Plant materials and growth condition
Seeds of an elite switchgrass line, HR8, originally selected from the lowland ecotype ‘Alamo’ was used in this study (33). In the qPCR experiment to study PvPIP2;9 expression pattern, 4-wk-old plants were cultured in 1/2 Hoagland solution and grown in a growth chamber with a 12-hour (hr) light/dark cycle and accurately controlled temperature [30/25°C (day/night)] and light intensity (photosynthetically active radiation at 750 µmol·photons m–2· s–1). In other experiments, switchgrass plants were grown in grown in clay loam soil mixed with sand (1:1) in 1.1×10-2 m3 pots in the greenhouse at Nanjing Agricultural University (Nanjing, China) with temperatures set at 30/20 ± 3 °C (day/night) and the photoperiod set at 14/10 hr (day/night).
The second fully expanded leaves from the top were sampled for relative gene expression level and physiological parameter analyses. To detect the diurnal oscillation of PvPIP2;9, the first sampling time was set at the dawn for consecutive 40 hr with four hr internals in-between. For stress treatments, plants were grown in 1/2 Hoagland solution containing 20% polyethylene glycol (PEG) 6000 (Huada, Shantou, China) and 100 μM ABA according to Yuan et al. (34), and sampled after 0, 0.5, 1, 2, 4, 8 and 12 hr after the treatment.
The total RNA was isolated using OMEGA E.Z.N.A.® plant RNA Kit. The first strand cDNA was synthesized with 1 μg RNA using the PrimeScriptTM RT reagent Kit (Takara, Dalian, China) with the Perfect Real Time gDNA Eraser (TaKaRa). The qRT-PCR was performed using SYBR Green Master Mixes on a Roche LightCycler®480 II machine. The qRT-PCR was performed with three biological replicates and two technical replicates, and the qPCR program set as follows: 10 min at 95 °C for initial denaturation, and 40 cycles (95 °C for 15s, 58 °C for 15 s, and 72 °C for 20 s). Relative expression levels of PvPIP2;9 were calculated using the 2-ΔΔCT method with PvACTIN as the reference gene (35). Primers used in this study were shown in Additional file 1: Table S1.
Gene cloning and vector construction
According to the switchgrass genomic sequence information (Panicum virgatum v4.1, DOE-JGI, http://phytozome.jgi.doe.gov), we cloned the gene from gDNA for its functional characterization. In brief, the gene was amplified from switchgrass genomic DNA using PCR, cloned into the vector pENTR/D and sequenced. Then we sub-cloned the gene into the Gateway-compatible binary vector pVT1629 (35) using LR reaction (Invitrogen). The resultant vector, pVT1629-PvPIP2;9, harboring the PvPIP2;9 driven under maize ubiquitin promoter and the UidA (GUS) reporter gene under CaMV 35S promoter, was transformed into the Agrobacterium tumefaciens strain ‘AGL1’ through electroporation.
Switchgrass genetic transformation
Switchgrass line ‘HR8’ was used for Agrobacterium-mediated genetic transformation and the transformation procedure was the same as reported before (33). Hygromycin B (Sigma) at 50 mg/L was used to select against the non-transformed calli. Regenerated plants from independent calli were regarded as putative transgenic lines which were further verified by GUS staining and PCR for the detection of HPTⅡ gene present in the T-DNA.
Drought treatment of WT and transgenic plants
Two independent transgenic lines and tissue culture-regenerated wild-type (WT) plants were propagated by splitting single tillers grown at the optimum condition. Plants grown from a single tiller for two and a half months reached E4 stage (36) and were used for drought treatment by withdrawing water. After 28d of drought, the treated plants were re-watered to observe their re-growth status. At the same time period, normally-watered plants were used as controls. A soil water content detector (Mini Trase Kit 6050X3; Soil Moisture Equipment Corp., Santa Barbara, CA) was used to monitor the soil water content (SWC) in 0-8 cm deep soil layer of each pot. And soil water potential was determined using ERS-Ⅱwater potential and temperature meter (Yibaiyi Mechanical and Electrical Equipment Co., Ltd., Wenzhou, China). The correlation between soil water content and soil water potential was shown in Additional file 2: Figure S1.
Biomass feedstock quality analysis
After 21 d of treatment, the above ground of WT and transgenic plants were collected and dried in a 70℃ oven and then ground for feedstock quality analysis. The amount of total sugar was determined by the phenol sulfuric acid reagent method (37) with slight modifications. In brief, 0.05 g samples were added to the mixture of 10 ml ddH2O and 3 ml HCl and incubated in water bath set at 100℃ for 1 h. Then 0.5 ml supernatant was transferred to a 10 ml tube and mixed with 0.5 ml ddH2O, 1 ml 5% phenol, and 5 ml sulfuric acid. The reaction mixture was incubated at 30℃ for 20 min in a water bath and then the absorbance was quantified at 490 nm. The quantity of total sugar was based on a standard curve generated with known sugar concentrations.
Cellulose content was estimated using the anthrone method (38). Briefly, 0.05 g samples were mixed with 35 ml 60% H2SO4 in a 50 ml tube and incubated at ice bath for 30 min. After ten times dilution, the absorbance of supernatant was quantified at 620 nm. Microcrystalline cellulose (Avicel) was used as a standard for the standard curve generation.
Hemicellulose and lignin content were measured by hydrochloric acid hydrolysis method and sulfuric acid method, respectively (39). For hemicellulose analysis, 0.1 g sample was mixed with 10 ml 80% calcium nitrate and boiled on a heater for 5 min. The mixture was then centrifuged and the supernatant was discarded. After rinsed three times with ddH2O, 10 ml 2 M HCl was added to the mixture and boiled for another 45 min. The supernatant was neutralized using NaOH and mixed with DNS reagent. The mixture was incubated in water bath at 100℃ for 5 min and then absorbance of supernatant was measured at 520 nm. For lignin, 0.1 g sample was washed using 10 ml 1% acetic acid and the mixture of ethanol and ether (1:1) and then was dried in a water bath set at 100℃. The sample was incubated with 72% H2SO4 for at least for 16 h to remove cellulose. The precipitate was then mixture with 10 ml 10% H2SO4 and 0.1 M potassium dichromate and incubated in water bath set at 100℃ for 15 min. The lignin content was then quantified after mixed with 5 ml 20% KI and 1 ml 0.5% starch solution by titration using 0.2 M sodium thiosulfate.
The Kjeldahl procedure was used to determine the total nitrogen (TN) content, and the crude protein content was calculated by multiplying TN by 6.25 (40).
Measurement of physiological parameters
Leaf membrane stability was evaluated by measuring the electrolyte leakage (EL) (41) according to a method described before (42). In brief, leaves were excised and cut into 3 cm segments. Then the leaves were incubated in 35 ml distilled deionized water. Centrifuge tubes were shaken on a shaker for 24 hr at room temperature, and the initial level of EL (Ci) was measured using a conductance meter (Thermo Scientific, Beverly, USA).Then the leaf tissue was killed by autoclaving at 121°C for 15 min, and then incubated for 24 h on a shaker for measuring the maximum conductance (Cmax) of the solution. Relative EL was calculated as EL = (Ci/Cmax) × 100%.
The leaf relative water content (RWC) was determined according to the method described by Hu et al. (43) with modifications. In brief, RWC was determined using fresh fully expanded leaves (~0.2 g). Leaf samples were detached from the plants and immediately weighed to determine the fresh weight (FW). Samples were placed into covered centrifuge tubes filled with water for leaves to reach full hydration. After approximately 24 h at 4 °C, leaf samples were blotted dry with paper towels and weighed to determine the saturated weight (SW). Leaf tissue was then dried in an oven at 65 °C for 72 hr to determine dry weight (DW). Leaf RWC was calculated as RWC = (FW – DW) / (SW – DW) × 100.
The ratio of the variable ﬂuorescence (Fv) to the maximal ﬂuorescence (Fm) (Fv/Fm) was used to represent leaf photochemical efficiency (Oxborough and Baker, 1997). The Fv/Fm ratio was determined using a ﬂuorescence meter (Dynamax, Houston, TX, USA) as described before (42). And chlorophyll content were measured using the DMSO extraction method as described before (42).
Leaf instantaneous WUE was calculated by measuring leaf net photosynthetic rate (Pn) and transpiration rate (Tr) using the LI-6400 portable photosynthesis system (LI-COR, Lincoln, NE). The area of leaves enclosed in the leaf chamber was determined on a scanner, which was then used to calculate the Pn and Tr values. The WUE was calculated as Pn/Tr.
Data in this study were statistically analyzed using one-way ANOVA, and their means were compared by Duncan test at the significance level of 0.05 by using SPSS20.0.