MAP20, a microtubule-associated protein in the secondary cell walls of hybrid aspen, is a target of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile

Abstract

We have identified a gene, denoted PttMAP20, which is strongly up-regulated during secondary cell wall synthesis and tightly coregulated with the secondary wall-associated CESA genes in hybrid aspen (Populus tremula x tremuloides). Immunolocalization studies with affinity-purified antibodies specific for PttMAP20 revealed that the protein is found in all cell types in developing xylem and that it is most abundant in cells forming secondary cell walls. This PttMAP20 protein sequence contains a highly conserved TPX2 domain first identified in a microtubule-associated protein (MAP) in Xenopus laevis. Overexpression of PttMAP20 in Arabidopsis (Arabidopsis thaliana) leads to helical twisting of epidermal cells, frequently associated with MAPs. In addition, a PttMAP20-yellow fluorescent protein fusion protein expressed in tobacco (Nicotiana tabacum) leaves localizes to microtubules in leaf epidermal pavement cells. Recombinant PttMAP20 expressed in Escherichia coli also binds specifically to in vitro-assembled, taxol-stabilized bovine microtubules. Finally, the herbicide 2,6-dichlorobenzonitrile, which inhibits cellulose synthesis in plants, was found to bind specifically to PttMAP20. Together with the known function of cortical microtubules in orienting cellulose microfibrils, these observations suggest that PttMAP20 has a role in cellulose biosynthesis.

Figure 1.

Coexpression of poplar secondary cell wall-associated CESA genes and MAP20. A, Relative mRNA abundance of PttCESA1, PttCESA3, PttCESA9 (nomenclature according to Djerbi et al. [2004]), and PttMAP20 in different tissues and organs as analyzed on cDNA microarrays. B, The relative mRNA abundance of PtCESA1 (♦), PtCESA3-2 (▴), PtCESA9-2 (○), and PtMAP20 (•) as quantified by qPCR in eight samples across developing xylem tissues, from functional phloem toward maturing wood cells. The radial width of each sample is indicated in the figure (top), and the corresponding sampling positions are indicated on the anatomical section (bottom). The tissue types of the samples were as follows: functional phloem (sample 1), cambial meristem (sample 2), expanding wood cells (sample 3), and secondary cell wall forming and maturing wood cells (samples 4–8). Scale bar = 50 μm.

Figure 2.

Immunolocalization of PttMAP20 and microtubules in the wood-forming tissues of hybrid aspen using confocal microscopy. A to C, Affinity-purified polyclonal anti-PttMAP20 antibodies. B, Monoclonal anti-α-tubulin antibodies. C, Preimmune serum, a negative control for the MAP20 antibodies. Transverse sections through the wood-forming tissues show developing and mature phloem (dPH and mPH), vascular cambium (CA), radial expansion zone (RE), and the secondary wall deposition zone (SW). The arrow in B points to the sieve tube cell.

Figure 3.

A, Full-length protein coding sequence of PttMAP20 aligned with the Arabidopsis At5g37478_alt protein sequence (see text for an explanation) and the M. truncatula hypothetical protein ABE90152. The TPX2 domain region is boxed. B, Phylogenetic analysis of the TPX2 domain sequences used in Supplemental Figure S3. Populus genes are indicated in bold, and the relative position of the TPX2 domain in each protein sequence is indicated by a black box.

Figure 3.

A, Full-length protein coding sequence of PttMAP20 aligned with the Arabidopsis At5g37478_alt protein sequence (see text for an explanation) and the M. truncatula hypothetical protein ABE90152. The TPX2 domain region is boxed. B, Phylogenetic analysis of the TPX2 domain sequences used in Supplemental Figure S3. Populus genes are indicated in bold, and the relative position of the TPX2 domain in each protein sequence is indicated by a black box.

Figure 4.

SDS-PAGE stained with Coomassie Blue demonstrating the binding of recombinant PttMAP20 to in vitro-assembled, taxol-stabilized BMTs. Twenty microliters of supernatant (S) or 5 μL of resuspended pellet (P; see text) containing the different proteins was loaded in the gel as follows: 1, BSA and BMTs; 2, BSA; 3, BMTs; 4, BMTs and PttMAP20; 5, PttMAP20.

Figure 5.

Phenotypic analysis of transgenic lines of Arabidopsis overexpressing PttMAP20. A, RT-PCR analysis of wild type (wt) and transgenic (PttMAP20-OE) plants using primers specific for PttMAP20 and actin. B, Two-week-old wild-type (wt) and transgenic (PttMAP20-OE) Arabidopsis seedlings, the latter showing left-handed twisting of cotyledon petioles. C, Etiolated hypocotyls from 4-d-old wild-type (i) and two independent PttMAP20-overexpressing lines (ii and iii). Helical right-handed twisting of epidermal cells was consistently observed in the two transgenic lines.

Figure 6.

Confocal microscopy images of tobacco leaf epidermal cells transiently expressing PttMAP20 fused to YFP. The images were recorded 3 to 5 d after the agroinfiltration. Scale bar = 20 μm. Shown is the decoration of the tobacco microtubules by the PttMAP20-YFP fusion proteins in vivo.

Figure 7.

A, Labeling of recombinant PttMAP20 by [³H]DCPA. Lanes 1 to 5, 1 ng, 10 ng, 100 ng, 1 μg, and 10 μg (respectively) of purified PttMAP20 were labeled in the presence of 0.75 μm [³H]DCPA as described in “Materials and Methods”; lanes 6 to 8, control experiments performed using 1 μg of PttMAP20 but omitting UV irradiation (lane 6), [³H]DCPA (lane 7), or both (lane 8). B, Inhibition of the binding of [³H]DCPA to PttMAP20 by competition with DCPA. The experiments were performed using 0.75 μm [³H]DCPA, 1 μg of PttMAP20, and increasing concentrations of DCPA as indicated in the plot. The amount of bound [³H]DCPA was determined by measuring the radioactivity in the PttMAP20 bands excised from SDS-PAGE gels (liquid scintillation; see “Materials and Methods” for experimental details). C, As in B, but using DCB as a competing molecule. A maximum of 5% variation was obtained for each point in B and C after replicate experiments. D, Effect of DCB on the binding of PttMAP20 onto BMT and on the polymerization of BMT. Increasing concentrations of DCB were incubated in the presence of 1 μg of recombinant PttMAP20 for 20 min. The mixture were subsequently added to preassembled BMT and subjected to the spin-down binding assay (see “Materials and Methods”). The presence of tubulin and PttMAP20 in the pellet and supernatant was assessed by SDS-PAGE analysis. 1, No DCB; 2 to 4, 2 μm, 20 μm, and 200 μm DCB, respectively. E, The effect of DCB on the polymerization of microtubules as evidenced by SDS-PAGE, stained by Coomassie Blue. Increasing concentrations of DCB (0.75 μm, 7.5 μm, 75 μm, 750 μm, and 7,500 μm, lanes 3–7, respectively) were mixed with monomeric α- and β-tubulins before initiating the polymerization reaction as described in “Materials and Methods.” Control reactions with buffer or methanol replacing DCB are shown in lanes 1 and 2, respectively. Both supernatant and pellet fractions are shown for all samples.