Accepted Article 1

Differential responses of plasma membrane aquaporins in

2

mediating water transport of cucumber seedlings under

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osmotic and salt stresses1

4

ZHENG-JIANG QIAN1,2#, JUAN-JUAN SONG1#, FRANÇOIS CHAUMONT3 and

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QING YE1,3*

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1

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South China Botanical Garden, Chinese Academy of Sciences, 723 Xingke Road,

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Tianhe District, Guangzhou 510650, PR China

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2

Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems,

University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, P R

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China

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3

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4-L7.07.14, B-1348 Louvain-la-Neuve, Belgium

Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud

13 14

#

These two authors contributed equally to this work.

15 16

*Author for Correspondence:

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Qing Ye

18

Tel: +86-020-37083320

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Fax: +86-020-37252615

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E-mail: [email protected]

21 22

Running head: Role of aquaporins in plant water transport

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pce.12319 1

This article is protected by copyright. All rights reserved.

ABSTRACT

24

It has long been recognized that inhibition of plant water transport by either osmotic

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stress or salinity is mediated by aquaporins (AQPs), but the function and regulation of

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AQPs are highly variable among distinct isoforms and across different species. In this

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study, cucumber seedlings were subjected to PEG or NaCl stress for duration of 2h or

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24h. The 2h treatment with PEG or NaCl had non-significant effect on the expression

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of plasma membrane AQP (CsPIPs) in roots, indicating the decrease in hydraulic

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conductivity of roots (Lpr) and root cells (Lprc) measured in these conditions were due

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to changes in AQP activity. After both 2h and 24h PEG or NaCl exposure, the

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decrease in hydraulic conductivity of leaves (Kleaf) and leaf cells (Lplc) could be

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attributed to a down-regulation of the two most highly expressed isoforms, CsPIP1;2

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and CsPIP2;4. In roots, both Lpr and Lprc were further reduced after 24h PEG

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exposure, but partially recovered after 24h NaCl treatment, which were consistent

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with changes in the expression of CsPIP genes. Overall, the results demonstrated

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differential responses of CsPIPs in mediating water transport of cucumber seedlings,

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and the regulatory mechanisms differed according to applied stresses, stress durations,

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and specific organs.

Accepted Article 23

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Key-words: aquaporin; hydraulic conductivity; osmotic stress; plasma membrane

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intrinsic protein; pressure probe; salinity

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-2-

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Accepted Article 44

Abbreviations:

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CPP

cell pressure probe

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gs

stomatal conductance

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w

leaf water potential

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Kleaf

leaf hydraulic conductivity

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Lplc

leaf cell hydraulic conductivity

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Lpr

root hydraulic conductivity

51

Lprc

root cell hydraulic conductivity

-3-

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INTRODUCTION

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Aquaporins (AQPs) are trans-membrane proteins that facilitate rapid and passive

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water transport across cell membranes in virtually all living organisms (Maurel et al.

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2008; Gomes et al. 2009; Alleva et al. 2012). Plant AQPs show a large diversity of

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isoforms which can be divided into seven subfamilies: plasma membrane intrinsic

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proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26 like intrinsic proteins

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(NIPs), small intrinsic proteins (SIPs), GlpF-like intrinsic proteins (GIPs), hybrid

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intrinsic proteins (HIPs) and X-intrinsic proteins (XIPs) (Johanson et al. 2001;

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Gustavsson et al. 2005; Danielson & Johanson 2008). Among them, the PIPs

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constitute the largest number of AQPs and are further divided into PIP1 and PIP2

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subgroup according to their amino acid sequence similarity. When heterologously

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expressed in Xenopus laevis oocytes or yeast, PIP2s rather than PIP1s display a high

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water permeability (Chaumont et al. 2000; Suga & Maeshima 2004), but

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co-expression of inactive PIP1s with active PIP2s induced a significant increase in the

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oocyte membrane permeability compared to the cells expressing PIP2s alone (Fetter

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et al. 2004). It has been found that a number of internal (metabolic) factors including

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cytosolic pH and pCa, protein phosphorylation, reactive oxygen species (ROS) and

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plant stress hormone abscisic acid (ABA) regulate the water transport activity of

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AQPs (Guenther et al. 2003; Tournaire-Roux et al. 2003; Verdoucq et al. 2008; Ye &

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Steudle 2006; Boursiac et al. 2008). On the other hand, the activity and expression of

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AQPs can be affected by many external (environmental) stimuli such as osmotic

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stress, salinity, temperature, hypoxia and heavy metals (Zhang & Tyerman 1999; Ye,

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Wiera & Steudle 2004; Lee et al. 2012; Hachez et al. 2012; Muries et al. 2013). Of

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these environmental factors, both osmotic stress and salinity can directly induce water

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deficit to plants, thus great attention has been paid to their effects on plant water

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transport and AQP expression.

Accepted Article 52

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A large number of experimental studies have shown that plant water transport is

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usually inhibited by osmotic stress (Aroca et al. 2012; Wan 2010b). For instance,

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Mahdieh et al. (2008) reported that the sap flow rate and osmotic hydraulic -4-

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conductance decreased in Nicotiana tabacum roots after 24h of polyethylene glycol

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(PEG) treatment, and that NtPIP1;1 and NtPIP2;1 mRNA levels were significantly

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down-regulated. By contrast, Hachez et al. (2012) observed that root hydyraulic

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conductivity (Lpr) of maize was inhibited after 2h of PEG treatment, but the

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expression of ZmPIPs mRNA and protein and root cell hydraulic conductivity (Lprc)

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were increased. Similarly, Zhang et al. (2007) found that the exposure of Jatropha

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curcas to different concentrations of PEG for 24h resulted in a reduction in Lpr and an

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accumulation of JcPIP2 protein.

Accepted Article

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In parallel, salinity (salt stress) has long been recognized as an inhibiting factor of

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plant water uptake (Shalhevet et al. 1976; Azaizeh & Steudle 1991). Boursiac et al.

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(2005) found that Lpr of Arabidopsis rapidly decreased after 1 h of salt (NaCl) stress,

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a process accompanied with a down-regulation of the mRNA and protein level of

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most AQPs. A similar reduction in Lpr and the expression of most HvPIPs was

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observed in Hordeum vulgare treated with NaCl (Horie et al. 2011). In contrast to the

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down-regulation of AQPs, salt treatment up-regulated maize ZmPIPs mRNA levels,

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while Lpr decreased (Marulanda et al. 2010). In broccoli plants, Lpr was inhibited by

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salt stress, but BoPIP2 protein abundance increased (Muries et al. 2011).

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Although it is evident that both osmotic and salt stresses have inhibiting effects

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on plant water transport in common, the associated responses, namely the function

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and regulation of AQPs to these stresses are highly variable among distinct isoforms

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and across different plant species. In order to have a straightforward comparison of

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how plants respond to osmotic and salt stresses in terms of water transport and AQP

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mediation, we applied in the present study both osmotic and salt stresses to the same

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plant species, i.e., Cucumis sativus. In brief, cucumber seedlings were treated with

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PEG- or NaCl-induced stress at equal osmotic strength (i.e. osmotic concentration) for

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2h or 24h time period. Hydraulic conductivity of both roots and leaves in response to

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the stresses were measured using a pressure chamber technique, and water

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permeability across cell membranes was monitored using a cell pressure probe. In

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parallel to plant hydraulic measurements, the alterations of PIP expression at both

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transcript and protein levels were investigated. Finally, a subsequent 24h recovery -5-

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experiment was carried out with plants that were already under osmotic or salt stress

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for 24h, both hydraulic properties and AQP expression were re-examined to ensure

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that effects caused by the stresses were completely reversible.

Accepted Article

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MATERIALS AND METHODS

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Plant material and culture condition

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Seeds of cucumber (Cucumis sativus L. “Yuexiu 3”, Guangzhou, China) were

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germinated in wet filter paper in covered petri dishes for 3 days at room temperature

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in the dark. Then the seedlings were transferred to a hydroponic culture plastic boxes

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(7 L) filled with modified Hoagland solution (pH ~6.1; 795 M KNO3, 603 µM

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Ca(NO3)2, 270 µM MgSO4 and 109 M KH2PO4; micronutrients: 40.5 M

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Fe(Ⅲ)-EDTA, 20 M H3BO3

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Na2MoO4). The nutrient solution was aerated with the aid of aquarium diffusers. After

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one week, the young plants were transferred to 37 L boxes (15 plants per box) filled

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with the same solution as described above. Nutrient solution was completely replaced

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weekly. The growing conditions in a growth chamber were 14 h light/10 h dark cycle,

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25/21℃, 65% humidity and a photon flux density of 200-300 µM m-2s-1.

.0 M MnSO4, 0.085 M ZnSO4 and 0.25M

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Plant treatments

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Thirty to thirty five days old cucumber plants were subjected to water stress induced

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by the addition of PEG 6000 (Sanland, Los Angeles, USA) (140 mM) or NaCl (70

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mM) to the hydroponic culturing solution, such that the osmotic strength of solutions

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containing either PEG or NaCl was identical as confirmed using an osmometer

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(OM806, Loser, Giessen, Germany). Plants were treated by solutions with either PEG

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or NaCl for a 2h and 24h time periods. After the 24h treatment, a subsequent 24h

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recovery experiment was conducted by placing the stress-treated plants into normal -6-

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growing solution (without addition of PEG or NaCl). To prevent the potential effects

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of diurnal rhythm on hydraulic properties and AQP expression (Hachez et al. 2012),

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we used plants growing in normal culturing solution as control at each time point of

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the treatments (i.e., 2h, 24h treatment, and subsequent 24h recovery).

Accepted Article

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141 142

Measurements of root and leaf hydraulic conductivity

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Pressure chamber technique was used to determine hydraulic conductivity of roots

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(Lpr) and leaves (Kleaf) as described by Javot et al. (2003) and Postaire et al. (2010)

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with slight modifications.

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For Lpr measurements, shoots were cut off using a razor blade from the first node of

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the base, and the whole root system was inserted into a pressure chamber (PMS,

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Corvallis, Oregon, USA) filled with either normal growing solution for control plants,

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or solution containing PEG or NaCl for stress-treated plants. The hypocotyl was

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carefully threaded through the soft plastic washer of the metal lid. Using air gas,

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pressure (P) was slowly applied to the chamber in steps of 0.1 MPa up to 0.5 MPa.

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Exuded sap was collected in Eppenddorf tubes over 5 min periods for each stabilized

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pressure, and the rate of exuded sap water flow (Jv) was determined. When Jv was

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plotted against the applied P, a linear relationship was observed for P values between

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0.15 and 0.35 MPa (Fig. 1A). At the end of the measurement, the root system was

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removed and root dry weight (DW) was measured after oven-dried at 70 °C for 72h

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using a balance (FA2104N, Shanghai, China). Lpr (l s-1g-1MPa-1) of the roots was

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calculated from the slope of a linear regression line of Jv versus P, divided by the DW of

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the root system (Table S1).

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For Kleaf measurements, a single detached mature young leaf was inserted into a

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pressure chamber (PMS, Corvallis, Oregon, USA) filled with distill water. The petiole

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was carefully threaded through the soft plastic washer of the metal lid. Pressure (P) was

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applied to the chamber in steps of 0.1 MPa up to 0.5 MPa, using air gas. This resulted in

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a flow of liquid (Jv) entering through the leaf surface and exiting from the petiole -7-

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section. Jv was determined over successive 5 min periods for each stabilized pressure.

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When Jv was plotted against P, a linear relationship was observed for P values between

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0.2 and 0.4 MPa (Fig. 2A). At the end of the measurement, leaf surface area (S) was

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measured using a leaf area meter (Li-3000A; Li-Cor, Lincoln, Nebraska, USA). Kleaf (µl

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s-1m-2MPa-1) was calculated from the slope of a linear regression line of Jv versus P,

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divided by the S of the leaf (Table S1).

Accepted Article

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171 172

Measurements of cell hydraulic parameters

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Cell pressure probe (CPP) measurements were performed essentially as described by

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Javot et al. (2003) and Lee et al. (2012). Pulled glass micro-capillaries were beveled

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to a tip diameter of 5-7 µm, filled with silicon oil (type AS4; Wacker, Munich,

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Germany), and mounted vertically on a pressure probe.

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For root cortical cell hydraulic conductivity (Lprc) measurements, root segment was

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excised from plants grown in hydroponic conditions, and was placed on a metal

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sledge which was covered with filter paper. An aerated plant culturing solution (with

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or without the addition of PEG or NaCl) was circulated along the root segment to

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maintain hydration. Cortical cells from 2nd to 4th layer and at 5-8 cm distance from the

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root apex were punctured using a CPP. As cells were punctured, cell sap entered the

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oil-filled micro-capillary forming a meniscus between cell sap and oil. Cell turgor was

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restored by gently pushing the meniscus to a position close to the surface of the root,

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and the values of cell turgor pressure (P) were recorded by a computer. The half time

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(T1/2) of hydrostatic water flow across cell membrane, which is inversely proportional

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to cell hydraulic conductivity (T1/2  1/Lp) was obtained from pressure relaxation

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curves with the aid of the probe (Ye et al. 2006). The use of detached root segments in

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Lprc measurements was for the sake of easy manipulation in cell probing experiments.

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In this study, T1/2 measurements for a given cell were usually finished within 10min

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after root excision, and no difference in water uptake was found between cells of

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excised root segments and roots of intact plants (Fig. S1). Lprc was calculated from

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the measured T1/2 according to the following equation: -8-

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Accepted Article

Lp =

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V ln(2)  w . A T1/2 (   i )

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Here, V = cell volume; A = cell surface area; i = osmotic pressure of cell sap; = cell

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elastic modulus. iwas calculated from the initial cell turgor (P0), as P0 = i - 0 (0 =

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osmotic pressure of the medium as measured with an osmometer); elastic modulus (

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was determined from relative change of cell volume (ΔV/V) and the instantaneous

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change of cell turgor (ΔP) as:

 =V 

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P . V

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Water exchange across cell membrane so fast that this interfered with determination

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ofΔP/ΔV and the pressure change induced by an imposed volume change through

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the pressure probe was underestimated, since water started to exchange across

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membranes before the volume change was completed. Therefore,  should be

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corrected for fast water flow. Following Volkov et al. (2007), the corrected  ( corr)

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was calculated as:

 corr =  

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ln(2)  (t / T1/2 ) , 1  exp-[ ln(2)  (t /T1/2 ) ]

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where t was the time required to complete a pressure change prior to the pressure

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relaxation used to determine T1/2. The Lp of cells was calculated using  corr (Table S2,

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S3).

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For leaf cell hydraulic conductivity (Lplc) measurements, a mature young leaf

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blade (still attached to the plant) was fixed onto a metal support and leaf cells were

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punctured using a CPP (Ye et al. 2008). Upon a successful puncture, cell turgor was

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restored by gently moving the meniscus to a position close to the surface of the leaf.

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Water relation parameters such T1/2, ,and Lplc were determined as described above

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for root cell measurements (Table S2,S3).

217 218

Measurements

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conductance

of

leaf

water

potential

and

stomatal

-9-

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At each time point of the PEG or NaCl treatment, leaf water potential (w) was

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determined on the second fully expanded young leaf, using a pressure chamber (PMS,

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Corvallis, Oregon, USA). Stomatal conductance (gs) was measured on the abaxial

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surface of the second fully expanded young leaf with a portable leaf porometer

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(Delta-T Devices, Cambridge, UK).

Accepted Article

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225 226

RNA extraction and identification of CsPIP genes

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Total RNA from cucumber roots was extracted using TRIZOL Reagent (Invitrogen,

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USA) according to the manufacturer’s instructions. The concentration of RNA was

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quantified by spectrophotometrical measurement at λ=260 nm, and its integrity was

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checked on agarose gels. First strand cDNA was synthesized from 2 µg of total RNA

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and amplified by 3’ rapid amplification of cDNA ends polymerase chain reaction

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(RACE-PCR) such that a full-length of cDNA including 3’ untranslated region can be

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isolated. The RACE-PCR products were cloned and sequenced. Multiple sequence

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alignment of the deduced amino sequence was performed on Clustalx 1.83, and a

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phylogenetic tree was constructed using Neighbor-Joining method in Mega 5 (Fig.

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S2).

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Heterologous expression of CsPIPs cDNA in Xenopus oocytes

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The cDNAs encoding CsPIPs were amplified by PCR using isoform specific primers,

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containing USER cloning tails (Nour-Eldin et al. 2006), i.e., forward primer:

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(5’-GGCTTAAU + sequence complementary to target DNA-3’); reverse primer:

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(5’-GGTTTAAU + sequence complementary to target DNA-3’), and cloned into PacI

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cassette-containing pSP64T-derived BS vector carrying the 5’- and 3’-untranslated

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sequences of the b-globin gene from Xenopus laevis (pXbGev2). Clones with an

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insert in the correct orientation were identified by restriction mapping and sequencing.

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In vitro capped RNA (cRNA) synthesis, oocyte isolation, microinjection of cRNA and - 10 -

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osmotic water permeability (Pf) measurements (oocyte swelling assay) were

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performed as described in Fetter et al. (2004).

Accepted Article

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249 250

Quantitative real-time PCR (q-PCR) analyses

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Roots or leaves of cucumber plants were collected after each time period of PEG or

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NaCl treatments (2h and 24h treatments, and subsequent 24h recovery), and samples

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were immediately frozen in liquid nitrogen and stored at –80 °C until use. Total RNA

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extraction, concentration and integrity were determined as described above.

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First-strand cDNA for q-PCR analyses was synthesized from 2 µg of total RNA using

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PrimeScript RT reagent Kit (TaKaRa, Dalian Division) according to the

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manufacturer’s instructions, including a special step for genomic DNA digestion.

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Quantitative real-time PCR (qPCR) experiments were conducted on ABI 7500

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Real-Time PCR system using SYBR Premix Ex TaqTM Ⅱ Kit (TaKaRa, Dalian

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Division) with CsPIP gene specific primers (Table S5). The reaction mixture had a

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final volume of 20 µl, containing 10 µl 2 × SYBR Premix Ex TaqTMⅡ, 0.4 µM of

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each primer 0.4µl 50 × ROX Reference Dye Ⅱ and 2 µl of 10 fold dilution cDNA.

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The PCR conditions were as follow: 30 s at 95 ℃ for pre-denaturation; 40 cycles of

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5 s at 95 ℃, 34 s at 60 ℃. The melt-curve analysis was conducted using the method

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as recommended by the manufacturer of ABI 7500 system. For each qPCR

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experiment, no cDNA-template controls and no reverse transcript RNA-template

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controls were performed to ensure that reagents and RNA samples were free of

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genomic DNA contamination (Beaudette et al. 2007). The amplifications were

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performed on three independent samples for each treatment (biological replicates) and

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triplicate reactions were carried out for each sample (technical replicates), in 96-well

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plates. To ensure equivalent PCR efficiency for the amplification of each CsPIPs,

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standard curves (log of cDNA dilution vs. Ct) using serial10-fold dilution of cDNA

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were built for each pair of primers (Hachez et al. 2006). The results were normalized

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by the geometric mean of the expression level of three reference genes, i.e., alpha

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tubulin (TUB; gi: 54287294), elongation factor 1- (EF; gi: 127951062) and ubiquitin - 11 -

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(UBI; gi: 34581768), according to Wan et al. (2010a). The relative expression of

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CsPIPs was calculated using the 2-△△Ct method (Livak & Schmittgen 2001; Pfaffl

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2001).

Accepted Article

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279 280

Protein extraction and western blotting assay

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Plasma membrane proteins were extracted as described by Hachez et al. (2012). After

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each time period of osmotic or salt treatment, roots or leaves of cucumber seedlings

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were collected and were immediately frozen in liquid nitrogen and stored at –80 °C

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until use. The samples were homogenized in ice-cold extraction buffer (250 mM

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sorbitol, 50 mM Tris–HCl pH8.0, 0.2 mM EDTA). All subsequent steps were

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performed at 4 °C. The homogenate was centrifuged for 5 min at 5,000g, and the

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resulting supernatant was centrifuged for 10 min at 10,000g. The second supernatant

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was then centrifuged at 100,000g for 30 min and the resulting pellet was re-suspended

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in suspension buffer (5 mM H2PO4, 330 mM saccharose, 3 mM KCl, pH 7.8). The

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protein concentration of the extraction was determined by Bradford assay (GENIOS,

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Tecan, Austria), using BSA as standard.

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Equal amounts (15 µg) of proteins were loaded for 12% sodium dodecyl

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sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein samples had

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been previously denatured by incubating them at 80℃ for 5 min in loading buffer (50

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mM Tris/HCl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 1%

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β-mercaptoethanol). After electrophoresis, the gel was incubated for 5 min in transfer

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buffer (25mM Tris, 192mM glycine, 20% methanol) before transferring the protein to

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a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The PVDF membrane

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had been previously incubated for 1 min in pure methanol and 5 min in transfer buffer,

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separately. Protein transfer was performed at 300 mA for 80 min. Following protein

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transfer, the membrane was blocked for 2h at room temperature in Tris-Buffered

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saline with 0.1% Tween 20 (TBST), containing 5% non-fatted dry milk (blocking

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solution). After that, the membrane was incubated for 2h at room temperature in

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TBST blocking solution with antibodies raised against 16 amino acid N-terminal - 12 -

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peptides of CsPIP1s (MEGKEEDVRLGANKFN) and 15 amino acid C-terminal

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peptides of CsPIP2s (SFGAAVIFNKEKAWD). The dilutions used were 1/4000 and

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1/2000 for anti-CsPIP1s and anti-CsPIP2s antisera, respectively. After 3×10min

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washes in TBST solution, the membrane was incubated for 1h at room temperature

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with horseradish peroxidase coupled goat anti-mouse IgG antibody (dilution 1/10000

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in TBST blocking solution). The immune-blotting signals were detected using a

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chemiluminescent substrate (West-Pico, Super Signal; Pierce, Rockford). Film with

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signals were scanned and protein band was quantified using Gel-Pro analyzer 4.0

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(Media Cybernetics, USA).

Accepted Article

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314 315

Statistical analyses

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Statistical analyses were performed with the SPSS version 13.0 software 261 package

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(SPSS, Chicago, Illinois, USA) for Windows. Data are presented as mean values ±

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SD of three independent experiments. Differences between treatments were analyzed

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by one way ANOVA, taking P < 0.05 as a significant difference.

320 321

RESULTS

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Changes in root and leaf hydraulic properties in response to

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osmotic and salt stresses

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Two and 24h exposure of cucumber plants to PEG induced a significant reduction in

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Lpr by 43.2 and 74.3%, respectively, as compared with control plants. By contrast, Lpr

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of plants treated with NaCl for 2h decreased by 46.9%, while upon 24h NaCl

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treatment, Lpr showed a reduction of 39.7% (Fig. 1B). Similar patterns were found for

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Lprc. As compared with control plants, Lprc was inhibited by 40.2% and 69.0% for 2h

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and 24h PEG treatment, respectively. While NaCl exposure resulted in a reduction in

330

Lprc by 44.9% and 20.1% after 2h and 24h, respectively (Fig. 1C). Regardless of PEG - 13 -

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or NaCl treatment, both Lpr and Lprc recovered to the control level upon a subsequent

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recovery application by placing stress-treated plants back into normal culturing

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solution for 24h (Fig. 1B, C).

Accepted Article

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334

Concerning Kleaf, a reduction of 25.1% and 39.2% was observed after 2 and 24h

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PEG exposure, respectively, compared with control plants. Similarly, plants treated

336

with NaCl for 2 and 24h showed a reduction of Kleaf by 22.6% and 35.3%,

337

respectively (Fig. 2B). As compared with control plants, Lplc was inhibited by 13.7%

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and 37.0% for 2h and 24h of PEG treatment, respectively. While NaCl exposure

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resulted in 12.5% and 32.9% reduction of Lplc after 2h and 24h, respectively (Fig. 2C).

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Placing stress-treated plants back into the normal culturing solution restored both the

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Kleaf and Lplc to the control levels (Fig. 2B, C).

342 343

Changes in leaf water potential, stomatal conductance and leaf

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cell turgor in response to osmotic and salt stresses

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Compared with non-stressed plants, 2h treatment with PEG or NaCl induced a decline

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in w from -0.30 MPa to -0.56 and -0.55 MPa, respectively. However, the decrease in

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w was reduced after 24h treatment with PEG (-0.50 MPa) or NaCl (-0.44 MPa) as

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compared to control (-0.32 MPa) (Fig. 3A). The gs decreased to 47.2% and 52.4% of

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the control plants after 2h of osmotic and salt stresses, respectively. After 24h of

350

treatment, gs of PEG-treated plants further decreased to 37.8% of the non-stressed

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plants, while gs of salt-treated plants was partially recovered and reached 67.3% of the

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control plants (Fig. 3B). Leaf cell turgor displayed a significant drop to 51.5% and

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52.3% of the control plants after 2h PEG and NaCl exposure, respectively. A partial

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restoration of leaf cell turgor was observed in response to PEG (69.1% of the control

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plants) and NaCl (72.9% of the control plants) after 24h treatment (Fig. 3C).

356

Regardless of osmotic or salt stress applied to the plants, w, gs and leaf cell turgor

357

were restored to the level of non-stressed plants after 24h of recovery experiments

358

(Fig. 3).

359 - 14 -

This article is protected by copyright. All rights reserved.

Expression of CsPIPs in roots and leaves and functional tests

361

in Xenopus laevis oocytes

362

Based on the cucumber genome (Huang et al. 2009), ten CsPIP genes of the PIP

363

family were identified as full-length sequences, and according to their amino acid

364

sequence similarity, the isolated CsPIP genes in this study are three CsPIP1s and

365

seven CsPIP2s (Fig. S2). Quantitative real-time PCR (q-PCR) analyses with gene

366

specific primers (Table S5) revealed that the ten CsPIP genes were expressed in

367

cucumber roots and leaves. Of these genes, CsPIP1;2 and CsPIP2;4 were the most

368

highly expressed ones that accounted for about 81% and 73% of the total expression

369

of CsPIPs in roots and leaves, respectively (Table. 1).

Accepted Article 360

370

To determine the water transport activity of each CsPIP, the membrane osmotic

371

water permeability coefficient (Pf) of the oocytes expressing each CsPIP cRNA was

372

obtained from the rate of oocyte swelling. As shown in Fig. 4, oocytes injected with

373

CsPIP1 cRNAs showed no change in Pf as compared with water-injected oocytes. By

374

contrast, CsPIP2s expressing oocytes displayed significant increases in Pf, indicating

375

that CsPIP2s formed functional water channels in the cell membranes. As it was

376

previously shown that PIP1s required PIP2s to be targeted to the plasma membrane

377

and act as active water channels (Fetter et al. 2004), we co-expressed CsPIP1;2 with

378

either CsPIP2;4 or CsPIP2;5 genes in oocytes, and observed significant increase of Pf

379

in oocytes co-injected with cRNAs encoding the two isoforms (Fig. 4), implying a

380

positive interaction between PIP1s and PIP2s.

381

382

Changes in CsPIP mRNA levels in response to osmotic and

383

salt stresses

384

To investigate the responses of CsPIP genes to osmotic and salt stresses, the

385

expression of the ten CsPIP genes at transcriptional level were quantified using

386

q-PCR with specific primers (Table S5). As compared with control plants, the mRNA - 15 -

This article is protected by copyright. All rights reserved.

levels of the ten CsPIP genes in roots were not significantly altered after 2h PEG

388

treatment. Similar expression patterns of CsPIP genes were found in plants treated by

389

NaCl for 2h, with the exception of CsPIP2;2 and CsPIP2;3, which showed a two fold

390

increase in expression compared with that of control plants (Fig. 5A). By contrast, a

391

significant decrease in most CsPIP transcript levels was observed after 24h PEG

392

treatment, while the 24h NaCl exposure induced a significant increase in most CsPIP

393

mRNA levels (Fig. 5A). In leaves, 2h treatment of PEG or NaCl resulted in a

394

significant increase in CsPIP1;1, CsPIP2;1 and CsPIP2;3 expression levels, a

395

significant decreases in CsPIP1;2 and CsPIP2;4 expression, and no significant

396

changes for the rest of the isoforms compared with control plants (Fig. 5B). The

397

expression pattern of CsPIP genes after 24h treatment with PEG or NaCl was similar

398

to that of 2h treatment, with the exception of CsPIP1;3 and CsPIP2;7, whose

399

expressions exhibited a significant increase (Fig. 5B). The subsequent recovery

400

experiments resulted in the restoration of CsPIP mRNA levels to the control values in

401

both roots and leaves (Fig. 5A, B).

Accepted Article

387

402

403

Changes in CsPIP protein abundance in response to osmotic

404

and salt stresses

405

The effects of the osmotic and salt stresses on CsPIP protein abundance in both roots

406

and leaves were investigated by western blotting using antibodies raised against

407

CsPIP1s and CsPIP2s, respectively. Immunoblot analyses of total root and leaf protein

408

extracts revealed a major band with a molecular mass around 30 kD, corresponding to

409

the monomeric form of the CsPIP proteins (Figs. 6A, 7A). As compared with control

410

plants, CsPIP1 and CsPIP2 protein abundance in roots was not significantly altered

411

when plants were treated for 2h with PEG or NaCl (Fig. 6). Likewise, the abundance

412

of CsPIP1 protein remained unchanged after 24h of PEG and NaCl treatments.

413

However, the abundance of CsPIP2s showed a significant reduction (by 33.4%) in

414

response to 24h PEG treatment, but a significant increase (by 57.9%) upon 24h of - 16 -

This article is protected by copyright. All rights reserved.

NaCl exposure as compared with control plants (Fig. 6B). In leaves, 2h treatment of

416

PEG or NaCl had no significant effects on protein abundance of CsPIP1s and CsPIP2s

417

(Fig. 7). After 24h of PEG or NaCl exposure, CsPIP1 abundance was maintained as

418

compared with control plants, whereas CsPIP2 abundance decreased by 34.6% (PEG)

419

and 24.5% (NaCl), respectively (Fig. 7B). Consistent with the expression patterns of

420

the transcripts, protein abundance of CsPIPs recovered to the control level after the

421

subsequent recovery experiments (Figs. 6 and 7).

Accepted Article

415

422 423

DISSCUSSION

424

Our results showed that 2h treatment with PEG or NaCl led to a significant

425

reduction of cucumber plant hydraulic conductivity to a similar extent at both the

426

organ (root and leaf) and the cell level as compared with that of non-stressed plants

427

(Figs. 1 and 2). The results are consistent with a number of recent studies with

428

different plant species. For instance in barley, a similar reduction in Lpr was found

429

when plants were treated with NaCl or sorbitol solution having similar osmotic

430

strength (Horie et al. 2011). By applying salt or osmotic stresses with an equal

431

osmotic strength to corn seedlings, Wan (2010b) found a similar reduction in Lp of

432

root cortical cells. Salinity may have two adverse effects on plants: osmotic stress and

433

ionic toxicity (Munns & Tester 2008). Since the decline of plant hydraulic

434

conductivity occurred fairly quickly in response to salt stress, it was proposed that the

435

effect of salt stress on plant water transport might be through the osmotic component

436

(i.e., osmotic stress), when using low salinity (Silva et al. 2008) and at the early stage

437

of the salt treatment (Wan 2010b). Besides the similar reductions of plant hydraulic

438

conductivity, we showed that the w, gs and leaf cell turgor decreased to a similar

439

level in response to 2h exposure to PEG and NaCl (Fig. 3), indicating that a

440

short-term salt treatment may have the same adverse effect on cucumber plants as a

441

PEG incubation, possibly through an osmotic shock caused by the stresses

442

(MacRobbie 2006; Aroca et al. 2012; Shavrukov 2013).

443

Despite the significant reductions in hydraulic conductivities of both root system - 17 -

This article is protected by copyright. All rights reserved.

and root cortical cells (Fig. 1B, C), the expression of CsPIPs in roots at both mRNA

445

and protein level was not significantly altered after the 2h treatment of PEG and NaCl

446

(Figs. 5A, 6). Thus, declines in Lpr could be attributed to changes in AQP activity

447

induced by the short-term treatments. To verify the involving of AQPs in water flow

448

through roots, Lpr was calculated from the value of Lprc (measured with a cell

449

pressure probe) according to the “concentric membrane model” (Bramley et al. 2009;

450

Jones et al. 1988). It turned out that the calculated Lpr is not significantly different

451

compared with Lpr measured using the pressure chamber technique (Table S4),

452

indicating that the majority of water flow in roots is indeed through the cell-to-cell

453

pathway, as demonstrated in leaves that the cell-to-cell pathway (mediated by AQPs)

454

dominates leaf water transport from xylem to epidermis (Ye et al. 2008). A variety of

455

mechanisms affecting AQP activity have been identified in plants subjected to

456

osmotic or salt stress, including phosphorylation/dephosphorylation (Johansson et al.

457

2000; Guenther et al. 2003; Horie et al. 2011), internalization (Boursiac et al. 2005;

458

Vera-Estrella et al. 2004), and oxidative gating by reactive oxygen species (ROS)

459

(Boursiac et al. 2008; Leshem et al. 2006). In addition, osmotic shock resulting from

460

the addition of PEG or NaCl might be sensed by AQPs, which in turn reduced cell

461

hydraulic conductivity (Hill et al. 2004).

Accepted Article

444

462

Unlike in roots, the expression patterns of CsPIP transcripts were variable in the

463

leaves after the 2h PEG or NaCl treatment, with an up-regulation of CsPIP1;1,

464

CsPIP2;1 and CsPIP2;3, a down-regulation of CsPIP1;2 and CsPIP2;4, and virtually

465

no change for the other CsPIPs tested in this study (Fig. 5B). Quite similar results

466

were found in the leaves of Arabidopsis (Jang et al. 2004) and rice (Lian et al. 2006)

467

under osmotic or salt stresses. Nevertheless, down-regulation of CsPIP1;2 and

468

CsPIP2;4, the two most highly expressed CsPIPs (Table 1) may have a great

469

contribution to the reduction of hydraulic conductivities at both the whole leaf (Fig.

470

2B) and cell level (Fig. 2C). However, it should be noted that the variable expression

471

patterns of CsPIP transcripts in leaves were not consistent with the CsPIP protein

472

abundance, the latter being quite stable after 2h PEG or NaCl treatment as compared

473

with that of non-stressed plants (Fig. 7). A number of studies have shown the - 18 -

This article is protected by copyright. All rights reserved.

alterations of AQP expression in response to osmotic or salt stress were not

475

synchronous between mRNA and protein levels (Boursiac et al. 2005; Parent et al.

476

2009; Hachez et al. 2012). This discrepancy might be due to post-transcriptional

477

regulations of AQPs (Prak et al. 2008; Santoni et al. 2006), resulting in different

478

turnover and recycling rates of mRNA and/or proteins (Lee et al. 2009; Li et al. 2011;

479

Muries et al. 2011; Hachez et al. 2012). In addition, the PIP antibodies used in this

480

study might recognize other isoforms than the ones we studied at the transcript levels,

481

potentially leading to an apparent discrepency between CsPIP expression at mRNA

482

and protein levels. Hence, it would be necessary to generate and use specific

483

antibodies that can discriminate the different CsPIP isoforms in future experiments.

Accepted Article

474

484

In contrast to further decreases of root Lp at both organ and cell levels in

485

response to 24h PEG treatment, the 24h exposure to NaCl resulted in a partial

486

recovery of Lp, especially at the cell level (Fig. 1). The expression patterns of CsPIP

487

transcripts were tightly correlated with the fluctuations of Lprc, namely the majority of

488

CsPIP genes were down-regulated in response to 24h PEG treatment, but

489

up-regulated in response to 24h NaCl exposure (Fig. 5A). Consistent correlations

490

were observed at the protein level, i.e., NaCl exposure resulted in a significant

491

accumulation of CsPIP2 proteins (up to 157.9% of the control value) and a marginal

492

increase in CsPIP1 proteins, whereas a further decrease of CsPIP protein amount was

493

induced by PEG treatment (Fig. 6). The up-regulation of CsPIP transcripts and the

494

accumulation of CsPIP proteins after 24h NaCl exposure may represent an effective

495

regulatory mechanism to restore and compensate water uptake in roots (Lopez-Perez

496

et al. 2009; Muries et al. 2011). It has been shown that an increase in the expression

497

of AQPs could contribute to root water uptake and leaf growth recovery of salinized

498

plants (Fricke et al. 2006; Zhu et al. 2005). A further support to this scenario was the

499

partial recovery of stomata conductance (gs) in response to 24h NaCl treatment (Fig.

500

3B), which could be due to the increase of Lpr (Fig. 1). Howerer, it should be noted

501

that CsPIP2 protein level in roots was actually higher after 24h NaCl treatment

502

compared with control plants (Fig. 6B), yet Lpr was still considerably lower after 24h

503

NaCl exposure compared with control plants (Fig. 1B). Possible reasons could be the - 19 -

This article is protected by copyright. All rights reserved.

accumulation of CsPIP2 protein alone in response to 24h NaCl exposure was

505

insufficient to increase the Lpr to a level higher than that of control plants. On the

506

other hand, the expression of CsPIP1 proteins was virtually not altered after 24h NaCl

507

treatment compared with control plants (Fig. 6B), which might result in the

508

insufficiency of interaction between PIP1s and PIP2s. Moreover, the non-specific

509

antibodies used in this study might recognize different CsPIP isoforms, resulting in an

510

overestimation of the contribution of CsPIP2 protein level to the Lpr recovery in

511

response to 24h NaCl treatment. In addition, long-term exposure to NaCl may result

512

in the accumulation of Na+ and/or Cl- in plants, which in turn showed adverse effects

513

on plant growth, development, and physiological metabolism (Munns & Tester 2008).

514

For instance, the elevated salt levels inhibited both primary and lateral roots growth

515

(He et al. 2005; Duan et al. 2013), and the reductions in leaf elongation, stomatal

516

density, and photosynthetic capacity were associated with the accumulation of Na+

517

and/or Cl- in plants (Fricke et al. 2006; Orsini et al. 2012; Tavakkoli et al. 2010).

518

Therefore, the detrimental effects of Na+ and/or Cl- could potentially influence Lpr,

519

despite the significant accumulation of CsPIP2 protein after 24h NaCl treatment.

Accepted Article

504

520

In leaves, 24h PEG or NaCl treatment caused further decreases of Lpl and Lplc

521

(Fig. 2). Although up-regulations of transcripts were found for CsPIP1;1, CsPIP1;3,

522

CsPIP2;1, CsPIP2;3, and CsPIP2;7, the most highly expressed CsPIP isoforms in

523

leaves (CsPIP1;2 and CsPIP2;4) were significantly down-regulated (Fig. 5B),

524

suggesting that these two CsPIPs play an important role in mediating leaf water

525

transport under osmotic and salt stresses, and that the down-regulation of these two

526

genes may assoiciated with the drecrease in CsPIP proteins in leaves after 24h PEG or

527

NaCl treatment (Fig. 7B).

528

It is worth noting that, as a consequence of the 24h recovery experiments,

529

hydraulic properties of cell, leaf and root, and whole plant (e.g., w and gs), as well as

530

the expression of CsPIPs at mRNA and protein levels were restored to the control

531

levels regardless of osmotic or salt stress (Figs. 1, 2, 3, 5, 6 and 7), suggesting the

532

strength and duration of the two stresses applied in the present study had no severe

533

adverse effects on the plants. Nevertheless, we noted that the plant materials used in - 20 -

This article is protected by copyright. All rights reserved.

this study are rather young cucumber seedlings (30-35 days old only). From an

535

application point of view, the physiological importance of current findings deserves to

536

be tested with extended experiments using adult plants.

Accepted Article

534

537

The swelling assays performed with CsPIP expressing Xenopus laevis oocytes

538

revealed that CsPIP2s had greater water transport activity than CsPIP1s (Fig. 4), as

539

found in a number of previous studies (Chaumont et al. 2000; Fetter et al. 2004; Suga

540

& Maeshima 2004). However, when co-expressed with PIP2;4 or PIP2;5 isoform,

541

PIP1;2 also showed significant water channel activities due to a positive interaction

542

between PIP1s and PIP2s (Fig. 4), as demonstrated in Fetter et al. (2004). Hence,

543

although a distinct response in protein abundance of CsPIP2s rather than CsPIP1s was

544

observed in parallel with the changes of hydraulic conductivities in both roots and

545

leaves after 24h PEG or NaCl exposure (Figs. 6 and 7), both CsPIP1s and CsPIP2s are

546

probably involved in regulating water transport in cucumber seedlings under osmotic

547

and salt stresses.

548

There is accumulating evidence that long-distance communications between roots

549

and shoots (and vice versa) play an important role for the adaptation of plants as a

550

whole to water stress (Schachtman & Goodger 2008; Sakurai-Ishikawa et al. 2011).

551

For instance, Christmann et al. (2007) found a rapid decline in leaf mesophyll cell

552

turgor pressure when Arabidopsis roots were subjected to sorbitol-induced osmotic

553

stress. The auhors attributed this phenomenon to a hydraulic signal that transmitted

554

from roots to shoots and consequently elevated leaf abscisic acid (ABA) content

555

causing the closure of stomata. Applying a shoots-topping treatment to grapevine and

556

soybean plants, Vandeleur et al. (2014) observed a significant reduction in root

557

hydraulic conductance that mediated by changes in AQP expression and activity, and

558

proposed a hydraulic signal propagated rapidly from shoots to roots potentially due to

559

the decline of plant transpiration and the release of xylem tension. It has been shown

560

that alterations of leaf transpiration may trigger rapid changes in root hydraulics

561

across multiple species (Vandeleur et al. 2009; Kudoyarova et al. 2011;

562

Sakurai-Ishikawa et al. 2011). We found no significant differences in Lprc measured

563

on excised root segments and roots of intact plants (Fig. S1), indicating the changes in - 21 -

This article is protected by copyright. All rights reserved.

root hydraulics caused by shoot decapitation may not involve changes in Lprc

565

(Vandeleur et al. 2014), at least within the time (10 min) of the cell pressure probe

566

measurements in the present study. Nevertheless, both osmotic and salt stresses

567

applied to roots promoted significant reductions in w, gs and leaf cell turgor pressure

568

(Fig. 3), which occurred rather rapidly following the imposition of the stresses (within

569

minutes). In parallel with the changes in w, gs and leaf cell turgor, significant

570

reductions in hydraulic conductivity of both leaf organ and cells were observed (Fig.

571

2), which were significantly correlated with AQP expression and/or activity in leaves

572

(Fig. 5B). The results implied that under osmotic or salt stress, a hydraulic signal

573

sensed by the roots was rapidly conveyed to the shoots and initiated the changes of

574

leaf hydraulics, although the chemical or physical identity of signal remains to be

575

elucidated.

Accepted Article

564

576 577

Conclusion

578

The integration of hydraulic measurements in cucumber roots and leaves at both

579

organ and cell levels and the expression patterns of PIP aquaporins from mRNA to

580

protein profiles, allowed comprehensive insights into the function and regulation of

581

AQP on plant water transport in response to osmotic and salt stresses. The short-term

582

(2h) exposure to PEG or NaCl resulted in a significant decline in Lpr and Lprc that

583

might be due to changes in AQP activity, whereas the decreases of Kleaf and Lplc could

584

be attributed to the down-regulation of two most highly expressed AQP isoforms in

585

leaves, CsPIP1;2 and CsPIP2;4. The 24h exposure of both PEG and NaCl induced

586

further reductions of Kleaf and Lplc, and the down-regulation of CsPIP1;2 and

587

CsPIP2;4 further supported the crucial role of these two isoforms in mediating leaf

588

hydraulics. In contrast to greater inhibitions of Lpr and Lprc after 24h PEG exposure,

589

the 24h NaCl treatment resulted in partial recovery of water transport in roots, which

590

were tightly correlated with the expression of CsPIP genes. The subsequent recovery

591

experiments restored both plant hydraulics and AQP expression to the control level,

592

indicating that the strength and duration of the stresses applied did not exert severe - 22 -

This article is protected by copyright. All rights reserved.

adverse effect to the plants. Our results demonstrated that CsPIPs are indeed involved

594

in mediating water transport of cucumber seedlings, and the regulatory mechanisms

595

differed depending on stress types (osmotic or salinity), stress durations (2h or 24h),

596

and specific organs (leaf or root).

Accepted Article

593

597

- 23 -

This article is protected by copyright. All rights reserved.

Accepted Article 598

ACKNOWLEDGEMENTS

599

This study was initiated in the laboratory of FC at Université catholique de Louvain,

600

and QY was supported by an individual Marie Curie European fellowship. This work

601

was funded by the National Natural Science Foundation of China (31070231), the

602

Chinese Academy of Sciences through its Hundred Talent Program, a South China

603

Botanical Garden-Shanghai Institute of Plant Physiology & Ecology Joint project, and

604

the Belgian National Fund for Scientific Research, the Interuniversity Attraction Poles

605

Programme-Belgian

606

Belgique-Actions de Recherches Concertées”. This paper is dedicated to the memory

607

of Ernst Steudle, an international leader in the research field of plant water relations

608

and our mentor, colleague, and friend.

Science

Policy

and

the

“Communauté

française

de

609 610

611

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A.R. & Kjellbom P. (2001) The complete set of genes encoding major

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834 - 31 -

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Table 1 Expression of each CsPIP isoform as a percentage of the total CsPIPs

836

expression in roots and leaves of cucumber seedlings. The data (mean ± SD) were

837

calculated from the q-PCR results of control plants with three independent

838

experiments.

Accepted Article

835

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CsPIP1-1 CsPIP1-2 CsPIP1-3 CsPIP2-1 CsPIP2-2 CsPIP2-3 CsPIP2-4 CsPIP2-5 CsPIP2-6 CsPIP2-7

Percentage contribution to total CsPIP expression (%) roots leaves 1.2 ± 0.7 0.1 ± 0.1 21.7 ± 7.5 14.0 ± 2.0 2.0 ± 0.8 8.0 ± 2.2 6.1 ± 3.7 0.5 ± 0.1 1.1 ± 0.3 3.1 ± 1.2 1.4 ± 0.3 1.1 ± 0.5 59.4 ± 3.7 59.1 ± 8.1 6.5 ± 1.8 10.9 ± 3.9 0.1 ± 0. 1 2.8 ± 0.6 0.5 ± 0.2 0.4 ± 0.1

- 32 -

This article is protected by copyright. All rights reserved.

Accepted Article

FIGURE LEGENDS Fig.1 Effects of osmotic (PEG) or salt (NaCl) stress on root hydraulic properties of cucumber plants. (A) Representative pressure-to-flow relationship measured in roots of control plant (○), PEG ( ) or NaCl ( ) treated plants. (B) Relative changes in hydraulic conductivity of roots in responses to PEG or NaCl exposure as compared with control plants. (C) Relative changes in hydraulic conductivity of root cortical cell in responses to PEG and NaCl treatments as compared with control plants. X axis represents time duration of stress (2h, 24h) and recovery (Re 24h) experiments. Results were expressed as a percentage of the mean value of control plants. Different letters above columns represent a significant differences between stress-treated and control plants (P

Differential responses of plasma membrane aquaporins in mediating water transport of cucumber seedlings under osmotic and salt stresses.

It has long been recognized that inhibition of plant water transport by either osmotic stress or salinity is mediated by aquaporins (AQPs), but the fu...
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