Wim Van Camp

Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants

Hydrogen peroxide (H2O2) has been implicated in many stress conditions. Control of H2O2 levels is complex and dissection of mechanisms generating and relieving H2O2 stress is difficult, particularly in intact plants. We have used transgenic tobacco with ∼10% wild‐type catalase activity to study the role of catalase and effects of H2O2 stress in plants. Catalase‐deficient plants showed no visible disorders at low light, but in elevated light rapidly developed white necrotic lesions on the leaves. Lesion formation required photorespiratory activity since damage was prevented under elevated CO2. Accumulation of H2O2 was not detected during leaf necrosis. Alternative H2O2‐scavenging mechanisms may have compensated for reduced catalase activity, as shown by increased ascorbate peroxidase and glutathione peroxidase levels. Leaf necrosis correlated with accumulation of oxidized glutathione and a 4‐fold decrease in ascorbate, indicating that catalase is critical for maintaining the redox balance during oxidative stress. Such control may not be limited to peroxisomal H2O2 production. Catalase functions as a cellular sink for H2O2, as evidenced by complementation of catalase deficiency by exogenous catalase, and comparison of catalase‐deficient and control leaf discs in removing external H2O2. Stress analysis revealed increased susceptibility of catalase‐deficient plants to paraquat, salt and ozone, but not to chilling.

Comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress

The molecular mechanisms by which plants acclimate to oxidative stress are poorly understood. To identify the processes involved in acclimation, we performed a comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress. Combining mRNA differential display and cDNA array analysis, we estimated that at least 95 genes alter their expression in tobacco leaves acclimated to oxidative stress, of which 83% are induced and 17% repressed. Sequence analysis of 53 sequence tags revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular protection and detoxification, energy and carbohydrate metabolism, de novo protein synthesis, and signal transduction showed altered expression. Expression of most of the genes was enhanced, except for genes associated with photosynthesis and light-regulated processes that were repressed. During acclimation, two distinct groups of coregulated genes (“early-” and “late-response” gene regulons) were observed, indicating the presence of at least two different gene induction pathways. These two gene regulons also showed differential expression patterns on an oxidative stress challenge. Expression of “late-response” genes was augmented in the acclimated leaf tissues, whereas expression of “early-response” genes was not. Together, our data suggest that acclimation to oxidative stress is a highly complex process associated with broad gene expression adjustments. Moreover, our data indicate that in addition to defense gene induction, sensitization of plants for potentiated gene expression might be an important factor in oxidative stress acclimation.

Molecular identification of catalases from Nicotiana plumbaginifolia (L.)

We have isolated three different catalase cDNAs from Nicotiana plumbaginifolia (cat1, cat2, and cat3) and a partial sequence of a fourth catalase gene (cat4) that shows no discernible expression based on Northern analysis. The catalase sequences were used to determine the similarity with other plant catalases and to study the transcriptional response to paraquat, 3‐aminotriazole, and salicylic acid. 3‐Aminotriazole induces mRNA levels of cat1, cat2 and cat3, indicating that a reduction in catalase activity positively affects catalase mRNA abundance. Salicylic acid that binds catalase in vitro, had no effect on catalase transcript levels at physiological concentrations. Paraquat resulted in the induction of cat1.

Redox-Responsive Degradable PEG Cryogels as Potential Cell Scaffolds in Tissue Engineering

A Michael addition strategy involving the reaction between a maleimide double bond and amine groups is investigated for the synthesis of cryogels at subzero temperature. Low-molecular-weight PEG-based building blocks with amine end groups and disulfide-containing building blocks with maleimide end groups are combined to synthesize redox-responsive PEG cryogels. The cryogels exhibit an interconnected macroporous morphology, a high compressive modulus and gelation yields of around 95%. While the cryogels are stable under physiological conditions, complete dissolution of the cryogels into water-soluble products is obtained in the presence of a reducing agent (glutathione) in the medium. Cell seeding experiments and toxicologic analysis demonstrate their potential as scaffolds in tissue engineering.

Highly structured pH-responsive honeycomb films by a combination of a breath figure process and in situ thermolysis of a polystyrene-block-poly(ethoxy ethyl acrylate) precursor

In the present work, we show that Cu(0)-mediated controlled radical polymerization is a suitable method to synthesize high molar mass polystyrene-b-poly(ethoxy ethyl acrylate) PS-b-PEEA diblock copolymers. This method, applied at room temperature, is mandatory for complete preservation of ethoxy ethyl protecting groups during the course of polymerization. The synthesized PS-b-PEEA diblock copolymers were subsequently used for the elaboration of pH sensitive hierarchically structured honeycomb (HC) films through the Breath Figure (BF) process. The PS-b-PEEA hydrophobic honeycomb films were characterized by optical microscopy and atomic force microscopy (AFM) to reveal the hexagonal array of pores at the micrometer length scale, together with the phase segregation of the diblock copolymer. Similar to highly structured natural materials, the biomimetic honeycomb polymer films displayed intense iridescence. Moreover, the increase of surface roughness by peeling off the top layer of the PS-b-PEEA HC films produced superhydrophobic surfaces exhibiting a water contact angle of 155°. Subsequent deprotection of PEEA into pH-responsive poly(acrylic acid) (PAA) was performed in situ from the PS-b-PEEA honeycomb film by a simple thermolysis step carried out at 90 °C. The resulting PS-b-PAA honeycomb films showed a clear pH-responsive behavior with a water contact angle gap of 65° between a pH of 3 and 10.

Morphological transition during the thermal deprotection of poly(isobornyl acrylate)-b-poly(1-ethoxyethyl acrylate)

A set of poly(isobornyl acrylate)--poly(1-ethoxyethyl acrylate) polymers has been prepared by atom transfer radical polymerization. The 1-ethoxyethyl protecting group can be removed by a mild thermal treatment yielding the poly(acrylic acid) segment. The thin film morphological behavior of selected block copolymers was studied for as well as deprotected block copolymers using atomic force microscopy (AFM). Low-temperature thermal treatment yielded strongly phase-separated cylindrical features whereas treatment at temperatures above the glass transition temperature of the individual polymer blocks resulted in the initial generation of a similar phase contrast followed by a decrease in phase contrast caused by selective surface enrichment of the more hydrophobic poly(isobornyl acrylate) segment.

From Novel Block-Like Copolymers to Reactive Nanoparticles: ATRP and “Click” Chemistry as Synthetic Tools

In this chapter, we report on the synthesis and characterization of well-defined amphiphilic block copolymers, ‘block-like’ copolymers and star copolymers composed of poly(isobornyl acrylate) (PiBA) and poly(acrylic acid) (PAA) by ATRP. As PiBA polymers exhibit interesting physical characteristics, we report first a detailed study of the homopolymerization of iBA. The precursor monomers 1-ethoxyethyl acrylate as well as tert-butyl acrylate have been used to synthesize the precursor polymers for the PiBA-co-PAA block copolymers. Furthermore, a combination of ATRP and ‘click’ chemistry was used to prepare block and graft copolymers using a modular approach. The PiBA-PAA block and ‘block-like’ copolymers were investigated as pigment stabilizers for aqueous pigment dispersions. In the second part of the research, well-defined PiBA star copolymers were prepared, and reactive nanoparticles were obtained by end group modification of the PiBA star polymers with reactive moieties. Finally, the control of the visco-elastic properties by the incorporation of these nanoparticles in an acrylate polymer matrix was investigated.