JXB Advance Access originally published online on May 31, 2005
Journal of Experimental Botany 2005 56(417):1867-1875; doi:10.1093/jxb/eri176
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
G-protein-coupled 
2A-adrenoreceptor agonists differentially alter citrus leaf and fruit abscission by affecting expression of ACC synthase and ACC oxidase
University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA
* To whom correspondence should be addressed. Fax: +1 863 956 4631. E-mail: jkbu{at}ufl.edu
Received 5 November 2005; Accepted 31 March 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Temporal and spatial expression patterns of genes encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS1 and ACS2) and ACC oxidase (ACO), ACC concentration, and ethylene production in leaves and fruit of ‘Valencia’ orange (Citrus sinensis [L.] Osbeck) were examined in relation to differential abscission after treatment with 2-chloroethylphosphonic acid (ethephon) alone or in combination with guanfacine or clonidine, two G-protein-coupled
2A-adrenoreceptor selective agonists. Guanfacine and clonidine markedly reduced ethephon-enhanced leaf abscission, but had little effect on ethephon-enhanced fruit loosening. Ethephon-enhanced fruit and leaf ethylene production, and ACC concentration in fruit abscission zones, fruit peel, leaf abscission zones, and leaf blades were decreased by guanfacine. Guanfacine reduced ethephon-enhanced expression of ACS1 and ACO genes in leaf abscission zones and blades, but to a lesser extent in fruit abscission zones. The expression pattern of the ACS2 gene, however, was not associated with abscission. The results demonstrate that differential expression of ACS1 and ACO genes is associated with reduction of ethephon-enhanced leaf abscission by guanfacine, and suggest a link between G-protein-related signalling and abscission. Key words: 1-Aminocyclopropane-1-carboxylic acid, clonidine, ethephon, ethylene, guanfacine
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Abscission is a developmentally regulated, complex, and genetically programmed process whereby multicellular organs such as leaves, flowers, or fruit become detached from the parent plant body (Osborne, 1989
; Bleecker and Patterson, 1997; Taylor and Whitelaw, 2001; Roberts et al., 2002). Abscission occurs at predetermined sites referred to as abscission zones, which consist of a few layers of small, densely packed cells that respond in different ways from neighbouring cells to the same hormonal or environmental cues (Osborne, 1989; Roberts et al., 2002). Once abscission is initiated, cells in the abscission zone begin to enlarge, followed by increased expression of genes and the activity of enzymes associated with cell wall degradation such as ß-1,4-glucanase and polygalacturonase (Tucker et al., 1988; Brown, 1997; Bonghi et al., 2000; Roberts et al., 2002). As a result, the middle lamellae of abscission zone cells dissolve and, ultimately, the organ abscises. Other genes such as pathogenesis-related genes and those involved in secondary metabolism and signal transduction are also up-regulated during the abscission process (Burns, 2002; Roberts et al., 2002; Wu and Burns, 2003, 2004). Thus, once initiated, downstream events that ultimately lead to abscission are similar among diverse plants.
The presence and balance of plant hormones have been shown to affect abscission of leaves, flowers, and immature and mature fruit (Osborne, 1989; Brown, 1997; Roberts et al., 2002; Yuan et al., 2003). Developmental cues such as those occurring during fruit ripening, or stresses including drought, temperature extremes, wounding, chemical exposure, and pathogen attack promote abscission by decreasing abscission-inhibiting hormones such as auxin and cytokinin and increasing abscission-accelerating hormones such as ethylene and abscisic acid (Osborne, 1989; Taylor and Whitelaw, 2001; Yuan et al., 2003). In general, the application of ethylene or ethylene-releasing compounds such as ethephon promotes abscission, whereas ethylene biosynthesis or perception inhibitors prevent or delay abscission (Abeles et al., 1992; Brown, 1997). Although recent work with ethylene-insensitive and delayed abscission mutants of Arabidopsis demonstrated that ethylene does not initiate abscission (Bleecker and Patterson, 1997; Patterson and Bleecker, 2004), ethylene has an important role in accelerating leaf and fruit abscission once abscission is initiated (Brown, 1997; Roberts et al., 2002; Taylor and Whitelaw, 2001). The factor(s) that initiates and controls differential removal of an abscising organ from a plant remains unknown.
Recently, [(2,6-dichlorophenyl)acetyl] guanidine (guanfacine) and 2-[(2,6-dichlorophenyl)amino]-2-imidazoline (clonidine), pharmacological compounds known as 2A-adrenoreceptor selective agonists in animals, were reported to reduce leaf abscission caused by ethephon, without significantly affecting the ability of ethephon to promote mature fruit abscission in citrus [Citrus sinensis (L.) Osbeck] (Burns et al., 2003). Guanfacine was shown to be more effective than clonidine in reducing ethephon-enhanced leaf abscission. The physiological and molecular mechanisms by which citrus mature fruit and leaves differentially respond to the combination of ethephon and guanfacine or clonidine are unknown. Guanfacine is unlikely to be an ethylene perception inhibitor, because in the presence of ethylene, the triple response was observed in wild-type Arabidopsis seedlings germinated in guanfacine. Adventitious root length and number were increased in tomato and coleus cuttings treated with either agonist, suggesting that the compounds had an auxin-like effect. Guanfacine and clonidine can alter neurotransmission by activating heterotrimeric G-protein-coupled 2A-adrenoreceptors in animals (Feldman et al., 1997; Song et al., 2004). In plants, pharmacological studies have shown that heterotrimeric G-proteins play an important role in various signal transduction pathways, such as for auxin, abscisic acid, gibberellin, blue light, red light, and pathogen responses (Assmann, 2002). G-proteins are also involved in the regulation of stomatal apertures and activation of a plasma membrane Ca2+ channel. Genes encoding G-protein and ß subunits have been isolated and characterized in several dicots and monocots including Arabidopsis, tomato, rice, and tobacco.
In this study, the basis of the differential abscission response associated with ethephon was examined in combination with G-protein-coupled 2A-adrenoreceptor selective agonists, guanfacine and clonidine, by focusing on components of ethylene biosynthesis in leaves and mature fruit. The conversions of SAM to ACC and ACC to ethylene are the rate-limiting steps in ethylene biosynthesis, and are catalysed by ACS and ACO, respectively (Alexander and Grierson, 2002; Wang et al., 2002). Genes encoding ACS and ACO are members of multigene families, and their expression is differentially regulated by a variety of biotic and abiotic factors (Kende, 1993; Wang et al., 2002). In this work, two ACS genes, ACS1 and ACS2, and one ACO gene, ACO, were isolated from citrus and their expression and products of their encoded enzymes were examined in various leaf and fruit tissues after application of ethephon alone or in combination with guanfacine or clonidine. The results show that differential expression of genes encoding ACS1 and ACO in fruit abscission zones and leaf abscission zones is associated with selective reduction of ethephon-enhanced leaf abscission by guanfacine. The effect of guanfacine and clonidine suggest a link between G-protein-related signalling and abscission.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and treatments
Twenty-eight uniform 12-year-old ‘Valencia’ orange trees grafted on rough lemon (Citrus jambhiri Lush) rootstock were selected from a grove located at the Citrus Research and Education Center, Lake Alfred, FL, USA, and blocked into four groups of seven trees each during the 2001 and 2002 growing seasons. A randomized complete block design with four replications was used. One tree in each block was sprayed with 2 mM guanfacine (Tocris, Ellisville, MO, USA), 2 mM clonidine (Sigma Chemical, St Louis, MO, USA), 200 mg l–1 ethephon (as Ethrel, Aventis Crop Science, Research Triangle Park, NC, USA) alone or in combination with 2 mM guanfacine or clonidine, and 200 mg l–1 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP), a selective abscission agent used as a positive control. One tree in each block was sprayed with water and served as a negative control. Kinetic (Setre Chemical Co., Memphis, TN, USA) was included in all solutions as an adjuvant at 0.125% to allow compound dispersion. Solutions were applied to the canopy until run-off.
Determination of ethylene evolution, leaf abscission and fruit detachment force
Ethylene evolution of mature fruit and leaves in situ, fruit detachment force (FDF), and leaf abscission was determined 0, 24, 48, 72, 96, and 168 h after treatment as described in Yuan and Burns (2004). Briefly, fruit were enclosed in a 0.87 l Rubbermaid plastic container and maintained on the tree. At each sampling time, containers with fruit inside were closed with the plastic lid, and incubated for 2 h. One millilitre of gas sample was withdrawn from the sealed container through the rubber septum affixed to the lid, and ethylene concentration was measured with a gas chromatograph (Hewlett-Packard, Avondale, PA, USA). For the measurement of leaf ethylene evolution, two leaves were selected from each tree, and the petiole of each leaf was carefully slipped through a slit into the hole (5 mm in diameter) of a rubber stopper. The open spaces between the petiole and the rubber stopper were sealed with non-phytotoxic 3145 Mil-A-46146 RTV adhesive/sealant (Dow Corning Co., Midland, MI, USA). Each leaf was enclosed in a 60 ml syringe by inserting the rubber stopper into the end of the syringe and affixing a rubber septum to the head of syringe, and incubated for the times indicated. One millilitre gas sample was withdrawn from the sealed syringe through the rubber septum after 2 h of container closure, and ethylene concentrations were measured as described above. FDF, a force (Newton) necessary to separate the fruit from the parent plant at the abscission zone site, was measured on 30 randomly selected fruit/tree using a digital force gauge at the times indicated (Force Five, Wagner Instruments, Greenwich, CT, USA). Mature fruit with stems attached were clipped about 2.5 cm above the fruit abscission zone, inserted into the gauge, and the stem pulled parallel to the fruit axis until it separated from the fruit.
Measurement of ACC concentration
Mature fruit and leaves were collected from each tree 0, 24, 48, and 72 h after treatment and were immediately separated into peel, fruit abscission zone, leaf blade, and leaf abscission zone tissues. Fruit abscission zones were removed using a sharpened 4 mm diameter cork borer. The borer was slipped over the pedicel and pushed through the calyx and fruit peel. The location of the fruit abscission zone was visually determined in this cylinder of tissue and further trimmed to 6 mm in length by 4 mm in width using a razor blade. Leaf abscission zones at the petiole/blade junction were collected by cutting 2 mm at each side of the abscission fracture plane. Leaf blade tissues were removed from mid-blade and did not include midrib tissues. Harvested tissues were immediately frozen in liquid nitrogen and stored at –80 °C for future use.
The method of Wong et al. (1999) was modified to extract plant material for ACC. In brief, plant tissue was ground to a fine powder in liquid nitrogen using a prechilled mortar and pestle. The powdered tissue was transferred to a centrifuge tube and 10 ml of 80% ethanol was added. The homogenate was centrifuged at 10 000 g for 30 min after incubating the powdered tissue in ethanol at 65 °C for 15 min. The residue was re-extracted in 10 ml of 80% ethanol at 65 °C for 15 min. The supernatants were combined and dried under vacuum. The dry pellet was dissolved in 1 ml of water and extracted once with an equal volume of chloroform. The aqueous phase was collected by centrifugation, dried under vacuum, and redissolved in 0.7 ml of water. ACC was quantified according to Lizada and Yang (1979). A known amount of ACC was added as internal standard to plant extracts to determine the efficiency of conversion of ACC to ethylene.
Total RNA extraction and construction of ACS1, ACS2, and ACO gene-specific probes
Total RNA was extracted from tissues treated with test compounds and collected as described above using the phenol/SDS method as described by Wu and Burns (2003). Concentration and purity of total RNA were determined by spectrophotometry. Two citrus ACC synthase genes (ACS1 and ACS2) and one ACC oxidase gene (ACO) were previously isolated and characterized (Wong et al., 1999; Burns, 2002; Katz et al., 2004). Based on the sequences of citrus ACS1 (accession numbers AJ011095 and AJ012696) and ACS2 (accession numbers AJ276295 and AJ012551), gene-specific primer pairs were designed as follows: ACS1, ACS1L: 5'-GCTTTTCTTGTGCCTTCACC-3' and ACS1R: 5'-GAGAGGCGACTGGGGAAT-3'; ACS2, ACS2L: 5'-TCGCGTTGAGTAACCAAGTG-3' and ACS2R: 5'-CACCTTGGCTTCCACATTCT-3'. Total RNA extracted from mature fruit abscission zones or peel 48 h after CMNP application was used to synthesize first-strand cDNA using SuperScript II RNase H-Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. First-strand cDNA was used as template for PCR and products were viewed on a 1.0% agarose gel stained with ethidium bromide, cleaned using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA), cloned into pGEMT-easy vector (Promega, Madison, WI, USA), and sequenced at the DNA sequencing core laboratory, University of Florida, Gainesville. Citrus ACO was cloned and sequenced previously (Burns, 2002). 32P dATP-labelled DNA probes for ACS1, ACS2, and ACO were generated using a Random Primed StripAbleTM DNA Probe Synthesis and Removal Kit (Ambion Inc., Austin, TX, USA).
Northern blot analysis
Ten micrograms of total RNA were separated by electrophoresis in a 1.2% (w/v) formaldehyde-containing agarose gel and blotted overnight onto a positively charged nylon membrane (HybondTM-N+; Amersham, USA) using the capillary Rapid Downward Transfer system (Schleicher & Schuell, USA). After RNA was cross-linked onto the membrane using a Stratalinker UV Crosslinker (Stratagene, La Jolla, CA, USA), the blot was prehybridized in PerfectHybTM Plus hybridization buffer (Sigma, St Louis, MO, USA) at 65 °C for at least 2 h and then hybridized with 32P dATP-labelled DNA probes for ACS1, ACS2, or ACO overnight at 65 °C. After hybridization, the blot was washed once in low stringency wash buffer (2x SSC, 0.1% SDS) at room temperature for 5 min, and twice in high stringency wash buffer (0.5x SSC, 0.1% SDS) at 65 °C for 20 min. The blot was exposed to BioMax MS Film (Eastman Kodak, NY, USA) using a BioMax transcreen HE intensifying screen overnight at –80 °C. Northern blots were repeated with two biological replicates with similar results.
Effect of guanfacine and AVG on ethylene evolution and abscission caused by ethephon
To investigate whether ethylene biosynthesis was involved in mature fruit and leaf abscission caused by ethephon application, 16 uniform ‘Calamondin’ (Citrus madurensis) trees in 12 l pots were selected and blocked into four groups of four trees each. A randomized complete block design with four replications was used. Combinations of ethephon at 0 or 400 mg l–1 and aminoethoxyvinylglycine (AVG), an ethylene biosynthesis inhibitor, at 0 or 2.0 mM were applied to one tree from each block in November 2002. Ethephon at 400 mg l–1 was used because ‘Calamondin’ trees are less sensitive than ‘Valencia’ orange trees to ethephon (Burns et al., 2003). Ethephon was applied approximately 1 h after application of AVG. Leaves and mature fruit were dry when ethephon was applied. Kinetic at 0.125% was included in all solutions as an adjuvant. Solutions were applied to the canopy until run-off. Similarly, combinations of ethephon at 0 or 400 mg l–1 and guanfacine at 0 or 2.0 mM were applied to four of the 16 ‘Calamondin’ trees. Ethylene evolution of mature fruit and leaves in situ was determined 48 h after treatment as described previously. Cumulative leaf abscission and FDF were determined 168 h after treatment.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Effect of ethephon, clonidine, guanfacine, and CMNP on ethylene evolution and abscission
Ethephon reduced FDF and increased fruit ethylene evolution of ‘Valencia’ oranges (Fig. 1A, B). Addition of guanfacine or clonidine reduced ethephon-enhanced fruit ethylene evolution but had minimal effect on the ability of ethephon to reduce FDF. Clonidine had less effect in reducing ethephon-enhanced fruit ethylene evolution than guanfacine. Ethephon markedly enhanced leaf ethylene evolution and caused approximately 80% leaf abscission over a 7 d period, whereas ethephon-enhanced leaf ethylene evolution and leaf abscission were markedly reduced by the addition of guanfacine or clonidine (Fig. 1C, D). Ethylene was reduced approximately 32% by the addition of guanfacine in mature fruit, whereas 74% of the ethylene measured was decreased by the addition of guanfacine in leaves. The mature fruit selective abscission agent CMNP markedly enhanced fruit ethylene evolution and reduced FDF, but had no effect on leaf ethylene evolution and leaf abscission. Two additional
2A-adrenoreceptor selective agonists, guanabenz and UK14,304, also reduced ethephon-associated leaf abscission (data not shown); however, these compounds had lower water solubility than guanfacine and clonidine.
|
Effect of ethephon, guanfacine, and CMNP on ACC concentration
ACC concentration in fruit abscission zones, leaf abscission zones, fruit peel, and leaf blades was also examined following treatment applications. ACC concentration in fruit and leaf abscission zones was increased by ethephon treatment, and the addition of guanfacine reduced ACC concentrations by approximately 30% and 61% in fruit abscission zones and leaf abscission zones, respectively (Fig. 2A, B). Similarly, ethephon-enhanced ACC concentration was also decreased in fruit peel and leaf blades by addition of guanfacine (Fig. 2C, D). CMNP markedly enhanced ACC concentration in fruit abscission zones and fruit peel, but had no or minimal effect on ACC concentration in leaf abscission zones and leaf blades.
|
Temporal and spatial expression of ACS and ACO genes after application of ethephon, guanfacine and CMNP
ACS1 gene expression:
The expression of genes involved in ethylene biosynthesis in fruit abscission zones, leaf abscission zones, fruit peel, and leaf blades was investigated in response to ethephon, guanfacine, and CMNP (Fig. 3). Northern blot analysis demonstrated that ACS1 mRNA was increased in both fruit abscission zones and leaf abscission zones by ethephon. However, ethephon-enhanced ACS1 expression was suppressed by guanfacine in leaf abscission zones, but only slightly reduced in fruit abscission zones. Ethephon also significantly enhanced ACS1 mRNA levels in leaf blades, but had no effect on ACS1 mRNA levels in fruit peel. Ethephon-enhanced ACS1 accumulation in leaf blades was markedly suppressed by the addition of guanfacine. CMNP increased ACS1 mRNA levels in fruit abscission zones and fruit peel but not in leaf abscission zones and leaf blades.
|
ACS2 gene expression:
The pattern of transcript accumulation for ACS2 was different from ACS1 in the tissues examined. ACS2 appeared to be induced by the adjuvant used in fruit tissue-associated controls, especially 48 h and 72 h after application. Interestingly, ACS2 expression was suppressed by ethephon, but not by guanfacine alone at these times in fruit abscission zones and after 24 h in peel. No consistent trend in ACS2 expression was seen in leaf abscission zones or in leaf blades with ethephon or guanfacine. CMNP had no effect on ACS2 expression in fruit abscission zones, but increased in fruit peel. CMNP increased levels of ACS2 mRNA in leaf abscission zones but not in leaf blades.
ACO gene expression:
Gene expression of ACO was increased in fruit abscission zones and leaf abscission zones by ethephon, but the addition of guanfacine inconsistently affected ACO expression in fruit abscission zones. By contrast, the addition of guanfacine effectively reduced ethephon-enhanced ACO gene expression in leaf abscission zones. In fruit peel and leaf blades, ACO gene expression was enhanced by ethephon application. The addition of guanfacine markedly decreased ethephon-enhanced ACO transcript accumulation in leaf blades, but not in fruit peel. ACO expression was increased in fruit abscission zones and fruit peel by CMNP. However, CMNP had little or no influence on ACO expression in leaf abscission zones and leaf blades.
Effect of AVG and guanfacine on ethylene evolution and abscission caused by ethephon
The addition of AVG decreased the ability of ethephon to reduce FDF and reduced leaf abscission in ‘Calamondin’ trees, whereas the addition of guanfacine markedly reduced ethephon-induced leaf abscission as in ‘Valencia’ trees, but had a minimal effect on the ability of ethephon to reduce FDF (data not shown). Both AVG and guanfacine reduced ethephon-induced ethylene evolution in mature fruit and leaves of ‘Calamondin’ trees (Table 1). In the AVG experiment, the ethylene measured in fruit and leaves inhibited by the addition of AVG (52% and 59%, respectively) reflected ethylene synthesized by the tissues as a result of ethephon. Conversely, the portion in both tissues increased by ethephon, but was not inhibited by the addition of AVG (36–37%), originated from the breakdown of ethephon. In the guanfacine experiment, inhibition of ethylene production by the addition of guanfacine was comparable to that by the addition of AVG in leaves of ‘Calamondin’ trees. Thus, the ethylene produced (53%) that was inhibited by addition of guanfacine probably reflected ethylene synthesized by this tissue as a result of ethephon treatment. Ethylene production that was increased by ethephon, but not inhibited by the addition of guanfacine (39%), was possibly from the breakdown of ethephon. However, the percentage of ethylene in fruit (28%), evolved as a result of ethephon-induced biosynthesis, was markedly less than that of leaves and ethylene originating from the breakdown of ethephon was greater (63%). The possibility cannot be ruled out that a portion of this ethylene was derived from ethephon-induced biosynthesis, since this study's results showed that guanfacine only partially reduced expression of ACO in mature ‘Valencia’ orange fruit (Fig. 3). Treatment of ‘Valencia’ with ethephon+guanfacine resulted in similar trends; namely, ethylene production was more effectively inhibited in leaves than fruit. The higher percentage of ethylene that was inhibited by the addition of guanfacine in leaves of ‘Valencia’ than in ‘Calamondin’ may be attributed to a difference in temperature at the time of application and to the sensitivity of the variety. A small portion of the ethylene measured in both citrus types was not inhibited by AVG or guanfacine alone (3–16%) and probably reflected a basal level of ethylene production.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ethylene plays a regulatory role in mediating the abscission response to various stresses in many higher plants including citrus (Brown, 1997
; Bonghi et al., 2000; Taylor and Whitelaw, 2001; Roberts et al., 2002). However, little information is available about the basis of differential abscission response of fruit and leaves to biotic or abiotic stresses. In this study, treatments that resulted in the differential abscission of mature fruit and leaves had a differential effect on ACC accumulation and ethylene evolution. ACC accumulated and ethylene production increased in tissues or organs that abscised such as leaves and fruit treated with ethephon. On the other hand, abscission failed to occur in tissues where treatments had little or no effect on ACC accumulation and ethylene evolution, such as leaves treated with ethephon+guanfacine. These results suggest that reduction of ethephon-enhanced abscission by guanfacine is associated with ethylene biosynthesis in citrus.
When absorbed by plants, ethephon breaks down into ethylene, hydrochloric acid, and phosphate (Warner and Leopold, 1969). Ethylene derived from the decomposition of ethephon is thought to accelerate abscission; however, phosphate alone, but not hydrochloric acid, can also accelerate abscission by promoting ethylene biosynthesis of mature fruit and leaves in olives (Olea europaea L.) and citrus (Banno et al., 1993; Burnik-Tiefengraber et al., 1994; Goren et al., 1998). Involvement of ethylene biosynthesis in ethephon-induced abscission was supported by the fact that AVG, an inhibitor of ethylene biosynthesis, comparably reduced ethephon-induced ethylene evolution and abscission in both citrus leaves and mature fruit. The results also demonstrated that guanfacine markedly reduced leaf ethylene evolution by largely inhibiting ethephon-induced ethylene biosynthesis. Ethephon-induced ethylene production in fruit was less affected by guanfacine, and more ethylene was presumably derived from ethylene biosynthesis. Guanfacine was more effective in reducing ethephon-induced abscission in leaves than in fruit. This differential effect of guanfacine on ethylene evolution and abscission in mature fruit and leaves is unlikely to have resulted from differential penetration of guanfacine in these two tissues because the authors' unpublished work indicated that guanfacine reduced expression of the gene encoding the G-protein ß-subunit in mature fruit but not in leaves, suggesting that guanfacine does penetrate the fruit cuticle. Similarly, 1-methylcyclopropene, a readily diffusible ethylene binding inhibitor, reduced ethephon-associated leaf abscission, but was unable to alter the ability of ethephon to reduce FDF (Pozo et al., 2004).
Regulation of ethylene biosynthesis by various stresses and endogenous signals is mainly through the differential expression of ACC synthase and ACC oxidase (Kende, 1993; Tang et al., 1994; Zarembinski and Theologis, 1994; Wang et al., 2002; Nakano et al., 2003). Although citrus fruit are non-climacteric and have low basal rates of ethylene production (system I), mature fruit can produce an autocatalytic burst of system II-like ethylene (Katz et al., 2004). ACS1 is mainly involved in system II-like ethylene biosynthesis whereas ACS2 is constitutively expressed and involved in system I ethylene production in citrus. In this study, increased expression of ACS1 and ACO, but not ACS2, in mature fruit and leaf abscission zones was associated with ethephon-induced abscission, whereas the addition of guanfacine suppressed ethephon-enhanced accumulation of ACS1 and ACO transcripts in leaf abscission zones or leaf blades but to a lesser extent in mature fruit abscission zones. This suggests that guanfacine is involved in regulation of abscission through differential suppression of ACS1 and ACO transcript accumulation in leaf and mature fruit abscission zones. By contrast with the measured increase in ACS1 and ACO gene expression in this work, Katz et al. (2004) reported that expression of ACS1 and ACO in mature fruit was not affected by ethylene or propylene. This discrepancy could be due to the stimulatory effect of breakdown products of ethephon other than ethylene (Warner and Leopold, 1969; Banno et al., 1993; Burnik-Tiefengraber et al., 1994; Goren et al., 1998).
Since ethylene derived from ethephon, and guanfacine or clonidine are different in molecular structure and bind to apparently different receptors [ethylene acting upon a receptor histidine kinase (Wang et al., 2002) and guanfacine or clonidine on a G-protein coupled 7-transmembrane receptor (Assmann, 2002)], it is unlikely that they share the same site of action. Unlike the ethylene binding and perception inhibitor 1-MCP, guanfacine did not eliminate the Arabidopsis triple response in germinating seedlings or prevent wilting of Petunia flower petals in the presence of ethylene (Burns et al., 2003), suggesting that ethylene receptors are not a direct binding target. Further work will be necessary to determine if an adrenoreceptor-like receptor and natural ligand(s) exist.
The mode of action of guanfacine or clonidine has been extensively researched in animals but not in plants. Guanfacine and clonidine are pharmacological compounds belonging to the antihypertensive class of drugs (Feldman et al., 1997; Scahill et al., 2001) that act as selective agonists of 2A-subtype of G-protein-coupled adrenoreceptors (Duman and Nestler, 1995; Song et al., 2004). As a result of interaction between G-protein-coupled 2A-adrenoreceptors and the agonists, release of second messengers such as cyclic AMP, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca2+ (Duman and Nestler, 1995; Munnik et al., 1995; Song et al., 2004) can lead to cycles of phosphorylation and dephosphorylation of intracellular proteins, thereby regulating the activity of a variety of signalling processes (Duman and Nestler, 1995; Feldman et al., 1997). GPA1, AGB1, and AGG1, encoding prototypical subunits of G-proteins, have been reported in Arabidopsis (Assmann, 2002; Ullah et al., 2003), and it is likely that heterologues will be found in other plants. Protein phosphorylation and dephosphorylation were reported to be involved in the regulation of stress-enhanced ethylene production by increasing expression of ACS and ACO genes and/or post-transcriptional regulation of ACS (Spanu et al., 1994; Wang et al., 2002; Kim et al., 2003). In citrus, activation of G-protein-coupled receptors by the application of guanfacine or clonidine may lead to phosphorylation signalling cascades that directly or indirectly suppress the expression of ACS and ACO genes and/or their post-transcriptional regulation, ethylene production, and the abscission response. The differential abscission response mediated by guanfacine or clonidine could be due to its ability preferentially to target heterotrimeric G-protein-related receptors in leaves, and to a lesser extent in fruit, or to the difference in quantity or quality of these G-protein-related receptors between these two tissues. Although it is possible that the effect of guanfacine and clonidine on ethephon-induced abscission could be due to activity on processes unrelated to G-protein signalling, the agonists' effect on increasing adventitious root length and number of tomato and coleus cuttings (Burns et al., 2003) is consistent with an auxin-like effect reported for processes associated with G-protein signalling (Assmann, 2002).
While the effect of the mature fruit-selective abscission compound CMNP on ACC accumulation, ethylene production, and ACS1, ACS2, and ACO gene expression are in some ways similar to those mediated by ethephon+guanfacine, the reason for its differential abscission action in fruit and leaves may be different. Research in mature citrus fruit has shown that CMNP acts primarily as a protonophore in the peel, resulting in reduced cellular energy charge, increased membrane breakdown, and abscission (Alferez et al., 2005). Lower ACS1 and ACO expression in CMNP-treated leaf blades and leaf abscission zones may be related to lower sensitivity of leaves to CMNP and/or rapid detoxification upon uptake. Increased expression of ACS2 in CMNP-treated leaf tissues suggests that CMNP can effectively penetrate leaf tissue. Similarly, CMNP had no effect on ethylene evolution and abscission of young fruit in ‘Valencia’ oranges, even though more CMNP penetrates young fruit tissue than mature fruit tissue (Wheaton et al., 1977). Much information related to its mode of action remains to be discovered.
In conclusion, this work provides pharmacological evidence that G-protein-related signalling affects citrus abscission through reduction of ACS1 and ACO expression, ACC accumulation, and ethylene evolution.
|
Acknowledgements |
|---|
This research was supported by the Florida Agricultural Experiment Station and a grant from Florida Department of Citrus (no. 03-12) to JKB, and approved for publication as Journal Series No. R-10243.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Abeles FB, Morgan PW, Saltveit ME (eds). 1992. Ethylene in plant biology. New York: Academic Press.
Alexander L, Grierson D. 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. Journal of Experimental Botany 53, 2039–2055.
Alferez F, Singh S, Umbach AL, Hockema B, Burns JK. 2005. Citrus abscission and Arabidopsis plant decline in response to 5-chloro-3-methyl-4-nitro-1H-pyrazole are mediated by lipid signaling. Plant, Cell and Environment (in press).
Assmann SM. 2002. Heterotrimeric and unconventional GTP-binding proteins in plant cell signalling. The Plant Cell 14, S355–S373.
Banno K, Martin GC, Carlson RM. 1993. The role of phosphorus as an abscission-inducing agent for olive leaves and fruit. Journal of the American Society for Horticultural Science 118, 599–604.
Bleecker AB, Patterson SE. 1997. Last exit: senescence, abscission, and meristem arrest in Arabidopsis. The Plant Cell 9, 1169–1179.[CrossRef][Web of Science][Medline]
Bonghi C, Tonutti P, Ramina A. 2000. Biochemical and molecular aspects of fruitlet abscission. Plant Growth Regulation 31, 35–42.
Brown KM. 1997. Ethylene and abscission. Physiologia Plantarum 100, 567–576.[CrossRef]
Burnik-Tiefengraber T, Weis KG, Webster BD, Martin GC, Yamada H. 1994. Phosphorus effects on olive leaf abscission. Journal of the American Society for Horticultural Science 119, 765–769.
Burns JK. 2002. Using molecular biology tools to identify abscission materials for citrus. HortScience 37, 459–464.
Burns JK, Pozo LV, Yuan R, Hockema B. 2003. Guanfacine and clonidine reduce defoliation and phytotoxicity associated with abscission agents. Journal of the American Society for Horticultural Science 128, 42–47.
Duman RS, Nestler EJ. 1995. Signal transduction pathways for catecholamine receptors. In: Bloom FE, Kupfer DJ, eds. Psychopharmacology: the fourth generation of progress. New York, NY: Raven Press Ltd., 303–320.
Feldman RS, Meyer JS, Quenzer LF. 1997. Catecholamines. In: Bloom FE, Kupfer DJ, eds. Principles of neuropsychopharmacology. Sunderland, MA: Sinauer Assoc, Inc., 277–344.
Goren R, Huberman M, Martin GC. 1998. Phosphorus-induced leaf abscission in detached shoots of olive and citrus. Journal of the American Society for Horticultural Science 123, 545–549.
Katz E, Lagunes PM, Riov J, Weiss D, Goldschmidt EE. 2004. Molecular and physiological evidence suggests the existence of a system II-like pathway of ethylene production in non-climacteric Citrus fruit. Planta 219, 243–252.[CrossRef][Web of Science][Medline]
Kende H. 1993. Ethylene biosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 44, 283–307.[CrossRef][Web of Science]
Kim CY, Liu Y, Thorne ET, Yang H, Fukushige H, Gassmann W, Hildebrand D, Sharp RE, Zhang S. 2003. Activation of a stress-responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants. The Plant Cell 15, 2707–2718.
Lizada MCC, Yang SF. 1979. A simple and sensitive assay for 1-aminocyclopropane-1-carboxylic acid. Annals of Biochemistry 100, 140–145.
Munnik T, Arisz SA, de Vrije R, Musgrave A. 1995. G protein activation stimulates phospholipase D signalling in plants. The Plant Cell 7, 2197–2210.[Abstract]
Nakano R, Ogura E, Kubo Y, Inaba A. 2003. Ethylene biosynthesis in detached young persimmon fruit is initiated in calyx and modulated by water loss from the fruit. Plant Physiology 131, 276–286.
Osborne DJ. 1989. Abscission. Critical Reviews in Plant Sciences 8, 103–129.
Patterson SE, Bleecker AB. 2004. Ethylene-dependent and -independent process associated with floral organ abscission in Arabidopsis. Plant Physiology 134, 194–203.
Pozo L, Yuan R, Kostenyuk I, Alferez F, Zhong G, Burns JK. 2004. Differential effects of 1-methylcyclopropene on citrus leaf and mature fruit abscission. Journal of the American Society for Horticultural Science 129, 473–478.
Roberts JA, Elliott KA, Gonzalez-Carranza ZH. 2002. Abscission, dehiscence, and other cell separation process. Annual Review of Plant Biology 53, 131–158.[CrossRef][Medline]
Scahill L, Chappell PB, Kim YS, Schultz RT, Katsovich L, Shepherd E, Arnsten AF, Cohen DJ, Leckman JF. 2001. A placebo-controlled study of guanfacine in the treatment of children with tic disorders and attention deficit hyperactivity disorder. American Journal of Psychiatry 158, 1067–1074.
Song ZM, Abou-zeid O, Fang YY. 2004. 2A Adrenoceptors regulate phosphorylation of microtubule-associated protein-2 in cultured cortical neurons. Neuroscience 123, 405–418.[CrossRef][Web of Science][Medline]
Spanu P, Grosskopf DG, Felix G, Boller T. 1994. The apparent turnover of 1-aminocyclopropane-1-carboxylate synthase in tomato cells is regulated by protein phosphorylation and dephosphorylation. Plant Physiology 106, 529–535.[Abstract]
Tang X, Gomes AMTR, Bhatia A, Woodson W. 1994. Pistil-specific and ethylene-regulated expression of 1-aminocyclopropane-1-carboxylate oxidase genes in petunia flowers. The Plant Cell 6, 1227–1239.[Abstract]
Taylor JE, Whitelaw CA. 2001. Signals in abscission. New Phytologist 151, 323–339.[CrossRef][Web of Science]
Tucker ML, Sexton R, Campillo ED, Lewis LN. 1988. Bean abscission cellulase. Plant Physiology 88, 1257–1262.
Ullah H, Chen JG, Temple B, Boyes DC, Alonso JM, Davis KR, Ecker JR, Jones AM. 2003. The ß-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. The Plant Cell 15, 393–409.
Wang KLC, Li H, Ecker JR. 2002. Ethylene biosynthesis and signalling networks. The Plant Cell 14, S131–S151.
Warner HL, Leopold AC. 1969. Ethylene evolution from 2-chloroethylphosphonic acid. Plant Physiology 44, 156–158.
Wheaton TA, Wilson WC, Holm RE. 1977. Abscission response and color changes of ‘Valencia’ oranges. Journal of the American Society for Horticultural Science 102, 580–583.
Wong WS, Ning W, Xu PL, Kung SD, Yang SF, Li N. 1999. Identification of two chilling-regulated 1-aminocyclopropane-1-carboxylate synthase genes from citrus (Citrus sinensis Osbeck) fruit. Plant Molecular Biology 41, 587–600.[CrossRef][Web of Science][Medline]
Wu Z, Burns JK. 2003. Isolation and characterization of a cDNA encoding a lipid transfer protein expressed in ‘Valencia’ orange during abscission. Journal of Experimental Botany 54, 1183–1191.
Wu Z, Burns JK. 2004. A ß-galactosidase gene is expressed during mature fruit abscission of ‘Valencia’ orange. Journal of Experimental Botany 55, 1483–1490.
Yuan R, Burns JK. 2004. Temperature factor affecting the abscission response of mature fruit and leaves to CMN Pyrazole and ethephon in ‘Hamlin’ oranges. Journal of the American Society for Horticultural Sciences 129, 1–7.
Yuan R, Kender WJ, Burns JK. 2003. Young fruit and auxin transport inhibitors affect the response of mature ‘Valencia’ oranges to abscission materials via changing endogenous plant hormones. Journal of the American Society for Horticultural Sciences 128, 302–308.
Zarembinski TI, Theologis A. 1994. Ethylene biosynthesis and action: a case of conservation. Plant Molecular Biology 26, 1579–1597.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Malladi and J. K. Burns CsPLD{alpha}1 and CsPLD{gamma}1 are differentially induced during leaf and fruit abscission and diurnally regulated in Citrus sinensis J. Exp. Bot., October 1, 2008; 59(13): 3729 - 3739. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||