Constitutive overexpression of soybean plasma membrane intrinsic protein GmPIP1;6 confers salt tolerance
- Lian Zhou†1, 2,
- Chuang Wang†1,
- Ruifang Liu1,
- Qiang Han1,
- Rebecca K Vandeleur3,
- Juan Du1,
- Steven Tyerman3 and
- Huixia Shou1Email author
© Zhou et al.; licensee BioMed Central Ltd. 2014
Received: 12 May 2014
Accepted: 30 June 2014
Published: 7 July 2014
Under saline conditions, plant growth is depressed via osmotic stress and salt can accumulate in leaves leading to further depression of growth due to reduced photosynthesis and gas exchange. Aquaporins are proposed to have a major role in growth of plants via their impact on root water uptake and leaf gas exchange. In this study, soybean plasma membrane intrinsic protein 1;6 (GmPIP1;6) was constitutively overexpressed to evaluate the function of GmPIP1;6 in growth regulation and salt tolerance in soybean.
GmPIP1;6 is highly expressed in roots as well as reproductive tissues and the protein targeted to the plasma membrane in onion epidermis. Treatment with 100 mM NaCl resulted in reduced expression initially, then after 3 days the expression was increased in root and leaves. The effects of constitutive overexpression of GmPIP1;6 in soybean was examined under normal and salt stress conditions. Overexpression in 2 independent lines resulted in enhanced leaf gas exchange, but not growth under normal conditions compared to wild type (WT). With 100 mM NaCl, net assimilation was much higher in the GmPIP1;6-Oe and growth was enhanced relative to WT. GmPIP1;6-Oe plants did not have higher root hydraulic conductance (Lo) under normal conditions, but were able to maintain Lo under saline conditions compared to WT which decreased Lo. GmPIP1;6-Oe lines grown in the field had increased yield resulting mainly from increased seed size.
The general impact of overexpression of GmPIP1;6 suggests that it may be a multifunctional aquaporin involved in root water transport, photosynthesis and seed loading. GmPIP1;6 is a valuable gene for genetic engineering to improve soybean yield and salt tolerance.
KeywordsSoybean Aquaporins Salt tolerance Ovexpression Transformation GmPIP1;6
A significant proportion of cultivated land is salt affected representing about 2% of dry-land and 20% of irrigated agriculture (FAO Land and Plant Nutrition Management service, http://www.fao.org/nr/aboutnr/nrl/en/). Soil salinity arises from natural or human-induced processes that inhibits plant growth via osmotically induced water deficit and/or ion toxicity if excessive sodium (Na+) and chloride (Cl−) accumulate in the shoot via transpiration . Osmotic stress reduces the ability of the plant to extract water from the soil and growth will reduce rapidly and significantly as salt concentration around the roots increases past a threshold level. Ion toxicity occurs when salt (Na+ and Cl− ) gains entry via the transpiration stream and accumulates in the shoot to toxic concentrations resulting in injury to cells and causing further reductions in growth [1, 2]. Salt tolerance/sensitivity is indicated by the relative degree of biomass reduction in saline soil compared to plants in a non-saline soil, over an extended period of time . Clearly water flow is linked to both types of stresses induced by salinity, yet the role of water transport in plant salt tolerance is not yet clearly defined.
Plants have evolved three distinct mechanisms of salinity tolerance including osmotic adjustment to allow turgor to be maintained, Na+ and Cl− exclusion from leaf blades, and compartmentalization of Na+ and Cl− at cellular or intracellular sites . Numerous transporters have been identified as likely to be involved in Na+ and Cl− exclusion and compartmentation [1, 2, 4–7], but the proteins that transport water across membranes, the aquaporins, are not considered to be directly involved in these processes, though indirect effects could occur through their impact on osmotically driven water flow and pathways for water and solute flow in roots and leaves .
The radial flow of water from soil solution toward the root xylem encounters a relatively high resistance compared to subsequent axial flow in the xylem to the shoot. The radial flow pathway in the root consists of the apoplastic pathway along the intracellular spaces and the cell-to-cell pathway, in which water moves through plasmodesmata or across membranes . Apoplastic water flow can be blocked by Casparian bands and suberin lamellae at key cellular barriers such as the endo and exodermis [10, 11] where water transport across membranes occurs. Depending on the plant species and conditions, as well as the position along the root, there are variable contributions of the apoplast pathway compared to the cell-to-cell pathway . The conductance of the cell-to-cell pathway can be largely determined by the activity of aquaporins (AQPs) . AQPs are suggested to play a key role in plant water balance and water use efficiency [8, 13–17].
Aquaporins are members of the major intrinsic protein (MIP) family, which in plants are divided into five subfamilies that include the plasma membrane intrinsic proteins (PIPs). These are considered as the main water transport pathway across plasma membranes in root and leaf tissues that play important roles in plant water relations [8, 16–19]. According to the N terminal length of the proteins, the PIPs are further divided into two subclasses (PIP1 and PIP2). PIP1s require co-expression of PIP2s to show high water permeability in Xenopus laevis oocytes [20–27]. PIP1s and PIP2s interact affecting targeting to the plasma membrane [20, 21] and forming hetero-tetramers of variable stoichiometry that appears to affect their transport efficiency . Plant genomes have variable numbers of aquaporin genes, ranging from 35 in Arabidopsis thaliana, 33 in Oryza sativa and 66 in soybean, including 22 PIPs. Compared with other species, little is known about the function of AQP genes in soybean.
Aquaporins are clearly involved in water transport in roots and leaves  and have been linked to water uptake required for cell expansion [18, 26, 31–35]. Water is the carrier of Na+ and Cl− in the transpiration stream contributing to shoot ion toxicity, and in salinity-induced osmotic stress, free energy gradients need to be developed to drive water diffusion to the sites of cell expansion. In this context aquaporins could affect the root’s ion selectivity by determining the proportion of water that flows via membrane pathways relative to the apoplast, while in osmotic stress, they could allow continued water supply under diminished osmotic and pressure gradients by increasing membrane hydraulic conductivity.
Abiotic stresses such as salt, drought and cold influence the water balance of plants and the expression of AQP genes . Overexpression of several AQP genes in plants confers abiotic stress resistance. Overexpressing NtAQP1 in tobacco increased photosynthetic rate, water use efficiency and yield under salt stress . Overexpression of several wheat AQPs, including TaNIP, TaAQP8 and TaAQP7 genes in Arabidopsis or tobacco also increased salt tolerance or drought tolerance of the transgenic plants [37–39]. Recently, overexpression a MusaPIP1;2 in banana displayed high tolerance to multiple abiotic stresses including salt, cold and drought .
Soybean is a major source of protein and oil for humans and animals, yet relatively mild salt stress significantly reduces soybean growth, nodulation, seed quality and yield . Recently it was found that the expression of GmPIP1;6 in roots correlated with rapid and longer term changes in root Lo in response to shoot treatments and appeared to be more concentrated in stellar tissue . These results indicated that GmPIP1;6 may be important in the control of root water transport particularly in response to shoot signals. In this study, GmPIP1;6 was cloned and functionally characterized. Overexpression of GmPIP1;6 significantly increased salt tolerance of soybean by improving root Lo and Na+ exclusion.
Subcellular localization of GmPIP1;6
Expression patterns of GmPIP1;6
Generation of transgenic soybean overexpressing GmPIP1;6
Overexpression of GmPIP1;6enhances salt tolerance in soybean
Plant length and biomass of the WT and transgenic plants under normal and salt stress conditions
Plant length (cm)
Plant fresh weight (g)
22.2 ± 1.5ab
38.0 ± 2.1a
3.10 ± 0.13a
1.15 ± 0.09a
22.8 ± 0.8a
38.2 ± 2.5a
3.08 ± 0.08a
1.16 ± 0.11a
22.7 ± 0.5a
37.7 ± 2.2a
3.10 ± 0.15a
1.15 ± 0.06a
Salt stress condition
15.7 ± 1.0d
33.0 ± 4.0ab
1.72 ± 0.18c
0.67 ± 0.10b
19.0 ± 1.3bc
28.8 ± 3.7b
2.39 ± 0.17b
0.76 ± 0.10b
18.5 ± 1.6c
29.0 ± 3.1b
2.28 ± 0.07b
0.71 ± 0.07b
Overexpression of GmPIP1;6increased photosynthesis and root water conductance in soybean under salt stress conditions
Photosynthetic and root hydraulic characteristics of WT and transgenic plants under normal and salt stress conditions
Salt stress condition
gs (mol water m−2 s−1)
0.14 ± 0.01b
0.18 ± 0.01a
0.17 ± 0.02a
0.04 ± 0.01d
0.09 ± 0.01c
0.07 ± 0.01c
Tr (mmol water m−2 s−1)
6.02 ± 0.18b
6.93 ± 0.52a
6.76 ± 0.34a
1.83 ± 0.46e
3.99 ± 0.42c
3.12 ± 0.53d
AN (μmol CO2 m−2 s−1)
9.73 ± 1.26 b
13.29 ± 0.24a
12.17 ± 1.12a
3.31 ± 1.41d
8.26 ± 0.56bc
7.21 ± 1.74c
Ci (μmol CO2 mol−1)
295 ± 13a
271 ± 9b
278 ± 17b
279 ± 25d
243 ± 7c
236 ± 17c
Stomata pore aperture (μm)
3.48 ± 0.27b
3.88 ± 0.42a
3.86 ± 0.39a
1.93 ± 0.14d
3.00 ± 0.25c
2.96 ± 0.19c
Stomata density (0.1 mm2)
19 ± 3a
19 ± 3a
19 ± 4a
19 ± 3a
20 ± 4a
19 ± 3a
IWUE (mmol CO2 mmol−1 water)
1.61 ± 0.16c
1.92 ± 0.15b
1.88 ± 0.24b
1.73 ± 0.35bc
2.17 ± 0.09a
2.29 ± 0.22a
Under normal conditions, the substomatal concentration of CO2 (Ci) of GmPIP1;6-Oe was lower than that of WT though no significant difference was observed. In contrast, the Ci of GmPIP1;6-Oe was significantly lower than that of WT under salt treatment (Table 2, Figure 6D). This is in accordance with the higher rate of net photosynthesis of GmPIP1;6-Oe compared to WT plants under saline conditions. Instantaneous water use efficiency (IWUE = A/T) was significantly increased in GmPIP1;6-Oe plants under both normal and salt stress conditions compared with WT (Table 2). Changing stomata density and/or pore area will influence the gs and Tr. Examination of the abaxial leaf surface revealed a significantly wider stomatal aperture in GmPIP1;6-Oe plants under both normal and salt stress conditions while the stomata density was not changed (Table 2, Additional file 1: Figure S4A). As a result, the water loss rate was increased in the transgenic plants compared with WT plants (Additional file 1: Figure S4B).
We also measured root hydraulic conductance (Lo), normalized to root dry weight, in GmPIP1;6-Oe and WT plants. Interestingly the Lo was similar between GmPIP1;6-Oe and WT plants irrigated with nutrient solution. However when irrigated with nutrient solution containing 50 mM NaCl, Lo of WT plants decreased almost 50% while Lo of GmPIP1;6-Oe plants remained unchanged (Figure 5).
Overexpression of GmPIP1;6affects Na uptake and exclusion of transgenic plants under salt stress
Overexpression of GmPIP1;6increased yields of soybean in the field
Agronomic characteristics of WT, null transgenic and overexpression transgenic soybean plants in field
Plant height (cm)
83 ± 3a
87 ± 8a
83 ± 8a
89 ± 10a
83 ± 15a
87 ± 6a
4.6 ± 1.1a
5.6 ± 2.1a
4.7 ± 1.4a
4.8 ± 2.1a
5.4 ± 2.3a
5.0 ± 0.9a
24.6 ± 1.1a
24.7 ± 1.1a
23.7 ± 2.7a
24.3 ± 2.0a
23.2 ± 1.6a
22.7 ± 2.4a
175 ± 24ab
197 ± 40ab
195 ± 18ab
174 ± 28ab
200 ± 56a
166 ± 34ab
425 ± 62a
423 ± 78a
435 ± 45a
417 ± 69a
441 ± 175a
406 ± 93a
Seed weight (g)/plant
49.6 ± 12.1b
47.7 ± 14.2b
59.5 ± 6.5a
56.7 ± 8.2a
58.5 ± 23.0a
53.7 ± 12.4a
100 seed weight (g)
14.0 ± 0.5b
13.6 ± 1.6b
16.4 ± 0.8a
17.4 ± 1.8a
16.8 ± 1.7a
16.7 ± 1.0a
10 seed length (cm)
7.20 ± 0.07c
7.14 ± 0.11c
8.02 ± 0.08a
7.72 ± 0.08b
7.71 ± 0.08b
7.80 ± 0.09b
10 seed width (cm)
6.28 ± 0.13c
6.36 ± 0.11c
7.26 ± 0.05a
6.90 ± 0.07b
7.02 ± 0.08b
6.95 ± 0.10b
Recently, 66 AQP genes were identified in soybean by a genome wide analysis . The GmPIP subfamily contained 8 PIP1 genes and 14 PIP2 genes, all of which were predicted to localize on the plasma membrane. It is found that PIP2 aquaporins when expressed in Xenopus oocytes have high water permeability while PIP1 aquaporins do not. However, PIP1 aquaporins can work cooperatively with PIP2s in targeting to the plasma membrane and in water permeation as heterotetramers [20–27]. This is accordance with the fact that GmPIP1;6 protein fused with GFP localized on the plasma membrane (Figure 1).
GmPIP1;6 is the ortholog of AtPIP1;2, NtAQP1, HvPIP1;6/1;1 and TaAQP8 in Arabidopsis, tobacco, barley and wheat (Additional file 1: Figure S1). AtPIP1;2 and NtAQP1 play a key role in regulating root hydraulic conductance (Lo) in Arabidopsis and tobacco [17, 43, 44], respectively. In situ PCR showed that GmPIP1;6 was highly expressed in the stellar region of the root , which is similar as NtAQP1. Shoot topping rapidly decreased root hydraulic conductance (Lo) by 50% to 60%, which is correlated with the reduced expression of GmPIP1;6 in roots of soybean. Therefore, GmPIP1;6 was suggested to control the Lo as AtPIP1;2 and NtAQP1.
Water stress caused by drought, salt or cold has a complex effect on the expression of AQP genes . In summary, the expression of AQP genes could be divided into two stages. In the early stress response, the plant usually suppresses the expression of PIP genes, which is hypothesised to avoid water flow from the root to the soil when the soil water potential decreases [45, 46]. After a few days of acclimation, the expression of PIP genes recovers or even increases and is correlated with increased hydraulic conductance [47–49]. The expression of GmPIP1;6 in both roots and leaves showed this two stage response under salt stress (Figure 2B, Additional file 1: Figure S2), indicating GmPIP1;6 may be involved in the salt stress acclimation of soybean.
Overexpression of several PIP1 genes increased the hydraulic conductance and salt tolerance of the transgenic plants, such as NtAQP1, OsPIP1;1, TaAQP8 and MusaPIP1;2[17, 37, 40, 50]. Here we show that GmPIP1;6 conferred salt tolerance, but also under normal conditions the overexpression resulted in higher growth and greater yield under field conditions compared to WT plants (Figure 4A, Table 3). However, the mechanism of how these PIP1 genes can improve plant growth and salt tolerance is largely unknown, though a high K+/Na+ ratio was mentioned with overexpression of TaAQP8.
It is highly unlikely that GmPIP1;6 can transport Na+, therefore salt tolerance of transgenic GmPIP1;6 plants is more likely to occur through indirect mechanisms: First, improvement in water uptake by roots and leaf cell hydration, could improve energy capture and conversion by leaves. Greater energy availability in turn could improve Na+ exclusion by roots and improve tissue Na+ compartmentalization . We compared the root Lo of WT and GmPIP1;6-Oe plants under normal and salt stressed conditions (Figure 7). As expected, NaCl treatment decreased Lo by 50% in WT plants. In contrast, GmPIP1;6-Oe plants maintained Lo under salt stress conditions. Therefore, GmPIP1;6-Oe plants may have a higher water uptake activity than WT plants under saline conditions. Secondly, Na+ is transported to shoots in the transpiration stream through the xylem, but it can return to root via the phloem [43, 51–53]. Export of Na+ from leaves in the phloem could conceivably help to maintain low salt concentration in the leaves and may be enhanced by greater water permeability in phloem cells. Also we show that net assimilation and gas exchange are enhanced in the GmPIP1;6-Oe plants, and especially so under saline conditions compared to WT. This would potentially translate to a higher capacity to exclude Na+ via energy demanding salt exclusion mechanisms in the roots and the leaves. Thirdly, we measured the expression of GmNHX1 to analyze the effect of Na+ compartmentalization in the vacuole (Figure 8). Salt treatment induced the expression of GmNHX1 in the leaves and roots of WT but not in GmPIP1;6-Oe plants. This is accordance with the lower Na+ concentration of GmPIP1;6-Oe plants and indicated that vacuole compartmentalization of GmPIP1;6-Oe plants was not necessarily enhanced.
Another possibility that may account for reduced Na+ transport to the shoot in the over expressing plants could be that more water flow occurs radially across roots via the cell-to-cell (membrane) pathway, as opposed to the apoplast pathway. This would occur because of the higher activity of GmPIP1;6 in root membranes under salinity stress demonstrated by the higher root Lo compared to WT. A higher proportion of water flow via the membrane pathway in roots would confer a greater degree of ion selectivity relative to flow in the apoplast pathway. Altogether, we clarified that overexpression of GmPIP1;6 increased soybean salt tolerance by maintaining water uptake ability and Na+ exclusion.
In addition to function as a water channel, AtPIP1;2 and NtAQP1 may function to facilitate CO2 transport and enhance photosynthesis by increasing the mesophyll conductance to CO2 diffusion [55–59]. Overexpression of NtAQP1 in tobacco and tomato increased the AN, which resulted in increased WUE. The overexpression of NtAQP1 produced higher dry biomass and yield under normal irrigation and salt stressed conditions . GmPIP1;6-Oe plants also exhibited higher AN, gs and IWUE than WT under both normal and saline conditions (Figure 6, Table 2). However, the growth of GmPIP1;6-Oe plants was only enhanced under saline conditions compared to WT plants (Figure 4A, Table 1). Whether GmPIP1;6 has a similar function as NtAQP1 to facilitate CO2 diffusion across leaf cell membranes requires further research.
Importantly, GmPIP1;6-Oe plants showed higher yield in the field than WT because the seed weight and size of GmPIP1;6-Oe were increased (Table 3, Additional file 1: Figure S6). This may be reflecting the higher net assimilation, but also may indicate sink limitation of seed loading that could be enhanced by greater water permeability in the seed loading process . In addition to being highly expressed in roots and stems, the transcripts of GmPIP1;6-Oe were abundant in flower and pod, which supports a role of GmPIP1;6-Oe in seed loading of assimilates via enhanced water permeability.
In this study, the function of GmPIP1;6 was analyzed by constitutive expressing in the soybean plants. The expression of GmPIP1;6 was influenced by salt stress. Overexpression of GmPIP1;6 improved salt tolerance of transgenic plants by increasing water transport, photosynthesis and Na+ exclusion. Moreover, the yield of GmPIP1;6 overexpression plants was improved in the field indicating the potential of GmPIP1;6 in genetic engineering of soybean.
Plant materials, growth conditions and treatments
Soybean cultivar Williams 82 was used for all physiological experiments and soybean transformation. Seeds were germinated in nursery pots with sand. Five days after germination, the seedlings grown uniformly were transferred into pots with nutrient solution or soil. 1/2 Hoagland solution was used for hydroponic culture containing 2.5 mM KNO3, 2.5 mM Ca(NO3)2, 0.5 mM KH2PO4, 0.25 mM K2SO4, 1 mM MgSO4, 0.1 mM Fe-EDTA(Na), 4.57 μM MnCl2, 3.8 μM ZnSO4, 0.09 μM (NH4)6Mo7O24, 23 μM H3BO3, 1.57 μM CuSO4. Plants were grown in green house under 12 h light/12 h dark photoperiod with light intensity of 1000 μmol m−2 sec−1 and day/night temperatures of 30/22°C. Humidity of the growth room was controlled at approximately 30%.
Ten-day-old seedlings were transferred into nutrient solution with or without 100 mM NaCl. The nutrient solution was changed every two days. In the soil experiments, plants were irrigated nutrient solution every three days.
Subcellular localization of GmPIP1;6
Full length cDNA of GmPIP1;6 without stop code was amplified via PCR using the primers in Supplementary Additional file 2: Table S1. The PCR product was cloned into vector pCAMBIA1302 under the control of the CaMV 35S promoter. The resulting construct (pCAMBIA1302:GmPIP1;6) placed GmPIP1;6 in-frame, upstream of the sGFP. Plasmids DNA of pCAMBIA1302:GmPIP1;6 and CD3-1007 (AtPIP2A::mCherry fusion) was mix with 50 μl gold particles and bombarded into onion inner epidermal cells using the Biolistic PDS-1000/He particle delivery system (BIO-RAD). Fluorescence was observed by confocal laser scanning microscopy (LSM700; Carl Zeiss) after incubation at 25°C for 16-18 h on MS medium in dark.
Construction of transgenic plants
Full-length cDNA of GmPIP1;6 was amplified by PCR with cDNA of Williams 82 and ligated into pMD-18 T vector (Takara). After sequencing, the correct GmPIP1;6 was digested from pMD-18 T vector using BamHI and XbaI restriction enzymes. GmPIP1;6 was then cloned into binary plasmid pTF101-35S which was modified by introducing CaMV 35S promoter and nos terminator into pTF101. The vector was transformed into Williams 82 via Agrobacterium tumefaciens media soybean cotyledon node transformation system as described .
Total RNA was isolated from tissues of soybean cultivar Williams 82 using TRIzol reagent (Invitrogen, Carlsbad, CA) according the manufacturer’s instruction. 50 mg soybean tissues with three bilogical replicate were quickly harvested, frozen in liquid nitrogen and stored at -80°C. Contaminating DNA was removed with DNaseI treatment for 20 min at 25°C (Takara), and RNA was stored at -80°C. Total RNA was quantified with nanodrop.
Semi-quantitative RT-PCR and quantitative real-time PCR
First-strand cDNAs were synthesized from total RNA using SuperScript II reverse transcriptase (Invitrogen). Semi-quantitative RT-PCR was performed using a pair of gene-specific primers. The housekeeping gene GmACTIN was used as an internal control. Quantitative real-time PCR was performed using a SYBR Green I on a Light Cycler 480 II machine (Roche Diagnostics), according to the manufacturer’s instructions. The amplification program for SYBR Green I was performed at 94°C for 10 sec, 58°C for 10 sec and 72°C for 10 sec. Triplicate quantitative assays were performed on each cDNA sample. The relative level of expression was calculated using the formula 2 -△(△cp). All primers used for RT-PCR are given in Supplementary Additional file 2: Table S1.
Homozygous lines were selected from the T2 generation of transgenic GmPIP1;6 overexpression plants and used for the physiology experiment. AN, gs, Tr and Ci were recorded in GmPIP1;6 overexpression and control plants in green house on fully expanded leaves, using an Li-6400 portable gas-exchange system (LI-COR). All measurements were conducted between 8:00 AM and 4:00 PM. Photosynthesis was induced in saturating light (1000 μmol m−2 s−1) with 400 μmol mol−1 CO2 surrounding the leaf. The leaf-to-air VPD was kept at around 2 to 4 kPa and leaf temperature was approximately 30°C (ambient temperature) during all measurements. For each treatment, there were four biological replicates.
Stomata aperture and density
Epidermis of soybean abaxial leaf was separation by forceps. All samples were collected around 2:00 PM (at peak transpiration). Counting and photographing were performed with a bright-field microscope (80i; Nikon) mounted with a camera. Stomata images were later analyzed to count the number per 0.1 mm2 area and determine aperture using the microscope software (NIS elements) measurement tool. A microscopic ruler was used for the size calibration.
Leaves and root from 17-day old transgenic lines and WT were sampled and dried at 80°C for 3 days. 50 mg of the material was weighed and dissolved with 3 ml of nitric acid and 2 ml of H2O2 (30%). The digested samples were diluted to a total volume of 50 ml with ultrapure water and transferred into new tubes before analysis by using an inductively coupled plasma-mass spectrometer (ICP-MS, ELAN DRC-e).
To analyze the relative Na+ exclusion, Ten-day-old WT and GmPIP1;6-Oe transgenic soybean plants in hydroponics were treated with 100 mM NaCl for 7 days. Soybean plants after treatment were transferred into narrow neck flask individually, which filled with same volume of normal nutrient solution, and cultured for 24 hours. Na+ concentration of solution was measured by ICP-MS described above. The relative Na+ exclusion was calculated by the formula: relative Na+ exclusion = (Na+ concentration of solution × Volume of solution)/(Na+ concentration of shoot × DW of shoot).
Root hydraulic conductance
Root hydraulic conductance were measured with a hydraulic conductance flow meter (HCFM) (Dynamax, Houston, TX, USA) as described in Vandeleur . 5-week-old potted soybean plants grown in greenhouse. 1 day before root hydraulic conductance were measured, control plants were irrigated with normal nutrient solution and treatment plants were irrigated with nutrient solution containing 50 mM NaCl. Measurements were made between 10:00 AM to 12:00 AM. Hydraulic conductance (Lo) was normalized by dividing total root dry weight. The soil was washed from the roots, and baked at 80°C for 3 days.
This work was supported by National Science Foundation of China (31172024, 31201675), the Ministry of Science and Technology of China (2011CB100303, 2011ZX08004, 2014ZX0800401B ) and the Australian Research Council.
- Munns R, Tester M: Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008, 59: 651-681.View ArticlePubMedGoogle Scholar
- Munns R: Genes and salt tolerance: bringing them together. New Phytol. 2005, 167 (3): 645-663.View ArticlePubMedGoogle Scholar
- Colmer TD, Munns R, Flowers TJ: Improving salt tolerance of wheat and barley: future prospects. Aust J Exp Agri. 2005, 45 (11): 1425-1443.View ArticleGoogle Scholar
- Tyerman SD, Skerrett IM: Root ion channels and salinity. Sci Hortic. 1999, 78: 175-235.View ArticleGoogle Scholar
- Teakle NL, Tyerman SD: Mechanisms of Cl(-) transport contributing to salt tolerance. Plant Cell Environ. 2010, 33 (4): 566-589.View ArticlePubMedGoogle Scholar
- Horie T, Schroeder JI: Sodium transporters in plants. Diverse genes and physiological functions. Plant Physiol. 2004, 136 (1): 2457-2462.PubMed CentralView ArticlePubMedGoogle Scholar
- Tester M, Davenport R: Na + tolerance and Na + transport in higher plants. Ann Bot. 2003, 91 (5): 503-527.PubMed CentralView ArticlePubMedGoogle Scholar
- Chaumont F, Tyerman SD: Aquaporins: highly regulated channels controlling plant water relations. Plant Physiol. 2014, 164 (4): 1600-1618.PubMed CentralView ArticlePubMedGoogle Scholar
- Steudle E: Water uptake by plant roots: an integration of views. Plant Soil. 2000, 226 (1): 45-56.View ArticleGoogle Scholar
- Krishnamurthy P, Ranathunge K, Franke R, Prakash HS, Schreiber L, Mathew MK: The role of root apoplastic transport barriers in salt tolerance of rice (Oryza sativa L.). Planta. 2009, 230 (1): 119-134.View ArticlePubMedGoogle Scholar
- Hose E, Clarkson DT, Steudle E, Schreiber L, Hartung W: The exodermis: a variable apoplastic barrier. J Exp Bot. 2001, 52 (365): 2245-2264.View ArticlePubMedGoogle Scholar
- Steudle E: Water uptake by roots: effects of water deficit. J Exp Bot. 2000, 51 (350): 1531-1542.View ArticlePubMedGoogle Scholar
- Knepper MA: The aquaporin family of molecular water channels. Proc Natl Acad Sci U S A. 1994, 91 (14): 6255-6258.PubMed CentralView ArticlePubMedGoogle Scholar
- Tyerman SD, Niemieta CM, Bramley H: Plant aquaporins: multifunctional water and solute channels with expanding roles. Plant Cell Environ. 2002, 25: 173-194.View ArticlePubMedGoogle Scholar
- Maurel C: Plant aquaporins: novel functions and regulation properties. FEBS Lett. 2007, 581 (12): 2227-2236.View ArticlePubMedGoogle Scholar
- Kaldenhoff R, Ribas-Carbo M, Sans JF, Lovisolo C, Heckwolf M, Uehlein N: Aquaporins and plant water balance. Plant Cell Environ. 2008, 31 (5): 658-666.View ArticlePubMedGoogle Scholar
- Sade N, Gebretsadik M, Seligmann R, Schwartz A, Wallach R, Moshelion M: The role of tobacco Aquaporin1 in improving water use efficiency, hydraulic conductivity, and yield production under salt stress. Plant Physiol. 2010, 152 (1): 245-254.PubMed CentralView ArticlePubMedGoogle Scholar
- Maurel C, Verdoucq L, Luu DT, Santoni V: Plant aquaporins: membrane channels with multiple integrated functions. Annu Rev Plant Biol. 2008, 59: 595-624.View ArticlePubMedGoogle Scholar
- Hachez C, Besserer A, Chevalier AS, Chaumont F: Insights into plant plasma membrane aquaporin trafficking. Trends Plant Sci. 2013, 18 (6): 344-352.View ArticlePubMedGoogle Scholar
- Fetter K, Van Wilder V, Moshelion M, Chaumont F: Interactions between plasma membrane aquaporins modulate their water channel activity. Plant Cell. 2004, 16 (1): 215-228.PubMed CentralView ArticlePubMedGoogle Scholar
- Zelazny E, Borst JW, Muylaert M, Batoko H, Hemminga MA, Chaumont F: FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization. Proc Natl Acad Sci U S A. 2007, 104 (30): 12359-12364.PubMed CentralView ArticlePubMedGoogle Scholar
- Temmei Y, Uchida S, Hoshino D, Kanzawa N, Kuwahara M, Sasaki S, Tsuchiya T: Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation. FEBS Lett. 2005, 579 (20): 4417-4422.View ArticlePubMedGoogle Scholar
- Vandeleur RK, Mayo G, Shelden MC, Gilliham M, Kaiser BN, Tyerman SD: The role of plasma membrane intrinsic protein aquaporins in water transport through roots: diurnal and drought stress responses reveal different strategies between isohydric and anisohydric cultivars of grapevine. Plant Physiol. 2009, 149 (1): 445-460.PubMed CentralView ArticlePubMedGoogle Scholar
- Alleva K, Marquez M, Villarreal N, Mut P, Bustamante C, Bellati J, Martinez G, Civello M, Amodeo G: Cloning, functional characterization, and co-expression studies of a novel aquaporin (FaPIP2;1) of strawberry fruit. J Exp Bot. 2010, 61 (14): 3935-3945.PubMed CentralView ArticlePubMedGoogle Scholar
- Bellati J, Alleva K, Soto G, Vitali V, Jozefkowicz C, Amodeo G: Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression. Plant Mol Biol. 2010, 74 (1–2): 105-118.View ArticlePubMedGoogle Scholar
- Chen W, Yin X, Wang L, Tian J, Yang R, Liu D, Yu Z, Ma N, Gao J: Involvement of rose aquaporin RhPIP1;1 in ethylene-regulated petal expansion through interaction with RhPIP2;1. Plant Mol Biol. 2013, 83 (3): 219-233.View ArticlePubMedGoogle Scholar
- Yaneff A, Sigaut L, Marquez M, Alleva K, Pietrasanta LI, Amodeo G: Heteromerization of PIP aquaporins affects their intrinsic permeability. Proc Natl Acad Sci U S A. 2014, 111 (1): 231-236.PubMed CentralView ArticlePubMedGoogle Scholar
- Johanson U, Karlsson M, Johansson I, Gustavsson S, Sjovall S, Fraysse L, Weig AR, Kjellbom P: The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiol. 2001, 126 (4): 1358-1369.PubMed CentralView ArticlePubMedGoogle Scholar
- Sakurai J, Ishikawa F, Yamaguchi T, Uemura M, Maeshima M: Identification of 33 rice aquaporin genes and analysis of their expression and function. Plant Cell Physiol. 2005, 46 (9): 1568-1577.View ArticlePubMedGoogle Scholar
- Zhang DY, Ali Z, Wang CB, Xu L, Yi JX, Xu ZL, Liu XQ, He XL, Huang YH, Khan IA, Trethowan RM, Ma HX: Genome-wide sequence characterization and expression analysis of major intrinsic proteins in soybean (Glycine max L.). PLoS One. 2013, 8 (2): e56312-PubMed CentralView ArticleGoogle Scholar
- Maurel C, Javot H, Lauvergeat V, Gerbeau P, Tournaire C, Santoni V, Heyes J: Molecular physiology of aquaporins in plants. Int Rev Cytol. 2002, 215: 105-148.View ArticlePubMedGoogle Scholar
- Fricke W, Chaumont F: Solute and Water Relations of Growing Plant Cells. The Expanding Cell, Volume 6. Edited by: Verbelen JP, Vissenberg K. 2007, Berlin Heidelberg: Springer, 7-31.View ArticleGoogle Scholar
- Liu D, Tu L, Wang L, Li Y, Zhu L, Zhang X: Characterization and expression of plasma and tonoplast membrane aquaporins in elongating cotton fibers. Plant Cell Rep. 2008, 27 (8): 1385-1394.View ArticlePubMedGoogle Scholar
- Ma N, Xue J, Li Y, Liu X, Dai F, Jia W, Luo Y, Gao J: Rh-PIP2;1, a rose aquaporin gene, is involved in ethylene-regulated petal expansion. Plant Physiol. 2008, 148 (2): 894-907.PubMed CentralView ArticlePubMedGoogle Scholar
- Peret B, Li G, Zhao J, Band LR, Voss U, Postaire O, Luu DT, Da Ines O, Casimiro I, Lucas M, Wells DM, Lazzerini L, Nacry P, King JR, Jensen OE, Schaffner AR, Maurel C, Bennett MJ: Auxin regulates aquaporin function to facilitate lateral root emergence. Nat Cell Biol. 2012, 14 (10): 991-998.View ArticlePubMedGoogle Scholar
- Aroca R, Porcel R, Ruiz-Lozano JM: Regulation of root water uptake under abiotic stress conditions. J Exp Bot. 2012, 63 (1): 43-57.View ArticlePubMedGoogle Scholar
- Hu W, Yuan Q, Wang Y, Cai R, Deng X, Wang J, Zhou S, Chen M, Chen L, Huang C, Ma Z, Yang G, He G: Overexpression of a wheat aquaporin gene, TaAQP8, enhances salt stress tolerance in transgenic tobacco. Plant Cell Physiol. 2012, 53 (12): 2127-2141.View ArticlePubMedGoogle Scholar
- Zhou S, Hu W, Deng X, Ma Z, Chen L, Huang C, Wang C, Wang J, He Y, Yang G, He G: Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco. PLoS One. 2012, 7 (12): e52439-PubMed CentralView ArticlePubMedGoogle Scholar
- Gao Z, He X, Zhao B, Zhou C, Liang Y, Ge R, Shen Y, Huang Z: Overexpressing a putative aquaporin gene from wheat, TaNIP, enhances salt tolerance in transgenic Arabidopsis. Plant Cell Physiol. 2010, 51 (5): 767-775.View ArticlePubMedGoogle Scholar
- Sreedharan S, Shekhawat UK, Ganapathi TR: Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses. Plant Biotechnol J. 2013, 11 (8): 942-952.View ArticlePubMedGoogle Scholar
- Delgado MJ, Ligero F, Lluch C: Effects of salt stress on growth and nitrogen fixation by pea, faba-bean, common bean and soybean plants. Soil Biol Biochem. 1994, 26: 371-376.View ArticleGoogle Scholar
- Vandeleur RK, Sullivan W, Athman A, Jordans C, Gilliham M, Kaiser BN, Tyerman SD: Rapid shoot-to-root signalling regulates root hydraulic conductance via aquaporins. Plant Cell Environ. 2014, 37 (2): 520-538.View ArticlePubMedGoogle Scholar
- Tournaire-Roux C, Sutka M, Javot H, Gout E, Gerbeau P, Luu DT, Bligny R, Maurel C: Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. Nature. 2003, 425 (6956): 393-397.View ArticlePubMedGoogle Scholar
- Siefritz F, Tyree MT, Lovisolo C, Schubert A, Kaldenhoff R: PIP1 plasma membrane aquaporins in tobacco: from cellular effects to function in plants. Plant Cell. 2002, 14 (4): 869-876.PubMed CentralView ArticlePubMedGoogle Scholar
- Martinez-Ballesta MC, Aparicio F, Pallas V, Martinez V, Carvajal M: Influence of saline stress on root hydraulic conductance and PIP expression in Arabidopsis. J Plant Physiol. 2003, 160 (6): 689-697.View ArticlePubMedGoogle Scholar
- Boursiac Y, Chen S, Luu DT, Sorieul M, van den Dries N, Maurel C: Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression. Plant Physiol. 2005, 139 (2): 790-805.PubMed CentralView ArticlePubMedGoogle Scholar
- Marulanda A, Azcon R, Chaumont F, Ruiz-Lozano JM, Aroca R: Regulation of plasma membrane aquaporins by inoculation with a Bacillus megaterium strain in maize (Zea mays L.) plants under unstressed and salt-stressed conditions. Planta. 2010, 232 (2): 533-543.View ArticlePubMedGoogle Scholar
- Muries B, Faize M, Carvajal M, Martinez-Ballesta Mdel C: Identification and differential induction of the expression of aquaporins by salinity in broccoli plants. Mol Biosyst. 2011, 7 (4): 1322-1335.View ArticlePubMedGoogle Scholar
- Calvo-Polanco M, Sanchez-Romera B, Aroca R: Mild salt stress conditions induce different responses in root hydraulic conductivity of phaseolus vulgaris over-time. PLoS One. 2014, 9 (3): e90631-PubMed CentralView ArticlePubMedGoogle Scholar
- Liu C, Fukumoto T, Matsumoto T, Gena P, Frascaria D, Kaneko T, Katsuhara M, Zhong S, Sun X, Zhu Y, Iwasaki I, Ding X, Calamita G, Kitagawa Y: Aquaporin OsPIP1;1 promotes rice salt resistance and seed germination. Plant Physiol Biochem. 2012, 63C: 151-158.Google Scholar
- Durand M, Lacan D: Sodium partitioning within the shoot of soybean. Physiol Plant. 1994, 91 (1): 65-71.View ArticleGoogle Scholar
- Lauchli A: Salt exclusion: An adaptation of legumes for crops and pastures under saline conditions. Salinity Tolerance in Piants, Strategies for Crop Improvement. Edited by: Staples RC, Toenniessen GH. 1984, New York: John Wiley & Sons, 171-187.Google Scholar
- Lohaus G, Hussmann M, Pennewiss K, Schneider H, Zhu JJ, Sattelmacher B: Solute balance of a maize (Zea mays L.) source leaf as affected by salt treatment with special emphasis on phloem retranslocation and ion leaching. J Exp Bot. 2000, 51 (351): 1721-1732.View ArticlePubMedGoogle Scholar
- Li WY, Wong FL, Tsai SN, Phang TH, Shao G, Lam HM: Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells. Plant Cell Environ. 2006, 29 (6): 1122-1137.View ArticlePubMedGoogle Scholar
- Uehlein N, Lovisolo C, Siefritz F, Kaldenhoff R: The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. Nature. 2003, 425 (6959): 734-737.View ArticlePubMedGoogle Scholar
- Flexas J, Ribas-Carbo M, Hanson DT, Bota J, Otto B, Cifre J, McDowell N, Medrano H, Kaldenhoff R: Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in vivo. Plant J. 2006, 48 (3): 427-439.View ArticlePubMedGoogle Scholar
- Heckwolf M, Pater D, Hanson DT, Kaldenhoff R: The Arabidopsis thaliana aquaporin AtPIP1;2 is a physiologically relevant CO(2) transport facilitator. Plant J. 2011, 67 (5): 795-804.View ArticlePubMedGoogle Scholar
- Uehlein N, Sperling H, Heckwolf M, Kaldenhoff R: The Arabidopsis aquaporin PIP1;2 rules cellular CO(2) uptake. Plant Cell Environ. 2012, 35 (6): 1077-1083.View ArticlePubMedGoogle Scholar
- Evans JR, Kaldenhoff R, Genty B, Terashima I: Resistances along the CO2 diffusion pathway inside leaves. J Exp Bot. 2009, 60 (8): 2235-2248.View ArticlePubMedGoogle Scholar
- Patrick JW, Zhang W, Tyerman SD, Offler CE, Walker NA: Role of membrane transport in phloem translocation of assimilates and water. Func Plant Biol. 2001, 28 (7): 697-709.View ArticleGoogle Scholar
- Song ZY, Tian JL, Fu WZ, Li L, Lu LH, Zhou L, Shan ZH, Tang GX, Shou HX: Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability. J Zhejiang Univ Sci B. 2013, 14 (4): 289-298.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.