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Front Plant Sci
2018 Mar 26;9:530. doi: 10.3389/fpls.2018.00530.
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Roles of Soybean Plasma Membrane Intrinsic Protein GmPIP2;9 in Drought Tolerance and Seed Development.
Lu L
,
Dong C
,
Liu R
,
Zhou B
,
Wang C
,
Shou H
.
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Aquaporins play an essential role in water uptake and transport in vascular plants. The soybean genome contains a total of 22 plasma membrane intrinsic protein (PIP) genes. To identify candidate PIPs important for soybean yield and stress tolerance, we studied the transcript levels of all 22 soybean PIPs. We found that a GmPIP2 subfamily member, GmPIP2;9, was predominately expressed in roots and developing seeds. Here, we show that GmPIP2;9 localized to the plasma membrane and had high water channel activity when expressed in Xenopus oocytes. Using transgenic soybean plants expressing a native GmPIP2;9 promoter driving a GUS-reporter gene, it was found high GUS expression in the roots, in particular, in the endoderm, pericycle, and vascular tissues of the roots of transgenic plants. In addition, GmPIP2;9 was also highly expressed in developing pods. GmPIP2;9 expression significantly increased in short term of polyethylene glycol (PEG)-mediated drought stress treatment. GmPIP2;9 overexpression increased tolerance to drought stress in both solution cultures and soil plots. Drought stress in combination with GmPIP2;9 overexpression increased net CO2 assimilation of photosynthesis, stomata conductance, and transpiration rate, suggesting that GmPIP2;9-overexpressing transgenic plants were less stressed than wild-type (WT) plants. Furthermore, field experiments showed that GmPIP2;9-overexpressing plants had significantly more pod numbers and larger seed sizes than WT plants. In summary, the study demonstrated that GmPIP2;9 has water transport activity. Its relative high expression levels in roots and developing pods are in agreement with the phenotypes of GmPIP2;9-overexpressing plants in drought stress tolerance and seed development.
FIGURE 1. Subcellular localization of GmPIP2;9. (A,B) Onion epidermal cells expressing green fluorescent protein (GFP). (A) GFP signals in the nucleus, cytoplasm, and plasma membrane (PM). (B) Bright-field light image. (C) Onion epidermal cells expressing GmPIP2;9-GFP with GFP signals in the PM. (D) Onion epidermal cells expressing red fluorescent signal from the CD3-1007 PM marker. (E,F) Merged fluorescent and bright-field images of an epidermal cell expressing the GmPIP2;9-sGFP fusion protein and the CD3-1007 PM marker. Bars = 100 μm.
FIGURE 2. Expression of functional GmPIP2;9 in Xenopus oocytes. (A) YFP localization of nYFP-GmPIP2;9 in Xenopus oocytes. The oocytes were observed 2 days after injection as described in Section âMethods.â Bars = 100 μ m. Fluorescence (left), bright-field (middle), and merged images (right) showing that nYFP-GmPIP2;9 fusion protein is expressed in plasma membrane of oocytes. (B) Oocytes were injected with 23 ng GmPIP2;9 cRNA or water. The osmotic water permeability coefficients (Pf) of oocytes were determined from swelling kinetics. Data represent the mean ± SD measurements from 10 oocytes. ââP < 0.01.
FIGURE 3. Expression patterns of GmPIP2;9. (A) Relative expression levels of GmPIP2;9 in root (R), stem (S), unifoliate leaf (UL), trifoliolate leaf (TL), flower (F), and pod (P). All data are means of three biological replicates with error bars indicating SD. (B) β-glucuronidase (GUS) staining of root (a), stem (b), cross section of root (c), leaf (d), flower (e), pod (f), and developing seeds (g). Bar = 1 cm in a, b, d, e, and f. Bar = 1 mm in c and g.
FIGURE 4. Expression pattern of GmPIP2;9 in soybean plants under normal and drought treatments. Thirteen-day-old soybean seedlings were treated with or without 20% polyethylene glycol (PEG) in nutrient solution. Total RNA was extracted from (A) leaves and (B) roots of these seedlings at 1 h, 2 h, 6 h, 12 h, and 1 day (1â12h and 1d) of PEG treatment, and 2 h, 6 h, 1 day, and 3 days of the recovery (re2h, re6h, re1d, and re3d). Data represent the means ± SD. Three replicates were used. Expression levels of treated plants are relative to control plants at each time point.
FIGURE 5. Growth performance of wild-type (WT) and GmPIP2;9-overexpression (Oe) plants under normal and polyethylene glycol (PEG)-mediated drought conditions. (A) Thirteen-day-old WT and GmPIP2;9-Oe plants were treated with or without 20% PEG in culture media for 2 days, followed by a 5-day recovery. (B) Fresh shoot weight. (C) Fresh root weight. Data are means ± SD (n = 3). âP < 0.05 and ââP < 0.01.
FIGURE 6. Performance of wild-type (WT) and GmPIP2;9-overexpression (Oe) plants under drought stress conditions. (A) Drought stress treatment was applied to 2-week-old seedlings of WT, GmPIP2;9-Oe1, and -Oe2 lines by withholding water for 12 days and then re-watering for 3 days. (B) Water was withheld from 40-day-old WT and GmPIP2;9-Oe plants for 21 days, and then plants were re-watered for 7 days.
FIGURE 7. Daily (A) net CO2 assimilation (AN), (B) stomatal conductance (gs), and (C) transpiration rate (Tr) of wild-type (WT) and GmPIP2;9-overexpression (Oe) transgenic soybean plants under normal and drought stress conditions. Water was withheld from 40-day-old WT and GmPIP2;9-Oe transgenic soybean plants for 11 days. An LI-6400 was used to measure AN, gs, and Tr between 8 AM and 2 PM on day 11 of drought treatment. The parameters were measured in four different plants per treatment. Data are the means ± SD (n = 4). âP < 0.05 and ââP < 0.01.
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