Seed and Fruit Development
The seed develops from the ovule and contains the embryo and endosperm, surrounded by the maternally derived seed coat. The function of the seed is to protect the embryo, to sense environmental conditions favorable to germination and to nourish the germinating seedling.
Fruits develop from organs of the flower and thus involve differentiation or redifferentiation of preexisting organs. Evolutionarily, floral organs represent modified leaves and so the fruit is also a modified leaf. Fruits serve 2 functions: to protect the seeds during development, and then to disperse the seeds following maturation.
All mature seeds contain an embryo and a protective covering called a seed coat (testa). In early development all angiosperm seeds also contain an endosperm, but in many seeds the endosperm is completely absorbed by the developing embryo. The embryo and endosperm are products of fertilization while the seed coat develops from the integuments of the ovule.
The seed coat contains a variety of adaptations related to protection and dispersal mechanisms. The seed coat usually forms a dry tissue. It may contain waxes for water impermeability, mucilage to make seeds sticky, compounds resistant to digestion by animals, etc. In pomegranate, the seed coat forms the fleshy tissue that is consumed by humans. The seed coat often contains multiple layers with different characteristics.
Maternal tissues appear to have an important influence on seed development. An arabidopsis mutant called aberrant testa shape (ats) that lacks one of the 2 integuments also lacks several cell layers in the testa (3 layers vs. 5 normally). The seed are abnormally shaped in this mutant and seed shape shows maternal effect (ie. the genotype of the maternal parent determines the shape of the seed). Therefore, the seed coat and not the embryo determines the shape of the seed, and the embryo just grows to fill in the shape determined by the testa.
Another maternal gene called FBP7 is specifically expressed in the ovule and seed coat and is required for normal ovule development. Downregulation of this gene in transgenic plants resulted in degeneration of the endosperm that was dependent on maternal genotype. This demonstrates the interaction between maternal tissues and those produced by fertilization.
Several genes have been identified that negatively regulate seed development until fertilization has occurred. A mutant screen on a sterile line identified 3 genes that regulate seed development. Seeds develop on these mutants in the absence of fertilization. They are called fis for fertilization independent seeds. The genes appear important for control of seed development by fertilization. Several similar genes have been identified and cloned. They include:
FIE = fertilization independent endosperm, encodes a WD type POLYCOMB protein
MEDEA encodes a SET domain type POLYCOMB protein
FIS2 = fertilization independent seed2, encodes a zinc finger protein
POLYCOMB proteins are involved in chromatin structure and regulate (repress) the expression of genes in big portions of the genome. Therefore, the repression of large groups of genes is necessary to inhibit seed development until fertilization has occurred.
All three genes show parent-of-origin effects (imprinting). The maternally inherited gene is expressed and required but the paternally inherited gene is not expressed or required for seed development. (I.e. heterozygous mutants show 50% seed abortion, even when fertilized by wild type pollen [Luo, 2000 #167].
MEDEA and FIE proteins have been shown to interact by yeast 2-hybrid [Yadegari, 2000 #166].
Preparation for developmental arrest (seed/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. Acquisition of dessication tolerance is part of the seed maturation program. 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. Viviparous mutants fail to undergo the maturation program leading to seed dormancy but instead germinate directly. Morphogenesis in viviparous mutants 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. Viviparous mutants are either ABA deficient or insensitive. An ABA independent genetic program is also 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.
Contributions of different flower parts to the fruit
Most fruit develops from the ovary. In fact some schemes classify fruit derived from a single ovary as “true fruits” while “false fruits” are composed of tissues derived from flower parts other than the ovary or from more than one ovary.
In “true fruits” the outside of the fruit is called the pericarp and develops from the ovary wall. The pericarp can be dry and papery, like in maple or dandelions, woody like in nuts or fleshy as in berries (grapes and tomatoes) and stone fruits (cherries and peaches). These pericarp differences reflect adaptations to different dispersal mechanisms (eg. wind for papery pericarps, animal consumption for fleshy fruits). The fruit can contain a single seed as in corn, or many seeds like a pea pod or pumpkin. The pericarp of some fruits is further differentiated into specialized layers called exocarp, meso- and endocarp. For example in citrus the rind is the exocarp, the white covering is the mesocarp and the juice sacs are the endocarp.
Many fruits we consider berries, such as raspberries and strawberries, are botanically not classified as berries. Raspberries are examples of aggregate fruits. Each juicy little sphere is actually an individual fruit of the same class as cherries, and what we consider as the fruit is really an aggregation of fruits.
Strawberries and apples are examples of accessory fruits, where some of the fleshy tissue is derived from flower parts other than the ovary. Strawberry fruits are actually what we consider the seeds. They are called achenes, which are dry fruits in the same category as dandelions. The fleshy part that we eat develops from the receptacle. Most of the fleshy tissue in apples develops from the hypanthium which is a region of the flower where sepals, petals and stamens are all fused to the ovary. Thus all floral organs contribute to the fleshy portion of apples.
Phases of fruit development
Fruit development can generally be considered to occur in four phases: fruit set, a period of rapid cell division, a cell expansion phase, and ripening/maturation.
Fruit set involves the decision whether to abort the ovary or proceed with fruit development. Fruit set is normally dependent on pollination. Pollen triggers fruit development indicating that positive signals are generated during pollination. In the absence of these signals, the flowers abscise. Growing pollen produces GA and application of GA can induce parthenocarpic fruit, therefore it is believed that GA is a triggering signal. Lagging slightly behind the growing pollen tube is a wave of increased auxin production by the style and then the ovary. Auxin application can also induce parthenocarpy and so it is thought that GA acts by inducing auxin production. However, most GA deficient mutants are able to produce fruit indicating that this is not the sole mechanism to induce fruit development and in an auxin insensitive tomato mutant, fruit growth is normal.
Continued fruit development usually relies on the continued presence of developing seeds. Seed abortion or removal causes fruit abortion, which can be reversed with auxin application. For example. removal of strawberry “seeds” prevents the development of the receptacle as a “fruit” but if auxin is applied following seed removal, fruit development continues. Commercial crops that produce parthenocarpic (seedless) fruits, such as bananna, often show quantitaive or qualitative differences in GA or auxin content in the ovary when compared to nonparthenocarpic varieties.
The phase of rapid cell division involves all growing parts of the fruit. This is thought to be controlled by the developing seeds. The number of fertilized ovules in a fruit is correlated with both the initial cell division rate and the final size of the fruit. Also, fruits with an uneven distribution of seeds are often lopsided. There is a correlation between cytokinin levels in developing embryos and cell division in surrounding tissues but there is no direct evidence that embryo cytokinin in fact regulates fruit cell division. It is difficult to reconcile the complete development of parthenocarpic fruit with the requirement of embryos for cell division except to say that parthenocarpy represents an abnormal situation.
The cell division phase gradually shifts into the cell expansion phase. The rate and duration of cell division varies among fruits and also among tissues within a fruit. Tissues made up of many small cells at maturity continue dividing while tissues composed of large cells have begun expanding. In tomato the cell division phase lasts approximately 7-10 days while cell expansion lasts 6-7 weeks. Cell expansion accounts for the largest increase in fruit volume, often contributing in excess of a 100 fold size increase. Gibberellins are also associated with fruit expansion and removal of the seeds from pea pods inhibited GA biosynthesis in the pericarp. Many believe that auxins from seeds regulate cell expansion of the pericarp, but auxin application does not always compensate for seed removal, and in an auxin insensitive tomato mutant, fruit growth is normal.
Ripening represents the shift from the protective function to dispersal function of the fruit. Ripening occurs synchronously with seed and embryo maturation, as described in the lecture on embryo development. In dry fruits (cereals, nuts, dandelions) ripening consists of desiccation and is considered maturation. Ripening in fleshy fruits is designed to make the fruit appealing to animals that eat the fruit as a means for seed dispersal. Ripening involves the softening, increased juiciness and sweetness, and color changes of the fruit. Fleshy fruits are either climacteric or non-climacteric. Climacteric fruits produce a respirative burst with a concomitant burst in ethylene synthesis, as the fruits ripen. These include fruits with high degrees of flesh softening, like tomato, banana, avacado, peach etc.
Ripening has been most intensively studied in tomato. Ethylene is a major regulator of the ripening process. Inhibitioin of ethylene with inhibitors, transgenic approaches or mutants blocks ripening. Exogenous ethylene accelerates ripening. There are also developmental factors involved because fruit does not attain competence to respond to ethylene until near the end of the cell expansion phase (the mature green stage). Several genes associated with ripening are ethylene inducible. This occurs transcriptionally in most genes but at least one is known where mRNA accumulation is regulated post-transcriptionally. None of these genes are induced until competence for ethylene response is attained.
The tomato never-ripe mutation blocks fruit ripening and is insensitive to ethylene. The mutated gene is similar to the ethylene receptor isolated from arabidopsis, suggesting that never-ripe is an ethylene receptor mutant. NR mRNA is not expressed until the mature green stage, suggesting that lack of this ethylene receptor might be related to the lack of competence to respond to ethylene at earlier stages.
Ethylene production is autocatalytic. That is, exposure to ethylene stimulates the synthesis of more ethylene. This occurs because the genes for the biosynthetic enzymes (e.g. ACC SYNTHASE) are ethylene inducible. The result is a positive feedback loop. Furthermore, the Never-ripe gene is ethylene inducible, resulting in a positive feedback loop for ethylene sensitivity as well. Both these factors contribute to the dramatic burst of ethylene production during ripening.
Fruit softening involves a partial breakdown of cell walls. Several enzymes are known to be involved in this process. Polygalacturonase hydrolyzes bonds in pectins. The gene for this enzyme is ethylene inducible.
Changes in fruit color involve changes in the expression of pigment biosynthetic genes. The major pigment in tomato is a carotenoid. The first committed step in carotenoid biosynthesis is catalyzed by phytoene synthase, and the gene for this enzyme is induced by ethylene.
Seeds have mechanisms to ensure germination occurs only under favorable environmental conditions for seedling growth. The primary factors are water availability and season. All seeds must imbibe water to germinate and for some this is the only requirement. Some also contain growth inhibitors that must be leached out of the seed. Some have impervious seed coats that must be fractured by freezing or passage through the digestive tract of an animal. Yet others have light or photoperiod requirements. All these mechanisms ensure the seeds germinate in the correct season and when moisture is available.
Arabidopsis seeds have certain requirements for germination, including a period of dormancy (which can be substituted for by cold treatment) and light (a phytochrome response). Mutations in a gene called DAG1 (Dof Affecting Germination1) cause seeds that germinate in the dark without a dormancy period. Dof proteins are zinc finger transcription factors. The gene is expressed in the maternal tissues and all seeds of a mutant show this phenotype even if they result in pollination by a wild type (i.e. the embryo is wild type). Therefore, the maternal tissues during seed development control the dormancy behavior of the seed after being shed from the plant.
Upon imbibition, active metabolism resumes. Imbibed seeds contain high levels of GA. It is produced by the germinating embryo and stimulates the synthesis of hydrolytic enzymes by inducing the transcription of their genes. These enzymes appear after radicle elongation and are therefore postgerminative. The hydrolytic enzymes include proteases, amylases and lipases that break down storage compounds making building blocks available to the growing seedling. One enzyme of particular importance is a-amylase which cleaves starch into glucose and maltose molecules. This reaction is of economic importance to the malting industry and so the regulation of a-amylase gene expression has been carefully studied. It is transcriptionally induced by GA. Plants also contain a unique metabolic pathway called the glyoxylate cycle. This enables plants to convert fatty acids of the stored lipids into carbohydrates, specifically glucose and sucrose. In contrast, animals are unable to convert fatty acids to glucose.
GA and ABA act antagonistically to regulate the germination vs. maturation programs. ABA promotes maturation while GA promotes germination. As mentioned, ABA is necessary for seed maturation because ABA deficient mutants are viviparous and desiccation intolerant. Therefore, without ABA, seeds directly enter the germination program. Exogenous ABA can inhibit germination following dormancy. Conversely, promotes germination. GA is required for germination because GA deficient mutants are unable to germinate. Exogenous GA application to developing seeds can block maturation and induce vivipary. The VP1/ABI3 protein is a central regulator in these functions. This protein is a transcription factor that promotes the expression of maturation genes and inhibits the expression of germination genes. Mutants in this gene are ABA insensitive.