Stigma

At the top of the gynoecium, the stigma is comprised of a single layer of elongated papillar cells specialized for the germination of pollen (Figure 1B). The stigma is the first component of the transmitting tract, a set of cells that secrete a polysaccharide-rich extracellular matrix, which-forms a pathway for the growth and guidance of pollen tubes (Sessions and Zambryski, 1995; for reviews of pollen tube guidance see Lord and Russell, 2002 and Palanivelu and Preuss, 2000).

Style

Below the stigma, the style forms a solid cylinder around the central transmitting tract cells that guide the pollen tubes from the stigma to the ovary. In the style, a ring of vascular tissue surrounds the transmitting tract cells. The outer epidermis of the style consists of large rectangular cells with characteristic wax ridges (or crenulations) interspersed with stomata (Figure 1D) (Sessions and Zambryski, 1995).

Ovary

Below the style, the ovary, which houses the developing seeds, forms the majority of the Arabidopsis fruit. The ovary takes the form of a silique with two separate locules or compartments. The ovary consists of several distinct tissues including the valves (seedpod walls), replum (middle ridge), septum, and valve margins.

Valves

The seedpod walls, or valves, lie on the lateral sides of the ovary surrounding and protecting the developing seeds (Figure 3A). The outer epidermal layer of the valves, or exo-carp, consists of long rectangular cells interspersed with stomata (Figure 1F). Inside the exocarp, three layers of thin walled cells containing chloroplasts (chlorenchyma cells) establish the mesocarp (Figure 3). The lateral vascular strands run through the center of the valve and branch to form the secondary vasculature in the valve (Alvarez and Smyth, 2002). Two layers of endocarp cells form on the interior of the valves (Figure 3). The inner epidermis, the endocarpa (ena), consists of enlarged cells (Figure 3 and 8F), which break down later as the fruit matures (Figure 8E). The second endocarp layer, enb (or lignified valve layer), consists of very narrow highly elongated cells, which become lignified late in development (Figure 3 and 8D to 8F). Lignins are phenolic polymers that increase the strength and thickness of the cell walls in which they are deposited. The lignified enb layer is thought to be involved in the spring-loaded shattering mechanism of seed dispersal employed by Arabidopsis and its important crop relative, canola. During dehiscence, the valves separate from the replum and fall from the fruit so that the seeds can be dispersed (Figure 9B and 9G). It has been proposed that as the seedpod dries, the outer layers of thin walled valve cells contract, creating tension against this rigid lignified enb layer which helps to pop the valves off the fruit (Figure 9H; Spence et al., 1996).

Replum and septum

The ovary is divided into halves at the septum and the replum (Figures 1E and 3). The replum was originally defined as the structure that remains attached to the plant after fruit dehiscence, which includes the septum and the abaxial replum (Weberling, 1989). More recently, however, the term replum has come to refer to only the outer or abaxial portion and does not include the septum (Alvarez and Smyth, 2002). Each replum contains one of the medial vascular bundles. The ovules and funiculi arise from the placentae, which lie along each of the inner sides of each replum. The septum divides the fruit, stretching from the inner side of one replum to the other replum. In the middle of the septum, transmitting tract cells connect to the style forming a continuous tract for pollen tube growth.

Valve margins

The valve margins, the zones where the fruit opens, differentiate at the borders between the valves and the replum allowing the valve to separate from the replum in the mature fruit to disperse the seeds (Figure 9B and 9G). The valve margins constrict, forming an indentation, because the cells in the valve margins are smaller than the surrounding cells in the valve and the replum (Figure 1A to 1C, 1E, and 3). Each valve margin consists of two layers-a separation layer and a lignified layer (Figure 3B and Figure 8G to 8I). In the separation layer, or dehiscence zone, hydrolytic enzymes are secreted to break down the middle lamella between adjacent cells, allowing the cells to separate (Figure 9E; Meakin and Roberts, 1990). The lignified layer of the valve margin is thought to act in conjunction with the enb layer of the valve, creating tensions to assist in the seed dispersal process (Figure 9H; Spence et al., 1996).

Gynophore

At the base of the fruit there is a short stalk called the gynophore, which is also known as the internode or stipe (Figure 1A and 1C).

Stages 1–5

The flower first appears as a small bulge on the side of the shoot apical inflorescence meristem at stage 1 (Figure 4A). At stage 2, the floral primordium enlarges and becomes separated from the inflorescence meristem by a small groove (Figure 4A). The sepal primordia arise on the flanks of the floral meristem at stage 3 (Figure 4A). The sepals grow so that by stage 4 they partially cover the floral meristem (Figure 4A). At stage 5, the petal and stamen primordia are initiated. Late in stage 5, the floral meristem expands laterally to form an elliptical flattened platform where the gynoecium will develop (Figure 4B; Sessions, 1997).

Stage 6

When the sepals enclose the bud at stage 6, the gynoecium begins to develop as a raised ridge around a central cleft (Figure 4C to 4E; Hill and Lord, 1989; Sessions, 1997; Smyth et al., 1990). The cleft forms because the cells in the center of the meristem do not divide. The gynoecium consists of two congenitally fused carpels, which arise as a single cylinder because they are joined at the margins in the medial region (Okada et al., 1989). Based on the analysis of sectors, eight progenitor cells in the floral meristem give rise to each carpel (Bossinger and Smyth, 1996). The gynophore is formed from the layers of cells above the stamen primordia and below the tube of the gynoecium (Sessions, 1997). Because the gynoecium arises as a tube, both the exocarp and ena layers are derived from the epidermal layer of the meristem and thus can be described as the outer epidermis and the inner epidermis respectively.

Stage 7

Stage 7 begins when the medial, or long, stamens become stalked at their bases. The gynoecium continues to grow upward to form a continuous hollow cylinder (Figure 4F to 4H). At this stage the inner surfaces of the tube almost touch and the top of the tube is not level (Alvarez and Smyth, 2002).

Stage 8

The gynoecium continues to grow taller and wider during stage 8, which is marked by the formation of locules in the anthers. At the beginning of stage 8, the Ler gynoecium is about 80 µm tall, but by the end it is about 180 µm tall (Figure 5A and 5B; Sessions, 1997). The carpel walls are about 5 to 6 cell layers thick, suggesting that all of the cell layers of the gynoecium are already present (Figure 5B and 5C). The medial vascular bundles begin to develop, but there is no sign of the differentiation of style, replum, or valve cells in the epidermis. The septum is initiated during stage 8 when the inner medial surfaces form ridges. By the end of stage 8, the medial ridges become flanked by placentae (Figure 5C; Sessions, 1997).

Stage 9

Stage 9 is marked by the petal primordia becoming stalked at the base. The gynoecium continues to grow and by the end of stage 9, the apex starts to close (Figure 5D and 5E). Late in stage 9 the appearance of the first rounded cells at the top of the gynoecium suggest the development of the stigmatic papillae (Alvarez and Smyth, 2002). The style begins to become morphologically distinct from the ovary and the lateral vascular bundles start to differentiate (Ferrándiz et al., 1999). The septum forms when the leading edges of each medial ridge meet and fuse. The placenta produces ovule primordia (Figure 5F; Robinson-Beers et al., 1992).

Stage 10

Stage 10 begins when the petal primordia reach the length of the short stamens (Smyth et al., 1990). The upper end of the gynoecium closes and begins to produce immature stigmatic papillae (Figure 5G). The gynoecium continues to grow and reaches about 400 µm (Sessions, 1997). The ovule primordia grow to form finger-like projections, which mesh with those from the opposing placenta in an interlocking pattern (Figure 6C*; Robinson-Beers et al., 1992). The septa continue to grow out from the middle of the medial ridge and the cells at the middle begin to stain more darkly, suggesting that they are starting to differentiate as transmitting tract cells (Figure 5H; Alvarez and Smyth, 2002).

Stage 11

The stigmatic papillae cover the entire surface of the stigma during stage 11 (Figure 6A). The gynoecium reaches a length of about 600 µm (Sessions, 1997). The external surface of the style, characterized by its wax crenulations, begins to develop and continues to differentiate through stage 12 (Alvarez and Smyth, 2002). Cells in the enb layer begin to divide anticlinally to produce very narrow cells (Figure 6B). Two regions form in the septum: a pre-transmitting tract region with small cells that stain darkly lies in the center (first visible at stage 10) and a pocket with a few loosely spaced cells and ample air space forms closer to each edge of the fruit (Alvarez and Smyth, 2002). In the ovules, the funiculus, inner integument, and outer integument all start to differentiate (Figure 6C; Robinson-Beers et al., 1992).

Stage 12

At the beginning of stage 12, the petals reach the length of the long medial stamens. The gynoecium continues to grow and reaches about 800 µm (Figure 6D and 6E). Differentiation of the tissues starts to become apparent as the valves expand laterally to become clearly distinct from the narrow apical style. The valve margins and replum also become morphologically distinct. The gynoecium becomes ready for pollination when the stigmatic papillae elongate and fully differentiate. The transmitting tract simultaneously differentiates to guide the pollen tubes to the ovules (Figure 6E and 6F; Bowman et al., 1999). The integuments of the ovules grow and almost cover the nucellus (Figure 6G and 6H; Robinson-Beers et al., 1992). The secondary walls of the xylem thicken during stage 12 (Figure 6I; Alvarez and Smyth, 2002). Thickening of the medial vascular bundle cell walls begins at the base of the fruit and extends upward. Slightly later, cell wall thickening begins in the style and continues downward to meet with the upward-growing medial vasculature in the middle of the fruit. The lateral vascular bundle cell walls begin to thicken later than the medial bundles and the lateral vascular bundle cell walls thicken entirely from the bottom up. More xylem elements mature in the style and connect to the medial vascular bundles. A single strand of xylem in the funiculus thickens but does not connect to the medial xylem until stage 13 or 14.

Stage 13

Stage 13 is defined by anthesis, which is when the flower opens and self-pollinates (Figure 7A). After the pollen lands on the stigma and germinates, the pollen tube grows down the papilla cells between the inner and outer layers of the cell walls. The pollen tube takes 45 to 50 minutes to reach the extracellular matrix of the transmitting tract, where it grows between the transmitting tract cells until it reaches an ovule during stage 14 (Figure 7H; Kandasamy et al., 1994). By stage 13, the ovule is completely developed and ready for the pollen tube to reach it (Figure 7D; Robinson-Beers et al., 1992). The cells of the valve (the exocarp, mesocarp, endocarpb, and endocarpa) are small relative to the dramatic expansion they will undergo after fertilization as the fruit elongates to accommodate the developing seeds (Figure 7B and 7C, compare to 8F). Second order xylem strands begin to differentiate in the valves originating from the top of the lateral xylem (Figure 6I; Alvarez and Smyth, 2002).

Stage 14

At stage 14, the anthers extend above the top of the stigma (Figure 7E; Smyth et al., 1990). The exocarp cells of the valves and the replum divide and expand primarily along the longitudinal axis, contributing to the lengthening of the fruit. The mesocarp cells divide and elongate primarily longitudinally. Many chloroplasts develop in the mesocarp cells of the valves, whereas the mesocarp cells in the valve margin do not develop chloroplasts and do not divide or expand as much as their neighbors. This causes the valve margins to constrict, forming a small groove along the edge of the replum (Figure 7E and 7F). The enb cells become long and narrow as they expand longitudinally (Spence et al., 1996). The ena layer appears swollen as the cells expand in all directions (Figure 7F). As in the mesocarp, endocarp cells in the valve margins do not expand as much as their neighbors. The pollen tubes grow through the transmitting tract and along the funiculus to enter each ovule and fertilize the egg cell and endosperm (Figure 7G and 7H; Robinson-Beers et al., 1992). Second order vascular strands continue to develop in the valves and the vascular strand in each funiculus connects to the medial vasculature (Figure 6I; Alvarez and Smyth, 2002).

Stage 15

By the beginning of stage 15, the gynoecium has elongated such that the stigma extends above the top of the anthers (Figure 7I). The fruit continues to grow and the valve cells divide and expand (Figure 7J). Second order vasculature continues to mature in the valves in a reticulate pattern and has completed development by the end of stage 15 (Alvarez and Smyth, 2002).

Stage 16

The sepals, petals, and stamens start to wither during stage 16 (Smyth et al., 1990), and these organs fall from the fruit by the end of the stage. Concurrently, the fruit continues to elongate through cell division and cell expansion (Figure 8A and 8B).

Stage 17

Stage 17 is a long stage, which starts when the floral organs fall from the fruit and ends when the fruit start to yellow. Stage 17 has been divided into two sub-stages, 17A and 17B (Figure 8C). In stage 17A the fruit elongates and increases in width to its mature form. From anthesis onward, growth in width of the fruit is almost entirely due to cell expansion in all of the cell layers of the valves (Vivian-Smith and Koltunow, 1999). In contrast, the total elongation of the fruit is achieved through both cell expansion and cell division (Figure 8F, compare with Figure 7C). Cell division is especially prevalent in the mesocarp where the number of cells increases about threefold, while cell expansion is more pronounced in both the exocarp and ena (Vivian-Smith and Koltunow, 1999). Cell expansion is limited at the valve margin where cells remain relatively small, forming an indentation between the valve and the replum (Figure 8G).

Stage 17B starts when the fruit reaches its full length and ends when the fruit yellows before it dries. The valve margin begins to differentiate into the dehiscence zone, preparing the fruit to open at maturity. The cell walls of the valve margin lignified layer start to thicken and later lignify (Figure 8I). These cells form a connection with the enb layer of the valve, which also undergoes lignification (Figure 8D; Spence et al., 1996). The middle lamella between the cells in the valve margin separation layer breaks down (Figure 8H). In addition, the cells of the ena layer and the cells on the epidermis of the septum break down, leaving behind their thickened inner cell wall (Figure 8E; Spence et al., 1996).

Stage 18

Stage 18 begins about 12 days after pollination when the siliques turn yellow from top to bottom and start to dry (Figure 9A; Vivian-Smith and Koltunow, 1999). The separation layer cells disintegrate, leaving the valves free to detach from the replum at stage 19 (Figure 9E and 9F).

Stages 19 and 20

The valves separate from the replum and fall from the siliques during stage 19 (Figure 9B and 9G) and the seeds fall from the replum in stage 20 (Figure 9C and 9D; Smyth et al., 1990).

AGAMOUS encodes a MADS domain transcription factor

AGAMOUS was the first MADS-box gene cloned from Arabidopsis, and since that time dozens of related genes have been isolated and characterized (Yanofsky et al., 1990). Plant MADS-box genes typically have a canonical MIKC structure, where the M refers to the highly conserved DNA-binding MADS-domain at or near the amino terminus of the protein. The I refers to an intervening region that helps give each MADS protein its specificity and may contribute to dimer formation. The K-domain, which derives its name from the weak sequence similarity with keratin, likely forms a coiled coil domain that is important in protein-protein interactions. The C-terminus of plant MADS proteins is referred to as the C-domain, and although its precise functions have yet to be fully explored, it appears to play roles in protein-protein interactions as well as in transcriptional activation (for reviews of plant MADS-box genes see Reichmann and Meyerowitz, 1997 and Theissen et al., 2000).

Consistent with its role in stamen and carpel identity, the flower-specific AG RNA is first detected in the central domain of stage 3 flowers in cells that will later give rise to stamens and carpels (Figure 10D; Drews et al., 1991). AG RNA accumulates uniformly throughout stamen and carpel primordia during stages 4 through 7 (Figure 10D), but in more mature flowers it becomes localized to specific cell types within these organs (Bowman et al., 1991b). During stage 9, only low a level of AG RNA is detected in the valves of developing carpels, whereas a high level of AG RNA accumulates in ovule primordia. During stage 12, AG RNA is detected in all cells of developing ovules, in the septum, and in the stigmatic papillae, and no expression can be detected in the style and valves (Figure 10D). The specific localization of AG to these additional tissues within the carpels suggests that AG might have additional roles later in gynoecium development, and in fact AG is involved in specifying ovule identity (Pinyopich et al., 2003).

Ectopic expression of AG promotes carpel identity

Loss-of-function studies have shown that AG is required for stamen and carpel development. To determine if AG is sufficient to specify reproductive organ development in ectopic positions, gain-of-function transgenic plants were generated that ectopically expressed the AG gene from the constitutive CaMV 35S promoter (Mizukami and Ma, 1992; Mandel et al., 1992). 35S::AG plants produced flowers in which the first whorl sepals were converted into carpelloid organs and the second whorl petals were converted into staminoid organs (Figure 10C). Thus, within the context of the flower, ectopic AG expression is sufficient to promote reproductive organ development. It is also important to note that ectopic AG expression was unable to convert vegetative leaves into carpelloid organs, suggesting that additional factors are required. These factors include the SEPALLATA (SEP) genes, which are required for AG function (Honma and Goto, 2001; Pelaz et al., 2001). The SEP1,2,3 genes are also required for gynoecium formation and in the sep1 sep2 sep3 triple-mutant no gynoecium is formed (Pelaz et al., 2000).

ERECTA encodes a leucine rich repeat receptor-like kinase

ERECTA encodes a leucine rich repeat receptor protein kinase (Torii et al., 1996). Two hydrophobic domains in the ER protein are thought to form a signal peptide and a transmembrane domain. The C-terminal intracellular domain encodes the catalytic domain of a putative serine threonine kinase, while the extracellular domain contains twenty leucine-rich repeats (LRRs). ER is expressed strongly in the shoot apical meristem (Yokoyama et al., 1998). In addition, ER is expressed throughout the floral meristem from stages 1–3. In stages 4–6, expression is limited to the developing stamens and carpels (Figure 13D). By stage 8, expression is reduced throughout the flower. It is likely that ER acts to receive extracellular signals to promote cell division and expansion and thus controls the fruit shape.

The SHATTERPROOF genes encode redundant MADS box transcription factors

The SHATTERPROOF genes encode two very closely related members of the MADS-domain family of transcription factors (Liljegren et al., 2000). The 87% identical SHP proteins are functionally redundant because neither single mutant has any abnormal phenotype. Likewise, the SHP genes have almost identical expression patterns. Expression of the SHP genes is initially detected broadly in the developing gynoecium, but soon thereafter (stage 12) expression becomes limited to the valve margins and ovules (Figure 15A and 15B; Flanagan et al., 1996; Savidge et al., 1995; Roeder et al., 2003). This localized expression pattern coincides with the stage at which valve margins become distinct (Sessions, 1997). Expression of the SHP genes at the valve margins continues after fertilization through early stage 17 (Figure 15C). The SHP genes are also expressed in the nectaries, ovules, septum, and style, although no defect in any of these tissues is observed in the shp double mutant. The role of the SHP genes in these locations may be obscured by redundancy with other genes. In fact, the SHP genes have redundant roles in ovule development with the related SEEDSTICK (STK) MADS-box gene (Pinyopich et al. 2003). The observations that the SHP genes are not expressed in ag single-mutants and that AG can bind to a site in the SHP2 promoter in vitro suggests that the AG carpel identity gene plays a role in promoting SHP gene expression (Flanagan et al., 1996; Savidge et al., 1995). SHP in turn activates the expression of other genes involved in valve margin development, including ALCATRAZ and INDEHISCENT.

ALCATRAZ encodes a basic helix loop helix transcription factor

ALC encodes a basic helix loop helix (bHLH) transcription factor (Rajani and Sundaresan, 2001). Although ALC is broadly expressed in the gynoecium prior to fertilization, it becomes restricted to the dehiscence zone at stage 16 (Figure 16A and 16B; Liljegren, et al., 2004; Rajani and Sundaresan, 2001). At stage 17, ALC continues to be expressed in the valve margin, but expression expands into the outer replum cells (Figure 16C). When the fruit is starting to turn yellow at stage 18, ALC expression can be seen in the inner cells of the valve margin where it prevents the ectopic lignification of these separation layer cells (Figure 16D). ALC is also expressed in the stigma, ovules, nectaries, newly emerging leaves, lateral root primordia, and the fruit pedicel branch point, although it has no known function in these tissues.

INDEHISCENT also encodes a basic helix loop helix transcription factor

Like ALC, IND encodes a bHLH transcription factor. IND falls into an atypical class of bHLH proteins with an unusual amino acid sequence in the basic domain. Most bHLH proteins contain a critical glutamate residue in the basic domain that contacts the CA bases of the DNA binding site (Toledo-Ortiz et al., 2003), but in the wild-type IND protein this glutamate is replaced by an alanine (Liljegren et al., 2004). It would be interesting to investigate how this affects the DNA binding properties of the IND protein and whether it binds to a different consensus site or completely fails to bind DNA as has been suggested for this class of bHLH proteins (Toledo-Ortiz et al., 2003).

The helix loop helix domain of bHLH proteins is often involved in homo- or herterodimerization. In yeast, IND can interact with ALC, suggesting that these two proteins may heterodimerize to specify the separation layer (Liljegren et al., 2004).

IND is expressed in the valve margin. The GT140 molecular marker reports the expression pattern of IND in the valve margin from before fertilization (at stage 12) to early stage 17 (Figure 17A and 17B).

FRUITFULL encodes a MADS domain transcription factor

The FUL gene is a member of the extended family of MADS-box genes and is most closely related to the APETALA1 and CAULIFLOWER flower meristem identity genes. Interestingly, ap1 cal ful triple mutants show a dramatic non-flowering phenotype, indicating that these three genes share redundant roles in promoting flower meristem identity (Ferrándiz et al., 2000a).

The FUL gene is strongly expressed in the fruit valves, consistent with the location of the defects in ful mutants (Figure 19D and 21G; Mandel and Yanofsky, 1995; Gu et al., 1998). During flower development, FUL expression is first detected during stage 3 in a central region of the flower meristem in cells that will later form the carpels. As these carpels develop, FUL becomes restricted to the valves. FUL expression can also be detected in the style, consistent with the greatly elongated style that occurs in ful mutants. FUL is also expressed in the inflorescence meristem, where it presumably acts to promote flowering, and in leaves, which show developmental abnormalities in ful mutants.

REPLUMLESS encodes a homeodomain protein

RPL encodes a homeodomain protein in the BELL1 family (Roeder et al., 2003). RPL is also known as PENNYWISE (PNY), BELLRINGER (BLR), and VAAMANA (VAN) for its additional roles in meristem function and internode elongation (Smith and Hake, 2003; Byrne et al., 2003; and Bhatt et al., 2004).

RPL is expressed in the replum beginning very early in gynoecium development at stage 6 (Figure 23K). RPL::RPL-GUS is expressed in the replum at stage 12 when RPL negatively regulates SHP (Figure 23L to 23M). RPL is also expressed in the ovules, stem, leaves, roots, and shoot apical meristem.

Homeodomain transcription factors in the BELL family often heterodimerize with members of the KNOX family (Bellaoui et al., 2001; Smith et al., 2002). Smith and Hake, Byrne et al., and Bhatt et al. have found that the RPL protein interacts with certain members of the KNOX family including SHOOT MERISTEMLESS (STM), KNAT1, and KNAT6, but no interaction was observed with KNAT2, KNAT3, KNAT4, or KNAT5 (2003; 2003; 2004). Furthermore, Bhatt et al. have found that RPL becomes localized to the nucleus when it interacts with a KNOX partner (2004). Currently it is unknown whether these interactions have a role in fruit development.

Interestingly, Bao et al. have found that RPL also negatively regulates AG expression (2004). They have shown that the RPL protein can bind to AG regulatory elements in vitro, suggesting that the regulation of AG may be direct. Since AG is closely related to SHP, it would be interesting to determine whether the role of RPL in negatively regulating SHP is also direct.

The CUP-SHAPED COTYLEDON genes encode NAC domain transcription factors

CUC1 and CUC2 encode two closely related members of the plant-specific family of NAC domain transcription factors (Aida et al., 1997; Takada et al., 2001, Duval et al., 2002). CUC1 and CUC2 also act redundantly with CUC3, a third closely related member of the NAC family (Vroemen et al., 2003), although the role of CUC3 in gynoecium development has yet to be investigated.

As expected, the CUC genes are expressed in the boundaries between organs. In addition, CUC1 and CUC2 are expressed in the gynoecium during stage 7 at the inner edge of the medial ridge where the septum will form (Ishida et al., 2000; Takada et al., 2001). As the septum primordia grow, CUC1 and CUC2 continue to be expressed on the inner ridge and in the middle of the septum until stage 10 or 11. Since the CUC genes are not expressed in the replum, it is surprising that the replum is also affected in the cuc1 cuc2 double mutant.

LEUNIG (LUG) encodes a transcriptional corepressor

LUG encodes a putative transcriptional corepressor related to the yeast protein Tup1 and the Drosophila protein Groucho with two glutamine rich regions and seven WD repeats (Conner and Liu, 2000). It is likely that LUG interacts with other transcription factors because it does not contain a DNA binding domain. The LUG protein is localized to the nucleus, consistent with its proposed role in transcription.

LUG is highly expressed in stage 1 to 2 floral primordia and subsequently in each of the floral organ primordia, including the carpels as they arise (Figure 24E; Conner and Liu, 2000). LUG is weakly expressed in the carpel valves, but it is strongly expressed in the vasculature and the developing placenta and ovules (Figure 24E).

The TOUSLED kinase

The TSL protein belongs to a family of a nuclear localized serine threonine kinase that is conserved in plants and animals (Roe et al., 1993; Roe et al., 1997a; Ehsan et al., 2004). Studies of TOUSLED-like kinases in animal systems suggest that they are involved in chromatin metabolism, particularly in chromatin assembly, DNA repair, and the regulation of gene expression (Ehsan et al., 2004). The Arabidopsis TSL protein has been shown to transautophosphorylate and to phosphorylate histone H3 and Asf1b/SGA1 (an Arabidopsis relative of yeast Asf1 which recruits histone H3 and H4 during nucleosome assembly) in vitro, suggesting that the Arabidopsis TSL may also have a role in regulating chromatin metabolism (Roe et al., 1997a; Ehsan et al., 2004). Based on northern blot analysis, TSL is expressed strongly in inflorescences and expression continues at moderate levels in flowers through anthesis stage to the beginnings of fruit development. TSL transcript is also present in shoots and leaves although it is absent in stems (Roe et al., 1993). Within the gynoecium, TSL::GUS is expressed in the apical half of the gynoecium at stage 11 (Figure 25H). During stage 13, TSL::GUS expression becomes restricted to the style (Figure 25I). The expression of TSL in the style has been confirmed by in situ hybridization (Roe et al., 1997b). The broad pattern of TSL expression and the multiple phenotypes of tsl mutants as well as the phenotypes caused by mutations in different chromatin regulatory genes including gymnos (see section GYMNOS encodes a chromatin-remodeling enzyme) show the importance of chromatin remodeling in plant development.

tousled leunig double mutants have reduced apical tissues

Strong synergistic affects are seen when tsl mutants are combined with leunig (lug) mutants. As in tsl mutants, floral organ number is reduced in lug mutants and lug gynoecia are unfused at the top (Figure 26A; see section 5.6.1 LEUNIG (LUG) and fusion), suggesting that lug might have a role in the same genetic pathway as tsl. Most tsl lug double-mutant gynoecia have only one carpel with extensions of tissue occasionally topped with stigmatic papillae (Figure 26B). The placenta tissue in these single carpel gynoecia is greatly reduced. When two carpels are formed in tsl lug double mutants, they are largely unfused with long extensions devoid of stigmatic tissue (Figure 26C). The extreme severity of both of these phenotypes suggests that the developmental pathways in which TSL and LUG function overlap.

tousled perianthia double mutants form unfused gynoecia

Strong synergistic affects are also seen in tsl perianthia (pan) double mutants. Gynoecium development is only minimally affected in pan single mutants, which occasionally have three carpels and/or an incompletely fused septum (Figure 26D). However, in the tsl pan double mutant, the gynoecium degenerates into two or three completely unfused carpelloid organs with very little style, stigma, or placenta (Figure 26E and 26F). TSL and PAN act redundantly in gynoecium development. Only in the tsl mutant is the role of PAN in the gynoecia uncovered. tsl mutants also interact synergistically with ett mutants (see section ETTIN interacts synergistically with TOUSLED).

The STYLISH genes encode RING finger proteins

STY1 and STY2 encode closely related C3HC3H zinc finger proteins (Kuusk et al., 2002). The zinc finger domains are similar to RING finger motifs, which are involved in protein-protein interactions. The STY proteins also have nuclear localization signals and IGGH domains. The STY genes belong to a family of ten genes in Arabidopsis including SHORT INTERNODES (SHI) and LATERAL ROOT PRIMORDIUM1 (LPR1).

Both STY1 and STY2 are expressed in the apex of the developing gynoecium where the stigma and style form (Figure 27K to 27M; Kuusk et al., 2002). STY1 expression begins in the gynoecium at stage 6, while STY2 is not observed in apex of the gynoecium until stage 9. The absence of STY2 expression in the earliest stages of gynoecium development may account for its incomplete redundancy with STY1.

SPATULA encodes a bHLH transcription factor

The SPATULA gene encodes a basic helix-loop-helix (bHLH) transcription factor (Heisler et al., 2001). SPT is expressed in a dynamic pattern within the developing gynoecium paralleling its role in the development of medial tissues. Within stage-4 floral meristems, SPT RNA is first detected in a pie-wedge-shaped domain that appears to mark the cells that will later form the gynoecium. By stage 6, SPT transcript is strongest in the medial regions of the gynoecium (Figure 28K) and by stage 7, SPT expression is limited to the inner medial regions of the gynoecium where the septum will form. At the apex of the gynoecium, SPT expression spreads where the future stigma will develop (Figure 28K). SPT expression is further restricted to only a few cells at the leading edge of the medial ridge during stage 8 (Figure 28K). From stage 9 to 11, SPT is expressed throughout the septum and the stigmatic papillae, but this expression ceases in stage 12 (Figure 28K). This early expression pattern correlates directly with the abnormal phenotypes observed in spt mutants. Beginning in stage 12, SPT is expressed throughout the valves except the epidermis and the vascular bundles (Figure 28K). After fertilization as the fruit expands, SPT is restricted to the valve margins (Figure 28K). Currently, no role for SPT in the valves or valve margins has been uncovered.

SPT is also expressed in petals, stamens, ovules, the seed attachment site, young leaves, stipules, maturing pith cells in the stem, differentiating vascular cells, and lateral root caps although no abnormal phenotype is observed in these tissues in spt mutants.

The expression of SPT in many tissues where no mutant phenotype has been observed raises the possibility that additional factors may act redundantly with SPT. Specifically, bHLH proteins often heterodimerize, so other interacting bHLH proteins may redundantly mediate these functions. For example, SPT is specifically expressed in areas where cell separation will occur, such as the valve margin, the stomium of the anther, and the seed abscission zone, although currently this expression has no known function. It will be interesting to test whether SPT heterodimerizes with the ALCATRAZ or INDEHISCENT bHLH proteins, which are also expressed in the valve margin (see sections 5.1.2 alcatraz (alc) mutants keep the seeds imprisoned and 5.1.3 INDEHISCENT (IND) is required for valve margin specification).

CRABS CLAW encodes a YABBY transcription factor

CRC is the founding member of the YABBY gene family, a family of six transcription factors each with a zinc finger and a helix loop helix domain (Bowman and Smyth, 1999). CRC is expressed in the developing nectaries and gynoecium paralleling the disruptions in the mutant. CRC is expressed in the two lateral sides of the gynoecium primordium at stage 6 (Figure 29H). During stages 7 and 8, two domains of expression become apparent: epidermal and internal. The epidermal expression is primarily in the outer epidermis although weak expression can be seen in the ena layer during stages 7 through 9. Expression in the outer epidermis completely encircles the fruit including the replum until stage 10 when expression in the replum begins to decline (Figure 29H). By stage 12, expression in the outer epidermis is not detected. Simultaneously, during stages 7 through 10, CRC is expressed internally in four stripes adjacent to the cells that will form the placentas (Figure 29H). Both the epidermal and internal expression domains run the whole length of the gynoecium, including the future style.

Constitutive expression of CRC (35S::CRC) produces solid cylindrical gynoecia composed primarily of style tissue topped with stigmatic tissue, which is consistent with the role of CRC in the development of the style (Eshed et al., 1999). In addition carpelloid tissues including stigma and ovules develop on the sepal margins supporting the role of CRC in the carpel specification pathway (see section 2.2 Carpel development in the absence of AGAMOUS).

6.1.1 Role of CRABS CLAW in Abaxial/adaxial Polarity

Members of the YABBY gene family generally specify abaxial cell fate (Siegfried et al., 1999). Although CRABS CLAW (CRC) is a member of the YABBY family and is expressed in the abaxial side of the gynoecium (see section CRABS CLAW encodes a YABBY transcription factor), the crc-mutant phenotype shows little evidence of altering abaxial/adaxial polarity (Figure 31B). Does CRC have a hidden role in establishing polarity in the fruit? To uncover the role of CRC in the abaxial/adaxial polarity of the gynoecium, Eshed et al. screened for genetic enhancers of crc (1999).

6.1.2 Ovules Form on the Outside of gymnos (gym) Crabs Claw Gynoecia

Mutations in gymnos (gym) (also referred to as pickle (pkl) Ogas et al., 1999) enhance the crc phenotype producing twenty to thirty external ovules on the double-mutant gynoecia (Figure 31D; Eshed et al., 1999). During stage 8, these external ovule primordia are formed in a row on the edges of the replum mirroring the development of the ovule primordia from the placenta on the inside of the fruit. As the gynoecium continues to develop, secondary asynchronous waves of ovule development occur on the outside of the gynoecium. The loss of abaxial identity is incomplete in crc gym double mutants because an external septum does not develop and the abaxial valves remain. However, large cells of aberrant morphology are scattered throughout the valve epidermis.

Although gym single mutants show a number of abnormalities (Ogas et al., 1997), no defect in the abaxial/adaxial polarity of the fruit has been observed. The gym gynoecia are shorter and narrower than wild type and seem to mature slowly (Figure 31C; Eshed et al., 1999).

GYMNOS encodes a chromatin-remodeling enzyme

GYM, also known as PICKLE (PKL) (Ogas et al., 1999), encodes a CHD3/4 DNA-dependant ATPase in the family of SWI2/SNF2 chromatin-remodeling enzymes (Eshed et al., 1999). A PHD finger and a MYB DNA-binding domain are also predicted in the GYM protein. GYM is related to Drosophila Mi-2, which forms complexes with histone deacetylases to repress transcription of target genes. GYM, likewise, may form complexes with histone deacetylases to repress the transcription of genes involved in maintaining an undifferentiated state.

GYM is expressed in undifferentiated tissue including embryos, meristems and organ primordia. Also, GYM is expressed throughout gynoecia during stages 5–7, but becomes limited to the medial ridge and ovule primordia during stage 8 (Eshed et al., 1999). Finally GYM expression becomes restricted to the developing ovules (Figure 31I).

Why are ectopic ovules produced on the outside of gym crc double-mutant fruit? Eshed et al., hypothesize that in addition to the roles of CRC in establishing carpel identity and promoting style formation, CRC specifies abaxial cell fate within the gynoecium, while GYM mediates the repression of genes that promote an undifferentiated state (1999). It is possible that mutations in gym prolong the expression of meristematic genes in the gynoecium extending the period of indeterminacy and allowing placenta to develop. Simultaneously, mutations in CRC lift the normal restriction of placenta development to the inside (adaxial side) of the fruit. However, the reversal in abaxial/adaxial polarity appears to be limited to the medial regions in crc gym. One possible reason that the valves are not as affected in crc gym mutants is that other YABBY genes expressed in the valve may act redundantly with CRC. FILAMENTOUS FLOWER (FIL), YABBY2 (YAB2), and YABBY3 (YAB3) are all expressed in the abaxial side of the valves, but not in the medial regions (Siegfried et al., 1999). Other genes specifying abaxial cell fate could act redundantly with CRC as well. One such gene, KANADI1, was also isolated as an enhancer of crc.

6.1.3 Inside Out kanadi (kan) Gynoecia

Mutations in both KAN1 and KAN2 nearly abolish abaxial/adaxial polarity throughout the plant. Adaxial cell types replace abaxial ones suggesting that the KAN genes redundantly promote abaxial cell fate. The effect on the gynoecium is monstrous; ovules cover the outside of kan1 kan2 gynoecia replacing all of the abaxial valve tissue (Figure 31G; Eshed et al., 2001). It is interesting that the inside of the fruit is not merely reproduced on the outside; instead, the placenta expands to cover the surface suggesting that kan1 kan2 gynoecia also have a defect in medial/lateral patterning.

KAN1 and KAN2 act redundantly and kan2 mutants look completely normal. In kan1-mutant gynoecia, a ridge of tissue grows out from the replum and a few ectopic ovules are found on the exterior base of the gynoecium hinting at a slight alteration in abaxial/adaxial polarity (Figure 31E; Kerstetter et al., 2001; Eshed et al., 2001).

kanadi1 enhances crabs claw abaxial/adaxial defects

The kan1 mutant was identified in a screen to find enhancers of the crc mutant (see section 6.1.1 Role of CRC in abaxial/adaxial polarity; Eshed et al., 1999). The crc kan1 double mutant produces two complete rows of ovules on the exterior of the gynoecium and the replum grows out to produce an exterior septum including transmitting tract tissue (Figure 31F). The development of the external ovules and septum coincides with the development of the internal ones. Ectopic carpel tissue is also formed from the bottom of the gynoecium. In contrast to kan1 kan2, the defect in abaxial/adaxial polarity is largely limited to the medial regions and the base of the crc kan1 fruit.

KANADI genes encode GARP transcription factors

The closely related KANADI genes encode members of the GARP family of putative transcription factors (Kerstetter et al., 2001; Eshed et al., 2001). In Arabidopsis, the GARP family comprises more than fifty members including two additional genes closely related to KAN1/2. The expression pattern of KAN1 is consistent with its role in promoting abaxial identity (Kerstetter et al., 2001). KAN1 is expressed in the abaxial side of young leaves and floral organs. Within the gynoecium, KAN1 is first expressed on the abaxial side (Figure 31J).

The KANADI genes have a central role in specifying abaxial polarity as is demonstrated by the severity of the mutant phenotypes. It is likely that the KANADI and YABBY genes act in separate, but overlapping pathways in specifying abaxial fate (Eshed, et al., 2001).

6.1.4 PHABULOSA (PHB) Promotes Adaxial Fate

While the KANADI and YABBY genes promote abaxial fate, PHABULOSA (PHB), PHAVOLUTA (PHV), and REVOLUTA (REV), three members of the class III homeodomain leucine zipper (HD-ZIP) family, promote adaxial fate (Emery et al., 2003; McConnell et al., 2001). Gain-of-function alleles of PHB, PHV, or REV cause lateral organs to become adaxialized, whereas loss-of-function mutations in all three genes (phb phv rev) cause the cotyledons to become radialized and abaxialized and block formation of the shoot apical meristem. In the fruit, the affects of PHB gain-of-function alleles are rather minor causing only a few ovules to be produced on the outside of the fruit at the base (Figure 31H; McConnell and Barton, 1998).

The gain-of-function alleles of PHB, PHV, and REV block a microRNA-binding site thereby disrupting the spatial regulation of transcript accumulation (Emery et al., 2003; Rhoades et al., 2002). Consequently, in the gain-of-function alleles, PHB is ectopically expressed throughout developing leaves instead of being limited to the adaxial side as in wild type (McConnell et al., 2001). It has been proposed that PHB, PHV, and REV may also receive a signal from the meristem signaling adaxial localization since these proteins contain START domains which are putative sterol binding sites.

6.2.1 ETTIN (ETT) Establishes Boundaries Along the Apical/basal Axis

Mutations in the ETTIN (ETT) gene cause spectacular alterations in gynoecium morphology, especially along the apical/basal axis (Sessions and Zambryski, 1995; Sessions, 1997). The ovary in the middle of the fruit is greatly reduced in size whereas the gynophore at the bottom of the fruit and the stigma and style at the top of the fruit increase in size (Figure 32A to 32H). In the ovary, the valves show the greatest reduction, but the placenta and septum are also reduced. Abaxial/adaxial polarity is also affected in ett gynoecia where an everted transmitting tract tissue develops in outgrowths on the exterior of the gynoecium (Figure 32H). The everted transmitting tract is stylar in origin and is evidence of the expansion of stylar tissues basally. Also, the ett stigma and style are split and the vascular tissue is affected (Sessions and Zambryski, 1995). Additionally, ett flowers tend to have one extra sepal and one extra petal, but tend to be missing a stamen.

The severity of the ett phenotype ranges along an allelic series. In ett-2, a weak allele, the fruit is fertile and valves are present although they are reduced in size (Figure 32B and 32F). Often the valves are uneven, suggesting that the development of one valve is independent of the other. In ett-3, an intermediate allele, small valves develop that are completely surrounded by stylar and stigmatic tissue (Figure 32C). The everted transmitting tract occurs in both medial and lateral outgrowths (Figure 32G). The basal stalk is covered in style-like epidermal cells. In the strong ett-1 allele, valves are often completely absent and the gynoecia are sterile (Figure 32D). The base of the ett-1 gynoecia consists of a solid stock (Figure 32H). At the top of the stalk region, the center opens into a narrow space filled with abnormal ovules. Style-like epidermal cells cover the stalk. The top of the ett-1 gynoecium is covered with everted transmitting tract. Although these descriptions typify the gynoecia from each allele, the ett phenotype is somewhat variable and gynoecia from a single plant will vary in severity of the phenotype (Sessions and Zambryski, 1995; Sessions, 1997).

Defects in ett-mutant morphology appear early in flower development (Sessions, 1997). At stage 5, the meristem elongates more than wild type, producing extra layers of cells below the gynoecium that likely contribute to the elongated gynophore. As the ett gynoecium grows during stages 7 and 8 it forms a trumpet shape instead of a cylinder. Differentiation begins prematurely in stage 8 when stigmatic papillae become visible.

The ett phenotype has been interpreted as altering two hypothetical boundaries along the apical/basal axis of the fruit (Figure 33; see also section 6.2.4 Relationship of ETTIN and SPATULA to polar auxin transport; Sessions, 1997; Nemhauser et al., 2000). One boundary marks the apex of the gynoecium and the other lies at the base of the ovary delineating it from the gynophore. In ett mutants, the basal boundary is pulled upward leading to the elongated gynophore, while the apical boundary is pulled downward, increasing the stigma and style tissue. In ett-1, the basal boundary is interpreted as being above the apical boundary and so no valves are formed (Sessions, 1997). The basal boundary must be independent on each side of the fruit because the size of the valve on each side is independent. The ETT gene may be involved in establishing or interpreting these boundaries within the gynoecium (see section 6.2.4 Relationship of ETT and SPT to polar auxin transport).

ETTIN encodes an auxin response factor

The ETTIN gene encodes a putative transcription factor with a DNA binding domain similar to that of Auxin Response Factors (ARF; Sessions et al., 1997). ARFs bind to auxin response elements (AuxREs) in the promoters of auxin-regulated genes to control their transcription. In addition to the DNA binding domain, ARFs contain a protein-protein interaction domain, which is not present in ETT. The ETT protein has a predicted nuclear localization sequence and two serine rich regions. The ETT protein probably functions to mediate an auxin response in the gynoecium (Sessions et al. 1997). Like other ARF transcription factors, ETT expression is not auxin inducible (Nemhauser et al., 2000).

ETT has a dynamic expression pattern that matches its many functions during flower development (Sessions et al., 1997). The ETT gene is first expressed in early floral meristems before stage 1. At stage 2 the ETT transcript becomes localized to an apical patch in the floral meristem and to the provascular tissue in the developing pedicel. ETT RNA is not detected in sepal primordia, but is found in both the developing petals and stamens during stages 3 to 9 (Figure 32I). ETT forms a thick ring around the terminal meristem preceding the formation of the gynoecium in stage 5. ETT RNA is localized abaxially in the gynoecium from stages 5 to 8 when it becomes restricted to the four vascular strands in cells between the phloem and the xylem during stages 8 to 12 (Figure 32I).

The expression pattern of ETT in the gynoecium coincides with the hypothesis that ETT acts to establish or interpret boundaries within the apical/basal axis of the gynoecium. At stage 5, the upper edge of the ETT transcript ring could establish the hypothetical apical boundary. Likewise the bottom of the ETT expression ring delineates the bottom of the ovary and could form the basal boundary. Alternatively ETT could act throughout the gynoecium at these early stages to interpret positional information (Sessions et al., 1997).

6.2.2 ETTIN Negatively Regulates SPATULA Expression

Mutations in spt suppress the ett phenotype by restoring valves and eliminating the overgrown gynophore and everted transmitting tract (Figure 34A to 34D; Alvarez and Smyth, 1998; Heisler et al., 2001). ETT negatively regulates SPT expression restricting SPT to the developing septum and transmitting tract. In ett mutants, SPT is ectopically expressed in the locations where ETT transcript would normally be present. At stage 6, SPT transcript is expanded into lateral regions of ett gynoecia where it is not found in wild type (Figure 34E). In stage 7, SPT is ectopically expressed in the abaxial cells of ett mutants. During stage 8, these abaxial cells undergo periclinal divisions similar to those that normally occur to form the septum and placenta. These periclinal divisions start to form the everted transmitting tract showing that the ectopic SPT expression is responsible for the formation of the everted transmitting tract. In summary, ETT negatively regulates the expression of SPT excluding it from the lateral and the abaxial zones of the gynoecium (Heisler et al., 2001). It will be interesting to determine whether ETT directly represses SPT since the SPT promoter contains several putative AuxRE sites that could serve as binding sites for ETT protein.

ETT patterns the gynoecium largely by restricting SPT activity. The ectopic expression of SPT in the abaxial domain clearly explains the defect in abaxial/adaxial polarity seen in ett mutants. However, ett spt double mutants also eliminate the apical/basal defects seen in ett; the apical and basal boundaries in the ett spt double mutant appear to form in the correct locations (Heisler et al., 2001).

ETTIN interacts synergistically with TOUSLED

In ett tsl double mutants, the gynoecium forms a fantastical structure composed only of placenta and ovules on a stalk (Figure 35A; Roe et al., 1997b). This extreme structure lacking valves, stigmatic tissue, and style results even when tsl is combined with the weak ett-2 allele, which itself has fairly large valves. This structure arises from a stalk with two laminar sheets producing ovule primordia at stage 10 (Figure 35B).

TSL is ectopically expressed in ett mutants stretching not just into the ectopic stylar medial tissue, but also into the valves of weak ett-2 mutants (Figure 35C to 35D compared to Figure 25I). Therefore, ETT limits the expression of TSL to the apex of the gynoecia establishing the apical boundary. The tsl ett double mutant would be expected to partially suppress the ett phenotype by eliminating the ectopic TSL. Instead, the phenotype is enhanced. One explanation is that TSL regulates other proteins involved in gynoecium development, which enhance the ett phenotype when they are misregulated in the double mutant. The observation that mutations in the spt gene can suppress the ett phenotype raises the question of whether spt mutants can also suppress the ett tsl double-mutant phenotype.

6.2.3 Auxin as a Morphogen in the Formation of the Apical/basal Axis of the Gynoecium

The plant hormone auxin is proposed to act as a morphogen forming a gradient that directs patterning in the Arabidopsis gynoecium (Nemhauser et al., 1998; Nemhauser et al., 2000). Generally auxin is thought to be synthesized in young apical tissues and transported basally throughout the plant. Members of the PIN family of proteins control the polarized transport of auxin and are thought to act as efflux carriers. The PIN proteins are dynamically localized to a particular side of the cell, so that transport has a net flow in the direction of their localization (Friml, 2003). This polarized transport of auxin is crucial for development of the plant. Application of polar auxin transport inhibitors is thought to disrupt the auxin gradient causing auxin to accumulate in high concentrations near the source and depriving the downstream cells of auxin. N-1-naphthylphthalamic acid (NPA) inhibits polar auxin transport and can be used to study the effects of disrupting auxin transport on development.

Application of auxin transport inhibitors throughout plant development has severe effects on the plant and greatly reduces the number of floral meristems produced (Okada et al., 1991). Therefore, to study the effects on gynoecium development, Nemhauser et al. (2000) devised a system of transient application of NPA to the inflorescence meristem. Even transient application of NPA caused the inflorescence meristem to become tapered and produce few floral meristems. The effect was temporary and after 13 days, resumption of normal flowering occurred. The flowers that developed within the period during which NPA affected development were studied in detail. In these flowers, the number of sepals and petals was variable and the number of stamens was reduced. The sepals and petals tended to be narrower than wild type. These phenotypes mimic those observed in pinformed (pin1) mutants confirming the disruption of polar auxin transport by NPA (see section 6.2.5 Mutations in PIN-FORMED (PIN1) disrupt polar auxin transport and the apical/basal axis of the gynoecium). In the gynoecium, application of low levels of NPA caused a slight increase in the stigma and style and a slight decrease in the ovary length (Figure 36A and 36B). Application of higher levels of NPA caused a more dramatic decrease in the valves while the stigma, style and gynophore increased in size (Figure 36C). In NPA treated gynoecia, stigmatic papillae developed earlier than normal and the replum protruded instead of being set in an indentation. These phenotypes caused by transient application of NPA to wild-type gynoecia mimicked weak ett phenotypes.

6.2.4 Relationship of ETTIN and SPATULA to Polar Auxin Transport

Since NPA treatment promoted ett-like phenotypes, Nemhauser et al. examined the effect of NPA on ett mutants (2000). Weak ett alleles were sensitive to application of NPA, which dramatically enhanced the severity of the ett phenotype. Application of low levels of NPA, which had only a slight effect on wild-type gynoecia, completely eliminated the formation of valves in ett-2 gynoecia similar to the phenotype of strong ett-1 alleles (Figure 36D and 36E). Increasing the NPA dosage further did not increase the effect (Figure 36F). Application of NPA to ett-1 mutants, only slightly enhanced the already severe phenotype. For both ett-2 and ett-1, NPA treatment prevented the formation of ovules. NPA treatment seemed to completely disrupt the boundaries between style and ovary and the ovary and gynophore in weak ett alleles.

Based on the phenotypes of NPA treated wild type and ett gynoecia, Nemhauser et al. (2000) have proposed a model suggesting that auxin forms a morphogen gradient within the gynoecium and ETT interprets the gradient establishing the boundaries between regions of the gynoecium (Figure 33). The auxin gradient is thought to form with high concentrations at the apex of the developing gynoecium and decreasing levels toward the base. In normal gynoecia, these thresholds lie at the top and the bottom of the ovary. If NPA is applied, it is thought that the gradient becomes very steep with pools of auxin in the style and very low levels of auxin below. In this case, extra stigma and style tissue develop as the apical boundary moves downward and extra gynophore develops as the basal boundary moves upward, all at the expense of the valves. In ett mutants, the interpretation of the auxin gradient is affected and the boundaries become destabilized causing the apical boundary to move down while the basal boundary moves up. Application of NPA to ett mutants further destabilizes the boundaries increasing the severity of the phenotype.

In contrast to the enhancement of ett phenotypes with NPA, treatment of spt-mutant gynoecia with NPA rescued the spt phenotype allowing complete fusion of the style and increased development of stigmatic papillae (Figure 36G and 36H). According to the model, addition of NPA increases the concentrations of auxin at the top of the gynoecium rescuing this phenotype (Figure 33). In spt mutants treated with NPA, the valve and gynophore remained unaffected in contrast to wild type suggesting that the effects of auxin polar transport probably act through a pathway mediated by SPT (Nemhauser et al., 2000). Since ETT negatively regulates SPT, the enhanced sensitivity of ett to NPA is likely to result from increases in the ectopic SPT expression (Heisler et al., 2001).

6.2.5 Mutations in PIN-FORMED (PIN1) Disrupt Polar Auxin Transport and the Apical/basal Axis of the Gynoecium

Mutations in the PINFORMED (PIN1) gene lead to a decrease in polar auxin transport to less than 15% of that seen in wild-type plants (Okada et al., 1991). The pin inflorescence initially forms a barren stalk without any floral meristems, but later becomes fasciated and forms flowers. The number of sepals and petals tends to be increased, but stamens are almost absent in pin flowers. The petals tend to fuse at their bases. In pin flowers, a conversion of organs toward carpels is seen including carpelloid sepals, carpelloid petals, and carpelloid stamens. Flowers at the top of the fasciated inflorescence tend to have more than one gynoecium. The pin gynoecia display a range of phenotypes from almost normal structures with two valves, style and stigma, to stalk like gynoecia with an elongated gynophore topped with a style and stigma (Figure 37A; Goto et al., 1991). The few stamens that are present do not produce pollen and ovules fail to develop in the gynoecia, making the pin mutant completely sterile (Goto et al., 1991; Okada et al., 1991). The phenotypes of pin mutants can be mimicked by the application of polar auxin transport inhibitors throughout the life of the plant (Okada et al., 1991). The amount of auxin synthesized in pin plants is dramatically reduced which could be a secondary effect of the lack of transport either through a feedback mechanism or because the flower buds which normally synthesize a large part of the auxin are absent (Okada et al., 1991).

PIN-FORMED encodes a transmembrane protein

The PIN1 gene is the founding member of the PIN family of putative auxin efflux carriers (Gälweiler, et al., 1998). PIN1 encodes a protein with 8 to 12 putative transmembrane domains surrounding a hydrophilic core, which has been proposed to function as a transmembrane carrier. PIN1 is expressed throughout the plant in cotyledons, flowers, roots, rosette leaves, seedlings, inflorescences, and siliques. In situ hybridization shows that the transcript accumulates in parenchymatous xylem and cambial cells. PIN1 protein is localized to one edge of the cell and this polar localization is often interpreted to indicate the direction of auxin transport (Reinhardt, et al., 2003; Benková, et al., 2003). Consistent with its role in basipetal auxin transport, PIN1 protein localizes to the basal cell membrane in the stem. Further characterization of the localization of PIN1 and the other PIN family members in the gynoecium should shed light on the flow of auxin in this tissue.

6.2.6 pinoid (pid) Gynoecia Are Missing the Central Ovary

Like pin1, the pinoid (pid) mutant forms very few flowers and the meristem forms a barren pin-like stalk. In the few flowers that arise, the pid gynoecia form a long gynophore capped by a style and stigma (Figure 37B and 37C). Therefore, the pid gynoecia appear similar to the most severe phenotypes seen in the pin gynoecia. About one third to one half of the pid gynoecia have small valves often only on one side of the gynoecium (Figure 37C). A small cone of growth occasionally continues up through the center of the stigma (Figure 37B). The defects in pid-mutant gynoecia are evident early in development when the pid gynoecia form as stalks with a small trumpet shaped opening at the top (Bennett et al., 1995). The pid gynoecium is similar to that of ett except that the abaxial/adaxial polarity is not affected (Alvarez and Smyth, 1998). Mutations in PID also affect the rest of the flower, reducing the number of stamens and sepals, but increasing the number of petals. The petals are often fused together forming a heart shape.

PINOID encodes a kinase

PID encodes a serine threonine protein kinase, which autophosphorylates in vitro (Christensen et al., 2000). In the developing gynoecium, PID is transiently expressed in the vascular tissues (Benjamins et al., 2001). PID expression is induced by the application of auxin and the promoter contains a single AuxRE. PID controls the polar localization of PIN protein within the cell and thus regulates polar auxin transport (Friml et al., 2004). High levels of PID expression (i.e. 35S::PID) lead to apical PIN localization while low levels of PID (i.e. in the pid mutant) lead to basal PIN localization. The targets of the PID kinase activity and the mechanism through which PID controls the polar localization of PIN remain elusive.

7.2.1 Hormone Induced Parthenocarpic Fruit

In Arabidopsis and many agricultural species, parthenocarpic fruit development can be triggered by application of gibberellins (GA), auxins, and cytokinins to unfertilized gynoecia (Figure 38A; Vivian Smith and Koltunow, 1999). These growth hormones applied to the entire gynoecium are thought to substitute for those normally provided by the seeds. Vivian-Smith and Koltunow (1999) have found that in Arabidopsis, gibberellic acid (GA₃) applied to the gynoecium of an emasculated flower at stage 13 caused a significant increase in the length of the gynoecium (Figure 38A and 38E) and the fruit shattered upon desiccation indicating that the fruit had matured, not just expanded. Similarly, application of cytokinin (benzyl adenine BA) or auxin (indoleacetic acid IAA or naphthylacetic acetic acid NAA) also generated parthenocarpic fruit (Figure 38A, 38F and 38G; Vivian-Smith et al., 1999). Although none of these parthenocarpic fruit treated with single growth hormones came close to reaching the length of pollinated fruit, fruit treated with both GA and cytokinin or both GA and auxin elongated as much as pollinated fruit (Vivian-Smith et al., 1999). These studies suggest a complex set of hormonal interactions occur during normal fruit development.

Of the parthenocarpic fruit treated with single hormones, those treated with GA₃ were the largest and the valve development most closely resembled that of pollinated fruit (Figure 38; Vivian-Smith and Koltunow, 1999). The number of cells and size of the cells in the valves of GA treated parthenocarpic fruit parallel those in wild type except that there are fewer cells in the mesocarp (Figure 38E compared with 38D). It is possible that normally GA acts fairly early after fertilization and is capable of inducing almost all of the downstream components for fruit expansion and maturation.

In contrast, the valve cells of auxin stimulated parthenocarpic siliques differed from those of pollinated siliques. The enlargement of the auxin treated gynoecia is primarily due to expansion of the valve cells especially in width. Cell division is reduced and only contributes a small amount to the total expansion of the silique (Figure 38G). The auxin treated siliques appear wider than pollinated siliques and the width of the valve wall is greater. The sclerenchymous cells of the enb layer wall do not thicken as much as in pollinated siliques although the dehiscence zone is functional and the fruit open. These differences between the auxin induced parthenocarpic siliques and pollinated siliques suggest that auxin is not sufficient to completely trigger normal fruit maturation. The expansion of the valve cells hints that auxin may primarily cause cell expansion during normal fertilized fruit development.

7.2.2 fruit without fertilization (fwf) Mutants Produce Parthenocarpic Fruit

A major advance to our understanding of parthenocarpy has come with the discovery of the fruit without fertilization (fwf) mutant of Arabidopsis. The fwf mutant is a facultative parthenocarp, setting seed like normal when pollinated, but also forming short seedless fruit when left unpollinated (Figure 40A; Vivian-Smith et al., 2001). The unpollinated fruit are 40% shorter than wild-type pollinated fruit. In the valves of fwf parthenocarpic fruit, greater cell expansion and reduced cell division are evident in the mesocarp layer than in pollinated wild-type fruit (Figure 40B to 40E). The vegetative fwf plant is indistinguishable from wild type, although a few other defects are seen in the flowers and the ovules.

Vivian-Smith et al. made an interesting discovery when they tested different methods of blocking pollination in fwf mutants and found that parthenocarpic fruit of different lengths resulted (2001). Longer fruit were produced when all of the floral organs were removed from the fwf flower prior to anthesis than when fwf was combined with a male sterile mutant, which only blocked pollination. Therefore, the other floral organs may have an inhibitory role on fruit development. In normal fruit development, this inhibition is removed when the floral organs abscise during stage 16. Therefore Vivian-Smith et al., postulate that a signal inhibiting fruit development travels from the floral organs to the gynoecium.

It is likely that FWF acts as an inhibitor of fruit development and that this inhibition is released after fertilization. Better understanding of FWF function awaits cloning of the gene.

7.2.3 Overexpression of Cytochrome P450 CYP78A9 in an Activation Tagged Line Promotes Parthenocarpy

Another parthenocarpic mutant was isolated in an activation-tagging screen in which CaMV35S enhancers were randomly inserted into the genome to drive ectopic expression of the surrounding genes (Ito and Meyerowitz, 2000; Weigel et al., 2000). Serendipitously, a plant that failed to self-pollinate and produced parthenocarpic fruit was identified. This dominant activation tagged parthenocarpic mutant was designated 28-5. At anthesis, 28-5 gynoecia are already slightly longer and twice as wide as the wild-type gynoecium, suggesting that fruit development has already begun before fertilization can take place. Without fertilization, the 28-5 fruit elongate 2.5 times as long and 1.7 times as wide as emasculated wild-type gynoecia. When pollinated with wild-type pollen, 28-5 siliques elongate to become 10 to 20% longer than wild-type fruit.

The 28-5 phenotype is caused by the overexpression of CYP78A9, a cytochrome P450 gene (Ito and Meyerowitz, 2000). Cytochrome P450 proteins synthesize or degrade secondary products such as brassinosteroids, flavonoids, and lignin. The endogenous CYP78A9 gene is not expressed in vegetative tissue and is first detected in the funiculi of stage 14 ovules. Signals from the ovules are thought to stimulate fruit development after fertilization and CYP78A9 might be involved in production of a signal that activates fruit development (Ito and Meyerowitz, 2000).

7.2.4 knuckles Mutants Produce Indeterminate Parthenocarpic Fruit

Although knuckles (knu) mutants are male sterile (at 25°C), the fruit develop parthenocarpically and form a large bump, which appears similar to the knuckle of a finger, protruding from the silique (Payne et al., 2004). This knuckle consists of ectopic stamens and carpels that initiate from the placental tissue in the base of the fruit. The development of ectopic organs from the placenta occurs reiteratively such that multiple layers of carpel tissue can be present in the fruit. About half of the knu gynoecia produce knuckles and only the gynoecia with knuckles form weakly parthenocarpic fruit in the absence of fertilization (at 25°C; Payne et al., 2004). The correlation between parthenocarpic fruit development and production of ectopic organs within the gyneocium is also observed in tomato plants when TM29, a SEPALLATA homologue, is downregulated (Ampomah-Dwamena et al., 2002). Payne et al. speculate that the ectopic organs growing within the fruit might produce growth regulators or proliferative signals that substitute for those normally produced in the ovules causing fruit development in the absence of fertilization. Production of ectopic organs within gynoecia could be a strategy for the production of parthenocarpic fruit in diverse agriculturally important fruit crops.

In addition to the production of parthenocarpic fruit, knu mutants have defects in patterning the basal elements of the fruit (Payne et al., 2004). Elongated gynophores often form at the base of knu fruit. Furthermore, the knuckle structure arises from the basal placental tissue. The knu mutation is temperature sensitive and homozygous seeds can be produced when the plants are grown at 16°C.

KNUCKLES encodes a C2H2 zinc finger protein

KNU encodes a small C2H2 zinc finger protein with an EAR-like active repression domain and is thought to act as a transcriptional repressor (Payne et al., 2004). KNU is expressed in a large patch at the base of the developing gynoecium during stages 6–9 as well as the developing pollen and ovules. No expression of KNU is detected after stage 13, suggesting that KNU acts early in gynoecium formation before fertilization. Payne et al. propose that KNU acts as a transcriptional repressor of the proliferation of cells from the placenta into non-ovule floral organs.