Application of Nuclear Volume Measurements to Comprehend the Cell Cycle in Root-Knot Nematode-Induced Giant Cells

Abstract

Root-knot nematodes induce galls that contain giant-feeding cells harboring multiple enlarged nuclei within the roots of host plants. It is recognized that the cell cycle plays an essential role in the set-up of a peculiar nuclear organization that seemingly steers nematode feeding site induction and development. Functional studies of a large set of cell cycle genes in transgenic lines of the model host Arabidopsis thaliana have contributed to better understand the role of the cell cycle components and their implication in the establishment of functional galls. Mitotic activity mainly occurs during the initial stages of gall development and is followed by an intense endoreduplication phase imperative to produce giant-feeding cells, essential to form vigorous galls. Transgenic lines overexpressing particular cell cycle genes can provoke severe nuclei phenotype changes mainly at later stages of feeding site development. This can result in chaotic nuclear phenotypes affecting their volume. These aberrant nuclear organizations are hampering gall development and nematode maturation. Herein we report on two nuclear volume assessment methods which provide information on the complex changes occurring in nuclei during giant cell development. Although we observed that the data obtained with AMIRA tend to be more detailed than Volumest (Image J), both approaches proved to be highly versatile, allowing to access 3D morphological changes in nuclei of complex tissues and organs. The protocol presented here is based on standard confocal optical sectioning and 3-D image analysis and can be applied to study any volume and shape of cellular organelles in various complex biological specimens. Our results suggest that an increase in giant cell nuclear volume is not solely linked to increasing ploidy levels, but might result from the accumulation of mitotic defects.

Cytotoxicity Assessment of Heavy Metal Arsenic (Arsenic Trioxide) Using <i>Nigella sativa</i> L. (Black Cumin) as Test System

“…Results show that As 2 O 3 at the different duration of treatments can induce clastogenic effect (chromosome fragmentations, ring configuration of chromosomes, lagging fragments, bridges, and micronuclei) as well as induce spindle aberrations (polyploidy cell formation and tripolarity) and cellular metabolic defects (formation of variable shaped giant cells). Giant cells are the resting cells with larger dimensions compared to the other routine cells encountered in squash preparations (Shrestha et al 2014, Kumbhakar et al 2018 and maybe the outcome of accumulation of cellular defects caused due to disturbed mitotic activity under different physiological (Hayashi et al 2013) as well as pathological conditions (Antonino de Souza Junior et al 2017). In the present investigation, polyploidy and giant cell formations are the most prevalent aberration types noted among treatments and their frequencies are enhanced with an increase in the duration of treatments.…”

The plant WEE1 kinase is involved in checkpoint control activation in nematode‐induced galls

“…For nuclear structure and morphology observation, thick gall slices were cleared and stained with DAPI essentially as described by Antonino de Souza Junior et al (2017). Galls were mounted in 90% glycerol and imaged using a Zeiss LSM 880 confocal microscope.…”

“…ut.ee/~markkom/volumest/) from the public domain IMAGEJ software. Analyses were performed as described by Antonino de Souza Junior et al (2017).…”

“…These nematode-induced feeding sites (NFSs) enclose around seven giant-feeding cells (GCs) surrounded by asymmetrically dividing neighboring cells (NCs). Their development is characterized by a mitotic phase illustrating defective spindles and absence or aborted phragmoplasts (giant cells contain more than one nuclei) in GCs with aborted cytokinesis, followed by an endocycle phase resulting in enlarged and highly invaginated nuclei (de Almeida Engler et al, 2004;Antonino de Souza Junior et al, 2017). Early host cell cycle hyperactivation in NFSs is distinguished by the expression of core cell cycle genes like cyclin-dependent kinases (CDKs) and cyclins (CYCs) (de Almeida Engler et al, 1999).…”

Plant Cyclin-Dependent Kinase Inhibitors of the KRP Family: Potent Inhibitors of Root-Knot Nematode Feeding Sites in Plant Roots

“…The possible consequences of such KRP accumulation could involve defective DNA replication, erroneous chromosome segregation, or defects on chromosome organization within the nuclear envelope. Recent nuclear analysis of the aberrant nuclei seen in mature GC upon ectopic KRP3 and KRP5 expression revealed that the increased nuclear volume may not only be linked to increased ploidy levels, but also to be the result of accumulation of mitotic defects ( Antonino de Souza et al, 2017 ).…”