Lipopolysaccharide mobility in leaf tissue of Arabidopsis thaliana

Abstract

Bacterial lipopolysaccharides (LPS) are triggers of defence responses in plants, and induce local as well as systemic acquired resistance. Arabidopsis thaliana plants pretreated with LPS show an increased resistance to the virulent bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000. To investigate the mobilization and transport of LPS in Arabidopsis leaves, fluorescently labelled LPS (Alexa Fluor® 488 conjugate) from Salmonella minnesota was used. Leaves were pressure infiltrated with fluorescein-labelled LPS and fluorescence microscopy was used to follow the movement and localization of LPS as a function of time. The observation of leaves 1 h after supplementation with fluorescein-labelled LPS revealed a fluorescent signal in the intercellular space. Capillary zone electrophoresis was used for the detection and analysis of the labelled LPS in directly treated leaves and systemic leaves. In addition, gel electrophoresis was used to confirm LPS mobilization. The results indicated that LPS mobilization/translocation occurs through the xylem from local, treated leaves to systemic, untreated leaves. Consequently, care should be taken when ascribing the observed biochemical responses and induced resistance from LPS perception as being uniquely local or systemic, as these responses might overlap because of the mobility of LPS in the plant vascular system.

Figure 1

Lipopolysaccharides (LPS) induce systemic acquired resistance against Pseudomonas syringae DC3000 (Pst) and the accumulation of salicylic acid (SA). (a) Arabidopsis thaliana Col‐0 plants were pretreated with LPS from an endophytic strain of Burkholderia cepacia (LPS_B.cep.) for 2 days, and systemic leaves were then inoculated with Pst. The diagrams indicate the number of colony‐forming Pst bacteria extracted from systemic leaves 0, 1, 2 and 5 days after infection (dpi). (b) SA accumulation in local and systemic leaves of LPS_B.cep.; treated and untreated control plants were analysed at the indicated time points. Values (nmol/g fresh weight) are displayed in relation to control leaves and represent a mean of three biological replicates. c, control; loc, treated leaves; sys, systemic leaves.

Figure 5

Visualization of lipopolysaccharide (LPS) in midribs of Arabidopsis thaliana leaves with sodium dodecylsulphate‐polyacrylamide gel electrophoresis. Gels were stained with an LPS‐specific stain (a), a protein‐specific stain (b) or a nonspecific stain (c). Lanes: 1, LPS standard (100 µg/mL); 2, LPS standard (10 µg/mL); 3, untreated control; 4, whole treated leaf directly after LPS inoculation; 5, midrib after 1 h; 6, midrib after 6 h; 7, midrib after 24 h.

Figure 2

Investigation of lipopolysaccharide (LPS) mobilization in Arabidopsis thaliana leaves using fluorescein‐labelled LPS from Salmonella minnesota (LPS_S.min.). After pressure infiltration of 100 µg/mL fluorescein‐labelled LPS_S.min., images were obtained from the abaxial leaf side at the indicated time points under bright field (a, d, g, j) and fluorescent (green light filter, 505–530 nm; c, f, i, l) light. Chlorophyll autofluorescence was captured with a long‐pass filter (585 nm; b, e, h, k). Scale bar, 10 µm.

Figure 3

Investigation of lipopolysaccharide (LPS) mobilization in cross‐sections of Arabidopsis thaliana leaves using fluorescently labelled LPS from Salmonella minnesota (LPS_S.min.). After pressure infiltration of 100 µg/mL fluorescein‐labelled LPS_S.min., images of cross‐sections were obtained after 3 h under fluorescent light (green light filter, 435–485 nm; red light filter, 653–695 nm). (a) LPS fluorescence (green) and autofluorescence of chloroplasts (red); (b) green fluorescence of injected LPS; (c) toludine blue staining shows pectin and pectic substances (pink to purple); (d) phloroglucinol test shows lignin (red–violet); (e) autofluorescence of ferulic acid bound to lignin‐ or cutin‐containing cells (blue). Staining was performed to characterize tissue‐specific properties. CZ, cambial zone; P, phloem; X, xylem.

Figure 4

Investigation of lipopolysaccharide (LPS) mobilization using capillary zone electrophoresis. After treatment of Arabidopsis leaves with 100 µg/mL fluorescently labelled LPS from Salmonella minnesota (LPS_S.min.), leaves were harvested at the indicated time points and veins (local leaves) or whole leaves (systemic leaves) were investigated. The LPS signal (dark red arrows) became visible after 1 and 6 h in local midribs and after 24 h in systemic leaves. An additional peak indicative of nonbound fluorescein was also observed (orange arrow). rfu, relative fluorescence units.