Monday, 20 May 2019



In higher plants, fertilization of the ovule leads to the formation of the embryo. Zygotic embryogenesis, the way in which the fertilized ovule develops, has been studied extensively; changes that occur have been described for many plant species at the level of morphology, metabolism, protein composition, and gene expression. Interestingly, plant embryos can also develop in the absence of a fertilized ovule, from somatic cells in callus culture (Sung et al, 1984), from differentiated cells such as leaf mesophyll (Conger et al., 1983) and, perhaps most surprisingly, from immature haploid male gametes termed microspores (Nitsch, 1969; Dunwell, 1985). These alternative routes to the formation of an embryo illustrate both the means by which plants use environmental stimuli as developmental signals and the plasticity of plant development that is maintained throughout growth.

This article is not a comprehensive review of embryogenesis but rather a brief introduction to some interesting key issues in the area, and an outline of the ways in which we have been approaching the subject at Leeds. Two principal issues about plant embryogenesis will be discussed. The first concerns embryo formation and the second concerns the way in which an embryo, fully capable of germinating within days of organ primordia differentiation, is nevertheless prevented from doing so until seed development has been completed.

Embryo formation
In theory, embryogenesis induced in culture should provide an ideal experimental system to analyze the factors that induce, determine and regulate embryo formation. For example, somatic embryogenesis in carrot suspension cultures has been studied extensively since its discovery in the  950s (Reinert, 1958; Steward, Mapes & Hears, 1958), but in practice, the system has proved to be more complex than first envisaged.

Current understanding of the carrot system
Studies have shown that cultures maintained in 2,4-D as proliferating cells could be converted to cultures containing embryos by the removal of 2,4-D from the medium (Nomura & Komamine, 1986). However, few changes in protein composition or translatable mRNAs were detected between the two populations, i.e. the genes expressed in the 'embryo' cultures appeared to be near-identical to those expressed in the 'non- embryo' cultures (Sung & Okimoto, 1981, 1983; Choi & Sung, 1984; de
Vries et al, 1988).

It has since been suggested that the 'non-embryo' cultures were nevertheless 'embryonic' through the existence of structures initially described as pro-embryogenic masses (Halperin, 1966). The pro-embryogenic masses were found to be derived from a specific cell-type (Type 1 cells) under the influence of 2,4-D and were suggested to be stable intermediates in the developmental pathway linking single somatic cells to embryos (Nomura & Komamine, 1985). 2,4-D is therefore required to initiate embryogenic development but inhibits further progress along the pathway at the stage of pro-embryogenic masses. Removal of 2,4-D was considered to release the embryogenic potential of the pro-embryogenic masses and lead to a continuation of the pathway beyond the stage of the stable intermediate. Subsequently, by using an alternative means of producing pro-embryogenic masses, it has been shown that patterns of gene expression in the cell clumps are near-identical to those of heart-shaped (Thomas & Wilde, 1987) or torpedo-stage somatic embryos (Wilde et al, 1988).

These results highlight the problems of callus cultures since although they are often assumed to consist of a homogeneous population of cell types, in reality, they are probably composed of a range of cell types with a range of developmental potentials, each responding in different ways to the prevailing conditions of culture. One of the interesting features of the carrot system is the relationship
between vacuolation and the potential to become embryogenic. The proembryogenic masses were first detected through their typical morphology: small, rounded cells, dense cytoplasm and non-vacuolate (Halperin & Jensen, 1987). In the presence of 2,4-D, most of the carrot cells in culture had a different appearance, exhibiting typical patterns of cell extension growth and vacuolation. Highly vacuolate cells were not embryogenic, suggesting a prerequisite for induction of embryogenesis was the prevention of cell enlargement. Cell lines of carrot have been described that fail to form embryos when 2,4-D is removed from the culture medium (Lo Schiaco, Giuliano & Terzi, 1985). In one cell line that has been studied, the mutant cells were vacuolate: the mutants could be 'rescued' by addition of a glycoprotein to the culture that had been isolated from the medium of cells undergoing embryogenesis (de Vries, 1989). Rescue resulted in a changed morphology: the cells became rounded with dense cytoplasm. The glycoprotein was found to have peroxidase activity and it was suggested that the altered growth properties might arise from peroxidase-mediated cross-linking of wall polymers (e.g. isodityrosine cross-links) thereby preventing turgor-driven cell expansion. Do these results suggest, therefore, that to become embryos, cells must be restrained from expansion, and that once vacuolation has become initiated, embryogenesis is a non-starter?

The carrot system also highlights the profound influence of the extracellular matrix in determining the properties of the cells that the wall surrounds. Tunicamycin prevents N-glycosylation of proteins but, at sufficiently low concentrations, certain isomers of the antibiotic have little effect on protein synthesis (Elbein, 1987). Tunicamycin abolished the ability of carrot cells to become embryos, and the tunicamycin-treated cells could be rescued by the addition of extracellular glycoproteins from the medium of embryogenic cultures (de Vries et ai, 1988). On the assumption that all of the non-glycosylated proteins were still secreted in tunicamycin-treated cells, the results are surprising, because they imply that TV-glycans may play a role in acquisition of embryogenic potential.

Very recently, it has been found that the addition of exogenous peroxidase to the cells could lead to the partial rescue. This finding suggests that, at least in part, the tunicamycin effect was related to changes in secreted peroxidase. However, peroxidase activity in the medium of tunicamycin-treated cells was as high as in untreated cells, and since inactivated peroxidase (acetone-treated) could similarly rescue the situation, even more, the emphasis was placed on the role of the Af-glycan(s) attached to the enzyme (S.C. de Vries, personal communication). Glycans of the cell wall are increasingly recognized to be important regulatory molecules both in defense gene activation and at some level in the determination of morphogenetic programming (Bowles, 1990). But the endogenous glycans identified to date have been fragments of polysaccharides (pectins or xyloglucans), not components of extracellular glycoproteins. Since N- glycosylation is a common occurrence in all eukaryotic cells, a regulatory role for N-glycans would have startling implications! Recent studies have also implicated extracellular arabinogalactan proteins as markers for cell identity during somatic embryogenesis in carrot (Stacey, Roberts & Knox, 1990). Withdrawal of 2,4-D from the medium led to a dramatic increase in expression of the epitope recognized by the monoclonal antibody JIM 4. The epitope, a component of arabinogalactan proteins (Knox, Day & Roberts, 1989), was differentially expressed by cells at all stages from pro-embryogenic masses to mature embryos. This led to the suggestion that the arabinogalactan proteins identified may be functionally concerned with the position of cells in relation to plant form.

Finally, the carrot system has also thrown into question the correlation between patterns of gene expression induced during embryogenesis in vitro and in vivo. This has arisen through the detection of a product encoded by a gene, Dc3 (Thomas & Wilde, 1987; Wilde et ai, 1988). A transcript complementary to Dc3 was found to be highly-expressed in lobular and torpedo embryos, as well as pro-embryogenic masses (Wilde et al., 1988; de Vries et al., 1988). Because the gene was not expressed in hypocotyl nor leaf tissue of carrot, it was suggested to represent a marker for embryogenic potential. However, the peptide sequence of the polypeptide, predicted from sequencing Dc3 cDNA, showed the protein could be classified as a late embryogenesis abundant (LEA) product, which by definition would not normally be expressed until late stages of development. LEAs have been suggested to be stress proteins, correlated with desiccation tolerance (Dure et ah, 1989).

Overall, therefore, the results suggest that the temporal pattern of gene expression of cultured embryos may not correlate with that of zygotic embryos, and the culture per se may lead to stress conditions not normally experienced by zygotic embryos in planta until much later during their maturation. This, in turn, leads to the idea that the control on morphogenesis may be uncoupled from that on gene expression per se: two structures may appear morphologically identical, but express quite different patterns of gene expression. Similarly, the genes required for maintaining an actively dividing cell are most probably not going to differ much between meristematic cells of the root, shoot, or young embryo, yet the eventual form of the structures derived from cell division in each case is clearly different.

The culture system in use at Leeds to study
embryogenesis in vitro
In collaboration with the group of Dr. Rob Lyne at Shell Research Ltd, Sittingbourne, we have investigated the route to embryogenesis that involves microspores: immature pollen. Anther culture leading to the direct formation of embryos from microspores has been established for a number of dicot species, such as tobacco (Raghavan, 1984). However, in cereals such as wheat or rice, the more typical route to embryogenesis from anther culture involves an intervening callus stage and the proliferation of secondary embryoids from the embryonic callus (Hu, 1986). In contrast, work at Sittingbourne has optimized a means of direct embryo formation from barley anther culture that does not involve callus (Lyne, Bennett & Hunter, 1984).

Within the defined parameters of the procedure, the culture system leads to the formation of well-formed, doubled haploid embryos at high efficiency (400 green plants per 100 anthers). In another culture, proliferation is clearly visible by 14 d from plating out, and embryonic structures can be detected from 18 d onwards. Probably as a consequence of nutrient and growth-factor gradients within the anther (Hunter, 1985), production of embryos from microspores is not synchronous; a typical culture dish contains young embryos of different sizes as well as preco- ciously germinated seedlings. Direct culture of microspores, i.e. removal from the anther before plating out, leads to a much more synchronous development of embryos (R.L. Lyne, unpublished results).

The similarity of embryos formed in culture and those formed
in planta
We were interested to compare zygotic embryos of barley with those formed from microspores. This initially involved studies of patterns of translatable mRNA populations, in which we compared zygotic and microspore-derived embryos and barley callus (Higgins & Bowles, 1990). RNA extracted from the structures was translated in vitro within a rabbit reticulocyte lysate in the presence of [ 35 S]methionine, and the newly synthesized polypeptides were analyzed by two-dimensional gel electrophoresis and visualized by autoradiography. The results indicated that the patterns of genes expressed in zygotic and microspore-derived embryos of the comparable developmental stage were not identical. There were common features, but there were also very noticeable differences. In particular, a number of gene products found in the cultured embryos, but not in the zygotic embryos, were also detected in callus. Superficially, this result is similar to that obtained from carrot somatic embryogenesis, although the similarities in carrot embryos and callus were subsequently resolved by the detection of pro-embryogenic masses in the callus. The barley callus analyzed in our studies was derived from scutellar tissue of embryos, but the callus had been subcultured over a period of several years before its use in the analyses. The similarities in the microspore-derived embryos and callus involved a range of gene products that were highly abundant, suggesting that they were not derived from only a small percentage of the cells analysed. An alternative explanation to the existence of proembryogenic cells in the barley callus is that the embryos formed in culture reflect that origin, through the expression of genes typical of cultured cells.

Equally, it is possible that the gene products found in zygotic embryos, but not in cultured embryos, reflect the influence of the particular environment in planta within which the fertilized ovule develops. In these instances therefore, the gene products common to the two classes of embryo could provide more insight into what is required for the construction of an embryo per se, outside of the impact of the environmental conditions within which it develops.

Molecular markers for barley embryogenesis
We have used two approaches to identify gene products that can be used as molecular markers for embryogenesis: an immunological strategy, and one involving the construction and differential screening of a cDNA library. The projects are in relatively early stages; in the following discussion, results describing two 'markers', one produced by each approach, will be described. Conclusions from the work completed to date will then be outlined.

Characterization of an embryo-specific polypeptide of
molecular mass 17 kDa
Extracts prepared from barley zygotic embryos (0.5-1.0 mm in length) were used for immunization. The antiserum was found to interact with a wide range of polypeptides on Western blots, many of which were common to embryos, leaf-base (meristematic tissue) and callus. However, from these preliminary analyses, an immunoreactive polypeptide of molecular mass 17 kDa appeared to be located specifically in embryos (Higgins, 1989). An immunoaffinity system was used to prepare monospecific antibodies to the product, i.e. the antiserum was passaged through immobilized proteins extracted from leaf-base and callus. The effluent from the matrix was assayed on Western blots and contained antibodies that reacted only with the 17 kDa polypeptide (Clark et al., 1991). The antigen is a low-abundance gene product, undetectable in SDS-PAGE profiles or total embryo extracts by either Coomassie blue or silver staining. Despite this high antigenicity, we can find no evidence for glycosylation, and the product does not cross-react with MAC 207, a monoclonal antibody that detects a carbohydrate determinant common to all arabinogalactan proteins (Pennell et al., 1989). By using Triton X-114 partitioning, we find that the protein is amphiphilic in character. During zygotic embryogenesis, the product appears at mid-stages of development, at 18 d post-anthesis (dpa) (growth temperature 12 °C), and remains at similar levels up to 6 d post-germination of the seedling. The timing differs in cultured embryos: the appearance of the polypeptide precedes the appearance of morphologically distinct embryos. Tissue-printing revealed that the antigen was distributed evenly throughout the embryo tissues, and within grains, the polypeptide could also be detected in the aleurone layer but not in the endosperm.

The low abundance of the product and the timing of its appearance and disappearance during zygotic embryo development and germination suggests that it is not an LEA (Dure et ai, 1989). However, the culture of young zygotic embryos on either ABA (10~ 6 M) or mannitol (9% w/v) led to the precocious appearance of the polypeptide. Preliminary analyses have shown that the protein is N-terminally blocked, but the sequencing of peptide fragments produced by V-8 digestion is underway. We anticipate that the peptide sequence information gained will be used to design amplimers for a polymerase chain reaction, thereby providing a way to obtain information on the gene encoding this embryo-specific product. 

Control of precocious germination
Once organ primordia have differentiated, the young immature embryo is capable of germination. This potential can be demonstrated in zygotic embryos through dissection of the embryo from the grain and culture in vitro (Norstog, 1972). Precocious germination occurs within several days, a process noted as early as 1904 by Hannig and subsequently described for a wide range of plant species. For embryos formed in culture, such as those derived from barley microspores, precocious germination occurs as a matter of course, once primordia have differentiated (Nitsch, 1969). Thus in culture, full maturation of the embryo does not occur and many genes characteristic of mid-late stages of embryo development are not expressed; for example, the gene encoding barley germ agglutinin is not expressed during embryogenesis in microspore culture (P.C. Morris & D.J. Bowles, unpublished results). 

Since the zygotic embryo does not germinate precociously in planta but does germinate immediately on removal from the plant, it follows that the environment surrounding the zygote influences the events that take place. The time period over which this germination potential is suppressed depends on varietal and environmental factors but can be quite considerable. For example, in wheat or barley, the differentiated embryo is prevented from germinating for 50-60 d, the time taken to construct the desiccated, fully mature grain (Rogers & Quatrano, 1983). During germination, growth occurs initially by rapid cell expansion following water uptake, and then by cell division within the meristems of the organ primordia. If this pattern of growth is initiated in precociously germinated immature embryos, it is clearly very different from that undertaken by the embryo that matures normally, in which all the cells are maintained at a similar size and shape.

The factors regulating the switch between embryogenic and germinative pathways of development have been studied in a number of laboratories worldwide, including research from my own group. In the following discussion, results obtained at Leeds on wheat and barley will be placed within the context of data from other laboratories. 

The relationship between abscisic acid and osmotic stress
Culture of immature zygotic embryos on nutrient media leads to precocious germination. Inclusion of abscisic acid, or an osmotic agent such as mannitol, in the medium leads to the inhibition of precocious germination (Morris & Bowles, 1987). It seems highly relevant that the concentration of these two agents must be progressively increased to prevent the germination of progressively 'older' immature embryos. This phenomenon has been shown for both dicots and monocots (see, for example, Morris et al., 1985; Finkelstein & Crouch, 1986). For example, abscisic acid at 10~ 6 M or mannitol at 4.5% (w/v) is sufficient to suppress precocious germination of young barley or wheat embryos (0.2 - 0.7 mg fresh mass), but 10~ 4 M abscisic acid and 9% (w/v) mannitol is required for 'older' embryos, e.g. 1.5 mg fresh mass (Morris et al, 1985). This is not due to problems of uptake, since embryos on ABA-containing media take up the growth regulator to the concentration supplied (Walker- Simmons, 1987; Morris et al, 1990).

Interestingly, studies of Schopfer & Plachy (1984,1985), on prevention of germination in imbibed Brassica seeds, found the effects of exogenous ABA and osmotic agent to be additive, i.e. a low concentration of growth regulator could be compensated for by a higher concentration of osmotic agent and vice versa. The effect of exogenous ABA was found to be on the control of cell wall extensibility and the minimum threshold turgor pressure for expansion. A prerequisite for turgor-driven cell expansion is the possibility that polymers within the extracellular matrix can slide past one another to accommodate the expansion of the protoplast. Exogenous ABA could very readily affect apoplastic pH: it is known, for example, to inhibit the action of acid hydrolases in the cell wall, and its effect is reversible by the application of fusicoccin (Labrador, Rodriguez & Nicolas, 1987), a toxin known to affect proton transport. Thus, from the work on Brassica napus, it is possible to envisage that exogenous ABA inhibits germination by affecting cell wall extensibility, whereas high external osmotica would affect expansion simply by preventing internal turgor pressure.

If we return to the problem of suppression of germination potential during embryo development, it is equally possible that ABA and the water relations of the embryo and grain, play two distinct roles in the process. This is an alternative explanation to the one more generally considered, namely that endogenous ABA is the sole direct mediator of the switch between continued embryo development and precocious germination. This latter viewpoint suggests that osmotic stress may be the environmental stimulus in culture or in planta, but the transduction pathway linking that stimulus to gene activation or suppression involves ABA. Measurement of endogenous ABA in cultured embryos of dicots (Finkelstein et al, 1985; Finkelstein & Crouch, 1986; Eisenberg & Mascarenhas, 1985), or cereals (Morris et al, 1988; Walker-Simmons, 1987) suggests that there is more to the story than a simple increase of internal ABA in response to stress imposed by the external osmoticum.

Suppression of germination in planta
A number of studies have determined the changes in level of ABA during seed development, for example wheat and barley (McWha, 1975; Quarrie, Tuberosa & Lister, 1988; Quarrie et al., 1988), soybean (Akerson, 1984), and Brassica napus (Finkelstein et al., 1985). The precise pattern of change is dependent on species, genotype and environment, but follows a general trend: ABA levels steadily increase during the early stages of development to a maximum value and then decline as the seed dehydrates. Viviparous mutants of maize have provided good evidence for an involvement of ABA in control of vivipary, since embryos from the mutant plants have been shown to be insensitive to exogenous ABA (McDaniel, Smith & Price, 1977) or to contain abnormally low levels of the growth regulator (Brenner, Burr & Burr, 1977). Recently, we have followed a number of parameters in parallel during cereal embryo development in planta, in order to examine endogenous ABA levels within the context of the water relations of the embryo and the grain. Thus, ABA levels, fresh mass, dry mass, and water potential were assayed for embryos and for the remainder of the grain, at intervals from shortly after anthesis through to desiccation and full maturation at 65 dpa (Morris et al, 1990).

The overall concentration of ABA, determined by immunoassay using standardized procedures (Morris et al., 1988), fluctuated only 0.25 - 2.0 pmol mg" 1 fresh mass during development, whether in the grain or embryo, and followed the general trend described in earlier studies. Thus, at early stages of embryo formation, precocious germination could in practice be prevented by the endogenous levels of ABA, since they correspond to the values known to inhibit the germination of young, immature cultured embryos (e.g. 0.2 mg fresh mass). However, by later stages of development, for example by the time embryos are 1.5 mg fresh mass, it is known that 50-100 ^IM exogenous ABA is required to prevent their precocious germination in culture. This value clearly exceeds the level found in embryos or grains from the plant.

Interestingly, as development proceeded from 18-20 dpi onwards, a progressive increase in water potential difference was set up between the embryo and the rest of the grain. Thus, at the earliest time point measured (2 dpa), the grain (at this stage, consisting of a coenocyticum with very high levels of low-molecular-mass metabolites) had a negative water potential of —5 to —6 MPa. By 7 dpa, the water potential changed to —3 to —2 MPa, perhaps reflecting a decreasing osmotic potential
through the conversion of metabolites to polymers and an increasing turgor pressure through the deposition of cell walls within the endosperm. This value of water potential for the grain was then maintained through to 21 dpa and was found to be near identical to the water potential of the embryo. However, from 21 to 50 dpa, the water potential of the embryo remained constant, whereas that of the rest of the grain decreased steadily to —8 MPa. This therefore led to an increasing water potential difference between the embryo and the grain, such that cell expansion of the developing embryo would be negligible through lack of water uptake. Only by very late stages of maturation, at 50 dpa, did the water potential of the embryo again resemble that of the rest of the grain, when values
changed from —2 to —8 MPa in less than 7 days and the seed approached full desiccation.

These results suggest that, in planta, both ABA and the water relations of the system may control the switch between continued embryo development or germination. At the early stages, endogenous ABA levels are those predicted from in vitro studies as sufficient to suppress germination potential. By mid-stages of maturation, the water potential difference that is progressively established between the embryo and the rest of the grain is sufficient to ensure that cell expansion is inhibited by prevention of water uptake. It is possible that, in vitro, the unphysiological high levels of exogenous ABA required to prevent germination of the larger embryos act in a manner suggested by Schopfer's work, i.e. indirectly mimic the in planta control, through blocking cell-wall extensibility and thereby inhibiting water uptake.

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