Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar

Abstract

Laccases are encoded by multigene families in plants. Previously, we reported the cloning and characterization of five divergent laccase genes from poplar (Populus trichocarpa) xylem. To investigate the role of individual laccase genes in plant development, and more particularly in lignification, three independent populations of antisense poplar plants, lac3AS, lac90AS, and lac110AS with significantly reduced levels of laccase expression were generated. A repression of laccase gene expression had no effect on overall growth and development. Moreover, neither lignin content nor composition was significantly altered as a result of laccase suppression. However, one of the transgenic populations, lac3AS, exhibited a 2- to 3-fold increase in total soluble phenolic content. As indicated by toluidine blue staining, these phenolics preferentially accumulate in xylem ray parenchyma cells. In addition, light and electron microscopic observations of lac3AS stems indicated that lac3 gene suppression led to a dramatic alteration of xylem fiber cell walls. Individual fiber cells were severely deformed, exhibiting modifications in fluorescence emission at the primary wall/middle lamella region and frequent sites of cell wall detachment. Although a direct correlation between laccase gene expression and lignification could not be assigned, we show that the gene product of lac3 is essential for normal cell wall structure and integrity in xylem fibers. lac3AS plants provide a unique opportunity to explore laccase function in plants.

Figure 1

Structures of poplar laccase (lac1AS, lac3AS, lac90AS, and lac110AS) AS constructs. P35S CaMV, CaMV 35S RNA promoter; Tnos, termination sequence of the nopaline synthase gene.

Figure 2

Molecular screening of laccase AS poplar transformants by northern-blot analysis. Twenty-five micrograms of total RNA from lac3AS, lac90AS, and lac110AS stems (A, B, and C, respectively) were electrophoresed on denaturing gels and probed with the corresponding radiolabeled lac3, lac90, or lac110 3′-untranslated region (UTR). In A, histograms represent levels of endogenous (white bars) and AS transgene (black bars). In B and C, gray bars represent endogenous + transgene expression. In all cases, signals were normalized with respect to 28S ribosomal RNA content. Controls, In vitro-propagated untransformed poplars (C1) and poplars transformed with empty pJR1 vector (C2).

Figure 3

Soluble phenolic content in AS laccase and control poplar plants. Data are presented as means (±sd) of three separate measurements. In abscissa, numbers (3, 90, and 110) before the dot refer to the name of the laccase clone introduced in AS orientation. As in Figure 2, C1 and C2 are controls (untransformed poplars and poplars transformed with empty pJR1 vector, respectively).

Figure 4

HPLC profiles of total soluble phenolics of control (A), lac3.2AS (B), and lac3.4AS (C) plants. The numbered peaks were identified by HPLC-mass spectrometry (MS). The corresponding chemical structures of the peaks identified by HPLC-MS are indicated in D and are: salicin (1), salicortin (2), salireposide (3), tremuloidin (4), and tremulacin (5; Clausen et al., 1989). Peaks marked with an asterisk could not be identified by MS. An arrow indicates a peak that was present in AS lines but not in controls. Bz, Benzoate.

Figure 5

Transverse stems sections of poplar visualized by UV fluorescence microscopy (A–C) and phloroglucinol staining (D–F). A and D, Control (transformed with empty vector); B and E, lac3.2AS; C and F, lac3.4AS. v, Xylem vessel; rp, ray parenchyma; f, xylem fiber. Note the deformed cell contour of xylem fibers and an absence of fluorescence emission (arrows) at the middle lamella/primary wall region in lac 3.2AS (B) and lac3.4AS (C) as compared with control fibers (A). Scale bars = 50 μm.

Figure 6

A and B, Transverse sections of poplar stained with toluidine blue. A, Control; B, lac3.4AS. Note the accumulation of dark-blue stain in xylem ray parenchyma cells and the detachment of cell walls (indicated by black and white arrows, respectively). Scale bars = 50 μm. Abbreviations are as in Figure 5. C through F, Electron micrographs of xylem fibers in control (C) and lac3.4AS (D–F) plants. Note detachment of primary (D) and secondary (E and F) cell walls (indicated by open arrows). CWI, Primary wall; CWII, secondary wall. Scale bars = 2 μm in C and 1 μm in D through F.