Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants

Abstract

The biosynthesis of lignin in vascular plants is regulated both developmentally and environmentally. In the inflorescence stems of Arabidopsis, lignin is mainly deposited in the walls of xylem cells and interfascicular fiber cells during normal plant growth and development. The mechanisms controlling the spatial deposition of lignin remain unknown. By screening ethyl methanesulfonate-mutagenized populations of Arabidopsis, we have isolated two allelic elp1 (ectopic deposition of lignin in pith) mutants with altered lignin deposition patterns. In elp1 stems, lignin was ectopically deposited in the walls of pith parenchyma cells in addition to its normal deposition in the walls of xylem and fiber cells. Lignin appeared to be deposited in patches of parenchyma cells in the pith of both young and mature elp1 stems. The ectopic deposition of lignin in the pith of elp1 stems was accompanied by an increase in the activities of enzymes in the lignin biosynthetic pathway and with the ectopic expression of caffeoyl coenzyme A O-methyltransferase in pith cells. These results indicate that the ELP1 locus is involved in the repression of the lignin biosynthetic pathway in the pith. Isolation of the elp1 mutants provides a novel means with which to study the molecular mechanisms underlying the spatial control of lignification.

Figure 1

Lignification in the inflorescence stems of wild-type Arabidopsis plants. Inflorescence stems from 6-week-old plants were sectioned and sections were stained for lignin with phloroglucinol-HCl. Lignin staining is red. Pictures were taken under a dissection microscope with dark-field illumination. A, Section from the top part of the stem. Lignin was only seen in xylem cells. B, Section from the middle part of the stem. Lignin staining was heavy in xylem cells but light in interfascicular fibers. C, Section from the basal part of the stem. The lignin staining was heavy in both xylem cells and interfascicular fibers. co, Cortex; f, interfascicular fiber; pi, pith; x, xylem. Magnification, ×52.

Figure 2

Ectopic deposition of lignin in the pith of elp1 stems. Basal parts of the stems of 6-week-old plants were sectioned and sections were stained for lignin with phloroglucinol-HCl (left column) and the Mäule reagent (right column). Phloroglucinol-HCl stains total lignin; the Mäule reagent stains syringyl lignin. Lignin was stained red with both reagents. A and B, Sections from a wild-type stem. Lignin was present in both xylem cells and interfascicular fibers but absent in the pith. Note that xylem cells were stained orange with the Mäule reagent due to the lack of syringyl lignin. C and D, Sections from an elp1-1 stem. Lignin was seen in the pith in addition to xylem cells and fibers. Like the lignin in the interfascicular fibers, the lignin in the pith was rich in syringyl lignin units, as revealed by staining with the Mäule reagent. E and F, Sections from an elp1-2 stem. Lignin was evident in the pith in addition to xylem cells and fibers. Similar to the elp1-1 mutant, the lignin in the pith was rich in syringyl lignin units, as revealed by staining with the Mäule reagent. co, Cortex; f, interfascicular fiber; pf, phloem fiber; pi, pith; x, xylem. Magnification, ×52.

Figure 3

Lignification pattern in the pith throughout an elp1-2 stem. A stem from the elp1-2 mutant plant was divided into five equal segments, and thin sections from each segment were stained for lignin with phloroglucinol-HCl. Lignin staining is red. A, Section from the top segment of the stem. B and C, Sections with 1-cm apart from the second segment of the stem. D, Section from the third segment of the stem. E. Section from the fourth segment of the stem. F, Section from the bottom segment of the stem. Although lignin was seen in the pith cells in all sections, lignin distribution in the pith appeared to be mosaic. f, Interfascicular fiber; pf, phloem fiber; pi, pith; x, xylem. Magnification, ×52.

Figure 4

Lignin content and enzyme activity in elp1 stems. Mature inflorescence stems of 50 individual plants were pooled and assayed for Klason lignin content. Klason lignin in the mutants was expressed as a percentage of the wild type. Data for Klason lignin are the means ± se from three separate assays. Inflorescence stems from 6-week-old plants were collected and assayed for the activities of PAL, CCoAOMT, and CCR. The enzyme activity in the wild type was taken as 100, and the activity in the mutants was expressed as a percentage of the wild type. Data for enzyme activity are the means ± se from 10 plants.

Figure 5

Immunolocalization of CCoAOMT in elp1 stems. Stem sections were first probed with polyclonal antibodies against zinnia CCoAOMT, and then incubated with gold-labeled secondary antibody. After silver enhancement, the signal was visualized under UV epifluorescent illumination. The CCoAOMT signal is seen as yellow under UV epifluorescence. Lignin shows red autofluorescence under UV light. A and B, Stem sections from the elp1-1 (A) and elp1-2 (B) mutants, respectively, probed with the CCoAOMT antibodies. Note that the CCoAOMT signal was evident in lignified pith cells. C, Stem section from the wild type probed with the CCoAOMT antibodies. Note that the CCoAOMT signal was seen only in xylem cells and fibers but not in pith cells. D, Stem section from the elp1-2 mutant incubated with preimmune serum. No signal was observed in any cells. f, Interfascicular fiber; pi, pith; x, xylem. Magnification, ×625.

Figure 6

Electron microscopy of the walls of pith cells. A, Primary walls of the pith cells from a wild-type stem. B, Primary walls of the pith cells from an elp1-2 stem. No secondary wall thickening was observed in the pith cells of the elp1-2 mutant. pw, Primary wall. Magnification, ×12,000.

Figure 7

Genetic mapping of the elp1 locus. A total of 464 F₂ mapping plants were used for mapping with markers on the left side of the elp1 locus, and a total of 449 F₂ mapping plants were used with markers on the right side of the elp1 locus. All markers used for mapping were CAPS markers. The markers shown on chromosome 1 were not positioned on scale.