Table 1.
PCR primers used in the current study.
Fig 1.
Nucleotide sequence of the ZmGAPP promoter.
The “A” of the translation initiation code “ATG” of ZmGAPP was designated as “+1”. Putative cis-acting elements underlined or shown in the border. See Table 2 for descriptions of the elements. The arrow above the sequence indicates the start point of different deletion fragments (D1–D9).
Table 2.
Identification of cis-acting elements in the ZmGAPP promoter sequence using the PLACE and PlantCARE databases.
Fig 2.
GUS histochemical assays of tissues of D1–D9 and CaMV 35S transgenic tobacco plants.
Twenty-day-old seedlings (A), and flowers, fruits and seeds (B) were incubated in staining solution at 37°C. The D1–D3 and D4–D9 fragments were stained for 24 h and 6 h, respectively, following which the samples were observed and photographed after decolorization. Scale bar: 0.5 cm.
Fig 3.
GUS activity assays of D1–D9 transgenic tobacco plants under normal conditions.
Values are means ± SD from 15 independent transgenic plants (5 individual plants/ line, 3 lines for each construct). Different lowercase letters above the bars indicate significant differences at P < 0.05.
Fig 4.
GUS staining of detached leaves of transgenic tobacco under normal and salt-stress conditions.
Ninety leaf discs (diameter 0.5 cm) from 15 individual plants (5 individual plants/ line, 3 lines for each construct) of D1–D9 and CaMV 35S transgenic tobacco plants were incubated in liquid 1/2 MS medium supplemented with 200 mM NaCl for 1, 3, 6, 12, 16, 24, 48, and 72 h; leaf discs floated in liquid 1/2 MS medium were used as control. The leaf discs of D1–D3 plants were then incubated in staining solution at 37°C for 24 h, whereas the leaf discs of D4–D9 and CaMV 35S transgenic plants were stained for 6 h. Finally, the samples were observed and photographed after decolorization.
Fig 5.
GUS staining of detached leaves of transgenic tobacco under normal and PEG treatment conditions.
Ninety leaf discs (diameter 0.5 cm) from 15 individual plants (5 individual plants/ line, 3 lines for each construct) of D1–D9 and CaMV 35S transgenic tobacco plants were incubated in liquid 1/2 MS medium supplemented with 18% PEG 6000 (w/v) for 1, 3, 6, 12, 16, 24, 48, and 72 h; leaf discs floated in liquid 1/2 MS medium were used as control. The leaf discs of D1–D3 plants were then incubated in staining solution at 37°C for 24 h. The leaf discs of D4–D9 and CaMV 35S transgenic plants were stained for 6 h. Finally, the samples were observed and photographed after decolorization.
Fig 6.
Analysis of different ZmGAPP promoter deletion constructs in transgenic tobacco plants under normal and NaCl treatment conditions.
The D1–D9 and CaMV 35S transgenic tobacco plants were incubated in liquid 1/2 MS medium supplemented with 200 mM NaCl for 24 h; plants grown in liquid 1/2 MS medium were treated as control. (A) qRT-PCR analysis. The tobacco α-tubulin (AJ421411) was used as an internal control. (B) GUS histochemical staining. The leaves of D1–D3 plants were incubated in staining solution at 37°C for 24 h; leaves of D4–D9 and CaMV 35S transgenic plants were stained for 6 h. Samples were then observed and photographed after decolorization. (C) GUS activity assays. Values represent the means ± SD from 15 independent transgenic plants (5 individual plants/ line, 3 lines for each construct). Different lowercase letters above the bars indicate significant differences at P < 0.05.
Fig 7.
Analysis of different ZmGAPP promoter deletion constructs in transgenic tobacco plants under normal and PEG treatment conditions.
The D1–D9 and CaMV 35S transgenic tobacco plants were incubated in liquid 1/2 MS medium supplemented with 18% PEG 6000 (w/v) for 24 h; plants grown in liquid 1/2 MS medium were treated as control. (A) qRT-PCR analysis. The tobacco α-tubulin (AJ421411) was used as an internal control. (B) GUS histochemical staining. The leaves of D1–D3 plants were incubated in staining solution at 37°C for 24 h; leaves of D4–D9 and CaMV 35S transgenic plants were stained for 6 h. Samples were then observed and photographed after decolorization. (C) GUS activity assays. Values represent the means ± SD from 15 independent transgenic plants (5 individual plants/ line, 3 lines for each construct). Different lowercase letters above the bars indicate significant differences at P < 0.05.
Fig 8.
GUS transient assays in tobacco leaves.
(A) The plasmids used in the transient assay. The CaMV 35S represents the full-length 35S promoter; p-mini35S represents the truncated 35S (–46 to +10 bp) promoter. The test construct consisted of the p-71bp-mini35S, in which the 71-bp region (–219 to –148 bp) identified in the ZmGAPP promoter was fused to the p-mini35S promoter to drive the GUS expression. (B) GUS activity in the transiently transformed tobacco leaves with constructs p-mini35S and p-71bp-mini35S under both normal and 200 mM NaCl or 18% (w/v) PEG 6000 treatment for 24 h. Results are mean ± SD from three experiments (n = 15). Different lowercase letters above the bars indicate significant differences at P < 0.05.
Fig 9.
Diagrams of the D8 fragment of the ZmGAPP promoter and the 71-bp region for NaCl/PEG stress response.
Putative cis-regulatory elements in the 71-bp (–219 to –148 bp) sequence of the ZmGAPP promoter predicted by PlantCARE and PLACE are shown in the border. CAAT box: common cis-acting element in promoter and enhancer regions; TGACG motif: cis-acting element involved in MeJA-responsiveness; GT-1 motif: cis-acting element involved in pathogen- and NaCl-induced gene expression of SCaM-4 in soybean and Arabidopsis.