Map-based cloning of <i>ALT1</i> and complementation analysis.

<p>(A) Fine mapping of the <i>ALT1</i> locus. Numbers below the horizontal line are the number of recombinants. The <i>ALT1</i> locus was fine mapped to a 35.2-kb region between markers M1 and M6. Indicating three putative ORFs contained in this region. (B) Gene structure of <i>ALT1</i>. Black boxes indicate exons and lines between boxes indicate introns. There is 1-bp deletion in the 10^th exon in the <i>alt1</i> background. (C) Phenotypic analysis of two-leaf stage seedlings of WT, <i>alt1</i> and <i>alt1</i>-c under normal (left) and alkaline (right, pH 9.5) stress conditions, respectively. Bar = 2 cm. (D) Protein structure of ALT1 and alt1. (E) Transcript levels of <i>ALT1</i> in the roots of two-leaf-stage <i>alt1</i> and WT seedlings. <i>Actin</i> was used as an internal control. Data shown are mean values of three biological repeats with SD.</p

<i>alt1</i> showed intact root morphology under alkaline stress.

<p>Two-leaf stage <i>alt1</i> and WT seedlings were subjected to alkaline solution (pH 9.0) and evaluated over a time course. Left: WT; Right: <i>alt1</i>. (A) Root morphology of <i>alt1</i> and WT under alkaline treatment. (B) Propidium iodide staining of root cells of <i>alt1</i> and WT plants subjected to alkaline treatment. Bars = 2 mm.</p

Expression analysis of oxidative stress-related genes.

<p>qRT-PCR was conducted on the roots of hydroponically grown two-leaf stage <i>alt1</i> and WT seedlings. <i>Actin</i> was used as an internal control. Data shown are mean values of three biological repeats with SD. (A) Genes related to ROS producing.- (B) Genes related to ROS scavenging. (C) Genes related to DNA repair.</p

Expression and subcellular localization of ALT1.

<p>(A) Transcriptional response of <i>ALT1</i> to alkaline stress. Two-leaf stage WT seedlings were treated with alkaline solution (pH 10.0), and <i>ALT1</i> expression was monitored at the indicated time points by qRT-PCR analysis. <i>Actin</i> was used as internal control. Data shown are mean values of three biological repeats with SD. (B) Subcellular localization of ALT1. GFP and the ALT1-GFP fusion under the control of the maize <i>Ubi</i> promoter were transiently expressed in onion epidermal cells. Indicating the ALT1-GFP fusion protein was specifically expressed in the nucleus. Bars = 100 µm.</p

ALT1, a Snf2 Family Chromatin Remodeling ATPase, Negatively Regulates Alkaline Tolerance through Enhanced Defense against Oxidative Stress in Rice

<div><p>Alkaline salt stress adversely affects rice growth, productivity and grain quality. However, the mechanism underlying this process remains elusive. We characterized here an alkaline tolerant mutant, <i>alt1</i> in rice. Map-based cloning revealed that <i>alt1</i> harbors a mutation in a chromatin remodeling ATPase gene. <i>ALT1</i>-RNAi transgenic plants under different genetic background mimicked the <i>alt1</i> phenotype, exhibiting tolerance to alkaline stress in a transcript dosage-dependent manner. The predicted ALT1 protein belonged to the Ris1 subgroup of the Snf2 family and was localized in the nucleus, and transcription of <i>ALT1</i> was transiently suppressed after alkaline treatment. Although the absorption of several metal ions maintained well in the mutant under alkaline stress, expression level of the genes involved in metal ions homeostasis was not altered in the <i>alt1</i> mutant. Classification of differentially expressed abiotic stress related genes, as revealed by microarray analysis, found that the majority (50/78) were involved in ROS production, ROS scavenging, and DNA repair. This finding was further confirmed by that <i>alt1</i> exhibited lower levels of H₂O₂ under alkaline stress and tolerance to methyl viologen treatment. Taken together, these results suggest that <i>ALT1</i> negatively functions in alkaline tolerance mainly through the defense against oxidative damage, and provide a potential two-step strategy for improving the tolerance of rice plants to alkaline stress.</p></div

Suppression of <i>ALT1</i> resulted in enhanced tolerance to alkaline stress.

<p>(A) Transcript levels of <i>ALT1</i> in the three selected RNAi transgenic lines and the vector control. <i>Actin</i> was used as an internal control. Data shown are mean values of three biological repeats with SD. (B) Morphology of two-leaf stage seedlings of the three RNAi transgenic lines and the control cultured in tap water under natural conditions. Bar = 2 cm. (C) Phenotypic analysis of alkaline tolerance. Two-leaf stage seedlings of the three RNAi transgenic lines and the vector control were treated with alkaline solution (pH 9.5), and photographed at 0 day and 13 day after the start of treatment, respectively.</p

Phenotypic analysis of <i>alt1</i> and WT plants under oxidative stress.

<p>Two-leaf stage <i>alt1</i> and WT seedlings grown hydroponically were subjected to 20 µM MV treatment. The left is WT and right is <i>alt1</i> for (A) and (B), respectively. (A) Phenotypes of <i>alt1</i> and WT at the indicated time points during MV treatment. (B) Leaf morphology of <i>alt1</i> and WT during MV treatment. (C) DAB staining of <i>alt1</i> and WT leaves from plants under normal (left) and stressed (right, pH 9.5) conditions, respectively. (D) Quantitative measurement of H₂O₂ in <i>alt1</i> and WT leaves during pH 9.5 treatment. Values are means ± SD (n = 3).</p

Phenotypic analysis of the <i>alt1</i> mutant.

<p>Left part in each pot of (A) and (B): WT; Right part: <i>alt1</i>. (A) Two-leaf stage <i>alt1</i> and WT seedlings were subjected to alkaline treatment with values 9.0, 9.5 and 10.0, respectively, and photographed at 7 (left) and 12 (right) days after treatment. (B) Time-course observation for tolerance phenotype of <i>alt1</i> and WT at pH 9.5. (C) Comparison of relative plant height of <i>alt1</i> and WT grown under pH 9.5 for the indicated number of days. Values are means ± SE (n = 20). **: <i>P</i>≤0.01. (D) Comparison of relative dry weight of <i>alt1</i> and WT after 10 days of treatment at pH 9.5. Values are means ± SE (n = 3, with 15 plants in each repeat). **: <i>P</i>≤0.01. (E) Comparison of survival rate of <i>alt1</i> and WT after 12 days of treatment at pH 9.5. Values are means ± SE (n = 3, with 50 plants in each repeat). **: <i>P</i>≤0.01. (F) Phenotypes of two-leaf stage seedlings of <i>alt1</i> and WT. Bar = 2 cm.</p

Metal ion quantification.

<p>Two-leaf stage <i>alt1</i> and WT seedlings grown hydroponically were subjected to alkaline solution (pH 9.5), and quantification of metal ions was carried out in the shoots of <i>alt1</i> and WT plants on days 0, 3, 6 and 10, respectively. Values are means ± SE (n = 3). Asterisks denote significance compared with the control plants of day 0. *: <i>P</i>≤0.05, **: <i>P</i>≤0.01.</p