Consequences of antisense down-regulation of a lignification-specific peroxidase on leaf and vascular tissue in tobacco lines demonstrating enhanced enzymic saccharification.

Tobacco plants expressing an antisense construct for a cationic peroxidase, which down-regulated lignin content at the presumed level of polymerisation, have been further analysed. T(1) plants were derived from a large-scale screen of T(0) mutant lines, previously published, which identified lines demonstrating consistent lignin down-regulation. Of these, line 1074 which had the most robust changes in lignin distribution through several generations was shown to have accompanying down-regulation of transcription of most lignin biosynthesis genes, except cinnamoyl-CoA reductase. The consistent 20% reduction in lignin was not accompanied by significant gross changes in vascular polysaccharide content and composition, despite a modest up-regulation of transcripts of genes involved in cellulose and hemicellulose synthesis. Morphologically, 1074 plants have under-developed xylem with both fibers and vessels having thin cell walls and limited secondary wall thickening with an abnormal S2 layer. However, they were not compromised in overall growth. Nevertheless, these and other lines showed improved potential industrial utility through a threefold increase in enzymic saccharification efficiency compared with wild-type (wt). Therefore, they were profiled for further un-intended effects of transgenesis that might compromise their value for industrial or biofuel processes. Other phenotypic changes included increased leaf thickness and bifurcation at the tip of the leaf. wt-Plants had smaller chloroplasts and higher stomatal numbers than mutants. Transgenic lines also showed a variable leaf pigment distribution with light-green areas that contained measurably less chlorophyll a, b, and carotenoids. Changes in epidermal pavement cells of mutant lines were also observed after exposure to various chemicals, while wt leaves retained their structural integrity. Despite these changes, the mutant plants grew and were viable indicating that lignification patterns can be manipulated considerably through targeting polymerisation without serious deleterious effects.

Comparison of rhizosphere bacterial communities in Arabidopsis thaliana mutants for systemic acquired resistance.

Systemic acquired resistance (SAR) is an inducible systemic plant defense against a broad spectrum of plant pathogens, with the potential to secrete antimicrobial compounds into the soil. However, its impact on rhizosphere bacteria is not known. In this study, we examined fingerprints of bacterial communities in the rhizosphere of the model plant Arabidopsis thaliana to determine the effect of SAR on bacterial community structure and diversity. We compared Arabidopsis mutants that are constitutive and non-inducible for SAR and verified SAR activation by measuring pathogenesis-related protein activity via a beta-glucoronidase (GUS) reporter construct driven by the beta-1-3 glucanase promoter. We used terminal restriction fragment length polymorphism (T-RFLP) analysis of MspI- and HaeIII-digested 16S rDNA to estimate bacterial rhizosphere community diversity, with Lactobacillus sp. added as internal controls. T-RFLP analysis showed a clear rhizosphere effect on community structure, and diversity analysis of both rhizosphere and bulk soil operational taxonomic units (as defined by terminal restriction fragments) using richness, Shannon-Weiner, and Simpson's diversity indices and evenness confirmed that the presence of Arabidopsis roots significantly altered bacterial communities. This effect of altered soil microbial community structure by plants was also seen upon multivariate cluster analysis of the terminal restriction fragments. We also found visible differences in the rhizosphere community fingerprints of different Arabidopsis SAR mutants; however, there was no clear decrease of rhizosphere diversity because of constitutive SAR expression. Our study suggests that SAR can alter rhizosphere bacterial communities, opening the door to further understanding and application of inducible plant defense as a driving force in structuring soil bacterial assemblages.

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.

Transcripts for genes encoding soluble acid invertase and sucrose synthase accumulate in root tip and cortical cells containing mycorrhizal arbuscules.

Arbuscule formation by the arbuscular mycorrhizal fungus Glomus intraradices (Schenck & Smith) was limited to cortical cells immediately adjacent to the endodermis. Because these cortical cells are the first to intercept photosynthate exiting the vascular cylinder, transcript levels for sucrose metabolizing-enzymes were compared between mycorrhizal and non-mycorrhizal roots. The probes corresponded to genes encoding a soluble acid invertase with potential vacuolar targeting, which we generated from Phaseolus vulgaris roots, a Rhizobium-responsive sucrose synthase of soybean and a cell wall acid invertase of carrot. Transcripts in non-mycorrhizal roots were developmentally regulated and abundant in the root tips for all three probes but in differentiated roots of P. vulgaris they were predominantly located in phloem tissues for sucrose synthase or the endodermis and phloem for soluble acid invertase. In mycorrhizal roots increased accumulations of transcripts for sucrose synthase and vacuolar invertase were both observed in the same cortical cells bearing arbuscules that fluoresce. There was no effect on the expression of the cell wall invertase gene in fluorescent carrot cells containing arbuscules. Thus, it appears that presence of the fungal hyphae in the fluorescent arbusculated cell stimulates discrete alterations in expression of sucrose metabolizing enzymes to increase the sink potential of the cell.

The apoplastic oxidative burst in response to biotic stress in plants: a three-component system.

The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant-pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species. NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca(2+) and proton influxes and the K(+) efflux common to most elicitation systems as one of the earliest responses. The third component, the actual reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC-MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H(2)O(2) production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H(2)O(2) production revealed by cerium chloride staining together with the cross-linked wall proteins and callose and callose synthase. The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.