In Vitro Cell. Dev. Biol.Plant 41:620644, September October 2005 q 2005 Society for In Vitro Biology 1054-5476/05 $18.00+0.

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DOI: 10.1079/IVP2005686

SEED COATS: STRUCTURE, DEVELOPMENT, COMPOSITION, AND BIOTECHNOLOGY

SE1, SHUYOU HAN1, LORETA GUDYNAITE JAIMIE A. MOI -SAVITCH1, DOUGLAS A. JOHNSON1,
1

AND

BRIAN L. A. MIKI2*

Ottawa-Carleton Institute of Biology, Department of Biology, University of Ottawa, P.O. Box 450, Station A, Ottawa, Ontario, Canada K1N 6N5 2 BioProducts & BioProcesses, Research Branch, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada K1A OC6
(Received 8 May 2005; accepted 17 May 2005; editor E. C. Pua)

Summary Although seeds have been the subject of extensive studies for many years, their seed coats are just beginning to be examined from the perspective of molecular genetics and control of development. The seed coat plays a vital role in the life cycle of plants by controlling the development of the embryo and determining seed dormancy and germination. Within the seed coat are a number of unique tissues that undergo differentiation to serve specic functions in the seed. A large number of genes are known to be specically expressed within the seed coat tissues; however, very few of them are understood functionally. The seed coat synthesizes a wide range of novel compounds that may serve the plant in diverse ways, including defense and control of development. Many of the compounds are sources of industrial products and are components of food and feeds. The use of seed coat biotechnology to enhance seed quality and yield, or to generate novel components has not been exploited, largely because of lack of knowledge of the genetic systems that govern seed coat development and composition. In this review, we will examine the recent advances in seed coat biology from the perspective of structure, composition and molecular genetics. We will consider the diverse avenues that are possible for seed coat biotechnology in the future. This review will focus principally on the seed coats of the Brassicaceae and Fabaceae as they allow us to merge the areas of molecular biology, physiology and structure to gain a perspective on the possibilities for seed coat modications in the future. Key words: biotechnology; Brassica; genes; legume; seed coat.

Introduction Seeds have played a fundamental role in the development of civilizations by supplying food, feed, natural products, and traditional medicines. The application of genetics resulted in breeding for quality and yield and the adoption of agricultural and industrial processes for harvesting their valuable components. Because cultivated seeds remain a vital link to health and prosperity, acquiring knowledge of plant seed biology has been a priority for most cultures. Today years of research have generated new domesticated varieties of plants that have diverged considerably from their wild ancestors in form and traits, yet we still understand little about the biological processes that govern these valuable traits. It is a goal of agricultural research to accelerate the development of seed quality and yield and to diversify their traits to satisfy more of our needs. Technologies such as genomics, proteomics, and metabolomics promise to accelerate our understanding of seeds and thus open new possibilities for uses. Emerging technologies for generating transgenic plants provide an

*Author to whom correspondence should be addressed: Email mikib@ agr.gc.ca The authors have contributed equally and are considered rst authors.

opportunity for enhancing existing seed traits and for adding value to seeds. Fundamental to the study of seed coat developmental genetics is a comprehensive understanding of seed coat morphology. Seed coats develop from the integuments that surround the ovule prior to fertilization. Before fertilization, cells of the integuments are relatively undifferentiated. However, development after fertilization can include extensive differentiation of the cell layers into specialized cell types. In addition, some cell layers in the seed coats may accumulate large quantities of certain substances, such as mucilage or pigments that can also contribute to overall seed morphology. A number of cell types are found in common in seed coats of both the Brassicaceae and Fabaceae. Some cell layers will not undergo any signicant differentiation and will remain parenchymatous. These are often crushed at maturity. Other cells will undergo a slight thickening of the cell wall and thus become collenchymatous. Some cell layers will undergo extensive secondary thickening of some parts of the cell walls and will, thus, become sclerotic. These layers are often called palisade layers, especially if the cells also become elongated in the radial plane. Figure 1 demonstrates the positioning of different cell layer types in Arabidopsis and soybean. By harboring the embryo, seed coats separate one generation of plants from the next and ensure the survival of the offspring. Strong 620

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(a)

mu p pa en

In this review, we will examine the recent literature on seed coats to review our understanding, particularly at the level of molecular biology, and to assess the potential for new seed coat modications and uses. We will pay attention to the legume seed, which represents a well-established system for studying metabolic control mechanisms, and Arabidopsis, which is the major system for understanding genetic control mechanisms. Finally, we will examine a wide range of novel compounds found in the seed coats of diverse plants and consider the biotechnological applications for diversifying our use of seed coats. Role of Seed Coats in Plant Development

(b) p

pa a em

FIG . 1. Schematic diagrams illustrating the general organization of the seed coat of Arabidopsis thaliana and soybean (G. max L. Merrill). a, A. thaliana. The seed coat at the torpedo stage. mu, Mucilaginous epidermal cells; p, palisade layer with thickened inner cell walls; pa, parenchymatous cells; en, endothelium layer. The mucilaginous cells and the palisade layer comprise the outer integument while the inner integument consists of parenchymatous cells and the endothelium layer. Adapted from Beeckman et al. 2000; b, Soybean. A mature seed coat. p, Palisade layer; h, hourglass cells; pa, partially crushed parenchyma; a, aleurone; em, crushed endosperm The palisade layer and the hourglass cells comprise the outer integument while the inner integument consists of parenchymatous cells. Adapted from Miller et al. (1999).

impermeable seed coats protect the embryos during dormancy and maintain an environment around the embryo that is conducive for quiescence (Bewley, 1997). During germination the seed coat must weaken and break open and may provide components that contribute to biotic- and abiotic-stress resistance. Recently, a large body of research has shown that the seed coat is vital for directing the nutrient supply to the embryo during seed development (Weber et al., 2005). By governing seed dormancy and germination the seed coat plays an important role in determining the optimal environmental conditions for the viability and growth of the next plant generation. An understanding of these roles is essential for developing crops suitable for agricultural production. The seed coat is also a rich source of many valuable naturally occurring compounds and the use of transgenic technologies can greatly expand and diversify them. Generally, the seed coat has not been characterized at the molecular level to the extent of the embryo and endosperm. It is now recognized that such knowledge is a prerequisite for the development of plants with modied seed coat traits.

Although the seed coats of different species vary greatly in structure and composition, they undergo similar phases of development in relation to the embryo and endosperm. For example, in legume seed development the seed coat and endosperm develop rst, followed by the development of the embryo, maturation of the seed coat, and maturation of the embryo (Weber et al., 2005). This will be discussed later in detail for Arabidopsis. The coordination of these events is governed by communication among the tissues of the seed organs. For example, communication between the seed coat and endosperm of Arabidopsis has shown to be particularly important by targeted cell ablation experiments (Weijers et al., 2003). Early embryo development and differentiation is controlled by the maternal tissues, therefore signals must be transmitted through the seed coat and endosperm before they can reach the embryo. For example, specialized cell types called transfer cells facilitate the transfer of nutrients within the seed (Thompson et al., 2001). An elaborate model for the maternal control of embryo development through sugar metabolism has been developed and well discussed in the literature (Wobus and Weber, 1999; Weber et al., 2005). Such models must also integrate a variety of other signaling pathways that involve phytohormones (Bewley, 1997), hypoxia (Rolletschek et al., 2002) and carbon dioxide recycling (Furbank et al., 2004) to mention a few. The phytohormone and sugar response pathways are known to converge in seed development and are well studied (Gibson, 2004). Cross talk among the various pathways must play a major role in the control of seed development and provide mechanisms for communication among the various seed organs, which undergo coordinated development. A number of recent reviews examine these in detail (Olszewski et al., 2002; Gibson, 2004; Weber et al., 2005). As we will show later, there is a wide range of seed coat morphologies and compositions, therefore the details of these processes will likely differ among species. Metabolic Controls Seed coat invertases of legumes play a central role in the maternal control of seed development. Cell wall invertases facilitate assimilate unloading by increasing the sucrose gradient in the unloading zones of the legume seed coat (Weber et al., 1995). The high hexose-to-sucrose ratios are believed to promote embryo growth by enhancing cell division (Borisjuk et al., 1998). This may be mediated through sensing pathways that use D-type cyclins (Weber et al., 2005). The growth of the embryo within the connes of the legume seed coat is correlated with the crushing of the inner seed coat cell layers and loss of cell wall invertase activity and the assimilate supply (Weber et al., 1995).

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The expression of yeast invertase in transgenic Vicia narbonensis to alter the sugar composition in the seed led to alterations in embryo development and partitioning of carbon into starch as a storage product (Weber et al., 1998). Data from transgenic tobacco with articially prolonged invertase activity revealed no comparable alterations in development or carbon partitioning to storage lipids, indicating that the switch in metabolism to storage product accumulation in the seed is not simply a direct response to the elevated hexose to sucrose ratio conferred by the invertase activity in all plants (Tomlinson et al., 2004). Dormancy and Abscisic Acid Through seed dormancy the progression of the embryo into a seedling or plantlet is arrested. This process plays a vital role in the plant life cycle by allowing progeny to survive adverse environmental conditions and to coordinate their growth with the most favorable conditions. In domesticated crops, this is less necessary and has been eliminated by breeding (Bewley, 1997). The seed coat is a major determinant of dormancy, particularly in species with seed coat-imposed dormancy in which the embryo is physically constrained from developing further. This differs from embryo dormancy where the embryos of certain species are dormant. A critical interplay between the seed coat and embryo occurs that involves the phytohormone abscisic acid (ABA), which reaches the seed in two phases. In Nicotiana, studies with ABA mutants revealed that the rst phase of ABA is synthesized in the maternal vegetative tissues and translocated to the seed to initiate early seed development. The second phase, which is needed for seed coat maturation, is synthesized in the seed coat itself (Frey et al., 2004). Similarly, ABA produced in the embryo was needed to complete embryo dormancy in Arabidopsis (Karssen et al., 1983) after the arrest of embryo growth (Raz et al., 2001). Mutations in the ABA synthetic genes can result in major disruptions in seed development, including dormancy, and, reciprocally, mutations in seed coat development can result in defects in dormancy (reviewed by Finkelstein et al., 2002). Control of seed coat development and dormancy are therefore fundamentally linked. The signaling networks controlled by ABA in Arabidopsis are very extensive and it has been estimated that 8 10% of the Arabidopsis genes on a partial chip are ABA-responsive (Finkelstein et al., 2002). Many of the ABA responses associated with embryo maturation and stress responses also occur in the seed coat. Interestingly, an Arabidopsis orthologue of the ABA-responsive gene rab 28 which is a late embryogenesis abundant protein (LEA), Atrab 28, is expressed selectively in the outer integuments of the seed coat, embryos and silique epidermis but is not responsive to ABA in somatic cells (Arenas-Mena et al., 1999). The storage proteins which accumulate in the embryo during ABA-induced maturation also accumulate in the seed coats of legumes where they may act as deterrents to certain predators and provide selection pressure for the evolution of pests (Silva et al., 2004). Germination and Gibberellin Germination, which involves factors that break dormancy, is initiated with the imbibition of water and resumption of respiration and metabolic activity. Gibberellin signaling appears to play a role in this activity and may act antagonistically to ABA (Bewley, 1997).

In soybean, which has a very hard, impermeable seed coat, water enters through small cracks in the seed coat (Ma et al., 2004a). Germination is completed once the radicle breaks through the seed coat. In species with coat-enhanced dormancy, such as Nicotiana, the constraints imposed by the seed coat and underlying endosperm must be weakened enzymatically. This occurs sequentially through the rupture of the seed coat followed by rupture of the endosperm. It appears that enzymes such as class I b-1,3-glucanase I (bGluI) are involved in both processes by weakening of the cell walls (LeubnerMetzger, 2002). De novo expression of bGluI in the inner seed coat of dry seeds appears to be among the rst steps in releasing coatenhanced dormancy (Leubner-Metzger, 2005). The signals for the induction of bGluI appear to involve the decline in ABA and the onset of GA signaling (Olszewski et al., 2002). Genes coding for other cell wall-weakening enzymes, such as a-amylase, have been shown to be induced in seed coats of morning glory coordinately with the induction of gibberellin 3-oxidases (Nakajima et al., 2004). Control of GA:ABA balance is likely to have an effect on the embryo in addition to the seed coat, in that GA may stimulate the resumption of embryo growth to achieve radical protrusion (Debeaujon and Koornneef, 2000). In species such as radish and soybean, reactive oxygen intermediates such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals are synthesized by the seed coat and embryos as by-products of the metabolic processes that accompany germination and presumably provide resistance to pathogens during seedling emergence (Schopfer et al., 2001). Oxidases such as peroxidases, oxylate oxidases, or amine oxidases can be potential sources of hydrogen peroxide. Peroxidases are commonly found in seed coats, particularly soybean seed coats (Gijzen, 1993; Welinder and Larsen, 2004) and seed coat-specic forms of oxylate oxidase have been found in barley (Wu et al., 2000). Structure and Development of Brassicaceae Seed Coats The seed coats of the Brassicaceae are becoming very important for understanding the genetic mechanisms that govern seed coat development, particularly with the emergence of Arabidopsis as a model genetic system for plants (Kuang et al., 1996; Beeckman et al., 2000; Western et al., 2000; Windsor et al., 2000). In the past, the seed coat has also served as a useful characteristic for taxonomy (Vaughan and Whitehouse, 1971; Bouman, 1975; Zeng et al., 2004). Today, it is understood that the family includes a large number of important crops and that seed coat characteristics are associated with important agronomic traits, motivating studies on seed coat structure and development (Bouman, 1975; Van Caeseele et al., 1981, 1982; Kuang et al., 1996; Beeckman et al., 2000; Western et al., 2000; Windsor et al., 2000). From these studies, a common seed coat structure has been identied within the Brassicaceae, consisting of four distinct layers (Vaughan and Whitehouse, 1971; Bouman, 1975). The outermost, or epidermal layer of the outer integument is most frequently one celllayer thick and may or may not contain mucilage (Vaughan and Whitehouse, 1971; Bouman, 1975). Below the epidermal layer, in the middle of the outer integument, is a subepidermal layer that may consist of one, or more cell layers and is typically parenchymatous, although it may be collenchymatous, or sclerotic (Vaughan and Whitehouse, 1971; Bouman, 1975). In some species, including Arabidopsis, this cell layer is entirely absent. The innermost layer of the outer integument is most often one-cell thick and forms a sclerotic

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layer, sometimes known as the palisade layer. This layer is often characterized by thickened inner tangential and radial walls (Vaughan and Whitehouse, 1971; Bouman, 1975). These rst three layers are derived from the outer integument of the ovule, while the fourth layer is derived from the inner integument. The fourth layer, formed of parenchyma cells compressed at maturity, is referred to as the pigment layer, as pigments most often accumulate within this layer (Vaughan and Whitehouse, 1971; Bouman, 1975). At maturity, the outer layer of the endosperm is closely associated with the inner integument and forms an aleurone layer (Bouman, 1975). The epidermal layer of Brassicaceae seed coats may contain mucilage, a pectic polysaccharide that may contribute to seed hydration and seed dispersal. In Arabidopsis, mucilage can contribute to efcient germination and seedling establishment when the seeds are in an environment of low water potential (Peneld et al., 2001). Mucilage is present in a dehydrated form in the mature seeds, but, upon imbibition, it expands, rupturing the outer cell wall of the epidermal cells enveloping the entire seed. Mucilage is also present in the seed coats of some Solanaceae, Linaceae, and Plantaginaceae species. In some agricultural species, such as yellow mustard, mucilage is benecial, as it contributes to the quality of the nal product. In other species, the presence of mucilage is an economical disadvantage, as it impedes the removal of the seed coat during processing. Another important feature of Brassicaceae seeds is the presence of avonoids found in the inner integument. The role of avonoids in plants is diverse and includes protection against UV-B light, regulation of auxin transport, signaling between plants and microbes, male fertility, and plant defense through antimicrobial activity and decreased palatability (Winkel-Shirley, 2001). Furthermore, avonoids have recently gained importance for their role in preventing bloat and controlling internal parasites in ruminants (Aerts et al., 1999). Some avonoids are also known to have antioxidant activity and may, therefore, be benecial for human health (Ross and Kasum, 2002). In seeds, avonoids have important functions in the induction of seed coat-imposed dormancy, as well as in seed longevity and quality (Debeaujon et al., 2000). In many Brassicaceae species, the absence of avonoids in yellow-seeded cultivars is correlated with greater oil yield, higher protein content, and a reduction in undesirable characteristics such as ber content (Simbaya, 1995). Arabidopsis The mature seed coat of Arabidopsis is mostly composed of ve cell layers of maternal origin (Beeckman et al., 2000). The outer two cell layers of the seed coat, which are derived from the outer integument, form the epidermal and palisade layers. A subepidermal parenchyma layer is absent in the Arabidopsis seed coat. The inner integument has three cell layers, although the middle layer is only present in the curving body, such that the inner integument is composed of two cell layers near the chalaza and micropyle (Beeckman et al., 2000). Outer and inner integuments. In Arabidopsis, two integuments, both epidermal in origin, initiate close to the chalaza and grow to surround the ovule (Robinson-Beers et al., 1992; Schneitz et al., 1995). The outer integument has two cell layers with prominent vacuoles (Schneitz et al., 1995). Cells of the outer layer of the outer integument are wider on their convex side, which, in addition to the

greater proliferation of cells on this side, contributes to the curvature of the integument. Furthermore, the convex side of the ovule experiences a more rapid growth of the cells of the outer integument, which further increases the curvature (Robinson-Beers et al., 1992; Schneitz et al., 1995). This asymmetrical growth of the outer integuments initiates the abaxial adaxial axis of the developing ovule (Balasubramanian and Schneitz, 2002). Initially, the inner integument is also composed of two cell layers (Schneitz et al., 1995). The outer layer of the inner integument undergoes vacuolization and ultimately appears most similar to the two cell layers of the outer integument (Schneitz et al., 1995). The inner layer of the inner integument, however, develops into a distinctive endothelium, which is closely connected to the embryo sac. The endothelium has cuboid cells with little vacuolization and a compact appearance (Schneitz et al., 1995). Just prior to fertilization, a third cell layer of the inner integument is initiated and becomes the middle layer of the inner integument. This new layer, however, only surrounds part of the embryo sac (Schneitz et al., 1995). Ultimately, these cells are also vacuolated, to an even greater extent than the outer integument and the outer layer of the inner integument (Schneitz et al., 1995). The epidermal layer. Following fertilization, cells of the outer integument initially have a large central vacuole (Beeckman et al., 2000; Western et al., 2000; Windsor et al., 2000). Both layers also begin to accumulate starch grains early in development, around the globular stage of embryo development (Beeckman et al., 2000; Western et al., 2000). They accumulate near the outer cell wall in the outer layer and near the inner cell wall of the inner layer (Windsor et al., 2000). The outer cell layer, or epidermis initially increases in size immediately following fertilization, as does the central vacuole. Around the torpedo stage, the epidermal layer begins to produce mucilage, which is deposited between the primary radial cell wall and the protoplasm. The mucilage is pectinaceous and mostly composed of unsubstituted rhamnogalacturonan I (RGI), with rhamnose and uronic acid being the main sugars (Goto, 1985; Peneld et al., 2001). Other monosaccharides are present in lower ratios. As mucilage deposition continues, the protoplasm is compressed into a column located in the center of the cell, a structure known as the columella (Western et al., 2000; Windsor et al., 2000). The primary cell wall remains attached to the plasma membrane along the inner tangential wall, as well as the center of the outer tangential wall and the inner sections of the radial walls. As the protoplasm continues to compress, the vacuole shrinks and disappears. The starch grains also begin to disappear, coincident with the deposition of a secondary cell wall around the columella (Western et al., 2000; Windsor et al., 2000). This temporal sequence likely indicates that the starch granules are the precursors to the polysaccharides required for the reinforcement of cell walls. At maturity, the columellae appear to entirely consist of cell wall material and spread along the inner tangential wall and up parts of the radial walls, providing reinforcement in these areas (Western et al., 2000). The cytoplasm is typically no longer observable in this layer (Kuang et al., 1996; Western et al., 2000). At maturity, the mucilage has dried into a thin, compressed layer under the primary cell wall, causing the cell wall to drape over the columella (Western et al., 2000; Windsor et al., 2000). During imbibition, the tangential primary cell walls likely break rst as the mucilage swells, while the radial walls and the top of the columellae remain attached to the outer wall (Western et al., 2000; Windsor et al., 2000).

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The palisade layer. In the inner layer of the outer integument, known as the palisade layer, the starch granules rst enlarge, while the central vacuole divides into two or three smaller vacuoles. As the starch granules begin to shrink during the torpedo stage, the inner tangential cell walls are thickened or reinforced (Beeckman et al., 2000; Western et al., 2000; Windsor et al., 2000). Again, the starch granules likely contribute to cell wall reinforcement. At maturity, the palisade layer is largely collapsed, although it may persist in some areas (Beeckman et al., 2000). The pigment layer. At fertilization, the innermost layer of the inner integument, known as the endothelium, is composed of isodiametric cells with dense cytoplasm and few starch grains (Kuang et al., 1996). Immediately following fertilization, this layer then becomes vacuolated (Beeckman et al., 2000). During the onecell embryo stage, the endothelial cells form large central vacuoles, which are subsequently lled with light yellow pigments during the two-cell embryo stage. The majority of these pigments are proanthocyanidins (PAs) (Devic et al., 1999). The pigments initially ll the vacuole, but ultimately accumulate in the cytoplasm as well. Pigment accumulation is maximal at the early torpedo stage of embryo development. In the late torpedo stage, the pigment begins to disappear from the center of the cells, remaining only at the periphery (Beeckman et al., 2000). At maturity, the endothelial layer is broken down, such that only dead cells remain, although these may be crushed and indistinguishable from the layers above (Beeckman et al., 2000). The other two layers of the inner integument are highly vacuolated, especially on the abaxial side of the seed (Beeckman et al., 2000). Beginning in the bent-cotyledon stage, these layers begin to shrink and disintegrate as the embryo grows (Kuang et al., 1996; Beeckman et al., 2000). At maturity, these layers are completely crushed and, with the remains of the inner layer, form a brown pigment layer (Beeckman et al., 2000). Flavonoids accumulate in all the three layers of the inner integument (Debeaujon et al., 2000). At maturity, the seed coat is desiccated, allowing the oxidation of the avonoids found in these layers. The oxidation process causes the avonoids to turn brown, which is the source of the color of the mature seed coat. Other Brassicaceae Many Brassica species have a seed coat structure that is similar to that of Arabidopsis. Seed coat development also proceeds through many of the same steps. Brassica campestris. The outer integument of the seed coat of Brassica campestris L. cv. Candle has been studied in great detail (Van Caeseele et al., 1981, 1982). The palisade layer develops a secondary thickening of the inner tangential cell wall throughout development, much like Arabidopsis (Van Caeseele et al., 1982). Unlike Arabidopsis, some parts of the radial cell walls are also thickened, becoming progressively thinner as they approach the outer tangential cell walls. As thickening occurs, the inner tangential cell walls of the palisade layer become closely associated with the outer tangential wall of the outer layer of the inner integument (Van Caeseele et al., 1982). The palisade cells elongate along the radial plane, in regions that have retained the primary cell wall, and these areas are associated with numerous vacuoles (Van Caeseele et al., 1982). At maturity, sections of the radial walls without secondary thickening collapse into folds. The thickened

tangential walls appear fused to the adjacent region of the inner integument (Van Caeseele et al., 1982). B. campestris, like Arabidopsis, has a mucilaginous epidermal layer; however, unlike Arabidopsis, the epidermal cells do not form the distinct columella structure and, at maturity, this layer is compressed, much like the parenchyma cells of the inner integument (Van Caeseele et al., 1981). Initially following pollination, there is little change in the epidermal cell structure, with the exception of the amyloplasts, which further develop starch grains. However, by 15 d after pollination, degraded organelles, membranes, and possibly endoplasmic reticulum and tonoplasts are sometimes visible in the cytoplasm. As the compression of the epidermal layers continues, mucilage becomes deposited and appears stratied, likely as a result of layered deposition (Van Caeseele et al., 1981). The cytoplasm becomes strongly disorganized by 25 d after pollination and the radial cell walls of the epidermis begin to collapse. Ultimately, cytoplasm is no longer visible and mucilage deposition is completed, forming a meniscuslike structure in the middle of each elongated cell. Along the inner tangential wall, the mucilage appears to be thickest but the entire lumen of the cell is lled (Van Caeseele et al., 1981). At maturity, the outer tangential cell walls of the epidermal layer are easily ruptured. Capsella bursa-pastoris. The mature seed coat of C. bursapastoris is almost identical to that of Arabidopsis. It has a mucilaginous epidermal layer that forms central columns (Bouman, 1975). The subepidermal layer is absent and the outer integument remains two-cell layered throughout development. Adjacent to the epidermal layer, therefore, is the palisade layer, which is sclerotized (Bouman, 1975). A minor difference from Arabidopsis is that both the inner tangential cell walls and all of the radial walls of the palisade layer are secondarily thickened, and these cells are elongated along the tangential plane (Vaughan and Whitehouse, 1971). All three layers of the inner integument are crushed at maturity and partially resorbed, although the outermost two layers are crushed before the pigmented endothelial layer (Bouman, 1975). Sinapis alba and Brassica nigra. The seed coat of S. alba and B. nigra are very similar with the exception of the inner integument, which does not accumulate pigments in B. nigra (Bouman, 1975). In these two species, the outer integument originates subdermally into three-cell layers and even four-cell layers in some areas (Bouman, 1975). The epidermal layer is mucilaginous, although it does not form central columns, as seen in Arabidopsis and C. bursa-pastoris (Bouman, 1975). A subepidermal layer is present in the outer integument and is formed of parenchyma cells that may appear collenchymatous at times. Like C. bursa-pastoris, the palisade layer displays secondary thickening of both the inner tangential and the radial cell walls, although the cells remain isodiametric (Vaughan and Whitehouse, 1971; Bouman, 1975). The inner integument has two cell layers in some areas and multiple cell layers in others and is completely crushed at maturity, such that cells are no longer distinguishable (Bouman, 1975). Lunaria annua. The seed coat of Lunaria annua exhibits a rare vascularization within the innermost layer of the inner integument (Bouman, 1975). The innermost layer, in this species, demonstrates characteristics of an integumentary tapetum, which may play a special role in nutrition in the seed (Bouman, 1975). The somewhat mucilaginous epidermal layer of the L. annua seed coat is composed of large cells that likely collapse at maturity. Cells of the palisade

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layer are cubic and the walls are thickened on both the inner and the outer tangential walls (Vaughan and Whitehouse, 1971; Bouman, 1975). Both the subepidermal layer and the outer layers of the inner integument are crushed and partially resorbed at maturity (Bouman, 1975). The innermost layer, however, forming the integumentary tapetum, develops into sclereids, with cell wall thickening along the inner tangential wall and parts of the radial walls (Bouman, 1975). On the dorsal side of the seed, the crushing of the parenchyma layers leads to the formation of a seed wing (Bouman, 1975).

A list of some of the seed coat genes studied in Arabidopsis is given in Table 1.

Genes Involved in Ovule Integument Development Structure and patterning of the mature seed coat is often dependent upon the correct initiation, growth, and differentiation of the integuments. In Arabidopsis, the proximal distal and abaxial adaxial poles found in the mature seed coat are formed during integument development. Furthermore, after fertilization there is no change in the number of cell layers in the seed coat, such that the development of the seed coat is largely a process of differentiation as opposed to proliferation. Therefore, the regulation of development of the integuments prior to fertilization can have profound effects on nal seed coat structure, composition, and function. The control of ovule development has been reviewed in a number of papers (Grossniklaus and Schneitz, 1998; Schneitz, 1999; Skinner et al., 2004). The following section will focus on genes specically involved in the development of the integument, with an emphasis on recent work highlighting the importance of these genes for future seed and seed coat development. Integument identity. BELL1 (BEL1) is an Arabidopsis homeodomain protein that contributes to ovule integument identity (Reiser et al., 1995). bel1 mutant plants produce only a single integumentlike structure (Robinson-Beers et al., 1992; Modrusan et al., 1994).

Molecular Genetics of Brassicaceae Seed Coats Several genetic systems specic to the seed coat in Arabidopsis were rst identied by mutations followed by the identication of the genes. As a model, Arabidopsis has become very important for understanding the genetic processes that govern development of the seed coat and that are responsible for seed coat qualities, such as mucilage production and pigment accumulation. These genes are very important for future studies that aim to alter or modify seeds through biotechnological strategies. It is assumed that the most basic genetic regulatory systems governing Arabidopsis seed coat development will also apply to the other Brassicaceae species; however, each will have to be functionally assessed individually.

TABLE 1 EXAMPLES OF GENES EXPRESSED IN THE ARABIDOPSIS SEED COAT Category Ovule integument development Gene BEL1 INO SUP ANT NZZ SIN1/DCL1 TT1 TT2 TT3 TT4 TT5 TT6 TT7 TT8 TT12 TT16 TT18/ TDS4 TT19 TTG1 TTG2 BAN AHA10 EGL3 GL2 MYB61 AtMYB23 MUM4/ RHM2 AP2 dVPE Locus AT5G41410 AT1G23420 AT3G23130 AT4G37750 AT4G27330 AT1G01040 AT1G34790 AT5G35550 AT5G42800 AT5G13930 AT3G55120 AT3G51240 AT5G07990 AT4G09820 AT3G59030 AT5G23260 AT4G22880 AT5G17220 AT5G24520 AT2G37260 AT1G61720 AT1G17260 AT1G63650 AT1G79840 AT1G09540 AT5G40330 AT1G53500 AT4G36920 AT3G20210 Encodes Homeodomain protein Member of the YABBY family of putative transcription factors Zinc nger domain protein AP2 domain protein Novel nuclear protein Multidomain ribonuclease Zinc nger domain protein MYB domain protein Dihydroavonol 4-reductase Chalcone synthase Chalcone isomerase Flavonone 3-hydroxylase Flavonone 30 -hydroxylase bHLH domain protein Multidrug secondary transporter-like protein MADS box protein Leucoanthocyanidin dioxygenase Putative glutathione S-transferase WD40 repeat protein WRKY domain protein Anthocyanidin reductase Plasma membrane H-ATPase bHLH domain protein Homeodomain-leucine zipper protein MYB domain protein MYB domain protein Putative NDP-L -rhamnose synthase Homeotic regulatory protein Putative asparaginyl endopeptidase References Reiser et al., 1995 Villanueva et al., 1999 Sakai et al., 1995 Elliott et al., 1996; Klucher et al., 1996 Schiefthaler et al., 1999 Golden et al., 2002 Sagasser et al., 2002 Nesi et al., 2001 Shirley et al., 1992 Feinbaum and Ausubel, 1988 Shirley et al., 1992 Wisman et al., 1998 Schoembohm et al., 2000 Nesi et al., 2000 Debeaujon et al., 2001 Nesi et al., 2002 Abrahams et al., 2003; Shikazono et al., 2003 Kitamura et al., 2004 Walker et al., 1999 Johnson et al., 2002 Devic et al., 1999; Xie et al., 2003 Baxter et al., 2005 Zhang et al., 2003 Di Cristina et al., 1996 Peneld et al., 2001 Matsui et al., 2005 Usadel et al., 2004; Western et al., 2004 Jofuku et al., 1994 Nakaune et al., 2005

Flavonoid biosynthesis

Mucilage production

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BEL1 may function by suppressing expression of AGAMOUS (AG), a homeotic gene that promotes oral meristem, carpel and stamen identity (Ray et al., 1994; Reiser et al., 1995). In the initial stages of ovule development, BEL1 is restricted to the region from which the integuments initiate. Although most bel1 mutants are female-sterile, viable seed is produced occasionally and these seeds have altered seed coat morphology, indicating the dependence of seed coat structure on proper integument development (Modrusan et al., 1994). bel1 mutants also have altered embryo sac development, although it is not clear if this is a result of altered integument development, or a pleiotropic phenotype of the bel1 mutant (Robinson-Beers et al., 1992; Modrusan et al., 1994; Western and Haughn, 1999). Integument patterning. A number of genes expressed in the integuments have been found to play important roles in ovule patterning. These genes include BEL1, INNER NO OUTER (INO), SUPERMAN (SUP), AINTEGUMENTA (ANT), and NOZZLE (NZZ). BEL1 and NZZ redundantly specify the chalazal region from which the integuments initiate, contributing to the proximal distal axis of the ovule primordium (Balasubramanian and Schneitz, 2000). ANT encodes an AP2 domain protein and its expression is limited to the distal chalaza and funiculus just prior to integument initiation and then to the integument primordial and distal funiculus once integument cell divisions begin (Elliott et al., 1996; Klucher et al., 1996; Balasubramanian and Schneitz, 2000). This spatial pattern of expression indicates that it may play an important role in forming the proximal distal axis. INO encodes a member of the YABBY family of transcription factors, and mutations in this gene result in ovules with an inner integument but no outer integument (Baker et al., 1997; Villanueva et al., 1999). In wild-type plants, INO is expressed in the ovule primordium in the cells that will give rise to the outer integument, but only on the abaxial side and is, therefore, important for the formation of the abaxial adaxial axis (Villanueva et al., 1999). Also important for the abaxial adaxial axis is SUP, which encodes a zinc nger transcription factor (Sakai et al., 1995). sup mutants develop an outer integument that grows equally on both the abaxial and adaxial side and thus may play a role in the maintenance of the abaxial adaxial axis as opposed to its initiation (Gaiser et al., 1995; Sakai et al., 1995). NZZ, in addition to specifying the proximal distal axis, plays a role in the regulation of INO expression, restricting it spatially to the abaxial epidermis (Balasubramanian and Schneitz, 2000, 2002). NZZ, which encodes a novel nuclear protein, may represent a molecular link between the proximal distal axis and the abaxial adaxial axis during ovule development (Schiefthaler et al., 1999; Balasubramanian and Schneitz, 2002). A recent study found that NZZ could directly bind INO, indicating an additional level of regulation that may also contribute to the formation of the abaxial adaxial polarity (Sieber et al., 2004). Morphogenesis and differentiation. INO also appears to function in the growth of the outer integument, as weak ino mutants can initiate an outer integument but the integument exhibits reduced growth compared to wild type (Villanueva et al., 1999). Another gene, SHORT INTEGUMENTS2 (SIN2) also plays a role in integument growth by promoting cell division (Broadhvest et al., 2000). SIN1, important for integument growth, is also known as DICER-LIKE1 (DCL1). Mutations in SIN1 lead to the development of integuments that are shorter than wild-type integuments, largely

as a result of reduced cell elongation as opposed to cell division (Robinson-Beers et al., 1992). Recently, SIN1 was cloned and found to encode a protein related to the Drosophila melanogaster gene Dicer, which functions in RNA silencing and plays an important role in animal development on a temporal scale (Golden et al., 2002). Dicer encodes a multidomain ribonuclease that specically digests double-stranded RNA. Interestingly, sin1 mutants demonstrate defects in embryo development that are maternally inherited, indicating that expression of SIN1 in the maternal sporophyte is controlling some aspects of embryo development (Ray et al., 1996). However, it is not clear if it is the expression of SIN1 in the integuments or in the funiculus that ultimately affects embryonic growth. A mutant exhibiting heart-shaped seeds, as opposed to the wild ontype oval shape was named aberrant testa shape (ats) (Le Kloosterziel et al., 1994). Microscopic analysis indicated that at the apical end of the seed, ats mutants have only three cell layers, as opposed to the ve normally visible in wild-type seeds and, within the three layers, there is no clear distinction between the outer and the inner integument. In addition, ATS may also function in postfertilization development as the epidermal layer of the seed coat of ats mutants is abnormal, composed of fewer larger cells compared to wild type. These epidermal cells also produce very little mucilage on-Kloosterziel et al., 1994). and the columellae are absent (Le The ats mutation is maternally inherited and, therefore, demonstrates the importance of the seed coat in determining seed on-Kloosterziel et al., 1994). Mutant ats plants also shape (Le exhibit reduced dormancy, supporting a role for the seed coat in on-Kloosterziel et al., controlling this aspect of development (Le 1994). Genes Involved in Flavonoid Biosynthesis The Arabidopsis seed coat has served as a model for the study of avonoid biosynthesis. This largely arose because Arabidopsis mutants defective in avonoid biosynthesis could be easily identied by changes in seed color. Mature Arabidopsis seeds are brown as a result of the accumulation and subsequent oxidation of PAs, also known as condensed tannins, within the endothelial layer of the seed coat. To date, 21 such mutants, known as transparent testa (tt), have been identied. Two of these are known as transparent testa glabra (ttg) because they also demonstrate a hairless phenotype. An additional six mutants named tannindecient seed (tds) have also been identied (Abrahams et al., 2002). Many of these mutations affect the dormancy of the seeds, indicating the important role of pigments in this aspect of development (Debeaujon et al., 2000). Flavonoid biosynthesis. Genetic analysis of the tt mutants has identied a number of enzymes in the avonoid biosynthesis pathways. TT4 and TT5 encode chalcone synthase (CHS) and chalcone isomerase (CHI), two enzymes at the beginning of the biosynthesis pathways (Feinbaum and Ausubel, 1988; Shirley et al., 1992). TT3 encodes dihydroavonol 4-reductase (DFR), TT6 encodes avonone 3-hydroxylase (F3H), and TT7 encodes avonoid 30 -hydroxylase (F30 H) (Shirley et al., 1992; Wisman et al., 1998; Schoenbohm et al., 2000). TT18 encodes leucoanthocyanidin dioxygenase (LDOX) (Shikazono et al., 2003). tds4, the only tds mutant characterized to date, was also found to be mutated at this locus (Abrahams et al., 2003). Mutations in all of these genes also

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affect anthocyanin production in vegetative tissues as the enzymes are common to both the anthocyanin and PA pathways. Arabidopsis plants with seeds exhibiting purple coloration were found to have a mutation in the gene BANYULS (BAN) which encodes anthocyanidin reductase (AR), an enzyme located at a major branch point between anthocyanin and PA production (Devic et al., 1999; Debeaujon et al., 2003; Xie et al., 2003). Besides BAN, no other biosynthetic genes specic to the PA pathway have been identied. In Brassica carinata, yellow-seeded cultivars have reduced pigments in the seed coat and the seeds have increased oil concentrations and ber, all of which are desirable agronomic traits in this species (Simbaya et al., 1995). Comparing brown-seeded and yellow-seeded cultivars of B. carinata revealed that the yellowseeded lines have reduced or absent expression of DFR, indicating that this locus is important in determining pigmentation levels (Marles et al., 2003a). The identication of this relationship has important implications for the generation of yellow-seeded, low ber cultivars of B. napus, which has proven difcult in typical breeding approaches (Marles et al., 2003a). Regulation of avonoid biosynthesis. Proteins that regulate the avonoid biosynthesis pathway are encoded by another set of TT genes. These regulatory proteins include TTG1, a WD40-repeat protein; TT1, a protein belonging to the WIP subfamily of zinc nger proteins; TT2, an R2R3 MYB domain protein; TT8, a basic helix loop helix (bHLH) domain protein; TTG2, a WRKY transcription factor; and TT16, an ARABIDOPSIS BSISTER MADS domain protein (Walker et al., 1999; Nesi et al., 2000, 2001, 2002; Johnson et al., 2002; Sagasser et al., 2002). Another regulator subsequently identied is ENHANCER OF GLABRA3 (EGL3), a bHLH domain protein (Zhang et al., 2003). These regulatory proteins control a number of the biosynthetic genes discussed above. Largely through gene expression studies, it has been determined that TT2, TT8, and TTG1 regulate DFR; TT2, and TTG1 also regulate LDOX; and TT2, TT8, TTG1, and TT16 regulate BAN (Shirley et al., 1995; Nesi et al., 2000, 2001, 2002; Debeaujon et al., 2003). In addition, some of these genes may play a function in seed coat endothelial cell differentiation. For example, tt16 and tt1 mutant endothelial cells both display aberrant morphology, which suggests that they may regulate differentiation of these cells as well as regulating PA production (Nesi et al., 2002; Sagasser et al., 2002). ttg2 mutants have reduced seed size, due to a reduced endosperm size and this phenotype is sporophytic maternal (Garcia et al., 2005). The reduction in size is suggested to be the result of reduced cell elongation in the integuments, possibly resulting from changes in cell wall composition because of the PA deciency in these mutants (Garcia et al., 2005). TTG1 appears to play an important role in the regulation of epidermal cell morphogenesis in a number of tissues. Some recent studies have demonstrated the interplay between TTG1, bHLH domain proteins, and MYB domain proteins. In the control of seed coat pigmentation, TTG1 interacts with the bHLH protein TT8 and the R2R3 MYB protein TT2 (Zhang et al., 2003). There is evidence that TTG1, TT8, and TT2 can form a ternary complex and that their combined activity is required for maximum BAN expression (Baudry et al., 2004). The bHLH proteins do not appear to be specic to one function. TT8 also regulates anthocyanin production in vegetative tissues, as does EGL3, a bHLH protein that functions with TTG1 to regulate mucilage production (Zhang et al., 2003). However, there

is evidence that it is mainly through these bHLH proteins that TTG1 acts (Baudry et al., 2004). The MYB proteins, however, are often associated with only one pathway and may account for the specicity of each of the various pathways (Zhang et al., 2003). TTG1 also regulates anthocyanin production, as well as trichome and root development, and, for each of these functions, it appears to interact with different combinations of bHLH proteins and MYB proteins (Zhang et al., 2003). Vacuolar transport. During Arabidopsis seed coat development, pigments are stored in vacuoles and two other tt mutants apparently have defects in machinery for the vacuolar sequestration of the PAs. TT12 was found to encode a member of the multidrug and toxic compound extrusion (MATE) family (Debeaujon et al., 2001). TT19 was found to encode a member of the glutathione S-transferase (GST) gene family (Shikazono et al., 2003; Kitamura et al., 2004). In addition, mutations in the gene AHA10, encoding a plasma membrane H-ATPase, were also found to result in a reduction in seed coat PAs (Baxter et al., 2005). When all three of these genes are mutated, multiple small vacuoles, as opposed to a single large vacuole, accumulate, indicating that these mutants are also defective in vacuole biogenesis (Kitamura et al., 2004; Baxter et al., 2005). Genes that are specic to the PA branch of avonoid biosynthesis, or that regulate this branch, are typically expressed exclusively within the seed coat, particularly the endothelium. These include the transcription factors TT1 and TT2 (Nesi et al., 2001; Sagasser et al., 2002), the vacuolar sequestration proteins TT12 and AHA10 (Debeaujon et al., 2001; Baxter et al., 2005), and the biosynthetic enzyme BAN (Devic et al., 1999; Debeaujon et al., 2003). This is likely due to the fact that PAs are only produced in the seed coat in Arabidopsis plants. Accordingly, homologues of many of these genes found in other species are not exclusively expressed in seed coats (Winkel-Shirley, 2001; Marles et al., 2003b). For example, the genetics of avonoid biosynthesis have also been studied in petunia and snapdragon owers and maize aleurone. Genes Involved in Mucilage Production As mentioned above, the epidermal cells of the Arabidopsis seed coat produce mucilage that is excreted upon imbibition. Several genes involved in this process have been identied. TTG1, TTG2, TT8, EGL3 and GL2. Interestingly, a number of mutants with defects in avonoid biosynthesis also demonstrate defects in the mucilage-secreting epidermal cells. These include TTG1, TTG2, and TT8. TTG1 affects mucilage production in the Arabidopsis seed coat, possibly through interactions with EGL3 and a MYB domain protein (Koornneef, 1981; Peneld et al., 2001; Zhang et al., 2003). TTG1, EGL3 and the MYB domain protein may control mucilage production through GL2 (Western et al., 2001, 2004). Mutations in GL2, a homeodomain-leucine zipper protein, produce defects in mucilage production that are very similar to those of ttg1 mutants (Koornneef 1981; Di Cristina et al., 1996). Furthermore, GL2 expression in the seed coat is reduced in ttg1 mutants, supporting the role of TTG1 in regulating GL2 (Western et al., 2004). The role of TT8 in mucilage production was only identied recently, through the analyses of double mutants. Mucilage production is normal in tt8 mutants, partially reduced in egl3

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mutants, but completely absent in tt8 egl3 double mutants, which also demonstrate collapsed columellae (Zhang et al., 2003). This indicates that TT8 and EGL3 likely have redundant roles in mucilage production. ttg2 mutants also lack mucilage and columellae (Johnson et al., 2002). In the seed coats of ttg1 mutants, TTG2 expression is reduced, suggesting that TTG2 acts downstream of TTG1 in mucilage production (Western et al., 2004). TTG2 expression is not affected by mutations in GL2, however, indicating that TTG2 and GL2 may regulate mucilage production through independent pathways (Johnson et al., 2002; Western et al., 2004). This is supported by the nding that expression of MUM4, another gene involved in mucilage production that is discussed below, is reduced in ttg1 and gl2 mutants but not in ttg2 mutants (Western et al., 2004). MYB61. The myb61 mutant lacks mucilage extrusion and the columellae have a reduced stature (Peneld et al., 2001). The seeds have reduced levels of polysaccharides as well as rhamnose and galacturonic acid, indicating a reduction in pectin levels (Peneld et al., 2001). During development, mucilage deposition is abnormal, with dense mucilage deposited only against the outer tangential cell wall and the rest of the space lled with more diffuse mucilage (Peneld et al., 2001). It has been suggested that MYB61 may interact with TTG1 and EGL3 in the regulation of mucilage production (Zhang et al., 2003). However, the myb61 mutant phenotype is unique from that of the ttg1 mutant and myb61 ttg1 double mutants demonstrate an additive effect, indicating that they may function in independent pathways (Pendeld et al., 2001). Furthermore, as with ttg2 mutants, expression of MUM4 is not affected in myb61 mutants, as it is in ttg1 and gl2 mutants, again suggesting independent pathways (Western et al., 2004). AtMYB23. Another MYB protein, AtMYB23, also regulates mucilage production in epidermal seed coat cells. A chimeric AtMYB23 repressor inhibited the deposition of mucilage in transgenic Arabidopsis plants, in addition to a number of other defects (Matsui et al., 2005). It has been suggested that AtMYB23 may be a better candidate than MYB61 for the MYB domain protein that interacts with TTG1 and EGL3 to regulate mucilage production (Western et al., 2004). This was supported in the study by Matsui et al. (2005) by the nding that the AtMYB23 repressor was able to suppress GL2 expression in Arabidopsis siliques but did not affect expression of TTG1. MUM1 5. Five other mutants were identied because of the absence of mucilage after imbibition and were named mum1 mum5 (Western et al., 2001). mum1 and mum2 are decient in mucilage extrusion. The composition and amount of mucilage is comparable to wild type, but upon imbibition, the mucilage is not excreted. There is, however, an increase in the levels of methylation of the mucilage and possibly the cell walls, as well. This increased methylation, or some other as yet unidentied alteration, likely alters the cell walls, or the mucilage, preventing release of mucilage upon imbibition (Western et al., 2001). mum4, as well as ttg1 and gl2, have reduced peaks for galacturonic acid, rhamnose, fucose, and galactose, the major components of pectin, as well as a reduction in the overall amount of mucilage produced (Western et al., 2001). The secondary cell wall in these three mutants is deposited, but in a peaked dome over the vacuole, as opposed to the volcano-shaped deposition visible in wild-type cells. These mutants appear to have defects in

cytoplasmic rearrangement and possibly construction, as well as defects in mucilage production (Western et al., 2001). MUM4, also known as RHM2, has been cloned and found to encode a putative NDP-L -rhamnose synthase, required for the synthesis of RGI (Usadel et al., 2004; Western et al., 2004). MUM4 belongs to a family of putative nucleotide sugar interconversion factors, which includes two other members, RHM1 and RHM3. MUM4 gene expression was found to be regulated by AP2, TTG1, and GL2, which is also supported by the fact that mutations in all of these genes result in very similar seed coat epidermal cell mutant phenotypes (Western et al., 2004). mum3 and mum5 have mucilage with altered staining properties (Western et al., 2001). Reduction in the levels of rhamnose and fucose, or other defects, may affect the basic structure of the mucilage in these mutants, and, therefore, its branching and crosslinking properties (Western et al., 2001). APETALA2 gene Mutations in the APETALA2 (AP2) gene give rise to many different phenotypes, resulting from defects in many different aspects of ower and ovule development, including mucilage production. Epidermal cells of ap2 mutants show altered morphology, including lack of a columella, larger size, and irregular shape (Jofuku et al., 1994; Western et al., 2001). A study of the development of the seed coat in ap2 cells indicates that the outer integument begins development normally, but halts before mucilage production and cytoplasmic rearrangements begin. At maturity, the pigmented layer is the only discernable cell layer in the seed coats. The epidermal and palisade layers appear absent, and only crushed remnants of the cells surround the pigmented layer (Western et al., 2001). The AP2 gene was rst cloned by Jofuku et al. (1994), and found to encode an unknown protein with a number of interesting domains. These include a serine-rich acidic domain, a putative nuclear localization signal, and two copies of a 68 amino acid direct repeat. The last domain was named the AP2 domain and can be found in transcription factors in many different plant species (Jofuku et al., 1994). The AP2 domains may be able to form amphipathic a-helical structures that could potentially mediate protein protein interactions (Jofuku et al., 1994). AP2 is a gene of particular interest because recent studies have indicated that ap2 mutant seeds demonstrate increased seed mass accompanied by increases in both total seed protein and total seed oil (Jofuku et al., 2005; Ohto et al., 2005). This is unusual because typically total seed protein and total seed oil vary inversely. ap2 embryos have altered cell number and cell size, which contributes to the overall increase in seed mass (Ohto et al., 2005). AP2 may affect seed mass by controlling source-sink relations in the seed (Jofuku et al., 2005; Ohto et al., 2005). Ohto et al. (2005) examined hexose and sucrose accumulation in ap2 mutants and found that ap2 seeds accumulated higher levels of hexose that decreased more slowly compared to wild-type seeds (Ohto et al., 2005). It has been suggested that the changes in hexose to sucrose ratios may result in extended cell division, leading to the increase in embryo size (Ohto et al., 2005). AP2 controls seed mass through the maternal sporophyte and endosperm (Jofuku et al., 2005; Ohto et al., 2005). However, there is some evidence that AP2 activity in the maternal sporophyte, potentially including the seed coat, may be more

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important than endosperm AP2 activity in controlling seed mass (Ohto et al., 2005). Genes Involved in Programmed Cell Death As described above, many of the seed coat layers of Arabidopsis, and other Brassicaceae species, are compressed at maturity, as a result of the expansion of the embryo. Cells of the outer two layers of the inner integument are compressed during seed development in Arabidopsis, such that the seed coat diameter decreases by at least half by maturity (Nakaune et al., 2005). DAPI staining indicates that cells in these layers undergo programmed cell death while being compressed (Nakaune et al., 2005). This process has not been extensively studied in seed coats, but some initial studies have highlighted the importance of cysteine proteinases. dVPE. A vacuolar processing enzyme, dVPE, from the cysteine proteinase family, was found to be expressed exclusively in the outer two layers of the inner integument of the Arabidopsis seed coat (Nakaune et al., 2005). Its expression increases rapidly and transiently during early embryogenesis, coordinate with the process of programmed cell death. dVPE is likely synthesized as an inactive protein and was subsequently found to be inhibited by caspase-1 inhibitors, indicating that it has caspase-1 like activity (Nakaune et al., 2005). These results indicate that dVPE may be involved in the process of programmed cell death in the inner integument (Nakaune et al., 2005). BnCysP1. Cells in the inner integument of B. napus seed coats also undergo programmed cell death (Wan et al., 2002). The gene BnCysP1 encodes a protein with similarity to cysteine proteinases and its expression is temporally correlated with programmed cell death in the inner integument (Wan et al., 2002). Its similarity to papaya papain indicates it may be synthesized as a preproprotein, much like dVPE (Wan et al., 2002). These results again support a role for a cysteine proteinase in the process of programmed cell death in the seed coat. Structure of Legume Seed Coats The seed coats of legumes such as soybean, common bean and pea are relatively large and complex. As discussed above, they are well suited to the study of the physiological and biochemical processes that govern embryo nutrition and composition (Rochat and Boutin, 1991; Zeng et al., 2004; Wang and Grusak, 2005); for example, the control of the aqueous and gaseous environment around the embryo (Gijzen et al., 1999b; Souza and Marcos-Filho, 2001), or the protection of the embryo against pests and diseases (Ndakidemi and Dakora, 2003). The morphological features of the legume seed coat are relatively insensitive to environmental conditions and therefore are sufciently distinctive to be used for taxonomy (Souza and Marcos-Filho, 2001). Yet, information on the differentiation and development of specic cells and tissues of seed coats is generally lacking. Soybean seed coat development is one of the few that have been examined in detail (Miller et al., 1999). The features of the mature seed coat have been described by Corner (1951). Outermost is the epidermal layer, which consists of a single layer of palisade cells (macrosclereids). They are elongated perpendicular to the surface of the seed. Inside the palisade layer is an hourglass cell layer, which is composed of thick-walled osteosclereids. The innermost portion of the seed coat proper is a

multicellular layer of partially attened parenchyma. The aleurone layer is immediately inside the inner parenchyma. Each of the above layers is maternally derived from the outer, or inner integuments (Fig. 1). The next innermost layer is the endosperm, derived by double fertilization. As shown in Fig. 1, it is tightly compressed against the seed coat by the expansion of the embryo cotyledons (Miller et al., 1999). Recently, the seed coat structure of the legume Medicago truncatula has been characterized (Wang and Grusak, 2005). It is similar to that of soybean. It features an epidermal layer of macrosclereids, a subepidermal layer of osteosclereids, and 2 5 rows of internal parenchyma cells. The parenchymal layer is thinnest at the end of the seed coat opposite the hilum. As discussed later, M. truncatula is an important model legume that may facilitate the functional identication of legume genes involved in seed coat development and composition. Cuticle and Epidermal (Palisade Cell) Layer The outermost layer of the legume seed coat is the waxy cuticle, which represents the rst barrier to imbibition. It is variable in thickness. Actually, two layers of waxy deposits, one very stable and the other environmentally labile are suggested for soybean seeds (Ragus, 1987; Souza and Marcos-Filho, 2001). In several Glycine species the membranous inner endocarp epidermis of the pod wall detaches and adheres to the seed coat surface, becoming part of the mature seed coat (Gijzen et al., 1999b). The epidermis arises from the outer cell layer of the outer integument (Zeng et al., 2004). It is composed of a layer of thick-walled cells. Only one palisade layer is found, except under the hilum, where two can occur. The external layer is called the counter-palisade and originates from the funiculus (Souza and Marcos-Filho, 2001). In Trifolium pratense L., the cytoplasm of the macrosclereids contains small and medium-sized vacuoles and several organelles such as mitochondria, rough endoplasmic reticulum, and ribosomes. The cell vacuoles are completely or incompletely lled with tannins, indicating that the macrosclereid cells may play a role in the hardening of seed coats. Studies showed that cell walls of the palisade layer contribute to the mechanical yu kkartal, 2000). strength of the seed coat (Algan and Bu Hourglass Cell Layer The hypodermis is formed from a single layer of cells called hourglass cells, pillar cells, osteosclereids, or lagenosclereids, depending on their pattern of cell-wall thickness and shape. The hourglass cells of the seed coat arise from the outer-cell layer of the inner integument (Zeng et al., 2004). They are usually larger than adjacent cell layers and are separated by wide intercellular spaces, except under the hilum cleft where they are absent (Souza and Marcos-Filho, 2001). The osteosclereid layer is composed of large vacuolated cells and is densely cytoplasmic. The presence of numerous starch grains in the hourglass cells during embryogenesis indicates that the seed coat could synthesize nutrients for the yu kkartal, 2000; Wang and developing embryo (Algan and Bu Grusak, 2005). Like palisade cells, hourglass cell walls also play a role in the mechanical strength of the seed coat. The hourglass cells in the soybean seed coat are interesting in that they appear to serve as a reservoir for proteins. A single

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isozyme of seed coat peroxidase accumulates in large amounts (5% total soluble protein) in the hourglass cell vacuoles (Gillikin and Graham, 1991; Gijzen et al., 1993). Mutants (epep) decient in this peroxidase produced normal-looking hourglass cells and did not under-perform compared with wild-type (EPEP) soybean varieties. Parenchyma Layers Adjacent to the hourglass cells is the interior parenchyma, formed by 6 8 layers of thin-walled, tangentially elongated parenchyma cells. They are uniformly distributed throughout the seed coat, except in the area of the hilum, where a smaller number of layers can be distinguished. In mature seed coats, the interior parenchyma is often crushed or partially crushed (Miller et al., 1999) as the embryo expands. An important function of the seed coat is to deliver nutrients to the embryo. In the parenchyma layer, three sublayers can be discerned: chlorenchyma, ground parenchyma, and branched parenchyma in pea (Pisum sativum L.). Light- and cryo-scanning electron microscopy (cryo-SEM) from the late pre-storage phase to the end of seed lling showed that solutes imported by the phloem moved into the chlorenchyma and ground parenchyma, but not into the branched parenchyma (van Dongen et al., 2003). It is believed that chlorenchyma and ground parenchyma may be involved in the post-phloem symplasmic transport of nutrients in the seed coat. In the developing red clover seed coat, the parenchyma cells contain numerous organelles, such as amyloplasts, rough ER, ribosomes, plastids, dictyosomes, mitochondria, lipids, and protein bodies (Algan yu kkartal, 2000), suggesting that this tissue is in a metabolically and Bu active state. The ER system in the parenchyma cells suggests that these cells may produce nutrients destined for the developing embryo. It was shown that many intracellular spaces in the seed coat parenchyma are lled with an aqueous solution, suggesting that it facilitates the diffusion of nutrients from the site of unloading towards the cotyledons (van Dongen et al., 2003). The parenchyma cells degenerate later and become a major source of nutrients for the embryo. It is uncertain whether the seed coat vascular system lies within the parenchyma layer. It should be noted that seed coat vascular systems, which are responsible for transporting the nutrients from the maternal organs to the embryo, vary structurally in the legumes. Some species possess extensive vascular systems that anastomose to form reticulated networks throughout the entire seed coat; for example, soybean. Other species have relatively simple vascular systems with only a single chalazal vascular bundle and two lateral branches extending into the seed coats; for example, M. truncatula. The nutrients are almost exclusively imported through the phloem and include the main organic nutrients, sucrose, and amino acids, as well as potassium and micronutrients (Patrick and Ofer, 2001). Sucrose leaves the seed coat unaltered, but it is hydrolyzed by extracellular invertases during the pre-storage phase. Amino acids imported via the phloem undergo elaborate metabolic conversion in the seed coat before release into the apoplast (van Dongen et al., 2003). Aleurone Cell Layer and Compressed Endosperm Layer As the seed coat matures, the endosperm cell layers adjacent to the embryo degenerate and eventually appear as compressed wall materials at seed maturity but the outermost endosperm layer remains intact and differentiates into what has been known as the

aleurone layer, which is the only portion of the endosperm that is clearly visible (Miller et al., 1999). The aleurone layer of soybean and a few other legumes is known for its role in the enzymatic mobilization of seed reserves, such as carbohydrates, during germination (Ma et al., 2004b); however, its role during seed development is not known. Yaklich et al. (1992) observed that aleurone cells in a developing soybean seed have features of secretory cells and speculate that they are able to convert photosynthate into substances for export to the milieu (apoplast) bathing the embryo during seed ll. A mature soybean seed contains endosperm remnants as a vestigially crushed layer inside the aleurone and by convention it is considered to be part of the seed coat (Miller et al., 1999). The endosperm is formed initially as a coenocytium around the young embryo and starts to compartmentalize into cells when the embryo is at its late globular stage (Chamberlin et al., 1994). Its function is to serve as a nutrient source. The antipit of seed coat located in the abaxial center of each cotyledon is composed of additional endosperm cells situated between the aleurone layer and the compressed endosperm tissue. It was postulated that the hilum of the legume seed is a weak spot and, thus, can be a potential site for bacterial invasion. However, Ma et al. (2004b) suggested that the pit region might be resistant to pathogen attack due to the thick cuticle along with thick periclinal walls of its epidermal cells. Alternatively, this thickened area of seed coat endosperm might have a physical role in helping to pin the embryo in position within the seed coat (M. Gijzen, personal communication). Molecular Characterization of the Legume Seed Coats Legumes comprise one of the most important agricultural taxa worldwide, providing a major source of protein and oil. Soybean accounts for over 50% of the world oilseed production each year and alfalfa (Medicago sativa) ranks high in acreage planted and dollar value among forage crops (Cook, 1999; Harrison, 2000). However, both have limitations for molecular genetic studies, such as large genome sizes, abundant repetitive DNA, complex ploidy, and difculties with regeneration of transgenic plants for functional studies of genes (Cook, 1999; Maguire et al., 2002). Arabidopsis, as a model plant, has accelerated the development of plant genetics in general but it is inadequate for addressing many of the legumespecic properties of legume seeds (VandenBosch and Stacey, 2003). M. truncatula and Lotus japonicus have been recommended as more appropriate models (Harrison, 2000; Bell et al., 2001; Young et al., 2003). Attributes of M. truncatula includes its small, diploid genome (5 108 bp); self-fertility; prolic seed production; and rapid generation time (Cook, 1999). The legume information system (LIS) (http://www.comparative-legumes.org) has been set up to integrate comparative genetic and molecular data from multiple legume species, which include Medicago, Lotus, and soybean, and Arabidopsis (Gonzales et al., 2005). In the NCBI database, as of March 2005, researchers have deposited 355 863 ESTs for Glycine max, 216 645 ESTs for M. truncatula and 111 471 ESTs for Lotus corniculatus (http://www. ncbi.nlm.nih.gov/dbEST/dbEST_summary.html). It was shown that 2525 legume-specic ESTs contigs were found after comparing unigene sets from Medicago, Lotus, and soybean with and rice and Arabidopsis genomic sequences (Graham et al., 2004). Soybean seed coat ESTs have also been generated and analyzed (Shoemaker et al.,

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2002). In an immature seed coat cDNA library (Gm-c1019), 644 contigs were assembled, and the unigene percentage was 71%. In a mature seed coat cDNA library (Gm-c1023), 188 contigs were assembled and the unigene percentage was 82%. The occurrence of a large number of seed-coat-specic genes was conrmed by microarray analyses performed with different soybean organs, such as seed coat, embryo, ower, and pod (Maguire et al., 2002; Thibaud-Nissen et al., 2003). According to the NSF soybean functional genomics website (http://soybeangenomics.cropsci.uiuc. edu/les/NSFWeb Overview.pdf) 2163 unigenes from soybean seed coat were identied. Alkharouf and Matthews (2004) have set up the the soybean genomics and microarry database (SGMD), which contains genomic, EST and microarray data with embedded analytical tools, allowing correlation of soybean ESTs with their gene expression proles (http://psi081.ba.ars.usda.gov/SGMD/ default.htm). Legume Seed Coat Composition Seed coats possess a wide assortment of novel compounds. Upon imbibition, a complex mixture of chemicals is released from the seed coat, which consists of avonoids, proteins, peptides, amino acids, alkaloids, terpenoids, steroids, etc. (Ndakidemi and Dakora, 2003). Of these, the isoavonoids are best known for their role as anti-microbial phytoalexins and phytoanticipins (Dakora and Phillip, 1996). It is known that proanthocyanins and the isoavonoid, glycitin, contribute to resistance to legume weevils by inhibiting their reproduction (Oigiangbe and Onigbinde, 1996). Thus, many seed coat components play important roles in defense. Moreover, the components of seed coat tissues affect the overall quality and value of legume food or feed products, and are the sources of novel compounds for industry and medicine. In this section, we discuss legume seed coat composition and the importance of some compounds to agriculture, food quality and human health. A list of some of the genes involved in these processes is given in Table 2. Polyphenolics Functions and uses. Polyphenolic compounds, including phenolic acids and derivatives, tannins, and avonoids, represent the largest group of natural products in the plant kingdom. They confer color to fruit and seed. They play a signicant role in plant disease resistance. They have diverse roles in plant development and interactions with the environment (Salunkhe et al., 1982; Harborne, 1988; Dixon and Paiva, 1995; Paiva, 2000). For example, seed and seed coat phenolic compounds participate in nodulation by acting as chemoattractants; promoting rhizobial growth; and inducing transcription of nodulation genes in symbiotic bacteria (Ndakidemi and Dakora, 2003). The developing seed is a sink for many products synthesized in other plant tissues. However, Dhaubhadel et al. (2003) demonstrated that IFS1 and IFS2, genes encoding 2-hydroxyisoavonoid synthase, are expressed not only in a variety of plant vegetative organs, but also in developing embryos and seed coats. Moreover, reciprocal crosses of cultivars, with high and low isoavonoid content revealed that the biosynthesis of isoavonoids in maternal tissues of soybean seeds may contribute substantially to total seed isoavonoid content at maturity (Dhaubhadel et al., 2003).

In recent years, polyphenols in many edible plant products have received increasing attention due to their inuence on the nutritional and aesthetic quality of foods and pharmacological implications. As they possess both antioxidant and antimutagenic activity, they are implicated in the prevention of many diseases, including atherosclerosis and cancer (Stavric, 1994; Karakaya and nez et al., 2003). The majority of Kavas, 1999; Hou, 2003; Mart phenolic compounds found in the seeds of legumes are located in the seed coat. Phenolics of the cotyledon and the seed coat of lentils and peas have been used in studies of dietary intake of these as et al., compounds (Bekkara et al., 1998; Wang et al., 1998; Duen 2002, 2004). In soybean and common bean, the concentration of phenolic compounds, such as avonoids and anthocyanins, correlates with seed coat color (Hungria and Phillips, 1993; Yaklich and BarlaSzabo, 1993; Nakamura et al., 2003; Benitez et al., 2004). Legumes with dark seed coats, such as soybean, broad beans, faba beans, lentils, and peas, possess high antioxidant activity mainly due to the proanthocyanidins (Cardador et al., 2002; nez et al., 2003). They are as et al., 2002, 2004; Mart Duen natural sources of antioxidants that could replace the synthetic antioxidants in foods. Tannins, such as proanthocyanidins, might be less favored because they form complexes that may inactivate enzymes, or precipitate proteins, thereby, reducing food protein quality (Tan et al., 1983; Cabrera and Martin, 1986). Furthermore, a high content of tannins in sorghum was shown to be associated with a marked degree of resistance to preharvest seed germination (Harris and Burns, 1970). Tannins are also responsible for retarding seedling growth by decreasing the rate of starch and protein degradation in germinating high-tannin seeds, possibly by inactivating hydrolytic enzymes (Salunkhe et al., 1982). The accumulation of phenolics can decrease the agronomic value of soybean due to increased cracking of the seed coat (Yaklich and Barla-Szabo, 1993; Nakamura et al., 2003; Benitez et al., 2004). Two types of cracking have been reported: Type I with irregular cracks, and Type II with net-like cracks. Both result from separation of epidermal and hypodermal tissues exposing the underlying parenchyma tissue (Wolf and Baker, 1972; Yaklich and BarlaSzabo, 1993). Genetic control of cracking has been well characterized and closely linked to the pigmented seed coat which results from the accumulation of anthocyanin in epidermal cells (Yaklich and Barla-Szabo, 1993; Nakamura et al., 2003; Benitez et al., 2004). Genetics of seed coat color. Several independent genetic loci, including I, T, R and O, control the color and distribution of pigments (Bernard and Weiss, 1973; Palmer and Kilen, 1987). The best characterized I locus (for inhibitor) inhibits the production and accumulation of anthocyanins and proanthocyanidins in the epidermal layer of the seed coat. It consists of four alleles with the following phenotypes: I results in complete absence of seed coat pigment; i i limits pigment to the narrow hilum area where seed and pod are attached; i k restricts pigment to a saddle-shaped region over two-thirds of seed coat, and i results in a completely pigmented seed coat (Bernard and Weiss, 1973). Most cultivated soybean varieties are homozygous for a dominant form of the I gene resulting in a yellow seed coat. However, spontaneous mutations from yellow seed to dark-colored seed with i/i genotype arise frequently within highly inbred soybean varieties (Wilcox, 1988).

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SE ET AL. MOI TABLE 2 EXAMPLES OF GENES EXPRESSED IN LEGUME SEED COATS

Category Seed coat color

Gene, Accession number CHS1 BG509422 CHS2 X65636 CHS3 X16186 CHS4 X52097 CHS5 L07647 CHS6 L03352 CHS7 BE800791

Plant species Glycine max G. max G. max G. max G. max G. max G. max

Gene product Chalcone synthase Chalcone synthase Chalcone synthase Chalcone synthase Chalcone synthase Chalcone synthase Chalcone synthase

Function(s) Flavonoid biosynthesis, response to UV light and elicitors Flavonoid biosynthesis, promoter contains sugar response elements Flavonoid biosynthesis Flavonoid biosynthesis Flavonoid biosynthesis, response to UV-B irradiation Flavonoid biosynthesis, response to UV-B irradiation Flavonoid biosynthesis, responsible for seed coat pigmentation; promoter contains nodule-development-specic regulatory element Flavonoid biosynthesis, responsible for seed coat pigmentation Hydroxylation of 30 position in B-ring of avonoids, control of pubescence color Developmentally regulated, with organ-specic and stage-specic expression. Possible role in plant development Developmentally regulated, with organ-specic and stage-specic expression. Possible role in plant development Expressed late in seed development, involved in plant defense against pathogens Protection against pathogens, or predators, attachment of the endocarp to the seed surface Cleavage of the incoming sucrose within the apoplast at a stage when mitosis is active in the embryo Sucrose synthesis Aquaglyceroprotein which plays a role in water absorption during seed imbibition Aquaporin, expressed in the cotyledons of developing and germinating seed, involved in the release of water from seed coat symplast Aquaporin, expressed in the cotyledons of developing and germinating seed, involved in the release of water from seed coat symplast Only detected in seed coat. Encodes aquaglyceroporin, involved in water absorption and formation of water and glycerol channels Developmental regulation possibly via callose hydrolysis, release of oligosaccharides and/or degradation of thin-walled parenchyma and endosperm layers during development Expressed in the developing soybean seed coat, plays a role in the differentiation of seed coat parenchyma cells Expressed in thick-walled parenchyma, is involved in a signal transduction, and differentiation of soybean seed coat cells Possibly defense via oxidative processes

References Akada et al., 1991; Shimizu et al., 1999 Akada et al., 1993a Wingender et al., 1989 Akada et al., 1990 Akada et al., 1995; Shimizu et al., 1999 Akada et al., 1993b; Shimizu et al., 1999 Akada et al., 1993c; Tuteja et al., 2004 Akada et al., unpublished; Tuteja et al., 2004 Shoemaker et al. 1999a; Toda et al., 2002 Shoemaker et al., 1999b; Keller, 1993 Shoemaker et al., 1999c; Keller, 1993 Gijzen et al., 2001 Gijzen et al., 1999b; 2003 Weber et al., 1995 Dejardin et al., 1997 Schuurmans et al., 2003 Schuurmans et al., 2003

CHS8 AY237728 SF3 0 H1 BF069512 Prolinerich proteins SbPRP1 AW704950 SbPRP2 BG882833 Chitinase Allergens Invertases Sucrose synthase Aquaporins CHIA1 AF202731 HPS AF100159 VfVINV1 Z35162 SUS X98598 PsPIP1-1 AJ548795 PsPIP2-1 AJ24330 PsTIP1-1 AJ243309 PsNIP-1 AJ243308 Glucanases PsGNS2 AJ251646 AJ251199

G. max G. max G. max G. max G. max G. max Vicia faba Pisum sativum P. sativum P. sativum

Chalcone synthase Flavonoid 30 -hydroxylase Proline-rich cell wall protein 1 precursor Proline-rich cell wall protein 1 precursor Chitinase class I Hydrophobic seed protein precursor Cell wall invertase I Sucrose synthesis Plasma membrane intrinsic protein Plasma membrane intrinsic protein Tonoplast intrinsic protein Nodulin26-like intrinsic protein b-1,3-Glucanase

P. sativum

Schuurmans et al., 2003

P. sativum

Schuurmans et al., 2003

P. sativum

Buchner et al., 2002

BURPdomain proteins Subtilisin Peroxidases

SCB1 AY075133 AF467554 SCS1 A276710 A276407 SBP(Ep) AF014502

G. max G. max G. max

Seed coat BURP domain protein Putative subtilisin precursor Seed coat peroxidase precursor

Bachelor et al., 2002 Bachelor et al., 2000 Gijzen, 1997

SEED COATS TABLE 2 Continued Gene, Accession number PRX2, AF039027 MADS box proteins Leginsulin peaMTF1, AJ223318 Leginsulin gene, A223037 lup-leg1, U74383 Plant species G. max P. sativum G. max Lupinus angustifolius

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Category

Gene product Cationic peroxidase 2 MADS box transcription factor Leginsulin Leginsulin-like protein

Function(s) Function unknown Possible regulation of development of seed coat Signaling pathways for the transport of nutrients to the embryo Signaling pathways for the transport of nutrients to the embryo

References Gijzen et al., 1999a Buchner and Boutin, 1998 ncio et al., 2003; Vena Oliveira et al., 2004 Ilgoutz et al., 1997

At the molecular level the I locus has been extensively analyzed and shown to be a naturally occurring duplication of chalcone synthase (CHS) genes (Wang et al., 1994; Todd and Vodkin, 1996; Senda et al., 2002a, b). CHS is the rst committed enzyme of the multibranched pathway of avonoid/ isoavonoid biosynthesis and it also plays a signicant role in the synthesis of secondary metabolites functioning as UV protectants, phytoalexins, insect deterrents, and symbiosis initiators in various plant tissues. Three of the seven members of the multigene family (CHS1, CHS3, and CHS4) are located in tandem within a 10-kb duplication region (Akada and Dube, 1995). Paradoxically, deletions of the CHS promoter sequences allow higher levels of CHS mRNA accumulation and restore pigmentation to the seed coat (Todd and Vodkin, 1996). The restoration upon deletion points to the involvement of the homology-dependent gene silencing processes (Todd and Vodkin, 1996; Senda et al., 2004; Tuteja et al., 2004). Tuteja et al. (2004) demonstrated that the presence of the yellow dominant (I) allele signicantly decreases CHS mRNA levels in seed coat but not in pods, leaves, stem, roots, and cotyledons. Moreover, their investigations on the relative expression prole of CHS gene family members show that the three CHS genes comprising the I locus are transcribed at comparable although low levels in both the pigmented and the non-pigmented isolines. The increase of total CHS mRNA levels in the seed coats of I to i mutations is primarily because of an increase in CHS7/CHS8 transcript levels (Tuteja et al., 2004). Thus, the dominant alleles inhibit pigmentation in a trans-dominant manner. It is possible that relative orientation of the two genes generates inverted repeat, between CHS3 and CHS4, resulting in dsRNA (Tuteja et al., 2004). If cleaved into small siRNAs (Hamilton and Baulcombe, 1999), sequence-specic post-transcriptional degradation of CHS7/CHS8 could be generated in a tissue-specic manner to inhibit pigmentation of the seed coats (Tuteja et al., 2004). A detailed understanding of this system may reveal mechanisms of tissuespecic gene silencing which could be applied to the targeted silencing of other seed coat genes. Another locus, T, consists of two alleles, the dominant T and the recessive t, which produce brown and gray pubescence, respectively (Palmer and Kilen, 1987). Further, T generally darkens hilum and/or seed coat color in combination with other genotypes, and thus, induces seed coat cracking (Palmer and Kilen, 1987). The T locus encodes avonoid 30 -hydroxylase (F30 H), which is necessary for the formation of quercetin from kaempferol and is responsible for the hydroxylation of the 30 position of avonoids,

leading to the production of cyanidin-based pigments (Forkman, 1991). The SF3 0 H1 cDNA from soybean represents the only fulllength sequence of F30 H from leguminous plants (Toda et al., 2002). The sequence analysis of this gene from a pair of isogenic lines for T (TT brown and tt gray) revealed that they are different only by a single C deletion in the coding region of SF3 0 H1 (Toda et al., 2002). The truncated protein lacks the conserved region of F30 H and the heme-binding domain and probably lacks F30 H activity. This may result in the absence of quercetin and its derivatives in tissues and consequently produces the grey pubescence color (Toda et al., 2002). Seed coat color is a commercially important trait for other legumes such as common bean, lentil, vetch, or pea. A common vetch with low toxins could provide a valuable source of proteins for animals. However, the linkage of color and other important agronomic traits in such species is not yet evident. Five genes in common bean, at least two genes in lentil, and one gene in common vetch were shown to be involved in the control of seed coat color (Brady et al., 1998; Emami and Sharma, 2000; Chowdhury et al., 2004). However, these data were obtained from genetic crosses and little is known about regulation of pigmentation in these species at the molecular level. Involvement of proline-rich proteins. Seed coat cracking in pigmented soybean lines is also correlated with the accumulation of proline-rich proteins (PRP1 and PRP2) in the cell walls. PRPs represent a class of plant cell wall proteins characterized by high proline content. They are composed of small tandem repeats, such as PPPVYK, or PPVEK, where the second proline is often hydroxyproline (Marcus et al., 1991; Keller, 1993; Showalter, 1993). PRPs exist as a soluble form but can also be insolubilized in the wall over time (Kleis-San Francisco and Tierney, 1990; Bradley et al., 1992). The two genes are expressed in different stages of developing seed coats, hypocotyls and roots of soybeans. Expression of the SbPRP1 gene is high in young seed coats and later during seed desiccation (Lindstrom and Vodkin, 1991). The abundance of PRP1 protein is affected by the I locus in soybean which controls distribution of pigment in the seed coat. Interestingly, pigmented varieties possessing lower levels of soluble PRP1 also display a netlike pattern of seed coat cracking (Lindstrom and Vodkin, 1991, Nicholas et al., 1993). Both SbPRP1 and SbPRP2 cytoplasmic mRNA were found in the net-defective seed coat, suggesting that the absence of soluble PRP polypeptides in the defective net lines is due to post-translational regulation, or due to a more rapid and/or premature insolubilization of PRP polypeptides within the cell wall matrix (Percy et al., 1999).

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Globulins. Legume seed resistance to pests and pathogens may also involve factors other than the phenolics. For example, in the seed coat of common bean (Phaseolus vulgaris L.) neither thickness nor the levels of phenolic compounds such as tannins and tannic acids alone were important for resistance (Silva et al., 2004). Vicilin-like 7S storage globulins, such as canavalin, concanavalin A, canatoxin and phaseolin, reported in Jack bean (Canavalia ensiformis), Lima bean (Phaseolus lunatus) and common bean (P. vulgaris) have been implicated (Oliveira et al., 1999a; Moraes et al., 2000; Silva et al., 2004). Canatoxin was shown to be toxic to some insects (Carlini et al., 1997) and plant pathogenic fungi (Oliveira et al., 1999a). Canavalin inhibits spore germination of several fungi. Furthermore, both phaseolin and canavalin have detrimental effects on larval development in bruchids (Oliveira et al., 1999a, b; Moraes et al., 2000; Silva et al., 2004). Albumins. The insecticidal properties of the pea albumin 1b peptide(s) has opened new possibilities for seed protection against cereal weevils. Although the mechanism of action of this toxin is still unknown, binding to insect protein extracts occurs (Gressent et al., 2003). This albumin is the rst entomotoxic cystine-knot peptide identied. It might belong to a multi-gene family, as at least ve isoforms of the peptide exist within a single pea genotype. Moreover, it seems to be widespread among legumes (Higgins et al., 1986; Gressent et al., 2003). The cystine-knot structural motif is present in peptides and proteins from a variety of species and appears to be a highly efcient motif for structure stabilization (Craik et al., 2001). Chitinase. Gijzen et al. (2001) isolated a class I chitinase from soybean seed coat. Although chitin is absent in plants it is a major component of fungal cell walls; therefore, chitinase may play a role in plant defense against pathogens (Schlumbaum et al., 1986). The seed coat chitinase is expressed late in seed development, with particularly high expression levels in the seed coat. Moreover, expression is associated with senescence, ripening and response to pathogen infection (Gijzen et al., 2001). Polysaccharides. Applebaum et al. (1970) demonstrated that a polysaccharide fraction isolated from P. vulgaris seeds, present at a level of c. 1% dry weight, increases larval mortality and reduces rate of larval development. Gatehouse et al. (1987) observed that carbohydrates from P. vulgaris seeds reduced Acanthoscelides obtectus adult emergence and this activity was due, at least in part, to the presence of a heteropolysaccharide which has an unusually high content of arabinose and fructose. Recent work of Oliveira et al. (2001) demonstrated the presence of the polysaccharide galactorhamnan in the innermost cell layer of the seed coat and also in cotyledons of Jack bean (C. ensiformis). The concentration of this polysaccharide in the seed coat (c. 2%) is sufcient by itself to protect the seeds from attack by Callosobruchus maculatus (Oliveira et al., 2001). Allergens Soybean is known to have at least 15 allergenic proteins (Ogawa et al., 1991). The majority of these allergens are oil body proteins (Ogawa et al., 1993). Surface proteins associated with the seed coat luster phenotype are also responsible for seed dust allergenicity (Gonzalez et al., 1995; Gijzen et al., 1999b). Recently, the

hydrophobic protein (HPS) which is synthesized in the pod endocarp and subsequently deposited to the seed surface was found to be a major soybean dust allergen (Swanson et al., 1991; Gonzalez et al., 1995; Gijzen et al., 1999b). The hydrophobicity and topography of the surface of soy varieties expressing high levels of HPS could affect pathogen attachment and penetration, inuence the water-absorptive properties of the seed (Gijzen et al., 1999b) and/or mediate the attachment of the endocarp to the seed surface (Gijzen et al., 2003). Recently, Herman et al. (2003) demonstrated that transgene silencing could be used to remove a major soybean allergen, the Gly m Bd 30 K protein found in the seed globulin protein fraction. This approach may prove to be useful for the reduction, or elimination of allergens deposited upon the seed coat. Insulin-like Proteins Collip (1923) discovered the presence of insulin-like hypoglycemic activity in plant materials such as green tops of onions, lettuce and bean leaves, barley and beet roots, and others. A few decades later proteins from plant tissues were found which were potentially benecial to diabetic patients and had properties similar to those of insulin (Khanna et al., 1981; Collier et al., 1987). The insulin-like peptide and its receptor were isolated from a number of legume seeds, such as soybean, cowpea, Jack bean, winged bean, French bean, and lupin (Komatsu and Hirano, 1991; Ilgoutz et al., ncio et al., 2003; Yamazaki et al., 1997; Hanada et al., 2003; Vena 2003; Hanada and Hirano, 2004). The nding that insulin-like protein and insulin receptor-like protein with tyrosine protein kinase activity were localized to the innermost region of the seed coats may indicate that they are involved in signaling pathways for ncio et al., 2003; the transport of nutrients to the embryo (Vena Oliveira et al., 2004). Interestingly, insulin has been shown to stimulate the activity of enzymes involved in the conversion of stored fat to carbohydrates in the fat-storing seed of sunower, watermelon, and cucumber, as well as the legume Jack bean (Goodman and Davis, 1993; Oliveira et al., 2004). The presence of insulin-like antigens in the leaves of several plants, from bryophytes to angiosperms, as well as in a red alga, fungi, and cyanobacterium, suggests that insulin-dependent sugar transport may have been conserved during evolution (Silva et al., 2002; Xavier-Filho et al., 2003). Proteins Involved in Cellular Differentiation Invertases. As reviewed extensively (Weber et al., 2005), seed coats play a very important role in the metabolic control of seed development. Not surprisingly, many seed coat proteins are associated with sucrose metabolism (Kuo et al., 1997), including invertase which regulates the hexose:sucrose ratios (Weber et al., 1995; Borisjuk et al., 1998), or sucrose synthase, which is responsible for 37% of neosynthesized sucrose in pea seed coat cells. There are two kinds of invertase found in the maternal tissue of young legume seeds. Soluble invertase, in the vacuole, is important for the control of the hexose:sucrose ratio. Insoluble invertase, embedded in cell walls, is active in growing zones and extending tissues (Weber et al., 1996). Sequential expression of rst soluble and then insoluble invertases has been found during legume seed development (Weber et al., 1995). In Vicia faba, the gene

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VfVINV1 is expressed in the unloading area of the seed coat and functions in the cleavage of incoming sucrose within the apoplast at a stage when mitosis is active in the embryo. High levels of hexoses in the cotyledons and the apoplastic endospermal space are correlated with activity of cell wall-bound invertase in the seed coat during the prestorage phase. In situ hybridization showed that the gene, VfCWINV1, is expressed exclusively in the chalazal vein and the inner rows of the thin-walled parenchyma of the seed coat. These cells represent the end of the sieve element system. VfWINV1 plays an important role in providing developing embryos with hexoses and contributing to the establishment of sink strength in young seeds. Aquaporins. Unloading of nutrients from the seed coat is a vital function and must be associated with a wide range of other processes such as release of water from the seed coat during the transfer of the solutes. These processes may involve the family of major intrinsic proteins (MIP) which include the aquaporins. Four full-length aquaporin-related cDNAs were cloned and sequenced from a cDNA library of developing pea seed coats (P. sativum L.). The cDNA of PsPIP1-1 appears to be a turgor-responsive gene. PsNIP-1 is only detected in the seed coat, while PsPIP1-1, PsPIP21, and PsTIP 1-1 are expressed in cotyledons of developing and germinating seeds, and seedling roots and shoots. In mature dry seeds, strong hybridization signals were detected with the probe for PsPIP1-1, but no transcripts of PsPIP2-1, PsTIP1-1, and PsNIP-1 were found. Functional analysis in Xenopus oocytes conrmed that PsPIP2-1 and PsTIP1-1 are aquaporin whereas PsNIP-1 is an aquaglyceroporin. It is suggested that PsPIP1-1 could play a role in water absorption during the seed imbibition. PsPIP2-1 together with PsPIP1-1 could be involved in the release of water from the seed coat symplast that is nally related to release of nutrients for the embryo (Schuurmans et al., 2003). Glucanases. Plant b-1,3-glucanases represent a highly diverse family of hydrolytic enzymes, which are generally induced in response to pathogen attack or environmental stress (Kauffmann et al., 1987; Simmons et al., 1992). Nevertheless, tissue-specic and developmentally regulated but non-pathogen-induced expression of b-1,3-glucanase genes have also been reported (Memelink et al., 1990; Hird et al., 1993). The expression of PsGNS2, encoding b-1,3-glucanase, was demonstrated to be spatially and temporally regulated in P. sativum seed coats (Buchner et al., 2002). The high abundance of the transcripts observed when the embryo reached the late heart stage, remained until the mid seed-lling stage and was restricted to a strip of the inner parenchyma tissue of the seed coat (Buchner et al., 2002). It was suggested that PsGNS2 could function in maintaining routes for lateral transport and cell cell communication through plasmodesmata possibly via callose hydrolysis (Buchner et al., 2002). Alternatively, it might be associated with the release of oligosaccharides which are important for embryogenesis (Berger, 1999; Buchner et al., 2002). Finally, both seed coat thin-walled parenchyma and endosperm layers are tissues subjected to partial degradation during development (Weber et al., 1995; Berger, 1999; Miller et al., 1999) and these apoptotic processes may also involve glucanases (Buchner et al., 2002). BURP-domain proteins. Expressed sequence tags (ESTs) exhibiting homology to a BURP domain-containing gene family were identied in G. max (L.) Merr (Granger et al., 2002). These ESTs were assembled into 16 contigs of variable sizes and lengths.

The soybean family members exhibit 35 98% similarity in a , 100-amino acid C-terminal region. Soybean BURP-domain proteins have diverse expression patterns. Batchelor et al. (2002) investigated SCB1, a BURP-domain protein gene, which is expressed in developing soybean seed coats. It is known that changes in the cell wall structure are a distinguishing features of cell differentiation in the seed coats (Miller et al., 1999). The BURP domain may represent a general motif for localization of proteins within the cell wall matrix. SCB1 mRNA accumulates rst within the developing thick-walled parenchyma cells of the inner integument and later in the thick- and thin-walled parenchyma cells of the outer integument. This occurs prior to the period of seed coat maturation and seed lling and before either of the layers start to degrade. In addition, the SCB1 protein appears to be located within cell walls, and thus, may play a role in the differentiation of the seed coat parenchyma cells. Seed coat subtilisin. Another seed coat specic gene is SCS1 (seed coat subtilisin 1) that belongs to a small gene family of genes with sequence similarity to subtilisin, a serine protease (Bachelor et al., 2000). Northern blot analysis and in situ hybridization studies revealed that accumulation of SCS1 mRNA is restricted to thick-walled parenchyma cells of the soybean seed coat and reaches maximal levels at 12 d post-anthesis, preceding the nal stages of seed coat differentiation. The thickwalled parenchyma, which is derived from the inner integument, is very prominent during the rst week but is rapidly degraded by the second week. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. As serine proteases are often involved in signal transduction, SCS1 could play a role in cell differentiation in seed coats. Seed coat peroxidases. Soybean seed coat peroxidase (SBP) is another important enzyme found in seed coats. This enzyme belongs to class III plant peroxidases that function in cell wall biosynthesis, defense, and other oxidative processes (Welinder, 1992). Class III plant peroxidases can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide (Henriksen et al., 2001). Peroxidase activity in the seed coat of soybean is controlled by the Ep locus. Such activity from seed coats of EpEp cultivars is 100fold higher than that from epep cultivars. In seed coat extracts, peroxidase is the most abundant soluble protein in EpEp cultivars, whereas this enzyme was present only in trace amounts in epep cultivars (Gijzen et al., 1993). No obvious difference in the structure of the seed coat was associated with the Ep locus (Gijzen et al., 1993). The mutation in the peroxidase-decient line was a deletion in the promoter region of the Ep peroxidase genes (Gijzen, 1997). Histochemical localization of peroxidase activity revealed that the enzyme accumulates predominately in the hourglass cells of the subepidermis. A single isozyme of the seed coat peroxidase constitutes about 5% of total soluble protein in the seed coat of EpEp cultivars (Gillikin and Graham, 1991; Gijzen et al., 1993). No putative plant peroxidases are orthologous to soybean peroxidase. Soybean peroxidase shows more than 70% amino acid sequence identity to peroxidases from other legumes involved in various defense responses (Welinder and Larsen, 2004). For instance, the soybean sequence is very closely related to four peroxidase cDNAs isolated from alfalfa, with 65 67% identity at the amino acid level (Gijzen, 1997).

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It was once thought that soybean seed coat peroxidase might be important in lignication, or suberization, hardness, and permeability (Gillikin and Graham, 1991; Gijzen et al., 1993). Now, the similar phenotypes of EpEp and epep cultivars, and the existence of vacuolar targeting signals on the protein argue against the involvement of the peroxidase in the synthesis or modication of extracellular polymers. It may have a function in defense, when released from hourglass cells during seeds imbibition (M. Gijzen, personal communication). Due to its high stability and activity, SBP provides an alternative to horseradish peroxidase in many industrial applications. SBP is present at high concentration in the soybean seed coat depending on the cultivar (Gijzen, 1997; Gijzen et al., 1993). Due to its high thermo- and pH-stability it can be easily puried from soybean hulls (Henriksen et al., 2001; Nissum et al., 2001; Welinder and Larsen, 2004). Gijzen et al. (1999a) isolated a second peroxidase gene, Prx2 that is also highly expressed in developing seed coat tissues. Sequence analysis of Prx2 cDNA indicates that this transcript encodes a cationic peroxidase. By comparing the abundance and localization of the EP and Prx2 transcripts, it was shown that the expression of EP begins in a small number of cells anking the vascular bundle in the seed coat, spreads to encircle the seed, and then migrates to the hourglass cells as they develop. Expression of Prx2 occurs throughout development in all cell layers of the seed coat, and is also evident in the pericarp and embryo. The Prx2 enzyme is either insoluble in a catalytically inactive form, or is subjected to degradation during seed maturation. MADS box proteins. MADS box genes represent a large family of highly conserved transcription factors. In plants, most of these factors seem to be involved in the control of oral development. However, the pea gene paeMFT1-encoding MADS box transcription factor was shown to be specically expressed in the seed coats during seed development with weak oral expression (Buchner and Boutin, 1998). Maternally controlled defects in seed development after downregulation of MADS box genes in petunia (Colombo et al., 1997) suggest that MADS box proteins might play important roles in the seed coat development (Buchner and Boutin, 1998).

Seed Coat Biotechnology The modication of seed quality and use of seeds for the expression and storage of foreign proteins has been widely investigated in plant systems, including crop species. Seeds have several major advantages, including the availability of regulatory signals derived from studies of seed storage proteins and their genes, and the agricultural systems designed to harvest, store, and process them. The types of foreign proteins that have been expressed include proteins to improve the amino acid content, enzymes, antigens, and biopharmaceuticals among others. In theory, any protein could be expressed in the seed, as could non-protein materials such as vitamin A precursors in rice (Xudong et al., 2000) if the metabolic pathways in the developing seed were modied. Many of the advantages relating to seeds (e.g., harvesting, storage, processing) would also accrue to seed coats. Although seed coats in soybean may constitute , 8% of the seed by weight, this amount still represents a very substantial source of material for processing when considering that the production of soybean seed in 2004 was estimated to be , 190 106 Mt (http://www.fas.usda.gov/ default.asp). Although most proteins expressed in seeds could also be expressed in seed coats, including pharmaceuticals, the greatest potential for the technology may be in the use of biotechnology to solve problems in industrial, environmental (white biotechnology), or agricultural processes (green biotechnology). Genetically modied seed coats may provide a biodegradable, environmentally friendly source of enzymes and biochemicals either in a puried or partially puried form. Many uses for seed coat technology have been described and these can be scrutinized on government, industry and growers web sites (http://www.uspto.gov/, http://www. pnpi.com/welcome.htm, http://www.soy2020.ca/index.ph, http:// www.asasoya.org/home.htm, and many more) but here we will focus on examples of non-medical applications, such as industrial processing, pollution control, and feed modication. Fig. 2 provides an overview linking together many of the traditional seed traits that can be improved through biotechnology, and new opportunities that exist for generating novel bioproducts and bioprocesses.
Defense Germination & dormancy Nutrition & allergenicity Processing qualities Seed color

Mutations/ breeding

Seed size & shape Agriculture Seed coat specific expression Environment Hulls Novel products Health Industry

Soybean

Cloned genes Embryos Composition yield

FIG . 2. Modication of soybean seed coats by transgenic routes including value-added properties.

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Industrial and Environmental Applications Peroxidases and laccases are oxidoreductases that have potential applications in industrial processes and environmental technologies. Peroxidases are heme-containing enzymes that use hydrogen peroxide as the electron donor, while copper-containing laccases employ molecular oxygen as the electron donor. These enzymes are widespread in nature and catalyze one-electron oxidations of suitable substrates. Many potential uses for these enzymes have been identied, ranging from bleaching denim, to pulp and paper bleaching, waste water treatment, soil remediation, food conditioning, and diagnostic kits (http://www.novozymes.com/, http://www.vtt. /bel/indexe.htm, http://www.pnpi.com/Welcome.htm). And there is evidence that SBP can substitute for formaldehyde in the formation of phenolic resins used for the production of plywood and particle board (Enzymol International, USA, US patent 5,491,085). SBP is better suited for industrial applications than most peroxidases, having a relatively high thermal stability (active at 708C) and being active over a broad pH range. Many laccases have been isolated from fungi and other organisms, their properties investigated and shown to be potentially useful. The major factor that limits the adoption of these technologies is the cost of the enzymes. The seed coat expression system for the production of heterologous enzymes of potential industrial relevance offers great promise to overcome this limitation by lowering the cost. Further advantages include the targeting of enzymes to the hourglass cells that inherently store proteins, thus avoiding potential problems with proteolytic degradation. Soybean seed coats are already harvested as part of the processing of soybeans, so there is no additional cost associated with harvesting. The potential for high yield, low cost downstream processing is good, since the target enzyme will likely be the predominant protein in the extracts when the host plant does not express the endogenous Ep peroxidase. Since the amount of hulls produced worldwide approaches , 15 106 Mt yr21, a cheap, stable source of raw materials can be provided through biotechnology. The feasibility of such applications is illustrated by the work of Bassi and Gijzen (Flock et al., 1999; Geng et al., 2004) who studied the degradation of phenols and chlorophenols. Their work demonstrated that crude hulls were superior to puried SBP for the degradation of pollutants in batch reactors and removed 96% of the phenol within 20 min and 98.5% of the 2-chlorophenol within 15 min. These studies point to the potential cost savings by the use of unpuried, or partially puried enzymes for industrial/ environmental purposes. Feed Modication Soybean in the form of meal, oil, or pellets is a major source of protein and nutrient in animal feed for livestock, poultry, and pets. This market is growing and could expand to include sh, exotic animal farming, and many other applications. Seed coats can be added back to feed as a source of ber and nutrients. Thus modied seed coats could be the means to improve the nutritional quality of the feed by introducing enzymes, or compounds that aid digestion, specic micronutrients, or proteins with antibiotic activity. For example, PNPI (http://www.pnpi.com/Welcome.htm) has proposed that seed coats could be a source of phytase, an enzyme that improves phosphorus utilization in feed for poultry, swine and other

monogastric animals by breaking down the phytin that binds phosphorus in plant material. The improved efciency of phosphate utilization has an additional benet, i.e., reduced phosphorus pollution when the manure is used as a fertilizer (Golovan et al., 2001). This example illustrates a general strategy by which a seed coat supplement containing digestive enzymes could be used to modify feed stocks and improve nutrition. Animal feeds are sometimes used as a means to introduce antibiotics and other growth-promoting compounds. Recent concerns about antibiotic resistance have spurred a search for alternatives. Antimicrobial peptides (AMPs) expressed in seed coats could provide a viable substitute. AMPs are small (12 45 amino acids) cationic peptides that at low concentrations (0.5 4.0 mg ml21) have antimicrobial activity against bacteria, fungi, and viruses (Hancock and Lehrer, 1998). Their antibacterial mode of action is to assemble within the bacterial or fungal membrane to create pores, leading to leakage of the cell contents and death. Naturally occurring AMPs are encoded by genes and are produced as a precursor protein that is processed to give the nal active polypeptide. They are non-immunogenic when ingested but may stimulate an immune response if injected. Several groups have demonstrated that plants can express AMP transgenes at low levels and that the AMPs retain their biological activity (Osusky et al., 2004; P.G. Arnison, SynGene Biotek Inc., personal communication). Since seed coats have reduced digestibility when compared to meal, the active AMP inside the hourglass cell may be protected from proteolytic activity while the seed coat slowly breaks down, releasing AMP in a manner similar to a slow-release pill. The recent in vitro experiments (Geng et al., 2004) described above suggest that this may be a viable strategy for feed supplements. Vectors for Transformation Soybean can be effectively transformed with a variety of vector types, including the CAMBIA series (http://www.cambia.org/). For expression of the transgene in specic cell types, such as the HGCs of seed coats, these vectors must be modied by the addition of an HGC-specic promoter. If accumulation of the protein in a particular location (vacuole, endoplasmic reticulum, cell wall) is desired, then additional protein targeting signals must be added to the transgene to create a fusion protein with these signals. Ep peroxidase is expressed in HGCs and the enzyme is translocated to the protein storage vacuole (PSV) via the endomembrane system with concomitant removal of the appropriate signal peptides (Welinder and Larsen, 2004). Thus sequences for an HGC-specic promoter, an amino-terminal ER signal peptide and a carboxy-terminal VSP signal may be supplied from the Ep gene. While the addition of these signals requires more steps in transgene construction, the extra effort of targeting the Escherichia coli Lt-B toxin to the PSV resulted in a higher expression level in maize (Hood, 2004). The choice of target will depend on many factors including the properties of the protein and its ease of purication. Preliminary experiments suggest that the Ep peroxidase promoter region can direct the HGC-specic expression of GUS in transgenic soybean (Gijzen and Simmonds, unpublished) but the ability of the signal peptides to correctly target a foreign protein to the vacuole has not yet been veried experimentally. Many potential cis-acting elements within the Ep promoter can be identied by bioinformatics tools, but a functional analysis of the promoter has not yet been

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performed. From the point of view of optimizing the expression of transgenes, the identication of sequences that determine tissuespecic expression would be a necessary prelude to the construction of hybrid promoters that would retain elements for tissue-specicity while introducing elements from stronger promoters, such as tCUP (Malik et al., 2002). Future experiments employing CHIPs to analyze the soybean transcriptome (Stacey et al., 2004) will most certainly identify more seed coat-specic genes and their regulatory sequences, including promoters and enhancers. The availability of such a toolbox would allow much more exibility in the design and expression of transgenes. Targeting of heterologous proteins may also lead to problems, and again SBP can be used as an example. In the mature SBP, 18% of the mass is heterogeneous glycan (Welinder and Larsen, 2004) attached though N-linkages at asparagines residues within the consensus sequence Asn-X-Ser/Thr. They are added in the ER and modied in the Golgi during protein processing (Helenius and Aebi, 2001). While expression of the transgene in the cytoplasm does not lead to this type of modication, targeting to the ER, vacuole and cell wall most certainly will, and could change the properties of the protein, including activity and antigenicity. It would be necessary to carefully analyze each protein product to investigate whether modications will inuence the desired properties. Additional Considerations for Exploitation To this point the discussion has centered on the use of HGCs in soybean as a vehicle for the expression of a single gene. The expression of multiple genes (gene stacking) would extend the utility of this system and allow the accumulation of combinations of enzymes for industrial or environmental purposes, or the construction of metabolic pathways for the synthesis of novel compounds. For some purposes, high levels of SBP normally stored in the HGCs may prove to be problematic and epep lines in which the SBP is not expressed (Gijzen, 1997) could be substituted. Worldwide, soybeans are the major legume crop and thus could potentially generate the greatest amount of seed coat for processing. Indeed large-scale production of seed, oil, and feed implies that a processing system is in place. Other legumes are signicant sources of seed and forage in many countries around the world and potential for the use of other minor legumes as alternatives to soybean should be considered for certain geographic regions. Hourglass cells (or osteosclereids, pillar cells, lagenosclereids) have been described in the seed coats from a large number of legumes such as lupin (Clements et al., 2004), pea (Harris, 1984), milkvetch (Miklas et al., 1987), common bean (Yeung, 1990), white popinac (Serrato-Valenti et al., 1995), yam bean (Ene-Obong and Okoye, 1993), pulse (Gupta et al., 1985), and many others (Irving, 1984; Manning and van Staden, 1987; Trivedi and Gupta, 1987; Pandey and Jha, 1988; Izaguirre et al., 1994; Sharma and Sharma, 1994; Paria et al., 1997; Sornsathapornkul and Owens, 1999). Their wide distribution suggests that legumes have similar seed coat anatomy and that the regulatory signals for transcription and protein targeting that function in soybean may also function correctly in closely related species. If such speculation is in fact true in practice, then the use of alternatives to soybean becomes not a technical problem but a socio-economic one. The choice of system depends upon the ability of the local economy to supply seed coats, the presence of the appropriate processing and purication systems and

the demand for the products. Where seed legumes are grown, their future use as a bioreactor must successfully compete with the present use for seed coats. For example, although the price of seed coats may be low, if they are added back to feed after crushing to remove the oil, then their effective price is the price of meal ($160 170 US/ton in 2004 , http://usda.manlib/cornell/edu/data-sets/ crops/89002/ .). As new uses for legumes are developed then thought should be given to the potential of the seed coat system and to the merging of technologies, e.g., lupin seeds could be processed in such a way that allows the easy isolation of seed coats for further exploitation (http://www.grdc.com.au/growers/res_upd/west/ 04/ sands.htm). Conclusions Not only has the seed coat developed highly sophisticated processes for the protection of embryos and germinating seedlings from biotic and abiotic stresses, but it also plays a pivotal role in the control of development. The ability to manipulate any of these processes with cloned genes has the potential to dramatically alter the yield and composition of seeds for traditional uses and for the production of novel products for new uses (Fig. 2). In this review we have tried to summarize our understanding of seed coat form, composition, and genetics. It is clear that our knowledge of the genes that regulate seed coat development or the pathways that produce their valuable compounds is very poor, and inadequate to realize the full potential of biotechnology at this time. However, many of the biological tools needed for biotechnology are well developed. Arabidopsis is an important model species that may accelerate our acquisition of knowledge but it may not be sufcient to provide the information that is specic to many of our crops. There is a need for more basic research on the seed coat, a novel and fascinating developmental system in its own right that has been ignored for too long. Acknowledgments
The authors are grateful to Drs Mark Gijzen, Shea Miller, and Tamara Western for reviewing the manuscript prior to submission. The authors are grateful to C.L. Johnson for drawing the gures. J. M. is the recipient of an NSERC postgraduate scholarship. S. H. is the recipient of an Ontario Graduate Scholarship. The research was supported in part by an NSERC Strategic Grant awarded to D. A. J. and by Agriculture and Agri-Food Canada. Eastern Cereal and Oilseed Research Centre publication number 05520.

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