Abstract
The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton.
Keywords: actin cytoskeleton, Arabidopsis, development, microfilament analyzer, root epidermis
During the plant’s life cells undergo different developmental stages. They are formed through division in the meristem, massively increase their volume during elongation and finally differentiate and acquire their mature functions. During these stages, the cells’ cytoskeleton undergoes drastic rearrangements and it plays a central role in the control of cell growth and development. The root provides a useful system to visualize changes in cytoskeletal arrangement related to cell growth because it continuously maintains a gradient of cells in the different developmental stages.1 Especially the microtubular rearrangements during root development were studied extensively. They form mitotic figures during division, they align parallel and orthogonal to the cell’s future long axis and become oblique or longitudinal when cells differentiate.2-4 These (re)arrangements are rather easily distinguishable, but they become less evident when changes are small or more subtle. Furthermore, visual interpretations and manual measurements of structures on digital images are prone to errors by the researcher and usually they require a great amount of time. We therefore recently introduced MicroFilament Analyzer, a tool that enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way and utilized it to study microtubular arrangements during root gravitropic bending.5
The MFA software was exploited to analyze the organization of the actin cytoskeleton in root epidermal cells of Arabidopsis thaliana at different stages of development. For the visualization of the actin cytoskeleton transgenic GFP-FABD2-GFP plants6 were used. A (Nikon C1) confocal equatorial section image and a projection of a z-stack (16 optical sections, step-size 0.5 µm, 20× objective) were collected from the division zone on toward the differentiation zone. This was repeated for 30 different roots, resulting in more than 400 cells that were subsequently analyzed by the MFA software. Figure 1 represents a combined confocal image of actin filaments in the root (upper panel) together with the identified cells (middle panel) and the filament detection using MFA (lower panel). The output of the MFA analysis identified different orientations of detected filaments, grouped into transverse, oblique and longitudinal according to the angle limits that are set and described in Figure 2. A random orientation is defined as containing more than three identified orientations, while a combined organization summarizes combinations of two orientations.
Figure 1. Actin visualization and detection using MFA. Upper image: a combined image of successive projections of a z-stack of actin filaments in root epidermal cells. Middle image: selection of 77 cells for actin detection during MFA analysis. Lower image: detected actin filaments in the 77 selected cells, indicated by yellow lines.
Figure 2. Schematic representation of the limits in longitudinal, transversal and oblique orientations related to the positioning of the virtual polarizer (MFA detection module) on the image.
According to their area the root epidermal cells are clustered into six different groups. From the relative abundance of the different actin patterns within each group (Fig. 3), it is obvious that the actin organization changes according to the area and, consequently, to the age of the cells. In the greater share of the youngest and smallest cells (0–400 µm2), the actin filaments are organized in a random way. With increasing cell area, this organizational pattern decreases in relative abundance while a longitudinal orientation gains more importance, reaching a value of 50.75% in cells with an area of more than 1,500 µm.2 Over all cell sizes, however, small fractions of the cell population display different orientations (transversal, oblique) or combinations of orientations that are quite constant in frequency.
Figure 3. Root epidermal cells (n = 430) are analyzed and clustered into six groups of increasing cell area. (n = 71.66 ± 18.52 cells per range). According to the long axis of the root, five different organizational patterns of actin could be distinguished. The relative abundance of all five patterns is displayed for all ranges of cell area.
Therefore, it can be concluded that F-actin is organized in a mainly random pattern during the early stages of cell development. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. Over all ranges of cell area, an approximately even distribution is present for the three other organizational patterns. These results confirm earlier descriptions of the cortical actin pattern in root epidermal cells, based on visual analysis.7
Actin filaments are known to regulate cell elongation by controlling vesicle transport to areas where cell walls grow.8 A longitudinal array, seen here in elongating cells, would indeed deliver the vesicles in the most efficient way all over the cell’s surface, as the wall grows uniformly. Furthermore, the use of MFA could quantify changes in the actin cytoskeleton upon treatments with for example aluminum,9 sorbitol or other osmotic agents10 or in mutant backgrounds.11
Acknowledgments
This work was supported by grants from the Research Foundation Flanders (FWO), the University of Antwerp and the Inter-university Attraction Poles Programme/Belgian State/Belgian Science Policy (IUAP VI/33).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Footnotes
Previously published online: www.landesbioscience.com/journals/psb/article/24821
References
- 1.Verbelen JP, De Cnodder T, Le J, Vissenberg K, Baluška F. The Root Apex of Arabidopsis thaliana consists of four distinct zones of growth activities: meristematic zone, transition zone, fast elongation zone and growth terminating zone. Plant Signal Behav. 2006;1:296–304. doi: 10.4161/psb.1.6.3511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Sainsbury F, Collings DA, Mackun K, Gardiner J, Harper JD, Marc J. Developmental reorientation of transverse cortical microtubules to longitudinal directions: a role for actomyosin-based streaming and partial microtubule-membrane detachment. Plant J. 2008;56:116–31. doi: 10.1111/j.1365-313X.2008.03574.x. [DOI] [PubMed] [Google Scholar]
- 3.Sugimoto K, Williamson RE, Wasteneys GO. New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis. Plant Physiol. 2000;124:1493–506. doi: 10.1104/pp.124.4.1493. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Le J, Vandenbussche F, Van Der Straeten D, Verbelen J-P. Position and cell type-dependent microtubule reorientation characterizes the early response of the Arabidopsis root epidermis to ethylene. Physiol Plant. 2004;121:513–9. doi: 10.1111/j.1399-3054.2004.00342.x. [DOI] [Google Scholar]
- 5.Jacques E, Buytaert J, Wells DM, Lewandowski M, Bennett MJ, Verbelen JP, et al . Microfilament Analyzer identifies changes in microtubule orientation during root gravitropic bending in Arabidopsis thaliana. Plant J. 2013 [Google Scholar]
- 6.Wang YS, Yoo CM, Blancaflor EB. Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2. New Phytol. 2008;177:525–36. doi: 10.1111/j.1469-8137.2007.02261.x. [DOI] [PubMed] [Google Scholar]
- 7.Baluška F, Barlow PW, Volkmann D. Actin and myosin VIII in developing root apex cells. In: Staiger CJ, Baluška F, Volkmann D, Barlow PW. Actin: a dymanic framework for multiple plant cell functions. Dordrecht: Kluwer Acdemic: 457-476. [Google Scholar]
- 8.Blancaflor EB. Cortical actin filaments potentially interact with cortical microtubules in regulating polarity of cell expansion in primary roots of maize (Zea mays L.) J Plant Growth Regul. 2000;19:406–14. doi: 10.1007/s003440000044. [DOI] [PubMed] [Google Scholar]
- 9.Amenós M, Corrales I, Poschenrieder C, Illés P, Baluška F, Barceló J. Different effects of aluminum on the actin cytoskeleton and brefeldin A-sensitive vesicle recycling in root apex cells of two maize varieties differing in root elongation rate and aluminum tolerance. Plant Cell Physiol. 2009;50:528–40. doi: 10.1093/pcp/pcp013. [DOI] [PubMed] [Google Scholar]
- 10.Wojtaszek P, Baluška F, Kasprowicz A, Luczak M, Volkmann D. Domain-specific mechanosensory transmission of osmotic and enzymatic cell wall disturbances to the actin cytoskeleton. Protoplasma. 2007;230:217–30. doi: 10.1007/s00709-006-0235-6. [DOI] [PubMed] [Google Scholar]
- 11.Zhu L, Zhang Y, Kang E, Xu Q, Wang M, Rui Y, et al. MAP18 regulates the direction of pollen tube growth in Arabidopsis by modulating F-actin organization. Plant Cell. 2013;25:851–67. doi: 10.1105/tpc.113.110528. [DOI] [PMC free article] [PubMed] [Google Scholar]