Abstract

Cortical microtubules (MTs) are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA) and β-tubulins (TUB), which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM). While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem where high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, non-tyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of P. deltoides and P. tremula ×alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isotypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes.

Highlights

  • Microtubules (MTs) are filamentous cytoskeleton components made up of α- and β-tubulins

  • We focused on developing xylem because it undergoes extensive MT-dependent secondary cell wall thickening (Funada, 2008) and exhibits very high tubulin transcript levels especially in tension wood (TW; Oakley et al, 2007)

  • Populus possesses a relatively large tubulin gene family composed of 8 TUAs and 20 TUBs (Oakley et al, 2007)

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Summary

Introduction

Microtubules (MTs) are filamentous cytoskeleton components made up of α- and β-tubulins. MTs play critical roles in regulating intracellular trafficking, morphogenesis, and cellulose microfibril deposition during cell wall formation (Wasteneys, 2002). In the woody perennial Populus, α- (TUAs) and β-tubulins (TUBs) are encoded by relatively large multi-gene families, with highly conserved amino acid sequences (88–98% identities), except for the hypervariable C-terminus. The C-terminal tails of tubulin are of interest because they are hotspots for posttranslational modifications (PTMs) in animals and protists (MacRae, 1997; Westermann and Weber, 2003; Magiera and Janke, 2014). Tubulin gene expression (Oakley et al, 2007) and transgenic manipulation (Swamy et al, 2015) have been reported in Populus, characterization at the protein level has been largely unexplored.

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