“Rhizogenesis in vitro” from leaf explants as a model for studying root cell differentiation under real and simulated microgravity
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1
Institute of Botany (NAN Ukraine), Ukraine
A new model of “Rhizogenesis in vitro” from A. thaliana leaf explant petioles was investigated under the simulated microgravity conditions (linorotation). Some ultrastructural changes were revealed in the cells of the roots formed de novo under clinorotation. Auxin distribution as the physiological parameter showed that in vitro roots did not perceive the gravity stimulus under clinorotation, but they reacted to gravity after clinorotation. Based on the experimental data, we consider that in vitro roots are gravisensitive and propose the model of “Rhizogenesis in vitro” for using in spaceflight experiments.
Introduction
Numerous spaceflight biological experiments have shown the essential changes in cell behavior of multicellular and unicellular organisms in comparison with that on Earth (Halstead and Dutcher, 1987; Kordyum, 2014; Kittang et al., 2015). In our investigations, we used the model “Rhizogenesis in vitro” to study cell differentiation in the root cap and growth zones under clinorotation. An advantage of this model is the possibility to study the influence of clinorotation at the beginning of root initiation de novo and next morphogenetic processes unlike experiments in vivo with embryonal roots formed in seeds in the stationary conditions.
Material and methods
Arabidopsis thaliana wild type (Columbia), GFP-MAP4 and DR5rev::GFP transgenic plants were selected. Rhizogenesis in vitro from leaf explants of the plants growing in the sterile condition in vivo was reached by using the modified Murashige-Skoog medium contained 1/10 of MS mineral salts, without vitamins and hormones. The structure of roots formed in vitro was investigated by using the methods of light microscopy (Axioscope, Carl Zeiss, Germany), electron transmission microscopy (JEM 1230, Jeol, Japan) and confocal microscopy (LSM 5 Pascal, Carl Zeiss, Germany).
Results and discussion
Anatomical investigations of A. thaliana wild type roots formed de novo in the stationary conditions showed that they consisted of a root cap with differentiated statocytes and growth zones (meristem, distal elongation zone (DEZ), central elongation zone (CEZ), and mature zone). Under clinorotation, roots formed in vitro preserved their anatomical structure. A cell number in the meristem and DEZ, as well as a length and a cell size of these growth zones did not also differ from control samples.
Electron-microscopic investigation showed that graviperceptive cells - statocytes were fully differentiated and preserved their polarity in the stationary conditions. Under clinorotation, amyloplasts revealed mainly a tendency to group in the cell center, rarely – in the cytoplasm whole volume. Under clinorotation, the ultrastructural organization of cells in protodermis and epidermis of DEZ was in common similar to control. At the same time, a size of mitochondria changed in protodermis cells. In the epidermis of DEZ, changes were observed in a size of mitochondria, vacuoles and ER-bodies.
For vital visualization of the tubulin cytoskeleton orientation in roots formed in vitro, we firstly obtained and used roots from leaf explants of A.thaliana transgenic plants GFP-MAP4. Cortical microtubules were found in root cells in control and under clinorotation. In the meristem and DEZ cells in control, cortical microtubules oriented more or less perpendicularly and shifted to oblique orientation in the CEZ. Under clinorotation, cortical microtubules preserved their orientation in the meristem and CEZ. In cells of the DEZ, the disorientation of microtubules was observed that may indicate gravisensitivity of the tubulin cytoskeleton in this zone. This process is probably connected with specific physiological properties of the DEZ (Ishikawa and Evans, 1995; Kalinina, 2006).
Auxin (IAA) plays an important regulatory role in the plant life cycle and takes part in a root gravitropic reaction. We firstly regenerated roots in vitro from leaf explants of A. thaliana DR5rev::GFP transgenic plants, in which DR5rev green fluorescent protein marks auxin localization, and checked auxin distribution in them. In roots growing vertically (Figure 1A), auxin-dependent reporter DR5rev green fluorescent protein was revealed in the root central cylinder and in the cap columella. Under clinorotation (Figure 1B), DR5rev::GFP signal was only noted in cap cells of roots which had not a visible bending. Under gravistimulation of clinorotated roots (Figure 1C), the DR5rev::GFP localization was revealed in the columella and epidermis. Thus, our data demonstrate that roots formed de novo in vitro under clinorotation preserve their sensitivity to gravity.
On the whole, the results of performed experiments clearly showed the similarity of the anatomical structure and differentiation of cells, specialized and not specialized to gravity perception, in roots of A. thaliana wild type formed in vitro to those of embryonal roots. Structural and functional reactions of roots formed de nono demonstrated their gravisensitivity. Thus, a model of rhizogenesis from leaf explants in vitro is proposed for using in plant space biology, especially in spaceflight experiments.
Figure 1. Auxin distribution in roots formed de novo in vitro: A – control, B – clinorotation, C – gravistimulation after clinorotation.
References
Halstead, T.W., and Dutcher, F.R. (1987). Plants in space. Annu. Rev. Plant. Physiol. 38, 317‒345.
Ishikawa, H. and Evans, M.L. (1995). Specialized zones of development in roots. Plant Physiol. 109, 725–727.
Kalinina, Ya. M. (2006) Microtubules in epidermal and cortical root cells of Brassica rapa under clinorotation. Cyt. Gen. 40, 21–27.
Kittang J. A-I., Hoson, T., and Iversen, T.-H. (2015). The utilization of plant facilities on the international space station − the composition, growth, and development of plant cell walls under microgravity conditions. Plants 4, 44–62.
Kordyum, E.L. (2014). Plant cell gravisensitivity and adaptation to microgravity. Plant. Biol. (Stuttg). Suppl. 1, 79–90. doi: 10.1111/plb.12047.
Keywords:
A.thaliana,
Leaf explants,
Rhizogenesis in vitro,
Structure,
Clinorotation
Conference:
39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.
Presentation Type:
Extended abstract
Topic:
Plants
Citation:
Bulavin
I
(2019). “Rhizogenesis in vitro” from leaf explants as a model for studying root cell differentiation under real and simulated microgravity.
Front. Physiol.
Conference Abstract:
39th ISGP Meeting & ESA Life Sciences Meeting.
doi: 10.3389/conf.fphys.2018.26.00033
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Received:
02 Dec 2018;
Published Online:
16 Jan 2019.
*
Correspondence:
PhD. Iliya Bulavin, Institute of Botany (NAN Ukraine), Kiev, Ukraine, iliyavbulavin@ukr.net