LECTURE 3. PLANT EMBRYOGENESIS

PLANT EMBRYOGENESIS 1. From fertilization until dormancy

One-cell zygote→ embryogenesis→mature embryo.

2. The basic body plan of the sporophyte is established

plan is repeated several times over and elaborated after dormancy is broken.

3. Challenges of embryogenesis are:

a. Form basic body plan: apical-basal (shoot-root) axis; radial patterning- produces three tissue systems, axial patterning formation of cotyledons.

b. Form meristematic tissue for post-embryonic development of structure (leaves, roots, flowers, etc.).

c. Establish a food reserve for the germinating embryo until it becomes autotrophic.

 

A. MORPHOGENETIC STAGES 1. Single Cell-

The zygote undergoes the first division-an asymmetric cell division

Result: to dissimilar cells- a smaller apical or terminal cell and a larger basal cell. The apical cell receives most of the cytoplasm, and the basal cell receives the vacuole (Figure 9).

2. Polarity is established during this first asymmetric division. Most of the plant embryo develops from the apical (terminal) cell.

3. Basal cell divides to form the suspensor. Cells of the suspensor divide transversely to form a longitudinal file of cells.

Function of suspensor:

i) anchors the embryo to the endosperm

ii) nutrient conduit for the developing embryo

iii) The distal end of the suspensor is called the hypophysis contributes to gives rise to the root. (The other suspensor cells divide to form a filamentous or spherical organ that degenerates later in embryogenesis).

4. Two celled pro-embryo. The apical cell divides longitudinally to form two cells.

5. Four celled pro-embryo. The two cells each divide again longitudinally in a plane perpendicular to the first division to form a quadrant or 4-celled filament (Figure 9).

6. Octant stage. These four cells then divide transversely to form an octant (Figure 9, 10). 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.

7. Globular stage. All cells then divide periclinally to form the first histologically distinct tissue, the protoderm (Figure 11a). This stage is called the globular or dermatogen stage. The three basic tissue systems (dermal, ground, and vascular) can be recognized at this point based on characteristic cell division patterns.

8. Heart shaped stage. As cotyledons begin to form the globular shape of the embryo changes to a heart-shaped appearance in dicots. In monocots, only a single cotyledon forms.

 

a. Transition to heart stage begins when two groups of cells divide periclinally causing bulges that emerge as cotyledon lobes.

b. This represents a shift from radial to bilateral symmetry.

c. This also delineates the 2 main embryonic organ systems: the cotyledons and the shoot axis.

d. The root apical meristem (RAM) begins organizing at this stage.

e. 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.

f. At this stage, procambium (vasculature) initials can first be discerned (Figure 11)

NB. Thus differentiation of most major tissue systems has begun by early heart stage.

9. Torpedo stage. Continued cell division, growth and differentiation lead to the late heart stage and then to the torpedo stage.

a. By this point the suspensor is degenerating

b. And the shoot apical meristem (SAM) and root apical meristem (RAM) are established.

c. These meristems will give rise to the adult structures of the plant upon germination

10. Walking stick stage. Further growth of the cotyledons results in the walking stick stages. At this point, embryogenesis is arrested, and the mature seed desiccates and remains dormant until germination.

 

 

Figure 9. Diagrammatic representation of the different stages embryogenesis.

 

Figure 10. Stages of development in an angiosperm the embryo upto dormancy

B. MORPHOGENETIC CHANGES 1. Establishment of body plan.

2. Establishment of meristems

3. Establishment of food reserves; Dormancy

1. Establishment of body plan. Two developmental patterns have been identified

a) Shoot-root axis determination.

1. Begins -asymmetric cell division to form terminal and basal cell. 2. Establishes polarity. The apical/terminal cell →embryo proper while the basal cell →

suspensor (more in lecture 4).

3. Hypophysis -distal end of the suspensor contributes to root formation. Rest degenerates

 

Function of the suspensor:

orients the embryo toward food source; in angiosperms serve as a nutrient conduit for the developing embryo.

hormones

Evidence:

1. Isolate embryos with and without suspensor and culture them.

Result. Embryos without suspensor: do not survive at heart shape stage while embryos with a suspensor are twice as likely to survive at the heart-shape stage.

Conclusion: need for a suspensor through the heart-shape stage in dicots (Yeung and Sussex 1979).

Possible reason: The suspensor may be a source of hormones.

Further evidence: In scarlet runner beans, younger embryos without a suspensor can survive in culture if they are supplemented with gibberellic acid (Cionini et al. 1976).

b) Axial and Radial Symmetry.

1. Cell division and differentiation continue to give radial and axial and patterns. The cells of the embryo proper divide in transverse and longitudinal planes to form a globular-stage embryo with tiers of cells (Figure 11).

2. The emerging shape of the embryo depends on regulation of the patterns of cell division, since the cells are not able to move and reshape the embryo (see lecture 4).

3. Cell division planes in the outer layer become restricted and this layer, called the protoderm, becomes distinct.

4. Radial patterning emerges in the globular stage as the three tissue systems (dermal, vascular, and ground) of the plant are initiated.

a. The dermal system forms from the protoderm and contributes to the outer protective layers of the plant.

b. Ground tissue forms from the ground meristem and surrounds the developing vascular tissue;

c. The vascular system forms from the procambium cells that differentiate in the center of the globular embryo are for support and transport.

 

NB. The ground and vascular systems form independently.

Evidence Knolle mutants of Arabidopsis have their epidermal (L1) layer disrupted by misoriented cell divisions from the early globular stage while the Keule mutants have bloated and irregularly arranged epidermal cells recognizable at the globular stage. In both mutants the inner tissue systems develop normally (Mayer et al. 1991).

 

1. Axial (bisexual) patterning.

Becomes evident after the cotyledons, the first leaves, begin to form at end of the globular stage. Dicots have two cotyledons, which gives the embryo a heart-shaped appearance as they form.

Hormones (specifically, auxins) may mediate the transition from radial to bilateral symmetry (Liu et al. 1993). In monocots such as maize, only a single cotyledon emerges.

Function of cotyledons:

i. Become photosynthetic after germination and aid in nourishing the plant (although some never emerge from the ground).

ii. Where food reserve in the endosperm is used up before germination, the cotyledons become the nutrient source for the germinating seedling e.g. pea.

Cotyledons store food reserves such as starch, lipids, and proteins e.g. maize.

Figure 11. Radial and axial patterning. (a) Radial patterning in angiosperms begins in the globular stage and results in the establishment of three tissue systems. (b) The axial pattern (root-shoot axis) is established by the heart-shape stage. Adapted from http://zygote.swarthmore.edu/phyto1.html

1.Establishment of meristems

a. The shoot apical meristem and root apical meristem formed: are clusters of embryonic cells that persist in post-embryonic development and give rise to most of the sporophyte body.

b. The root meristem is partially derived from the hypophysis (the uppermost cell of the suspensor) in some species. All other parts of the sporophyte body are derived from the embryo proper. (More during shoot and root formation).

c. Genetic evidence indicates that the formation of the shoot and root meristem are regulated independently.

 

Evidence from observations of mutants of Arabidopsis:

i. The shootmeristemless (STM) mutant in Arabidopsis, is able to form a root meristem but fails to initiate a shoot meristem (Clark and Sheridan 1986; Barton and Poethig 1993).

ii.The STM gene is expressed in the late globular stage, before cotyledons form.

iii.STM's role appears to be to repress cell differentiation in the shoot apical meristem so that the cells maintain their indeterminate state (Long et al. 1996).

iv.The shoot apical meristem will initiate leaves and ultimately the transition to reproductive development after germination.

1.Establishment of food reserves; Dormancy

1.As the embryo reaches a maturation phase there is a shift from constructing the basic body plan to creating a food reserve by accumulating storage carbohydrates, proteins, and lipids.

2.The high level of metabolic activity in the developing embryo is fueled by continuous input from the parent plant into the ovule.

3.Eventually metabolism slows and the connection of the ovule (seed) to the ovary is severed by the degeneration of the adjacent supporting sporophyte cells.

4.The seed loses water (dessication) and the integuments harden to form a tough seed coat and Dormancy sets in ending embryogenesis. Embryo can persist in a dormant state for weeks or years.

5.Maturation → dormancy is due to precisely regulated program.

NB. The hormone abscisic acid is important in maintaining dormancy in many species. Gibberellins, another class of hormones, are important in breaking dormancy.

 

References

Arabidopsis book (TAB) an electronic book published by ASPB at http://www.aspb.org/publications/arabidopsis/toc.cfm

Graham, C.F., and Wareing, P.F. Eds. Developmental biology of plants and animals. Blackwell Scientific Publications, Oxford.

Lecture 16

Embryogenesis

 

Overview

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

Arabidopsis:

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.

Maize:

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.

Axis Formation

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

axis formation

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

axis fixation

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).

Pattern mutants

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).

Axial pattern:

gurke apical deletion

fackel central deletion

monopterous basal deletion

gnom (emb30) terminal deletion (both termini)

Radial pattern

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 embryogenesis

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

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.