Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis

Abstract

Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric 14 N/ 15 N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/ RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.

Two Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel Development

“…First, we confirmed that the downregulation of CSI1/POM2 caused aberrant secondary wall deposition in proto-and metaxylem trans-differentiating cell suspension cultures (Derbyshire et al, 2015;Supplemental Figures 1A and 1B). Using confocal microscopy, we quantified the occurrence of spiral, reticulate, and pitted secondary wall patterns and the percentage of calcofluor-stained irregular deposits in the secondary walls of nontransgenic and CSI1 downregulated cell lines (Supplemental Figures 1C and 1D).…”

“…MAP65-1, AIR9, MAP70-1, and MAP70-5 (Pesquet et al, 2010;Derbyshire et al, 2015). These proteins contribute to the MTbundling/stabilization and are important to achieve the MT array rearrangements during secondary wall synthesis.…”

“…reported no defects on secondary walls nor decreased cellulose content in csi1/pom2 mutant Arabidopsis stems. By contrast, Derbyshire et al (2015) showed that induction of tracheary elements in Arabidopsis cell cultures was impaired in cells with reduced CSI1/POM2 expression. The role of CSI1/ POM2 in secondary wall cellulose production therefore remains unclear.…”

Patterning of plant cell wall deposition by cortical microtubules

“…In these systems, non-xylem plant cells can be triggered to transdifferentiate into xylem-like cells by hormonal treatment 45,46 . Using trans-differentiation systems, xylem formation has been studied in cell cultures of Zinnia elegans [46][47][48] and Arabidopsis thaliana (Arabidopsis) 49,50 . These studies resulted in the discovery of the master regulators of proto-and metaxylem formation Vascular NAC Domain 7 (VND7) and VND6 respectively in Arabidopsis 45 .…”

“…The discovery of transdifferentiating cell cultures, triggering non-xylem cells to develop into xylem like cells, has greatly helped to identify cellular processes involved in xylem formation. The benefits are that these transdifferentiation cells cultures can be synchronised and are well suitable for analysis by microarrays 45,48 , proteomics 50 and experimental cell biology 46,47,49,129 . Microarray analysis followed up by overexpression experiments revealed the master regulators for proto-and metaxylem formation, respectively VND7 and VND6 45 .…”