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
Cell pressure probe (CPP) measurements were performed as described in Steudle (1993). In brief, pulled glass micro-capillary were beveled to a tip diameter of 5–7 µm, filled with silicone oil (type AS4; Wacker, Munich, Germany), and mounted to the CPP. To measure root cell hydraulic conductivity (Lprc), root segment from plants grown in hydroponic condition was fixed by magnetic bars on a metal sledge which was covered with wet filter paper. An aerated nutrient solution was circulated along the root segment to maintain moisture. Root cells were punctured using a CPP, and cell sap entered the oil-filled micro-capillary forming a meniscus between cell sap and oil. Cell turgor pressure was restored by gently pushing the meniscus to a position close to the surface of the root, and the values of cell turgor pressure (P) were recorded by a computer (Ye et al. 2004). Half time of water exchange (T1/2) across cell membranes was obtained from hydrostatic pressure relaxation curves with the aid of the probe. After CPP measurements, average values of cell volume and surface area were obtained through microscopic analyses with root sections, assuming that cells had a cylindrical shape. Lprc was calculated according to the following equation:
$$ {\text{Lp}} = \frac{{V}}{{A}} \times \frac{\ln (2)}{{T_{1/2}^{w} (\varepsilon + \pi^{i} )}} $$
(1)
Here, V = cell volume; A = cell surface area; πi = osmotic pressure of cell sap; ε = cell elastic modulus. πi was calculated from the initial cell turgor (P0), as P0 = πi − π0 (π0 = osmotic pressure of the medium measured with an osmometer); elastic modulus was determined from relative change of cell volume (ΔV/V) and the instantaneous change of cell turgor (ΔP):
$$ \varepsilon = V \times \frac{\Delta P}{\Delta V} $$
(2)
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.
Statistical analysis
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.
