NITRILASE1 regulates the exit from proliferation, genome stability and plant development

Abstract

SummaryNitrilases are highly conserved proteins with catabolic activity but much less understood functions in cell division and apoptosis. To elucidate the biological functions of Arabidopsis NI-TRILASE1, we characterized its molecular forms, cellular localization and involvement in cell proliferation and plant development.We performed biochemical and mass spectrometry analyses of NITRILASE1 complexes, electron microscopy of nitrilase polymers, imaging of developmental and cellular distribution, silencing and overexpression of nitrilases to study their functions.We found that NITRILASE1 has an intrinsic ability to form filaments. GFP-NITRILASE1 was abundant in proliferating cells, distributed in cytoplasm, in the perinuclear area and associated with microtubules. As cells exited proliferation and entered differentiation, GFP-NITRILASE1 became predominantly nuclear. Nitrilase silencing dose-dependently compromised plant growth, led to loss of tissue organization and sustained proliferation. Cytokinesis was frequently aborted, leading to enlarged polyploid cells. In reverse, independently transformed cell lines overexpressing GFP-NITRILASE1 showed slow growth and increased rate of programmed cell death.Altogether, our data suggest that NITRILASE1 homologues regulate the exit from cell cycle and entry into differentiation and simultaneously are required for cytokinesis. These functions are essential to maintain normal ploidy, genome stability and tissue organization.

Lipid Peroxide-Derived Short-Chain Carbonyls Mediate Hydrogen Peroxide-Induced and Salt-Induced Programmed Cell Death in Plants

“…In Arabidopsis, changes in the polymer structure and the intracellular localization of Nit1 are closely associated with the transition from proliferation to differentiation (Doskocilová et al, 2013). It is unclear, however, whether the HNE modification of Nit1 causes such changes.…”

A DEK Domain-Containing Protein Modulates Chromatin Structure and Function inArabidopsis

“…Among the other proteins copurifying with DEK3 were the cohesion protein SCC3 involved in mitotic and meiotic cell division (Chelysheva et al, 2005;Schubert et al, 2009) and a putative homolog of the cohesion-associated protein PDS5. Nitrilase1, a multifunctional protein implicated in cytokinesis and in maintaining genome stability (Dosko cilová et al, 2013), was also identified in all experiments. Further, histone deacetylase 3 (HDA3/HDT1) copurified with DEK3, confirming a similar interaction observed in human cells (Hollenbach et al, 2002).…”

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

“…Flow cytometry has also been used to sort nuclei prior to transcriptomic analysis [Macas et al, 1998;Zhang et al, 2008;Bourdon et al, 2012] and for cell cycle analysis [Binarová et al, 1993;Kotogány et al, 2010;Petrovská et al, 2012;Doskočilová et al, 2013]. A protocol aimed at yielding suspensions of intact chromosomes [Doležel et al, 1992] is also suitable for the isolation of nuclei; the mild fixation with formaldehyde does not compromise the quality of the DNA in the context of preparing DNA libraries [Šafář et al, 2004;Kasprzak et al, 2006].…”