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 [5]. 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 [9]. Transcripts of AtPIPs are generally down-regulated in leaves upon gradual drought stress, but AtPIP1-4 and AtPIP2-5 transcript levels are up-regulated [12]. 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 [20]. 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 [10]. Salt and drought tolerance are enhanced in transgenic plants overexpressing either OsPIP1-1 or OsPIP2-2 [13]. 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 [21]. 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 [22].
Most of PIPs subfamily members in Arabidopsis thaliana are down-regulated by cold treatment, but AtPIP2-5 is up-regulated [9]. Overexpression of AtPIP2-5 alleviates the inhibition of low temperature on plant growth in transgenic Arabidopsis [23] and facilitates seed germination under cold stress [20]. 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 [24]. OsPIP1-3 plays an important role in chilling tolerance through interacting with members of OsPIP2 subfamily and improving water balance [8].
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 [27]. 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 [32], 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.