Virus-induced plasma membrane aquaporin PsPIP2;1 silencing inhibits plant water transport of Pisum sativum
© The Author(s) 2016
Received: 2 March 2016
Accepted: 13 July 2016
Published: 6 August 2016
Aquaporins (AQPs) are known to facilitate water transport across cell membranes, but the role of a single AQP in regulating plant water transport, particularly in plants other than Arabidopsis remains largely unexplored. In the present study, a virus-induced gene silencing (VIGS) technique was employed to suppress the expression of a specific plasma membrane aquaporin PsPIP2;1 of Pea plants (Pisum sativum), and subsequent effects of the gene suppression on root hydraulic conductivity (Lpr), leaf hydraulic conductivity (K leaf ), root cell hydraulic conductivity (Lprc), and leaf cell hydraulic conductivity (Lplc) were investigated, using hydroponically grown Pea plants.
Compared with control plants, VIGS-PsPIP2;1 plants displayed a significant suppression of PsPIP2;1 in both roots and leaves, while the expression of other four PIP isoforms (PsPIP1;1, PsPIP1;2, PsPIP2;2, and PsPIP2;3) that were simultaneously monitored were not altered. As a consequence, significant declines in water transport of VIGS-PsPIP2;1 plants were observed at both organ and cell levels, i.e., as compared to control plants, Lpr and K leaf were reduced by 29 %, and Lprc and Lplc were reduced by 20 and 29 %, respectively.
Our results demonstrate that PsPIP2;1 alone contributes substantially to root and leaf water transport in Pea plants, and highlight VIGS a useful tool for investigating the role of a single AQP in regulating plant water transport.
KeywordsCell pressure probe Hydraulic conductivity Plant water relations VIGS Water channels
Plant water relations are continually challenged by diverse environmental stimuli, such as light, temperature, soil water availability, and atmospheric humidity. To keep water homeostasis, plants need to respond promptly to the ever-changing environments via regulating water transport at cellular, tissue, organ, and whole plant level (Aroca et al. 2012; Bramley et al. 2009; Chaumont and Tyerman 2014; Chevalier and Chaumont 2015; Henry et al. 2012; Luu and Maurel 2005). Aquaporins (AQPs) are trans-membrane proteins that facilitate rapid and passive water transport across cell membranes. According to sequence homology and sub-cellular localizations, plants AQPs can be classified into seven subfamilies, i.e., plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic protein (TIP), NOD26-like intrinsic proteins (NIPs), small intrinsic proteins (SIPs), X-intrinsic proteins (XIPs), GlypF-like intrinsic proteins (GIPs), and hybrid intrinsic proteins (HIPs) (Anderberg et al. 2011; Gustavsson et al. 2005; Johanson et al. 2001; Li et al. 2014). Among them, PIPs constitute the largest number and can be further divided into two subgroups named PIP1 and PIP2 (Ayadi et al. 2011; Chaumont et al. 2000; Johansson et al. 2000).
The role of AQPs in regulating plant water transport has been abundantly documented, and PIPs represent the most likely candidates for protein-mediated hydraulic conductivity in plants (Heinen et al. 2009; Maurel et al. 2008). The contribution of AQPs to plant hydraulic conductivity has been tested by variable approaches. The first notion that AQPs involving in plant water transport was raised from experiments showing that root water transport can be substantially inhibited by AQP blocker, i.e., mercurial regents (Javot and Maurel 2002; Maggio and Joly 1995; Zhang and Tyerman 1999). Because mercury compound showed inhibitive effects in general on other physiological processes besides blocking AQPs, more specific approaches involved the use of transgenic plants with altered expression of targeted PIPs were employed (Jang et al. 2007; Javot et al. 2003; Lee et al. 2012; Postaire et al. 2010; Secchi and Zwieniecki 2014; Yu et al. 2005). For instance, over-expression of Arabidopsis PIP1b in tobacco improved plant vigor under favorable growth condition (Aharon et al. 2003). Low temperature induced reductions in cell hydraulic conductivity was alleviated by over-expressing AtPIP2;5 in Arabidopsis plants (Lee et al. 2012). In grapevine, it was found that the over-expression of a root specific AQP VvPIP2;4N enhanced water transport at the whole plant level (Perrone et al. 2012). By contrast, hydraulic conductivity of root cortex cell was reduced by 25–30 % in PIP2;2 knockout mutant of Arabidopsis plants (Javot et al. 2003), and a reduction of about 20 % in the relative water flux into rosette leaves was found in these mutants (Da Ines et al. 2010). Similarly, disruption of AtPIP1;2 resulted in a significant decrease (by 20–30 %) in root hydraulic conductivity of Arabidopsis (Postaire et al. 2010), while PIP1 and PIP2 double antisense Arabidopsis plants had a threefold decrease in the root hydraulic conductivity (Martre et al. 2002). All these pioneer findings pointed to the important roles of AQPs in regulating water transport across diverse species, while the contribution of a single AQP to hydraulic conductions in plants other than Arabidopsis remains to be explored.
Virus-induced gene silencing (VIGS) is a reverse genetics technology that can produce a rapid, sequence-specific knockdown phenotype for the target gene (Burch-Smith et al. 2004). To this end, a fragment of the target gene is inserted into a viral delivery vector which is used to infect plants. During the inoculation, virus replication triggers the natural defense mechanisms of plants to suppressing virus replication, which is also result in specific degradation of mRNAs from the endogenous gene that is targeted for silencing (Baulcombe 1999; Lu et al. 2003). Therefore, compared with other transgenic methods, VIGS technology represents a simple but attractive reverse-genetics tool for gene functional studies (Pflieger et al. 2013). In addition, VIGS does not need to develop stable transformants, thus can be used to study the function of genes that might be fatal for plants when such functions are impaired in stable transformed lines (Burch-Smith et al. 2004; Purkayastha and Dasgupta 2009). With these advantages, VIGS technology has been broadly applied for functional studies of specific genes across a number of plant species including Tobacco, Arabidopsis, Tomato, Rice, and Pea plants (Constantin et al. 2004; Fragkostefanakis et al. 2014; Purkayastha et al. 2010; Senthil-Kumar and Mysore 2014).
Results and discussion
Cell pressure probe measurements of root cortex cells and leaf epidermal cells of control plants and virus induced PsPIP2;1 silencing plants (VIGS-PsPIP2;1)
Root cortical cell
Turgor pressure, P (MPa)
0.38 ± 0.08 a
0.37 ± 0.06 a
Cell volume, V (m3)
1.7 ± 0.3E−13 a
1.6 ± 0.2E−13 a
Cell surface area, A (m2)
2.2 ± 0.4E−08 a
2.1 ± 0.3E−08 a
4.0 ± 1.0 a
3.9 ± 1.2 a
1.8 ± 0.1 a
2.6 ± 0.6 b
Leaf epidermal cell
Turgor pressure, P (MPa)
0.34 ± 0.05 a
0.32 ± 0.08 a
Cell volume, V (m3)
1.1 ± 0.2E−13 a
1.2 ± 0.1E−13 a
Cell surface area, A (m2)
1.7 ± 0.5E−08 a
1.6 ± 0.4E−08 a
2.2 ± 1.1 a
2.3 ± 1.2 a
1.2 ± 0.2 a
1.7 ± 0.4 b
Our results demonstrated that the expression of PsPIP2;1 in Pea plants was specifically suppressed through the VIGS method. As a result, both root and leaf hydraulic conductivities were significantly reduced in PsPIP2;1-silenced plants compared with control plants. Consistent with previous findings that PsPIP2;1 showed marked water transport activity when expressed in Xenopus oocytes, and displayed a tight correlation with the diurnal change in root hydraulic conductivity, our results provided further evidence that PsPIP2;1 play an important role in regulating Pea plant water transport. However, precise mechanisms by which this AQP mediates plant water transport remain to be explored. For instance, whether PsPIP2;1 had a tissue specific expression pattern in root endodermis and/or leaf bundle sheath that are proven to be critical in the pathway of plant water transport, as well as the responsiveness of PsPIP2;1 to abiotic stresses (e.g., drought stress), all merit future investigations.
Plant material and growth conditions
Pea plants (P. sativum L. line JI992) used in this study was obtained from National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China. Seeds were germinated in wet filter paper in covered Petri dishes for 3 days at room temperature in the dark. Then seedlings were transferred to a hydroponic culture plastic box (7 L) filled with modified Hoagland solution (pH = 6.0; 1.25 mM Ca(NO3)2, 1.25 mM KNO3, 0.5 mM MgSO4, 0.25 mM KH2PO4; micronutrients: 10 µM H3BO3, 1 µM MnSO4, 0.5 µM ZnSO4, 0.05 µM (NH4)6Mo7O24 and 0.4 µM CuSO4) following Jelali et al. (2010). The nutrient solution was aerated with the aid of aquarium diffusers. One week later, seedlings were transferred to 37 L boxes (15 plants per box) filled with the same nutrient solution that was replaced weekly. Growing conditions in the growth chamber were 16 h light/8 h dark photoperiod, 18/20 °C, 65 % humidity, and a photon flux density of 200–300 µM m−2 s−1. Plants used in the experiments were six- to seven-week old.
RNA extraction and PsPIP genes identification
Total RNA was extracted from roots and leaves of Pea plants using Trizol regent (Invitrogen, Grand Island, NY, USA) following the manufacturer’s instructions. The concentration of RNA was quantified by spectrophotometrical measurement at λ = 260 nm, and its integrity was checked on agarose gels. First strand cDNA was synthesized from 2 µg of total RNA using GoScript reverse transcription regent Kit (Promega, Madison, WI, USA).The synthesized cDNA was amplified by polymerase chain reaction (PCR) using oligo(dT) and degenerate oligonucleotide primers (Additional file 1: Table S1) which were designed from the known sequences of different plant PIP genes. The PCR products were gel-purified and sub-cloned into pMD18-T vector (Takara, TAKARA Biotechnology Co. Ltd, Dalian, China), and the constructed plasmids were transformed into E. coli DH5α. The positive clones were sequenced and analyzed. Next, 5′-rapid amplification of cDNA ends (RACE) was applied to clone the 5′-end sequences of the PIP genes. Sequences analyses with database were performed at NCBI (http://www.ncbi.nlm.nih.gov/) using the BLAST network services, and a phylogenetic tree was generated in MEG5.1 software (http://www.megasoftware.net) to test the evolutionary relationships.
The Silencing of PsPIP2;1 in Pea plants
To optimize the VIGS method, Constantin et al. (2004) transferred the RNA1 and RNA2 expression cassettes of a Pea early browning virus (PEBV) to the binary agrobacterium vector pCAMBIA1300. Then, pCAMBIA1300-derived plasmid with the expression cassette of RNA1 was named as pCAPE1, and pCAMBIA1300-derived plasmid with the expression cassette of RNA2-GFP was named as pCAPE2-GFP. In the present study, sequence of GFP in pCAPE2-GFP was replaced with cDNA fragment of P. sativum phytoene desaturase (PDS) gene and with partial encoding region of PsPIP2;1 plus 3′UTR sequence to obtain pCAPE2-PDS and pCAPE2-PsPIP2;1, respectively. Also, a vector control plasmid, pCAPE2-Con was constructed by replacing the GFP sequence of pCAPE2-GFP with a fragment derived from the cDNA of Bean yellow mosaic virus (AJ622899). Next, the constructed plasmids including pCAPE1, pCAPE2-PDS, pCAPE2-PsPIP2;1, and pCAPE2-Con were transformed separately into Agrobacterium tumefaciens GV3101 using the freeze–thaw method (Hofgen and Willmitzer 1988). Two-week old Pea plants were infiltrated at the abaxial side of the youngest pair of leaves with agrobacterium cultures carrying pCAPE1 and the pCAPE2-derived plasmids at a 1:1 ratio. Plants were separated into three groups that were subsequently inoculated with three different agrobacterium cultures: (1) pCAPE2-PDS, which served as an indicator of gene silencing, in that PDS silenced plants had photo-bleached leaves (as a result of lacking carotenoids and destruction of chlorophyll by photo-oxidation), and this phenotype was associated with a significant reduction in PsPDS mRNA (Kumagai et al. 1995); (2) pCAPE2-PsPIP2;1 to silence the target PsPIP2;1 gene; and (3) pCAPE2-Con as the control. When the target gene was silenced, as indicated by the photo-bleached leaves of PDS silenced plants, the shoots of PsPIP2;1 silenced plants were labeled at the position where the photo-bleached phenotype began to appear (Additional file 2: Figure S1). Meanwhile, roots of PsPIP2;1 silenced plants were cut back to approximately 3 cm and root growth was allowed to re-initiate. Then plants were grown in the growth chamber for additional 2–3 weeks to allow the production of newly emerged leaves and regenerative roots, which were used in subsequent experiments.
Quantitative real-time PCR (q-RT-PCR) analyses
Total RNA extraction, concentration and integrity were determined as described above. First strand cDNA was synthesized using primeScript RT regent Kit (TakaRa, TAKARA Biotechnology Co. Ltd, Dalian, China) following manufacturer’s instructions, including a special step for genomic DNA elimination. Quantitative PCR analysis was conducted on an ABI 7500 Real-Time system using a SYBR Green Premix Ex-Taq™II Kit (TakaRa, TAKARA Biotechnology Co. Ltd, Dalian, China) with PsPIP gene specific primers (Additional file 3: Table S2). The reaction mixture had a final volume of 20 µL, containing 10 µL 2× SYBR Premix Ex Taq™II, 0.4 µM of each primer, 0.4 µL 50× ROX Reference Dye II and 2 µL of tenfold dilution cDNA. The PCR conditions were: 30 s at 95 °C for pre-denaturation; 40 cycles of 5 s at 95 °C, 34 s at 60 °C. The melt-curve analysis was conducted using the method recommended by the manufacturer. The results were normalized by the geometric mean of the expression of three reference genes, i.e., elongation factor 1-alpha (EF1α, X96555), 18 s ribosomal RNA (18 s, X52575) and beta-tubulin 3 (TUB, X54846). The relative expression of PsPIPs was calculated using the 2−ΔΔCt method (Pfaffl 2001; Schmittgen and Livak 2008).
Root and leaf hydraulic conductivity measurements
Root and leaf hydraulic conductivity (Lpr and K leaf , respectively) was measured using the pressure chamber technique following Javot et al. (2003) and Postaire et al. (2010), with slight modifications. For Lpr measurements, shoots were cut off below the first node of the plants, and the whole roots were bathed in nutrient solution in a pressure chamber (PMS, Corvallis, OR, USA). The hypocotyl was carefully threaded through the soft plastic washer of the metal lid. Pressure (P) that was generated by compressed air in steps of 0.1 MPa (up to 0.5 MPa) was slowly applied to the chamber, and the rate of exuded sap flow (J v) was determined. When J v was plotted against the applied P, a linear relationship was observed for P values between 0.2 and 0.4 MPa (Fig. 4a). At the end of the measurement, the root system was removed and dry weight (DW) of the roots (after oven-dried at 70 °C for 72 h) was measured using a balance (FA2104N, Minqiao Instrument Co. Ltd, Shanghai, China). Lpr (µL s−1 g−1 MPa−1) was calculated from the slope of the exuded sap flow rate versus pressure, divided by DW of the roots.
Similarly, for K leaf determination, a detached mature compound leaf was inserted into a pressure chamber (PMS, Corvallis, OR, USA) filled with distilled water. The common petiole was carefully threaded through the soft plastic washer of the metal lid. Pressure was applied to the chamber in steps of 0.1 MPa (up to 0.5 MPa), using compressed air gas. This resulted in a flow of liquid (J v) entering through the leaf surface and exiting from the common petiole. When J v was plotted against P, a linear relationship was observed for P values between 0.3 and 0.5 MPa (Fig. 5a). At the end of the measurement, leaves were scanned and the surface area (S) was measured using Image J software v1.42 (Bethesda, MD, USA). K leaf (μL s−1 m−2 MPa−1) was calculated from the slope of the exuded sap flow rate versus pressure, divided by S of the leaves.
Cell pressure probe measurements
Where the change in cell volume (ΔV) was induced by moving the meniscus with the aid of the CPP, which was calculated from the length of meniscus movement in the micro-capillary using the eyepiece reticule of the microscope under a given magnification, and from the inner diameter of the capillary where the meniscus located (Steudle 1993).
For leaf cell hydraulic conductivity (Lplc) measurements, a mature young leaf blade (still attached to the plant) was fixed onto a metal sledge. Leaf cells were punctured using a CPP, and water relation parameters such as T1/2, ε, and Lplc were determined as described above for root cell measurements.
Results were presented as mean ± SD of three independent experiments. Statistical analyses were performed using SPSS 13.0 program (Chicago, IL, USA). Statistical significant differences were determined by t test at P < 0.05.
cell pressure probe
- K leaf :
leaf hydraulic conductivity
- Lplc :
leaf cell hydraulic conductivity
- Lpr :
root hydraulic conductivity
- Lprc :
root cell hydraulic conductivity
plasma membrane intrinsic protein
quantitative real-time PCR
- T1/2 :
half-time of water exchange
virus-induced gene silencing
JS and QY conceived and designed the project. JS, GY and ZQ conducted the experiments, JS and QY analyzed the results. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.
We are grateful to two anonymous Reviewers for their insightful comments and constructive suggestions on an earlier version of this article. We thank Dr. J Yang (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China) for providing seeds of P. sativum L. This work was funded by Natural Science Foundation of Guangdong Province, China (2014A030313787), the National Natural Science Foundation of China (31070231), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry of China.
The authors declare that they have no competing interests.
Availability of supporting data
All supporting data are included as additional files.
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