Leaf Abscission Zones Research Articles

Gene expression profiles for cell wall-modifying proteins associated with soybean cyst nematode infection, petiole abscission, root tips, flowers, apical buds, and leaves

Changes in transcript accumulation for cell wall-modifying proteins were examined in excised soybean root pieces colonized by soybean cyst nematodes (SCN), Heterodera glycines, using RT-PCR and soybean Affymetrix GeneChips. Sequence-specific PCR primer pairs were prepared from sequence data for core sequences in the GenBank soybean database and consensus sequences derived from the assembly of soybean ESTs. In addition, to identify previously uncharacterized soybean transcripts, degenerate primers were prepared for conserved motifs in cellulases (endo-1,4-beta-glucanases, EGases) and polygalacturonases (PGs) and these were used to amplify segments of transcripts that were then extended with 3' and 5' RACE. Several novel EGase and PG transcripts were identified. Gene expression patterns were determined by real-time RT-PCR for 11 EGases, three expansins (EXPs), 14 PGs, two pectate lyases (PLs), and two xyloglucan endotransglucosylase/hydrolases (XTHs) in soybean roots inoculated with SCN, non-inoculated roots, serial dissections of root tips, leaf abscission zones, flowers, apical buds, and expanding leaves. A large number of genes associated with cell wall modifications are strongly up-regulated in root pieces colonized by SCN. However, in contrast to most of the transcripts for cell wall proteins, two XTH transcripts were specifically down-regulated in the colonized root pieces. Gene expression in serial dissections of root tips (0-2 mm, and 2-7 mm) and whole roots indicate that the SCN up-regulated genes are associated with a wide range of developmental processes in roots. Also of interest, many of the cDNAs examined were up-regulated in petiole abscission zones induced to abscise with ethylene.

G-protein-coupled α2A-adrenoreceptor agonists differentially alter citrus leaf and fruit abscission by affecting expression of ACC synthase and ACC oxidase

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 alpha(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.

A beta-galactosidase gene is expressed during mature fruit abscission of 'Valencia' orange (Citrus sinensis).

beta-galactosidases have been detected in a wide range of plants and are characterized by their ability to hydrolyse terminal non-reducing beta-D-galactosyl residues from beta-D-galactosides. These enzymes have been detected in a wide range of plant organs and tissues. In a search for differentially expressed genes during the abscission process in citrus, sequences encoding beta-galactosidase were identified. Three cDNA fragments of a beta-galactosidase gene were isolated from a cDNA subtraction library constructed from mature fruit abscission zones 48 h after the application of a mature fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMN-pyrazole). Based on sequence information derived from these fragments, a full-length cDNA of 2847 nucleotides (GenBank accession number AY029198) encoding beta-galactosidase was isolated from mature fruit abscission zones by 5'- and 3'-RACE approaches. The beta-galactosidase cDNA encoded a protein of 737 amino acid residues with a calculated molecular weight of 82 kDa. The deduced protein was highly homologous to plant beta-galactosidases expressed in fruit ripening. Southern blot analysis demonstrated that at least two closely related beta-galactosidase genes were present in 'Valencia' orange. Temporal expression patterns in mature fruit abscission zones indicated beta-galactosidase mRNA was detected 48 h after treatment of CMN-pyrazole and ethephon in mature fruit abscission zones. beta-galactosidase transcripts were detected in leaf abscission zones only after ethephon application. The citrus beta-galactosidase was expressed in stamens and petals of fully opened flowers and young fruitlets. The results suggest that this beta-galactosidase may play a role during abscission as well as early growth and development processes in flowers and fruitlets.

Functional analysis of peach ACC oxidase promoters in transgenic tomato and in ripening peach fruit

To investigate the transcriptional regulation of two 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) genes of peach, chimeric fusions between β-glucuronidase (GUS) reporter gene, and Pp-ACO1 and Pp-ACO2 promoters have been constructed and introduced in tomato (cv Microtom). Pp-ACO1 promoter is able to induce in transgenic tomato plants the same pattern of expression observed in peach. In fact, Pp-ACO1–GUS activity was localized in leaf blade, ovary, leaf and fruit abscission zones and pericarp, and it is up-regulated by propylene and wounding. A gradient of transcript accumulation has been observed in ripening fruit tissues: in tomato it decreased moving from the inner to the outer pericarp, while in peach the opposite occurred. This different behavior could be related to the fruit type (berry vs. drupe). In order to identify cis-acting element involved in ethylene induction of Pp-ACO1, portions of its promoter fused with the GUS gene have been constructed and used in peach fruit transient activity assay. The deletion analysis has shown that a region located between −716 and −346 bp, containing an ethylene-responsive element (ERE), is responsible for the higher stimulation by the gas. In addition, two auxin-responsive elements (AUXre), probably responsible for the auxin suppression of the propylene induction of Pp-ACO1 gene expression, are present upstream from EREs. Pp-ACO2 promoter is able to drive the expression in vascular bundles of immature and ripe fruit, senescent leaf blade, and in fruit and leaf abscission zones. In peach, Pp-ACO2 mRNA is detected only in immature fruit, epicotyl and root of seedling. This discrepancy might be imputed to a lesser stability and translation of Pp-ACO2 mRNA in comparison to that of chimeric one.

Phenylalanine ammonia lyase gene expression during abscission in citrus.

Phenylalanine ammonia lyase (PAL) gene expression was examined in fruit and leaf abscission zones of Valencia orange for periods up to 72 h after induction of abscission with Ethrel(R) (CEPA) or the citrus mature fruit-specific abscission material 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMN-P). PAL gene expression was detected in mature fruit abscission zones beginning 6 and 24 h after CEPA and CMN-P application, respectively, and continued up to 72 h. PAL gene expression was detected in leaf abscission zones 6 h after CEPA application and continued throughout the remainder of the study. In contrast, PAL gene expression was not detected in leaf abscission zones treated with CMN-P. 2-aminoindan-2-phosphonic acid (AIP), a specific inhibitor of PAL enzyme activity, decreased CMN-P-induced PAL expression in fruit abscission zones, and this was accompanied by a lack of fruit drop. PAL transcripts were low in abscission zones of immature fruit; however, the transcript was induced by CMN-P but less by CEPA application. The data suggest that downstream products of PAL activity may be important not only for wound healing and defense reactions that occur at the abscission zone fracture plane, but also for regulation of PAL gene expression during abscission.

Overexpression of polygalacturonase in transgenic apple trees leads to a range of novel phenotypes involving changes in cell adhesion.

Polygalacturonases (PGs) cleave runs of unesterified GalUA that form homogalacturonan regions along the backbone of pectin. Homogalacturonan-rich pectin is commonly found in the middle lamella region of the wall where two adjacent cells abut and its integrity is important for cell adhesion. Transgenic apple (Malus domestica Borkh. cv Royal Gala) trees were produced that contained additional copies of a fruit-specific apple PG gene under a constitutive promoter. In contrast to previous studies in transgenic tobacco (Nicotiana tabacum) where PG overexpression had no effect on the plant (K.W. Osteryoung, K. Toenjes, B. Hall, V. Winkler, A.B. Bennett [1990] Plant Cell 2: 1239-1248), PG overexpression in transgenic apple led to a range of novel phenotypes. These phenotypes included silvery colored leaves and premature leaf shedding due to reduced cell adhesion in leaf abscission zones. Mature leaves had malformed and malfunctioning stomata that perturbed water relations and contributed to a brittle leaf phenotype. Chemical and ultrastructural analyses were used to relate the phenotypic changes to pectin changes in the leaf cell walls. The modification of apple trees by a single PG gene has offered a new and unexpected perspective on the role of pectin and cell wall adhesion in leaf morphology and stomatal development.

Temporal and spatial expression of a polygalacturonase during leaf and flower abscission in oilseed rape and Arabidopsis.

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZAT isolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5'-upstream sequences. Fusion of a 1,476-bp 5'-upstream sequence of PGAZAT to beta-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals.

Temporal and Spatial Expression of a Polygalacturonase during Leaf and Flower Abscission in Oilseed Rape and Arabidopsis

During leaf abscission in oilseed rape (Brassica napus), cell wall degradation is brought about by the action of several hydrolytic enzymes. One of these is thought to be polygalacturonase (PG). Degenerate primers were used to isolate a PG cDNA fragment by reverse transcriptase-polymerase chain reaction from RNA extracted from ethylene-promoted leaf abscission zones (AZs), and in turn a full-length clone (CAW471) from an oilseed rape AZ cDNA library. The highest homology of this cDNA (82%) was to an Arabidopsis sequence that was predicted to encode a PG protein. Analysis of expression revealed that CAW471 mRNA accumulated in the AZ of leaves and reached a peak 24 h after ethylene treatment. Ethylene-promoted leaf abscission in oilseed rape was not apparent until 42 h after exposure to the gas, reaching 50% at 48 h and 100% by 56 h. In floral organ abscission, expression of CAW471 correlated with cell separation. Genomic libraries from oilseed rape and Arabidopsis were screened with CAW471 and the respective genomic clones PGAZBRAN and PGAZATisolated. Characterization of these PG genes revealed that they had substantial homology within both the coding regions and in the 5′-upstream sequences. Fusion of a 1,476-bp 5′-upstream sequence ofPGAZAT to β-glucuronidase or green fluorescent protein and transformation of Arabidopsis revealed that this fragment was sufficient to drive expression of these reporter genes in the AZs at the base of the anther filaments, petals, and sepals.

Failed expression of an endo-β-1,4-glucanhydrolase (cellulase) in a non-abscinding mutant of Lupinus angustifolius cv Danja

Cellulase expressions in a normal shedding wild-type and a non-abscinding single gene mutant of Lupinus angustifolius have been studied during ethylene treatments of leaf abscission zone explants. Of the range of different glycohydrolases investigated only the abscission cell-specific β-1,4-glucanhydrolase (cellulase) was not produced in the non-abscinding mutant. An endo-polygalacturonase was induced equally in both wild-type and mutant and other glycohydrolases were equally up-regulated. The abscission cell-specific cellulase induced at shedding of wild-type is antigenically similar to the Phaseolus vulgaris induced leaf abscission pI 9.5 cellulase but with a higher molecular mass (50 kD compared with 48 kD) and like the bean abscission-specific cellulase that of lupin is not glycosylated. Causes of the loss of function of cellulase expression in the non-shedding mutant are discussed.

Molecular characterization of a tomato polygalacturonase gene abundantly expressed in the upper third of pistils from opened and unopened flowers.

A polygalacturonase (PG) gene, TPG7 (Lyces;Pga1;8), has been cloned from tomato (Lycopersicon esculentum Mill., cv. Rutgers). RNA blot analysis reveals that TPG7 is highly expressed in pistils (ovary removed) from unopened and fully open flowers. Dissection of mature pistils demonstrated that TPG7 expression is limited to the top third (stigmatic region) of the pistils. This is contrasted with another tomato PG, TAPG4, which is also expressed in the same region of the pistil but only in mature pistils from fully open flowers. Hybridization of the TPG7 probe to anther RNA was nil to none and was barely detectable in RNA from leaf and flower abscission zones. The TPG7 polypeptide shares 39% sequence identity with the tomato fruit PG and between 63% and 73% sequence identities with six other tomato PGs.

662 Molecular Mechanisms and Potential Control of Abscission in Horticultural Crops

The process of abscission results in shedding of plant parts such as leaves, fruit, flowers, and in citrus, shoot tips and entire shoots. Growers must successfully manage abscission in their operations to avoid unnecessary defoliation or loss of yield due to floral abscission or preharvest fruit drop. Conversely, abscission enhancement may be desired during harvest. Yet despite its importance to horticulture, little is known about mechanisms that control abscission. We know that abscission can be induced by ethylene and altered to some extent by auxin. Over the years, many physiological and anatomical events of abscission have been described. For example, cellulase, polygalacturonase and pectin methylesterase genes are induced during abscission, and they are thought to have a role in alteration and depolymerization of middle lamella polysaccharides located in the abscission zone area. Other genes, such as those associated with the process of pathogen resistance, are also induced during abscission. We are interested in using tools of molecular biology to examine abscission-related gene expression prior to organ separation in Florida field-grown Valencia orange (Citrus sinensis L. Osbeck) and greenhouse-grown calamondin (Citrus madurensis Loureiro) citrus trees. Subtractive cDNA library screening and differential display were used to examine gene expression in fruit, leaf and floral abscission zones 6, 24 and 48 h after induction of abscission with 5-chloro-3-methyl-4-nitro-1H-pyrazole or Ethrel® (Rhone-Poulenc, [2-chloroethyl] phosphoric acid). Some isolated cDNAs encoded polypeptides with no significant matches in the database or share significant similarities with unknown proteins isolated from Arabidopsis. Other cDNAs encoded polypeptides with similarity to cell wall modifying proteins such as polygalacturonases and expansin, PR proteins such as chitinase, proteins associated with secondary and xenobiotic metabolism such as amine oxidase, benzoquinone reductase, caffeic acid methyltransferase, phenylalanine ammonia lyase and squalene synthase, and proteins associated with signal transduction such as several serine/threonine kinases. Temporal and spatial expression of these genes and others will be presented. Use of this information to target potential points of abscission control will be discussed.

Characters and origin of vessels with heterogenous structure in leaf and flower abscission zones

The lifetime of many plant organs does not exceed a few weeks or a few months. These organs separate from the plant at the level of specialized abscission zones. The observation of xylem vasculature in abscission zones, a largely neglected subject, revealed original features when a vessel casting method was used. In all species of dicotyledons examined so far, flower and leaf abscission zones possessed heterogenous metaxylem vessels adjoining protoxylem and secondary xylem vessels with homogenous patterns of lignified thickenings. Heterogenous metaxylem vessel thickenings were helical, reticulate, or scalariform elements when in the abscission zone and pitted elements on the proximal and the distal sides. The origin and possible role of these vessels are considered. Data obtained on the flower abscission zone of tomato (Lycopersicon esculentum Mill.) suggest that formation of heterogenous vessels results from localized changes in the rhythm of cell differentiation and cell maturation inside the procambium-cambium continuum.Key words: abscission zone, cambium, differentiation, heterogenous vessels, procambium, vessel cast.

Molecular characterization of a tomato endo-beta-1,4-glucanase gene expressed in mature pistils, abscission zones and fruit.

A cDNA (TAC1) and genomic clone (cel5) encoding an endo-beta-1,4-glucanase (EGase) were identified from tomato (Lycopersicon esculentum Mill., cv. Rutgers). The cel5 gene is expressed in pistils, flower pedicel and leaf abscission zones, and ripening fruit. The genomic sequence includes a 22 bp 5' upstream sequence that is conserved in a closely related peach EGase gene, ppEG1.

Characters and origin of vessels with heterogenous structure in leaf and flower abscission zones

Characters and origin of vessels with heterogenous structure in leaf and flower abscission zones

Genomic organization of six tomato polygalacturonases and 5' upstream sequence identity with tap1 and win2 genes.

Recently, three polygalacturonase (PG) cDNAs (TAPG1, TAPG2, and TAPG4) were identified in a library prepared from tomato (Lycopersicon esculentum cv. Rutgers) leaf abscission zones. Genomic clones encoding these three cDNAs have been identified. Moreover, the genomic clones include three additional PG genes, TPG3, TAPG5 and TPG6, which have not been previously reported. A transcript for TAPG5 was detected in the RNA from leaf and flower abscission zones; however, transcripts for TPG3 and TPG6 were not. DNA sequence analysis revealed that TAPG1, TAPG2, and TPG3 are linked in a close tandem array. TAPG4, TAPG5 and TPG6 are also closely linked to each other but in divergent and inverted orientations and are not closely linked to TAPG1, TAPG2, or TPG3. TAPG4, TAPG5 and TPG6 map to the middle of chromosome 12. TPG6 contains two introns. The other five PG genes include four exons and three introns. The relative positions of introns 1 and 2 are shared by all six PG genes. The position of intron 3 is conserved in the other five. The structure of the tomato fruit PG gene, which contains 8 introns, is compared with that of the six PG genes described above. Of interest is an approximately 300 bp inverted repeat found in TAPG1, TAPG2 and TAPG4 that shares significant sequence identity with sequence in the first intron of the tomato anionic peroxidase gene, tap1. RNA blot analysis indicates that the transcript for an anionic peroxidase increases during abscission. In addition, a 250 bp sequence found in TPG3 shares high sequence identity with a 5' upstream region in a wound-induced win2 gene from potato. Potential sites of transcriptional regulation in these genes are discussed.

Endo‐β‐1,4‐glucanases are involved in peach fruit growth and ripening, and regulated by ethylene

During peach (Prunus persica[L.] Batsch) fruit development and ripening the cell wall undergoes several structural and biochemical changes driven by several hydrolases. Among these, the endo‐β‐1,4‐D‐glucanase (EGase, EC 3.2.1.4), or cellulase, may play a crucial role. Involvement of EGase throughout development and ripening of the fruit of cv. Redhaven was assessed by monitoring enzyme activity, specific polypeptide accumulation and gene transcription. During the four stages of growth EGase activity was high during S1 and in the early S2, declined during S3, and increased with the onset of ripening (S4). Two isoforms with isolectric points of 6.5 and 9.5 were identified. The pI 6.5 EGase was the only form present during the early stages of growth. whereas the pI 9.5 EGase was most abundant during ripening. The same isoforms were present in leaf and fruit abscission zones. The antibody raised against the pI 9.5 EGase, purified from leaf abscission zones, cross‐reacted with a protein of 54 kDa. A cDNA clone of 753 bp encoding peach EGase was obtained by RT‐PCR. EGase transcripts, detectable only after amplification of total RNA by RT‐PCR, were observed during S1, and at the preclimacteric and climacteric stages. However, the strongest hybridisation occurred at ripening, in correspondence with the maximal enzyme activity and polypeptide accumulation, which took place before the ethylene climacteric and in the early stage of flesh softening. Propylene treatments reduced EGase activity during the early stage of fruit growth but dramatically enhanced enzyme activity and the related transcript accumulation at ripening, and accelerated the loss of firmness. In fruit treated with 2,5‐norbornadiene the softening process was strongly inhibited and the rise in EGase transcripts and activity did not take place. The results point to the EGases being involved in early fruit growth and the initial phases of softening. The presence of two isoforms and the dual effect of propylene on enzyme activity suggest that different EGase genes operate during the early and late developmental stages in peach fruit.

Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern.

Abscission, or organ separation, is accompanied by a marked increase in hydrolases, which are responsible for the degradation of the middle lamella and the loosening of the primary cell wall surrounding cells in the separation layer. We recently reported on the cloning of a tomato (Lycopersicon esculentum) polygalacturonase (PG) cDNA, TAPG1, expressed during leaf and flower abscission. In addition to TAPG1, we have cloned two more PG cDNAs (TAPG2 and TAPG4) that are also expressed during leaf and flower abscission. The peptide sequences for the three abscission PGs are relatively similar (76-93% identity) yet different from the those of tomato fruit PG (38-41% identity). None of the three abscission PG mRNAs are expressed in fruit, stems, petioles, or anthers of fully open flowers. An RNase protection assay revealed that all three PGs are expressed in leaf and flower abscission zones and in pistils of fully open flowers. TAPG4 mRNA is detected much earlier than TAPG1 and TAPG2 mRNA during both leaf and flower abscission.

Characterization and expression of an mRNA encoding a wound-induced (Win) protein from ethylene-treated tomato leaf abscission zone tissue

A cDNA clone (TAB7) encoding a putative wound-induced (Win) protein has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) leaf abscission zone cDNA library using a differential screening strategy. The clone has a high degree of homology at the amino acid level to both the potato win1 and 2 genes, Hevea brasiliensis hevein and Nicotiana tabacum PR-4a and PR-4b proteins. The mRNA encoded by TAB7 is up-regulated within 12 h of exposure to ethylene (10 μl l -1 ) and its expression increases steadily within the cells comprising the leaf abscission zone and to a lesser extent in the adjacent non-zone tissue. This rise precedes the onset of cell separation. Southern analysis indicates that the mRNA is encoded by either a single gene or a small gene family. The role of the protein during abscission is discussed.

The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase.

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues.

Dominant mutations in the Arabidopsis ETR1 gene block the ethylene signal transduction pathway. The ETR1 gene has been cloned and sequenced. Using the ETR1 cDNA as a probe, we identified a cDNA homologue (eTAE1) from tomato. eTAE1 contains an open reading frame encoding a polypeptide of 754 amino acid residues. The nucleic acid sequence for the coding sequence in eTAE1 is 74% identical to that for ETR1, and the deduced amino acid sequence is 81% identical and 90% similar. Genomic Southern blot analysis indicates that three or more ETR1 homologues exist in tomato. RNA blots show that eTAE1 mRNA is constitutively expressed in all the tissues examined, and its accumulation in leaf abscission zones was unaffected by ethylene, silver ions (an inhibitor of ethylene action) or auxin.