There are 3 major objectives for plant embryogenesis:
1. To establish the body plan (axis formation) and major organ systems of the plant.
2. Establishing meristems (populations of cells that will continue postembryonic growth).
3. To prepare the embryo for dormancy and subsequent germination. This involves storage of compounds for nutrition of the germinating seedling, and acquiring tolerance to desiccation.
Embryonic development can be divided into 3 overlapping stages:
1. A period of morphogenesis and histogenesis in which the major organ and tissue systems of the plant are established. This period is characterized by a high rate of cell division and differentiation.
2. A maturation phase characterized by cell expansion and the massive accumulation of storage reserves. Fresh weight can increase 100 fold with little to no cell division.
3. Acquisition of desiccation tolerance and the loss of water associated with the onset of developmental arrest.
1. Establishment of the plant body.
Description of embryogenesis
At the time of fertilization the egg cell is polarized, with a large vacuole toward the micropylar end and the nucleus and most of the cytoplasm toward the chalazal end. After fertilization, this polarity is maintained in the zygote. The first division is perpendicular to the axis of polarity and is asymmetric. The apical cell receives most of the cytoplasm, and the basal cell receives the vacuole. From this point on the pattern of cell division varies among species.
The basal cell gives rise to the suspensor. Cells of the suspensor divide transversely to form a longitudinal file of cells. The suspensor pushes the embryo proper into the nutrient-rich endosperm and is also important for nutrient transport into the developing embryo. Only the distal-most cell of the suspensor will contribute to the embryo proper.
The apical cell divides longitudinally. Both cells then divide again longitudinally in a plane perpendicular to the first division to form a quadrant. These four cells then divide transversely to form an octant. The transverse walls of these 4 cells divides the embryo in half along what is known as the O’ line. This becomes a boundary between distinct domains of the embryo (see below). All cells then divide periclinally to form the first histologically distinct tissue, the protoderm. This stage is called the globular or dermatogen stage. Afterward, protodermal cells divide predominantly anticlinally to form a distinct cell lineage. Inner cells divide both anticlinally and periclinally cause growth as a mass. In the globular stage embryo, there is expression of a lipid transfer protein gene specifically in the protodermal cells. The distinct pattern of cell division and differential gene expression are the first indications of histological differentiation.
Transition to heart stage begins when two groups of cells divide periclinally causing bulges that emerge as cotyledon lobes. This represents a shift from radial to bilateral symmetry. This also delineates the 2 main embryonic organ systems: the cotyledons and the shoot axis. The root apical meristem (RAM) begins organizing at this stage. The apical-most suspensor cell, called the hypophysis, becomes incorporated into the embryo proper. Both the hypophysis and apical cell derivatives contribute to the formation of the RAM. At this stage, procambium (vasculature) initials can first be discerned. Thus differentiation of most major tissue systems has begun by early heart stage. Continued cell division, growth and differentiation lead to the late heart stage and then to the torpedo stage.
Differentiation of the shoot apical meristem (SAM) less obvious than the RAM. As the cotyledon lobes begin to grow, a group of cells between them is seen to divide infrequently. Later, the cotyledons and subtending tissues begin expressing genes for storage proteins. The group of quiescent cells does not express these genes and the lower boundary of the non-expressing cells in at a prominent cell wall corresponding to the O’ line.
After the first division, there is no set pattern of divisions. However apical cell derivatives divide actively while basal cell derivatives (the suspensor) divide infrequently. The suspensor is multiple cells thick. In the late proembryo stage a protoderm differentiates, first over the apical region of the embryo, then progressively downward. Like dicots, the protoderm divides anticlinally and represents the first evidence of histodifferentiation.
At transition stage, a small wedge of cells becomes densely cytoplasmic and begins rapidly dividing. These cells continue dividing and form a protuberance that becomes the apical meristem. Internal to and basal to the developing apical meristem, another group of cells assumes meristematic characteristics (dense cytoplasm, rapid division). This becomes the root apical meristem. Subsequently, procambial strands differentiate to connect the shoot and root primordia and this defines the plant axis. A collar bulges up around the apical meristem, but is not formed by the apex. This becomes the coleoptile which ensheathes the shoot, protecting it as it pushes through the soil during germination. As the embryo continues to grow, the apical meristem initiates several leaf primordia (typically 6). This is in contrast to dicots which form no leaf primordia prior to germination.
Plant tissues contain an intrinsically fixed polarity. Stem segments will retain their original apical-basal polarity no matter what the treatment. If the stem segment is grafted in reverse orientation to a plant, roots will form at the original basal end. The egg cell shows marked polarity prior to fertilization and this polarity is manifest in the formation of the plant axis. Because the polarity is always present in the plant tissues, it is difficult to study how the polarity is established. The in vitro fertilization system described by Digonnet (Digonnet et al., 1997) has promise in this regard. However most of the research to date has made use of the brown algae Fucus or Pelvetia See the following for review: (Fowler and Quatrano, 1997).
Fucus—marine brown algae
zygotes free floating APOLAR
1st division is assymetric—gives rise to a thallus cell and rhizoid
rhizoid gives rise to holdfast, thallus to fronds
direction of light determines the axis of polarity—shade side becomes the rhizoid
axis determination is a stepwise process
1. axis formation: 4-10 hours after fertilization
requires a gradient—most convenient is light
shine directional light for 1 hour and then darkness or uniform light—axis of polarity forms according to the light direction :: treatment sufficient to induce axis formation
shine directional light for 1 hour, then shine light from a different direction—up to 10 hours after fertilization, axis of polarity forms according to direction of second pulse :: axis of polarity still labile
2. axis fixation : 10-12 hours after fertilization
changing direction of light will no longer change the axis of polarity
3. rhizoid formation : 14-18 hours after fert.
on side away from light
1st detectable sign of asymmetry is an electrical current flowing inward at presumptive rhiziod site
requires monovalent cation--medium containing only K+ ions sufficient to support axis formation—it’s not clear if the electrical current is actually required for axis formation;; it’s also not clear whether the ion is required for this current or other events in axis formation or both
cortical clearing—localized cortical vesicle exocytosis—hypothesized to incorporate ion channels into the plasma membrane at presumptive rhizoid site and contribute to the electrical current
requires microfilaments—treat zygotes with cytochalasinB during directional light treatment, wash and place in dark, rhizoids form randomly relative to light treatment
no detectable redistribution of microfilaments during axis formation
experimental approach--give zygotes directional light (light 1), then during axis fixation period treat with inhibitors and give light from a different direction (light 2)—if inhibitor interfered with axis fixation, rhizoids will form according to the second light direction, if not they will form according to the first.
requires microfilaments—treat zygotes with cytochalasinB, wash out and rhizoids form according to second light direction
requires cell wall—digest cell wall with enzymes to make protoplast
give light 1, remove wall during axis fixation, then no light 2—rhizoid forms according to light 1 therefore the cell wall is not required for axis formation
give light1, remove wall during axis fixation, then give light 2—rhizoid forms according to light 2—therefore the cell wall is required for axis fixation
Involvement of auxin in embryo morphogenesis
Treatment of developing embryos with auxin polar transport inhibitors shows a requirement for auxin polar transport, however, zygotic and somatic embryos respond differently. Zygotic embryos treated at globular stage develop coyledons in a fused cylinder. The embryos remain radially symmetrical thus auxin polar transport is required to establish bilateral symmetry. Somatic embryos when treated with the inhibitor fail to progress to the next stage of morphogenesis. Eg. globular embryos continue spherical growth but no axis elongation or cotyledon formation.
Positional information along the embryo axis
Torpedo stage embryos have recognizable cytological characteristics at different points along their longitudinal axis. Torpedo stage embryos were transected at various points along their longitudinal axis and allowed to regenerate root poles. Regeneration involved the sequential production of cells with the correct characteristics for their position along the axis. For example, embryos transected near the apical pole first regenerated a hypocotyl, then a radicle and finally a RAM. Thus regenerating cells respond to positional information that specifies cellular characteristics along the longitudinal axis (Schiavone and Racusen, 1990).
The major pattern elements of the embryo include components of the longitudinal axis, and the radial pattern of tissues found in stem cross sections. The longitudinal elements include SAM, cotyledons, hypocotyl and root (including RAM). The radial pattern consists of the epidermis, cortex (ground tissue) and vasculature. A saturation mutagenesis for alterations in pattern revealed a small group of genes controlling these elements. Reviewed by: (Jurgens et al., 1994).
gurke apical deletion
fackel central deletion
monopterous basal deletion
gnom (emb30) terminal deletion (both termini)
knolle and keule no epidermis
gurke deletes tissues that roughly correspond to those derived from cells above the O’ line suggesting that the first transverse division of the embryo proper is important for establishing the apical domain.
fackel mutants have severely shortened hypocotyls, suggesting the central domain deletion, but also show abnormal numbers of cotyledons, shoot apicies and other defects. The FACKEL gene is involved in the synthesis of a previously unknown sterol compound (Jang et al., 2000; Schrick et al., 2000).
monopterous mutants lack a root and hypocotyl and show defects in vasculature. MONOPTEROUS encodes a transcription factor that binds auxin-inducible gene promoters and the mutants show defects in auxin polar transport (Hardtke and Berleth, 1998).
gnom mutants (also called emb30) have abnormal cell division, expansion and adhesion. The first cell division in the zygote is equal instead of assymetric. GNOM encodes a protein related to a secretory yeast protein Sec7 which is involved in golgi vesicle secretion (Shevell et al., 1994). It functions as an ARF GEF (ADP-ribosylation factor G-protein Guanine exchange factor) that is required for the polar localization of the auxin efflux carrier PIN1 (Steinmann et al., 1999). Whether mislocalization of PIN1 is directly responsible for the pattern defects is not clear.
knolle and keule mutants display abnormal cytokinesis leading to polyploidy and multinucelate cells. Incomplete cytokinesis might prevent segregation of pattern determinants between epidermal and internal cells. KNOLLE encodes a syntaxin related protein (Lauber et al., 1997). Syntaxins are target membrane receptors that interact with vesicle membrane proteins to promote secretory vesicle membrane fusion. KEULE encodes a Sec1 protein that interacts with KNOLLE (Assaad et al., 2001).
Segregation of cell wall determinants
Early Fucus embryo development resembles plant embryogenesis. The first cell division results in a thallus cell that will form the shoot and a rhizoid cell that will form the holdfast. If the rhizoid cell is completely ablated the thallus cell will form a thallus only. If the thallus cell is completely ablated, the rhizoid will form a rhizoid only. If protoplasts are made of the rhizoid cell, they will regenerate an entire embryo indicating they still possess full totipotency. If the rhizoid cell is ablated but fragments of rhizoid cell wall material remain attached to the apical cell, apical cell derivatives that come in contact with the rhizoid wall material will acquire a rhizoid fate. Similarly, if rhizoid derivatives contact wall material from an ablated apical cell, they will acquire a thallus fate. This demonstrates the presence of cell wall determinants in Fucus embryos (Berger et al., 1994).
Coupling of pattern formation, morphogenesis and differentiation
Several mutants that altered embryo shape without causing pattern deletions also arose from the same pattern mutant screen discussed above. One of these called fass causes a variety of morphological abnormalities, including variable numbers of cotyledons, but contained all the basic elements of the axial and radial patterns. This indicates that pattern formation and morphogenesis are separable programs. fass mutants have abnormal patterns of cell division indicating that the normal cell division orientation is not required for pattern formation (Torres-Ruiz and Jurgens, 1994).
Another mutant called raspberry is arrested early in the globular embryo stage. Accumulation cell type specific molecular markers indicated that tissue (cell) differentiation proceeded beyond that normally seen at the stage of morphogenesis at which the embryo was blocked. This indicates that cell differentiation and embryo morphogenesis are controlled by separable programs (Yadegari and al, 1994).
Somatic cells can be induced to undergo embryogenesis. This was one of the earliest and clearest demonstrations of totipotency, and therefore genomic equivalence. Somatic tissue is induced to form callus. In the presence of auxin, the callus will form pre-embryogenic masses (PEMs)—small clumps of cells. Upon removal of auxin the PEMs organize into a typical globular embryo which then progresses through heart stage and torpedo stage. These embryos are the same size and morphology as normal zygotic embryos. At the cotyledon stage, instead of entering developmental arrest (like normal) the embryos initiate a shoot meristem and seedling growth.
That embryo development can occur outside the environment of maternal tissue indicates that the developmental program is intrinsic to the embryo per se. It does not require maternal gene products, signals or spatial constraints for normal morphogenesis. That is not to say the maternal environment is not important because it is likely that the tissue culture conditions mimic some of the conditions within maternal tissue.
The plant hormone, auxin, is required to induce competence of the PEMs to undergo embryogenesis following removal of auxin. What the auxin or removal from auxin actually does to induce embryo formation is unknown.
Embryonic development vs. seedling development (germination)
The plant hormone, abscisic acid (ABA) is intimately involved in controlling embryogenesis and maturation. ABA deficient mutants show vivipary (ie. the seeds germinate directly instead of going into dormancy following embryogenesis. Furthermore, germination of dormant seeds can be inhibited by exogenously applied ABA. Another hormone, gibberellic acid (GA) acts antagonistically to ABA and promotes germination. GA deficient mutants germinate poorly and exogenously applied GA can block dormancy and promote vivipary.
Chemical environment of the endosperm: Raghavan and Torrey (1963) Late stage embryos (cotyledon stage) placed in culture with a simple medium (inorganic salts, vitamins and 2% sucrose) grew into normal seedlings with no dormancy period. Early stage (globular-heart) embryos will not grow on simple medium. They cease embryonic development and undergo cellular changes such as rapid elongation and vacuolation associated with germination. Precise concentrations of the plant hormones auxin and cytokinin allowed progression of embryonic development. Alternatively, high osmoticum (10% sucrose or mannitol) allowed embryonic development. Progressively lower concentrations of osmoticum are required in later stages to complete embryogenesis. The osmoticum acts independently of ABA because precocious germination of ABA deficient mutant embryos can be inhibited.
Genetic control of developmental program: leafy cotyledons (lec) is a recessive mutation that causes the cotyledons to acquire characteristics of leaves; they have trichomes (hairs), leaf-like vasculature and lack storage proteins typical of cotyledons. The mutant also displays vivipary and lacks desiccation tolerance. The phenotype suggests that LEC normally regulates embryonic development and in mutants, embryonic development is disrupted allowing the default program of seedling growth to proceed. It also suggests homology between cotyledons and leaves. This relationship would not otherwise be obvious because leaves and cotyledons have very different ontogenies. Leaves are produced by apical meristems whereas cotyledons arise prior to the meristem during embryogenesis. Ectopic expression of the LEC gene in adult vegetative tissues induces embryo formation (Lotan et al., 1998).
The suspensor is clearly involved in transport of nutrients to the embryo proper. In some species the suspensor cells form haustoria or cell wall invaginations typical of transfer cells. It is very metabolically active and thought to also provide growth regulators to the embryo proper.
Removal of the embryo proper allows the suspensor to proliferate into a mass of cells. In some plants, it will develop into another embryo. Thus the developmental potential of the suspensor is greater than it’s fate and the embryo proper is inhibitory to further growth.
2. Preparation for developmental arrest (embryo maturation).
Most cell division is complete by the beginning of the maturation phase of embryo development, but the embryo can increase in size up to 100 fold. This is by cell expansion and accompanies a massive accumulation of storage compounds. The major storage compounds are proteins, starch and lipids. These storage compounds are what give nutritional value to important crops such as cereals and beans. They are also valuable for other uses such as production of vegetable oil and starch which are used in a wide variety of ways ranging from cooking to industrial lubricants and plastics. Therefore there is a huge economic interest in seed storage compounds.
Accumulation of storage products
Storage proteins represent an important source of amino acids, nitrogen and carbon for the germinating seedling. Storage protein mRNAs represent up to 20% of the total mRNA found in a maturation phase embryo. They are synthesized on the RER and accumulate in the vacuole or as membrane bound vesicles called protein bodies. The storage proteins are encoded by several multigene families with up to 55 different genes coding for a given storage protein. Synthesis is controlled at the transcriptional level, with a few regulatory genes each controlling particular classes of storage proteins. An example is the opaque2 gene of maize which codes for a transcription factor.
The regulation of starch and lipid accumulation, although no less important, is less well understood. These compounds are produced by complex enzymatic pathways. Each class of compound is a mixture of molecules with different chain lengths, chain branching characteristics, levels of saturation and other chemical modifications. Thus the synthesis of these compounds is much less straight forward than storage proteins.
Acquisition of dessication tolerance
At the end of embryonic development, most seeds dehydrate to about 5% moisture content. Such severe dehydration is lethal to most plant tissues and embryos express a developmental program that allows them to survive. Two problems faced by desiccated cells are high ionic concentrations and membrane stresses. At such low moisture levels, solutes would tend to crystallize and precipitate. Hydrophobic interactions with the aqueous solution are important for maintaining the integrity of the lipid bilayer. With no aqueous phase, the membrane becomes unstable and leaky.
A group of proteins called dehydrins are expressed in late maturation. The role for these proteins in desiccation tolerance is supported by their induction by drought stress in vegetative tissues and during desiccation of the resurrection plant, one of the few plants that can tolerate desiccation of postembryonic tissues. They are hypothesized to function in ion sequestration and in forming a protective layer for stabilizing membranes.
Coupling of morphogenetic and maturation programs
Morphogenesis and maturation appear to be controlled by independent developmental programs. In viviparous mutants which fail to undergo maturation, morphogenesis is normal whereas other mutants arrested at various stages of morphogenesis undergo normal maturation as evidenced by the absence of necrosis following desiccation and the accumulation of storage proteins.
Integration of these programs involves both hormonal mechanisms and genetic programs. ABA is necessary to induce the expression of genes involved in maturation and desiccation tolerance. ABA deficient viviparous mutants are desiccation intolerant. An ABA independent genetic program is necessary to confer ABA sensitivity to the embryo and mutants in this program show ABA insensitive vivipary. The LEC gene, in which mutants both display seedling instead of embyro morphological characteristics and bypass embryo maturation are likely candidates for coordinating the two different programs.
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