Laccases direct lignification in the discrete secondary cell wall domains of protoxylem

Abstract

Plants precisely control lignin deposition in spiral or annular secondary cell wall domains during protoxylem tracheary element (TE) development. Because protoxylem TEs function to transport water within rapidly elongating tissues, it is important that lignin deposition is restricted to the secondary cell walls in order to preserve the plasticity of adjacent primary wall domains. The Arabidopsis (Arabidopsis thaliana) inducible VASCULAR NAC DOMAIN7 (VND7) protoxylem TE differentiation system permits the use of mutant backgrounds, fluorescent protein tagging, and high-resolution live-cell imaging of xylem cells during secondary cell wall development. Enzymes synthesizing monolignols, as well as putative monolignol transporters, showed a uniform distribution during protoxylem TE differentiation. By contrast, the oxidative enzymes LACCASE4 (LAC4) and LAC17 were spatially localized to secondary cell walls throughout protoxylem TE differentiation. These data support the hypothesis that precise delivery of oxidative enzymes determines the pattern of cell wall lignification. This view was supported by lac4lac17 mutant analysis demonstrating that laccases are necessary for protoxylem TE lignification. Overexpression studies showed that laccases are sufficient to catalyze ectopic lignin polymerization in primary cell walls when exogenous monolignols are supplied. Our data support a model of protoxylem TE lignification in which monolignols are highly mobile once exported to the cell wall, and in which precise targeting of laccases to secondary cell wall domains directs lignin deposition.

Figure 1.

Lignin autofluorescence in secondary cell walls of endogenous and VND7-induced Arabidopsis seedling TEs. A, In a control, uninduced, 10-d-old Arabidopsis hypocotyl, only protoxylem TEs (arrow) emit autofluorescence when excited by UV light. B, In an Arabidopsis hypocotyl carrying VND7-VP16-GR, after induction with DEX, epidermal cells transdifferentiate into protoxylem TEs (arrows) and emit autofluorescence. C to E, UV autofluorescence of protoxylem TEs imaged under identical conditions in VND7-VP16-GR alone or in plants expressing both VND7-VP16-GR and artificial microRNAs targeting monolignol biosynthetic genes C4H (D) or CCR1 (E). Protoxylem TE differentiation and secondary cell wall (arrowheads) formation was not inhibited by constitutive expression of artificial microRNAs (insets in D and E). Maximum projection images of z-stacks are shown. Bars = 50 μm.

Figure 2.

C4H-GFP and CCR1-GFP are localized between secondary cell wall domains during VND7-induced protoxylem TE differentiation. A, Bright-field image of a differentiating protoxylem TE induced in VND7-VP16-GR plants, showing secondary cell wall thickenings (arrowheads). B, Maximum projection image of optical sections through the same cell shown in A, in which C4H-GFP localization is observed in the ER network that is excluded below developing secondary cell walls (arrowheads) and is adjacent to primary cell wall domains. C, Transmission electron micrograph showing laminar ER, Golgi, and mitochondria in relation to secondary cell walls of root protoxylem TEs. D and E, Maximum projection images of optical sections showing cytoplasmic localization of CCR1-GFP (D) and cytGFP (E) in control hypocotyl epidermal cells of VND7-VP16-GR seedlings without DEX induction. F to I, After VND7-VP16-GR protoxylem TE induction for 24 h, both CCR1-GFP (F) and cytGFP (G) were predominately localized in the cytoplasm between the secondary cell wall thickenings and as shown in a single median optical slice in CCR1-GFP (H) and cytGFP (I). er, Endoplasmic reticulum; G, Golgi; m, mitochondria; n, nucleus; scw, secondary cell wall. Bars = 12 μm in A, B, and D to G; 500 nm in C.

Figure 3.

ABC transporters are evenly localized in plasma membranes during protoxylem TE differentiation in VND7-VP16-GR seedlings. A, Maximum projection image of cotyledon epidermal cells undergoing protoxylem TE differentiation, expressing GFP-ABCG33. B, Cellulose stain, Pontamine S4B, counterstained image of cells shown in A, highlighting secondary cell wall domains. C, Median optical slice of induced protoxylem TE, showing invaginations of GFP-ABCG33 (green), counterstained with Pontamine S4B (red) demonstrating secondary cell wall domains, with inset image of the optical cross section of one secondary wall thickening. D, Induced protoxylem TE from plants expressing GFP-ABCG29, with inset showing plasma membrane localization over primary and secondary wall domains. Bars = 15 μm.

Figure 4.

Mobility of monolignols in the apoplast: Fluorescently tagged monolignols are specifically incorporated into the secondary cell walls of VND7-induced protoxylem TEs. A, Green fluorescence of incorporated NBD-CA specifically in secondary cell walls (arrowhead) and not primary cell walls (arrows) of epidermal cells induced to transdifferentiate into protoxylem TEs. B, Cellulose-specific Pontamine S4B counterstained image of cells shown in A, demonstrating secondary cell walls (arrowhead) and primary cell walls (arrow). C, Merged image of A and B showing colocalization of signal in secondary cell walls. D, In an area in which only one leaf mesophyll cell differentiated into a protoxylem TE, exogenously added NBD-CA was specifically incorporated in secondary wall thickenings and not in the surrounding neighbors. E, Pontamine S4B counterstained image of protoxylem TE shown in D, demonstrating background stain of primary cell wall domains of surrounding leaf cells, and strong secondary cell wall domains in lone protoxylem TE. F, Merged image of D and E. Bars = 15 μm.

Figure 5.

LACs are necessary and sufficient to direct lignin polymerization in Arabidopsis cell wall domains. A, Maximum projection image of hypocotyl epidermal cells induced to transdifferentiate into protoxylem TEs in VND7-VP16-GR seedlings, showing NBD-CA fluorescence in the secondary cell walls (arrowheads). B, VND7-induced protoxylem TE showing the following: i, cellulose deposition (Pontamine S4B stain); ii, whole mount immunolocalization of xylan (LM10 antibody); and iii, merged images. C, Maximum projection image of epidermal cells induced to transdifferentiate into protoxylem TE, in VND7-VP16-GR seedlings in lac4 lac17 double-mutant backgrounds showing that NBD-CA was not incorporated into the secondary cell walls (arrowheads). D, VND7-VP16-GR induced protoxylem TE in lac4 lac17 double mutants showing identical deposition patterns for the following: i, cellulose (Pontamine S4B stain); ii, xylan (LM10 immunolocalization); and iii, merged images, as observed in VND7-VP16-GR alone shown in B. E and F, Constitutive expression of LAC4 (E) or LAC17 (F) catalyzes lignin polymerization of NBD-CA in primary cell walls of cotyledon epidermal cells. G, Control wild-type cotyledon epidermal cells do not incorporate NBD-CA into their cell walls. H, Corresponding bright-field image of control wild-type cotyledons with one cell outlined. Bars = 25 μm.

Figure 6.

LAC4 localizes specifically to protoxylem TE secondary cell walls. A and B, LAC4 (LAC4-mCherry) localization in endogenous protoxylem (A) and metaxylem (B) TE from primary control roots. C, Median optical slice of an induced protoxylem TE, in hypocotyl epidermis, after 18 h of VND7-VP16-GR induction showing LAC4-mCherry localization, specifically in developing secondary cell walls. D, Higher magnification of developing secondary cell wall outlined in C. E, Maximum projection image showing that LAC4-mCherry localization persists in secondary cell walls after extended exposure to DEX and programed cell death. F, Immunolocalization of xylan (LM10 antibody) on a sectioned differentiating protoxylem TE showing identical deposition patterns as observed for LAC4-mCherry. G, Higher magnification of the developing secondary cell wall outlined in F. Bars = 5 μm in A and B; 12 μm in C to F.