Overexpression of MfPIP2-7 from Medicago falcata promotes cold tolerance and growth under NO3 − deficiency in transgenic tobacco plants
© The Author(s). 2016
Received: 1 December 2015
Accepted: 19 May 2016
Published: 14 June 2016
Plasma membrane intrinsic proteins (PIPs), which belong to aquaporins (AQPs) superfamily, are subdivided into two groups, PIP1 and PIP2, based on sequence similarity. Several PIP2s function as water channels, while PIP1s have low or no water channel activity, but have a role in water permeability through interacting with PIP2. A cold responsive PIP2 named as MfPIP2-7 was isolated from Medicago falcata (hereafter falcata), a forage legume with great cold tolerance, and transgenic tobacco plants overexpressing MfPIP2-7 were analyzed in tolerance to multiple stresses including freezing, chilling, and nitrate reduction in this study.
MfPIP2-7 transcript was induced by 4 to 12 h of cold treatment and 2 h of abscisic acid (ABA) treatment. Pretreatment with inhibitor of ABA synthesis blocked the cold induced MfPIP2-7 transcript, indicating that ABA was involved in cold induced transcription of MfPIP2-7 in falcata. Overexpression of MfPIP2-7 resulted in enhanced tolerance to freezing, chilling and NO3 − deficiency in transgenic tobacco (Nicotiana tabacum L.) plants as compared with the wild type. Moreover, MfPIP2-7 was demonstrated to facilitate H2O2 diffusion in yeast. Higher transcript levels of several stress responsive genes, such as NtERD10B, NtERD10C, NtDREB1, and 2, and nitrate reductase (NR) encoding genes (NtNIA1, and NtNIA2) were observed in transgenic plants as compared with the wild type with dependence upon H2O2. In addition, NR activity was increased in transgenic plants, which led to alterations in free amino acid components and concentrations.
The results suggest that MfPIP2-7 plays an important role in plant tolerance to freezing, chilling, and NO3 − deficiency by promoted H2O2 diffusion that in turn up-regulates expression of NIAs and multiple stress responsive genes.
Aquaporins (AQPs) form a superfamily of intrinsic channel proteins and function as diffusion facilitators for water and small molecules such as CO2, glycerol, ammonium, and urea cross plasma and intracellular membranes in plant cells [1–4]. Plant AQPs are divided into five subgroups consisting of the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins, nodulin 26-like intrinsic proteins, small basic intrinsic proteins, and X intrinsic proteins . The PIPs can be further subdivided into PIP1 and PIP2, based on sequence similarity. Several PIP2s function as water channels, while PIP1s have low or no water channel activity, but are associated with water permeability through interacting with PIP2 [6–8].
Responses of PIP expression to abiotic stresses are variable, with up-, down- or no regulation, depending on species or tissues [9–14]. Most of AtPIPs are less affected by salinity, except for AtPIP1-5 and AtPIP2-6 which are down-regulated in roots and shoots respectively . Transcripts of AtPIPs are generally down-regulated in leaves upon gradual drought stress, but AtPIP1-4 and AtPIP2-5 transcript levels are up-regulated . Osmotic water permeability of protoplasts is decreased by down-regulation of certain PIP, which leads to a higher susceptibility to drought and osmotic stress [15–17], while overexpression of PIP genes generally increases root osmotic hydraulic conductivity and transpiration in transgenic plants [10, 18, 19]. The transgenic tobacco and Arabidopsis plants overexpressing AtPIP1-4 or AtPIP2-5 display enhanced water loss under dehydration stress . The responses of PIPs to water stress and ABA are different between upland rice and lowland rice [10, 11]. For example, OsPIP1-3 is up-regulated by osmotic stress in highland rice, while OsPIP1-3 transcript is unaltered in lowland rice, indicating that OsPIP1-3 is associated with the differential avoidance to drought in the two varieties . Salt and drought tolerance are enhanced in transgenic plants overexpressing either OsPIP1-1 or OsPIP2-2 . GhPIP2-7 expression is up-regulated in leaves after drought treatments, and overexpression of GhPIP2-7 in Arabidopsis leads to an enhanced drought tolerance in transgenic plants . TaAQP8, a wheat PIP1 gene, is induced by NaCl, which involves ethylene and H2O2 signaling. Overexpression of TaAQP8 in tobacco increases root elongation under salinity, with increased K+/Na+ ratio and Ca2+ content and reduced oxidative damages .
Most of PIPs subfamily members in Arabidopsis thaliana are down-regulated by cold treatment, but AtPIP2-5 is up-regulated . Overexpression of AtPIP2-5 alleviates the inhibition of low temperature on plant growth in transgenic Arabidopsis  and facilitates seed germination under cold stress . Chilling results in decreased expression of some PIPs in rice seedlings, but higher transcript levels of OsPIP1-1, OsPIP2-1, OsPIP2-7 in shoots and OsPIP1-1, OsPIP2-1 in roots were observed in a chilling-tolerant variety than a chilling-sensitive one during the recovery at room temperature, indicating an important role of PIPs in re-establishing water balance after chilling conditions . OsPIP1-3 plays an important role in chilling tolerance through interacting with members of OsPIP2 subfamily and improving water balance .
Medicago falcata is closely related to alfalfa (Medicago sativa), the most important perennial forage legume, with better cold tolerance [25–27]. Higher levels of sucrose, myo-inositol, galactinol, and raffinose family oligosaccharides (RFOs) are accumulated in falcata than in alfalfa during cold acclimation . Transcript levels of myo-inositol phosphate synthase (MIPS), galactinol synthase (GolS), and myo-inositol transporter-like (INT-like) genes are accordingly induced in falcata [27–29]. In addition, expression of S -adenosylmethionine synthetase (SAMS) and a temperature induced lipocalin (TIL) are also induced by low temperature, and these genes are associated with cold tolerance in falcata plants [30, 31].
In our previous investigation a fragment encoding a PIP was harvested in a cDNA library of falcata responsive to cold , and no other PIP genes was found in the library, implying a potential role of the PIP in cold tolerance of falcata. We isolated the cold responsive PIP from falcata, which was highly homologous to MtPIP2-7. However, there is no report on the role of plant PIP2-7 in regulation of cold tolerance. The objective of this study was to investigate the role of the PIP2-7 gene (MfPIP2-7) in cold tolerance of falcata. MfPIP2-7 transcript in response to low temperature was analyzed, and transgenic tobacco plants overexpressing MfPIP2-7 were generated for examining tolerance to abiotic stresses such as cold and nitrate reduction.
Characterization of MfPIP2-7
A cDNA sequence of MfPIP2-7 (910-bp) was cloned from falcata leaves. It contains an open reading frame (ORF) of 864 bp (GenBank accession number FJ607305) and encodes a deduced polypeptide of 30.9 kDa (GenBank accession number ACM50914). Sequence blast showed that MfPIP2-7 was most homologous (97.2 %) in AA sequence to a PIP2-7 (MTR_2g094270) in M. truncatula. A phylogenetic tree of MfPIP2-7 and all PIPs from Arabidopsis showed that MfPIP2-7 is most similar to AtPIP2-7 (Additional file 1: Figure S1). A multiple alignments of three PIPs indicated that six amphipathic channels/transmembrane helices and two signature motifs, which characterize major intrinsic protein, were found in MfPIP2-7 protein (Additional file 1: Figure S2). MfPIP2-7 was predicted to be localized in plasma membrane using PSORT Prediction (http://psort.hgc.jp/form.html) and cross checking with CELLO v.2.5 prediction software (http://cello.life.nctu.edu.tw/).
MfPIP2-7 transcript in response to abiotic stress
Analysis of transgenic tobacco plants
Freezing tolerance was evaluated using survival rate and LT50. Most of the wild type plants could not survive after freezing treatment, while 52 to 69 % transgenic plants could survive (Fig. 2c, d). Compared to a −1.3 °C of LT50 in the wild type, lower levels of LT50 were observed in transgenic lines than in the wild type (Fig. 2d). Moreover, the difference in LT50 was blocked by pretreatment with dimethylthiourea (DMTU), a scavenger of H2O2, and DMTU treatment resulted in increased LT50 in all plants (Fig. 2e), indicating that the differential LT50 between transgenic plants and the wild type was associated with H2O2.
Sensitivity of yeast cells expressing MfPIP2-7 to externally supplied H2O2
Abiotic stress responsive genes were induced in transgenic plants
Amino acid levels were altered in transgenic plants
Analysis of free amino acid levels (nmol g -1 FW) in transgenic plant line (3-1) in comparison with the wild type (WT)
γ-Amino butyric acid
An ORF encoding MfPIP2-7 was cloned from falcata. MfPIP2-7 has the highest AA similarity with MtPIP2-7 or AtPIP2-7 among PIP proteins in M. truncatula or Arabidopsis. MfPIP2-7 transcript was induced by cold and ABA treatment. ABA is signaling in regulation of downstream stress responses, including expression of multiple down-stream genes with relevance to abiotic stress tolerance [35–37]. ABA is also involved in cold acclimation of falcata . In this study, ABA was demonstrated to be involved in MfPIP2-7 expression induced by cold, suggesting that ABA-regulated MfPIP2-7 plays an important role in cold tolerance in falcata.
The role of MfPIP2-7 in cold tolerance was documented using transgenic plants. Overexpression of MfPIP2-7 resulted in enhanced tolerance to freezing and chilling stresses in transgenic tobacco plants, suggesting that MfPIP2-7 expression is associated with elevated cold tolerance. Similarly, expression of GhPIP2-7 leads to an improved growth under osmotic stress in transgenic Arabidopsis , while transgenic Arabidopsis overexpressing OsPIP1-1 or OsPIP2-2 showed improved root growth under osmotic or salt stress . The altered osmotic or drought stress in transgenic plants up- or down-regulating PIP genes expression is associated with the increase or decrease in hydraulic conductivity and transpiration [10, 15–17]. In addition, plant PIPs function to facilitate H2O2 diffusion across plasma membrane apart from as water channels [3, 34]. In this study MfPIP2-7 was found to facilitate H2O2 diffusion through expressing in yeast cells. Transgenic plants had higher levels of NtERD10B, NtERD10C, NtNIA1, NtNIA2, NtDREB1, and NtDREB2 transcripts, which were blocked by scavenger of H2O2, suggesting that the high transcript levels in transgenic plants were associated with elevated H2O2. Intercellular H2O2, mainly produced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, is an important signal in regulating expression of multiple genes associated with abiotic stress tolerance [38, 39]. H2O2 is also involved in cold and/or drought induced gene expression, such as MfMIPS and MfSAMS, in falcata [27, 30] and NIA1 in tobacco plants . NtDREB1, 2, 3, and 4 belong to DREB1/CBFs (C-repeat binding factors) transcription factors, which are induced in response to cold in tobacco plants , while CBFs regulate cold acclimation and expression of cold responsive genes. ERD10B and ERD10C belong to the dehydrin (DHN) family . They are induced by drought and cold , and protect plant cells against stress induced damages by potent chaperone activity and membrane-binding capacity for increased stabilization of diverse proteins and membrane systems . Nevertheless, the higher transcript levels of NtERD10B, NtERD10C, NtDREB1, and NtDREB2 in MfPIP2-7 transgenic plants are associated with the elevated cold tolerance.
It is interesting that NtNIA1 and NtNIA2 transcript levels were up-regulated in transgenic plants with dependence upon H2O2, which led to enhanced NR activity in both leaves and roots. NR is a key enzyme in nitrate reduction and nitrogen metabolism . The elevated NR activity resulted in alterations in free amino acid components and concentrations in transgenic plants, indicating that expression of MfPIP2-7 influences N metabolism. An elevated concentration of total free amino acids in roots may provide transgenic plants with more nitrogen under NO3 −-deficiency and thus promote NO3 −-deficiency tolerance in transgenic tobacco plants. In addition, NR-dependent NO production is involved in cold acclimation and freezing tolerance by modulating proline accumulation in Arabidopsis . Apart from proline, many free amino acid concentrations, such as argine, ornithine, and γ-amino butyric acid, were higher in transgenic tobacco than in the wild type. Many free amino acids can modulate membrane permeability and ion uptake and function as osmolyte in plants . γ-Aminobutyric acid is involved in cold acclimation and freezing tolerance in barley and wheat . Ornithine and argine are the precursor of polyamine biosynthesis, while polyamines are involved in cold tolerance . Thus the alterations in free amino acid are proposed to be associated with the elevated cold tolerance in transgenic plants.
MfPIP2-7 was characterized in this study. MfPIP2-7 transcript level is induced by cold and ABA, while ABA is involved in the cold induced expression of MfPIP2-7. MfPIP2-7 showed facilitation of H2O2 diffusion in yeast cells. Overexpression of MfPIP2-7 led to enhanced cold tolerance in transgenic tobacco plants, which was associated with the induced expression of stress responsive genes, such as NtERD10B, NtERD10C, and CBF transcription factors. Moreover, the higher levels of NtNIA1 and NtNIA2 transcripts and NR activity led to alterations of free amino acid in components and concentrations which are associated with the elevated tolerance to NO3 −-deficiency and cold.
Isolation of MfPIP2-7 cDNA from falcata
Medicago sativa subsp. falcata (L.) Arcang. cv. Hulunbeir seeds were provided by Institute of Animal Science, Chinese Academy of Agricultural Sciences. Total RNA was isolated from leaves of cold-treated falcata plants (0.1 g) . cDNA was synthesized from two micrograms of total RNA in the presence of 160 U of M-MLV reverse transcriptase (Promega, Madison, WI, USA) and oligo (dT)18 in a 20 μl reaction mixture . Primers RT59 (5’-GAACACAAACATGGGCAAAGA-3’) and RT60 (5’-CAACTCATACATAATAATTGAAACCA-3’) were designed for amplification of MfPIP2-7, based on assembly of EST sequences from the GenBank using SeqMan (DNASTAR Inc, Madison, WI, USA). PCR reaction mixture contain the first-strand cDNA as the template, primers RT59 and RT60, and Ex Taq DNA polymerase (Takara Bio Inc., Dalian, China). After sequencing of the PCR product, the deduced amino acid sequence was analyzed using DNAMAN software.
Transgenic tobacco generation
An expression plasmid pBI-MfPIP2-7 was constructed by inserting the ORF of MfPIP2-7 into the pBI121 binary vector and used for generation of transgenic tobacco plants as described previously . Seeds of the wild type (Nicotiana tabacum L. cv. Zhongyan 90) were initially provided by Crops Research Institute, Guangdong Academy of Agricultural Sciences and harvested in our laboratory. Seeds of homozygous transgenic tobacco plants (T3) and the wild type used for investigation in this study were harvested at the same time.
Plant growth and treatments
Homozygous lines of transgenic tobacco and the wild type plants of tobacco and falcata were grown in a greenhouse for 2 months as described previously . Falcata plants were placed in a growth chamber at 5 °C for 4 days for cold treatment . In addition, the detached leaves were placed in distilled water for 1 h to eliminate the potential wound stress influence, followed by moving into new beakers: (1) containing 100 μM ABA for 12 h for detecting effect of ABA on MfPIP2-7 expression; (2) containing H2O or 1 mM NAP for 2 h, followed by transferring to a growth chamber at 5 °C for 8 h as cold treatment for detecting involvement of ABA in cold-induced expression of MfPIP2-7 as described previously , while those placed in beakers containing H2O under room temperature 8 h were used as a non-stressed control. In addition, tobacco leaf discs were placed in beakers containing H2O (control) or 5 mM DMTU for 2 h before determinations of freezing tolerance, gene expression, or NR activity. The experiments were repeated for three times.
DNA blot hybridization
Genomic DNA was extracted from tobacco leaves using hexadecyltrimethylammonium bromide (CTAB) as previously described . DNA samples (15 μg) were separated by electrophoresis on 0.8 % agarose gel after digestion overnight with HindIII, followed by transfer to Hybond XL nylon membrane (Amersham, GE Healthcare Limited, Buckinghamshire, UK). Hybridization was conducted using [α-32P] dCTP labeled fragment (407 bp) of MfPIP2-7 as probe. The hybridization signals were detected using Typhoon Trio (General Electric Company, Fairfield, CT).
Real time quantitative reverse transcription PCR (qRT-PCR)
One μg of total RNA was used for synthesis of first-strand cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Takara). After dilution the cDNAs were used as template in 10-μl PCR reactions containing 200 nM forward and reverse primers and 5 μl SYBR Premix Ex Taq (Takara), and qRT-PCR was conducted in MiniOption Real-Time PCR System (Bio-Rad, Hercules, CA) . Parallel reactions to amplify actin were used to normalize the amount of template. We use actin as reference gene because it had been demonstrated to be reliable in M. falcata and M. truncatula . The primers and their sequences used in this study are listed in Additional file 1: Table S1. Three technical and two biological replicates were conducted in each experiment.
Abiotic stress tolerance assessment
Survival rate and the temperature (LT50) that resulted in 50 % lethal were measured to evaluate freezing tolerance as previously described . For measurement of survival rate, 6-week-old tobacco plants were placed in a growth chamber under light of 700 μmol photon m−2 s−1, with decreasing temperature from 25 to −3 °C within 6 h and maintained for 3 h . The experiments contained five replicates and 20 plants each line per replicate. Plant survival rate was calculated 3 d after plants were moved to room temperature for recovery. LT50 was calculated using a fitted model plot based on ion leakage data after leaf discs detached from 6-week-old tobacco plants were treated with freezing [26, 30]. For assessment of chilling tolerance, 10-week-old pot plants were chilled at 3 °C for 4 d under light of 200 μmol photon m−2 s−1 in a growth chamber with a 12-h photoperiod. Ion leakage, F v/F m, and A were measured as previously described [28, 30]. For nitrogen-deprivation treatment, tobacco seeds were sterilized and germinated on half strength of MS medium, followed by transferring to a half strength of MS medium containing 0 or 0.2 mM NO3 − and growing in a growth room with a 12-h photoperiod under light of 200 μmol photon m−2 s−1 at 25 °C, while those growing on ½ MS medium were used as a control. Compared to ½ MS medium that contained 10 mM NO3 −, the nitrogen deprivation medium was made by replacing NH4NO3 with (NH4)2SO4 so that KNO3 was the sole nitrate source at 0 or 0.2 mM. The K+ concentration was adjusted to 10 mM by the addition of K2SO4 in all media . Plant fresh weight was weighed at the eighth week after transplanting.
Yeast growth assay
Yeast growth assay was conducted according to the method described by Bienert et al.  with modification. The Saccharomyces cerevisiae strain INVSc1 was transformed with either an empty pYES2 (Invitrogen) as control or derivate of pYES2 carrying MfPIP2-7 coding sequence. Yeast cells were grown on SD/-Ura synthetic medium containing 2 % glucose until an A600nm of 0.6 to 0.8, followed by two times washing with liquid SG/-Ura synthetic medium containing 2 % galactose to an A600nm of 0.6. After a series of dilution, 10 μl were spotted on solid SG/-Ura medium containing various concentrations of H2O2 as indicated. Differences in growth and survival were recorded after 4 days of incubation at 30 °C.
Measurement of NR activity
Tobacco leaves (0.5 g) were ground in a mortar with pestle in 5 ml of 50 mM phosphate buffer (pH 7.8) containing 2 % (w/v) polyvinylpyrrolidone (PVP), 2 mM EDTA and 5 mM dithiothreitol (DTT) at 4 °C. The homogenate was centrifuged at 12,000 × g for 15 min for recovery of the supernatant. Nitrate reductase activity and protein content were measured as described previously . The enzyme reaction mixture (2 ml) contained 50 mM K-phosphate buffer (pH 7.5), 60 mM KNO3 and 0.25 mM NADH. The reaction was started by addition with 400 μl of the supernatant and incubated at 25 °C for 30 min, followed by addition of 1 ml of 1 % sulphanilamide in 1.5 M HCl and 1 ml of 0.01 % 1-naphthylamine. After incubated for 15 min, the mixture was centrifuged for 5 min at 10,000 × g and absorbance at 540 nm of the supernatant was measured to determine nitrite production. One unit of NR was defined as the amount of enzyme required for catalyzing the production of one μmol NO2 − within one hour. Protein content in the enzyme extracts was determined using Coomassie Brilliant Blue G-250.
Analysis of free amino acids
Free amino acids were extracted from leaves (0.4 g) by grinding in 1 ml of 6 % (w/v) 5-sulfosalicylic acid at 4 °C. The extract was centrifuged for 15 min at 12,000 rpm. The supernatant was subjected to derivatization by phenyl isothiocyanate, followed by filtration (0.45 μm). 20 μl of the filtrate was injected into a Hitachi model L-8800 amino acid analyzer (Hitachi Co. Ltd., Tokyo, Japan), supplied with Hitachi chromatographic column 855–350, for measurement of amino acids.
A, net photosynthetic rate; ABA, abscisic acid; AQPs, aquaporins; CBFs, C-repeat binding factors; CTAB, hexadecyltrimethylammonium bromide; DHN, dehydrin; DMTU, dimethylthiourea; DREB, dehydration responsive element binding protein; DTT, dithiothreitol; ERD, early response to drought; GolS, galactinol synthase; INT-like, myo-inositol transporter-like; LEAs, late embryogenesis abundant proteins; MIPS, myo-inositol phosphate synthase; MS, Murashige and Skoog; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NAP, naproxen; NIA, nitrate reductase; NR, nitrate reductase; ORF, open reading frame; PIPs, Plasma membrane intrinsic proteins; PVP, polyvinylpyrrolidone; RFOs, raffinose family oligosaccharides; SAMS, S-adenosylmethionine synthetase; TIL, temperature induced lipocalin
This work was supported by the National Basic Research Program of China (2014CB138701) and the Natural Science Foundation of China (31472142, 30830081).
Availability of data and materials
All datasets supporting the results of this study are included in the article and the additional files.
CZ analyzed gene expression in falcata and performed all experiments using transgenic tobacco plants; TW generated transgenic tobacco and conducted DNA blot analysis; ZG designed the study, analyzed the data, and wrote the paper; SL designed the study with ZG. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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