G. Paul Bolwell

Role of active oxygen species and NO in plant defence responses

Research in the area of active oxygen species is going through a reflective stage. There is controversy whether multiple mechanisms for active oxygen species generation exist and some data may need reassessing since the discovery of a role for NO in defence responses. Important work concerning upstream and downsteam signalling in this area is emerging, and the stage is set for approaches utilising transgenic knockouts and mutants to resolve many questions.

The Apoplastic Oxidative Burst Peroxidase in Arabidopsis Is a Major Component of Pattern-Triggered Immunity

This article examines the role of apoplastic peroxidases in Arabidopsis thaliana pattern-triggered immunity (PTI). The reduced expression of two key peroxidase genes, PRX33 or PRX34, causes defects in PTI, and an rbohD mutant is also impaired in some PTI-related responses, but to a lesser degree than the peroxidase knockdown lines. In plants, reactive oxygen species (ROS) associated with the response to pathogen attack are generated by NADPH oxidases or apoplastic peroxidases. Antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase (FBP1) cDNA in Arabidopsis thaliana was previously shown to diminish the expression of two Arabidopsis peroxidases (peroxidase 33 [PRX33] and PRX34), block the oxidative burst in response to a fungal elicitor, and cause enhanced susceptibility to a broad range of fungal and bacterial pathogens. Here we show that mature leaves of T-DNA insertion lines with diminished expression of PRX33 and PRX34 exhibit reduced ROS and callose deposition in response to microbe-associated molecular patterns (MAMPs), including the synthetic peptides Flg22 and Elf26 corresponding to bacterial flagellin and elongation factor Tu, respectively. PRX33 and PRX34 knockdown lines also exhibited diminished activation of Flg22-activated genes after Flg22 treatment. These MAMP-activated genes were also downregulated in unchallenged leaves of the peroxidase knockdown lines, suggesting that a low level of apoplastic ROS production may be required to preprime basal resistance. Finally, the PRX33 knockdown line is more susceptible to Pseudomonas syringae than wild-type plants. In aggregate, these data demonstrate that the peroxidase-dependent oxidative burst plays an important role in Arabidopsis basal resistance mediated by the recognition of MAMPs.

Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants

In this study the antioxidant activities of the hydroxycinnamic acids, chlorogenic, caffeic, ferulic and p‐coumaric have been investigated in peroxidising lipid systems mediated by metmyoglobin. The results show that the order of effectiveness in increasing the resistance of LDL to peroxidation, in protecting LDL cholesterol from oxidation and preventing the oxidative modification of the LDL apoprotein B100 is caffeic = chlorogenic > ferulic > p‐coumaric acid. Assessment of the rates of reaction of the hydroxycinnamates with ferrylmyoglobin, a product of the reductive decomposition of lipid hydroperoxides, reveals that the compounds are more effective as peroxyl radical scavengers than reductants of ferryl myoglobin in peroxidising LDL systems mediated by haem proteins.

Plant cytochrome p450

The present status of plant cytochrome P450 research is reviewed. A comparison of the properties of this group of cytochrome proteins with those of other microsomal b-type haem proteins is made. The range of reactions catalysed by P450s is discussed as well as recent progress in improving purification and reconstitution. Molecular cloning approaches that have overcome the earlier block to accessing this gene superfamily are discussed and future prospects highlighted. Expression of the gene family is discussed in relation to regulation in response to environmental and developmental cues and tissue and subcellular localization. The biotechnological importance of this gene family is stressed.

Reactive oxygen species and their role in plant defence and cell wall metabolism

Harnessing the toxic properties of reactive oxygen species (ROS) to fight off invading pathogens can be considered a major evolutionary success story. All aerobic organisms have evolved the ability to regulate the levels of these toxic intermediates, whereas some have evolved elaborate signalling pathways to dramatically increase the levels of ROS and use them as weapons in mounting a defence response, a process commonly referred to as the oxidative burst. The balance between steady state levels of ROS and the exponential increase in these levels during the oxidative burst has begun to shed light on complex signalling networks mediated by these molecules. Here, we discuss the different sources of ROS that are present in plant cells and review their role in the oxidative burst. We further describe two well-studied ROS generating systems, the NADPH oxidase and apoplastic peroxidase proteins, and their role as the primary producers of ROS during pathogen invasion. We then discuss what is known about the metabolic and proteomic fluxes that occur in plant cells during the oxidative burst and after pathogen recognition, and try to highlight underlying biochemical processes that may provide more insight on the complex regulation of ROS in plants.

Phenylalanine ammonia-lyase gene organization and structure

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) genomic sequences were isolated from bean (Phaseolus vulgaris L.) genomic libraries using elicitor-induced bean PAL cDNA sequences as a probe. Southern blot hybridization of genomic DNA fragments revealed three divergent classes of PAL genes in the bean genome. Polymorphic forms were observed within each class. The nucleotide sequences of two PAL genes, gPAL2 (class II) and gPAL3 (class III), were determined. gPAL2 contains an open reading frame encoding a polypeptide of 712 amino acids, interrupted by a 1720 bp intron in the codon for amino acid 130. gPAL3 encodes a polypeptide of 710 amino acids showing 72% similarity with that encoded by gPAL2, and contains a 447 bp intron at the same location. At the nucleotide level, gPAL2 and gPAL3 show 59% sequence similarity in exon I, 74% similarity in exon II, and extensive sequence divergence in the intron, 5′ and 3′ flanking regions. S1 nuclease protection identified transcription start sites of gPAL2 and gPAL3 respectively 99 bp and 35 bp upstream from the initiation codon ATG, and showed that gPAL2 but not gPAL3 was activated by elicitor, whereas both were activated by wounding of hypocotyls. The 5′ flanking region of both genes contain TATA and CAAT boxes, and sequences resembling the SV40 enhancer core. gPAL2 contains a 40 bp palindromic sequence and a 22 bp motif that are also found at similar positions relative to the TATA box in 5′ flanking regions of other elicitor-induced bean genes.

A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification.

A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.

A Peroxidase-Dependent Apoplastic Oxidative Burst in Cultured Arabidopsis Cells Functions in MAMP-Elicited Defense

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

Dynamic aspects of the plant extracellular matrix

Publisher Summary This chapter discusses dynamic aspects of the plant extracellular matrix. It describes the current status of knowledge of biochemical regulation that leads to differential patterns of synthesis of wall components, their site of synthesis and deposition, and the changes that occur in response to developmental and environmental cues. The plant-cell wall, with its connotations of rigidity and maintenance of shape, is possibly considered as an extracellular matrix with dynamic properties. Various classes of polysaccharides of the extracellular matrix are well-conserved. Cellulose microfibrils are spun into a matrix consisting of both pectinaceous and hemicellulosic polysaccharides. In addition to cellulose microfibrils, plant walls also contain hemicelluloses and pectins. Phenolics are deposited mainly in the secondary wall but are also important constituents within the primary wall. Wall phenolics are based upon the phenylpropanoid unit and are found as both conjugated acids and, more familiarly, lignin alcohols. The cell wall is intimately involved as part of the sensing of the environment and in responding through the ensuing changes.

Synthesis of cell wall components: Aspects of control

Abstract The dynamic qualities of the plant cell wall are explored. Recent developments in the understanding of how membrane systems operate in the synthesis of wall components is discussed. Particular attention is paid to the synthesis of polysaccharides and wall-bound phenolics, while the identification of some wall bound proteins and the subsequent study of their synthesis and processing is also described. Probable molecular controls, both qualitative and quantitative, governing the appearance of newly synthesized components within the wall are discussed. The conditions under which sets of components are modified specifically, by the accumulation of newly synthesized material or extracellular enzyme action, are outlined. Some deficiencies in understanding how these aspects may be interrelated, especially with regard to mechanisms of signal transduction are highlighted. However the potentially rapid capacity for implementation of changes in the extracellular matrix of the cell, probably commencing with processes of activation of specific subsets of genes in response to particular signals, is striking.