ovary and gametophyte development are coordinately ... · the angiosperm flower represem the...

The Plant Cell, Vol. 5, 403-418, April 1993 O 1993 American Society of Plant Physiologists

Ovary and Gametophyte Development Are Coordinately Regulated by Auxin and Ethylene following Pollination

Xian Sheng Zhang and Sharman D. ONelll’ Division of Biological Sciences, Section of Botany, University of California at Davis, Davis, California 95616

The differentiation and development of ovules in orchid flowers are pollination dependent. To define the developmental signals and timing of critical events associated with ovule differentiation, we have examined factors that regulate the initial events in megasporogenesis and female gametophyte development and characterized its progression toward maturity and fertilization. Two days after pollination, ovary wall epidermal cells begin to elongate and form hair cells; this is the earliest visible morphological change, and it occurs at least 3 days prior to pollen germination, indicating that signals associated with pollination itself trigger these early events. The effects of inhibitors of ethylene biosynthesis on early morphological changes indicated that ethylene, in the presence of auxin, is required to initiate ovary development and, indirectly, subsequent ovule differentiation. Surprisingly, pollen germination and growth were also strongly inhibited by inhibitors of ethylene biosynthesis, indicating that male gametophyte development is also regulated by ethylene. Detailed characterization of the development of both the female and male gametophyte in pollinated orchid flowers indicated that pollen tubes entered the ovary and grew along the ovary wall for 10 to 35 days, at which time growth was arrested. Approx- imately 40 days after pollination, coincident with ovule differentiation as indicated by the presence of a single archesporial cell, the direction of pollen tube growth became redirected toward the ovule, suggesting a chemical signaling between the developing ovule and male gametophyte. Taken together, these results indicate that both auxin and ethylene contrib- ute to the regulation of both ovary and ovule development and to the coordination of development of male and female gametophytes.

INTRODUCTION

The angiosperm flower represem the culmination of reproduo tive evolution in plants. Within the reproductive structures of the flower are found the embryo sac and pollen grain, the female and male gametophyte generations, respectively. In recent years, a great deal has been learned about flower initiation and development using molecular genetic approaches. Specifically, changes in patterns of gene expression associated with floral initiation and transition of thevegetative shoot apex to reproductive growth have been described (Meeks-Wagner et al., 1989; Kelly et al., 1990; Melzer et al., 1990), and major advances have been made in our understanding of floral or- ganogenesis, especially in terms of specification of floral pattern and floral organ identity at the molecular, genetic, and cellular levels (Bowman et al., 1989,1991; Drews and Goldberg, 1989; Meyerowitz et al., 1989; Schwarz-Sommer et al., 1990; Sommer et al., 1990).

In addition, numerous studies have described the regulation and expression of genes within the developing male gametophyte. It has been determined that although the mature male gametophyte of flowering plants is morphologically simple, reflecting its progressive reduction in size over the course of sporophyte evolution, its development requires a series of discrete differentiation events programmed by a relatively

* To whom correspondence should be addressed.

large number of genes (reviewed by Mascarenhas, 1989,1990). Indeed, genes specifically expressed in the developing male gametophyte have been identified and characterized (Stinson et al., 1987; Hanson et al., 1989; Ursin et al., 1989; Brown and Crouch, 1990). An equally detailed leve1 of understanding has been achieved for tissues of the anther, the sporophyte tissue in which the male gametophyte is formed and retained until anthesis (Koltunow et al., 1990).

Although the female gametophyte (or embryo sac) has been the focus of many investigations as well, most of the research to date has focused primarily on descriptive anatomy and cy- tology, with little emphasis on the molecular genetic basis of development. Female gametophyte development involves many basic developmental processes that have parallels in other eukaryotic organisms, including lineage-specific nuclear division and establishment of polarity and pattern (Drubin, 1991; St. Johnson and Nüsslein-Volhard, 1992). Eventually, genetic approaches in model systems such as Arabidopsis or molecular biochemical approaches in useful physiological systems should lead to a better understanding of the molecular mechanisms underlying this fundamental aspect of plant reproduction (Mansfield and Briarty, 1991; Mansfield et al., 1991; Robinson-Beers et al., 1992).

In most flowers, the ovary matures during flower development and contains fully formed ovules prior to pollination that

404 The Plant Cell

are ready for fertilization which generally occurs soon after pollination (Bouman, 1984). In contrast, the ovary of some flowers is immature until pollination triggers maturation of the ovary and differentiation of ovules within. Because ovary maturation and ovule differentiation are separated in time from development of other floral structures, these flowers provide a unique opportunity to dissect the signals that evoke this developmental transition in the ovary and to elucidate the molecular mechanisms underlying female gametophyte development.

Orchid flowers provide the most extreme example of pollination-regulated ovary development and ovule differentiation. Unlike all other flowers, at anthesis the ovary is immature and lacks ovules (Wirth and Withner, 1959; Withner et al., 1974). Pollination rapidly induces ovule differentiation, growth, and development and embryo sac formation in preparation for arrival of the growing pollen tube and fertilization. This precise induction by pollination has allowed us to describe and dissect the regulation and progression of ovule and embryo sac development as a foundation for our eventual goal of elucidat- ing the underlying molecular basis of these developmental events.

A role of auxin in promoting ovary development after pollination was originally established through studies of partheno- carpy, which demonstrated that auxin application led to the development of fruit without fertilization (Gustafson, 1939; Van Overbeek et al., 1941; Muir, 1942; Crane, 1969). Indeed, orchid pollen contains high levels of indoleacetic acid (Müller, 1953; Arditti, 1971; Stead, 1992), leading investigators to as- sociate the response to pollination with auxin. Many studies have demonstrated that the application of a number of auxins to the orchid stigma partially substitutes for pollination in inducing the postpollination syndrome in orchid flowers (Curtis, 1943; Burg and Dijkman, 1967; Arditti and Knauft, 1969; Arditti, 1979; Strauss and Arditti, 1982).

Although there are numerous studies of the role of ethylene in fruit ripening (Theologis, 1992) and flower senescence (Halevy and Mayak, 1979,1981; Borochov and Woodson, 1989), the role of ethylene in early ovary development has received very little attention probably because most reports attribute the growth response of this organ to a direct effect of auxin. However, auxin in many instances may be acting indirectly via its stimulatory effect on ethylene production (Abeles, 1966; Burg and Burg, 1966; Hall and Forsyth, 1967). Furthermore, in many flowers the gynoecium is an active site of ethylene production (Hall and Forsyth, 1967), but few reports implicate ethylene in regulating early ovary development. In carnation, Nichols (1971) reported that 2-chloroethylphosphonic acid (metabolized to ethylene) as well as 2,4-dichlorophenoxyacetic acid (syn- thetic auxin) accelerated peta1 senescence and ovary development. The effect of ethylene on ovary growth was analogous to that observed following pollination and occurred in both excised flowers and flowers treated in the plant. Exog- enous ethylene was also reported to promote column swelling in Vanda orchid flowers (Burg and Dijkman, 1967).

There is a similar dearth of information about the role of ethylene in male gametophyte development after its arrival on the stigma. In studies of peach and pear pollen, Search and Stanley (1968) and Buchanan and Biggs (1969) reported that both in vitro pollen germination and/or tube growth were stimulated by exogenous ethylene. Other studies do not support a role for ethylene in growth of pollen tubes in petunia styles (Sfakiotakis et al., 1972; Hoekstra and Van Roekel, 1988).

In this study, we present the detailed characterization of ovary development, ovule differentiation and development, and male and female gametophyte development that proceed at a slow rate for 12 weeks after the initial pollination event and prior to fertilization. The initiation of these events by pollination pro- vides a unique opportunity to study this process in plant reproduction. In addition, we present the results of studies to determine the role of pollination-associated factors in signaling the initiation of ovary and ovule development, which indicate that ethylene, with auxin, contributes to the regulation of female and male gametophyte development.

A parallel study has demonstrated that the postpollination syndrome of development in orchids, including ovary development, involves coordinated interorgan regulation of expression of ethylene biosynthetic genes (ONeill et al., 1993). Together, these results present the foundation of a molecular model for the regulation of ovary, ovule, and gametophyte development by pollination.

RESULTS

Figure 1 illustrates the effect of pollination on ovary development. The perianth tissue has been removed to reveal the dramatic changes taking place in the gynoecium (stigma and ovary together). Prior to pollination (Figures 1A and lB, left), the stigma forms an open cavity in which the pollen is depos- ited during insect-mediated pollination. The ovary, a short, grooved segment immediately below the stigma, is slightly curved. By 48 hr after pollination (Figures 1A and lB, right), the stigma has enlarged in diameter, enclosing the pollinia (orchid pollen masses) within the stigmatic cavity. The ovary undergoes similar changes, enlarging in diameter and length (Figures 1A and 18, right). These changes in the gynoecium are coordinately regulated with perianth senescence, which proceeds over a period of -6 days.

The long-term effect of pollination is shown in Figure lC, which compares an unpollinated flower (left) with a pollinated flower after 80 days (right) on the same inflorescence. Two points are significant. First, without pollination the unpollinated flower, now more than 80 days old (left), is as fresh as it was at the time of anthesis, illustrating the clear distinction between aging and pollination-induced development that is apparent in this species. The second point is that in spite of the obvious enlargement of the ovary in the pollinated flower after 80 days of development (right), fertilization has not yet occurred in this

Pollination-Regulated Flower Development 405

Figure 1. Changes in the Gynoecium of Phalaenopsis after Pollination.

(A) The perianth has been removed to reveal the straightening of theovary.(B) The swelling of the stigma enclosing the pollinia in pollinated (right)as compared to unpollinated (left) flowers.(C) At 80 days after pollination, the ovary has matured and is readyfor fertilization (right). At the same time, an unpollinated flower on thesame floral stalk remains fresh (left).

flower. In this context, it is important to note that the enlargedovary is not a fruit but represents the mature ovary containingmany thousands of differentiated ovules.

Figure 2 illustrates morphological changes that occur fol-lowing pollination. Growth rate, as indicated by ovary diameter,

demonstrates that it is a biphasic process, occurring over aperiod of ~ 85 days. Anatomical studies of these developingovaries indicated that the first peak of growth correspondedto the time of maximum ovary growth and ovule primordia dif-ferentiation from the placentae followed by a slowing of growthduring which time ovule differentiation occurred (42 to 56 days).The second peak of growth coincides with the differentiationand growth of the female gametophyte. The photographic se-ries shown in Figure 2 illustrates the gross morphologicalchanges occurring in the ovary during this prolonged periodof ovary development. Following fertilization at ~85 days af-ter pollination, a third phase of ovary development occurs,reflecting developmental changes associated with early em-bryogenesis and development of the fruit.

Pollination Induces Ovule Differentiation

To describe more completely the anatomical basis for the ob-served growth of the ovary following pollination but prior tofertilization, we conducted a series of anatomical studies using

ODays /Days 14 Days 28 Days 12 Days 56 Days 70 Days 84 Days 98 Days

40 60

DAYS AFTER POLLINATION

100

Figure 2. Rate of Ovary Growth following Pollination.

Measurement of growth rate in terms of ovary diameter indicates abiphasic growth pattern that can be correlated with internal structuralchanges. The first peak reflects the maximum rate of ovary growthand proliferation of ovule primordia from the placentae followed by ovuledifferentiation between 42 and 56 days. The second peak correspondsto the differentiation and growth of the female gametophyte (embryosac) just prior to fertilization, which does not occur until ~85 days af-ter pollination. The photographic series across the top of the graphillustrates the gross morphological changes in the ovary at variousdays after pollination.

406 The Plant Cell

Figure 3. Scanning Electron Micrographs of the Developing Ovary.(A) Placental ridge. Bar = 56 nm.(B) Enlarged view of (A). Bar = 16 urn.(C) Placental protuberances 14 days after pollination. Bar = 28 urn.(D) Immature ovules at 42 days after pollination. Bar = 28 urn.(E) Immature ovules at 49 days after pollination. Bar = 21 urn.(F) Immature ovules at 56 days after pollination. Bar = 42 nm.(G) Mature ovules at 84 days after pollination. Bar = 42 urn.(H) Pollen tubes growing along the ovary wall at 14 days after pollination. Bar = 21 |im.(A) and (B) show the interior view of an unpollinated ovary that reveals no significant ovule differentiation.

both scanning electron and light microscopy. Figure 3 illus-trates the developmental changes occurring along the placentalridge (site of ovule differentiation) in the pollinated ovary. Alongitudinal section through the unpollinated ovary revealedthe absence of ovules or ovule initials along the placental ridge(Figures 3A and 3B). At 14 days after pollination (Figure 3C),placental protuberances have differentiated from a singleepidermal layer of the placenta; immature ovules are evidentat 42, 49, and 56 days (Figures 3D to 3F); and mature ovulesare visible at 84 days after pollination (Figure 3G). Unpollinatedovaries show no similar differentiation along the placental ridgeover this same time period (data not shown). During thisprolonged period of ovule development, the pollen tubes havegrown into the ovary along the secretory epidermis of the ovarywall (Figure 3H) but do not grow toward the ovule until differ-entiation of the archesporial cell has occurred; perhaps thisis in response to a chemotrophic signal.

Early Ovary Development and Ovule Differentiation

Light microscopy reveals finer detail of pollination-regulatedovary maturation and ovule differentiation. Figure 4 presentsa series of transverse sections of the orchid ovary before andafter pollination and illustrates the early stages of ovule differ-entiation. Just prior to pollination, the ovary wall is made upof six elements: three sterile elements alternating with threefertile elements, with each of the latter bearing one placentawith two rows of ridges. The placentae appear "anchor-shaped"and fit close together, leaving little space in the central partof the ovary (Figure 4A). Figure 4B shows the placental ridgeprior to pollination, and no cell division is observed.

At 2 days after pollination, some cells of the ovary wall fac-ing between the placentae grow inward to form hair cells, asshown in Figure 4C. At the same time, cell division is initiatedin the placental ridge and in the inner face of the ovary wall

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Figure 4. Early Ovary Development and Ovule Differentiation.

(A) Transverse sections of an unpollinated ovary showing three placentae. Bar = 80 urn.(B) Enlargement of (A): a ridge of placentae is shown at the arrow. Bar = 40 urn.(C) Hair cells at 2 days after pollination. Bar = 40 urn.(D) Cell divisions of placental ridge (arrows) 2 days after pollination. Bar = 20 Mm.(E) Protuberances (arrow) from placental ridge 6 days after pollination. Bar = 80 nm.(F) Pollen tubes in ovary and protuberances (arrows) 14 days after pollination. Bar = 80 |im.(G) Branching protuberances at 28 days after pollination. Bar = 40 urn.(H) Ovule primordia and archesporial cells at 42 days after pollination. Bar = 20 nm.A, archesporial cell; H, hair cell; O, ovule; OW, ovary wall; P, placentae; PA, pollen tube area.

408 The Plant Cell

near the bases of the placentae (Figure 4D). Cell division in the placental ridges has no particular orientation; however, in the inner face of the ovary wall only anticlinal divisions occur. As the size of the ovary increases, the three placentae begin to separate. At 6 days after pollination, there is more space in the central part of the ovary, and protuberances have been produced from the placental ridge (Figure 4E). Figure 4F indicates that by 14 days after pollination, approximately Seven vascular bundles begin to differentiate in each placenta, many pollen tubes appear in the ovary, and the placental protuberances have further enlarged. At 28 days after pollination, cell division has stopped in the inner faces of the ovary wall, the placental protuberances have branched dichotomously once or twice (Figure 4G), and hair cells have filled the space in the ovary (data not shown). The growth of these hair cells is correlated with ovule differentiation.

From 35 to 42 days after pollination, the final branches of the placental protuberances differentiate ovular primordia, each composed of an axial row of cells covered by an epidermal cell layer (Figure 4H). A single archesporial cell, situated ter- minally in the axial row and distinguished by its large nucleus and rich cytoplasmic contents, is now present in each ovule primordium.

Ovule and Female Gametophyte Development

Figure 5 illustrates the latter phase of female gametophyte development. After the formation of the ovule primordia, the inner integument appears as a collarlike growth and is shortly followed by the initiation of the outer integument (Figure 5A). Each ovule becomes anatropous by 56 days after pollination. The archesporial cell enlarges further and functions directly as a megaspore mother cell that is evident in Figure 5A. It undergoes the first meiotic division to form two daughter cells: the micropylar cell of the dyad does not divide further and degenerates into a dark-staining mass, while the chalazal one undergoes the second division of meiosis resulting in the formation of two megaspores (Figures 58 to 5E). The chalazal megaspore is larger than the micropylar one, indicating the establishment of unequal cell division and early establishment of polarity.

The megaspore at the chalazal end develops into the func- tional megaspore giving rise to the one-nucleate embryo sac while the other one degenerates (Figures 5E and 5F). The embryo sac development is of the “polygonum-type” (Johri, 1963; Willemse and Van Went, 1984). At first, the one-nucleate embryo sac has no vacuoles (data not shown), the enlargement of this embryo sac is accompanied by increased vacuolation, and afew vacuoles are observed during its later stage (Figure 5F). The one-nucleate embryo sac stage is prolonged but then undergoes successive divisions to form the two- and four- nucleated stages (Figures 5G and 5H). At the same time, the vacuoles fuse to form a single, large vacuole in the central part of the embryo sac. By 85 days after pollination, the eight-nucleate, seven-celled embryo sac, i.e., the female

gametophyte, is mature and contains an egg cell, two synergids, two polar nuclei, and three antipodal cells (Figure 51). A filiform apparatus at the base of the synergids was observed. At 98 days after pollination, a two-celled proembryo, a primary endosperm nucleus, and three antipodal cells with degener- ating pollen tubes in the embryo sac were observed (data not shown).

Male Gametophyte Development after Pollination

Each Phalaenopsis orchid flower has a single pair of pollinia made up of loosely aggregated masses of pollen grains. These pollen grains are associated together in tetrads that in turn adhere to one another forming a single mass, i.e., a pollinium. Figure 6A illustrates that at the time of flower anthesis, each pollen or immature male gametophyte has two cells. At 48 hr after pollination, there is a noticeable increase in the size of the pollen. The generative cell can be seen as spindle-shaped, and a vacuole is observed in its cytoplasm; most of the generative cell volume is occupied by a nucleus with aggregates of chromatin (Figure 6B). Figure 6C shows the increased vacuolation of the vegetative cells just prior to pollen germination. Figure 6D illustrates that at 7 days after pollination, some pollen has germinated, and the generative cell and vegetative nucleus enter the pollen tube. In pollen tubes, the vegetative nucleus is positioned ahead of the generative cell (Figure 6E): both of these together constitute the “male germ unit” (Yu and Russell, 1992).

By 28 days after pollination, bundles of pollen tubes appear in the ovary distributed in the cavities of both sides of each placenta. The inner layer of cells of the inner ovary wall near the placenta is closest to the areaoccupied by the pollen tubes, and because this layer of cells is deeply stained by aniline blue black, these cells may contain more protein than the surround- ing cells (Figure 6F). Two sperm cells were observed in the pollen tube at 70 days after pollination (Figure 6G), and Fig- ure 6H shows a pollen tube with one vegetative nucleus and two sperms arriving near the ovule at 77 days after pollination. The pollen tube was observed in the embryo sac at 85 days after pollination; fertilization presumably occurs at this time.

Relationship between Female and Male Gametophyte Development

Table 1 compares the stages of female and male gametophyte development following pollination of orchid flowers and illustrates the coordinated development of the male and female gametophytes over a prolonged period following pollination. It is particularly interesting to note that the pollen tubes are visible in the ovary for a period of 70 days (between 14 and 85 days after pollination) in a state of apparent suspended development. Indeed, the final stages of development involving final cell divisions to reach maturity and the final growth of the waiting pollen tubes toward the embryo sacs suggest a

Figure 5. Female Gametophyte Development after Pollination.

(A) Immature ovule containing the megaspore mother cell at 56 days after pollination. Bar = 20 |im.(B) Late prophase I of meiosis at 70 days after pollination. Bar = 20 urn.(C) Metaphase I of meiosis. Bar = 20 urn.(D) Telophase II of meiosis. After the first meiotic cell division, the micropylar cell of the dyad degenerates (arrow). Bar = 20 urn.(E) Two megaspores (arrow) at 77 days after pollination. Bar = 20 |im.(F) Immature female gametophyte: one-nucleate embryo sac (arrow) at 77 days after pollination. Bar = 20 um.(G) Two-nucleate embryo sac. Bar = 20 um.(H) Four-nucleate embryo sac. Bar = 20 jim.(I) Mature female gametophyte: eight-nucleate embryo sac showing egg cell, synergids, polar nuclei, antipodal cells, and filiform apparatus (ar-row) at 84 days after pollination. Bar = 20 um.M, megaspore mother cell; E, egg cell; S, synergid; PN, polar nucleus; AC, antipodal cell.

Figure 6. Male Gametophyte Development after Pollination.

(A) Pollen grains in tetrads prior to pollination. Bar = 20 urn.(B) Two-celled male gametophyte containing vegetative cell and generative cell 2 days after pollination. Bar = 20 |im.(C) Increased vacuolation at 5 days after pollination. Bar = 20 urn.(D) Vegetative nucleus and generative cell in pollen tubes 7 days after pollination. Bar = 20 urn.(E) Pollen tubes in the ovary at 10 days after pollination. Bar = 20 urn.(F) Transverse section of an ovary showing pollen tubes in the ovary and one layer of cells with more protein (arrow) at 28 days after pollination.Bar = 80 nm.(G) Mature male gametophyte containing the vegetative nucleus and two sperm cells approaching the ovule 70 days after pollination. Bar = 40 urn.(H) Pollen tube approaching the micropyle of an ovule at 84 days after pollination. Bar = 20 urn.G, generative cell; O, ovule; PA, pollen tube area; PT, pollen tube; S, sperm; V, vegetative nucleus.

Pollination-Regulated Flower Development 41 1

Table 1. Relationship between Male and Female Gametophyte Development within the Same Flower after Pollination

Days after Pollination Male Gametophyte Female Gametophyte

Developmental Stage

7

14

28

42

56

70

a4

98

Pollen germination

Pollen tubes in ovary



Generative cell and vegetative nucleus

one vegetative nucleus

Pollen tube in embryo sac

Pollen tube degeneration

Two sperm and

Protuberance differentiation

Protuberance development

Protuberance branching

Ovule primordia differentiation:

Archesporial cell Megaspore mother cell

Prophase of meiosis I

Maturation of female

Two-celled proembryo gametophyte

gametic interaction that directs final development, growth, and fertilization. This may involve a chemical signaling, as has been discussed by Mascarenhas (1975).

Role of Ethylene in Regulating Postpollination Ovary Development

The association of ethylene with flower peta1 senescence is well established (Halevy and Mayak, 1979,1981; Borochov and Woodson, 1989), and it is also established that pollination ac- celerates this process by inducing ethylene production (Halevy, 1986). However, comparatively little is known about the relationship of ethylene to ovary development. We conducted a series of experiments to determine the effect of various pollination-associated factors and events on ovary development in comparison with senescence-related changes affecting the perianth and whole flower.

Figure 7 shows the effect of various pollination-associated factors and events on ethylene production by flowers (Figures 7A and 78) and the effect of an ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) (Yu and Yang, 1979). Control flowers left untreated and emasculated flowers did not pro- duce significant amounts of ethylene (Figure 7A). Following pollination, a dramatic increase in ethylene production occurred, with a peak after -36 hr (Figure 7A). In addition to pollination, auxin applied as naphthaleneacetic acid (NAA) stimulated ethylene production by the flower over a time course similar to that of pollination (Figure 78). 1-Aminocyclopropane- 1-carboxylic acid (ACC), which has been shown to be present

in petunia pollen (Whitehead et al., 1983), also stimulated ethylene production over a time course similar to that of pollination or NAA treatment (Figure 76). Pretreatment of the flower with AVG resulted in inhibition of ethylene production by all treatments. A small amount of ethylene was produced even when ACC was applied to AVG-pretreated flowers, but only at the earliest time points. A nearly identical response was observed when ACC was applied in combination with pollination to AVG- pretreated flowers (data not shown). This ethylene presumably resulted from direct conversion of ACC to ethylene by an ACC oxidase activity in the stigma that was already present and, therefore, unrelated to pollination-induced activity. The inhibition of ethylene production by AVG pretreatment in the case of ACC treatment (except at earliest time points) suggests that the small amount of ethylene produced by direct conversion

Pollinated I \

20 40 60 80 1 O0 Time After Treatment (hours)

Figure 7. Time Course of Ethylene Production by Orchid Flowers following Different Treatments.

(A) Ethylene production of pollinated (O), emasculated (A), and unpollinated control (D) flowers treated on the plant. (8) Ethylene production of unpollinated flowers treated with ACC (O), NAA (A), AVG plus ACC (O), or AVG plus NAA (A) and AVG-treated, pollinated flowers (O). Pollination, NAA, and ACC each induced ethylene production. AVG inhibited the production of ethylene in the ACC treatment except during the initial hours following treatment (at 6 hr) when an ACC oxidase activity that was unrelated to pollination might have been operative in converting it to ethylene. Ethylene production was measured in nanoliters per hour per gram fresh weight of tissue.

412 The Plant Cell

of applied ACC to ethylene is sufficient to induce ACC synthase activity and sustained endogenous production of ACC, and hence, autocatalytic ethylene production. Clearly, ethylene production is associated with pollination and its associated factors and, thus, is likely to play a role in pollination-associated responses. The remaining question is whether ethylene affects ovary development.

Experiments were konducted to determine the relative roles of auxin and ethylene in inducing components of the postpollination syndrome. Table 2 shows the effect of pollination- associated factors and mock pollination on early ovary development (stigma closure and ovary swelling) and perianth senescence. Flowers subjected to emasculation or physical contact with latex beads (mock pollination) were unaffected by treatment during the 6-day treatment period and exhibited physical characteristics similar to those of the flowers treated only with water (control). Pollination, on the other hand, resulted in severa1 morphological changes, including stigma closure, ovary swelling, and perianth (petal) senescence. Auxin (NAA) treatment fully substituted for pollination in inducing postpollination morphological changes, suggesting a key role for this hormone as a pollen-borne physiological trigger of the full postpollination response. Because auxin is a well-known stimulant of ethylene production in a number of plant organs, including orchid flowers, these experiments did not exclude the possibility of auxin acting indirectly by the stimulation of ethylene production.

ACC applied to the stigma induces ethylene production, as shown in Figure 78. As indicated in Table 2, when ACC was applied alone to the stigma, whole flower senescence was induced after 2 days. As observed in Figure 78 for ethylene production, the effect of ACC on perianth senescence was inhibited by AVG, again suggesting that the full effect of ACC

requires induction of ACC synthase activity and sustained endogenous production of ACC and ethylene. In the presence of AVG, pollination was less effective, inducing only partia1 stigma closure and ovary swelling, and perianth senescence was inhibited. When ACC was added to this treatment (AVG + ACC + pollination), a small amount of ethylene was produced at 6 hr only (data not shown), some of the gynoecium responses were restored, but perianth senescence was not. Thus, a small amount of ethylene produced by direct conversion of exogenous ACC to ethylene enhances the action of auxin in stimulating stigma closure and ovary growth but is not sufficient in itself to cause perianth senescence. We con- cluded from this experiment that ethylene must directly cause perianth senescence. This possibility was tested by applying exogenous ethylene to AVG-treated, pollinated flowers. In this case, the full postpollination syndrome was observed, including stigma closure, ovary development, and perianth senescence (data not shown). These results support the conclusion that ethylene has two roles in the postpollination response: (1) ethylene in the presence of auxin enhances developmental changes in the fertile reproductive organs of the flower, and (2) ethylene by itself is sufficient to cause senescknce-related degradative changes in all organs of the flower. Thus, stigma and ovary developmental processes are regulated by both auxin and ethylene, whereas petal senescence is regulated directly by ethylene.

Anatomical Basis for Early Ovary Development, Ovule Differentiation, and Pollen Tube Growth

Anatomical studies were conducted to further characterize the effects of auxin and ethylene on the earliest morphological

Table 2. Effect of Pollination-Associated Factors and Mock Pollination on Early Ovary Development and Perianth Senescence

1 Day 2 Days 4 Days 6 Days

Treatmenta Stigma Petal Stigma Petal Stigma Petal Ovary Stigma Petal Ovary

Water Ob F C O . F O F NSWd O F NSW Emasculation O F O F O F NSW O F NSW Beads O F O F O F NSW O F NSW Pollination (Poll) PCB F C S' C S swg Ch S sw NAA PC F C S C S sw C S sw ACC O F O S S S S S S S AVG + ACC O F O F O F NSW O F NSW AVG + ACC + Poll PC F C F C F sw C F sw AVG + Poll O F PC F PC F sw PC F sw

~ ~ ~ ~

a Flowers treated on the plant. b O, open.

F, fresh. NSW, not swollen.

e PC, partially closed. S, senescent.

9 SW, swollen. C, closed.

Figure 8. Regulation of Ovary and Male Gametophyte Development by Auxin and Ethylene.

(A) Transverse section of an ovary at 10 days after pollination, Bar = 80 urn.(B) Transverse section of an ovary at 4 days after treatment of the stigma with NAA. Bar = 80 urn.(C) Transverse section of an ovary at 10 days after treatment with pollen and AVG. Bar = 80 urn.(D) Transverse section of an ovary 10 days after treatment with pollen, AVG, and ACC. Bar = 80 |im.(E) Transverse section of an ovary 5 days after treatment with ACC. Bar = 80 urn.(F) Pollen tubes in the ovary 10 days after pollination. Bar = 80 urn.(G) With AVG treatment, no pollen tubes in the ovary 10 days after pollination. Bar = 80 urn.(H) Pollen tubes in the ovary 10 days after pollination following treatment with AVG and ACC. Bar = 80 urn.H, hair cells; OW, ovary wall; P, placentae; PA, pollen tube area.

414 The Plant Cell

120

100

80

60

40 h

E

5 v a 20

m

markers of pollination-induced ovary development. As indicated in Figure 4, one of the earliest observable anatomical events was the elaboration of hair cells from the ovary wall and the separation of placentae. Figure 8 shows the ovary in transverse section after 10 days of treatment. Pollination (Figure 8A) and pollination in the presence of AVG and ACC (Figure 8D) resulted in the greatest elongation of hair cells and differentiation of placenta1 protuberances as compared to pollination in the presence of AVG but without ACC (Figure 8C). In sharp contrast, ACC treatment alone did not trigger any hair cell growth and cell division; instead the cytoplasm of the ovary parenchyma cells disappeared by day 5, indicating that ACC alone leads to cell death (Figure 8E). These results clearly showed that the enhancement of ovary development and later ovule differentiation by ACC are dependent on the simultane- ous presence or action of another pollination-associated factor(s), most likely auxin. Auxin (NAA) alone can initiate hair cell growth and cell division after 4 days (Figure 8B), demon- strating the effect of auxin in initiating ovary development. An assessment at a later time point @e., at 10 days, as with other treatments) was not possible because NAA-treated flowers aborted before then.

The effect on male gametophyte development by blocking ethylene production with AVG was also examined. At 10 days after pollination, numerous pollen tubes were present in the ovary (Figure 8F). When ethylene production was blocked with AVG (Figure 8G), no pollen tubes were present in the ovary of the pollinated flower. The addition of ACC to the AVG- inhibited, pollinated flowers (Figure 8H) resulted in the pres- ente of pollen tubes in the ovary, indicating that the small amount associated with ACC addition in the presence of AVG stimulated male 'gametophyte development following pollination.

To determine the extent of the effects of pollination, AVG, and ACC on pollen development, we examined their effects on pollen germination and tube growth. As shown in Table 3, AVG inhibited both pollen germination and pollen tube growth, but upon the addition of ACC, which resulted in a small amount of ethylene production, both germination and tube growth were partially restored. Taken together, our data suggest that following pollination, ethylene regulates male gametophyte development.

-

-

-

-

-

-

Table 3. Quantitative Determination of Effects of Pollination, AVG, and ACC on Pollen Germination and Pollen Tube Growth

Pollen Length of Germination Pollen Tubes

Treatmenta ( 0 4 ("1 Pollination 1 O0 10.0 AVG + ACC + Pollination 80 5.5 AVG + Pollination 30 2.0

a Detached flowers at dav 6.

E Control Pollination Auxin ACC

B - - 8 L

'1 400 I

300

200

100

-

-

-

-

Control Pollination Pollination Pollination + AVG +AVG+ACC

Figure 9. Early Ovary Development as Assessed by Hair Cell Growth following Different Treatments.

(A) Effect of pollination, auxin, or ACC on hair cell growth at 4 days after treatment of flowers on the plant. (6) Effect of pollination with and without AVG or in the presence of AVG plus ACC on hair cell growth at 10 days after treatment of flowers on the plant.

Using hair cell length as a marker for the earliest detectable morphological change associated with ovary development, we evaluated the effects of auxin and ethylene on the initial stages of ovary development and ovule differentiation. Figure 9 shows the results 4 days after flowers were treated by pollination, auxin application, or ACC application (Figure 9A). 60th pollination and auxin treatment stimulated extensive hair cell growth, but ACC alone did not stimulate hair cell growth, indicating a re- quirement for auxin (Figure 9A). In longer term experiments (10 days), the presence of AVG inhibited pollination-induced hair cell growth, suggesting that ethylene is also required, along with auxin, for the initiation of ovary development (Figure 96). To test whether AVG was acting nonspecifically, we applied ACC with AVG and found that the effect of AVG could be partially reversed by the application of ACC, which bypasses the site of inhibition, resulting in a small amount of ethylene production (Figure 96). In related experiments, ethylene, rather than ACC, was used to overcome the AVG block on ethylene biosynthesis. This treatment was also effective in reversing AVG inhibition of hair cell growth, as shown in Figure 10. This result

Pollination-Regulated Flower Development 41 5

confirms that it is the small amount of ethylene produced by the inclusion of ACC, rather than ACC itself, that interacts with auxin to initiate ovary development. These results clearly dem- onstrate a hitherto unrecognized physiological response: initiation of both ovary development, and indirectly, ovule differentiation, and male gametophyte development requires ethylene as well as auxin.

DISCUSSION L,

Orchids are unusual among flowering plants in that in many orchid species the ovary is not mature at the time of pollination (Wirth and Withner, 1959; Withner et al., 1974). Following pollination, a prolonged period of reproductive development occurs during which time ovules are formed from rudimen- tary primordia in the ovary. The time elapsed between pollination and final fertilization among orchid species varies greatly from 7 to 130 days (Duncan and Curtis, 1942), reflecting the amount of time required for the formation of ovules and the mature embryo sacs within. The prolonged development of ovules only after pollination is a unique feature of the orchid flower and, as such, presents a unique opportunity to study the developmental processes of megasporogenesis and female gametophyte development. In spite of the obvious ad- vantages of the ovule development system in orchids, only a few comprehensive studies of ovule development have been conducted and in only a few orchid genera. Duncan and Curtis (1942) reported that following pollination in Phalaenopsis, the development of the ovary was intermittent and characterized by three well-defined phases of growth in diameter that could be correlated with interna1 structural development. Severa1 additional studies have examined reproductive development in orchids and provide a description of the process in several

600

2 500

400 C Q, - 300 a,

h

v

5

- y 200

= 100

.- m

Control Pollination Pollination Pollination +AVG +AVG+ETH

Figure 10. Relative Effects of Pollination, AVG, and Ethylene on Early Ovary Development.

Development was assessed by determining hair cell growth following pollination in the presence and absence of AVG and in the presence of AVG plus ethylene (ETH) 6 days after treatment in vitro.

orchid genera (Sagawa and Israel, 1964; Taylor et al., 1982; Fredrikson et al., 1988; Yeung and Law, 1989; Brown and Lemmon, 1991a, 1991b; Fredrikson, 1992; Yu and Russell, 1992).

One of the central regulatory processes in flower development is pollination, which induces a syndrome of postpollination development. The postpollination developmental syndrome is comprised of a number of processes, including perianth senescence, pigmentation changes, ovary maturation, ovule differentiation/development, and gametophyte development, components which are found in all flowering plants. The orchid flower system is particularly well suited for studying the developmental transition associated with pollination because it has the most highly developed and regulated response to pollination of all the flowering plants. Studies of pollination and postpollination development in orchids have an extensive history, providing an abundance of scientific information about the physiology of the postpollination response (Darwin, 1862; Hildebrand, 1863; Fitting, 1909; Laibach, 1933; Withner, 1959; Van de Pijl and Dodson, 1966; Arditti, 1969,1971, 1979; Dressler, 1982). Here we have focused on the developmental processes that are initiated by pollination in the ovary and on the hormonal signals that regulate these processes as a prelude to dissecting the molecular mechanisms underlying pollination-regulated ovule differentiation and gametophyte development.

Our experimental results showed that early characteristics of ovary development are hair cell growth and cell division at 48 hr after pollination, at which time the pollen grains have not germinated. These results indicate that the initial signals for ovary development come from the pollen and are not dependent on pollen germination or tube growth. Strauss and Arditti (1982) applied 14C-indoleacetic acid to the stigmas of orchid flowers, and by 24 and 72 hr following treatment, radio- activity could be detected in the flower, implicating auxin as a candidate for the pollination factor. Because NAA is the most effective of several auxins we tested, we applied NAA to the stigma of Phalaenopsis and after 48 hr determined its effect on hair cell growth as an early marker of ovary development. With NAA application alone, the ovary develops for a period of 4 to 5 days prior to abortion of the entire flower. We suggest that this reflects the exhaustion of the transported supply of NAA from the stigma. Under natural conditions, pollen and growing pollen tubes continuously supply auxin to the ovary, allowing the ovary to develop into a mature fruit.

Mock pollination using latex beads as a substitute for pollen grains has been reported (Sanders and Lord, 1989), and we used this approach to test the hypothesis that physical contact alone was sufficient to induce all or some components of the postpollination syndrome in orchid flowers. This was a reasonable hypothesis because by the time many pollination- induced changes are evident (e.g., 48 hr), the pollen grains have still not germinated. Germination does not occur until the fourth day after pollination (Figure 6). Thus, it was possible that mere placement of the pollinia on the stigmatic surface could in itself trigger the “pollination effect.” This possibility

416 The Plant Cell

also needed to be examined to test the hypothesis that a pollen-derived substance is responsible for triggering POstpOl- lination development. Our experimental results indicated that application of latex beads to the orchid stigma failed to induce any postpollination changes, and neither could bead treatment induce the expression of genes encoding ACC synthase and ACC oxidase (X. S. Zhang and S. D. ONeill, unpublished results), both of which are induced by true pollination and auxin treatment. Thus, the physical act of pollination is not sufficient to induce any component of the postpollination response.

Pollination of flowers stimulates ethylene production in the gynoecium and perianth, with ethylene production being initiated by auxin in the pollen. Indeed, a model of auxin as the pollination signal was proposed by Burg and Dijkman (1967) whereby auxin from the pollen diffuses through the column where it stimulates ethylene production that in turn triggers senescence of the perianth. We have shown that pollination and auxin induce the expression of genes encoding ACC synthase and ACC oxidase in the gynoecium (ONeill et al., 1993). The pattern of expression is similar for both genes with either of these two stimuli. Here we report that when AVG was used to block auxininduced ethylene production following either pollination or NAA treatment, ovary development was inhibited. If ACC was added to partially overcome the AVG block leading to ethylene production or if the whole flower was treated with exogenous ethylene, the initial stage of ovary development was restored. Pollen germination and pollen tube growth were affected in a similar manner. The addition of only ACC or ethylene without auxin only led to ovary senescence. These results clearly show that ethylene, with auxin, is required for ovary development and ovule differentiation following pollination, a hitherto unrecognized function of ethylene. Likewise, ethylene has an additional role in stimulating male gametophyte development.

METHODS

Plant Material

Orchid plants of the genus Phalaenopsis (cultivar SM9108, Stewart Orchids) were obtained as a clonal population of genetically identical mature individual plants and maintained under optimal growth conditions in a greenhouse at the University of California, Davis, Section of Botany. Plants were selected at random for each experiment.

Ovary Growth Measurements

For each time point, three flowers on each plant were self-pollinated. At various time intewals after pollination, flowers were excised, and the ovary diameter was determined at the middle of the ovary using calipers and a flexible millimeter ruler. Average values are presented, and the standard deviations at each time point did not overlap. The ovary growth experiment was repeated three times.

Light Microscopy

Ovary tissue was fixed at different stages after pollination in acetic acid/alcohol(1:3, v/v) for 5 hr or in 25% glutaraldehyde in 25 mM sodium phosphate buffer, pH 7.0, at 4OC for a minimum of 24 hr. After fixation, specimens fixed in acetic alcohol were stored in 70% ethanol at 4%. Specimens fixed in glutaraldehyde were rinsed in sodium phosphate buffer, dehydrated in a graded ethanol series to 70% ethanol, and stored at 4%. All specimens were embedded in JB-4 embedding medium (Polysciences, Inc., Warrington, PA). Sections were cut at 1.5 to 25 pm with glass knives on a Reichert-Jung 2050 microtome, mounted on slides, and stained with periodic acid-Schiffs reagent and 1% (w/v) aniline blue black in 7% (w/v) acetic acid. Stained sections were photographed using a Nikon OPTIPHOT 2 microscope.

Scanning Electron Microscopy

Samples of ovary tissue were mounted on a specimen holder with TissueTek(O.CT. 4583, Miles Laboratories Inc., Napewille, IL) and rapidly frozen in liquid nitrogen. After freezing, the samples were placed under a vacuum and moved to the cold stage of a preparative chamber maintained at --18oOC (EMscope SP2000, EMS, Fort Washington, PA) where they were given a thin coating of aluminum to provide con- ductivity. The samples were then moved under vacuum to a cold stage within a scanning electron microscope (Hitachi S-800) maintained at --18O0C where they were examined for surface morphology (second- ary electron mode images). lmages were recorded on P/N 55 Polaroid film.

Physiological Treatments

Parallel sets of experiments were conducted using detached or attached flowers. For experiments using detached flowers, flowers were har- vested by excision at the pedicel abscission zone and immediately inserted into floral tubes containing distilled H20. Otherwise, flowers were treated while attached to the plant. In both cases, whole flowers were pollinated, emasculated, or treated by applying a 15-pL volume of either distilled H20 or buffer as a control, or naphthaleneacetic acid (NAA, 25 pg per flower), 1-aminocyclopropane-lcarboxylic acid (ACC, 10 nmol per flower), aminoethoxyvinyl glycine (AVG, 0.5 mmol per flower), 5.85 pm polystyrene latex beads (Polysciences, Inc.) (15 pL per flower), or combinations thereof to the stigma. When AVG was used, flowers were pretreated with the inhibitor for 12 hr. When pollination was done in the presence of ACC, treatments were applied simultane- ously. For treatment with ethylene, whole flowers were placed in a sealed chamber, and pure ethylene gas was applied ata concentration of 10 pUL. Auxin and ethylene concentrations were chosen based on previous postpollination studies of orchids (Arditti and Knauft, 1969).

Ethylene Production

Ethylene production was determined as previously described (ONeill et al., 1993). Briefly, detached whole flowers were sealed in gas-tight containers equipped with septa for 2 hr. At least three flowers were used for each separate treatment. Fixed volume gas samples were withdrawn in triplicate, and ethylene concentration (microliters per Ii- ter) in the samples was determined by comparison with a standard

Pollination-Regulated Flower Development 417

(1 FUL ethylene) by gas chromatography using a Carle Analytical Gas Chromatograph 211 equipped with a flame ionization detector and an SP4270 integrator (Spectra-Physics Inc., San Jose, CA). Ethylene production (nanoliters per gram per hour) was calculated on the basis of the fresh weight of the flower.

ACKNOWLEDGMENTS

We thank the following people for their assistance: Dr. Elizabeth Lord for providing advice and material for the latex bead experiments, Susan Larson and Dr. Richard Falk for assistance with scanning electron microscopy and photographic work, and Debbie Van Blankenship (University of California, Davis, lllustration Services) for photography of flowers. Special thanks are given to Ned Nash (Stewart Orchids) for his generosity in providing plant material. This research was sup- ported by grants from the U.S. Department of Agriculture National Research lnitiative Competitive Grants Program (USDA 91-37304-6464) and the American Orchid Society to S. D. ONeill.

Received December 15, 1992; accepted February 25, 1993.

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DOI 10.1105/tpc.5.4.403 1993;5;403-418Plant Cell

X. S. Zhang and S. D. O'Neillfollowing Pollination.

Ovary and Gametophyte Development Are Coordinately Regulated by Auxin and Ethylene

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