Isolation, Expression Patterns And Role In Regulating Stomatal Movements Of An Aquaporin-like Gene In Vicia Faba

Stomatal pores in the epidermis of plant leaves are essential for CO2 influx into the leaves for photosynthetic carbon fixation. Stomatal closure and opening play an important role in regulating photosynthesis and transpiration, so many researchers are interested in the mechanism of stomatal movement, which are focused on signal transduction, osmotic substances in cytoplasm, ion channels and receptors in plasma membrane, cytoskeleton and so on. However, there are few studies on the regulation of water transport in stomatal movement. The discovery of plant aquaporins gives a new idea for exploring the regulation of water-transmembrane movement in stomatal movement.Aquaporins are channel proteins for water transport. A lot of aquaporins have been identified in organisms ranging from bacteria to animals and plants. The plant aquaporins could be divided into four classes: plasma membrane intrinsic proteins (PIPs) , tonoplast intrinsic proteins (TIPs) , NOD26-like membrane intrinsic proteins (NIPs) and small and basic intrinsic proteins (SIPs). However, their water channel activity, subcellular locations and functions in the signal transduction pathway need to be further studied.Although many kinds of aquaporin genes have been isolated, most of researches are still focused on the aquaporins in roots. Some evidence has previously indicated that aquaporin genes were expressed in Vicia faba guard cells and involved in the regulation of stomatal movement. However, which aquaporins are expressed in Vicia faba guard cells and function in stomatal movement are unclear. In this study, we isolated an aquaporin-like gene VfPlPl from Vicia faba leaf epidermis and analyzed its sequence, the expression and localization of this gene. At the same time, an over-expression vector for plants was constructed and transformed into Arabidopsis. In addition, the function and mechanism of VfPlPl in transgenic Arabidopsis were primarily studied. The results showed as followed.Two oligonucleotide degenerate primers were designed based on several conserved regions in the plant aquaporin amino acid sequences. Using RT-PCR and RACE-PCR, a full length cDNA of aquaporin-like gene VfPlPl was isolated from cDNA prepared from Vicia faba leaf epidermis. The analysis of the sequence shows that VfPlPl cDNA is 1039-bp in length and contains an open reading frame of 870-bp, flanked by 5' and 3'non-coding regions of 47- and 122-bp, respectively. The protein coding by 290 amino acids exhibits six predicted transmembrane helices, M1P family signature sequence SGxHxNPAVT, the special plasma membrane signature sequence GGGANXXXXGY and TGI/TNPARSL/FGAAI/VI/VF/YN, and a calculated molecular mass of about 30.7kD. As compared to PIPs identified from Arabidopsis , VfPIPl has the highest sequence identity at the amino acid level with AtPIPlb (84.48%), so VfPIPl should be a memberof PIP 1 subfamily and should have similar functions with PIPs.Northern blot analysis indicated that VfPIPl was expressed in roots and leaves more than in stems, and induced by ABA. Moreover, VfPIPl was strongly expressed in guard cells by in situ hybridization.To explore the function of VfPIPl, the gene was introduced into Arabidopsis and transgenic plants were obtained. In addition, the differences in drought tolerance and stomatal response to light, dark, ABA and CaCl2 between transgenic plant and wild type were studied.The results indicated that transgenic Arabidopsis decreased in transpiration rate and increased in drought tolerance. In addition, transgenic Arabidopsis can promote light-induced stomatal opening or dark-, ABA- and CaC12-induced stomatal closure. The above results revealed that VfPIPl can reduce transpiration to sustain water balance and play an important role in water movement under water deficiency by accelerating the rate of stomatal movement.This was the first report that plasma membrane aquaporins were involved in the regulation of stomatal movement. Although stomatal movement is controlled by many kinds of extracellular and intracellular factors, any route cannot do with...