NPQ assayed in leaf of well-watered potato plants.

<p>Potato WT (dashed line) and transgenic S-7 (solid line) grew in the walk-in growth chamber under controlled conditions and were watered to maintained FC at 65%. Performance of gross non-photochemical quenching (NPQ) were assayed on the first fully developed composite leaf from the top of plant at 4 hours after turning the light with Dual PAM-100. For measurement plants were adapted to dark for 20 minutes and then stimulated with repeated light pulses of actinic light (94 PPFD) for 5 minutes and once again subjected to dark for 6 minutes. Each point represents the mean ±SD (<i>n</i> = 3–4). Experiment was repeated three times and gave comparable results.</p

Profiling of annexin expression in WT potato leaves during drought.

<p>Potato WT plants grew in the walk-in growth chamber under controlled conditions. After 8–10 weeks irrigation was gradually reduced to decrease the field capacity (FC) to 25% (which took approximately 10 days) and then maintained at this level till 14^th day. Samples were collected from the first fully developed composite leaf from the top at indicated time points (D0 – beginning of drought, D6 – sixth day of drought, and D14 – fourteenth day of drought). RNA was isolated with Trizol and sq-RT-PCR was performed with primer sets specific for certain annexins. The level of expression was normalized against <i>EF1a</i> mRNA. Results are means ±SE (n≤4). Homogenic groups are determined by Tukey HSD (Honestly Significant Differences) test. The same letters designate values belong to the same homogenic group (p<0.05). Experiment was repeated twice.</p

Examination of leaf water status.

<p>Potato WT plants (white bars) and transgenic lines: S-2 (gray bars) and S-7 (black bars) were subjected to 14-day drought as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132683#pone.0132683.g002" target="_blank">Fig 2</a>. (A) Relative water content (RWC) analysis. Samples from the first fully developed undamaged leaf from the top of plant were collected at D0, D4, D7, D12 and 3 days after rewatering (RW3) and relative water content (RWC) was determined. Results are means ±SE (n = 3). (B) Stomatal conductance were measured in fully expanded, attached leaves at D0, D3, D6, D10 and RW3. After D10 the leaf surface was wrinkled to such an extent that further analysis was impossible. Measurements were done with a CI-510CF Chl fluorescence module, actinic light was provided by a CI-310LA light attachment. Results are means ±SE (n = 10). Experiment was performed three times and gave comparable results. Homogenic groups are determined by Tukey HSD (Honestly Significant Differences) test. The same letters designate values belong to the same homogenic group (p<0.05). Experiment was repeated 3 times and gave comparable results.</p

Annexin genes in potato genome.

<p>(A) Localization of annexin genes on potato chromosomes. The Roman numerals at the top denote the chromosome, digits in brackets indicate chromosome size. (B) Intron-exon organization of potato annexin genes. (C) Genomic PCR confirming the presence of predicted annexin genes in WT potato. Specific primers anneal to the 5’- and 3’- ends of coding sequence of certain annexin gene, hence the length of the resulting PCR product is a sum of the respective coding sequence with introns. (D) Schematic arrangement of <i>STANN3</i>.<i>1</i>, <i>STANN3</i>.<i>2</i>, <i>STANN3</i>.<i>3</i> and <i>STANN4</i> on chromosome I.</p

Accumulation of ROS (hydrogen peroxide and superoxide anion) and lipid peroxidation.

<p>Potato WT (white bars) and transgenic line S-7 (black bars) grew in walk-in growth chamber under controlled conditions. Leaf discs were expunged from the third, fourth and fifth upper fully expanded leaves and immediately vacuum infiltrated with methyl viologen (50 μM). After 1 hour incubation in dark discs were exposed to high light irradiance (850 PPFD) for indicated times (0.5–24 hours). Superoxide anion was determined colorimetrically with nitro blue tetrazolium chloride 9NBT). Hydrogen peroxide was stained in tissue with diaminobenzidine tetrahydrochloride (DAB) and quantified using the ImageJ. Lipid peroxidation was estimated spectrophotometrically with thiobarbituric acid (TBA). Results are means ±SE (n = 5). Homogenic groups are determined by Tukey HSD (Honestly Significant Differences) test. The same letters designate values belong to the same homogenic group (p<0.05). Experiment was repeated twice.</p

Drought tolerant phenotype of transgenic plants.

<p>Potato WT plants and transgenic lines (S-2, S-7) was subjected to drought as described above in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132683#pone.0132683.g002" target="_blank">Fig 2</a>. (A) Drought stress phenotype of WT (left column), S-2 (middle column) and S-7 (right column) plants. Photographs were taken on the beginning (D0), on eighth (D-8) and ninth (D-9) day of drought. Experiments were repeated twice in greenhouse and twice in growth chamber and gave similar results. (B) Regeneration of potato plants after prolonged drought. The procedure of drought imposition was the same as described above but the FC was maintained at 25% until the twenty first day of drought (D21). On D22 plants were rewatered and after draining of gravitationally bound water FC was kept up at 65%. Photograph was taken on the third day after rewatering. Left side—two WT plants; middle–two S-2 plants, and right–two S-7 plants. Experiments were repeated four times and similar results were obtained both in greenhouse and in growth chamber.</p

A simplify scheme depicting the interactions between cellular redox state and participation in ROS scavenging mechanisms.

<p>Oxidative stress is an unavoidable consequence of environmental stresses. ROS accumulation begins in chloroplasts and then it spreads throughout the whole cell. Activation of a secondary ROS sources e.g. NADPH oxidase complexes or photorespiration resulted in substantial H₂O₂ accumulation in cytosol. To avoid deleterious effects of ROS several compartment-specific mechanisms evolved, including accumulation of low-molecular-weight antioxidants (glutathione, ascorbate), scavenging enzymes (catalases CAT, ascorbate peroxidases APX, and sodium dismutases SOD) and protein thiols (peroxiredoxins PRX, glutaredoxins GRX, and thioredoxins TRX) that undergoes a reversible cycles the thiol-disulphide exchange. The redox-sensitive proteins sense, transduce, and translate ROS signals into appropriate cellular responses. Thus, precise regulation of size and redox status of the thiol pool is of essential importance for induction of appropriate responses. In plant cells glutathione is present in different compartments in milimolar concentrations and in quiescence it maintained largely in reduced state due to activity of glutathione reductases (GR) at expense of NADPH. Stress-induced ROS accumulation stimulates oxidation of glutathione (GSSG) and in the same time <i>de novo</i> synthesis of GSH. Disturbances in GSH/GSSG ratio might non-specifically influence several downstream pathways, e.g. by induction of thiol-disulfide exchange on target proteins. Cellular redox potential depends primarily on the total concentration of the total glutathione and the extend of its oxidation. GSSG accumulation did not disturb the redox potential if it is compensated by increasing the total glutathione concentration. However, if size of total pool remains unchanged when the GSH:GSSG ratio increased the cell redox potential in the cytosol become more positive. We propose that the improved stress tolerance of annexin STANN1-overexpressing potato plants results from amelioration of oxidative shift of the cytosolic glutathione redox potential. Elevation of STANN1 level had a pleiotropic effect on plant metabolism and physiology what suggested that not one specific but several downstream signaling pathways were touched. Disruption of the glutathione redox potential is sufficient to induce such effect; e.g., in transgenic tobacco with constitutive up-regulation of glutathione content MAPK and SA signaling pathways were modified. Annexin posses oxidation-sensitive cysteines and can act as a reductant influencing thus the redox potential. During stress in transgenic plants the capacity of cytosol redox buffer was more reducing compared to WT what prevents oxidation of downstream targets and modulate timing as well as magnitude of stress response. It had a beneficial effect on cell survival, photosynthesis and delay senescence. Similar effects were observed in tobacco and Arabidopsis plants and over-expressing particular elements of antioxidant systems.</p

STANN1 attenuated MeV-induced photooxidative stress.

<p>Confocal laser scanning image of the leaf epidermis of tobacco plant transiently expressing GFP (A-D and I-L) or STANN1_GFP (E-H and M-P). 3 days after infiltration leaf discs were excised and subjected to high light (850 PPFD) (A-H) or the combine treatment of high light (850 PPFD) and 50 μM MeV (G-L). The fluorescence was monitored with Nikon TE-2000E EZ-C1 exc. 488 nm and emission 515/30 and 605/75 for GFP and chloroplast, respectively. First column represent single focal plane, second–chloroplast autofluorescence acquired with the same excitation parameter for each construction to visualized the difference between responses to the same treatment, third–overlay of green and red fluorescence channels with GFP enhanced to visualized cells; right column–stack obtained with Volume Render program EZ-C1 combined with chloroplasts. Scale bar is 20 μm. Experiment was performed 3 times.</p

Photosynthetic pigment content during drought.

<p>WT (white bars) and transgenic line S-7 (black bars) were exposed to drought as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132683#pone.0132683.g002" target="_blank">Fig 2</a>. Samples were collected at the same time during the day at D0, D6, D14 and RW3 from third, fourth and fifth fully expanded leaves from top at 4 hours after turning the light. The level (A) chlorophyll <i>a</i>; (B) chlorophyll <i>b</i>; (C) zeaxanthin; and (D) violaxanthin were determined Non-polar lipids were separated on an ACQUITY UPLC system (Waters) and peaks were integrated at 436 nm. The level of xanthophylls is expressed as percent of the total carotenoids. The level of chlorophyll is expressed as mg mL^-1. Results are means ±SE (n = 3). Homogenic groups are determined by Tukey HSD (Honestly Significant Differences) test. The same letters designate values belong to the same homogenic group (p<0.05).</p

Diorganotin(IV) and triorganotin(IV complexes of meso tetra (4 sulfonatophenyl) porphine: do they bind DNA?

It was observed that organometallic porphyrin systems, where the Sn(IV) residue is in side chains, coordinated via sulphonatophenyl groups of porphyrin, show interesting and peculiar in vitro activity, in agreement with the anti-tumour activity of organotin complexes