Corrigendum Auxin is required for pollination-induced ovary growth in ...

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Abstract. In Dendrobium and other orchids the ovule becomes mature long after pollination, whereas the ovary starts growing within two days of pollination.
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Corrigendum Auxin is required for pollination-induced ovary growth in Dendrobium orchids Saichol Ketsa, Apinya Wisutiamonkul and Wouter G. van Doorn (Vol. 33, No. 9, pages 887–892) The published paper contained an error on p. 891 left-hand column, lines 35–36. The correct text is: However, ethylene by itself did not promote ovary growth (S Ketsa unpubl. data).

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Functional Plant Biology, 2006, 33, 887–892

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Auxin is required for pollination-induced ovary growth in Dendrobium orchids Saichol KetsaA,C , Apinya WisutiamonkulA and Wouter G. van DoornB A Department

of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand. University and Research Centre, PO Box 17, 6700AA Wageningen, The Netherlands. C Corresponding author. Email: [email protected]

B Wageningen

Abstract. In Dendrobium and other orchids the ovule becomes mature long after pollination, whereas the ovary starts growing within two days of pollination. The signalling pathway that induces rapid ovary growth after pollination has remained elusive. We placed the auxin antagonist α-(p-chlorophenoxy) isobutyric acid (PCIB) or the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) on the stigma, before pollination. Both treatments nullified pollination-induced ovary growth. The ovaries also did not grow after similar stigma treatment with 1-methylcyclopropene (1-MCP), AgNO3 (both inhibitors of ethylene action), aminooxyacetic acid (AOA) or CoCl2 (which both inhibit ethylene synthesis), before pollination. Pollination could be replaced by placement of the auxin naphthylacetic acid (NAA) on the stigma. All mentioned inhibitors nullified the effect of NAA, indicating that if auxin is the initiator of ovary growth, it acts through ethylene. The results suggest that the pollination effect on ovary growth requires auxin (at least auxin transport and maybe also auxin signalling), and both ethylene synthesis and ethylene action. Keywords: auxin, Dendrobium flower, ethylene action, ethylene production, ovary growth.

Introduction Most flowers are ready for fertilisation by the time of flower opening. In orchids, by contrast, the ovules are far from fully differentiated at anthesis. Depending on the orchid species, ovule differentiation only starts after pollination, or if some differentiation has already taken place, it is only finished after pollination (Hildebrand 1863; Heslop-Harrison 1957; O’Neill 1997). In Dendrobium nobile, for example, a clear beginning of ovule differentiation was observed only 2 months after pollination. The embryo sac was present by month four. Five months after pollination some embryos were found, showing that fertilisation had occurred (Hildebrand 1863). A long period between pollination and fertilisation was also observed in Phalaenopsis orchids. The megaspore mother cell had fully developed 2 months after pollination. The embryo sac was mature by month three after pollination, and fertilisation occurred shortly thereafter (Duncan and Curtis 1942; Zhang and O’Neill 1993). A similar difference between orchids and most other flowers is found in ovary development. In most species the

ovary is well developed by the time of flower opening. After fertilisation the ovary then shows further growth owing to the action of the developing seeds (Cox and Swain 2006). In orchids the initial phase of ovary growth starts only after pollination. Once fertilisation has taken place the ovary develops further, just as in other plants. Hildebrand (1863) reported that the ovary of Dendrobium nobile steadily grew in width and length, starting shortly after pollination. The diameter increased from 2 to 20 mm during the first 2 months, and increased further to 25 mm in the next 3 months. In the present paper we focus on the first 7 d of ovary growth in Dendrobium, as a response to pollination. Most orchids produce numerous pollen grains, which are packed into a few pollinia. Whole pollinia, rather than individual pollen, become deposited on the orchid stigma. It has remained unclear how the pollinium, once deposited on the stigma surface, causes rapid ovary growth. Since the ovary is at a distance of a several centimeters from the stigma, a signal is apparently transmitted to the ovary. In Dendrobium nobile, some pollen tubes were present in the developing

Abbreviations used: ACC, 1-aminocyclopropane-1-carboxylic acid; AOA, aminooxyacetic acid; AVG, aminoethoxyvinyl glycine; IAA, indoleacetic acid; 1-MCP, 1-methylcyclopropene; NAA, naphthylacetic acid; NOA, 2-naphthoxyacetic acid; PCIB, α-( p-chlorophenoxy) isobutyric acid; TIBA, 2,3,5-triiodobenzoic acid. © CSIRO 2006

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ovary only by day 20 after pollination (Hildebrand 1863) and in Phalaenopsis not before 14 d after pollination (Zhang and O’Neill 1993). This indicates that at least the first phase of ovary growth is not due to close interaction between the pollen tubes and the ovary cells. The data of Fitting (1909, 1910) on Phalenopsis amabilis indicate that pollination rapidly induced ovary growth, even before pollen germination. Fitting (1909) also showed that an aqueous extract of orchid pollinia, even if extracted with cold water and a short extraction time, placed on the stigma induced various post-pollination responses. This suggested that the effects of pollination were due to one or more chemicals, possibly present in the matrix embedding the pollen. There are at least two candidates for such a chemical factor: 1-aminocyclopropane-1-carboxylic acid (ACC), the direct precursor of ethylene, and auxin. ACC has been found in the pollinia of Dendrobium and other orchids (Ketsa and Luangsuwalai 1996; Porat et al. 1998; Ketsa et al. 2001). Auxin activity was present in the pollinia of numerous orchid species tested (Laibach and Maschmann 1933); unconjugated indoleacetic acid (IAA) was present in orchid pollinia (M¨uller 1953), including those of Dendrobium (Ketsa et al. 2001). As application of auxins such as naphthylacetic acid (NAA) to the stigma of several orchid species induced ovary growth (Arditti et al. 1971; Ketsa and Rugkong 2000; Zhang and O’Neill 1993), it has been suggested that pollination initiates ovary growth through auxin. However, this hypothesis has not been rigorously tested, for example by modulating gene expression, or by applying inhibitors of auxin synthesis, auxin transport, auxin signal transduction or auxin action. The purpose of the present study was to test the hypothesis that auxin is required as a signal for ovary growth. We applied the auxin antagonists α-( p-chlorophenoxy)-isobutyric acid (PCIB) and 2,3,5-triiodobenzoic acid (TIBA) to the stigma, before pollination. The exact mode of action of PCIB is unknown. It apparently impairs the auxin-signalling pathway by regulating Auxin / IAA protein stability (Frenkel and Haard 1973; Oono et al. 2003). TIBA inhibits polar auxin transport. This transport requires specific auxin influx and efflux carriers located on the plasma membrane of transporting cells. TIBA apparently inhibits auxin efflux carrier activity (Al-Hammadi et al. 2003). Materials and methods

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inflorescences were held in 15 mL centrifuge tubes containing 10 mL of distilled water. All flowers were held in a room with natural light (∼12 h per day; ∼15 µmol photons m−2 s−1 ). The temperature and relative humidity of the air-conditioned room were 25◦ C and 80%, respectively. Pollination Flowers were cross-pollinated by placing the pollinia from the open flowers of Dendrobium cv. Pompadour on the stigma of Dendrobium cv. Karen flowers. This was done without removal of the anther cap or removal of the pollinia from the test flowers. Pollen germination and pollen tube development Pollinated flowers were collected at intervals. After the petals had been excised the material was kept, for a few weeks, in FAA solution (formaldehyde 37% : acetic acid : alcohol 1 : 1 : 9 v / v). All four pollinia were removed from flowers kept in FAA and placed on a microscope slide. A few drops of safranin (0.1%) were added. The material was first squeezed with a glass rod and further squeezed with a cover glass, which was pressed on the sample. This procedure results in breaking of the pollinia and removal of most pollen. The preparation was examined under a light microscope. The number of germinated pollen grains was assessed by randomly choosing one field of view, and inspecting the 20 pollen grains closest to the ocular micrometer. To observe whether pollen tubes had left the pollinium, pollinia were left intact and inspected under the microscope. To determine whether pollen tubes entered the stigma, transverse sections through various parts of the stigma were cut by hand and examined by light microscopy. Chemicals Whole inflorescences bearing only open flowers were treated. Chemicals were obtained from Sigma (St Louis, MO), unless otherwise indicated. The chemicals were applied in aqueous solution (water or 50% ethanol) to the stigma surface, using one droplet of solution. Treatments were as follows: aminooxyacetic acid (AOA; 0.3 µmol per flower), CoCl2 (0.1, 0.2 and 0.4 nmol per flower), AgNO3 (0.3 µmol per flower), NAA (0.2 µmol per flower), and 2-naphthoxyacetic acid (NOA) at 0.025, 0.05 and 0.1 µmol per flower. Both PCIB (0.025, 0.05 and 0.1 µmol per flower) and TIBA (0.01, 0.015 and 0.02 µmol per flower) were dissolved in ethanol before mixing with water. Control treatments consisted of placing a drop of water or a drop of water : ethanol 1 : 1 (v / v) on the stigma surface. Pollination was done by hand and took place 24 h after the onset of these treatments. Other inflorescences were placed in a plastic chamber (37 × 47 × 35 cm) at 25◦ C. For the 1-methylcyclopropene (1-MCP) treatment, a quantity of EthylBlocr (Smartfresh, Springhouse, PA) powder was used which provides 500 nL L−1 of 1-MCP gas in the air. The powder was placed in a small glass bottle taped to a wall inside the chamber. 1-MCP gas is produced after contact with water. The chamber lid was sealed and 1 mL water was injected into the glass bottle by passing a hypodermic needle through a septum in the lid. Treatments were conducted at 25◦ C. The chambers remained sealed for 4 h. Control inflorescences were placed in identical chambers, without EthylBlocr . After 4 h the inflorescences were removed and individually placed in 15-mL centrifuge tubes containing distilled water.

Plant material Inflorescences of Dendrobium orchids of the cultivars Pompadour and Karen (also known as Sonia Bom #28) were purchased from a commercial grower near Bangkok. Inflorescences were harvested in the morning with 5–7 open flowers and 4–6 flower buds and were brought to the laboratory within 2 h of harvest. The upper parts of the stem with attached buds were cut off, leaving only five open flowers per inflorescence. Peduncles of individual inflorescences were recut in air, 12 cm from the lowermost open flower. Individual

Ovary diameter The largest diameter of the ovary is present at its distal side, subtending the column. The diameter was measured with an electronic digital caliper (Mitutoyo, Kawasaki, Japan). Statistical analysis In measurements of ovary diameter, five inflorescences were used in each treatment. Mean comparisons were made using Duncan’s

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multiple range test. All experiments were repeated at least once at a later date.

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before pollination. Dosages ranging from 0.01 to 0.02 µmol inhibited ovary growth (Fig. 2A). In a separate experiment ovary growth was nullified by 0.04 µmol TIBA (data not shown).

Time to pollen germination Pollen germination and pollen tube growth were studied in cv. Pompadour. No germinated pollen grains were observed on day one after pollination. About 10% germination was observed on day two. Pollen tubes had left the pollinium from about day four of pollination. By day seven of pollination no pollen tubes were yet observed to enter the stigma surface (data not shown). Effect of PCIB and TIBA on pollination-induced ovary growth In unpollinated Dendrobium flowers the ovary did not grow during the 7-d period of the experiments. After pollination, the ovary diameter had significantly increased by day 1–3, depending on the experiment. The increase in ovary diameter was apparently linear (Fig. 1), or exponential (Fig. 2). The auxin antagonist PCIB was applied to the stigma in aqueous solutions at 0.025 and 0.1 µmol per flower, 24 h before pollination. When applied without pollination it had no effect on ovary diameter (data not shown). When applied before pollination, the treatments considerably reduced ovary growth (Fig. 2B). Other stigmas were treated with aqueous solutions of TIBA, an auxin transport inhibitor,

Effects of ethylene inhibitors on pollination-induced ovary growth We used two inhibitors of ethylene synthesis, AOA and CoCl2 , and two inhibitors of ethylene action, 1-MCP and AgNO3 . AOA is an inhibitor of ACC synthase, while Co2+ inhibits ACC oxidase (Yang and Hoffman 1984). Both 1-MCP and Ag+ block the action of ethylene receptors (Guo and Ecker 2004). Application of 0.3 µmol AOA to the stigma, before pollination, significantly reduced the ovary growth of pollinated flowers (Fig. 3A). Application of 0.4 nmol CoCl2 to the stigma before pollination also significantly reduced ovary growth (Fig. 3B). Similarly, application of

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Days after pollination Fig. 1. Ovary growth of Dendrobium ‘Karen’ flowers, pollinated (䊐) or left unpollinated without ( ) and with 0.2 µmol NAA ( ) or with 0.2 µmol NAA + 0.05 µmol PCIB (䊏). Results are means of 25 flowers ± s.e. Significant differences (P>0.05) on day seven are shown by different letters.





Fig. 2. Ovary growth of Dendrobium ‘Karen’ flowers. (A) Unpollinated ( ) or pollinated with 0 µmol (䊐), 0.01 µmol (), 0.015 µmol (N ) or 0.02 µmol (䊏) TIBA. (B) Unpollinated ( ) or pollinated with 0 µmol (䊐), 0.025 µmol () or 0.1 µmol (䉬) PCIB. Results are means of 25 flowers ± s.e. Significant differences (P>0.05) on day seven are shown by different letters.





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Days after pollination Fig. 3. Effect of ethylene inhibitors, one in combination with the auxin NAA, on ovary growth in Dendrobium ‘Karen’ flowers. (A) Unpollinated ( ) or pollinated without (䊐) or with 0.3 µmol AOA (䊏), or unpollinated with 0.2 µmol NAA ( ) or with 0.2 µmol NAA + 0.3 µmol AOA (N ). (B) Unpollinated ( ) or pollinated with 0 nmol (䊐), 0.1 nmol (), 0.2 nmol (N ) or 0.4 nmol (䊏) CoCl2 . Results are means of 25 flowers ± s.e. Significant differences (P>0.05) on day seven are shown by different letters.







0.3 µmol AgNO3 to the stigma or 1-MCP fumigation of the inflorescences at 500 nL L−1 , before pollination, significantly reduced ovary growth (Fig. 4A). Application of NAA on the stigma of unpollinated flowers Application of the auxin NAA to the stigma surface of unpollinated Dendrobium cv. Karen flowers, at 0.2 µmol per stigma, partially (Fig. 3A) or fully (results not shown) replaced the effect of pollination on ovary growth. Application of PCIB to the stigma before NAA application substantially reduced the NAA-induced ovary growth, relative to treatment with NAA (Fig. 1). Application of AOA to the stigma unpollinated Dendrobium cv. Karen flowers, before NAA treatment, also significantly reduced the effect of NAA on ovary growth (Fig. 3A). Similarly, 1-MCP fumigation before NAA treatment clearly reduced ovary growth (Fig. 4B).

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Days after pollination Fig. 4. Ovary growth of Dendrobium ‘Karen’ flowers. (A) Unpollinated ( ) or pollinated without (䊐) and with 500 nL L−1 1-MCP () or pollinated with 0.3 µmol AgNO3 (N ). (B) Unpollinated ( ), pollinated (䊐), or unpollinated with 0.2 µmol NAA ( ) or with 0.2 µmol NAA + 500 nL L−1 1-MCP (䉬). Results are means of 25 florets ± s.e. Significant differences (P>0.05) on day seven are shown by different letters.







Comparison with Dendrobium cv. Pompadour In order to check whether these results were more generally applicable in Dendrobium we repeated several experiments with cv. Pompadour. Both PCIB and TIBA, tested at the same concentrations as in cv. Karen, inhibited ovary growth in Dendrobium cv. Pompadour. NAA treatment also had a similar effect in pollinated Dendrobium cv. Pompadour as in cv. Karen. We also tested the auxin NOA, which, at the concentrations used, stimulated ovary growth in Dendrobium cv. Pompadour (results not shown). Discussion Ovary growth was stimulated within about 2 d of pollination. This early effect of pollination did not relate to pollen tubes penetrating the ovary. By day two or three the pollen tubes had not even left the pollinia and so had not entered the stigma surface. The ovary growth might be related to pollen germination, which was observed from day two. The effect

Auxin and ovary growth in Dendrobium orchids

might therefore be due to a combination of the presence of the pollinia on the stigma and the emergence of the first pollen tubes. The results provide evidence in favour of the hypothesis that auxin is required for pollination-induced ovary growth in Dendrobium. The hypothesis is supported by the effects of the two auxin antagonists (PCIB and TIBA), which both prevented ovary growth if applied to the stigma before pollination. If NAA, an auxin, was applied to the stigma it induced ovary growth, an effect that was nullified by the two auxin antagonists used. This strongly suggests that the PCIB and TIBA did act as auxin antagonists in the system investigated, rather than acting through some other mechanism. Inhibitors of ethylene synthesis (AOA and cobalt chloride) and inhibitors of ethylene action (1-MCP and silver nitrate), applied to the stigma, also fully abolished pollination-induced ovary growth. This means that both ethylene action and ethylene synthesis are required for pollination-induced ovary growth. Since AOA is an antagonist of ACC synthase and cobalt chloride inhibits ACC oxidase, the data indicate requirement of at least the two last enzymes in the ethylene synthesis pathway. The effect of 1-MCP and silver nitrate indicate that ethylene perception is necessary. As outlined in the Results section, 1-MCP and silver ions are antagonists of the ethylene receptor proteins. A combination of auxin and ethylene is apparently required for ovary growth. NAA, if applied to the stigma, was adequate to promote ovary growth. When NAA application was combined with treatment with inhibitors of ethylene synthesis or ethylene action, its effect on ovary growth was nullified. This indicates that the effect of NAA on ovary growth is through both ethylene production and ethylene action. Auxins often act through ethylene (Abeles et al. 1992). However, auxin by itself did not promote ovary growth (S Ketsa unpubl. data). This suggests that a combination of auxin and ethylene is required. The data regarding ethylene requirement for ovary growth are similar to those in Phalaenopsis. ACC is present in Phalaenopsis pollinia (Porat et al. 1998). Application of aminoethoxyvinyl glycine (AVG), which blocks ethylene synthesis, inhibited (but did not prevent) pollination-induced ovary growth in Phalaenopsis (Zhang and O’Neill 1993). Whether Phalaenopsis ovary growth requires auxin has not been tested. The pollinia of Phalaenopsis had high auxinlike activity in the Avena bioassay (Laibach and Maschmann 1933), and application of NAA on the stigma of Phalaenopsis fully substituted for pollination with regard to ovary growth (Zhang and O’Neill 1993). It is not clear where the requirements for auxin and ethylene in stimulating pollination-induced ovary growth are located. It might relate, for example, to cells close to the stigma surface, those of any other part between the stigma and the ovary, and / or those in the ovary. Auxin

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might be at least one of the mobile factors that transfer the pollination signal from the stigma to the ovary. Auxin flow from the stigma to the ovary follows from application of 14 C-IAA in the orchid Angraecum. Considerable quantities of 14 C-auxin were found in the ovary, within 24 h of application (Strauss and Arditti 1982). Little label was found in the petals, indicating preferential flow to the ovary (Strauss and Arditti 1982). If such auxin transport from the pollinia to the ovary also occurs in Dendrobium, adequate auxin levels might be present, early on, in the ovary. This hypothesis would be consistent with the finding that auxin is adequate to induce all the steps necessary for complete ovule differentiation in an orchid species (Heslop-Harrison 1957). ACC has also been suggested to be mobile (Tudela and Primo-Millo 1992), but a role for ACC as mobile factor in inducing ovary growth is less clear than that of auxin. ACC application to the stigma did not induce ovary growth in Dendrobium (Ketsa et al. 2001) or in Phalaenopsis (O’Neill et al. 1993). The following picture emerges from the present tests: Auxin is required for the rapid, pollination-induced ovary growth in Dendrobium orchids. Auxin requirement at least involves auxin signalling and, depending on the precise mode of action of PCIB, probably auxin transport. Ethylene is also required. As the action of NAA on ovary growth depended on ethylene synthesis and action, the effect of auxin on ovary growth seems to involve a causal chain in which ethylene synthesis and action is necessary. Acknowledgments The research was financially supported by the Thailand Research Fund (TRF). References Abeles FB, Morgan PW, Saltveit ME (1992) ‘Ethylene in plant biology.’ (Academic Press: New York) Al-Hammadi ASA, Sreelakshmi Y, Negi S, Siddiqi I, Sharma R (2003) The polycotyledon mutant of tomato shows enhanced polar auxin transport. Plant Physiology 133, 113–125. doi: 10.1104/ pp.103.025478 Arditti J, Jeffrey DC, Flick BH (1971) Post-pollination phenomena in orchid flowers. III. Effect and interactions of auxin, kinetin or gibberellin. New Phytologist 70, 1125–1141. doi: 10.1111/j.14698137.1971.tb04595.x Cox CM, Swain SM (2006) Localised and non-localised promotion of fruit development by seeds in Arabidopsis. Functional Plant Biology 33, 1–8. doi: 10.1071/FP05136 Duncan RE, Curtis JT (1942) Intermittent growth of fruits of Phalaenopsis. A correlation of the growth phases of an orchid fruit with internal development. Bulletin of the Torrey Botanical Club 69, 167–183. doi: 10.2307/2481654 Fitting H (1909) Die Beeinflussung der Orchideenbl¨uten durch die Best¨aubung und durch andere Umst¨ande. Zeitschrift f¨ur Botanik 1, 1–86. Fitting H (1910) Weitere entwicklungsphysiologische Untersuchungen an Orchideenbl¨uten. Zeitschrift f¨ur Botanik 2, 225–267.

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Frenkel C, Haard NF (1973) Initiation of ripening in Bartlett pears with an antiauxin α-( p-chlorophenoxy) isobutyric acid. Plant Physiology 52, 380–384. Guo H, Ecker JR (2004) The ethylene signaling pathway: new insights. Current Opinion in Plant Biology 7, 40–49. doi: 10.1016/ j.pbi.2003.11.011 Heslop-Harrison J (1957) The physiology of reproduction in Dactylorchis. I. Auxin and the control of meiosis, ovule formation and ovary growth. Botaniska Notiser 110, 28–50. Hildebrand F (1863) Die Fruchtbildung der Orchideen, ein Beweis f¨ur die doppelte Wirkung des Pollens. Botanische Zeitung 21, 329–333. Ketsa S, Luangsuwalai K (1996) The relationship between 1-aminocyclopropane-1-carboxylic acid content in pollinia, ethylene production and senescence of pollinated Dendrobium orchid flowers. Postharvest Biology and Technology 8, 57–64. doi: 10.1016/0925-5214(95)00053-4 Ketsa S, Rugkong A (2000) The role of ethylene in enhancing the initial ovary growth of Dendrobium ‘Pompadour’ following pollination. Journal of Horticultural Science & Biotechnology 75, 451–454. Ketsa S, Bunya-atichart K, van Doorn WG (2001) Ethylene production and post-pollination development in Dendrobium flowers treated with foreign pollen. Australian Journal of Plant Physiology 28, 409–415. ¨ Laibach F, Maschmann E (1933) Uber den Wuchsstoff der Orchideenpollinien. Jahrb¨ucher f¨ur wissenschaftliche Botanik 78, 399–430. M¨uller R (1953) Zur quantitativen Bestimmung von Indolylessigs¨aure mittels Papierchromatographie und Papierelektrophorese. Beitr¨age zur Biologie der Pflanzen 30, 1–32.

O’Neill S (1997) Pollination regulation of flower development. Annual Review of Plant Physiology and Plant Molecular Biology 48, 547–574. doi: 10.1146/annurev.arplant.48.1.547 O’Neill S, Nadeau JA, Zhang XS, Bui AQ, Halevy AH (1993) Interorgan regulation of ethylene biosynthetic genes by pollination. The Plant Cell 5, 419–432. doi: 10.1105/tpc.5.4.419 Oono Y, Chiharu O, Rahman A, Aspuria ET, Hayashi K, Tanaka A, Uchimiya H (2003) p-Chlorophenoxyisobutyric acid impairs auxin response in Arabidopsis root. Plant Physiology 133, 1135–1147. doi: 10.1104/pp.103.027847 Porat R, Nadeau JA, Kirby JA, Sutter EG, O’Neill SD (1998) Characterization of the primary pollen signal in the postpollination syndrome of Phalaenopsis flowers. Plant Growth Regulation 24, 109–117. doi: 10.1023/A:1005964711229 Strauss MS, Arditti J (1982) Postpollination phenomena in orchid flowers X. Transport and fate of auxin. Botanical Gazette 143, 286–293. doi: 10.1086/337302 Tudela D, Primo-Millo E (1992) 1-Aminocyclopropane-1-carboxylic acid transported from roots to shoots promotes leaf abscission in cleopatra mandarin (Citrus reshni Hort, ex tan.) seedlings rehydrated after water stress. Plant Physiology 100, 131–137. Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Annual Review of Plant Physiololgy 35, 155–189. Zhang XS, O’Neill SD (1993) Ovary and gametophyte development are coordinately regulated following pollination by auxin and ethylene. The Plant Cell 5, 403–418. doi: 10.1105/tpc.5.4.403

Manuscript received 15 February 2006, accepted 18 May 2006

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