the third one, and its level increased

up to 5 cm from the root apex (Fig. 6), reflecting the

changes in mRNA levels. ZmPIP2;6, which had the

most stable transcript level of the ZmPIP genes

(Fig. 3), was already detected in the first segment and

its level did not vary significantly between the different

regions along the root axis. The ZmPIP1 subgroup

showed a strong developmental expression pattern, the

genes being expressed at low levels (or not at all in the

case of ZmPIP1;6) in the first segment, then at gradu-

ally higher levels, reaching a fairly steady state in the

third or fourth segment, corresponding to mature pri-

mary root tissue (Fig. 3). This trend was faithfully re-

flected at the protein level, the signal being virtually

absent in the first zone and gradually increasing to a

maximum in mature primary root tissues (Fig. 6).

Together, these data indicate a clear correlation be-

tween mRNA and protein levels in maize root grown in

aeroponic conditions. This correlation between AQP

mRNA and protein levels has not always been

observed, e.g. in roots exposed to chilling stress (Aroca

et al. 2005) or in other plant species (Suga et al. 2002);

(Alexandersson et al. 2005); (Boursiac et al. 2005).

However, the lack of isoform specificity of the antibody

and of spatial resolution within the analyzed tissues

made the interpretation of the data of these studies

much more difficult.

In situ immunolocalization of ZmPIP2;1

and ZmPIP2;5

ZmPIP2;5 and ZmPIP2;1 are two of the most highly

expressed ZmPIP mRNAs and proteins in the primary

maize root tip. To more precisely determine the

localization of both isoforms in developing roots,

immunocytochemistry was performed on transverse

sections taken at 5 mm (young tissue) and 5 cm

(mature tissue with suberized hypodermis and endo-

dermis) from the root tip (Figs. 7 and 8). The intensity

of the green color indicates the abundance of proteins.

As expected, the signals were observed around the

cells, indicating a plasma membrane localization of the

proteins. As shown above, the antibodies against

ZmPIP2;1 also recognize ZmPIP2;2

At the start of the elongation zone (Fig. 7A–F),

ZmPIP2;1/2;2 was mainly detected in the stele tissues,

the endodermis, and, to a lesser extent, the inner part

of the cortex (Fig. 7A, D, and E). No signal was

recorded in the epidermis (Fig. 7D). This labeling

contrasts with that for H+-ATPases, which were

uniformly detected in all root tissues, including the

PMA

ZmPIP2;1/2;2

ZmPIP2;5

ZmPIP2;6

ZmPIP1;2

RsPIP1

100

55

24

55

24

55

24

55

24

24

kD

0-5 5-10 50-6030-4010-20 100-110

Distance from the root apex (mm)

55

Fig. 6 ZmPIP protein expression in primary roots. Using 7-day-old primary roots, crude microsomal membranes were preparedfrom the same segments used in Fig. 3 and the proteins (15 lg)subjected to Western blotting using antibodies against PMA (H+-ATPases), ZmPIP2;1/2;2, ZmPIP2;6, ZmPIP1;2, or RsPIP1. ThePMA signal was used to control for gel loading and normalizethe signals. The positions of the molecular mass markers areindicated

Plant Mol Biol (2006) 62:305–323 315

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Fig. 7 Immunocytochemical localization of ZmPIP2;1/2;2 inmaize roots. A–M, Transverse sections at 5 mm (A–F) or 5 cm(G–M) from the root tip. A, Detection of ZmPIP2;1/2;2 in thestele, endodermis, and inner cortex. Note the absence of signal inthe epidermis and a strong signal in the stele and the innercortical cell layers. B, Negative control for A. Pre-immune serumwas used instead of anti-ZmPIP2;1/2;2 antibodies. C, Localiza-tion of plasma membrane H+-ATPases (PMA) used as control.A strong signal was recorded throughout the root. D, Localiza-tion of ZmPIP2;1/2;2 in the epidermis and parenchyma cells. Nosignal was detected in the epidermis, whereas cortical cells werelabeled. E, Localization of ZmPIP2;1/2;2 at the junction of thecortex and the stele; a strong signal was seen in the endodermis.F, Localization of PMA in the epidermis and parenchyma cells.

G, Localization of ZmPIP2;1/2;2 in transverse section 5 cm fromthe root tip. The protein was present in the epidermis, cortex,and endodermis. The signal was much fainter in the stele. H,Negative control for G (pre-immune serum). I, Same as G,except that the signal was stronger in the stele. J, Localization ofZmPIP2;1/2;2 in the epidermis and exodermis; strong labeling ofthese cell layers was seen. K, Localization of ZmPIP2;1/2;2 inroot hairs. L, Localization of ZmPIP2;1/2;2 at the junction of thecortex and the stele; strong labeling of the endodermis was seen.The signal in the cortex was higher than in the stele. M,Localization of PMA in a transverse section 5 cm from the roottip. A strong signal was recorded in the periphery of the cortexand in the stele. Bars = 80 lm in A, B, G, and H, 100 lm in Cand M, 25 lm in D, E, J, K, and L, 40 lm in F, and 110 lm in I

316 Plant Mol Biol (2006) 62:305–323

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epidermis (Fig. 7C and F). Interestingly, in the mature

zone of the primary root, the signal pattern was re-

versed, with stronger ZmPIP2;1/2;2 labeling in the

epidermis, exodermis, and cortex than the stele

(Fig. 7G–L), confirming the results obtained at the

transcriptional level by Zhu et al. (2005). The intensity

of the ZmPIP2;1/2;2 signal in the stele varied between

samples, but was always lower than that in the cortex

(Fig. 7G and I). The epidermal and endodermal cells

were strongly labeled, with high ZmPIP2;1/2;2 accu-

mulation at epidermal cell junctions (Fig. 7J). The root

hairs also showed labeling of their plasma membrane,

with some internal spots possibly corresponding to

fusing vesicles (Fig. 7K).

ZmPIP2;5 displayed some significant differences in

its expression pattern compared to ZmPIP2;1/2;2. At

the start of the elongation zone (Fig. 8A–F), the stele

tissue was strongly labeled (Fig. 8A and E), as with

ZmPIP2;1/2;2, but a stronger ZmPIP2;5 signal was seen

in the cortical cells (Fig. 8A and D). More remarkably,

contrary to ZmPIP2;1/2.2, a polarized signal was ob-

served in the epidermal cells of this zone, in which a

stronger ZmPIP2;5 signal was seen on the plasma

membrane exposed to the external medium, thus

exhibiting a characteristic U-shape (Fig. 8A and C). In

more mature tissue, about 1 cm from the root tip, the

endodermis was strongly labeled (Fig. 7F). In the ma-

ture zone, ZmPIP2;5 was highly expressed in the epi-

dermis/exodermis cell layers and at the endodermis

boundary (Fig. 7G, J, I, and K), suggesting that this

isoform is involved in facilitating transcellular water

movement in these regions. As with ZmPIP2;1/2;2, the

overall ZmPIP2;5 signal intensity was stronger in the

cortex than in the stele tissues (Figs. 8G, J, K), con-

firming the transcriptional data obtained by Zhu et al.

(2005).

Osmotic water permeability coefficients of root

protoplasts

To determine whether the increase in ZmPIP expres-

sion according to the root developmental stages cor-

related with an increase in osmotic water permeability

of the cell membrane (Pf), we analyzed the swelling of

the protoplasts in response to hypoosmotic challenge

as described previously (Moshelion et al. 2004). As

shown in Fig. 9, all the cells examined (N = 43) in the

zone at 0–5 mm (cap, meristem, and start of the elon-

gation zone) had a similar Pf value of < 10 lm/s, while,of the 50 cells examined in the zone at 10–20 mm (root

hair zone), some (N = 10, 20%) exhibited a Pf higher

than 10 lm/s, while the remainder (N = 40, 80%)constituted a ‘‘low Pf cell’’ population with Pf

values < 10 lm/s (Fig. 9B–C). The low Pf cells in the10–20 mm zone had a mean Pf similar to those of the

low Pf cells in the other zones. Moreover, the same

division between high and low Pf cells was observed for

the zone at 40–60 mm (data not shown). AQPs

appeared to control the water permeability, since the

Pf of the protoplasts was decreased by 67% after 5 min

incubation with 250 lM HgCl2, which inhibits AQPs(Fig. 9D).

Discussion

ZmPIP genes are widely expressed in the maize

primary root

Quantitative and in situ RT-PCR techniques were used

to monitor PIP gene expression in maize primary

roots. Since ZmPIP1;1, ZmPIP1;5, ZmPIP2;1, and

ZmPIP2;5 transcript abundance exhibits a diurnal

variation, with a peak at 2–4 h after light onset (Lopez

et al. 2003), all samples analyzed were collected during

this period of the day. We showed that all except

ZmPIP2;7 were transcribed at various levels. The four

most highly expressed genes were ZmPIP1;1,

ZmPIP1;5, ZmPIP2;1, and ZmPIP2;5. In general,

these data are in accordance with the number of ex-

pressed sequence tags obtained from maize root tissues

(Chaumont et al. 2001) and with DNA array hybrid-

ization studies recently performed on maize roots (Zhu

et al. 2005). However, we found ZmPIP1;5 to be less

abundantly expressed and ZmPIP2;5 more highly ex-

pressed than in the latter study. These discrepancies

might be explained by the different maize genotypes

and growing conditions used in the two studies. Our

seedlings were grown aeroponically in a system

exposing the roots to a humid atmosphere, while Zhu

et al. (2005) grew their plants in aerated hydroponic

medium.

The observation that almost all ZmPIP genes were

widely expressed in the primary root is quite puzzling,

but is not specific to maize. Similar results have been

reported in analyses of global AQP expression in

Arabidopsis, which demonstrated that all AtPIP genes

were expressed in roots, but showed differential regu-

lation according to the environmental conditions (Al-

exandersson et al. 2005; Boursiac et al. 2005; Jang

et al. 2004). This confirms the general view that AQPs

play an important physiological role in facilitating

water movement across cell membranes in roots.

Nevertheless, the fact that almost all ZmPIP genes

were transcribed simultaneously in the different root

tip tissues, as shown by in situ RT-PCR experiments

Plant Mol Biol (2006) 62:305–323 317

123

(Fig. 4), raises the question whether the ability to

conduct water is their sole physiological role. Evidence

is accumulating that plant AQPs exhibit diverse regu-

lation and transport properties and can behave as

multifunctional channels, suggesting that these pro-

teins may have different physiological roles, which

might explain the isoform diversity (Chaumont et al.

2005; Luu and Maurel 2005; Tyerman et al. 2002). In

addition, the observation that most ZmPIP genes from

both the PIP1 and PIP2 subfamilies were co-expressed

in the same cells suggests that they might physically

interact and form heterotetramers in planta. It is

Fig. 8 Immunocytochemical localization of ZmPIP2;5 in maizeroots. A–K, Transverse sections at 5 mm (A–E), 1 cm (F), or5 cm (G–K) from the root tip. A, Detection of ZmPIP2;5 in thestele, endodermis, cortex, and epidermis. A strong signal wasrecorded in the stele, endodermis and inner cortex. Note thepolarization of the signals in the epidermis. B, Negative controlfor A using pre-immune serum. C, Polarized ZmPIP2;5 signal inthe epidermis D, Detection of ZmPIP2;5 in the inner paren-chyma cells. E, Detection of ZmPIP2;5 in the xylem parenchymacells. F, Detection of ZmPIP2;5 in the stele and endodermis at1 cm from the root tip; strong labeling of the latter was seen. G,Detection of ZmPIP2;5 in a transverse section 5 cm from the

root tip. A strong signal was recorded in the epidermis, cortexand endodermis. The signal was much fainter in the stele. H,Negative control for G (pre-immune serum). I, Detection ofZmPIP2;5 in the epidermis and exodermis. The signal wasstronger in the exodermis than in the epidermis. J, Same as Gexcept that the signal was concentrated in two ‘‘rings’’, oneconsisting of the epidermis and exodermis and the other the cellsfacing the stele. K, Detection of ZmPIP2;5 at the junction of thecortex and the stele. The signal was stronger in the cortex thanthe stele. Bars = 90 lm in A, B, F, G, and H, 30 lm in C, I, K,18 lm in D and E, and 200 lm in J

318 Plant Mol Biol (2006) 62:305–323

123

interesting to observe that the ratio between PIP1 and

PIP2 gene expression in the different root segments is

relatively constant (Table 1). We previously showed

that co-expression of ZmPIP1;2 and ZmPIP2s in

Xenopus oocytes increases both the amount of

ZmPIP1;2 in the plasma membrane and the membrane

osmotic water permeability (Fetter et al. 2004). Inter-

estingly, Martre et al. (2002) pointed out that, when

comparing the water parameter data obtained from

PIP1, PIP2, or PIP1 + PIP2 antisense plants, the PIP

subgroup that was not down-regulated appeared inac-

tive. This observation further supports the theory that

PIP heteromerization regulates the cell Pf.

ZmPIP expression in the root is developmentally

regulated

Our data revealed that the content of all ZmPIPs

mRNAs except ZmPIP2;6 mRNA was strongly

dependent on the developmental stage of the root, with

a general increase in expression towards the elongation

and mature zones (Fig. 3). Two genes, ZmPIP1;5 and

ZmPIP2;5, showed the greatest increase in expression

(up to 11- and 17-fold, respectively) in the mature

zone, where they accounted for 50% of the total

expressed ZmPIPs, indicating an important role of

these two AQPs. The use of antibodies recognizing

ZmPIP2;1/2;2, ZmPIP2;5, ZmPIP2;6, ZmPIP1;2 or

ZmPIP1s showed that the amounts of protein detected

in the different root zones correlated well with the

transcript abundance (Fig. 6). This AQP mRNA-pro-

tein correlation has not always been observed in plant

tissues (Aroca et al. 2005; Boursiac et al. 2005; Lopez

et al. 2003), but this could be partly due to the general

lack of antibody specificity, which prevented the

accurate study of a specific isoform. This increase in

ZmPIP expression in maturing root zones was previ-

ously reported by Hukin et al. (2002), who showed a

huge increase in ZmPIP1;2 and ZmPIP2;4 gene

expression in the cell elongation zone compared to the

meristematic zone. Growing cells near the root tip are

symplastically connected to each other and to the

phloem by plasmodesmata (Bret-Harte and Silk 1994).

In contrast to the youngest cells, those located in the

root elongation zone loose symplastic continuity

(Hukin et al. 2002). The symplastic isolation may

extend to the more mature regions of the growing

zone, and may change under different solute avail-

0

2

4

6

8

10

12

14

16

0

5

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

zone between 0-5mm

zone between 10-20 mm

0-5

mm

10-2

0 m

m

(A)

(C) (D)

(B)

95

100

105

110

115

120

125

130

135

140

0 10 20 30 40 50 60

High PfLow Pf

Rel

ativ

e vo

lum

e

Time (s)

Pf at 60 s (µm/s)

Perc

enta

ge

P f (

µm/s

)

Control

Mercury

Fig. 9 Measurement of theosmotic water permeabilitycoefficient of root cells. A,Schematic diagram of the rootshowing the two zones studiedat 0–5 mm and 10–20 mm. B,Distribution (as a percentageof the cell population) of theosmotic water permeabilitycoefficient (Pf) in the twozones. Note the ‘‘high Pf’’population of cells in the zonebetween 10 and 20 mm. C,Comparison of swellingbehavior of sevenrepresentative root protoplastsfrom each of the high and lowPf cell populations from the10–20 mm zone. The meanand standard deviation areshown. D, Pf values of cellsfrom the 0 to 20 mm zonebefore, and after, incubationwith 250 lM HgCl2. The meanand standard deviation for 15controls and 18 mercurial-treated cells are shown

Plant Mol Biol (2006) 62:305–323 319

123

ability regimes (Patrick and Offler 1996). The increase

in ZmPIP transcript abundance along the longitudinal

root axis can be attributed to a decrease in symplastic

continuity along the growing and maturing zones

(Hukin et al. 2002) and therefore to a need for facili-

tated water transport through the cellular plasma

membrane.

Another physiological observation that could re-

quire higher AQP expression in mature regions of the

roots is that, in this zone, the xylem becomes more

conductive and water demand driven by the transpi-

ration stream is higher than in younger regions, where

water demand is essentially driven by cell elongation.

In addition, the development of suberized apoplastic

barriers in the mature zone might require higher AQP

expression in this zone to facilitate radial water

movement to the stele. Under aeroponic growth con-

ditions, the suberized endodermis was detected 3 cm

from the root apex, whereas the Casparian strips from

the exodermis were detected 1 cm further from the

apex (Fig. 2). Interestingly, the zones in which apo-

plastic barriers were suberized corresponded to regions

in which ZmPIP expression reached its maximum. The

development of barriers made of suberin and lignin

would force the plant to increase its membrane water

permeability at these crucial places by higher expres-

sion of AQPs.

On the other hand, ZmPIP2;6 gene (Fig. 3) and

protein (Fig. 6) expression did not vary during root

development, and its mRNA was detected in all root

cells by in situ RT-PCR (Fig. 4). ZmPIP2;6 expression

in the outer layers of the roots (epidermis, cortex, and

endodermis) and in the inner tissue (stele) is also

reported to be similar (Zhu et al. 2005). Together, these

data suggest that ZmPIP2;6 isoform is constitutively

expressed at similar level in maize primary root.

Functional assay in Xenopus oocytes showed that this

protein, like the previously characterized ZmPIP2;1,

ZmPIP2;4, and ZmPIP2;5 (Chaumont et al. 2000; Fet-

ter et al. 2004; Lopez et al. 2003), forms an active water

channel (Hachez and Chaumont, unpublished data).

Differential distribution of ZmPIP2;1/2;2

and ZmPIP2;5 in the primary root

Immunocytochemistry experiments allowed us to

localize ZmPIP2;1 and ZmPIP2;5, two of the most

highly expressed AQPs. The antibodies against

ZmPIP2;1 also recognize ZmPIP2;2, while those

against ZmPIP2;5 are isoform-specific. At 5 mm from

the root tip, both antisera mainly labeled the stele

tissues and the inner cell layers of the cortex. These

expression patterns might be explained by a phloem

unloading process. Growing root apices function as

heterotrophic sink organs that are dependent on the

continuous supply of photoassimilates from the leaves,

and these are delivered to the subapical region of the

root tip through the protophloem sieve elements

(Patrick and Offler 1996). Phloem unloading in the

root tips is usually believed to occur symplastically, as

indicated by the plasmodesmata linking the sieve

elements and meristematic cells (Bret-Harte and Silk

1994; Oparka et al. 1994). However, the possibility of

the symplastic and apoplastic pathways operating in

parallel during phloem unloading has been suggested

(Bret-Harte and Silk 1994). As sucrose is actively taken

up, the osmotic potential of the cells decreases, driving

water entry, and the phloem unloading mechanism

would then require facilitated water transport to

rapidly balance the osmotic potential, possibly

explaining the high PIP expression in the stele. A

similar function has been attributed to the tonoplast

AQP, ZmTIP1;1, which is highly expressed in the root

tip stele (Barrieu et al. 1998).

Whereas ZmPIP2;1/2;2 was not detected in the

epidermis of the root apices, ZmPIP2;5 was highly

expressed in these cells and was specifically localized in

periclinal plasma membrane domains facing the

external medium. The polarization of several mammal

AQPs in apical membranes has been reported (Agre

and Kozono 2003; Brown 2003) but, to our knowledge,

this is the first example in plant cells. Even if it is

estimated that 81% of the growth demand for water in

the root tip in maize plants growing in vermiculite

comes from the phloem, the remainder of the water

might come from inward transport from the root sur-

face (Bret-Harte and Silk 1994). This directional

movement of water could be facilitated by a polarized

localization of ZmPIP2;5 in the epidermis cells.

ZmPIP2;5 localization underlines the importance of

the non-apoplastic route in water uptake from the soil

into the developing tissues. In this zone, root hairs are

not yet present and a high amount of aquaporins in the

epidermis apical membrane might compensate for the

lack of these structures.

In the mature root zone, 5 cm from the root tip,

ZmPIP2;1/2;2 and ZmPIP2;5 were detected in the

epidermis, exodermis, cortex, and endodermis. Due to

the transpiration stream, water absorption is very

important in this region (Boyer 1985), and the need for

facilitated water uptake into the cells is suggested by

the ZmPIP2;1/2;2 and ZmPIP2;5 expression pattern.

The development of an exodermis substantially de-

creases the root hydraulic conductivity in hydrostatic

experiments (Zimmermann et al. 2000; Zimmermann

and Steudle 1998) and this effect could therefore be

320 Plant Mol Biol (2006) 62:305–323

123

partly compensated by the presence of a high level of

AQPs in the epidermis and exodermis membranes.

Similarly, the presence of a suberized endodermis

would require cell water uptake in the cortex, which

was strongly suggested by the localization of ZmPIP2;5

(Fig. 8J), which showed increased expression in two

rings of cells corresponding to the exodermis and cells

surrounding the endodermis. In the cortex, water could

move radially in the apoplast, but also use the trans-

cellular pathway, requiring high expression of ZmPIPs

(Zimmermann et al. 2000). This is in accordance with

the composite transport model (Steudle 2000). The

contribution of AQPs to the overall root hydraulic

conductivity is reported to account for up to 80% of

the Lpr in Arabidopsis (Boursiac et al. 2005).

The anti-ZmPIP2;1/2;2 antibodies also labeled the

root hair, confirming previous RT-PCR data (Fetter

et al. 2004). Whereas the whole plasma membrane was

labeled, cytoplasmic spots were detected in the hair tip,

possibly reflecting cytoplasmic accumulation of AQP-

containing vesicles that could, in turn, account for a

high turn-over of the protein in this region and the

rapid hair growth.

Root protoplasts display a low water permeability

It has been reported that the membrane diffusive water

permeability is around 10 lm/s, whereas AQP-mediated water transport results in a Pf ranging from

100 to 1000 lm/s (Tyerman et al. 1999). However,recent findings using different methods indicate that,

despite the presence of plasma membrane-associated

AQPs, the plasma membrane can display a low Pf(Chaumont et al. 2005; Moshelion et al. 2004). The low

water permeability of maize root protoplasts reported

by Ramahaleo et al. (1999) during the early stages of

root development are similar to our findings. They also

reported an increase in the Pf of root cells after 5 days

and proposed that the larger Pf could be due to

the developmental expression of AQPs. Such results

correlate well with our findings. However, protoplasts

may not ideally reflect the behavior of cells in planta, as

they are subjected to stressful conditions during their

preparation. The activity of AQPs in the membrane

is highly and reversibly regulated by post-translational

modifications and environmental conditions

(Chaumont et al. 2005; Luu and Maurel 2005). These

regulation mechanisms could explain the higher Pfvalues (between 30 and 200 lm/s) measured withpressure probe experiments on intact maize root cells

(Azaizeh et al. 1992; Zhu and Steudle 1991). A recent

study comparing the Pf of barley leaf cells measured

with a pressure probe in intact tissues or using the

protoplast swelling assay showed that the absolute Pfvalues were higher (up to 5-fold) using the former

method compared to those determined in protoplasts

(Volkov V, Hachez C, Moshelion M, Draye X,

Chaumont F, Fricke W, data not shown). However,

differences between the Pf of elongating and

non-elongating cells were similar whatever the method

used.

The contribution of AQPs was detectable even in

low Pf cells, since the Pf was reduced by 67% by incu-

bation of the protoplasts with 250 lM HgCl2 for 5 min(Fig 9D). Cells with a low water permeability were

found in the root tip, but also made up the majority of

the cells in the zones at 10–20 mm and 40–60 mm.

However, as the root tissues matured, the cells differ-

entiated and some acquired a significantly higher water

permeability, a phenomenon that we attributed to

developmentally regulated expression of PIPs (Fig. 9).

Given their size (data no shown), these high Pf cells

might correspond to cells adjacent to apoplastic barri-

ers with a high membrane permeability to allow cell

water loading or to cells with a specialized function,

such as xylem parenchyma cells involved in maintaining

the transpiration stream and refilling gas-filled xylem

vessels (Johansson et al. 2000). The existence of some

high Pf cells in mature parts of the root further supports

the theory of increased AQP activity in these zones due

to the symplastic isolation of the cells. This is in

accordance with the observation that 20 lM mercurycauses higher Lp inhibition in growing and mature cells

than in cells at the root tip (Hukin et al. 2002).

Conclusion

These results show the wide, but developmentally

regulated, expression of ZmPIPs in maize primary root

and highlight the importance of two ZmPIP genes,

ZmPIP1;5 and ZmPIP2;5, which, together, account for

more than 50% of all expressed ZmPIPs in mature root

tissue. AQPs provide the plant with the ability to

rapidly alter its membrane water permeability and this

is especially important in regions in which cell-to-cell

water transport acts as a bottleneck at sites of sym-

plastic isolation or in the vicinity of apoplastic barriers.

This theory is supported by the increase in AQP

expression seen during root maturation and the in-

creased expression of ZmPIP2;1/2;2 and ZmPIP2;5

proteins in the exodermis and endodermis. Finally the

polarized positioning of ZmPIP2;5 in the epidermis of

the root apex suggests a role in facilitating directional

water movement from the soil. More detailed mapping

of all the AQPs along the root and an examination of

Plant Mol Biol (2006) 62:305–323 321

123

the correlation between their localization and cell

water parameters will clarify their role in root growth

and plant water supply.

Acknowledgements This work was supported by grants fromthe Belgian National Fund for Scientific Research (FNRS), theInteruniversity Attraction Poles Programme—Belgian SciencePolicy, and the ‘‘Communauté française de Belgique—Actionsde Recherches Concertées’’. F.C. is a Senior Research Associateand C.H. a Research Fellow at the FNRS; E.Z. is a ResearchFellow at the Fonds pour la Formation à la Recherche dansl’Industrie et dans l’Agriculture. We thank R. Jung (PioneerHi-Bred International) for providing ZmPIP cDNAs,M. Maeshima (Nagoya University, Japan) and M. Boutry (Uni-versité catholique de Louvain) for supplying the anti-RsPIP1 andanti-PMA antibodies, respectively, and T. Trombik andE. Peeters (Université catholique de Louvain) for supplyingpGex-KG’ plasmid. We are very grateful to X. Draye andT. Lavigne for the use of the aeroponics facility and advices, andto M. Boutry and X. Draye for their critical reading of themanuscript.

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Localization and quantification of plasma membrane aquaporin expression in maize primary root: a clue to understanding their role as cellular plumbersAbstractIntroductionMaterials and methodsPlant materialIn situ reverse transcriptase-mediated PCRDetection of apoplastic barriersQuantitative RT-PCRRoot cell protoplast isolationGST-ZmPIP-H expressionSubcellular fractionationProtein analysisIn situ immunolocalizationResultsExpression of ZmPIP mRNAs in aeroponically grown maize rootsDevelopment of apoplastic barriersExpression of ZmPIPs according to root developmental stageFig1Fig2Expression of ZmPIP genes in the cap, meristem, and elongation zonesFig3Tab1Fig4ZmPIP protein levels in rootsFig5In situ immunolocalization of ZmPIP2;1 �and ZmPIP2;5Fig6Fig7Osmotic water permeability coefficients of root protoplastsDiscussionZmPIP genes are widely expressed in the maize primary rootFig8ZmPIP expression in the root is developmentally regulatedFig9Differential distribution of ZmPIP2;1/2;2 �and ZmPIP2;5 in the primary rootRoot protoplasts display a low water permeabilityConclusionAcknowledgementsReferencesCR1CR2CR3CR4CR5CR6CR7CR8CR9CR10CR11CR12CR13CR14CR15CR16CR17CR18CR19CR20CR21CR22CR23CR24CR25CR26CR27CR28CR29CR30CR31CR32CR33CR34CR35CR36CR37CR38CR39CR40CR41CR42CR43CR44CR45CR46CR47CR48CR49CR50CR51CR52CR53CR54CR55CR56CR57CR58CR59CR60CR61CR62

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