Plant microtubules reorganization under the indirect UV-B exposure and during UV-B-induced programmed cell death

Abstract

The role of microtubules in cellular pathways of UV-B signaling in plants as well as in related structural cell response become into focus of few last publications. As microtubules in plant cell reorient/reorganize (become randomized, fragmented or depolymerized) in a response to direct UV-B exposure, these cytoskeletal components could be involved into UV-B signaling pathways as highly responsive players. In the current addendum, indirect UV-B-induced microtubules reorganization in cells of shielded Arabidopsis thaliana (GFP-MAP4) primary roots and the correspondence of microtubules depolymerization with the typical hallmarks of the programmed cell death in Nicotiana tabacum BY-2 (GFP-MBD) cells are discussed.

Keywords: :

• UV-B
• signaling pathways
• cytoskeleton
• microtubules
• reorganization
• programmed cell death
• plant cell

Recently, the “paradigm shift” in understanding of cytoskeleton’s role in eukaryotic cell occurred. Apart from its “classical” functions such as cell division and growth, scaffolding, transport, microcompartmentation, etc,¹ the coordinated network of microtubules (MTs), actin filaments, microtubule-/actin-related proteins and others is definitely known to be intensely involved in cell signaling events (for review see refs. 2–4). The organization and the dynamic properties of plant cytoskeleton are regulated by a broad range of intracellular signaling molecules, namely phytohormones,^3,5,6 reactive oxygen (ROS) and nitrogen species (RNS),^4,7 Ca^{2+ 8,9}, by protein kinase/phosphatase activities^10-12 and others. Adaptive rearrangement of cytoskeleton in a response to both physiological environmental stimuli (gravity, touch, fluctuations of temperature, humidity, illumination regimes) and (a)biotic stress conditions (plant pathogens, toxic metals and herbicides pollution, salinization, high doses of UV irradiation) is determined by such intrinsic properties of cytoskeletal proteins as dynamic instability, threadmilling, bundling and abundant posttranslational modifications (for review see refs.13–16).

One of the constitutive abiotic environmental factors, UV non-ionising and mainly non-photosynthetically active radiation (UV, 200–400 nm)¹⁷ challenges adaptive morphogenic responses in plants¹⁸ as well as a set of destructive effects.^19,20 Its minor, but influential portion, UV B (UV-B, 280–315 nm) and, in a lesser extent, UV-A (315–400 nm) alters plant growth and morphology (lowering/increase of cell division rate, axillary branching, leaf thickening, cotyledon curling, number and/or diameter of inflorescence increase, root/shoot ratio shift, etc.).^18,19 Cytoskeleton role in such UV-induced morphological changes as their driving force remains poorly investigated, since only a few articles are focused on the cytoskeleton reorganization in vitro as one of the events underlying UV-B-induced responses of plant cell.^21-25 It was shown recently that the interphase and mitotic MTs in epidermal and cortex cells of all primary root zones of Arabidopsis thaliana L. seedlings expressing gfp-map4 (microtubule-assosiated protein 4) were randomized, depolymerized and/or stabilized in dose-dependent manner after the UV-B exposure (13.6–68 kJ/m²) in vivo that was accompanied by the cell swelling and excessive root hairs formation.²⁶ Our further experiments give additional evidences that plant MTs are involved in signal transduction under UV-B stress. The experimental system based on A. thaliana (GFP-MAP4) 4 d-old seedlings having their primary roots protected with aluminum foil downwards from the hypocotyls (hereinafter referred to as shielded seedlings) to avoid the direct UV-B exposure were designed. Thus, in 2 h after the UV-B irradiation (27.2 and 68 kJ/m²) of A. thaliana seedlings with shielded primary roots, randomization, depolymerization and/or bundling of MTs in epidermal cells of both shoots and roots occurred that was found in vivo by confocal microscopy (Fig. 1, (I)).

Figure 1. I. Cortical MTs organization in epidermal cells of A. thaliana in 2 h after the UV-B exposure: А − leaf, control; A' − leaf, 27.2 kJ/m2; A'' − leaf, 68 kJ/m2; B -hypocotyl, control; B' − hypocotyl, 27.2 kJ/m2; B'' − hypocotyl, 68 kJ/m2; C' − primary root transition zone, control; C' -primary root transition zone, 27.2 kJ/m2; C'' − primary root transition zone, 68 kJ/m2. Bar - 20 μm. II. A. thaliana primary roots growth after the direct exposure of both shielded and non-shielded seedlings to UV-B (27,2 and 68 kJ/m2). III. Primary roots morphology of shielded A. thaliana seedlings in 24 h after exposure to UV-B (27.2 and 68 kJ/m2): А − control; B - 27.2 kJ/m2; C − 68 kJ/m2. Bar - 200 μm.

The most resistant were cortical МTs in stomatal cells of adaxial leaf surface (Fig. One (I), A-A”) organized in radial network of toughly adjacent bundles. In contrast, cortical MTs of adaxial epidermal leaf and hypocotyl cells became randomized (27.2 kJ/m², Figure 1 (I), A’, B’) and partially depolymerized after the UV-B exposure (68 kJ/m², Figure 1 (I), A,” B”) as compared with the radially and obliquely oriented MTs in cells of non-irradiated roots (Fig. One (I), A, B). In cells of non-irradiated abaxial side the organization of cortical MTs remained unaltered similar to control (Fig. One (I), А). Although earlier it was shown that the UV-B-induced inhibition of A. thaliana leaf plates growth after chronic UV-B exposure is not supported by МТs reorganization in adaxial leaf surface epidermal cells,^25,27 we suppose that MTs could be a good candidate for UV-B-signal perception and its further transduction. However, their exact position in UV-B-related signaling cascades remains to be elucidated.

In the same time, in 2 h after the UV-B exposure cortical МTs in epidermal cells of primary root transition zone of shielded A. thaliana seedlings also became evidently randomized (Fig. One (I), C’) and depolymerized in dose-dependent manner (Fig. One (I), C”), while in the same cells of non-irradiated roots MTs oriented transversely (Fig. One (I), C). These observations are of special interest because the transition (distal elongation) zone is considered to be the main signaling-response nexus in the root as the inputs from hormonal and sensorial stimuli are integrated here and translated into signaling and motoric outputs.²⁸ Indeed, the transition zone cells are known to be sensitive to endogenous (auxin, ethylene, extracellular Ca²⁺) and exogenous factors (mechanical pressure, aluminum, pathogens).²⁹ In turn, cortical MTs in epidermal cells of the transition zone are exceptionally responsive to fluctuations of auxins³⁰ and nitric oxide content,⁴ protein kinase/phosphatase inhibitors³¹ and cold treatment as well.³² We have shown also that immediately after the UV-B exposure (13.6–68 kJ/m²) cortical MTs in both transition and elongation zones depolymerized rapidly.²⁶

Furthermore, UV-B-induced MTs reorganization in root cells of shielded A. thalianа seedlings was accompanied by primary root growth inhibition (Fig. 1, (II),,) and epidermal cells swelling together with the intense root hairs formation in differentiation zone (Fig. 1, (III), B, C) as compared with non-shielded irradiated ones (Fig. 1, (II), ,; Figure 1, (II), A, respectively) that points out to the activation of the morphogenetic processes.

As cytoskeleton reorganization was revealed mainly in vitro in protoplasts and other cell cultures,^21-24 we have obtained the complementary results on cells of Nicotiana tabacum BY-2 suspension culture expressing gfp-mbd (microtubule-binding domain of MAP4) as a suitable cell model for in vivo MTs visualization.³³ Since BY-2 cells are more resistant to UV-B exposure as compared with A. thaliana seedlings, the higher doses of UV-B (34, 81 and 135 kJ/m²) were used^33. In 3 h after the irradiation a dose-dependent depolymerization of both interphase (Fig. Two (I,II), А”-C”) and mitotic (not shown) MTs clearly corresponded to cytoplasm shrinkage (Fig. 2, I, А-C), chromatin condensation (Fig. 2, I, А'–C'), cytoplasm vacuolization (Fig. 2, II, А-В) and micronuclei formation (Fig. 2, II, А'-В').

Figure 2. Alterations of general ВУ-2 cells morphology, nuclei shape and interphase MTs organization in 3 h after the UV-B exposure (A - 34 kJ/m2; B - 81 kJ/m2;C -135 kJ/m2): I. A-C - cytoplasm shrinkage; A'-C'- chromatin condensation, DAPI (4',6-diamidino-2-phenylindole) nuclei staining; A”-C” - МТs randomization/depolymerization. II. A-C - cytoplasm vacuolization; A'-C'- micronuclei formation, DAPI staining; A”-C” - МТs randomization/depolymerization. Bar - 50 μm.

Hence, the key hallmarks of the programmed cell death (PCD) were found after UV-B exposure in BY-2 cells. Here we clearly demonstrate the involvement of MTs in UV-B response and an apoptosis development under UV-B stress in different plant cells. However, the involvement of MTs in PCD progression in plant cells is still scarce and need to be further investigated.

Conclusions

In summary, these data represent, to our knowledge, the first steps in understanding of the MTs involvement into signaling events under the UV-B response in plant cells. However, the detailed mechanisms of cytoskeleton reorganization during the UV-B response are complex and varied, and much still remain to be elucidated, especially in terms of the signaling molecules and related transduction pathways.

Abbreviations:
UV-B	=	ultraviolet irradiation with wavelengths in the range of 280−315 nm
MT	=	microtubules
PCD	=	programmed cell death

Disclosure of Potential Conflicts of Interest

There were no potential conflicts of interest to disclose.

Acknowledgments

This work was partially funded by the grant F47/072 of the State Fund of Fundamental Researches (Ukraine) for Dr. A. Yemets.