Overexpression of a maize plasma membrane intrinsic protein ZmPIP1;1 confers drought and salt tolerance in <i>Arabidopsis</i>

<div><p>Drought and salt stress are major abiotic stress that inhibit plants growth and development, here we report a plasma membrane intrinsic protein ZmPIP1;1 from maize and identified its function in drought and salt tolerance in <i>Arabidopsis</i>. ZmPIP1;1 was localized to the plasma membrane and endoplasmic reticulum in maize protoplasts. Treatment with PEG or NaCl resulted in induced expression of <i>ZmPIP1;1</i> in root and leaves. Constitutive overexpression of <i>ZmPIP1;1</i> in transgenic <i>Arabidopsis</i> plants resulted in enhanced drought and salt stress tolerance compared to wild type. A number of stress responsive genes involved in cellular osmoprotection in <i>ZmPIP1;1</i> overexpression plants were up-regulated under drought or salt condition. <i>ZmPIP1;1</i> overexpression plants showed higher activities of reactive oxygen species (ROS) scavenging enzymes such as catalase and superoxide dismutase, lower contents of stress-induced ROS such as superoxide, hydrogen peroxide and malondialdehyde, and higher levels of proline under drought and salt stress than did wild type. <i>ZmPIP1;1</i> may play a role in drought and salt stress tolerance by inducing of stress responsive genes and increasing of ROS scavenging enzymes activities, and could provide a valuable gene for further plant breeding.</p></div

Contents of superoxide and hydrogen peroxide and activities of reactive oxygen species scavenging enzymes in WT and transgenic <i>Arabidopsis</i> plants with drought or salt stress.

<p>Data are means of four biological replicates with error bars indicating SD. Asterisks indicate a significant difference between the WT and the transgenic lines (*P<0.01).</p

Phenotypes of <i>ZmPIP1;1</i> overexpression transgenic lines under drought treatment.

<p>(A) Growth of WT and <i>ZmPIP1;1</i>-Oe transgenic <i>Arabidopsis</i> seedlings under normal and dehydration conditions for 15 days. The re-watered photographs were taken after the plants were re-watered for another 7 days. (B) Survival rates of indicated genotypes upon re-watering for 1 week after dehydration treatment. Three independent repeats were performed, each data are means of 30 plants with error bars indicating SD. (C) Water loss from 0.5 g of detached leaves, from WT and <i>ZmPIP1;1</i>-Oe transgenic line, was measured at different times. Three independent repeats were performed, each data are means of 15 plants with error bars indicating SD, *P<0.01.</p

Expression pattern of <i>ZmPIP1;1</i> under normal and stress conditions.

<p>(A) Relative expression levels of <i>ZmPIP1;1</i> in root, stem, mature leaf, immature tassel (3–4 cm), immature ear (3–4 cm), 18 DAP endosperm and embryo. Maize seedlings were grown in nutrient solution. Total RNA was extracted from different tissues for qRT-PCR. (B) Trifoliolate maize seedlings were treated with or without 10% PEG in nutrient solution. (C) Trifoliolate maize seedlings were treated with or without 100 mM NaCl in nutrient solution. RNA was extracted from the root and leaf of these seedlings at different time after treatment. All data are means of three biological replicates with error bars indicating SD. Expression level of treated plants was relative to control plants at each time point.</p

Expression analysis of <i>Arabidopsis</i> osmotic stress responsive genes.

<p>The expression of <i>RD29A</i>, <i>RD29B</i>, <i>LEA18</i> and <i>TRX5</i> under normal, drought or salt stress conditions. RNA was extracted from shoots of two-week-old WT and <i>ZmPIP1;1</i> overexpression seedlings under normal condition dehydrated for 2 hours for drought treatment or watering with 100 mM NaCl for 6 hours for salt treatment. All data are means of three biological replicates with error bars indicating SD, *P<0.01. Expression of <i>AtUBQ10</i> was used as the internal control.</p

Subcellular localization of ZmPIP1;1 in maize protoplasts.

<p>ZmPIP1;1-GFP was colocalized with a mCherry-labeled plasma membrane marker (pm-rk; CD3-1007) and a mCherry-labeled endoplasmic reticulum marker (ER-rk; CD3-959), respectively. The vector pCAMBIA1302 that sGFP was under the control of the CaMV 35S promoter served as control. Bars = 20 μm.</p

Drought and salt stress tolerance of <i>ZmPIP1;1</i> overexpression transgenic lines.

<p>Five-day-old WT and <i>ZmPIP1;1</i>-Oe transgenic <i>Arabidopsis</i> seedlings were treated with 150 mM Manntiol or 100 mM NaCl in MS medium for 7 days. (A) Photographs of WT and <i>ZmPIP1;1</i>-Oe transgenic <i>Arabidopsis</i> seedlings with 150 mM Manntiol or 100 mM NaCl in MS medium for 7 days. Bar = 1 cm. (B) Primary root length in WT and <i>ZmPIP1;1</i>-Oe with 150 mM Manntiol or 100 mM NaCl in MS medium for 7 days. All data are means of 15 biological replicates with error bars indicating SD, *P<0.01. (C) qRT-PCR analysis of three representative <i>ZmPIP1;1</i> overexpression transgenic lines. RNA was extracted from the leaves of fourteen-day-old seedlings. All data are means of three biological replicates with error bars indicating SD. Expression of <i>AtACT2</i> was used as the internal control.</p

Contents of MDA and proline and analysis of <i>P5CS1</i> and <i>P5CS2</i> gene expression in WT and transgenic <i>Arabidopsis</i> plants with drought or salt stress.

<p>Data are means of four biological replicates with error bars indicating SD. Asterisks indicate a significant difference between the WT and the transgenic lines (*P<0.01).</p