Electron micrographs of chloroplasts from 1^st leaves and cotyledons of control and salt-grown seedlings.

<p>Biological samples were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082548#pone-0082548-g002" target="_blank">Figure 2</a>. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined by transmission electron microscopy. Shown are representative photographs for chloroplasts from (A) 1^st leaf of control plant; (B) 1^st leaf of salt-grown plant; (C) cotyledon of control plant; (D) cotyledon of salt-grown plant. g, grana; t, thylakoid; s, starch grain.</p

Sections of leaves and cotyledons of control and salt-treated seedlings.

<p>Two-week-old seedlings, germinated and grown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082548#pone-0082548-g001" target="_blank">Figure 1</a>, were fixed and embedded as described in Materials and Methods. Semi-thin (1 µm) sections were stained with 2% toluidine blue and examined under a light microscope. (A) 1^st leaf of control plant. (B) 1^st leaf of salt-grown plant. (C) Cotyledon of control plant. (D) Cotyledon of salt-grown plant. m, mesophyll cells. (E and F) Leaf thickness and chloroplast count per cell, respectively, in semi-thin sections of 1^st leaves and cotyledons of control (gray bars) and salt-grown (black bars) seedlings. Data shown are average ± SE. </p

Two-week-old seedlings germinated and grown on agar-solidified medium with or without added NaCl.

<p>Surface-sterilized cold-treated seeds were sown on 0.5X MS, 0.5% sucrose and 0.5% agar containing 0 or 0.1 M NaCl. </p

Relative gene copy number in control and salt-grown seedlings.

<p>DNA was prepared from 2-week-old seedlings grown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082548#pone-0082548-g001" target="_blank">Figure 1</a>, and used directly for gene-dose analyses by qPCR as described in Materials and Methods. The signal obtained for each gene in control grown seedlings was defined as 1. Gray bars, chloroplast-encoded genes; black bars, nucleus-encoded genes. Data shown are average ± SE. </p

Chloroplasts of Salt-Grown <i>Arabidopsis</i> Seedlings Are Impaired in Structure, Genome Copy Number and Transcript Levels

<div><p>The chloroplast is the most prominent and metabolically active plastid in photosynthetic plants. Chloroplasts differentiate from proplastids in the plant meristem. Plant plastids contain multiple copies of a small circular genome. The numbers of chloroplasts per mesophyll cell and of plastid genome copies are affected by developmental stage and environmental signals. We compared chloroplast structure, gene expression and genome copy number in <i>Arabidopsis</i> seedlings germinated and grown under optimal conditions to those in seedlings germinated and grown in the presence of NaCl. Chloroplasts of the NaCl-grown seedlings were impaired, with less developed thylakoid and granum membranes than control seedlings. In addition, chloroplasts of salt-grown <i>Arabidopsis</i> seedlings accumulated more starch grains than those in the respective control plants. Steady-state transcript levels of chloroplast-encoded genes and of nuclear genes encoding chloroplast proteins were reduced in salt-grown seedlings. This reduction did not result from a global decrease in gene expression, since the expression of other nuclear genes was induced or not affected. Average cellular chloroplast genome copy number was reduced in salt-grown seedlings, suggesting that the reduction in steady-state transcript levels of chloroplast-encoded genes might result from a decrease in template DNA.</p> </div

Expression of chloroplast- and nucleus-encoded genes in control and salt-grown seedlings.

<p>RNA was prepared from 2-week-old seedlings grown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082548#pone-0082548-g001" target="_blank">Figure 1</a>. cDNA was prepared and the indicated transcript levels were determined by qRT-PCR as described in Materials and Methods using cytosolic 18S rRNA as an internal reference. Steady-state levels of each gene transcript in control grown seedlings were defined as 1. (A) Chloroplast-encoded genes. (B and C) Nucleus-encoded genes. Data shown are average ± SE. </p

Activities of antioxidative enzymes in leaves and roots of adult tobacco plants.

<p>(A) SOD, (B) PPX, (C) APX, (D) CAT and (E) GR. For explanation of treatments see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087582#pone-0087582-g001" target="_blank">Fig. 1</a>. Vertical bars represent standard errors (n = 6). Columns marked with different lowercase letters indicate significantly different treatments (P<0.05) within leaf data set, while columns marked with different capital letters indicate significantly different treatments (P<0.05) within root data set (LSD multiple test).</p

Principal component analysis of roots data set.

<p>(A) loadings and (B) scores of the first two factors (PCs). Cd accumulation (Cd), Zn accumulation (Zn), MDA content (MDA), carbonyl content (carb), DNA damage (DNA) and activity of SOD, APX, PPX, GR and CAT represent variables. For explanation of treatments see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087582#pone-0087582-g001" target="_blank">Fig. 1</a>.</p

Principal component analysis of seedlings data set.

<p>(A) loadings and (B) scores of the first two factors (PCs). Cd accumulation (Cd), Zn accumulation (Zn), MDA content (MDA), carbonyl content (carb), DNA damage (DNA) and activity of SOD, APX, PPX, GR and CAT represent variables. For explanation of treatments see Fig. 1.</p

Cd and Zn content and their effects on MDA and protein carbonyl contents and DNA in tobacco seedlings after exposure to Cd (10 and 15 µM), Zn (25 and 50 µM) and their combinations.

<p>Values represent means ± standard errors (n = 6). Values marked with different letters indicate significantly different treatments (P<0.05) within seedling data set (LSD multiple test).</p