Induction of embryogenic competence in somatic ...

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Induction of embryogenic competence in somatic plant cells: a review

Attila FEHÉR*, Taras PASTERNAK, Krisztina ÖTVÖS, Pál MISKOLCZI and Dénes DUDITS Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt. 62., H-6726 Szeged, Hungary

* Author for correspondance

Keywords:

somatic

embryogenesis,

cell

cycle

reactivation,

auxin,

2,4-

dichlorophenoxyacetic acid, stress, cellular pH

Abstract Somatic embryogenesis may serve in many aspects as a model for zygotic embryo development with the advantage of unlimited source of biological material for cellular, molecular and biochemical studies. Due to this reason, several tissue culture systems have been developed to study the molecular and cellular biology of somatic embryogenesis. One of the basic questions considering the mechanism of somatic embryo induction is related to the importance of the first cell division in the determination of cell fate and embryo polarity. Alfalfa leaf protoplast-derived cells represent a suitable experimental system to study this question: at low 2,4-D

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concentrations cells become elongated, vacuolised and divide more or less equally forming unorganised callus tissue, while at ten-times higher concentrations small, densely-cytoplasmed cells preferentially divide asymmetrically developing into embryo-like structures. In addition to investigations of cell division characteristics, the system proved to be suitable to describe other cell physiological, molecular parameters associated with the transition of somatic cells to a dedifferentiated and embryogenic state. It was realised that the development of the embryogenic/non-embryogenic cell types can be influenced by different treatments (e.g. stress or pH manipulations, 2,4-D concentration and genotype combinations). As a result of the experiments with alfalfa leaf protoplast-derived cells, we could hypothetise that the following factors have key roles in the induction of embryogenic competence: the genotype (R15 non-embryogenic and A2 embryogenic genotypes); high 2,4-D concentration or low 2,4-D concentration plus stress (e.g. heavy metall stress); the pH across the plasma membrane; vacuolar pH regulation; chloroplast dedifferentiation and increased IAA content inside the cells. Herewith we give an overview about these early cellular changes during embryogenic cell development

comparing the results obtained using embryogenic alfalfa leaf

protplasts with similar results from other sytems used to study somatic embryogenesis.

Introduction

In vitro embryogenesis from somatic plant cells is probably the best example for the broad developmental flexibility of higher plants. Plants as sessile organisms have a developmental strategy which allows a continuous adaptation to the changing

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environment. This based on continuous organogenesis due to the presence of undifferentiated, organ-forming cell files, the meristems. The activity of meristems is dependent on co-ordinated responses to environmental and developmental signals mediated by plant hormones. Differentiated somatic plant cells under extrem conditions can regain their meristematic capability, became totipotent and regenerate into whole organs or even to somatic embryos. Somatic embryo induction requires the complete reorganization of cellular state including physiology, metabolism and gene expression. How totipotency is re-acquired by differentiated cells during this cellular reorganization still remains a miracle. Here we try to summarise the present knowledge on the early induction phase of embryogenic competence in somatic plant cells including some of our recent results using alfalfa as a modell system. We hope that these new data will give us better insigths into the basic mechanisms of plant development.

Alfalfa as a modell to study embryogenic induction

Carrot cells cultured in the presence of 2,4-D (2,4-dichlorophenoxyacetic acid) are the best studied somatic embryogenic system. Hypocotyl segments are cultured for several weeks in the presence of 2,4-D to induce the formation of the so-called proembryogenic cell mass (PEM) which can develop into real somatic embryos on removal of the inducer (2,4-D) (DE VRIES et al., 1988). The difficulty of this carrot system is that the induction period is long and in the established embryogenic culture a mixture of different cell types with different degrees of competence can be found (TOONEN et al., 1994).

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An other well characterized system is based on leaf explants of the Chicorium hybrid „474” (DUBOIS et al., 1991). In this system somatic embryos can be induced directly from the explants by growth regulators or by heat stress (35 oC) and embryo development can be synchronized by the addition of glycerol to the incubation medium. The disadventage of this experimental system is that embryogenic cells are embedded in a mass of unresponsive cells. Studying the acquisition of embryogenic competence requires a system with a well defined transition phase between somatic and embryogenic cell types in cultures of individual cells. Leaf protoplast-derived cells of embryogenic genotypes of Medicago sativa L. (like the genotype A2 used in our laboratory) provide a usefull experimental system to study the requirements for embryogenic competence (BÖGRE et al., 1990; DUDITS et al., 1991). Manipulating the composition of the culture medium (2,4-D and/or iron concentration, medium pH) cells can be forced to follow different developmental pathways (Fig.1.). This allowed the direct comparison of parallel cultures under embryogenic and non-embryogenic conditions at the single cell level in order to determine characteristics of competent and non-competent cells.

Morphological markers of embryogenic competence

Embryogenic cell formation can be correlated with characteristic morphological changes. In carrot, cells of the proembryogenic cell mass (PEM) are small, densely cytoplasmed, full of starch grains while non-embryogenic callus cells are large and highly vacuolated in appearance (YEUNG, 1995). This can be generalized for most embryogenic systems including alfalfa, where protoplast-derived cells cultured at

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different 2,4-D concentrations can develop into either embryogenic or non-embryogenic cell types with the above characteristic morphologies (BÖGRE et al., 1990; Fig.1.) NOMURA & KOMAMINE (1995) established a synchronous embryogenic system based on fractionation of carrot cells. They found that small, spherical cells with rich cytoplasm represent the competent cell population ("state 0") which finally form the embryogenic ("state 1") cell clusters in the presence of 2,4-D. Somatic embryogenesis, similarly to the division of many plant zygotes, frequently starts with a morphologically asymmetric division resulting in a small and a large daughter cell. This is also the case in the leaf protoplast-derived embryogenic cells of alfalfa (DUDITS et al., 1991). Video cell tracking demonstrated that in a heterogenous embryogenic carrot cell population different cells after either equal or unequal first division are capable to form somatic embryos (TOONEN et al., 1994). However, the differential fate of the daughter cells have been demonstrated in carrot cultures labelling the cells with the JIM8 antibody recognising an arabinogalactan protein epitope on the cell surface. It was shown that this labelling is asymmetrically distributed on the daughter cells following embryogenic division and the labelled daughter cell dye while the unlabelled one develop into somatic embryo (MCCABE et al., 1997). It can be generally stated that not morphological but physiological asymmetries (i.e. unequal distribution of molecules, cell constituents) are the important factors to determine differential cell fate (VROEMEN, 1999).

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Molecular biology of somatic embryo induction

Most of the genes activated durring the transition of somatic cells to an embryogenic state are stress genes or genes associated with general metabolism (protein synthesis) or cell division (DUDITS et al., 1995). This emphasizes the complexity of cellular reorganization required to erase the existing developmental information of the cells and to replace it with a new one. Although the expression of many of these genes is a good marker of cellular reorganization, they are often not specfic for embryogenesis. We have only very limited infomation about genes which can have a specific and regulatory role during the earliest phases of somatic embryo formation. One of a possible candidate for such a function is the gene which is responsible for the „leafy cotyledon” mutation in Arabidopsis (LOTAN et al., 1998). The lec1 gene has been identified as a mutation causing dessication intolarance of embryos and if the embryos were rescued the mutation caused defects during late embryogenesis and cotyledon identity (LOTAN et al., 1998). Interestingly, when the isolated gene coding for a transcription factor has been overexpressed in plants under the control of 35S promoter, on some of the transgenic lines ectopic embryo formation on leaf surfaces could be observed (LOTAN et al., 1998). This indicates that the lec1 gene/protein can activate genes required during not only late but at early embryogenesis as well, although it is likely not the one or the only one which is capable to do it in a normal Arabidopsis plant. Another protein exhibiting possible regulatory functions during somatic embryo induction but certainly serving as a marker of embryogenic cell fate is the somatic embryogenesis receptor kinase or SERK identified first in carrot cells (SCHMIDT et al.,

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1997). The expressionof the SERK gene was shown to mark individual cells which developed into somatic embryos (SCHMIDT et al., 1997). Its role can be hypothetised as being a member of the signal transduction cascade leading to somatic embryo formation. That is not know however, what is the signal inducing the expression of these proteins regulating early embryo development. We suggest that physiological changes in the cells can be responsible to switch cell delopment and induce the appropriate regulatory genes..

The physiology of embryogenic competent cells

Intracellular Ca2+. There are only few studies on the role of Ca2+ as a second messanger during somatic embryogenesis (OVERVOORDE & GRIMES, 1994; ANIL & RAO, 2000). During the conversion of sandalwood (Santalum album) PEM into globular embryoids a significant increase in intracellular Ca2+ concentration has been reported (ANIL & RAO, 2000). The role of Ca-dependent protein kinases during somatic embryo development is also hypothetised based on the accumulation of the protein and increased activity during early phases of somatic embryogenesis both in sandalwood and in alfalfa (DAVLETOVA et al., 2001; ANIL & RAO, 2000).

Cellular pH. An important mediator of external signals, marker of cellular state and regulator of general metabolism is the cellular pH. The embryogenically competent leaf protoplast-derived alfalfa cells could be characterized not only by morphological parameters like compact cell size, limited vacuolization and the dense appearance of the

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cytoplasm, but also by well defined changes in their cellular pH gradients (PASTERNAK et al., 2001). While cytoplasmic alkalinization paralleled cell reactivation and division under both embryogenic and non-embryogenic conditions, the pH values of the vacuoles as well as the chloroplasts were significantly higher in embryogenic versus non-embryogenic cells (PASTERNAK et al., 2001). A direct link between cell fate and and extracellular/cellular pH can be based on the experimental results where the embryogenic pathway of alfalfa leaf-protoplast-derived cells could be blocked by buffering of the medium by 10 mM morpholinoethanesulphonic acid (MES) (PASTERNAK et al., 2001). Numerous studies performed have demonstrated that changes in cytoplasmic pH (pHc) occure during metabolic and developmental transitions in a large veriety of cells (GROSS et al., 1983; ANAND & PRASAD, 1989; ZETTERBERG & ENGSTROM, 1981; for review FRELIN et al. 1988). In plants, however, there are only limited number of examples for the physiological role of long term changes in cellular pH (KURKDJIAN & GUERN, 1989). In Bidens pilosa, cytoplasmic pH was correlated with cell division (PICHON & DESBIEZ, 1994); in carrot, medium acidification (cytoplasmic alkalinization?) by NH4Cl resulted in the accumulation of preglobular stage proembryos which could develop further only if the medium pH was raised to app. pH 5.7 (SMITH & KRIKORIAN, 1990); in Arabidpsis, initiation of root hair cells could be characterized by the acidification of the apoplast and alkalinization of the cytoplasm (BIBIKOVA et al., 1998).

Endogenous auxin synthesis. Although pH gradients are clearly associated with cell fate in different eukaryotic cell types, it is unlikely that they are itself the triggers of

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developmental pathways. Hormones are the most likely candidates regulating developmental switches. Auxins and cytokinins are the main growth regulators in plants involved in the regulation of cell division and differentiation. The influence of exogenously applied auxin on the induction of somatic embryogenesis is also well recognized (DUDITS et al., 1991; YEUNG, 1995). Despite the absolute requirement of in vitro cultured plant cells for exogenous auxins for sustained growth, cultured plant cells produce substantial ammounts of the native auxin, indoleacetic acid (IAA). Higher endogenous IAA concentration has been shown in different species\explants being associated with an increased embryogenic response (IVANOVA et al., 1994; MICHALCZUK & DRUART, 1999; JIMENEZ & BANGERTH, 2001; RAJASEKARAN et al., 1987). In carrot cells, exogenous 2,4-D stimulated the accumulation of large amounts of endogenous IAA (MICHALCZUK et al., 1992a; 1992b). It was hypothetized that embryogenic competence of carrot cells is closely associated with the severalfold increase in endogenous IAA levels due to the presence of 2,4-D, and that 2,4-D likely acts on the cells not directly as a strong auxin but through disturbing endogenous auxin metabolism (MICHALCZUK et al., 1992b). In alfalfa leaf protoplasts cultured in the presence of 2,4-D the endogenous IAA levels increased considerably during the first four days of culture (PASTERNAK et al., 2001). Although this increase was transient and comparable under both embryogenic and nonembryogenic conditions, the appearance of the peak of endogenous IAA level showed an approximately one day of delay under non-embryogenic conditions. A similar peak of endogenous IAA level has been observed in immature zygotic embryos of sunflower induced to form somatic embryos (CHARRIÉRE et al., 1999). In both systems the peak more or less correlates with the reactivation of cell division.

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Reactivation of cell division during the acquisition of embryogenic competence

Dedifferentiation of somatic plant cells is associated with the activation of cell division. Both processes require the action of exogenous plant hormones, especially auxin and cytokinin. The organization of the nuclear chromatin (ZHAO et al., 2001) and consequently the morphology of the nucleus (PASTERNAK et al., 2000) are good markers of the reorganization of the general gene expression pattern in dedifferentiating plant cells. The ratio of nuclear and nucleolar volumes was found to be associated with cell activity dependent on the presence of auxin and/or cytokinin (PASTERNAK et al., 2000; Fig.2.). Both plant hormones are required for complete dedifferentiation as well as for the initiation of the cell division cycle in differentiated alfalfa leaf cells (PASTERNAK et al., 2000). Auxin is considered to be the main plant hormone required for the activation of cell division in differentiated plant cells both in vivo and in vitro. One of the possible targets of auxin action in this respect is the induction of the expression of the cdc2 gene coding for the key regulatory protein kinase of the cell cycle (HIRT et al., 1991). In alfalfa leaf protoplast-derived cells, it was shown that auxin alone can result in the accumulation of this protein in high ammounts but for the activation of the kinase the presence of cytokinin is required (PASTERNAK et al., 2000). This indicates that cytokinin play an indirect role in the posttranslational regulation of CDK activity either through inducing cyclin expression (RIOU-KHAMLICHI et al., 1999) or cdc25-like phosphatase action (ZHANG et al., 1996).

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Comparing embryogenic and non-embryogenic culture conditions no significant differeneces could be established in the activity of the Cdc2A-related kinase activities of leaf-protoplast derived cells (Fig. 3.), although the entry into the cell divison was app. half day earlier in the case of embryogenic cells PASTERNAK et al., 2001; Fig 4.). BÖGRE et al. (1990) also reported that leaf protoplast-derived cells of an embryogenic versus a non-embryogenic alfalfa genotype were activated earlier. In carrot cell cultures, 2,4-D concentration-dependent switch between elongation and division has been also observed and it was demonstrated that 2,4-D inhibited the elongation of cells not directly but as a consequence of promoting their division (LLOYD et al., 1980). These faster-dividing cells in all of the above cases exhibited a smaller size. In the apical regions of plants it has been also shown that files of smaller cells often divide faster as compared to their larger neighbours, although cell size and cell cycle time are not always correlated in the same manner in plants (for review, FRANCIS, 1998). Recently, nitric oxide (NO) has been hypothetised having a role in the reactivation of alfalfa leaf-protoplast-derived cells (ÖTVÖS et al., 2001). L-NMMA known as a potent inhibitor of nitric oxide synthase in animal cells blocked the division of alfalfa protoplast-derived cells. Further experiments are in progress to verify the role of this potentially new plant growth regulator in plant development.

Stress response and the induction of embryogenic competence

Although, 2,4-D is one of the most effective compounds to induce competence for embryogenic development in somatic plant cells in vitro, other hormones like cytokinins or very frequently stress treatments could be successfully used to initiate the

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process (DUDITS et al., 1995; NOMURA & KOMAMINE, 1995, YEUNG, 1995). DUDITS et al. (1995) hypothesized that in vitro somatic embryogenesis is indeed an extreme stress response of cultured plant cells grown under conditions, which are unfavourable for cell divisions. BÖGRE et al. (1990) showed that somatic embryo formation in alfalfa root explants was highest at 2,4-D concentrations, which already inhibited cell division and thus callus formation. In alfalfa leaf protoplasts, the link between somatic embryogenesis and stress could be strengthened by showing that embryogenic, "state 0", cells can also be formed as a response to different oxidative stress-inducing compounds (PASTERNAK et al., 2001).

While low 2,4-D

concentration resulted in the development of elongated, vacuolated cells, application of excess iron, alloxan, paraquat or menadion, at concentrations not decreasing cell viability ("mild oxidative stress") restored the embryogenic competent cell type (PASTERNAK et al. 2001; Fig. 1.). Heavy metal ions were reported to induce the embryogenic response in carrot cell suspensions as well (KAMADA et al., 1989; KIYOSUE et al., 1990). Wounding, high salt concentration or osmotic stress were also found to positively influence somatic embryo induction in diverse plant species evoking the hypothesis that somatic embryogenesis is an adaptation process of in vitro cultured plant cells (reviewed by (DUDITS et al., 1995)). A link between oxidative stress response, auxin signalling and cell cycle regulation can rely on MAPK phosphorylation cascades as it has been shown that the same tobacco MAPKKK, NPK1, can be involved in oxidative stress response, auxin signalling and cell cycle regulation as well (HIRT, 2000).

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Summary

The developmental phase during which somatic cells acquire embryogenic competence can not be well defined in most of the culture systems used to study somatic embryogenesis. Leaf protoplasts of the embryogenic alfalfa genotype Medicago sativa ssp. varia A2 can serve as a suitable system to study early phases of somatic embryogenesis including dedifferentiation and the acquisition of the embryogenic cell fate. Although some molecular markers have already been associated with embryogenic competence, the molecular basis of somatic embryo induction is poorly understood. Most frequently only morphological markers (cell size and density, asymmetry of cell division, starch accumulation etc.) are used to assign embryogenic competence to cultured cells. Using cultures of individual leaf protoplast-derived cells developing with a reliable synchrony, we have set up an experimental system where embryogenic and non-embryogenic pathways can be easily manipulated and swithed on and off by combining different 2,4-D concentration, stress conditions and medium pH. Based on the results obtained with this system, we propose the significance of hormonedependent cellular dedifferentiation, cell division reactivation and parallel stress responses in the reorganization of cell physiology (e.g. pH, auxin synthesis and level). The complex reorganization of cellular state itself may induce several parallel signaltransduction pathways leading to cellular totipotency (Fig. 5.). This is followed by (or parallel with) the activation of regulatory genes necessary for the organization of embryo-like, polarized structures capable of further development into a whole organisms. Up to now we have only limited data about the above processes. What are

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the true signals, the members of the signal transduction pathways and the key genes specifically involved in these processes still have to be revealed experimentally.

Acknowledgements

The presented work was supported by the INCO COPERNICUS grant IC15-CT960906, OTKA T034818 and the „Biotechnology 2000” grant BIO-00062/2000 from te Ministry of Education, Hungary. A.F. is thankfull for the János Bólyai fellowship for its support.

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Figure legends

Figure 1. Developmental fate of alfalfa leaf-protoplast-derived cells. Leaf protoplast-derived cells of the embryogenic genotype (Medicago sativa L. A2) form elongated, vacuolized cells if cultured at 1 M 2,4-D concentration (A) or small, densely-cytoplasmed cells in the presence of a ten-times higher concentration (B). The vaculized cells form only callus following cell divisions (C), while the compact cells can develop into somatic embryos (D). Cell fate can be altered by buffering of the culture medium pH by MES or applying stress like incresing iron concentration in the medium ten times.

Figure 2. Nuclear parameters are good markers of cell activation/dedifferentiation. The area of nucleoli (upper histogram) and nuclei (lower histogram) were determined during four days of protoplast culture at daily intervals in the presence or absence of auxin (2,4-D) but in the presence of cytokinin (0.5 mg/l zeatin). The area of nuclei and nucleoli has been calculated as described elsewhere (PASTERNAK et al., 2000. At least 15 nuclei were measured per time points as indicated on the figure. The standard errors were below ten percent in all case.

Figure 3. Histone H1 phosphorylation activity of the Cdc2Ms A/B kinase in leaf protplast-derived cells under non-embryogenic (1 M 2,4-D) or embryogenic (10 M 2,4-D) culture conditions.

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Figure 4. Alfalfa leaf protoplast-derived cells enter the division cycle earlier under embryogenic conditions. Alfalfa leaf protoplasts have been cultured in the presence of 1 M 2,4-D (nonembryogenic condition) and either 10 M 2,4-D or 1 M 2,4-D + 10 mM Fe-EDTA (embryogenic conditions). The frequency of the cells in the S-phase of the cell cycle (BudR incorporation) and those completing a division cycle as observed under a ligth microscope (division%) have been determined at different time points as indicated. Details are described in PASTERNAK et al., 2001.

Figure 5. Stress and plant hormones act together during the induction of the embryogenic pathway in somatic plant cells: a shematic model. Auxin and cytokinin are required to induce cellular dedifferentiation but parallel action of stress is required to induce cellular totipotency (embryogenic competence). Increases in cellular pH values and timing of endogenous auxin synthesis can serve as physiological markers of this specific cell state. Asymmetric cell division and removal of the stress factor are required for further development of embryogenic cells.