Robert Fluhr

Two Distinct Sources of Elicited Reactive Oxygen Species in Tobacco Epidermal Cells

Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, putatively acting as both cellular signaling molecules and direct antipathogen agents. A single-cell assay, based on the fluorescent probe dichlorofluorescein, was used to scrutinize the generation and movement of ROS in tobacco epidermal tissue. ROS, generated within cells, quickly moved apoplastically as H2O2 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the external addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase- or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as measured by dichlorofluorescein fluorescence. The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addition, induction of ROS by all elicitors was correlated with subsequent cell death.

Production of Reactive Oxygen Species by Plant NADPH Oxidases

NADPH oxidases (NOX) catalyze the production of superoxides, a type of reactive oxygen species (ROS). The dramatic induction of ROS production by human NOX2 in activated blood phagocytic cells and its role in promoting pathogen killing has long motivated research in this area ([Babior et al., 2002][

The role of calcium and activated oxygens as signals for controlling cross-tolerance

Plants are confronted on a regular basis with a range of environmental stresses. These include abiotic insults caused by, for example, extreme temperatures, altered water status or nutrients, and biotic stresses generated by a plethora of plant pathogens. Many studies have shown that the cellular responses to these environmental challenges are rather similar, which might be why plants resistant to one stress are sometimes cross-tolerant to others. To understand this phenomenon and to be able to take full advantage of it in agriculture, we must determine whether the individual biochemical pathways that make up the responses to each external stimulus are activated by unique, overlapping or redundant signalling systems. We discuss the potential role of signalling molecules, such as calcium and activated oxygen species, in underlying cross-tolerance.

Divergent evolution of plant NBS-LRR resistance gene homologues in dicot and cereal genomes.

Abstract. The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well.

The I2C family from the wilt disease resistance locus I2 belongs to the nucleotide binding, leucine-rich repeat superfamily of plant resistance genes.

Characterization of plant resistance genes is an important step in understanding plant defense mechanisms. Fusarium oxysporum f sp lycopersici is the causal agent of a vascular wilt disease in tomato. Genes conferring resistance to plant vascular diseases have yet to be described molecularly. Members of a new multigene family, complex I2C, were isolated by map-based cloning from the I2 F. o. lycopersici race 2 resistance locus. The genes show structural similarity to the group of recently isolated resistance genes that contain a nucleotide binding motif and leucine-rich repeats. Importantly, the presence of I2C antisense transgenes abrogated race 2 but not race 1 resistance in otherwise normal plants. Expression of the complete sense I2C-1 transgene conferred significant but partial resistance to F. o. lycopersici race 2. All members of the I2C gene family have been mapped genetically and are dispersed on three different chromosomes. Some of the I2C members cosegregate with other tomato resistance loci. Comparison within the leucine-rich repeat region of I2C gene family members shows that they differ from each other mainly by insertions or deletions.

Pathogenesis-related proteins are developmentally regulated in tobacco flowers.

The accumulation of pathogenesis-related proteins (PR) in tobacco leaves has been casually related to pathogen and specific physiological stresses. The known enzymatic function of some of these proteins is potentially antimicrobial. By using antibodies specific to three classes of pathogenesis-related proteins, we examined tobacco plants during their normal growth. The pathogenesis-related proteins accumulated during the normal development of the tobacco flower. The PR-1 class of proteins (biological function unknown) is located in sepal tissue. PR-P, Q polypeptides are endochitinases and are present in pedicels, sepals, anthers, and ovaries. A glycoprotein serologically related to the PR-2,N,O class is a (1,3)-beta-glucanase and is present in pistils. Differential appearance during flower development, in situ localization, and post-translational processing of floral pathogenesis-related proteins point to a hitherto unsuspected function these classes of pathogenesis-related proteins play in the normal process of flowering and reproductive physiology.

UV-B-Induced PR-1 Accumulation Is Mediated by Active Oxygen Species.

Depletion of the stratospheric ozone layer may result in an increase in the levels of potentially harmful UV-B radiation reaching the surface of the earth. We have found that UV-B is a potent inducer of the plant pathogenesis-related protein PR-1 in tobacco leaves. UV-B fluences required for PR-1 accumulation are similar to those of other UV-B-induced responses. The UV-B-induced PR-1 accumulation was confined precisely to the irradiated area of the leaf but displayed no leaf tissue specificity. A study of some of the possible components of the signal transduction pathway between UV-B and PR-1 induction showed that photosynthetic processes are not essential, and photoreversible DNA damage is not involved. Antioxidants and cycloheximide were able to block the induction of PR-1 by UV-B, and treatment of leaves with a generator of reactive oxygen resulted in the accumulation of PR-1 protein. These results demonstrate an absolute requirement for active oxygen species and protein synthesis in this UV-B signal transduction pathway. In contrast, we also show that other elicitors, notably salicylic acid, are able to elicit PR-1 via nonreactive oxygen species-requiring pathways.

Rapid Transcriptome Changes Induced by Cytosolic Ca2+ Transients Reveal ABRE-Related Sequences as Ca2+-Responsive cis Elements in Arabidopsis

The regulation of gene expression by cellular calcium is crucial for plant defense against biotic and abiotic stresses. However, the number of genes known to respond to specific transient calcium signals is limited, and as yet there is no definition of a calcium-responsive cis element in plants. Here, we generated specific cytosolic calcium transients in intact Arabidopsis thaliana seedlings and linked them to early transcriptome changes, followed by bioinformatic analysis of the responsive genes. A cytosolic calcium transient induced by calmodulin antagonists and blocked by lanthanides was characterized using aequorin-based luminometry and photon imaging. Analysis of transcriptome changes revealed 230 calcium-responsive genes, of which 162 were upregulated and 68 were downregulated. These include known early stress-responsive genes as well as genes of unknown function. Analysis of their upstream regions revealed, exclusively in the upregulated genes, a highly significant occurrence of a consensus sequence (P < 10−13) comprising two abscisic acid–specific cis elements: the abscisic acid–responsive element (ABRE; CACGTG[T/C/G]) and its coupling element ([C/A]ACGCG[T/C/A]). Finally, we show that a tetramer of the ABRE cis element is sufficient to confer transcriptional activation in response to cytosolic Ca2+ transients. Thus, at least for some specific Ca2+ transients and motif combinations, ABREs function as Ca2+-responsive cis elements.

Plant respiratory burst oxidase homologs impinge on wound responsiveness and development in Lycopersicon esculentum.

Plant respiratory burst oxidase homologs (Rboh) are homologs of the human neutrophil pathogen-related gp91phox. Antisense technology was employed to ascertain the biological function of Lycopersicon esculentum (tomato) Rboh. Lines with diminished Rboh activity showed a reduced level of reactive oxygen species (ROS) in the leaf, implying a role for Rboh in establishing the cellular redox milieu. Surprisingly, the antisense plants acquired a highly branched phenotype, switched from indeterminate to determinate growth habit, and had fasciated reproductive organs. Wound-induced systemic expression of proteinase inhibitor II was compromised in the antisense lines, indicating that ROS intermediates supplied by Rboh are required for this wound response. Extending these observations by transcriptome analysis revealed ectopic leaf expression of homeotic MADS box genes that are normally expressed only in reproductive organs. In addition, both Rboh-dependent and -independent wound-induced gene induction was detected as well as transcript changes related to redox maintenance. The results provide novel insights into how the steady state cellular level of ROS is controlled and portrays the role of Rboh as a signal transducer of stress and developmental responses.

Ethylene Signal Is Transduced via Protein Phosphorylation Events in Plants.

A plethora of abiotic and biotic environmental stresses exert their influence on plants via the gaseous hormone ethylene. In addition, aspects of plant development and climacteric fruit ripening are regulated by ethylene. Sensitivity to ethylene is presumably mediated by a specific ethylene receptor whose activation signal is then transduced via an unknown cascade pathway. We have used the plant pathogenesis response, exemplified by the induction of pathogenesis-related (PR) genes, as a paradigm to investigate ethylene-dependent signal transduction in the plant cell. Ethylene application induced very rapid and transient protein phosphorylation in tobacco leaves. In the presence of the kinase inhibitors H-7 and K-252a, the transient rise in phosphorylation and the induced expression of PR genes were abolished. Similarly, these inhibitors blocked the response induced by an ethylene-dependent elicitor, [alpha]-AB. Reciprocally, application of okadaic acid, a specific inhibitor of phosphatases type 1 and type 2A, enhanced total protein phosphorylation and by itself elicited the accumulation of PR proteins. In the presence of H-7 and K-252a, PR protein accumulation induced by okadaic acid was blocked. In contrast to the action of ethylene and [alpha]-AB, xylanase elicits the accumulation of PR protein by an ethylene-independent pathway. Xylanase-induced PR protein accumulation was not affected by H-7 and K-252a. The results indicate that responsiveness to ethylene in leaves is transduced via putative phosphorylated intermediates that are regulated by specific kinases and phosphatases.