Non-intrusive assessment of photosystem II and photosystem I in whole coral tissues

Reef building corals (phylum Cnidaria) harbor endosymbiotic dinoflagellate algae (genus Symbiodinium) that generate photosynthetic products to fuel their host's metabolism. Non-invasive techniques such as chlorophyll (Chl) fluorescence analyses of Photosystem II (PSII) have been widely used to estimate the photosynthetic performance of Symbiodinium in hospite. However, since the spatial origin of PSII chlorophyll fluorescence in coral tissues is uncertain, such signals give limited information on depth-integrated photosynthetic performance of the whole tissue. In contrast, detection of absorbance changes in the near infrared (NIR) region integrates signals from deeper tissue layers due to weak absorption and multiple scattering of NIR light. While extensively utilized in higher plants, NIR bio-optical techniques are seldom applied to corals. We have developed a non-intrusive measurement method to examine photochemistry of intact corals, based on redox kinetics of the primary electron donor in Photosystem I (P700) and chlorophyll fluorescence kinetics (Fast-Repetition Rate fluorometry, FRRf). Since the redox state of P700 depends on the operation of both PSI and PSII, important information can be obtained on the PSII-PSI intersystem electron transfer kinetics. Under moderate, sub-lethal heat stress treatments (33°C for ~20 min), the coral Pavona decussata exhibited down-regulation of PSII electron transfer kinetics, indicated by slower rates of electron transport from QA to plastoquinone (PQ) pool, and smaller relative size of oxidized PQ with concomitant decrease of a specifically-defined P700 kinetics area, which represents the active pool of PSII. The maximum quantum efficiency of PSII (Fv/Fm) and functional absorption cross-section of PSII (σPSII) remained unchanged. Based on the coordinated response of P700 parameters and PSII-PSI electron transport properties, we propose that simple P700 kinetics parameters as employed here serve as indicators of the integrity of PSII-PSI electron transfer dynamics in corals.This work was supported by the award of Australian Research Council Discovery Project (ARC DP120101360) to WC and Future Fellowship (FT130100202) to DS. IV and LS were partly supported by a grant from the Hungarian National Research, Development and Innovation Office (NN110960

Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform

Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress

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

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

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