Characterization of a tomato xyloglucan endotransglycosylase gene that is down-regulated by auxin in etiolated hypocotyls

Abstract

The reorganization of the cellulose-xyloglucan matrix is proposed to serve as an important mechanism in the control of strength and extensibility of the plant primary cell wall. One of the key enzymes associated with xyloglucan metabolism is xyloglucan endotransglycosylase (XET), which catalyzes the endocleavage and religation of xyloglucan molecules. As with other plant species, XETs are encoded by a gene family in tomato (Lycopersicon esculentum cv T5). In a previous study, we demonstrated that the tomato XET gene LeEXT was abundantly expressed in the rapidly expanding region of the etiolated hypocotyl and was induced to higher levels by auxin. Here, we report the identification of a new tomato XET gene, LeXET2, that shows a different spatial expression and diametrically opposite pattern of auxin regulation from LeEXT. LeXET2 was expressed more abundantly in the mature nonelongating regions of the hypocotyl, and its mRNA abundance decreased dramatically following auxin treatment of etiolated hypocotyl segments. Analysis of the effect of several plant hormones on LeXET2 expression revealed that the inhibition of LeXET2 mRNA accumulation also occurred with cytokinin treatment. LeXET2 mRNA levels increased significantly in hypocotyl segments treated with gibberellin, but this increase could be prevented by adding auxin or cytokinin to the incubation media. Recombinant LeXET2 protein obtained by heterologous expression in Pichia pastoris exhibited greater XET activity against xyloglucan from tomato than that from three other species. The opposite patterns of expression and differential auxin regulation of LeXET2 and LeEXT suggest that they encode XETs with distinct roles during plant growth and development.

Figure 1

Phylogenetic alignment of the tomato LeXET2-deduced amino acid sequence with other plant XETs. LeXET2 was aligned with 44 full-length deduced amino acid sequences using the ClustalX method, and a phylogenetic tree was generated using the neighbor-joining method and the TreeView program. Tomato genes are shown underlined. Details and GenBank accession numbers are described in “Materials and Methods.”

Figure 2

Southern-blot analysis of LeXET2 gene in tomato. Genomic DNA (10 μg lane⁻¹) was digested with the indicated restriction enzymes and the DNA gel blot hybridized with a radiolabeled LeXET2 3′-end cDNA as a probe. The blot was washed with 0.5× SSC and 0.5% (w/v) SDS at 65°C (14°C below the melting temperature).

Figure 3

Organ-specific expression pattern of tomato LeXET2. Poly(A⁺) RNA was isolated from different vegetative tissues (a) and tomato pericarp (b) at different stages of fruit development and was analyzed by northern blot (1 μg lane⁻¹). Blots were hybridized with a LeEXT2 cDNA probe and washed in 0.5× SSC at 65°C. The fruit blot was subsequently hybridized with a tomato actin probe as a loading control. Exposure to photographic film was 30 times longer for the fruit tissue blot than for the vegetative tissue blot. I, 0.5- to 1-cm diameter fruit; III, 4- to 6-cm diameter fruit; IG, immature green; MG, mature green; Br, breaker; LR, light red.

Figure 4

Differential expression and auxin regulation of the XET genes LeXET2 and LeEXT in etiolated tomato hypocotyls. a, RNA gel-blot analysis of LeXET2 and LeEXT expression along the tomato hypocotyl. Each lane contained 10 μg of total RNA isolated from consecutive 1-cm sections (A–D) of the hypocotyl. Gel blots were hybridized successively with the LeEXT2, LeEXT, and actin cDNA probes. b, Effect of auxin concentration on LeXET2 and LeEXT mRNA accumulation. Hypocotyl sections (1 cm) were cut immediately below the apical hook (A) or from a region 1.5 cm below the hook (B), incubated in the presence of the indicated concentration of 2,4-dichlorophenoxyacetic acid (2,4-d) for 24 h, and RNA was isolated. Total RNA (15 μg lane⁻¹) gel blots were hybridized with the LeXET2 and LeEXT cDNA probe. Ethidium bromide staining of ribosomal RNA is shown as a loading control.

Figure 5

Time course analysis of LeXET2 mRNA accumulation in tomato hypocotyl segments. Apical segments were incubated for the indicated times in buffer alone (control) or buffer plus 5 μm 2,4-d and RNA was isolated. Total RNA (15 μg lane⁻¹) gel blots were hybridized with the LeXET2 cDNA probe. Ethidium bromide staining of ribosomal RNA is shown as a loading control.

Figure 6

Hormonal regulation of LeXET2 mRNA levels in tomato hypocotyl segments. a, Effect of several plant hormones on LeXET2 gene expression. Apical segments were incubated for 20 h with buffer, with buffer plus 5 μm 2,4-d, 10 μm indole-3-acetic acid (IAA), 1 μm brassinolide (BR), 10 μm GA₃, 10 μm benzyladenine (BA), 10 μm abscisic acid (ABA), or 1 μm aminoethoxyvinyl Gly (AVG), or a combination of GA₃ plus 2,4-d or BA. b, Auxin regulation of LeXET2 mRNA accumulation in Nr and dgt hypocotyl segments. Apical segments from the ethylene-insensitive mutant Nr and the corresponding wild type (Pearson), or from the auxin-insensitive mutant dgt and its corresponding wild type (VFN8) were incubated in buffer alone or in buffer plus 5 μm 2,4-d for 20 h. c, Intact etiolated seedlings were treated with a continuous flow of air, air containing 10 μL L⁻¹ of ethylene, or were sprayed with a 1-mm solution of 2,4-d, and apical elongating (A) or mature (B) hypocotyl sections were cut after 48 h for RNA isolation. Total RNA (15 μg lane⁻¹) gel blots were hybridized with the LeXET2 cDNA probe. Ethidium bromide staining of ribosomal RNA is shown as a loading control.

Figure 7

Purification of recombinant LeXET2 from P. pastoris culture medium and activity of the recombinant protein. a, Proteins were separated on a SDS-polyacrylamide gel and were stained with Coomassie Blue. Proteins secreted from yeast transformed with pPIC3.5K vector only (1); proteins secreted from yeast transformed with LeXET2 in pPIC3.5K (2); eluate after SP-Sepharose chromatography of the culture media from yeast expressing LeXET2 (3); and purified LeXET2 after gel permeation chromatography (4). M_r markers are shown. The arrow indicates the band corresponding to LeXET2. b, Gel permeation chromatography showing transglycosylation between high-M_r tamarind xyloglucan and a xyloglucan oligosaccharide (XLLG) labeled with APTS, a fluorescent tag. Ten microliters of P. pastoris culture medium was incubated with 40 μg of tamarind xyloglucan and 282 pmol of XLLG-APTS in a total volume of 40 μL for 1 h at 25°C. The reaction mixtures were then analyzed by gel permeation chromatography using a Superdex-75 HR 10/30 column, as described in “Materials and Methods,” and fluorescent transglycosylation products were detected in the eluate with a fluorescent detector. XLLG-APTS alone (i); reaction containing high-M_r tamarind xyloglucan, XLLG-APTS, and culture medium from P. pastoris transformed with LeXET2 in pPIC3.5K (ii); and reaction containing high-M_r tamarind xyloglucan, XLLG-APTS, and culture medium from P. pastoris transformed with empty pPIC3.5K (iii). V_o, Void volume.

Figure 8

Effect of the molecular size of the xyloglucan donor substrate on LeXET2 activity. Xyloglucan fractions obtained after separation of arabinoxyloglucans (AXGs) from tomato suspension-cultured cells were used as a substrate in a reaction containing 100 μg of donor substrate, 282 pmol of XLLG-APTS, and 50 ng of enzyme in a total volume of 40 μL. Reactions were incubated for 1 h at 25°C and were analyzed by gel permeation chromatography on a Superdex-75 column as described in “Materials and Methods.” Prior to the reaction, the profiles of the xyloglucan donor substrates were analyzed on the same column measuring the total sugar content in the eluate by the anthrone assay (OD = 620 nm, dotted lines). Fluorescent transglycosylation products were detected in the eluate with a fluorescent detector (solid lines). A, Donor substrate with a molecular mass of 40 to 70 kD; B, donor substrate with a molecular mass of 10 to 40 kD; C, donor substrate with a molecular mass of 10 to 30 kD; D, donor substrate with a molecular mass around 10 kD; E, donor substrate with a molecular mass of 2.4 to 10 kD. V_o, Void volume. The elution time of a 10-kD average molecular mass Dextran and of a 2.4-kD xyloglucan oligosaccharide is indicated by an arrow.