Regulation And Function Analysis Of Three Soybean Plasma Membrane Intrinsic Proteins (PIPs)

Soybean is an important food and economic crop. Drought, salinity, pathogen, insect pests, and other stresses greatly affect soybean production. Identification and characterization of functional genes related to stress tolerance is essential for soybean improvement. Water channel proteins (Aquaporins, AQPs) are a group of water transporters, which directly participate in water uptake. Some of the family members have been reported to play important roles in drought and salt tolerance. There are 66 AQPs in the soybean genome, including 22 plasma membrane intrinsic protein (PIPs). The function of these PIPs has not been reported. In this research, regulation, expression, and function of three soybean PIP genes, including GmPIPl;6, GmPIP2; 1, and GmPIP2;9 were studied. Transient expression of GmPIP1;6-GFP and GmPIP 2;1-GFP fusion constructs on onion epidermal cells showed their localization on plasma membrane. Quantitative RT-PCR (qRT-PCR) showed that GmPIP 1;6 and GmPIP2;l mainly expressed in pods, flowers, roots, and stems, while there was almost no expression or low expression in leaves. GmPIP2;9 highly expressed in roots and pods and weakly expressed in stems and leaves. Analysis of the transgenic plants that expressed the endogenous PIP2;9 promoter::GUS construct revealed that GmPIP2;9 mainly expressed in roots, flowers, pods, and shoots, which was consistent with results from qRT-PCR analysis. Gus staining and section analysis showed that GmPIP2;9 was highly expressed in endodermis, exodermis, and stele in roots, and endodermis, exodermis and xylem in leaves. The expression of GmPIP 1; 6, GmPIP2; 1 and GmPIP2; 9 in leaves were all induced by salt stress. And GmPIP2; 9 expression can be induced by osmotic stress.To study the physiological function of these three PIP genes, transgenic soybean plants that overexpress GmPIP1;6, GmPIP2;1, and GmPIP2;9 were produced and designated as GmPIP1;6-oe, GmPIP2;1-oe, and GmPIP2;9-oe. Under salt stress condition, these transgenic soybeans showed better growth performance compared to the wildtype (WT) plants. The salt-treated transgenic plants showed better growth, greener leaf and lower leaf Na+concentration compared to those of the WT. Overexpression of GmPIP1;6 resulted in enhanced photosynthesis, transpiration rate, and leaf gas exchange compared to the WT. In addition, we also assayed the drought tolerance of GmPIP2;9 transgenic plants by 20% PEG8000 treatment. GmPIP2;9-oe plants recovered growth quicker and lost less water than those of the WT. In soil pot experiment, under both water sufficient and deficient conditions, the photosynthesis and transpiration rate of GmPIP2;9-oe lines are higher than those of the WT. GmPIP2;9-oe survived in the long-term drought stress while wild type died.In summary, the study showed that GmPIP1;6, GmPIP2; 9, and GmPIP2;1 are important genes that are involved in soybean water uptake and homeostasis. These genes have great potential for the improvement of stress tolerance in soybean breeding.