Arabidopsis seed coat development - Wiley Online Library

The Plant Journal (2000) 22(6), 483±493

Arabidopsis seed coat development: morphological differentiation of the outer integument J. Brian Windsor, V. Vaughan Symonds, John Mendenhall and Alan M. Lloyd* Molecular Cell and Developmental Biology Section, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1095, USA Received 20 January 2000; revised 17 March 2000; accepted 17 March 2000. *For correspondence (fax +512 232 3432; e-mail [email protected]).

Summary A morphological description of the differentiation of the outer integument of the Arabidopsis thaliana seed is presented. The period covered starts at about the octant embryo stage, extends to the mature seed, and concludes beyond that at the initial stages of seed imbibition. During this period the two-celllayered outer integument goes through a dramatic differentiation process. The outer cell layer secretes mucilage in a ring between the plasma membrane and the outer cell wall at the corners of the cell. This secretion forces the cytoplasm into a columnar shape in the center of the cell. Before and during this process, starch granules are produced, initially at the center of the outer wall and later within the column. Late in differentiation, the starch granules are degraded as the cell produces a highly reinforced wall surrounding the columnar protoplast and at the radial walls between adjacent cells. This results in a cell containing large amounts of mucilage surrounding and completely outside of a highly reinforced columella. The mucilage and outer wall then dehydrate to leave the columella and radial walls visible as the epidermal plateau and reticulations visible on the mature seed. The inner cell layer of the outer integument also produces and degrades starch granules concomitantly with the outer layer but produces no mucilage. In the mature dry seed the collapsed outer wall remains connected to the top of the columella and the radial walls, but these connections are rapidly broken as the mucilage fully hydrates.

Introduction The seed coat or testa is the protective outer covering surrounding the plant embryo. Important functions of the seed coat include protecting the embryo from mechanical damage and pathogen attack, maintaining the dehydrated dormant state of the embryo until proper germination conditions exist, and providing the means for initial water uptake. In addition, seed coats can provide dispersal mechanisms and protect the embryo from UV damage. Most seed coats are highly specialized and differentiate to assume several roles at maturity. For example, the cotton ®ber is a highly differentiated product of the outer or epidermal seed coat layer. The seed coat is an essential component in the higher plant life cycle and understanding its structure and development has been an important goal for biologists. A large body of literature exists describing the anatomy of mature seed coats of many species, primarily because seed coat morphology is an excellent taxonomic character (for example see Vaughan and Whitehouse, 1971; Werker, ã 2000 Blackwell Science Ltd

1997). However, there are very few descriptions of the developmental events that lead to the differentiation of mature structures. The process is well studied in cotton ®ber differentiation (Beasley, 1975; Stewart, 1975) and has been described for soybean (Miller et al., 1999). There have been a few reports describing the cytological and developmental changes that occur in the mucilage producing cells during seed coat maturation in genera such as Cydonia (Abeysekera and Willison, 1987), Collomia (Schnepf and DeichgraÈber, 1983a), Ruellia (Schnepf and DeichgraÈber, 1983b), and Plantago (Hyde, 1970). Within the Cruciferae, several aspects of seed coat development and maturation have been described, including testa initiation and development in Capsella bursa-pastoris, Sinapis alba, Brassica alba and Lunaria annua (Bouman, 1975); aleurone layer differentiation in Sinapis alba (Bergfeld and Schopfer, 1986); cytology of mucilage production (Van Caeseele et al., 1981) and palisade development (Van Caeseele et al., 1982) in Brassica campestris; and seed 483

484 J. Brian Windsor et al. coat maturation in Raphanus sativus (Harris, 1981). Because Arabidopsis thaliana has emerged as an important model for genetic and molecular studies of developmental and biochemical processes, we chose to investigate and characterize Arabidopsis seed coat development. An important prerequisite for understanding the development and function of the mature seed coat is a detailed morphological dissection of this process to de®ne the events and structures involved. At present, there is no detailed study of the developmental processes in the Arabidopsis seed coat that lead from the mature ovule to the mature seed. However, images of the exterior of mature Arabidopsis seed have been published numerous times. These mature seed coat images were primarily of interest to contrast wild type and mutant seed coats that display altered surface textures and a lack of releasable mucilage. However, the few publications that discuss where the mucilage is deposited and released from the Arabidopsis seed coat are largely incorrect (see Discussion). In Arabidopsis, mucilage is produced by the developing seed and deposited in the outer cell layer of the seed coat. Mucilage is a soluble hydrophilic polysaccharide produced at some time during development in various tissue types by most plant species. Mucilage can be pectic, hemicellulosic or cellulosic in nature. The monomer sugar composition of hydrolyzed Arabidopsis seed coat mucilage has been determined (Goto, 1985). It contains primarily rhamnose and galacturonic acid consistent with it being mostly pectic. However, the linkage and structure of the polymer(s) has not been reported. Upon imbibition or initial water uptake, the dehydrated mucilage is rapidly released from the seed coat providing a gelatin-like coating surrounding the mature seed. Like the seed coat itself, the mucilage is believed to be important for many reasons and is thought to play roles in seed germination and dispersal. The Arabidopsis testa is the product of the maternal parent and is formed from two integuments of epidermal origin that surround the mature ovule. The development of the integuments surrounding the Arabidopsis ovule have been well described (Gasser and Robinson-Beers, 1993; Robinson-Beers et al., 1992; Schneitz et al., 1995) and several mutants in integument development have been characterized. The integuments of a mature ovule at the time of anthesis consist of a two cell-layered outer integument and a mostly three cell-layered inner integument. At the micropyle end, an endothelium apparently does not form and the inner integument may be only one cell layer at that end grading to two and then three cells thick at the opposite, chalazal, end. In this paper, we describe the morphology and anatomical development of the cells and structures associated with events in normal Arabidopsis seed coat development concentrating on the

Figure 1. Toluidine blue-stained cross-section of a developing Arabidopsis seed in silique. This image shows the general organization of the developing seed coat and associated tissues fairly late in development, between the stages shown in Figure 2(c,d). SW = silique wall; OI = two cell-layered outer integument; II = three cell-layered inner integument; EN = cellular endosperm; EM = embryo. Bar = 20 mm.

differentiation of the outermost epidermal cells during the period of mucilage production, deposition and release. We seek to address questions of where and when mucilage is produced and deposited in the developing seed and what are the developmental pro®le and possible functions of the structures observable on and in the seed coat. This study uses light and electron microscopy to describe the key morphological events leading from the mature, fertilized ovule to the mature Arabidopsis seed coat and the subsequent release of the seed coat mucilage upon water imbibition. The period covered in this study is from mature ovule to mature seed and beyond to mucilage release upon initial seed imbibition. During this period, the outer cells differentiate from relatively non-distinct parenchymatous cells to a mucilage secretory phase where the cell is secreting mucilage between the plasma membrane and the primary cell wall. This secretion leads to cells with a greatly reduced protoplasm volume that occupies a columnar central position extending from the outer to ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 483±493

Arabidopsis outer integument differentiation

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Figure 2. Stages of outer integument differentiation. Toluidine-stained cross-sections of outer integument cells are shown along with a labeled cartoon of each and the associated embryo stage. The cartoon panels only include the two outer integument cell layers which are contiguous with the inner integument (Figure 1). (a) Row showing the earliest stage observed in this study. The outer and inner layer cells each have a large single vacuole with the cytoplasm compressed against the side of the cell and no starch granules or mucilage visible. (b) During this stage of differentiation both cell layers produce prominent starch granules. The outer cell layer characteristically produces granules distal to the embryo at the center of the outer periclinal wall and the cell inner layer produces them proximal to the embryo at the inner periclinal wall. (c) During this stage, the outer cells secrete mucilage to the space between the outer primary wall and the protoplast in a ring at the outer `corners' of the cell around the area where the starch granules are located. This secretion forces the protoplast to begin to assume the columella shape. The starch granules continue to enlarge during this period. The vacuole in the inner cells appears to break up into smaller vacuoles (it is possible that it forms a single convoluted tube of some sort that looks like more than one vacuole in cross section). The starch granules continue to enlarge here also and the inner cells begin to compress towards the outer cells. (d) By this stage, mucilage production, in terms of the space that it takes up, is complete. This has forced the protoplast to form a column in the center of the cell. The vacuole in the outer cells has disappeared and at this time the starch granules begin to be degraded as evidenced by a reduction in size in later sections. During processing of these sections the outer wall has slightly pulled away from the top of the column showing a ¯at-topped reinforced region. In the inner cells, the vacuoles are almost completely gone and the starch granules are beginning to be degraded. The inner cells are continuing to compress against the outer cell layer. (e) By this stage, a thick secondary wall has been laid down next to the column-shaped protoplast, the basal area of the cell, and the radial walls separating adjacent cells. The shape of this reinforced wall matches the shape of the cytoplasm as deduced from the GUS staining seen in Figure 3. The starch granules are almost entirely gone by this stage. The inner cell layer has completely compressed against the outer cells and at the same time reinforced its walls. The vacuoles and starch are undetectable and this layer appears as essentially a thick inner wall layer of the outer cells. This section is through three outer cells, off-center of a central cell that is ¯anked by two sections through the columella. At this stage it is dif®cult to keep the mucilage from fully hydrating during sample processing. The outer wall of the left cell is still connected to the top of the columella and the radial walls. Hydration of the mucilage in the two cells at the right have caused the outer wall to break free from its connections to the top of the columella and the radial walls. This section shows that the outer periclinal wall remains intact even after hydration of the mucilage. OW = thin outer wall of the outer cell; VA = vacuole; SG = starch granules; MU = mucilage; CO = columella; IL = inner cell layer of the outer integument; Bar = 10 mm in all panels.

ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 483±493

486 J. Brian Windsor et al. the inner periclinal walls and space adjacent to the inner wall. The cell then produces a spindle-shaped cellulosic secondary wall inside the mucilage layer. Large starch granules occupy much of the spindle-shaped protoplasm space and are degraded late in differentiation as the cell walls are reinforced. The mucilage then dehydrates to leave the thin, outer periclinal wall draped over the seed surface. The result of these processes is the distinctive reticulated Arabidopsis seed coat with reinforced radial walls and the spindle visible on the surface as the hexagonal array and the epidermal plateau or columella, respectively.

time the vacuole shrinks and eventually disappears as the mucilage forces the cell to reduce protoplasm volume. GUS histochemical staining (Jefferson, 1987) of WS wild-type Arabidopsis transformants containing a Glabrous2::GUS transcriptional fusion (Masucci et al., 1996; Szymanski et al., 1998) supports the anatomy observed in the sectioned material. GUS staining reveals

Results For the purposes of this paper we will use the following terminology. Tangential or periclinal walls are those that are more or less parallel to the outer surface of the seed. Radial or anticlinal walls are those that run more or less perpendicular to the surface of the seed. We are describing a two-cell-layered structure so the outer cell is on the outside of the developing seed and the inner cell is just inside of that, proximal to the embryo. The inner and outer cell walls are those that are proximal and distal to the embryo, respectively (Figure 1). Just past the point of the octant embryo, tangential and radial images of both layers of the outer integument reveal blocky cells, neither of which contains observable amounts of starch. Early in development, a single large vacuole occupies nearly the entire volume of the cells in each of these two layers (Figure 2a). It was not until TEM work that the intact cytoplasm was observed closely appressed against the cell walls (data not shown). The next major development in the maturation of these cells is the production of starch granules. Starch granules ®rst appear next to the outer and inner walls of cells in the outer and inner cell layers, respectively (Figure 2b). It is at this point that the developmental paths of these two cell layers diverge. Although starch continues to accumulate in both layers, only cells of the outer layer begin to produce mucilage and deposit it between the primary cell wall and protoplasm. Deposition begins at the outer region of each cell nearest to the radial walls. Because the cell membrane remains attached to the primary outer wall in the center of each cell, mucilage initially forms a doughnut-shaped ring surrounding the protoplasm and displaces the protoplasm from this area (Figure 2c). Through more mucilage production and deposition, the mucilage continues to displace and compress the protoplasm (which still contains starch granules) into a column in the center of each cell, eventually forming the columella-shaped protoplasm (Figure 2d). In addition, it also appears as though the cell membrane remains in contact with the primary cell wall along the inner tangential and some portion of radial walls. During this

Figure 3. Histochemical staining of GL2::GUS fusion expressed in the outer integument. (a) Several developing seeds are visible in this silique. This GUS fusion is strongly expressed in the outer cell layer of the outer integument late in the differentiation process and is highly speci®c to this cell layer. (b) When the outside seed surface is viewed, the GUS staining shows the columella and the radial walls probably because that is where the cytoplasm is thickest. (c) When a seed cross-section is viewed in close-up, the GUS staining shows the inverted umbrella shape of the cytoplasm in these cells at about the same stage as shown in Figure 2(d). This staining gradually disappears and is undetectable shortly after the stage shown in Figure 2(e). RW = radial walls; CO = columella; OW = outer wall.


Arabidopsis outer integument differentiation that, in terms of the seed coat, Glabrous2 (GL2) transcription is restricted to the outer cell layer of the outer integument late in the differentiation process (Figure 3a). More importantly, the staining shows the extent of the cytoplasm after the columella shape is attained (Figure 3b,c). What is apparent is that the cytoplasm is contained within the central column as expected but it occupies a thin layer next to the inner wall of the outer cell and continues up the radial walls to approximately half the height of the columella in an inverted umbrella shape. Thus the GUS staining prophecies what will later become the extent of the reinforced walls that are visible on the mature seed surface. Based on calca¯uor and toluidine stained sections and TEM, the outer cells appear to reinforce the outer wall in the central area where the starch granules reside fairly early in the process of attaining the column shape (Figure 2c). Later in the process, after the protoplast assumes the columella shape, starch degradation and wall reinforcement of the column commences. Reinforcement appears to take place at the cell membrane surface; however, in these outer cells the cell membrane is no longer in contact with the outer primary wall except at the center. Instead, secondary cell wall is deposited around the columella or umbrella-shaped protoplasm, located within both the mucilage and primary walls (Figures 2d,e and 4g,h). This process continues until there is no more starch detectable either in sections or by I2/KI staining and the columella is a highly reinforced cellulosic column. While the outer cell is producing mucilage, the starch granules of the second cell layer continue to enlarge and the large single vacuole appears to break up into two or three smaller vacuoles that continue to shrink (Figure 2b,c). During this process, the second cell layer becomes more and more compressed against the outer cell layer. The starch in these cells begins to degrade at about the same time that the staining of thick sections reveals a thickening or reinforcement of the inner cell's walls. At this point, the two cell layers appear to become somewhat synchronized, as the cells in the inner and outer layers undergo the same processes of starch degradation and wall reinforcement. The cells of the second layer continue to compress against the outer cells while the starch is completely degraded, the walls become highly reinforced, and the vacuoles disappear. At maturity, the second layer is not recognizable as cellular and appears in thick sections as an extremely thick inner wall of the outer layer (Figure 2e). After mucilage production, starch degradation and cell wall reinforcement are complete, the mucilage layer dries down compressing the mucilage into a thin layer on the seed surface under the relatively thin, outer primary cell wall. In the mature seed, the mucilage of each cell is contained in a kind of dry moat surrounding the columella. ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 483±493

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When mature dry seeds are placed in water, the mucilaginous layer imbibes water rapidly and the extent of mucilage expansion can be seen in Figure 4(a) by the displacement of India ink. Figure 4(b,c) shows mature, dry, untreated seed while Figure 4(d) shows the results of fully imbibing the seed mucilage and then allowing the seed to redry on a surface before sputter coating. When seeds are hydrated and allowed to redry in this manner, the mucilage and outer periclinal wall will again become a thin ®lm that conforms to the shape of the reinforced columella and radial walls. However, the mucilage does not necessarily go back to where it came from, as can be seen by the drapes of outer wall and mucilage that have stuck to the EM stub surface and extend up to the seed surface (Figure 4d). Figure 4(e) shows seeds that were water-imbibed, ®xed with glutaraldehyde and osmium tetroxide, dehydrated, critical point dried, and viewed with SEM. The full volume of the mucilage cannot be maintained with this procedure, however, this image shows that the outer surface of the seed loses cellular de®nition and that there is a contiguous outer wall that largely remains intact if it is not mechanically disrupted. The columella can be seen as the bumps underneath the outer wall. Small ®ssures can be seen in the surface where the wall is broken. The outer wall of the seed in the upper left has broken off from one end. Figure 4(f) shows the cut surface of a seed that was imbibed and ®xed. Although no columellas are visible in this image, the outer periclinal wall and ®brous mucilage are visible, as well as what we interpret as the inner cell layer of the outer integument, the inner integument, and the embryo. In order to help determine what wall connections are still in place in the dry mature seed, environmental SEM (ESEM) was used to look at the surface of a seed during the imbibition process (Figure 5). We had previously noted that when a drop of water was placed on one side of the seed, the water was wicked from cell to cell across the surface. As the cells took up the water, they swelled, but not necessarily to their fully hydrated volume. Images of these partially hydrated cells clearly show that the top of the columella and the radial walls remain attached to the outer wall in the dry seed and that the tangential walls are probably the ®rst to break during full hydration, at least when imbibed in this manner (Figure 5). Discussion We have described the morphological and developmental changes that occur during Arabidopsis outer seed coat or outer integument differentiation. The period covered begins when the embryo is in the octant stage and proceeds through seed maturation and beyond that to the initial water imbibition of the mature seed. We observe no cell division during this period and, as such, we are

488 J. Brian Windsor et al. Figure 4. Images of mature wild-type Arabidopsis seeds. (a) Mucilage will displace India ink allowing the extent of the mucilage expansion upon hydration to be observed. (b) This SEM of the surface of an untreated seed shows the characteristic reticulate pattern and the central plateau or columella. (c) Close up of seed in (b) showing detail. (d) If the seed is wetted and then allowed to dry completely, the dehydrated mucilage shrinks to again follow the shape of the reinforced secondary walls of the outer cell layer. However, where it contacts another surface it dries to form drapes `gluing' the seed to that surface (arrowheads). Seeds that have been wetted and dried, stick tenaciously to each other and any surface they dry in contact with. (e) These seeds were wetted and then ®xed prior to redrying. The ®xation inhibits but does not completely prevent the mucilage from shrinking during the drying process. Wrips and wrinkles in the thin outer wall are visible on the surface. The bumps are the tops of the columellas holding the outer wall away from the surface. The outer wall has torn off the end of the seed visible in the upper left. This image shows that the outer periclinal wall of the seed can remain intact even after complete hydration of the mucilage. This would imply that this wall possesses the ability to remain extremely plastic. (f) This seed was treated exactly as above and then hand-cut with a razorblade and re-sputter coated before imaging. The thin outer wall is visible above the ®brous-looking mucilage. What we are interpreting as the remnants of the inner integuments are visible above the embryo. No columellas are visible. It appears that we could not cut through a columella using this procedure. (g) TEM of cell prior to extensive secondary cell wall reinforcement. The arrow points to the side of what will become the columella at about the stage shown in Figure 2(c). At this stage only a thin cytoplasm and no secondary is visible. (h) TEM of highly reinforced columella wall. The arrow points to the side of the columella at about the stage shown in Figure 2(e). The walls are highly reinforced with secondary cell wall material at this stage. CO = columella; RW = radial walls; OW = outer wall; MU = mucilage; OI = outer integument; II = inner integument; EM = embryo. Bar in (a,c,d) = 50 mm; bar in (b,e) = 100 mm; bar in (f) = 10 mm; bar in (g,h) = 3 mm.

describing a dramatic cellular differentiation process that leads from relatively cuboid vacuolated cells, through massive starch and mucilage production stages, to starch degradation and wall reinforcement that leads to the distinctively shaped outer seed coat cells. The outer integument of the developing seed is maternally derived from the incipient ovule epidermis and remains two cells thick throughout its existence

(Schneitz et al., 1995). Thus, both of these cell layers can be considered to be epidermal. In fact, at least two mutants that affect other epidermal cell-fate and cell differentiation processes also affect the differentiation of the outer seed coat. These mutants are transparent testa glabra 1 (ttg1), affecting anthocyanin production, trichome cell-fate determination and position-dependent root hair production (Galway et al., 1994; Koornneef, 1981) and glabrous 2 ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 483±493

Arabidopsis outer integument differentiation

Figure 5. Environmental SEM of an imbibing seed. Water placed on one side of a seed will be wicked to adjacent cells partially hydrating the mucilage. The swelling of the mucilage in¯ates the thin outer periclinal wall out but the connections to the top of the columella and the radial walls remain intact. This is apparent as the `buttoned upholstery' look on the surface of the seed. WA = water drop; CO = top of columella connection; RW = radial wall connection. As can be seen in Figure 4(d,e), these connections can break and still leave the thin outer wall intact.

(gl2), affecting trichome differentiation and position-dependent root hair production (Masucci et al., 1996; Rerie et al., 1994). These two mutants, as well as apetela 2 (ap2) mutations which additionally affect ¯ower development (Jofuku et al., 1994), do not produce mucilage that is releasable upon imbibition and are missing a well-de®ned columella on the mature seed coat. However, they apparently are not missing any seed coat cell layer (data not shown). Many other genes and mutations affect integument initiation and early development and, as a result, ultimately seed coat development. For example, inner no outer (ino) mutants (Baker et al., 1997) do not produce an outer integument (and will produce seeds without mucilage or columella) while severe aintegumenta (ant) mutants (Baker et al., 1997; Klucher et al., 1996) produce no integuments at all. Aberrant testa shape (ats) (LeonKloosterziel et al., 1994) and bell1 (bel1) (Robinson-Beers et al., 1992) mutants produce structures that appear to be a fusion of the outer and inner integuments. These mutations all affect the initiation, development or differentiation of the integument layers early in development and most have pleiotropic effects on other aspects of ¯ower or ovule development. However, they have not been shown to speci®cally affect the processes we are analyzing here. The developmental pro®le of mucilage-producing seed coat cells has been characterized in a handful of species. In ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 483±493

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Figure 6. Cross-sections of starch metabolism mutants. (a) pgm1 mutant. (b) sex1 mutant (note the retention of the starch granules). These sections are at about the stage shown in Figure 2(e), with the inner layer completely compressed. Both mutants display an altered columella shape, wider at the center and bottom for both, and wider at the top for sex1. In addition, the secondary wall material does not stain as intensely as the wild type. OW = outer wall; MU = mucilage; CO = columella; IL = inner cell layer of outer integument; SG = starch granule. Bar = 10 mm.

all cases, mucilage is placed in the space between the plasma membrane and the outer periclinal wall of the epidermal cells resulting in the compression of the protoplast. In this respect, Arabidopsis thaliana seed coat development is very similar. However, the only noncruciferous species investigated whose epidermal mucilaginous cells were reported to contain large starch granules, like Arabidopsis, is Plantago ovata, where the granules precede mucilage production and eventually disappear (Hyde, 1970). In contrast to Arabidopsis, upon compression, none of the non-cruciferous species appear to form a cellulosic inner wall laid down next to the plasmalemma. However, Cydonia (Abeysekera and Willison, 1987), Collomia (Schnepf and DeichgraÈber, 1983a) and Ruellia (Schnepf and DeichgraÈber, 1983b) are all reported to contain cellulosic components in their mucilage and Plantago may contain cellulosic mucilage (Hyde, 1970). Our calca¯uor staining indicates that Arabidopsis mucilage contains substantial b 1,4-linked polysaccharides (data not shown) but the neutral sugar composition would imply that it is not cellulose (Goto, 1985). This requires further investigation.

490 J. Brian Windsor et al. Within the Cruciferae, many but not all species produce a mucilaginous epidermal cell layer during seed maturation (Buth et al., 1987; Vaughan and Whitehouse, 1971). In addition, many species produce a central column in the epidermal cells and most, probably all, of these columnproducing species also produce mucilage in these cells (Vaughan and Whitehouse, 1971). However, there are also many species that appear to produce mucilage and no column. In the few species that have been observed in some developmental detail, Brassica campestris (Van Caeseele et al., 1981), B. nigra, Lunaria annua and Sinapis alba (Bouman, 1975) are reported to produce epidermal mucilage but no columella. However, there are con¯icting reports and Vaughan and Whitehouse (1971) report that B. Campestris and L. annua do not produce mucilage. Furthermore, in their study of S. alba aleurone development, Bergfeld and Schopfer (1986) present a ®gure that appears to show a columella in the outer epidermis, although they do not discuss this aspect; Raphanus sativus reportedly produces no mucilage or columella (Harris, 1991; Vaughan and Whitehouse, 1971); and Capsella bursa-pastoris produces both a columella and mucilage (Bouman, 1975; Vaughan and Whitehouse, 1971). In this and other respects Capsella is most like Arabidopsis in its seed coat development. Both appear to have a two celllayered outer integument and a largely three cell-layered inner integument (Bouman, 1975). Most members of the Cruciferae have more cell layers in both integuments and in many species, the inner cell layer of the outer integument differentiates into a characteristic palisade layer. This layer is absent from both Arabidopsis and Capsella. Although the Capsella seed coat has not been studied in detail, we feel that it is almost certain that Capsella will be found to have the same outer integument developmental pro®le as Arabidopsis. Role of starch in seed coat differentiation The role of starch granules in Arabidopsis and other developing Crucifer seed coats has been considered to be that of a precursor to mucilage (Harris, 1991; Rerie et al., 1994). This would seem a logical hypothesis given that starch is present prior to and during mucilage synthesis and is completely degraded by the time the seed is mature. This would be consistent with the proposed role of starch in at least some other plant tissues, such as the maize root cap (Dauwalder et al., 1969). However, our investigations reveal two lines of evidence that cast doubt on a major role for starch as a reservoir for sugar to be used in mucilage production. First, three mutants in starch metabolism all apparently make wild-type amounts of mucilage. These mutants include phosphoglucomutase1 (pgm1) and ADP glucose pyrophosphorylase1 (adg1) neither of which can make

starch, and starch excess1 (sex1), which cannot degrade starch (Caspar et al., 1985; Caspar et al., 1991). The fact that adg1 and pgm1 lack starch production was con®rmed by our own feulgen and I2/KI staining of seeds and plants. However, both of these mutants release mucilage upon imbibition in an apparently normal fashion. We also con®rmed that sex1 lacks the ability to properly degrade starch resulting in an excess of starch granules easy to visualize with I2/KI staining. This mutant also appears normal regarding the release of mucilage (data not shown). This would indicate that starch is not a necessary precursor to Arabidopsis seed coat mucilage production and that, at the least, there must be an alternate pathway. Second, careful examination of seed coat development indicates a temporal correlation between starch granule degradation and the cellulosic reinforcement of the spindle shaped cell wall surrounding the shrunken protoplasm late in differentiation. Starch degradation and cell wall reinforcement correlate in time and space and occur largely or completely after mucilage production is complete. We have also observed that the columellas in the starch metabolism mutants have a different shape than wild type and do not appear to have the integrity or density of wildtype columellas (Figure 6). We propose that the major role of starch is as a precursor to the polysaccharides used to reinforce the spindle-shaped wall in the mature cell. We have also noted that the inner cell layer of the outer integument produces many starch granules yet produces no mucilage, but also undergoes wall reinforcement correlated with starch granule degradation. Therefore, a more likely role for starch in both cell layers of the outer integument is as a precursor to the reinforcing wall polymers produced late in the differentiation process. Placement of seed coat mucilage and column There has been confusion in the literature regarding the placement of the Arabidopsis seed coat mucilage and columella. Vaughan and Whitehouse (1971) state that there is a `column on inner periclinal wall protruding into the [mucilaginous] cell', implying that the column originated or grows from the inner periclinal wall. Our studies show that the column results from wall material laid down surrounding the protoplasm and forms a column in the cell's center that extends all the way through the cell to the outer periclinal wall. Koornneef (1981) states that `probably the elevating structures excrete mucilage upon imbibition' referring to the columnar structure. This statement has been propagated and cited by subsequent authors (Goto, 1982; Goto, 1985; Rerie et al., 1994). Rerie et al. (1994) go on to state, `The mucilage appears to accumulate on the seed surface in tufts that are linked by a network of strands'. Our work shows that the mucilage is laid down outside the columns/tufts/elevated structures ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 483±493

Arabidopsis outer integument differentiation and is not excreted from within the columns upon imbibition. The `strands' visible on the seed surface are folds in the thin outer periclinal wall that occur as the mucilage dehydrates. Kuang et al. (1996) are largely correct when they state, `At a later developmental stage, increased amounts of polysaccharides push the cytoplasm against the inner tangential wall, and ®nally occupy almost the entire volume of the cells'. Our work indicates that the cytoplasm is pushed against the inner tangential wall only at the cell's perimeter next to the anticlinal walls, not in the center where the columella forms. Kuang et al. (1996) never discuss the columella although a few are visible in their migrographs. The confusion surrounding the location of the dehydrated mucilage on the seed coat is a natural consequence of the phenotype of several mutations that affect mucilage production or release upon imbibition, ttg1, gl2 and ap2. The mature dry seed coats in these mutants do not have the characteristic columella, nor do they have releasable mucilage. This correlation led to the belief that the mucilage was contained within these raised structures. Rather than the missing columella resulting in an inability to release mucilage, we believe that the missing columella structure probably results from a lack of mucilage deposition in a characteristic ring between the cell membrane and the outer wall, which forces the protoplasm into the columella shape. Alternatively, it is possible that the mutants cannot reinforce the columella-shaped protoplasm with a secondary wall, resulting in a collapsed columella on the mature seed surface. Why this would result in no mucilage release upon imbibition is hard to imagine. Initial analysis of sections of mutant seed coats indicate that the mutants have all the normal testa cell layers but are defective in how these external cells differentiate (data not shown). Imbibition We examined the process of mucilage release upon imbibition in wild-type Arabidopsis seeds. Throughout development, the anticlinal walls as well as the top of the columella remain attached to the thin outer periclinal primary wall. Because of the role and placement of this wall, we wanted to ask how this thin exterior wall was connected to the seed surface in the mature dry seed. When seeds are immersed in water, the imbibition process proceeds more or less to completion in a matter of seconds, leaving a smooth outer surface with no pattern evident. We noticed that if we placed a small drop of water on one side of the seed, this water was slowly wicked across the surface from cell to cell. During this wicking process, it appeared that some cellular structure was maintained on the surface of seeds that were partially hydrated that looked very much like the surface of an ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 483±493

491

immature non-dehydrated seed. We used environmental SEM to document this observation by introducing a drop of water on the seed surface while the seed was contained in the sample chamber. These experiments showed that the outer wall remains connected to both the anticlinal walls and the columella. During complete imbibition, these connections are rapidly broken but this breakage does not necessarily disrupt the outer periclinal wall. In fact, we ®nd that if the imbibed seeds are treated gently, they allow the entry of water but exclude larger molecules such as Ruthenium Red. If the outer surface is then mechanically disrupted, the Ruthenium Red can enter the mucilage layer, staining the mucilage. It thus appears that the mature outer periclinal wall is semi-permeable and retains the ability to exclude at least some molecules. Cell layer identity The identity and function of the outer and inner layers of the outer integument are probably ®xed very early in integument development, before the stages observed in this study. The inner no outer (ino) mutant of Arabidopsis (Baker et al., 1997) is able to form an inner integument but no outer integument. This mutant indicates that the inner integument will not by default assume the function of the mucilaginous outer integument. The ino mutation also indicates that the outer integument is required for the curved shape of the embryo, which implies a physical boundary role for the outer integument. The expression of INO may prove to be very interesting in de®ning the time and place of outer integument differentiation from the chalazal region of the developing ovule immediately basal to where the inner integument initiates. The outer integument is a two cell-layered organ with essentially all cells of the outer layer assuming one fate and all cells of the inner layer assuming another. The late and highly polar differentiation process in these cell layers is synchronous and non-iterative on a developing seed with all cells on an ovule and all ovules within a given silique performing the same functions at the same time. This is in contrast to trichome or stomatal differentiation on a developing leaf surface, which proceeds basipetally. However, each silique on an in¯orescence represents a slightly different stage of differentiation so that one in¯orescence potentially includes all stages of integument differentiation. The differentiation of particularly the outer cell layer of the outer integument is quite dramatic and fairly easy to observe. Mutations that affect the differentiation of the outer integument after cell division in this layer is complete will most likely not affect embryo development adversely. This is certainly true for the mucilage defective mutants ttg1, gl2 and some alleles of ap2. As such, this late differentiation process should be amenable to genetic and

492 J. Brian Windsor et al.

The ecotypes used as wild type were Wassilewskija (WS) and Landsberg erecta (Ler). No seed coat differences were observed between the two ecotypes and the ®gures in this paper do not distinguish them. All plants were grown at 22°C under continuous ¯uorescent illumination.

sliced down one side with a double edged razor blade to allow easy penetration of the ®xative, the ®xative was vacuum in®ltrated and the siliques were left overnight. Four per cent osmium tetroxide was added to the glutaraldehyde-®xed siliques to make the solution 2% osmium and these were left for 2 h. Siliques were dehydrated through an ethanol series to 100% ethanol. The ethanol was gradually replaced with LR White resin (Pella) and the samples were baked. Thick sections (1 mm) were prepared on a MT1 microtome (Sorvall) using diamond knives. Sections were retrieved from the knife boat, placed on water drops on glass slides and dried on a hot plate. Cooled slides were stained with 2% toluidine blue with freshly added borax, placed on a hot plate for approximately 2 min until the stain just started to dry. The sections were then rinsed with distilled water and dried on a hot plate. A drop of immersion oil was placed over the specimen and a cover slip was added and sealed with nail polish. This protocol is modi®ed from Hayat (1972). Sections were photographed through an Olympus BX60 scope with an Olympus PM-C35DX camera.

Treatment of seed for SEM

Transmission electron microscopy

For the seed in Figure 4(b,c), mature, dry, untreated seed was ®xed to a standard SEM stub with a carbon conductive tab (Pella) and sputter coated as below. For the seed in Figure 4(d), mature, dry, untreated seed was ®xed to a standard SEM stub with a carbon conductive tab, the seed was covered with a drop of water and then allowed to air dry before sputter coating. For the seed in Figure 4(e), the seed was imbibed in water for several minutes in an Eppendorf tube. The water was replaced with 2% glutaraldehyde, 0.1% cacodylic acid and left overnight. 4% osmium tetroxide was added to the glutaraldehyde-®xed seeds to make the solution 2% osmium and these were left for 2 h. The seed was then dehydrated through a graded ethanol series to 100% ethanol and critical point dried, placed on a stub with a carbon conductive tab, sputter coated and imaged as below. The hydrated seed was treated as gently as possible throughout to avoid disrupting the outer periclinal wall and mucilage layer. For the cut seed in Figure 4(f), a glutaraldehyde/osmium-®xed seed on a stub, treated as in Figure 4(e), was sliced with a razor and then re-sputter coated and imaged as below.

Tissue was treated as above, sectioned at less than 100 nm thickness on a RMC MT6000-XL ultramicrotome, stained with uranyl acetate and lead citrate, and viewed on a Philips EM208.

Scanning electron microscopy

We wish to thank Helga Sittertz-Bhatkar of the Texas A & M Electron Microscopy Center for help with the environmental SEM; Barbara Goetgens for help with microscopy; Mike Windsor and Gwen Gage for help with the ®gures. This work was supported in part by the Texas Higher Education Coordinating Board, The Herman Frasch Foundation and the National Science Foundation.

molecular dissection. We feel that this process will be particularly attractive to investigators studying polysaccharide secretion and secondary wall deposition. Questions as to whether the different cell fates taken by the inner and outer cell layers are due to an asymmetric cell division or distance from some diffusable or physical signal or some other cause remain to be answered. Experimental procedures Plant strains and growth conditions

Where appropriate, specimens were critical point dried in a Tousimis Samdri-790 and sputter coated with a gold-palladium alloy using a Ladd instrument. Specimens were visualized with a Philips 515 scanning electron microscope and photographed with Polaroid P/N 55 ®lm.

Environmental scanning electron microscopy Dry, untreated seeds were attached to a standard EM stub with a carbon conductive tab. A small drop of distilled water was placed on one side of a seed through an injector while it was being observed through an Electroscan ESEM E-3 microscope. This water was wicked to adjacent cells and images of these cells were captured on video.

Plant sectioning and light microscopy Various aged siliques were harvested and placed in 2% glutaraldehyde, 0.1% cacodylic acid in a Petri dish. The siliques were

GUS histochemical staining Siliques were harvested, sliced longitudonally and placed in the GUS substrate, X-gluc (Sigma), prepared as per Jefferson (1987). After approximately 1 h, the siliques or seeds were photographed.

Image processing Photographs were scanned using an Apple ColorScanner or UMAX Astra2000U and the images converted to TIFF ®le format. Images were enhanced for color and clarity using Adobe Photoshop 4.0.

Acknowledgements

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Note added in proof While this paper was in review, a similar paper discussing Arabidopsis outer seed coat differentiation was published (Western, T.L., Skinner, D.J. and Haughn, G.W. (2000) Differentiation of mucilage secretory cells of the Arabidopsis seed coat. Plant Physiol. 122, 345±356).
