The heterochromatin as a marker for protoplast differentiation of Cucumis sativus
The protoplast cultures of Cucumis sativus in two culture systems were used to study heterochromatin reassembly during dedifferentiation of isolated protoplasts and their subsequent differentiation into calli and proembryos. Here we show that dedifferentiation of the cucumber mesophyll cells is accompanied by a dramatic reduction in size and numbers of nuclear chromocenters. Although chromocenters were newly established during protoplast culture, the measured relative heterochromatin content differed according to the culture system used. Protoplast culture leading to proembryo formation displayed a lower level of relative heterochromatin content than cultures resulting in calli and the relative heterochromatin content reached values close to those estimated for somatic embryos.
KeywordsCucumber Protoplasts Callus Proembryos Nuclear chromocenters Heterochromatin Decondensation
The relative heterochromatin content
The eukaryotic nucleus is a dynamic, highly organized organelle where chromatin is the substrate for most nuclear processes. Chromatin is directly influenced by modifications of DNA (methylation) and modifications of core histones (methylation, acetylation, etc.) (Jenuwein and Allis 2001; Verbsky and Richards 2001) and undergoes 3D organization in the nuclear space (Kozubek et al. 2002; Pečinka et al. 2004; Ondřej et al. 2008). This epigenetic system affects a variety of biological processes, such as cell differentiation, growth, development, abiotic and biotic stresses (Chen and Tian 2007). Studies on interphase nuclei by conventional microscopy reveal chromatin domains of different density representing highly dense heterochromatin and less dense euchromatin. Since a given gene expression program is governed by the portion of the genome that is transcribed (euchromatin) versus that which is repressed (heterochromatin), it is likely that, for a cell to change its differentiation fate, a new balance between euchromatin and heterochromatin must be established. In plants, the organizing element of the chromosome, the chromocenter, consists predominantly of centromeric and pericentromeric heterochromatin enriched with CpG methylated sites (reviewed in van Driel and Fransz 2004; Tessadori et al. 2004). These distinct heterochromatic domains are present in several organisms, such as mouse, Arabidopsis and many other plant species including cucumbers (Cucumis sativus).
In contrast to animal cells, which lose their pluripotency after cell determination, isolated plant protoplasts from different tissues are able to dedifferentiate and undergo regeneration processes resulting in new plantlets (Cocking 1960; Takebe and Otsuki 1969; Debeaujon and Branchard 1992). In Arabidopsis, the chromocenters of freshly isolated protoplasts totally decondensed followed by their reassembly during further protoplast culture and regeneration (Tessadori et al. 2007). Thus, the aim of this study was to show and test the chromocenter reassembly as a simple marker reflecting protoplast recalcitrance or regeneration capacity in the context of culture conditions. Although the protoplast culture has been used to develop plants with new agronomic and horticultural traits (Shahin and Spivey 1986; Veilleux and Johnson 1998; Rakoczy-Trojanowska 2002), the recalcitrance of protoplasts forbids its utilization in biotechnological applications for many plant species. An example of this phenomenon is cucumber protoplast culture (Fellner and Lebeda 1998; Gajdová et al. 2004, 2007). Nevertheless, some studies demonstrated overcoming of protoplast recalcitrance, not only for cucumber (C. sativus) but also for melon (Cucumis melo) (Trulson and Shahin 1986; Debeaujon and Branchard 1992). In the recent study, we used two culture systems (Gajdová et al. 2007; Trulson and Shahin 1986) to compare heterochromatin balance between them where first system shows low ability for the regeneration processes (Gajdová et al. 2007) while proembryos are formed in the second one (Trulson and Shahin 1986). The heterochromatin content of cultured protoplasts was also compared with heterochromatin content in the nuclei of young leaves and somatic embryos. Understanding of these processes and overcoming of cucumber protoplast recalcitrance should bring a valuable tool in cucumber breeding and improvement (Lebeda et al. 2007).
Material and methods
Protoplast isolation and culture
Seeds of Cucumis sativus L. (cv. Marketer, SEMO Smržice, Czech Republic) were sown under sterile conditions on half strength MS medium (Duchefa). After germination, the seedlings were planted on MS medium (supplemented with 20 g/l sucrose, 0.8% agar, 0.049 μmol/l IBA and 0.044 μmol/l BA) in plastic boxes. Plants were cultivated in culture room with a 16 h day (light intensity 32–36 μmol/m2s) and temperature 22 ± 2°C.
The leaves of the plantlets were chopped and digested in maceration solution containing 1% (w/v) Cellulase Onozuka R-10 (Duchefa) and 0.25% (w/v) Macerozyme R-10 (Duchefa) dissolved in PGly washing solution (Debeaujon and Branchard 1992). The pieces of leaves were incubated in enzyme solution for 16–17 h in the dark at 25°C. The protoplast suspension was filtered through nylon mesh (72 μm), mixed with PGly solution and centrifugated at 80g for 5 min three times and resuspended in culture media.
Two culture systems were used for protoplast culture. The first one was based on LCM media and as described Gajdová et al. (2007). Protoplasts were cultured in the dark at 25°C in LCM1 medium. Two weeks after protoplast isolation, the LCM2 medium was added to decrease osmolarity and sugars concentration. At this time the protoplast cultures were transferred into the culture room with 16/8 h day/night (light intensity 32–36 μmol/m2s). The growing microcalli were transferred onto medium used for induction of somatic embryogenesis (Vengadesan et al. 2005). The second culture system was based on series of CPM media (Trulson and Shahin 1986) where protoplast culture in CPM1a medium is adjusted with CPM1b medium to decrease sugar concentration 1 day after protoplast isolation. The protoplasts were cultured in the growth chamber with 16/8 h day/night period, however not on direct light. One week after protoplast isolation, the culture was transferred into CPM2 medium to induce somatic embryogenesis for 5 days. At this time, light intensity was increased up to 32–36 μmol/m2s. The microcalli were 12 days after protoplasts isolation transferred onto semi-solid CPM3 medium for somatic embryo growth.
Somatic embryos were also induced from young leaves sliced into 5 mm × 5 mm pieces and placed onto culture medium for induction of embryogenic callus (Vengadesan et al. 2005). The shiny pale green embryogenic calluses were transferred into liquid medium for somatic embryo development (Vengadesan et al. 2005). The torpedo-stage somatic embryos were fixed and used for heterochromatin analysis.
Fixation, DNA staining, image acquisition and quantification of RHC
Freshly isolated protoplasts were fixed in ethanol:acetic acid (3:1) (EAA) after the last centrifugation and directly stained by DAPI on slides after washing in 96% ethanol and drying. Microcalli (2 weeks after protoplast isolation), young leaves and somatic embryos in torpedo stage were also fixed in EAA, macerated in 1 N HCl for 1 min at 65°C, gently squashed in 45% acetic acid, washed in 96% ethanol, dried and stained with DAPI.
Image acquisition was carried out with a fluorescent microscope (Olympus BX 60) fitted with a CCD camera (Cool Snap, Photometrics). The images were analyzed by ImageJ freeware (http://rsb.info.nih.gov/ij/index.html). The relative heterochromatin content was calculated as a percentage of the area of chromocenters (intensively stained with DAPI) in relation to the total area of DAPI stained nucleus. 50 nuclei at each stage of differentiation were analyzed.
Protoplast isolation and differentiation system
Pericentric heterochromatin reassembly during protoplast differentiation
In the nuclei of protoplasts fixed immediately after isolation, chromocenters were largely decondensed resulting in their total disappearance from most nuclei (Fig. 2a) and RHC significantly decreased in contrast to leaves nuclei (two-tailed t-test, P < 0.0001). Nearly 90% of protoplast nuclei displayed a RHC between 0 and 5% (Fig. 2b).
We observed protoplasts during culture in two different culture systems. The first one was based on LCM1 medium containing 70 g/l of mannitol to increase osmolarity to 570 mOsm/l protecting protoplasts from disruption. The LCM2 medium containg 0.75 mg/ml BAP and no mannitol was added to the culture to decrease an osmolarity to 450 mOsm/l 2 weeks after protoplast isolation. The second culture system (CPM media) was also based on changing the osmolarity, but at 1 day after isolation and using just 4.56 g/l of mannitol in the initial, more osmotic, culture medium. In both types of culture, the first division of the protoplasts occurred 5 days after isolation. During the second week of culture microcallus formation followed. At this point, the RHC of microcallus nuclei was determined.
The microcalli from LCM medium showed various levels of the RHC ranging from 10 to 45%. The most frequent RHC values ranged between 15 and 20% 2 weeks after isolation (Fig. 2b). The nuclei also differed in size and number of chromocenters. The nuclei of microcalli from the CPM media culture system showed a higher number and smaller size of the chromocenters than was observed for LCM cultures (Fig. 2a). Also the RHC was significantly lower (two-tailed t-test, P < 0.05) than was estimated for the nuclei from LCM system. Nearly 50% of the nuclei had a RHC between 10 and 15% (Fig. 2b).
Although the CPM culture system displayed a lower level of heterochromatin condensation than the LCM system, mature somatic embryos were not achieved in either system. To get view of heterochromatin formation in the nuclei of somatic embryos and compare it with that in microcallus nuclei, we produced somatic embryos of cucumber from embryogenic calli originated from young leaves (Fig. 1g–h). Around 70% of the nuclei displayed a RHC between 5 and 10%, which lies between the most frequent intervals of RHC for protoplast and CPM microcalli (Fig. 2b).
Recent progress in the area of plant cytogenetics and genomics increases knowledge about the structural organization of the plant genome in the interphase nucleus and has extended the description of dynamic processes and the relationship between structure and function in the nucleus. Understanding of this relationship, the plant genome and nuclear architecture could bring new insights in biotechnology and crop breeding programs focusing on controlled regeneration and the formation of new synthetic poly- and alloploids. Plant protoplasts are commonly used for genetic manipulation, but they also represent an attractive experimental system to investigate dedifferentiation and differentiation processes (Exner and Hennig 2008). Here we show that dedifferentiation of cucumber mesophyll cells after protoplast isolation, accompanied by a dramatic decondensation of heterochromatin, resulted in the total disappearance of chromocenters in most nuclei of C. sativus (Fig. 2a). A similar decrease in the size and number of chromocenters during the isolation of Arabidopsis mesophyl protoplasts was shown by Tessadori et al. (2007). These authors also showed that decondensation of major tandem repeats occurred in chromocenters.
In this study, we used two culture systems for protoplast regeneration differing in media composition and in the timing of a decrease in osmolarity (Trulson and Shahin 1986; Gajdová et al. 2007). During cultivation of the protoplasts, chromocenters reform in both culture systems. However, they differ in the level of compaction. The condensation level of heterochromatin was calculated on RHC for microcalli and somatic embryos. We demonstrated that using a low mannitol content and decreasing the osmolarity immediately after cell wall formation led to a chromatin condensation level much closer to that measured for somatic embryos and prevented chromatin over-condensation, which was observed in the culture system with a high mannitol content and a late osmolarity decrease. Differentiated cells have characteristic gene expression patterns and corresponding chromatin states. This has been described during leaf development in Arabidopsis thaliana. During leaf development, mesophyll cells differentiate and chromocenters increase in size with the age of the leaf. Concomitantly, transcription from repeats, that is, minor 5S rDNA, gradually ceases (Mathieu et al. 2003; Tessadori et al. 2004). The heterochromatin fraction temporarily decreases during the transition from vegetative to reproductive growth in Arabidopsis (Sablowski 2007). Large-scale chromatin changes were also found in the nuclei of gametes after the karyogamy (Baroux et al. 2007).
We showed that a low level of heterochromatin content inside the microcallus nuclei indicated the formation of proembryos resembling zygotic proembryos observed in ovules (Ondřej et al. 2000), in contrast to microcalli with high level of chromatin condensation. A lot of the microcallus nuclei in the CPM system remain in a less condensed state, similar to the nuclei of somatic embryos. It becomes evident that this partial decondensation is necessary for further microcalli development to embryos and could support some not clear molecular mechanism associated with differentiation process.
Authors thanks to Dr. Heather MacDonald (UWE Bristol, School of Biosciences, UK) for reading and critical comments to the first draft of the manuscript. This work was supported by the Ministry of Education of the Czech Republic (MSM 6198959215).
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