Journal of Journal of Experimental , Vol. 48, No. 307, pp. 201-210, February 1997 Experimental Botany

REVIEW ARTICLE The role of ethylene in the development of form

Liam Dolan1

John Innes Centre, Colney, Norwich NR4 7UH, UK

Received 27 March 1996; Accepted 18 September 1996 Downloaded from https://academic.oup.com/jxb/article/48/2/201/652810 by guest on 02 October 2021

Abstract the number of parts determining their longevity and their ultimate fates. The fates of buds, for example, was largely Ethylene is a gaseous growth factor involved in a determined by their position on the tree. Buds formed diverse array of cellular, developmental and stress- within the last two years in the vicinity of the leader had related processes in . A number of examples of a greater chance of survival, of eventually developing as the role played by ethylene in the development of form long shoots or developing as inflorescences. Buds in more in plants are described; reaction wood formation, floral basal positions tended to be relatively dormant or more induction, sex determination, flooding-induced shoot likely to die (senesce or abscise) While many factors will elongation, and abscission. Recent advances in be involved in the regulation of this pattern of develop- the understanding of the molecular mechanism under- ment it is clear that ethylene plays a key role in the pinning post-pollination perianth wilting in orchids is co-ordination of these processes and as such plays a reviewed. This study indicates that the process of central role in the development of form in plants. The post-pollination perianth wilting involves an early role played by ethylene in the development of form will increase in sensitivity to endogenous levels of ethylene be described using examples gleaned from many species which set in motion a chain of events in which ethylene of angiosperms and gymnosperms. While it is not intended autocatalytically induces its own synthesis in the pistil. to review the literature exhaustively, it will be shown how Ethylene also induces the expression of ACO in the ethylene acts in some of the processes that Maillette perianth which converts pistil-derived ACC into ethy- (1982) showed to be important in the establishment of lene which drives the wilting process. Concepts drawn plant (tree) form: secondary thickening and reaction from this system are then applied to the Arabidopsis wood formation, floral development, sex determination, root epidermis in which ethylene is a positive regulator floral senescence, leaf abscission, shoot elongation, and of root hair development in an effort to come to a cell differentiation. mechanistic understanding of the process of pattern formation in this system. Understanding the molecular basis of the role of ethylene in these model systems will provide useful paradigms for examining the part Ethylene biosynthesis and signal transduction played by ethylene in the diverse array of processes Ethylene is a gaseous growth factor that was identified in which this growth factor is involved. by Neljubov as the causative agent for laboratory air- induced horizontal growth of pea seedlings (Neljubov, Key words: Ethylene, development, plant form. 1901). In recent years there have been great advances in our understanding of ethylene biosynthesis and signal transduction (see Ecker, 1995; Zarembinski and Introduction Theologis, 1994, for reviews). Ethylene is derived from The development of plants involves the progressive birth the , methionine: methionine is converted and death of their iterated parts: shoots, roots, , to 5-adenosyl methionine (SAM) by the action of , and buds (Maillette, 1982). In her analysis of the methionine adenosyl transferase; SAM is converted to development of tree form in silver birch, Maillette (1982) 1-aminocyclopropane-l-carboxylic acid (ACC) by ACC examined the life history of the buds (meristems) and synthase (ACS): ACC is converted to ethylene by ACC showed that tree shape could be examined by counting oxidase (ACO) (see reviews by Yang and Hoffman, 1984;

1 Fax: +44 1603 501771.

Oxford University Press 1997 202 Dolan Kende, 1993) (Fig. 1). ACS genes comprise a multi-gene the ethylene receptor (Chang et al, 1993). Its sequence family in most species examined to date (Liang et al, suggests that it is a histidine kinase similar to the bacterial 1992). Individual members of the gene family exhibit two component kinases and has recently been shown to tissue specific regulation or are transcriptionally activated bind ethylene. A similar gene, ERS which lacks the by a specific set of modulators such as , flooding or receiver domain, may also act as an ethylene receptor mechanical stress (Abel et al, 1995; Olson et al, 1995; since not all ethylene responses are blocked in etrl Botella et al, 1995). ACO genes also comprise a gene mutants (Hua et al, 1995). Epsisatic interactions have family (Tang et al, 1993). Three members have been shown that the CTR1 gene, a negative regulator of the identified in the orchid Phalaeopsis (Nadeau et al, 1993). ethylene response, acts downstream of the receptor Both ACS and ACO are positively regulated by ethylene (Kieber et al, 1993). CTR1 encodes a serine-threonine in a number of systems (O'Neill et al, 1993). kinase similar to members of the Raf kinase family ACC is soluble and has been shown to move (Kieber et al, 1993). It is possible that the role of this through the apoplast over great distances (from roots to gene is to modulate flux through the signalling pathway. shoots) in tomato plants and has been shown to move A number of other genes have been identified which act Downloaded from https://academic.oup.com/jxb/article/48/2/201/652810 by guest on 02 October 2021 from stigmatic surfaces to petals in a number of species downstream of CTR1 which are involved in specific (Bradford and Yang, 1980; Reid et al, 1984; Woltering, aspects of ethylene responses such as root elongation, 1990). Ethylene is 14 times as soluble in lipid as in water hypocotyl hook opening etc. (Roman et al, 1995). and other solutes present in the aqueous phase (as in the cytoplasm) will decrease its solubililty (see Abeles et al, 1992, for a description of the physical characteristics of Development of form: processes in which ethylene ethylene). Consequently, ethylene might be expected to plays a key role act locally in the absence of air spaces. Tree shape The genetic dissection of the ethylene signal transduc- tion pathway has provided powerful insights into the Upon germination the shoot meristem laid down in the molecular mechanism that underpin the ethylene response developing embryo produces a shoot system composed and has provided developmental biologists with a wealth of iterated units, leaf, node, internode, and axillary bud. of tools to examine specific aspects of plant development The pattern in which these units of construction are put (Ecker, 1995; Roman et al, 1995). Screening mutant together is highly invariant within a species, e.g. phyllo- populations for plants exhibiting abnormal ethylene taxy is generally invariant within a species although it responses has led to the identification of a number of may change with ontogeny. Nevertheless, the number and genes that are involved in ethylene signal transduction stature of the units that survive in long-lived plants (such (Bleecker et al, 1988; Guzman and Ecker, 1990; Kieber as trees) is largely determined by environmental factors etal, 1993; Roman et al, 1995; Huae/ al, 1995) (Fig. 1). such as light, water availability, physical stress, etc. That The ETHYLENE RESISTANT1 (ETR1) gene encodes physical stress is an important feature in the determination of plant form has been well documented. A striking example of the effect of mechanical stress not only on plant stature but the stature of an entire vegetation type, Biosynthesis Signal transduction is the elfin montane forests of central America. Methionine Ethylene Didymopanax pittieri is a tree that grows on and below + exposed hill crests in montane rain forests. Lawton (1982) 1 1 A9 clearly showed that plant stature is determined by proxim- SAM ETR1IEIN4 ity to the crest and, consequently, wind-induced mechan- 1 1 AM3 ical stress. Plants at the crest, have relatively thicker 1 trunks and finer twigs while those growing in less exposed ACC CTR1 areas are relatively taller with thicker twigs. He rules out a genetic basis for the morphological differentiation and, 1 1 therefore, highlights the importance of thigmmorphogen- Ethylene EIN2 esis, the morphological changes in response to physical stimuli, in the development of plant form. A\ In a classic set of experiments on the development of Various ethylene compression wood in Pinus strobus, Sinnott (1952) high- responses lighted the regulative role of reaction wood in response to physical stimulation. Reaction wood is a specialized Fig. 1. Simplified schematic diagram of the pathways of ethylene wood type that is formed in trees in response to physical biosynthesis and ethylene signal transduction. Adapted from figures in Zarembinski and Theologis (1994) and Roman et al. (1995). stress (Scurfield, 1973). Reaction wood in gymnosperms, Ethylene and plant form 203 such as P. strobus forms on the underside of stressed Leaf abscission branches, thereby serving to support or reorientate the Mailette's (1982) study showed that while the formation limb. In angiosperms, reaction wood forms on the upper of long and short shoots was a major factor in the side of the branch and serves to 'pull' the limb into development of birch tree form so also was the regulated position. Sinnott (1952) tied vertical shoot axes and loss (abscission) of these structures. The abscission of lateral branches in various abnormal positions, let them leaves in response to ethylene is one of the earliest develop for a further 2 years and examined the develop- documented effects of ethylene. Abscission results from ment of the reaction wood. Wood formed only in locations cell separation due to cell wall degradation in a differenti- in which it tended to restore the axis to its original ated group of cells that constitutes the abscission zone. position. The regulative nature of this reaction indicates Ethylene and ABA induce abscission while auxin (from that while the tree may be composed of many iterated the active leaf) represses the process (Burg, 1968; parts they constitute an integrated physiological unit with reviewed by Osborne, 1991). respect to reaction wood formation.

The bean (Phaseolus vulgaris) leaf has a proximal Downloaded from https://academic.oup.com/jxb/article/48/2/201/652810 by guest on 02 October 2021 Numerous experiments have implicated ethylene as abscision zone located at the stem- junction and a playing a role in the tree's response to physical stress. distal abscision zone at the petiole-lamina junction, prox- The induction of ethylene in response to physical stress imal to the leaf pulvinus. Ethylene-induced leaf abscission in pea seedlings (Goeschl et al, 1966) with consequent induces cellulolytic that break down cell walls morphological changes (decreased height, increased girth) in the abscision zone facilitating cell separation. Ethylene- led Leopold to examine the production of ethylene in induced abscission is associated with the expression of mechanically induced stress in pine, and apple polygalacturonase and endo-|S-l,4-glucan hydrolase in the branches (Leopold et al, 1972). The experiments were vicinity of the distal abscission zone. Endo-/3-l,4-glucan similar to Sinnott's since branches were bent and tied hydrolase has been purified and antibodies against the such that 90° curvature was obtained in 4-year-old wood. have been raised. Microinjection of the antibody Three days after the stress was removed ethylene levels into abscission tissue prevents abscission in bean (Sexton had increased 2-3-fold implicating ethylene in the et al., 1980). In situ localization using the antibody and response to mechanical stress (Robitaille and Leopold, using RNA probes indicates that endo-j8-l,4-glucan 1974). In a further investigation ACC was isolated from hydrolase is present not only in the stele and cortex of cambial tissue collected from the underside of a plagio- the abscission zone, but also in the stele of adjacent tropically-growing Pinus contorta branch where compres- pulvinal tissue (del Campillo et al, 1990). Examination sion wood forms, while none was detected in the cambium of the expression of the protein during the induction of from the upper side of the branch where no compression abscission indicates that the protein is initially expressed wood develops (Savidge et al, 1983). Such asymmetric in the stele of the abscission zone and only later is it distribution suggests that ethylene may be synthesized expressed in the abscission zone cortex. exclusively in the region of reaction wood development. To examine the role of the stele in regulating the Mechanical perturbation of field- and greenhouse- expression of the endo-j8-l,4-glucan hydrolase in response grown Abies fraseri results in an increase in girth and a to ethylene-induced abscission, surgical experiments were carried out (Thompson and Osborne, 1994). Firstly, decrease in height compared to control plants (Telewski removal of the abscission zone stele inhibits endo- and Jaffe, 1986a). The increase in girth results from /3-1,4-glucan hydrolase induction indicating that the stele increased cambial activity and tracheid development in is necessary for induction. Surgical separation of the direction of the perturbation. The decrease in height the abscission zone cortex from the stele soon after may be due to the decreased length of tracheids that ethylene treatment represses endo-j8-1,4-glucan hydrolase develop as a result of perturbation (Telewski and Jaffe, induction. Separating the tissues at a later time but before 1986a, b). Similar perturbations carried out on half-sibs endo-/3-l,4-glucan hydrolase activity is visible, permits of Pinus taeda indicate that there exists genetic variation enzyme induction. These results suggest that ethylene for ability to respond to the mechanical stress (Telewski induces a signal in the abscission zone stele which sub- and Jaffe, 19866, c). One half-sib that responded to the sequently moves to the cortex inducing the abscission mechanical stimulation produced 16 times more ethylene process. Insertion of dialysis membrane (molecular weight than the non-responding sib, clearly implicating ethylene cut-off of 12000-14000) between the stele and cortex as playing a central role in the morphological response blocks the movement of the signal suggesting that the to physical stress (Telewski and Jaffe, 1986c). Exogenous signal has a molecular weight greater than 12 000-14000. treatment of P. taeda with ethephon (an ethylene Insertion of dialysis membrane with small holes results compound) results in the development of many of the in the induction of endo-£-l,4-glucan hydrolase activity morphological characteristics associated with mechanical in the vicinity of these holes suggesting that the signal is perturbation (Telewski and Jaffe, 1986c). of limited mobility. 204 Dolan These studies indicate that ethylene induces abscission is higher in ethylene-treated corms than in controls, by inducing a factor in the stele that moves from the stele suggesting that ethylene stimulated the mobilization of into the abscission zone cortex which itself, is not compet- carbohydrate stores in the corm which are utilized ent to make the inducing signal in response to ethylene. during growth.

Floral development Sex determination The development of flowers from a subset of over- Once a meristem has become committed to forming a wintering buds in birch is an important factor in determin- in birch, its sex is then determined by its position ing the form of this tree since such buds are committed on the tree (Mailette, 1982). Male inflorescences (catkins) to the floral state and will contribute nothing to the future develop at the end of the growing season on long shoots body of the plant. While ethylene has not been implicated and female inflorescences develop in the spring on short in floral development in birch this gas is in widespread shoots. While the physiological basis of floral sex deter-

use as an inducer of floral development in a number of mination in birch is not understood, a great deal is known Downloaded from https://academic.oup.com/jxb/article/48/2/201/652810 by guest on 02 October 2021 species of horticultural importance. The most notable about the role of ethylene-regulated floral sex determina- example is the use of ethylene for the induction of tion in cucurbits (cucumbers and melons). flowering in pineapple (Ananas sativus). Treatment of Genetic variation exists in different cucumber lines that plants with AVG blocks induction (Mekers et a!., 1983). specify their modes of sexuality (Shifriss et al., 1964). Induction depends on the presence of leaves. No induction Many horticultural varieties are monoecious (male and takes place when all leaves are removed while induction female flowers are produced on the same plant), while takes place when a single leaf remains on the shoot others are gynoecious (female and hermaphrodite flowers (Traub et al., 1940). The effect of ethylene in Aechema produced on the same plant). Gynoecious plants grown victoriana has been shown to depend on the size of the under environmental conditions that promote female plant. Small plants less than 22 g fails to flower upon development produces more ethylene than monoecious ACC treatment while plants 23 g or over respond. The plants, indicating that ethylene may be associated with larger the plant at the time of treatment, the greater the the feminization of flowers(Rudic h et al., 1972). Removal number of flowers produced (De Greef et al., 1989). of endogenous gases (including ethylene) by growing The inhibitory effect of ethylene on flowering in short these all-female plants under hypobaric conditions, results day (long night) plants has been described for a number in the formation of hermaphrodite flowers, consistent of species (Abeles, 1967; Nitsch and Nitsch, 1969; with the feminizing role for endogenous ethylene in female Amagasa and Suge, 1987). Treatment of ethylene during flower development (Byers et al., 1972). Supplementing the dark inductive period in Japanese morning glory these plants (grown under hypobaric conditions) with (Pharbitis nil) abolishes the inductive effect (Amagasa exogenous ethylene results in the restoration of female and Suge, 1987). The inhibition remains until 30 h later floral development. Consistent with the feminizing role which is the earliest time in which a dark inductive period of ethylene in floral development is the observation that is successful in inducing flowering after an ethylene treat- inhibitors of ethylene biosynthesis resulted in the forma- ment. The application of ethylene in the light prior to the tion of either perfect or male flowers, depending on the dark, inductive period has no effect on floral induction. species and genetic background (Byers et al., 1972; Makus This suggests that the plant is sensitive to ethylene during et al., 1975; Atsmon and Tabbak, 1979; Den Nijs and the inductive period only. A molecular understanding of Visser, 1980). these events remains to be ascertained. While the role of ethylene in the feminization of flowers Ethylene is a potent inducer of flowering in geophytes, has been clearly shown, another growth factor, gibberellic plants with subterranean perennating organs, such as acid (GA) is known to promote the development of male corms and bulbs (discussed in Abeles et al., 1992). flowers. A role for GA in sex determination in maize has Although the use of smoke as a potent inducer of also been shown by genetic means (DeLong et al., 1993; flowering in Iris has been a traditional practice in various Dellaporta and Calderon-Urrea, 1993). A recent study in cultures for hundreds of years, little is known about the cucumber suggests that ethylene not only promotes mechanism involved. Treatment of corms and bulbs with femaleness, but inhibits maleness and, conversely, GA ethylene generally promotes early sprouting, early flower- both promotes maleness and inhibits femaleness (Yin and ing, more flowers per inflorescence, and increased size of Quin, 1995). Experiments in which plants were treated daughter corms or bulbs. Ethylene-induced changes in with different combinations of both growth factors and Triteleia laxa are accompanied by an increase in growth inhibitors of growth factor action and synthesis, led to rate and, consequently, a larger apical meristem, which the conclusion that ethylene acts more directly on sex may be responsible for the greater number of flowers expression than GA. An alternative model which suggests formed in treated plants (Han et al., 1990). Respiration that each growth factor promotes one sex only and does Ethylene and plant form 205 not inhibit the other, i.e. ethylene promotes femaleness Ethylene stem elongation in submerged deep water rice only and GA promotes maleness only, has also been Maillette (1982) showed that buds of birch trees may proposed. The application of molecular techniques in the form either short or long shoots depending on the bud's future may provide important insights into which of these position in the tree. Buds located near the leader shoot alternative models most nearly approximates reality. are exposed to high light levels and tend to form long shoots as a result of the high photosynthetic activity of Cellular basis of the ethylene change in form: ethylene, cell leaves in this position. Buds in older parts of the tree elongation, microtubules, and microfibrils form a higher proportion of short shoots since they are exposed to lower light levels and therefore produce lower Exogenous treatment of tissues, organs or whole plants amounts of expendable photosynthate for investment in with elevated ethylene results in a dramatic change in the shoot development. Shoot extension in the developing direction of cell expansion in growing cells. Root cells in birch tree is therefore tightly coupled with local modula- most terrestrial plants undergoing expansion usually do

tions in environmental factors such as light. Many aquatic Downloaded from https://academic.oup.com/jxb/article/48/2/201/652810 by guest on 02 October 2021 so in a direction parallel to the long axis of the root. and semi-aquatic plants respond to submergence by rapid Ethylene treatment of such roots decreases expansion in shoot elongation, resulting in the positioning of photosyn- the longitudinal direction and induces radial expansion thetic leaves at or above the water surface. Early studies (Chadwick and Burg, 1967). Mechanical perturbation has on Callitriche indicated that ethylene accumulation in been shown to induce radial swelling and inhibit elonga- submerged stems of the plant was responsible for shoot tion in etiolated pea seedlings (Goeschl et al, 1966). elongation (Jackson, 1985). Short and radially expanded cells characteristic of many Deep water rice is a variety of Oryza sativa that is soil-grown roots might also result from ethylene produced planted in flood plains and can become covered by up to as a result of such mechanical impedance (L Dolan, 5 feet of water during the wet season. Such flooding unpublished observations). Similar changes have been results in increased culm (stem) growth, achieving growth described in tracheids that differentiate in reaction wood rates of 25 cm d"1 (Raskin and Kende, 1984a). in response to physical stimulation in Pinus taeda Submergence-induced culm growth in deep water rice (Telewski and Jaffe, 1986a, b). This decrease in length results from the stimulation of ACC synthesis in response and increase in girth contribute, at least in part, to the to lowered O2 levels in the intercellular spaces of flooded change in stature that mechanical perturbation induces plants resulting in a two orders of magnitude increase in in trees. ethylene accumulation in the stem lacunae (Metraux and Cellulose microfibrils in the inner layers of elongating Kende, 1983). Treatment of deep water rice with ethylene cell walls are generally arranged perpendicular to the long induces submergence-like growth stimulation in the axis of the cell. Such an organization is considered to be absence of submergence (Metraux and Kende, 1983). mechanically favourable since the transverse stress in a Non-deep water rice varieties show no ethylene-induced cylinder is twice the axial stress (Green et al, 1970). The growth in response to submergence. Examination of the orientation of microtubules in elongating cells mirrors role of GA indicate that ethylene might act by increasing the alignment of the microfibrils: the cortical microtubules the activity of endogenous GA and thereby stimulating in elongating cells are largely transverse in orientation. growth (Raskin and Kende, 19846). As might be expected, the change in the dimensions of The primary effects of submergence in deep water rice growth associated with ethylene treatment is accompanied is an increase in cell number per cell file which results by both a reorientation of nascent cellulose microfibrils from a decrease in cell cycle time from 24 h to 7 h in the in the inner wall layer and reorganization of the microtub- intercalary meristem (Metraux and Kende, 1984; Bleecker ules in the cortical cytoplasm (Apelbaum and Burg, et al, 1986). The elongation zone above the intercalary 1971). Exposure of mung-bean hypocotyl cells to short meristem increases in extent resulting in a 3-fold increase pulses of ethylene lead to a predominantly longitudinal in final cell length. Tissue differentiation is suppressed in microtubule organization in less than 1 h (Roberts et al, the internode and growth of its associated leaf ceases. 1984). In addition, GA treatment of elongating cells has the opposite effect: cells elongate and both microfibrils and microtubules become relatively more transversely Molecular mechanism underpinning the oriented (Baluska et al., 1993). The molecular mechan- generation of form by ethylene isms that underpin such cell shape, microtubule and While the effects of ethylene on plant development have microfibril changes are not known. Examination of these been well documented, few are understood in a mechan- processes in Arabidopsis in which there is a wealth of istic way at the molecular level. Recent insights into the genetic, molecular and cellular tools available will be role played by ethylene in post-pollination-induced senes- invaluable for future advances in this area. cence in a number of species including Chrysanthemum, 206 Dolan Petunia and Phalaenopsis have been instructive (reviewed cesses, ethylene sensitivity was examined in unpollinated in Stead, 1992; Woltering and de Vrije, 1995). A mechan- flowers after treatment with 1 mM Ca2 + and the Ca2 + istic understanding of pollination-induced perianth wilt- ionophore A23187 (Porat et al, 1995). Ca2+ treatment ing in the orchid, Phalaenopsis, based on the molecular renders the unpollinated flowers as sensitive to ethylene analysis of this process, is presently emerging. The model as pollinated flowers. Ca2+ treatment of pollinated flowers involves developmental regulation of both ethylene bio- decreases the time to wilting by 30%. Consistent with role synthesis and sensitivity (O'Neill et al., 1993). In a second of Ca2+ in ethylene sensitivity is the observation that example, largely genetic studies of the development of the treatment with the Ca2 + -chelator, EGTA, or La3+ results root epidermis in Arabidopsis are described (Dolan et al., in an extension of wilting time, i.e these treatments render 1994; Tanimoto et al., 1995). The spatial regulation of flowers less sensitive to ethylene. These results suggest cell differentiation in this system is controlled again by that the induction of sensitivity is independent of ethylene developmentally regulated ethylene biosynthesis and sens- itself and the increased sensitivity renders cells in the itivity. Such model systems are providing mechanistic flower sensitive to the endogenous, low ethylene levels insights into the role of ethylene in the development of that exist in the floral tissue. This results in the induction Downloaded from https://academic.oup.com/jxb/article/48/2/201/652810 by guest on 02 October 2021 plant form and together with the recent advances in our of ethylene biosynthetic genes and, consequently, the understanding of the molecular basis of ripening (in production of ethylene in a tissue-specific manner. tomato) and of the role of ethylene in the wound response, Ethylene production increases drastically 8 h after pol- will illustrate how conserved or diverse is the mode of lination and continues to increase for a further 26-30 h ethylene action in plants. (O'Neill et al., 1993). Examination of the ethylene produc- tion from different portions of the flower indicate that Changes in ethylene sensitivity and mobile ACC regulate the stigma is responsible for almost all ethylene produced pollination-induced senescence in orchids in the first 12 h, at which time ethylene begins to be formed in petals. Examination of the pattern of expression Pollination represents a watershed in the life of many of the ethylene biosynthetic genes ACS and ACO reveals flowers. No more is their aim to attract pollinators or that both genes are expressed in the gynoecium approxi- pollen, but they embark on a new phase in which they mately 6 h after pollination. The petals on the other hand may shed themselves of pollen-attraction organs (petals show no ACC synthase expression and ACC oxidase etc.) and invest their energies in fertilization, embryo, expression is only apparent after approximately 12-24 h, , and fruit development. In many species, pollination reaching a peak at approximately 48 h after fertilization, results in the wilting or complete shedding of perianth coinciding exactly with the pattern of ethylene formation segments. Foxglove and cotton petals abscise while those described for this tissue. This pattern of gene expression of petunia and many orchids simply wilt (reviewed by suggests that ACC is synthesized in the gynoecium and Stead, 1992). Ethylene plays a central role in the events transmitted to the perianth where it is converted into that lead to the developmentally regulated abscission and ethylene by the action of ACO. Previous surgical experi- wilting of the perianth. ments in another orchid Cymbidium, indicate that ACC Recently, a model has been proposed for the mechanism might move through the flower to the perianth during of post-pollination wilting in the orchid, Phalaenopsis. the wilting process (Woltering, 1990). Incisions into the This model suggests that pollination induces ethylene base of the labellum inhibit the increase in anthocyanin sensitivity in cells of the gynoecium which then respond levels associated with wilting in this species and can be to endogenous (low) levels of ethylene, thereby activating restored by adding exogenous ACC to the tissue. the ethylene biosynthetic genes in a defined pattern that That the expression of the biosynthetic genes is posit- results in the eventual wilting of the flower (O'Neill ively regulated by ethylene is clearly shown by the fact et al., 1993). that treatment of flowers with the ACS inhibitor, AVG The first step in the pathway after pollination is the (O'Neill et al, 1993), blocks expression of both genes. In induction of ethylene sensitivity in the flower. This was addition, it is known that ethylene is necessary throughout examined by exposing pollinated flowers to ethylene at the induction period since the exposure of flowers that different times after pollination (Porat et al, 1994). It have been treated with AVG to short pulses of ethylene was found that sensitivity increases after 4 h, rising to a results in a short period of ethylene production which maximum between 8h and 12 h after pollination. That soon ceases. Similar results have been obtained in tomato, this change in sensitivity is independent of endogenous in which it was shown that ACS antisense plants must be ethylene levels was illustrated by exposing pollinated exposed to ethylene for at least 8 h before ripening can flowers to ethylene in the presence of amino-oxyacetic proceed (Oeller et al., 1991). This suggests that ethylene acid, which blocks ethylene biosynthesis (Porat et al., does not act as a simple developmental switch setting a 1994, 1995). Since numerous studies have indicated that cascade in motion, but rather is an inducer whose presence Ca2+ plays an important role in ethylene-mediated pro- is required for long periods of time. Ethylene and plant form 207 O'Neill et al. (1993) proposed a model which accounts for the delay in ethylene production in the perianth and the pattern of ACS and ACO expression (Fig. 2). Pollination induces ethylene sensitivity in cells of the gynoecium which then respond to the endogenous (low) levels of ethylene in this tissue by transcribing the ACS and ACO genes resulting in the burst of ethylene after 8-12 h. This increase in ethylene formation further stimu- lates gene expression and activates transcription of ACO in the perianth in which there is presently no ethylene being made since there is no substrate for ACC oxidase. Some time before 24 h post-fertilization, ACC moves from the gynoecium into the perianth where it is converted into ethylene by the action of ACO, the expression of Downloaded from https://academic.oup.com/jxb/article/48/2/201/652810 by guest on 02 October 2021 which has been activated by the early burst of ethylene from the gynoecium. Ethylene is formed in the perianth until approximately 48 h after fertilization by the action of ACC oxidase on ACC which was synthesized in the gynoecium, which coincides with the onset of senescence of this tissue.

Ethylene plays a key positive regulatory role in the development of pattern in the Arabidopsis root epidermis The root epidermis of Arabidopsis is composed of files of root hair cells spaced by one or more files of non-hair cells (Dolan et al., 1994). Transverse sections through the root reveal that the hair cell files are located above the anticlinal walls of two underlying cortical cells while non-hair epidermal cells are located over a single periclinal cortical cell wall. Roots grown in the presence of ACC, the ethylene precursor, develop hairs in the location normally occupied by non-hair cells. In addition, the formation of root hairs is blocked by AVG which blocks ethylene biosynthesis and by Ag2+ which blocks the perception of ethylene (Tanimoto et al, 1995). These results suggest that epidermal cells overlying cortical periclinal cells can differentiate as root hair cells if exposed to an ethylene signal. This suggests that only cells overly- Fig. 2. Regulation of ethylene biosynthesis during the process of ing the cortical anticlinal walls are exposed to the ethylene perianth wilting in a schematic diagram of an orchid based on that signal during development or that epidermal cells differ observed in Phalaeopsis. (A) The parts of the orchid flower, pi, ventral petal known as the labium or lip. p2 and p3, lateral petals, si, dorsal in their sensitivity to ethylene. sepal. s2 and s3, lateral sepals, pi, p2, p3, si, s2, and s3 constitute the Evidence suggesting that differential ethylene sensi- perianth. In this study pi (the labium was treated independently from tivity may account at least in part, for the pattern of the other perianth segments because of its different physiology and function), c. column, a modified structure characteristic of the orchids cell differentiation comes from experiments in which that is composed of modified anthers and stigmatic organs, st, stigmatic roots were grown in darkness. Dark-grown wild type surface, ov, ovary. (B) Soon after pollination ACS and ACO are (Columbia) produce few root hairs when grown in the induced in the gynoecium resulting in the formation of ethylene. This ethylene then induces expression of the ACO gene in the organs of the absence of physical stimulation to the hypocotyl (L Dolan, perianth (sepals and petals). (C) Later (48-72 h post-pollination) ACC unpublished results). Treatment of roots with 10 ftM ACC which is synthesized in the gynoecium (stigma/ovary) moves to the perianth where it is converted to ethylene by the activity of the ACO results in the development of an abundance of root hairs that was induced in (B). (D) Wilting of the perianth (p2, p3, si, s2. (L Dolan, unpublished results). The majority of root hair and s3) as a result of the induction of a burst of ethylene biosynthesis cells develop in the location where hair cells normally in the sepals and petals between 48 h and 72 h after pollination. develop in the light, i.e. over the anticlinal walls of under- Adapted from data of O'Neill et al. (1993). lying cortical cells. If all epidermal cells were equivalent, it might be expected that hair cells would develop with 208 Dolan equal frequency in either location. This clearly is not the Periodically throughout this review there has been a case. Additional evidence which suggests that differential return to this study to highlight the importance of certain sensitivity accounts, at least in part for the pattern of hair processes to the generation of plant form and the role cell development, comes from analysis of the Ctrl mutant that ethylene plays in these processes. No effort has been phenotypes (Dolan et al., 1994). Plants homozygous for made to be all-encompassing in this review. Such a putative complete loss of function Ctrl alleles develop an detailed treatise would require volumes not pages. The abundance of ectopic root hairs, but not all cells in the works of Matoo and Suttle (1991) and Abeles et al, ectopic locations form root hairs. This suggests that cells (1992) provide such detailed treatments. in each position are responding differently even though The recent advances that have been made in under- the negative regulator, CTR1, is absent, indicating that standing the molecular aspects of the regulation of ethy- differential sensitivity is independent of CTR1 activity. lene biosynthesis and signal transduction has provided While the genetic analysis of cell patterning has not yet not only physiologists, but also plant morphologists, with identified genes regulating the differential sensitivity of a new set of tools with which to determine underlying

the two epidermal cell types to ethylene, it has identified mechanisms in ethylene-related processes. That ethylene Downloaded from https://academic.oup.com/jxb/article/48/2/201/652810 by guest on 02 October 2021 a set of genes that act earlier than ethylene in the pathway. is a key player in the development of form is clear. It is Plants homozygous for loss of function alleles at the TTG also apparent that ethylene is involved in a diverse array anf GL2 loci develop hair cells in all files, suggesting that of seemingly non-related developmental phenomena. Such the wild-type genes are positive regulators of non-hair use of the same molecule in numerous non-related pro- cell fate (Galway et al., 1994; Rerie et al, 1994; L Dolan, cesses within an individual is to be observed in unpublished results). The inhibition of root hair develop- almost all kingdoms. For example, the Notch gene prod- ment in ttg and gl2 backgrounds in the presence of AVG uct of Drosophila is involved in a number of apparently and Ag2+ suggests that plants lacking TTG and GL2 non-related developmental processes, such as the estab- activity still require ethylene to make hair cells. Evidence lishment of embryo polarity, neural cell fate, wing devel- that the TTG and GL2 genes do not regulate differential opment, etc. Ethylene too, is being used by plants in a epidermal ethylene sensitivity comes from experiments in multitude of distinct developmental processes in which which ttg or gl2 plants are grown in the presence of low the gaseous molecule has evolved a role. Future studies concentrations of AVG. Ectopic hair cells that develop that utilize modern technologies to answer age-old ques- in these backgrounds are more sensitive to AVG treatment tions will provide valuable mechanistic insights into the than hairs in the correct location, indicating that cells role of ethylene in the development of plant form. overlying the periclinal walls are less sensitive to ethylene than those overlying cortical anticlinal walls. If GL2 or TTG were to mediate differential sensitivity it might be References expected that AVG treatment of mutant roots would Abel S, Nguyen MD, Chow W, Theologis A. 1995. ASC4, a repress hair development to the same extent in both cell primary indole acetic acid-responsive gene encoding types equally. Identification of mutations that alter the 1-aminocyclopropane-l-carboxylate synthase in Arabidopsis pattern of root hair cell development in the Arabidopsis thaliana. 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