Distribution, mobility, and anchoring of lignin-related oxidative enzymes in Arabidopsis secondary cell walls

Abstract

Lignin is an important phenolic biopolymer that provides strength and rigidity to the secondary cell walls of tracheary elements, sclereids, and fibers in vascular plants. Lignin precursors, called monolignols, are synthesized in the cell and exported to the cell wall where they are polymerized into lignin by oxidative enzymes such as laccases and peroxidases. In Arabidopsis thaliana, a peroxidase (PRX64) and laccase (LAC4) are shown to localize differently within cell wall domains in interfascicular fibers: PRX64 localizes to the middle lamella whereas LAC4 localizes throughout the secondary cell wall layers. Similarly, laccases localized to, and are responsible for, the helical depositions of lignin in protoxylem tracheary elements. In addition, we tested the mobility of laccases in the cell wall using fluorescence recovery after photobleaching. mCHERRY-tagged LAC4 was immobile in secondary cell wall domains, but mobile in the primary cell wall when ectopically expressed. A small secreted red fluorescent protein (sec-mCHERRY) was engineered as a control and was found to be mobile in both the primary and secondary cell walls. Unlike sec-mCHERRY, the tight anchoring of LAC4 to secondary cell wall domains indicated that it cannot be remobilized once secreted, and this anchoring underlies the spatial control of lignification.

Fig. 1.

Distinct cell-type distributions of lignin-related oxidative enzymes LAC4 and PRX64 in Arabidopsis stems. (A) Epifluorescence of a transverse section of stems from proLAC4:LAC4-mCHERRY plants, with red fluorescence in all secondary cell walls. (B) Blue UV autofluorescence from lignin of the section shown in (A). (C) Spinning disk confocal image of one vascular bundle from a LAC4-mCHERRY stem with red fluorescent protein (RFP) fluorescence in both vessels (V) and fibers (F). UV autofluorescence shows lignin, and the overlap of UV-RFP is shown in the merged image on the right. (D) Epifluorescence of a transverse section of stems from proPRX64:PRX64-mCHERRY plants, with red fluorescence in secondary cell walls of fibers. (E) Blue UV autofluorescence from lignin of the section shown in (D). (F) Spinning disk confocal image of one vascular bundle of from PRX64-mCHERRY plants with RFP fluorescence only in fibers (F), but not vessels (V). UV autofluorescence shows lignin, and the overlap of UV-RFP is shown in the merged image on the right. All plants were 8–9 weeks old, and sampled at the base of mature (25–30 cm) inflorescence stems. Scale bars: (A,B,D,E) = 1 mm; (C,F) = 40 μm.

Fig. 2.

The lignin-related oxidative enzymes LAC4 and PRX64 are found in different secondary cell wall domains in Arabidopsis stem fibers. (A) Spinning disk confocal images of interfascicular fibers from stems of proPRX64:PRX64-mCHERRY Arabidopsis plants with red fluorescence protein (RFP) fluorescence only in the middle lamella and cell corners. (B) UV autofluorescence shows lignin. (C) Overlap of UV and RFP in a merged image. (D) Spinning disk confocal images of interfascicular fibers from stems of proLAC4:LAC4-mCHERRY Arabidopsis plants with RFP fluorescence in secondary walls but not the middle lamella or cell corners. (E) UV autofluorescence shows lignin. (F) Overlap of UV and RFP in a merged image. (G) Spinning disk confocal images of interfascicular fibers from stems of control small secreted red fluorescent protein (sec-mCHERRY) in Arabidopsis plants with RFP fluorescence mainly in the middle lamella and cell corners. (H) UV autofluorescence shows lignin. (I) Overlap of UV and RFP in a merged image. All plants were 8–9 weeks old, and sampled at the base of mature (25–30 cm) inflorescence stems. Scale bars =40 μm.

Fig. 3.

Fluorescence recovery after photobleaching (FRAP) of cell wall proteins in Arabidopsis stems. (A) Mobility of a small secreted control protein (sec-mCHERRY) in primary cell walls of pith cells. (B) Lack of mobility of the laccase LAC4 in fiber secondary cell walls. All experiments were performed with fresh hand-sections of the base of mature (25–30 cm) stems from 8-9-week-old plants. Scale bars = 3 μm.

Fig. 4.

Laccases are immobile in the secondary cell wall of induced protoxylem tracheary elements (TEs). (A) LAC4-mCHERRY in secondary cell walls of apical hook cells of proLAC4:LAC4-mCHERRY/VND7-GR Arabidopsis seedlings 36 h after dexamethasone (DEX) induction. Left, pre-bleaching of the region of interest (ROI, white dashed circle); centre, at the time of bleaching; right, 60 s after bleaching. (B) Fluorescence recovery after photobleaching (FRAP) recovery curve for the sample shown in (A), showing the mobile fraction (F_m) and T_½ (vertical line). (C) LAC4-mCHERRY ectopically expressed in primary cell walls in proUBQ10:LAC4-mCHERRY/VND7-GR lines without DEX induction. Left, pre-bleaching the of ROI (white dashed circle); centre, at the time of bleaching; right, 60 s after bleaching. (D) FRAP recovery curve of the sample shown in (C), showing a large mobile fraction. (E) The mobility (F_m) of the mCHERRY-tagged laccase in the secondary cell walls (2CW) was significantly lower than in the primary cell walls (1CW) (Mann–Whitney U-test, *P≤0.0001). Data are means ±SD. Scale bars = 3 μm.

Fig. 5.

Secreted mCHERRY (sec-mCHERRY) is less mobile in the secondary cell wall than the primary cell wall. (A, C) Fluorescence recovery after photobleaching (FRAP) images taken of apical hook cells of Arabidopsis seedlings containing proUBQ10:sec-mCHERRY/VND7-GR pre-bleaching the region of interest (ROI, white dashed circles), at the time of bleaching, and 60 s after bleaching. (A) Rapid recovery of sec-mCHERRY in uninduced seedlings with primary cell walls. Scale bars = 3 μm. (B) The mobile fraction (F_m) and T_½ (vertical line) were calculated for the FRAP experiments done on the uninduced cells shown in (A). (C) Slow recovery of sec-mCHERRY in the secondary cell walls of protoxylem tracheary element cells, 36 h after induction of VND7-GR. (D) The mobile fraction (F_m) was calculated for the FRAP experiments done on the cells shown in (C). (E) Comparison of LAC4-mCHERRY and sec-mCHERRY mobility in primary cell walls (1CW) and secondary cell walls (2CW). Within 2CW environments sec-mCHERRY had statistically higher recovery than LAC4-mCHERRY (Mann–Whitney U-test, P≤0.05); however, the mobility of both LAC4-mCHERRY and sec-mCHERRY were much greater in 1CW than 2CW (Mann–Whitney U-test, *P≤0.0001). Data are means ±SD.

Fig. 6.

Laccases are highly immobile in the secondary cell wall. Fluorescence recovery after photobleaching (FRAP) in the primary cell walls (1CW) and secondary cell walls (2CW) of seedlings carrying proLAC4:LAC4-mCHERRY/VND7-GR (LACCASE4) or proUBQ10:sec-mCHERRY/VND7-GR (sec-mCHERRY). (A) Decreased levels of cellulose (10 μM 2,6-dichlorobenzonitrile, DCB, for 36 h), xylan (irx10/irx10-L mutants), or lignin (10 µM piperonylic acid, PA) did not change the low mobility of laccases. (B) FRAP mobile fraction when xylan and lignin were simultaneously disrupted using irx10/irx10-L mutants transformed with proLAC4:LAC4-mCHERRY/VND7-GR and treated with PA. (C) Although 1CW sec-mCHERRY mobility was higher than 2CW mobility, the lack of recovery in 2CW was reversed when lignin biosynthesis was inhibited with PA (Mann–Whitney U-test, **P≤0.0001, *P≤0.001). Data are means ±SD.