Figure 1.
Comparison of ZmPMP3s and plasma membrane protein 3 related proteins.
A) Alignment of deduced amino acid sequences of ZmPMP3s with Arabidopsis AtRCI2A/B (NP_187239.1, NP_187240.1), rice Oslit6a/b (AY607689.1, A2Y075.2), Aneurolepidium chinense AcPMP3-1/2 (BAD34658.1, BAD34659.1), Puccinellia tenuiflora PutPMP3-1/2 (BAG54793.1, BAG54794.1), Hordeum vulgare Blt101 (CAA80984.1). The amino acid identity in dark grey is larger than 75%, and the amino acid identity in light grey is larger than 50%. Straight arrows marked predicted transmembrane domains. The curved arrow indicated a putative loop structure. B) Phylogenetic analysis based on putative amino acid sequences of PMP3-related proteins in rice, Arabidopsis, Hordeum vulgare, Aneurolepidium chinense, and Puccinellia tenuiflora. The analysis was performed by using the MEGA 4.0 program with neighbor joining method and with 1000 replicates. Numbers on the figure 1B were bootstrap values.
Table 1.
Identification of the ZmPMP3 genes in maize.
Figure 2.
Subcellular localization of ZmPMP3-1-GFP fusion proteins in onion epidermal cells.
A) Fluorescent microscopic images of GFP protein. B) Fluorescent microscopic images of nonplasmolyzed cells transiently expressing ZmPMP3-1-GFP fusion protein. C) Fluorescent microscopic images of plasmolyzed cells in 30% sucrose solution.
Figure 3.
Expression patterns of the eight ZmPMP3 genes in CN165 under various stresses.
One-week old shoots and roots of maize seedlings, treated by 200 mM NaCl, 4°C, 30% PEG-6000, and 100 µM ABA, were harvested respectively. The qRT-PCR was performed to determine the relative expression levels of the eight genes under various treatment. The maize GAPDH gene was selected as internal control. Data from qRT-PCR experiments were analyzed according to the 2−ΔΔCT method. Data represents the average of three independent experiments ±SE. when the relative expression levels go up to 2 fold or down to 0.5 fold, the difference of the expression is regarded as significant (P value<0.05).
Figure 4.
Expression patterns of the ZmPMP3 genes in various tissues of maize.
Total RNA was isolated from various tissues (mature leaves, mature roots, young silks, ears, tassels and stalks) at V12 and VT stage. Maize GAPDH gene was selected as internal control. Data from qRT-PCR experiments were analyzed according to the 2−ΔΔCT method. Data represents the average of three independent experiments ±SE. All genes expression levels was assigned a value of 1 in leaf.
Figure 5.
Functional complementation in yeast mutant strains.
A) The 10-fold dilutions of YR31-1, YR93-31, and YR93-31M1-8 strains were spotted onto solid YPDA plates and YPDA supplemented with 25 mM NaCl. B) Dose- response growth curve of YR93-1, YR93-31, and YR93-31Ms (YR93-31 transferred by pAUR123-ZmPMP3s) strains of yeast under various concentrations of NaCl. C) The relative growth rates of YR93-1, YR93-31, and YR93-31M1-8 yeast strains under 5 mg/L hygromycin B treatment. D) The relative growth rates of YR93-1, YR93-31, and YR93-31M1-8 yeast strains under low calcium and high calcium conditions. Values are mean±SE (n = 3).
Figure 6.
Comparison of green cotelydon rates between ZmPMP3-1 transgenic plants and WT.
A) The performance of transgenic lines and WT in MS plate and 150 mM 175 mM NaCl plates. The L4, L6, L11 represent three different independent transgenic lines. B, C, and D) Comparison of the green cotyledon rates between transgenic lines (L4, L6 and L11) and WT. The green cotelydon rates were calculated 5-days after sowing under normal, 150 mM and 175 mM NaCl treatments. The number of plants is expressed as means SEM (n = 40 plants). A statistic difference measurement from three independent replicates. “*” indicates significant difference between transgenic plants and WT control with F-test (* P<0.05, ** P<0.01). E) Transcript level of ZmPMP3-1 in transgenic plants (L4, L6, and L11) and WT. Total RNA was extracted from leaves of 3-week old plants grown under normal conditions. The transcript level of ZmPMP3-1 was measured by RT-PCR. Thirty PCR cycles were used, and the Arabidopsis Actin2 gene was amplified as the control.
Figure 7.
Comparison of fresh weight between ZmPMP3-1 transgenic plants and WT.
A) The performance of transgenic lines (L4, L6, and L11) and WT in 150 mM, 175 mM NaCl plates and control. The photos were taken two-week after sowing. B) Comparison of fresh weights between transgenic lines (L4, L6 and L11) and WT. The fresh weights were calculated two-week after sowing. The number of plants is expressed as means SEM (n = 20 plants). A statistic difference measurement from three independent replicates. “*” indicates significant difference between transgenic plants and WT. F-test (* P<0.05, ** P<0.01).
Figure 8.
Comparison of the number of lateral roots between ZmPMP3-1 transgenic plants and WT.
A) The root growth performance of transgenic lines (L4, L6 and L11) and WT in MS and 200 mM NaCl plates. The photos were taken 10-day after transferring. B) Comparison of the lateral root number between transgenic lines (L4, L6, and L11) and WT. The 0ne-week old seedlings were transferred to MS and 200 mM NaCl plates. The numbers of lateral roots were determined 10-day after transferring. The number of plants is expressed as means SEM (n = 10 plants). A statistic difference measurement from three independent replicates. “*” indicates significant difference between transgenic plants and WT. F-test (* P<0.05, ** P<0.01).
Figure 9.
Analysis of the survival rate in ZmPMP3-1 transgenic plants and WT.
A) The performance of ZmPMP3-1 transgenic lines (L4, L6 and L11) and WT under salt condition and control. The 4 week-old transgenic and WT plants were irrigated by the 300 mM NaCl solution for one week. Here the transgenic plants grew well, but the WT plants exhibited chlorosis and sterility. B) The performance of the anthotaxy tips of transgenic lines (L4, L6 and L11) and WT under salt stress. After one week salt treatment, the almost anthotaxy tips of WT plants exhibited chlorosis. The red arrows marked the wilted anthotaxy tips of WT. C) comparison of the survival rate between transgenic lines and WT. Each value represents the average of 15 plants with three replicates. Values are mean±SE. “*” indicates significant difference between transgenic plants and WT. F-test (* P<0.05, ** P<0.01).
Figure 10.
Elucidation of the CMS and Photosynthetic rate of ZmPMP3-1 transgenic plants.
A) Comparison of maximum fluorescence ratios (Fv/Fm) between transgenic lines (L4, L6 and L11) and WT. The Fv and Fm values were measured to calculate the maximum fluorescence ratios (Fv/Fm). The Fv and Fm values of 4 week old Arabidopsis were measured after one week treatment by 300 mM NaCl. The number of plants is expressed as means SEM (n = 8 plants). A statistic difference measurement from three independent replicates. “*” indicates significant difference between transgenic plants and WT. F-test (* P<0.05, ** P<0.01). B) Comparison of the CMS between transgenic lines (L4, L6 and L11) and WT. The three-old plants were treated with 300 mM NaCl and the CMSs were determined at selected time points. The CMS values represented the average of six individual plants.
Table 2.
Up-regulated genes in ZmPMP3-1 overexpressed transgenic plants.