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Abstract

The cell wall is one major determinant of plant cell morphology, and is an attractive bioresource. Here, we report a novel strategy to modify plant cell wall property by small molecules. Lasalocid sodium (LS) was isolated by chemical screening to identify molecules that affect the cell morphology of tobacco BY-2 cells. LS treatment led to an increase in cell wall thickness, whilst the quantity and sugar composition of the cell wall remained unchanged in BY-2 cells. The chemical also disordered the cellular arrangement of hypocotyls of Arabidopsis plants, resulting in a decrease in hypocotyl length. LS treatment enhanced enzymatic saccharification efficiency in both BY-2 cells and Arabidopsis plants. Microarray analysis on Arabidopsis showed that exposure to LS upregulated type III peroxidase genes, of which some are involved in lignin biogenesis, and jasmonic acid response genes, and phloroglucinol staining supported the activation of lignification by the LS treatment. As jasmonic acid-mediated lignification is a typical reaction to cell wall damage, it is possible that LS induces cell wall loosening, which can trigger cell wall damage response. Thus, LS is a unique chemical for modification of cell wall and morphology through changes in cell wall architecture.

LS changed cell wall thickness without obvious alteration to wall quantity and sugar composition

To investigate the effect of LS on cell wall structure, we observed BY-2 cell walls by transmission electron microscopy after LS treatment (). Cell wall thickness increased in a dose-dependent manner, consistent with observations obtained from the confocal microscope. Thickness when treated with 1.0 μM LS was approximately twice that of non-treated cells (). Solid-state NMR analysis showed that LS treatment hardly changed the cellulose crystallinity of the cell wall (). This result indicated that change in wall thickness was not related to cellulose crystallinity.

LS changed cell wall thickness without obvious alteration to wall quantity and sugar composition

Next, we checked the quantity of the cell wall and its cellulose, hemicellulose and pectin content with and without LS treatment. No significant differences were found in the amount of cell wall fraction or in the contents of each cell wall component between these samples ( and ). Analysis of the sugar composition of the matrix polysaccharide fraction also showed that the amounts of sugars in the LS treated samples were comparable to the non-treated samples (). Therefore, increase in cell wall thickness by LS does not result from changes in the amount or the chemical composition of the cell wall.

LS increased saccharification efficiency

To further analyze the effect of LS on properties of the cell wall, we investigated enzymatic saccharification efficiency of the cell wall material. Preparations from BY-2 cells with and without chemical treatment were subjected to enzymatic saccharification analysis. Results showed that LS-treated samples released approximately 1.2 times as much glucose as non-treated samples (). This increase was found from early stages of incubation with the enzymes. Taken together with observations that LS affected neither cellulose crystallinity nor compositional properties of the cell wall ( and and ), the increase in enzymatic saccharification efficiency by the treatment is caused by changes in structural properties of cell wall components other than cellulose crystallinity.

Effects of LS treatment on Arabidopsis seedlings

To investigate effects of LS on plant growth, Arabidopsis thaliana seeds were germinated and grown continuously on MS agar medium containing 0, 0.1, 0.5, 1.0 or 5.0 μM of LS under dark conditions. Germination was severely inhibited by 5.0 μM, and elongation of hypocotyls was affected on medium containing 0.5 and 1.0 μM (upper panels in ). Abnormal-shaped cells and reduced cell length were observed in hypocotyls grown in the presence of 0.5 and 1.0 μM LS, (lower panels in ), suggesting that inhibition of hypocotyl elongation is mainly attributed to abnormal cell elongation and reduction of cell length. Moreover, we found that LS induced opening of cotyledons even in the dark, when cotyledons generally remain closed (). We further checked enzymatic saccharification efficiency of Arabidopsis samples treated with or without LS. As with the BY-2 cells, chemical-treated samples showed higher amounts of released glucose compared with non-treated samples (approximately 1.3 times) from early stages of incubation with the enzymes (). These results indicated that LS treatment can change cell wall properties not only in culture cells but also in plants.

LS induced cell wall damage response in Arabidopsis plants

To examine the effects of LS on gene expression, we performed microarray analysis using 3-day-old dark-grown Arabidopsis seedlings treated with or without the chemical for 24 hours. The results showed that 1,430 and 1,526 transcripts were significantly increased and decreased, respectively, by LS treatment (threshold value of fold change is 1.5 and p < 0.05). Gene ontology analysis showed that up-regulated genes were enriched in extracellular, membrane-related and cell wall-related components in “Cellular Component” categories and that genes annotated with enzymatic activities and transporter activity were enriched in the “Molecular Component” categories (). The most remarkable finding from these enrichment analyses is that, of the transcripts that were increased, 23 encode class III secretable peroxidase family proteins, a part of which are presumed to function in lignification,,, and 27 encode jasmonic acid (JA)-related proteins including JA biosynthetic enzymes and JA-mediated signaling proteins ( and ). Besides, some transcripts for lignin biosynthesis-related genes were also increased by LS treatment (). Previous works have suggested that reactive oxygen species (ROS) production and JA signaling are important cross-talk modules to regulate cell wall damage-induced lignin biosynthesis,. Thus, it was possible that LS triggers cell wall damage response in plants.

LS induced cell wall damage response in Arabidopsis plants

To check if high levels of peroxidase gene expression induced by LS are associated with increased peroxidase activity, mm2d Arabidopsis suspension cells were incubated with different concentrations of 0, 0.1, 0.5, 1.0 and 5.0 μM LS for 24 h, and then subjected to diaminobenzidine (DAB) staining analysis. The results showed that DAB staining signal increased as concentration of LS increased, indicating that the chemical activates peroxidase activity through up-regulation of peroxidase gene expression and releases ROS (). Next, we checked whether LS treatment induced lignin accumulation by staining with phloroglucinol-HCl, which dyes lignin-containing tissues a red-violet color. We observed that phloroglucinol staining was more apparent in vascular tissues of plants grown on medium containing 1 μM LS compared with control plants (). These facts show that LS increases peroxidase activity by up-regulation of class III peroxidase genes in plant cells, and that the increased peroxidase activity would increase lignification activity.