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Cloning and characterization of a xyloglucan endo-transglycosylase/hydrolase gene expressed in response to inclination in radiata pine seedlings

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Abstract

The cell wall is a dynamic structure that can be modified by different conditions during growth. Stem inclination induces biochemical changes and chemical modifications in the cell walls of conifers driving plant stem reorientation. Different enzymes take part in cell wall remodeling, including xyloglucan endotransglycosylase/hydrolase (XTH). This enzyme modifies xyloglucan, the main hemicellulose found in primary cell walls of pine trees. The full-length complementary DNA of PrXTH1 was isolated and characterized from radiata pine. PrXTH1 contains an open-reading frame of 882 bp encoding a protein of 294 amino acids, including an N-terminal signal peptide for secretion that targets the protein to the cell wall. Phylogenetic and gene structure analyses suggest that PrXTH1 belongs to group I of XTHs. The recombinant purified protein has only transglycosylase activity. A 3D model of PrXTH1 protein was built through comparative modeling, showing a β-sandwich structure with the catalytic site in the middle of the protein structure, composed of residues Glu79, Asp81, and Glu83. One-year-old half-sib seedlings were inclined at 45° and transcriptional analysis indicated that PrXTH1 transcripts are more abundant in the lower side of inclined stems. Additionally, along the stem, PrXTH1 transcript accumulation starts at the apex and moves down over time to the stem base. This gene shows regulation by ethylene, indicating that it could be a target in the hormonal cascade during the inclination response. Finally, in radiata pine seedlings PrXTH1 is induced during the inclination response, suggesting an active participation of this enzyme in the cell wall reorganization required during this phenomenon.

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Acknowledgments

This research was supported by Fondecyt 1120635 and 11121170 projects. C.V. thanks the Universidad de Talca for his doctoral fellowship. P.R. was supported by postdoctoral PBCT-PSD61 project. The authors would like to thank the reviewers for their highly valuable comments and also Thomas Lehe for critical reading of this manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Data archiving statement

The sequence used in this study has been deposited at GenBank. The sequence of PrXTH1 from Pinus radiata D. Don has the GenBank accession number KF151203.

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Correspondence to Raúl Herrera.

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Communicated by R. Sederoff

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Supplement Figure 1

Nucleotide and deduced amino acid sequence of PrXTH1 gene. In bold and underlined “ATG” indicate the putative translational start site. An asterisk (*) marks the putative stop codon. Capital letters within the open reading frame indicate the conserved regions to which primers were designed. The italic lower case letters indicate the intergenic zones. Bold letters indicate the sequence in PrXTH1 that corresponds to primer sequences used. (GIF 131 kb)

High Resolution Image (TIFF 641 kb)

Supplement Figure 2

Genomic sequence of Pr-XTH1. Grey dark boxes indicate exons while the light grey boxes indicate untranslated regions (UTRs). Horizontal lines connecting with the exons indicate introns. The number of nucleotides contained within each intron and exon is indicated. (GIF 9 kb)

High Resolution Image (TIFF 79 kb)

Supplement Figure 3

A) SDS/PAGE, B) y C) Western blot of PrXTH1 recombinacifnt protein expressed in P. pastori culture media with anti-histidine tag (Anti-His (C-Term)-HRP Invitrogen CA) and anti-xet polyclonal (Agrisera AS05 080) antibodies respectively. M, molecular weight protein markers; lane 1, crude protein extract; lane 2, protein fraction not bound to BD-Talon affinity resin; lane 3, recombinant protein purified through BD-Talon affinity resin. (GIF 186 kb)

High Resolution Image (TIFF 9014 kb)

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Valenzuela, C., Ramos, P., Carrasco, C. et al. Cloning and characterization of a xyloglucan endo-transglycosylase/hydrolase gene expressed in response to inclination in radiata pine seedlings. Tree Genetics & Genomes 10, 1305–1315 (2014). https://doi.org/10.1007/s11295-014-0762-9

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