Gunnar Wingsle

Cold acclimation and photoinhibition of photosynthesis in Scots pine

Cold acclimation of Scots pine did not affect the susceptibility of photosynthesis to photoinhibition. Cold acclimation did however cause a suppression of the rate of CO2 uptake, and at given light and temperature conditions a larger fraction of the photosystem II reaction centres were closed in cold-acclimated than in nonacclimated pine. Therefore, when assayed at the level of photosystem II reaction centres, i.e. in relation to the degree of photosystem closure, cold acclimation caused a significant increase in resistance to photoinhibition; at given levels of photosystem II closure the resistance to photoinhibition was higher after cold acclimation. This was particularly evident in measurements at 20° C. The amounts and activities of the majority of analyzed active oxygen scavengers were higher after cold acclimation. We suggest that this increase in protective enzymes and compounds, particularly Superoxide dismutase, ascorbate peroxidase, glutathione reductase and ascorbate of the chloroplasts, enables Scots pine to avoid excessive photoinhibition of photosynthesis despite partial suppression of photosynthesis upon cold acclimation. An increased capacity for light-induced de-epoxidation of violaxanthin to zeaxanthin upon cold acclimation may also be of significance.

MYB transcription factors are differentially expressed and regulated during secondary vascular tissue development in hybrid aspen

More than 120,000 poplar ESTs have been sequenced from 20 different cDNA libraries by the Swedish Centre for Tree Functional Genomics. We screened this EST collection for MYB transcription factors involved in secondary vascular tissue formation, and genes assigned as PttMYB3Ra, PttMYB4a and PttMYB21a were selected for further characterisation. Three MYB genes showed different expression patterns in various organs, tissues and stem sub-sections representing different developmental stages of vascular tissue formation. Furthermore, the analysis showed that PttMYB21a expression was much higher in secondary cell wall formation zone of xylem and phloem fibers than in other developmental zones. Transgenic hybrid aspen plants, expressing the 3′-part of the PttMYB21a gene in antisense orientation were generated to assess the function of PttMYB21a gene in vascular tissue formation and lignification. All transgenic lines showed reduced growth and had fewer internodes compared to the wild-type. The analysis of selected lines showed that acid soluble lignin present in the bark was higher in transgenic lines as compared to wild-type plants. Moreover a higher transcript level of caffeoyl-CoA 3-O-methyltransferase [CCoAOMT]; EC 2.1.1.104) was found in the phloem of the transgenic plants, suggesting that PttMYB21a might function as a transcriptional repressor.

Differential redox regulation by glutathione of glutathione reductase and CuZn-superoxide dismutase gene expression in Pinus sylvestris L. needles

Glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) are two well-known enzymes involved in the scavenging of reactive oxygen intermediates. However, little is known about the regulation of Gor and Sod genes in plant cells. To obtain information about hypothetical redox regulatory mechanisms controlling Gor and Sod gene expression we artificially enhanced the levels of reduced and oxidized forms of glutathione (GSH and GSSG) in Pinus sylvestris L. needles. Scots pine shoots were placed for 12 h in beakers containing 5 mM GSH, 5 mM GSSG or water. Increased levels of both GSSG and GSH were observed in the GSSG-treated needles after 3 h. In contrast, only the GSH level was increased by the GSH treatment. Thus, the GSH/GSSG ratio increased up to 15-fold during the GSH treatment and decreased approximately two-fold during the GSSG treatment. The GR activity was significantly higher (60%) when GSSG was applied, without any apparent change in the amount and isoform population of GR or accumulation of Gor gene transcripts. This indicates that the GR activity increased per se in the GSSG treatment. The level of cytosolic CuZn-Sod transcripts was decreased significantly by the GSH treatment without any change in enzyme activity. The chloroplastic CuZnSod gene generally showed a more stable transcript level in the different treatments. However, a similarity between the cytosolic and chloroplastic levels of CuZn-Sod transcripts could be observed in different treatments. This suggests that the redox state of glutathione plays an important role in the in vivo regulation of CuZn-Sod gene expression in plants.

The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

Cisplatin (cisPt), Pt(NH3)2Cl2, is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1’s metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.

Antagonistic Effects of Hydrogen Peroxide and Glutathione on Acclimation to Excess Excitation Energy in Arabidopsis

The redox status of the quinone B (QB) and plastoquinone (PQ) pools plays a key role in the cellular and systemic signalling processes that control acclimatory responses in plants. In this study, we demonstrate the effects of hydrogen peroxide and glutathione on acclimatory responses controlled by redox events in the proximity of the QB‐PQ pools. Our results suggest that the chloroplast is a sink for H2O2 and that, paradoxically, high concentrations of H2O2 in the chloroplast protect the photosynthetic apparatus and the plant cell from photoinhibition and photooxidative damage. Excess glutathione, however, caused an effect antagonistic to that observed for high H2O2. An explanation of this apparent paradox and a hypothetical redox‐signalling model are suggested.

Amyloid formation by the pro-inflammatory S100A8/A9 proteins in the ageing prostate

Background The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimers disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon − prostate tissue remodelling in middle-aged and elderly men. Methodology/Principal Findings By using multidisciplinary analysis of corpora amylacea inclusions in prostate glands of patients diagnosed with prostate cancer we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. In prostate protease rich environment the amyloids are stabilized by dystrophic calcification and lateral thickening. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro under native and acidic conditions and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions. Conclusions/Significance These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. The results provide strong support for the prediction that the generic ability of polypeptide chains to convert into amyloids could lead to their involvement in an increasing number of otherwise apparently unrelated diseases, particularly those associated with ageing.

Pinus sylvestris L. needles contain extracellular CuZn superoxide dismutase

Four new isoforms of superoxide dismutase (SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1.) were identified in extracellular washing fluid from Scots pine (Pinus sylvestris L.) needles. The isoforms had an apparent molecular mass of 33 kDa. No neutral carbohydrates were present in the enzymes. The enzymatic activities were inhibited by 3 mM NaCN. One of the putative extracellular SOD isoforms was purified and NH2-terminal-sequenced. The sequence contained the domain KAVAVL. The domains VEG and V(K/S)G, present in chloroplastic and cytosolic CuZn SODs of plants, respectively, were not detected. The enzyme was composed of two subunits of 17.8 kDa each. The isoelectric point was determined to be 6.5. The results suggest the existence of an extracellular SOD in Scots pine.

Complex phenotypic profiles leading to ozone sensitivity in Arabidopsis thaliana mutants

Genetically tractable model plants offer the possibility of defining the plant O(3) response at the molecular level. To this end, we have isolated a collection of ozone (O(3))-sensitive mutants of Arabidopsis thaliana. Mutant phenotypes and genetics were characterized. Additionally, parameters associated with O(3) sensitivity were analysed, including stomatal conductance, sensitivity to and accumulation of reactive oxygen species, antioxidants, stress gene-expression and the accumulation of stress hormones. Each mutant has a unique phenotypic profile, with O(3) sensitivity caused by a unique set of alterations in these systems. O(3) sensitivity in these mutants is not caused by gross deficiencies in the antioxidant pathways tested here. The rcd3 mutant exhibits misregulated stomata. All mutants exhibited changes in stress hormones consistent with the known hormonal roles in defence and cell death regulation. One mutant, dubbed re-8, is an allele of the classic leaf development mutant reticulata and exhibits phenotypes dependent on light conditions. This study shows that O(3) sensitivity can be determined by deficiencies in multiple interacting plant systems and provides genetic evidence linking these systems.

Integrated analysis of transcript, protein and metabolite data to study lignin biosynthesis in hybrid aspen.

Tree biotechnology will soon reach a mature state where it will influence the overall supply of fiber, energy and wood products. We are now ready to make the transition from identifying candidate genes, controlling important biological processes, to discovering the detailed molecular function of these genes on a broader, more holistic, systems biology level. In this paper, a strategy is outlined for informative data generation and integrated modeling of systematic changes in transcript, protein and metabolite profiles measured from hybrid aspen samples. The aim is to study characteristics of common changes in relation to genotype-specific perturbations affecting the lignin biosynthesis and growth. We show that a considerable part of the systematic effects in the system can be tracked across all platforms and that the approach has a high potential value in functional characterization of candidate genes.