Isolation and Functional Validation of Salinity and Osmotic Stress Inducible Promoter from the Maize Type-II H⁺-Pyrophosphatase Gene by Deletion Analysis in Transgenic Tobacco Plants

<div><p>Salinity and drought severely affect both plant growth and productivity, making the isolation and characterization of salinity- or drought-inducible promoters suitable for genetic improvement of crop resistance highly desirable. In this study, a 1468-bp sequence upstream of the translation initiation codon ATG of the promoter for <i>ZmGAPP</i> (maize Type-II H⁺-pyrophosphatase gene) was cloned. Nine 5´ deletion fragments (D1–D9) of different lengths of the <i>ZmGAPP</i> promoter were fused with the GUS reporter and translocated into tobacco. The deletion analysis showed that fragments D1–D8 responded well to NaCl and PEG stresses, whereas fragment D9 and <i>CaMV 35S</i> did not. The D8 segment (219 bp; -219 to -1 bp) exhibited the highest promoter activity of all tissues, with the exception of petals among the D1–D9 transgenic tobacco, which corresponds to about 10% and 25% of <i>CaMV 35S</i> under normal and NaCl or PEG stress conditions, respectively. As such, the D8 segment may confer strong gene expression in a salinity and osmotic stress inducible manner. A 71-bp segment (-219 to -148 bp) was considered as the key region regulating <i>ZmGAPP</i> response to NaCl or PEG stress, as transient transformation assays demonstrated that the 71-bp sequence was sufficient for the salinity or osmotic stress response. These results enhance our understanding of the molecular mechanisms regulating <i>ZmGAPP</i> expression, and that the D8 promoter would be an ideal candidate for moderating expression of drought and salinity response genes in transgenic plants.</p></div

GUS histochemical assays of tissues of D1–D9 and <i>CaMV 35S</i> transgenic tobacco plants.

<p>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.</p

Analysis of different <i>ZmGAPP</i> promoter deletion constructs in transgenic tobacco plants under normal and NaCl treatment conditions.

<p>The D1–D9 and <i>CaMV 35S</i> 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 <i>α-tubulin</i> (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 <i>CaMV 35S</i> 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 <i>P</i> < 0.05.</p

GUS transient assays in tobacco leaves.

<p>(A) The plasmids used in the transient assay. The <i>CaMV 35S</i> 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 <i>ZmGAPP</i> 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 <i>P</i> < 0.05.</p

GUS staining of detached leaves of transgenic tobacco under normal and PEG treatment conditions.

<p>Ninety leaf discs (diameter 0.5 cm) from 15 individual plants (5 individual plants/ line, 3 lines for each construct) of D1–D9 and <i>CaMV 35S</i> 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 <i>CaMV 35S</i> transgenic plants were stained for 6 h. Finally, the samples were observed and photographed after decolorization.</p

Identification of <i>cis</i>-acting elements in the <i>ZmGAPP</i> promoter sequence using the PLACE and PlantCARE databases.

<p>Identification of <i>cis</i>-acting elements in the <i>ZmGAPP</i> promoter sequence using the PLACE and PlantCARE databases.</p

Nucleotide sequence of the <i>ZmGAPP</i> promoter.

<p>The “A” of the translation initiation code “ATG” of <i>ZmGAPP</i> was designated as “+1”. Putative <i>cis</i>-acting elements underlined or shown in the border. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154041#pone.0154041.t002" target="_blank">Table 2</a> for descriptions of the elements. The arrow above the sequence indicates the start point of different deletion fragments (D1–D9).</p