Cell-specific and conditional expression of caffeoyl-coenzyme A-3-O-methyltransferase in poplar

Abstract

Caffeoyl coenzyme A-3-O-methyltransferase (CCoAOMT) plays an important role in lignin biosynthesis and is encoded by two genes in poplar (Populus trichocarpa). Here, we describe the expression pattern conferred by the two CCoAOMT promoters when fused to the gus-coding sequence in transgenic poplar (Populus tremula x Populus alba). Both genes were expressed similarly in xylem and differentially in phloem. In xylem, expression was preferentially observed in vessels and contact rays, whereas expression was barely detectable in storage rays and fibers, suggesting different routes to monolignol biosynthesis in the different xylem types. Furthermore, after wounding, fungal infection, and bending, the expression of both genes was induced concomitantly with de novo lignin deposition. Importantly, upon bending and leaning of the stem, the cell-specific expression pattern was lost, and both genes were expressed in all cell types of the xylem. CCoAOMT promoter activity correlated well with the presence of the CCoAOMT protein, as shown by immunolocalization. These expression data may explain, at least in part, the heterogeneity in lignin composition that is observed between cell types and upon different environmental conditions.

Figure 1

Phenylpropanoid and monolignol biosynthesis pathways. C3H, Coumarate 3-hydroxylase; CCoA3H, coumaroyl-CoA 3-hydroxylase; CCR, cinnamoyl-CoA reductase; F5H, ferulate 5-hydroxylase; CAld5H, coniferaldehyde 5-hydroxylase.

Figure 2

Histochemical analysis in transgenic poplar showing GUS activity or lignin deposition. A Thick transversal section of young stem of poplar transformed with PBINPOP1. B, Enlargement of A showing GUS activity in young xylem cells. C, Thin transversal section of the xylem in the middle part of the stem (PBINPOP1). D, Longitudinal section of the same stem as shown in C. E, Thin transversal section of the bark of the middle part of the stem (PBINPOP1). F, Thin section of the bark of the middle part of the stem (PBINPOP2). G, Thin transversal section of the bark showing GUS staining (PBINPOP1). H, (Legend continues on facing page.)Thick transversal section of a poplar stem transformed with a PCaMV35S-GUS construct. I and M, Thick transversal sections of young leaf and petiole stained with P-HCl. J and N, Thick transversal sections of young leaf and petiole (PBINPOP1). K and O, Thick transversal section of old leaf and petiole (PBINPOP1). L and P, Thick transversal section of old leaf and petiole (PBINPOP2) at the same developmental stage as sections in K and O. CC, Companion cell; Co, cortex; CR, contact ray cell; CZ, cambial zone; Ep, epidermis; MPF, mature phloem fibers; P, phloem; PF, phloem fibers; Ph, phellem; Pi, pith; Pt, pit; PX, primary xylem; SR, storage ray cell; V, vessel; X, xylem; XF, xylem fiber. Bars = 200 μm (in A and H–P) and 50 μm (in B–G).

Figure 3

CCoAOMT promoter activity in transgenic poplar upon biotic and abiotic stress conditions. A, Transversal section of a petiole stained for both lignin and GUS. B, GUS activity in a mechanically wounded leaf. C, M. pinitorca-infected leaf stained for both lignin deposition and GUS activity. D, Double staining of a thick transversal section of a bent stem for lignin and GUS activity. E, Thick transversal section of a non-bent stem stained by P-HCl as control. F, Enlargement of a thin transversal section of the bent stem, transformed with PBINPOP1. CZ, Cambial zone; F, site of fungal infection; P, phloem; PF, phloem fibers; Pi, pith; W, wound; X, xylem. Bars = 200 μm (in A, B, D, and E), 100 μm (in C), and 50 μm (in F).

Figure 4

Protein gel-blot analysis of poplar protein extracts. Crude protein extracts from poplar control leaf (CL) and wounded leaf (WL) separated by SDS-PAGE, immobilized on a nitrocellulose membrane, and incubated with anti-CCoAOMT antibodies. The protein molecular mass is shown at the left in kD.

Figure 5

Induction of GUS activity in transgenic poplar stems as a consequence of mechanical bending. GUS activity was measured in xylem, phloem, and pith tissues. The data represent the average of three independent experiments. The se is shown.

Figure 6

Immunolocalization of CCoAOMT in transversal sections of poplar stem by light microscopy. A, Young stem of 3-month-old greenhouse-grown poplar. B, Xylem tissue of the middle part of a 3-month-old greenhouse-grown poplar stem. C and D, Bark tissue of old and young stem. E, Stem of transgenic poplar down-regulated for CCoAOMT. F, Bent stem. G, Leaned stem. H, Control section treated with preimmune serum. Co, Cortex; CR, contact ray cell; CZ, cambial zone; Ep, epidermis; F, phloem; MPF, mature phloem fibers; Pe, periderm; PF, phloem fibers; Ph, phellem; Pi, pith; PX, primary xylem; SR, storage ray cell; V, vessel; X, xylem; XF, xylem fiber. Bars = 100 μm.

Figure 7

Subcellular localization of CCoAOMT in poplar stem by electron microscopy. A, Contact ray cell from a stem section of the middle part of a 3-month-old greenhouse-grown poplar. The labeling with alfalfa CCoAOMT antibodies is concentrated in the cytosol. B, control section treated with preimmune serum. CR, Contact ray cell; Cyt, cytoplasm; SW, secondary wall. Arrows indicate gold particles. Bars = 1 μm.