|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2009) 33, 524533 (Printed in Great Britain)
Establishing in vitro Zinnia elegans cell suspension culture with high tracheary element differentiation
Peter Twumasiabc*, Jan H.N. Schela, Wim van Ieperenb, Ernst Wolteringd, Olaf Van Kootenb and Anne Mie C. Emonsa
aLaboratory of Plant Cell Biology, Department of Plant Sciences, Wageningen University and Research centre, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
bHorticultural Production Chains Group, Department of Plant Sciences, Wageningen University and Research Centre, Marijkeweg 22, 6709 PG Wageningen, The Netherlands
cDepartment of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana
dAgrotechnology and Food Innovation, Wageningen University and Research Centre, Wageningen, The Netherlands
The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and for studying cellulose synthesis. There is, however, the problem of non-uniformity and significant variations in the yields of TEs (%TE). One possible cause for this variability in the %TE could be the lack of a standardized experimental protocol in various research laboratories for establishing the Zinnia culture. Mesophyll cells isolated from the first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were cultured in vitro and differentiated into TEs. The xylogenic culture medium was supplied with 1
Keywords: Cellulose, In vitro culture, Programmed cell death, Apoptosis, Tracheary element, Xylogenesis, Zinnia elegans.
*Corresponding author at: Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology (KNUST), PMB, Kumasi, Ghana. Tel.: +233 245 131806; fax: +233 516 4338.
Xylem vessels and tracheids are important structures in higher plants due to their water conducting abilities and mechanical support (Tyree, 2003). The xylem cells originate from the root and shoot procambium during the early developmental stages of the plant and also from the vascular cambium during the secondary growth periods of the plant. For over a century, extensive work has been done to unravel the complex mechanisms involved in xylem formation and its hydraulic function in the plant (Aloni, 1987; Chaffey, 1999; Dengler, 2001). In just over two decades, our knowledge about xylogenesis both at cellular and molecular levels has increased more than ever before (McCann et al., 2001). For instance, there is a great deal of studies involving xylem formation focusing on understanding the mechanism of cellulose synthesis (Haigler et al., 2001; Mellerowicz et al., 2001; Cano-Delgado et al., 2003). The availability of xylogenic cell culture systems, such as Zinnia, Arabidopsis and Populus, has provided essential tools for an in-depth understanding of the xylogenesis process (Ye, 2002). Programmed cell death (PCD) is essential during formation of certain functional structures, in both animals and plants. It is also involved in defense mechanisms as demonstrated in the hypersensitive response (Iakimova et al., 2008). In animals this type of self-induced cell death is referred to as apoptosis, a term relating to the apoptotic bodies (or membrane-bound structures) resulting from the breakdown of the membrane at the end of the death process (Yang et al., 1999; Ranganath and Nagashree, 2001; Sanmartin et al., 2005). In animals, these apoptotic bodies are later engulfed through phagocytotic activity in the organism. Such apoptotic bodies have, however, not been found in plant cells during programmed cell death. Because of the involvement of the PCD in TE differentiation in the xylem (Fukuda, 1996; Roberts and McCann, 2000), more attention is being focused on the use of this simpler xylogenic system to study regulation of PCD in plants.
The in vitro xylogenic cell culture is suitable for the study of xylem development and differentiation studies due to the easy accessibility for manipulation, microscopic analyses and production of one simple cell type isolated from the complexity of the tissue. On the other hand, the whole plant xylogenic process is preserved. Since many molecular studies require extraction of ample amounts of the molecule under study, a highly efficient and synchronous TE differentiating cell culture would be necessary. The Zinnia elegans xylogenic cell culture, although introduced long ago (Fukuda and Komamine, 1980), continues to show the highest efficiency and synchrony in TE differentiation better than any of the recently introduced xylogenic cultures (Arabidopsis: (Oda et al., 2005); Populus: (Ohlsson et al., 2006)). At the time of Zinnia xylogenic cell culture discovery, the yield of TE was just around 30% (Fukuda and Komamine, 1980). More recently, there have been records of TE differentiation as high as 60% (Church, 1993; Fukuda, 1996).
Despite these achievements, different laboratories report of low and varying TE yields formation in the xylogenic Zinnia cultures (Gabaldon et al., 2005; Tokunaga et al., 2005; Oda and Hasezawa, 2006). The inconsistencies in the protocol for establishing the Zinnia culture, ranging from plant material to phytohormonal induction, may account for these differences. The aim of this work therefore was to produce a standardized and reproducible protocol for establishing higher yields of TEs in Z. elegans in vitro cultures. Also, modified cytological and biochemical methods were to be designed for monitoring progress of TE differentiation.
2.1 Sequence of events during TE differentiation
Mechanically isolated Zinnia mesophyll cells have definite shapes, usually asymmetrically cylindrical and measuring 20–60
Time course for TE differentiation in Zinnia elegans suspension culture. The differentiation and lignification processes require 96
Changes in the viability of Zinnia elegans mesophyll cells in culture during tracheary element differentiation. Concentration of inductive hormones (NAA and BA) were set at 1
Rate of cell division in suspension cultures of Zinnia elegans upon differentiation (white bars) and in control (black bars).
Effect of tracheary element differentiation on the size of Zinnia mesophyll cells. Different letters indicate significant differences. (NICM, non-inductive culture medium, ICM, inductive culture medium; N
TE differentiation becomes visible 48
Comparison of different types of tracheary elements in the Zinnia in vitro culture and those in planta showing different thickening patterns (annular, spiral, reticulate and pitted).
Summary of events that occur during tracheary element differentiation in Zinnia elegans suspension culture. The entire process lasts for 96
2.2 Vital staining with FDA
We observed that high levels of dead mesophyll cells in starting Zinnia culture either inhibited TE differentiation completely or significantly reduced TE differentiation. Moreover, since mature TEs are dead and hollow cells with secondary cellulose thickenings, the TE differentiation itself eventually results in lowering of the initial viability. Cell viability is therefore a measure of the progress of TE differentiation. Differentiating Zinnia cultures with initial cell viability of 60% or higher (Fig. 7) was found to produce workable amounts of TEs.
Zinnia mesophyll cells stained with fluorescein-diacetate (FDA) for the calculation of cell viability. Viable cells are those emitting green fluorescence at 510
2.3 Nuclear condensation, DNA labelling with TUNEL and gel electrophoresis
Micrographs showing morphological changes in differentiating cells of Zinnia elegans in inductive culture medium. DIC image of freshly isolated mesophyll cells (A); DIC image of fully differentiated TE 96
The TUNEL positive nuclei (having DNA ladders or fragments) were observed in the differentiating cultures 36
a: DNA fragmentation in nuclei as detected by the TUNEL assay in Zinnia elegans mesophyll cells during TE differentiation – control at 72
Gel electrophoresis showing DNA fragmentation in Zinnia cell undergoing TE differentiation. DNA fragmentation is pronounced at 78
2.4 Lignin synthesis
Lignin is bluish-white under UV. The lignin was observed 96
2.5 Secondary cellulose deposition and pattern formation
Staining of the cell wall with Calcofluor white revealed a steady increase in cellulose deposition from 36
2.6 Vacuolar collapse
FDA staining showed clear distinction between the cytoplasm and the vacuole in TE differentiating cells. During autolysis when the tonoplast collapses, the cytosol and vacuoles become stained with FDA. At this stage, the differentiating and non-differentiating cells are easily recognized (Fig. 8G, H and Fig. 11).
Changes in the level of cell populations with collapsed vacuoles upon induction of TE differentiation. Statistically significant differences are indicated by different letters. (Bars
Xylogenic cultures of Z. elegans established from leaf mesophyll cells have high tracheary element (TE) differentiation (76%), and at the moment leads all the available xylogenic cultures including that from Arabidopsis thaliana (Oda et al., 2005). The associated high frequency and synchrony in TE differentiation of the Zinnia system, measuring over 76%, makes it useful especially for molecular studies involving isolation of ample amounts of molecular markers. Additionally, the Zinnia culture is suitable for studying mechanism involved in autonomous programmed cell death in plants, especially in the development of the vascular system. Another interesting feature of the Zinnia system is its high reproducibility in TE differentiation and the relatively short time necessary for the differentiation process. Nevertheless, the critical steps involved in setting up the culture must be carefully adhered to to maximize the potentials of the system. Also, the monitoring techniques applied to the TE differentiation process are fast and reliable, and therefore allowing the processes involved to be scrutinized.
Preconditioning of the isolated cells 24–48
Wounding of cells initiate signal molecules that help to differentiate native cells into TEs. This might explain why old or sub-cultured cells that have healed do not respond to the differentiation induction. Thus, it is difficult, if not impossible, to maintain a mother culture that can be sub-cultured over a longer period of time and still maintain same level of TE differentiation. This means that every experiment will require preparation of freshly isolated mesophyll cells to initiate the xylogenic culture. TE differentiation in fresh Zinnia cultures is associated with production of wound or stress signaling molecules. Others have shown in vivo that wounds inflicted on vascular bundle cells cause local accumulation of signal molecules such as auxin to initiate vascular regeneration through transdifferentiation of localized parenchyma cells. These cells form new tracheary or vessel elements and sieve elements which connect ends of the severed bundle (Aloni, 1992; Nishitani et al., 2002).
Development of techniques that would allow in vitro induction of wound signals or stress inducing compounds such as reactive oxygen species (ROS) and exogenous cytokinin in the old or sub-cultured cells, might help in keeping the Zinnia cultures over a longer period and still maintaining adequate levels of TE differentiation in the subculture or older culture.
Apoptosis, a term commonly used for programmed cell death (PCD) in animals (Yan et al., 2006), is attracting much attention from many laboratories as a result of the recent surge in researchers aiming at retarding aging (Li et al., 2006). The TE differentiation process also recruits PCD in building a functional cell remains required for water uptake in plants (Groover and Jones, 1999). This is shown by TUNEL staining and gel electrophoresis. The Zinnia system is therefore a useful plant model for studying programmed cell death.
It has been shown in this work that, with the right conditions and techniques as indicated in this paper, the xylogenic Zinnia culture can be improved by conditioning the culture. With this approach, percentage TE as high as 76% percent was achieved. It is therefore recommend that Z. elegans var Envy be used as model plant for cellulose, xylogenesis, programmed cell death and other molecular research. This would also require whole genome sequencing.
4 Experimental procedures
4.1 Plant material
60 seeds of the Z. elegans var Envy – a cultivar commonly used for cell culture work – (Muller Bloemzaden BV, Lisse, Netherlands), were germinated in 30
4.2 Cell isolation and culture
Isolation of mesophyll cells from leaves of the Z. elegans is a critical step as it influences the TE differentiation in the induced culture. An initial culture of mesophyll cells with cell viability of 60% or more was maintained throughout the experiment. We established that cultures that have initial cell viability below 40% either did not differentiate at all or differentiated with very low TEs (data not shown). Also, healthy mesophyll cells ensured high level of TE formation. The following protocol was therefore developed for isolation of cells with high percentage differentiation.
A schematic representation of the various steps involved in the culture is also shown in Fig. 2. Except for the first two steps (1 and 2), all the steps were carried out in a sterile airflow cabinet with benches sterilized with 70% ethanol. Sterile hand gloves were worn throughout the cell isolation and culture transfers. 1. 30 sets of first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were harvested by cutting the stalks connecting the leaves to stems. The right stage for the seedlings was when the first true leaves have just fully expanded and the primordium of the second set of leaves has just been initiated (Fig. 1). This stage is critical because older leaves tend to produce lower rates of TEs. 2. The leaves were surface sterilized in a 500 3. The leaves were rinsed three times in sterile Milli-Q water. 4. Gentle mechanical maceration of the leaves in 30 5. The mixture was then filtered through a sterile 50 6. The filtrate was transferred to a 10 7. The supernatant was carefully removed and discarded. The pellet made up of the mesophyll cells was washed 3× with NICM. 8. The cleaned mesophyll cells were resuspended in 10
30 sets of first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were harvested by cutting the stalks connecting the leaves to stems. The right stage for the seedlings was when the first true leaves have just fully expanded and the primordium of the second set of leaves has just been initiated (Fig. 1). This stage is critical because older leaves tend to produce lower rates of TEs.
The leaves were surface sterilized in a 500
The leaves were rinsed three times in sterile Milli-Q water.
Gentle mechanical maceration of the leaves in 30
The mixture was then filtered through a sterile 50
The filtrate was transferred to a 10
The supernatant was carefully removed and discarded. The pellet made up of the mesophyll cells was washed 3× with NICM.
The cleaned mesophyll cells were resuspended in 10
The mesophyll cell suspension were cultured in 3
4.3 Zinnia culture medium
The basic nutrient composition of the Zinnia culture medium (CM) is shown in Table 1. The composition is closely related to the one first formulated by Fukuda and Komamine (1980), but vary at concentrations especially with the phytohormones. In this work equimolar concentrations (1
Composition of culture medium used in establishing xylogenic Zinnia elegans mesophyll cell cultures.
4.4 Cytological and biochemical measurements
Similar to developmental processes of many other cells, the in vitro TE differentiation can be subjected to cytological, biochemical and microscopical techniques. In this work, the following parameters were measured on the established cultures: cell viability, cellulose deposition, lignification of the cellulose fibres, density and integrity of nuclei, vacuolar collapse, DNA laddering (using TUNEL method), %TE and TE anatomy.
4.4.1 Vital staining with fluorescent diacetate (FDA)
The viability of the mesophyll cells in the culture is an important parameter both at the beginning and during differentiation of TEs. FDA vital staining is based on the detection of the esterase enzyme activity which is exclusively associated with living cells. These esterases lose their activity once the cell dies. Hydrolysis of FDA by the esterases in living cells produces an acetate moiety and a yellow florescein. Two drops each of cell suspension and aqueous FDA solution (0.01%
4.4.2 Nuclear condensation, DNA labelling by TUNEL and gel electrophoresis
The morphology of the nuclei in control cells and in the differentiating TEs was studied at 12h time intervals. Cells were first fixed in 4% formaldehyde (FA) solution containing 0.025% glutaraldehyde (GA), followed by staining with 1
For in situ detection of nDNA fragmentation in cells undergoing xylogenesis, samples were collected at various time points and fixed in MSB buffer (100
Isolation of nDNA from Zinnia suspension culture at various time points for DNA laddering detection with gel electrophoresis was performed according to De Jong et al. (2000) with slight modifications. About 1
4.4.3 Staining of the cell wall
Cellulose deposition on the cells was measured by staining the cells or TEs with an aqueous Calcofluor white (CW) solution (0.0001%
4.4.4 Lignin measurement
Lignified TEs were detected by staining the lignin component with phloroglucinol-HCl (Siegel, 1953). Phloroglucinol is dissolved in 20% HCl, about 1% (w/v). Then a 1:1 mixture of the cell suspension and this solution are incubated on a glass slide for 10
4.4.5 Vacuolar staining
The intact vacuoles can be distinguished by staining with dyes that can enter the cytoplasm but are excluded from the vacuole. Here we stained the cells or TEs with FDA (0.01%
4.4.6 TE measurement
To be able to compare percentage TE differentiation of two or more cultures with different initial cell viability values, a correction factor must be included in the equation to calculate the actual %TE (%TE
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Received 3 January 2009; accepted 31 January 2009doi:10.1016/j.cellbi.2009.01.019