Effect of ethylene on auxin transport and metabolism in midrib sections in relation to leaf abscission of woody plants

Abstract The relationship between ethylene-induced leaf abscission and ethylene-induced inhibition of auxin transport in midrib sections of the leaf blade of Citrus sinensis L. Osbeck, Populus deltoides Bart, and Eucalyptus camaldulensis Dehn. was studied. These species differed greatly in their abscission response to ethylene. The kinetic trend of abscission resembled that of the inhibition of auxin transport in all three species. It is suggested that one of the main actions of ethylene in the leaf blade is to inhibit auxin transport in the veinal tissues, thus reducing the amount of auxin transported from the leaf blade to the abscission zone. Ethylene inhibited transport of both IAA (indole-3-acetic acid) and NAA (α-naphthaleneacetic acid) in the midrib sections. However, while ethylene enhanced the conjugation of IAA with aspartic acid and glucose in the apical (absorbing) segment of the midrib sections, it had little effect on the conjugation of NAA. The data indicate that auxin destruction through conjugation does not play a major role in the inhibition of auxin transport by ethylene.     

Effects of exogenous ethylene on ethylene production in citrus leaf tissue

Exogenous ethylene stimulated ethylene production in intact citrus (Citrus sinensis L. Osbeck cv. “Washington Navel”) leaves and leaf discs following a 24-hour exposure. Studies with leaf discs showed that ethylene production decreased when ethylene was removed by aeration. The extent of stimulation was dependent upon the concentration of exogenous ethylene (1-10 microliters per liter). Silver ion blocked the autocatalytic effect of ethylene at concentrations of 0.5 millimolar and lower, but increased ethylene production at higher concentrations. The stimulating effect of ethylene resulted from the enhancement of both 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. Whereas autocatalysis was evident following 24 hours incubation, autoinhibition of wound- and mannitol-induced ethylene production was observed during the first 24-hour incubation. Ethylene treatment during this period resulted in a marked decrease in ACC levels and ethylene production rates. Furthermore, in leaf discs treated for 24 hours with ethylene, ethylene production rates increased greatly during the first 2 hours after removal of exogenous ethylene by aeration. This increase was eliminated if the discs were transferred to propylene instead of air, indicating that the autocatalytic effect of ethylene is counteracted by its autoinhibitory effect. It is suggested that autocatalysis involves increased synthesis of ACC synthase and the enzyme responsible for the conversion of ACC to ethylene, whereas autoinhibition involves suppression of the activity of these two enzymes.     

Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants

Citrus ( Citrus sinensis L. Osbeck) leaf explants completely abscise within 48 h when exposed to saturating amounts of ethylene at 25°C. When 2,5-norbornadiene was added, 2000 μl 1⁻¹ reduced abscission of explants also exposed to 2 μl 1⁻¹ of ethylene to the level of the control, and 8000 μl 1⁻¹ reduced abscission in explants exposed to 10 μl 1⁻¹ of ethylene to the level of the control, but abscission was complete when 1 000 μl 1⁻¹ of ethylene was used in the presence of 8 000 μl 1⁻¹ of 2,5-norbornadiene. When explants were exposed to 2 μl 1⁻¹ of ethylene, 2000 μl 1⁻¹ of 2,5-norbornadiene prevented abscission if applied up to 10 h after exposure to ethylene. After 18 h, applied 2,5-norbornadiene had little effect on abscission at 48 h. A Lineweaver-Burk plot gave a 1/2 maximum value of 0.12 μl 1⁻¹ for ethylene on abscission, 2,5-Norbornadiene gave competitive kinetics with respect to ethylene with a K₁ value of approximately 120 μl 1⁻¹ of 2,5-norbornadiene. The presence of 2,5norbornadiene stimulated ethylene production, which progressively increased as the 2,5-norbornadiene concentration was increased from 250 to 8 000 μl 1⁻¹ 2,5-Norbornadiene also suppressed the induction of cellulase and polygalacturonase by ethylene. Together, 2,5-norbornadiene and 2,4-dichlorophenoxyacetic acid were more effective than either alone in reducing abscission. 2,5-Norbornadiene also was effective in preventing the reduction of indole-3-acetic acid transport induced by ethylene.     

Chilling-induced leaf abscission of Ixora coccinea plants. II. Alteration of auxin economy by oxidative stress

Chilling-induced leaf abscission of ixora ( Ixora coccinea ) plants was almost completely inhibited by α -naphthaleneacetic acid (NAA), even in the presence of exogenous ethylene, which enhanced the chilling effect on leaf abscission. Chilling reduced free indoleacetic acid (IAA) content, quantified immediately after chilling, in the abscission zone (AZ) and leaf blade. Free IAA content in chilling-treated plants continued to decrease gradually with time after chilling. Application of the antioxidant butylated hydroxyanisole (BHA) before or after chilling not only prevented the post-chilling decline in free IAA content, but also restored free IAA level during 6–48 h of the post-chilling period almost to the control level. No significant effect of chilling on the endogenous content of ester- and amide-conjugates of IAA or the metabolism of exogenous labeled IAA were observed. Chilling enhanced the decarboxylation of IAA, particularly in the AZ tissue. Auxin transport capacity was significantly inhibited by chilling, and this effect was counteracted by BHA applied before chilling. The data indicate that chilling reduces free IAA content in the AZ, an effect that may lead to increased sensitivity to ethylene. The chilling-induced reduction in IAA content in the AZ seems to result, at least in part, from increased IAA decarboxylation and reduced auxin transport capacity. These processes seem to be triggered by the oxidative stress imposed on the tissues by chilling.     

Differential growth dependency of normal and habituated sugarbeet cell lines upon endogenous ethylene production and exogenous ethylene application

A fully habituated (auxin‐ and cytokinin‐independent) nonorganogenic (HNO) sugarbeet ( Beta vulgaris ) callus produces very little ethylene as compared with a normal (N) hormone‐requiring callus of the same strain. Both callus types react by growth changes to application of inhibitors of ethylene biosynthesis and ethylene action, of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) as the immediate precursor of ethylene, to transfer from light to darkness, and also to application of exogenous ethylene or an ethylene trapper. This indicates their growth dependency upon their endogenously biosynthesized ethylene and also their sensitivity to exogenous gas. However, the sensitivity was generally higher for the HNO callus producing naturally less ethylene. The weaker reaction of the HNO callus to the exogenous ethylene was attributed to its hyperhydric status (a water layer surrounding the cells). Because low ethylene production appears as a general characteristic of habituated cell lines, the causal and/or consequential relationships of this low ethylene production with other characteristics of habituated tissues (absence of exogenous hormones in the culture media, deficiency of cell differentiation, accumulation of polyamines in neoplastic tissues) are discussed.     

Effect of exposure to subfreezing temperatures on ethylene evolution and leaf abscission in citrus

Citrus leaves exposed to subfreezing temperatures evolved ethylene at rates between 0.1 and 38.3 microliters per kilogram fresh weight per hour whereas untreated leaves evolved between 0.01 and 0.50 microliter per kilogram fresh weight per hour. Leaves not injured by freezing temperatures did not abscise, and ethylene evolution was near normal after 2 days. Freeze-injured leaves continued evolving high ethylene levels 4 or 5 days subsequent to freeze injury, and many of the freeze-killed leaves abscised. Supportive evidence suggested freeze-induced ethylene was involved in freeze-induced leaf abscission; whereas freeze-inhibited abscission was not due to a lack of ethylene but injury to other metabolic systems necessary for abscission.     

The role of endogenous auxin in root initiation

This paper is the second part of a review which considers evidence for the involvement of auxin in root initiation. Part II examines the research being carried out with transformed plant tissues. Agrobacterium rhizogenes causes abundant root initiation at the site of inoculation. Ri plasmid T-DNA contains several genes which encode enzymes involved in the biosynthesis and metabolism of indole-3-acetic acid. Transfer of various fragments of the Ri plasmid has also been reported to confer increased sensitivity to auxin upon plant cells. Controlled expression of these genes in the plant genome potentially offer an insight for developmental plant physiologists into the role of plant growth substances in the process of root initiation. The importance of absolute levels of IAA in the stimulation of root initiation is discussed.     

The role of endogenous auxin in root initiation

This paper describes the process of the formation of adventitious roots. There appears to be good agreement that this consists of four stages, defifferentiation coupled with the formation of a meristematic locus, cell division to form a radially symmetrical cluster of cells, further divisions coupled with organisation into a bilaterally symmetrical meristem and finally growth of cells in the basal part of the meristem which causes its protursion through the epidermis. Evidence for the involvement of auxins in these various stages is reviewed and the extent to which rooting of easy- and hard-to-root species can be accounted for in terms of auxin content discussed. Peaks of IAA occur soon after excision of cuttings in some species and there is some evidence suggesting that this is correlated with changes in peroxidase activity. The possible involvement of cytokinins with auxins is briefly considered.     

Seasonal changes in endogenous ABA and IAA and the influence of applied ABA and auxin in relation to shoot growth and abscission in Valencia Orange (Citrus sinensis (L.) Osbeck)

Seasonal changes in endogenous IAA and ABA were measured by gas chromatography/mass-spectrometry. Highest concentrations of ABA occurred in leaves. There was a major ABA peak in early spring (up to 1360 ng g^–1 dw). Levels were low in summer (90 ng g^–1 dw). There was a minor ABA peak in autumn. Endogenous IAA in leaves was highest in winter/spring (up to 76 ng g^–1 dw). Applied ABA promoted abscission of leaves and shoots while applied NAA delayed abscission. The main peak in leaf-ABA content was followed by extensive shoot abscission. The involvement of ABA and IAA in regulation of flush growth was not clear.This paper is dedicated to Michael G. Mullins, who died on 13 November 1990, for his outstanding contribution to horticulture.     

Endogenous and exogenous auxin in the control of root growth

The endogenous indol-3yl-acetic acid (IAA) of detipped apical segments from roots of maize (cv ORLA) was greatly reduced by an exodiffusion technique which depended upon the preferential acropetal transport of the phytohormone into buffered agar. When IAA was applied to the basal cut ends of freshly prepared root segments only growth inhibitions were demonstrable but after the endogenous auxin concentration had been reduced by the exodiffusion technique it became possible to stimulate growth by IAA application. The implications of the interaction between exogenous and endogenous IAA in the control of root segment growth are discussed with special reference to the role of endogenous IAA in the regulation of root growth and geotropism.Abbreviations IAA indol-3yl-acetic acid - GC-MS gas chromatography-mass spectrometry     

Carbohydrates stimulate ethylene production in tobacco leaf discs : I. Interaction with auxin and the relation to auxin metabolism

Various naturally occurring carbohydrates, applied at a concentration range of 1 to 100 mm, stimulated ethylene production for several days in indoleacetic acid (IAA)-treated or untreated tobacco (Nicotiana tabacum L. cv `Xanthi') leaf discs. The lag period for this sugar-stimulated ethylene production was 8 to 12 hours after excision in the untreated leaf discs, but less than 2 hours in the IAA-treated ones. Among the tested carbohydrates, 12 were found to increase synergistically ethylene production, with d-galactose, sucrose, and lactose being the most active; mannitol and l-glucose had no effect. The extent and duration of the increased ethylene production was dependent upon the type of sugar applied, the tissue's age, and the existence of both exogenous IAA and sugar in the medium. Sucrose appeared to elicit a continuous IAA effect for 48 hours, as expressed by increased ethylene production, even when IAA was removed from the medium after a 4-hour pulse. Sucrose stimulated both the uptake and decarboxylation of [1-¹⁴C]IAA, as well as the hydrolysis of the esteric and amide IAA conjugates formed in the tissue after application of free IAA. This gradual hydrolysis was accompanied by a further accumulation of a third IAA metabolite. Moreover, synthetic indole-3-acetyl-l-alanine increased ethylene production mainly with sucrose, and this effect was accompanied by its increased decarboxylation and turnover pattern suggesting that release of free IAA was involved. An esteric IAA conjugate, tentatively identified by GC retention time was found to be the major component (84%) of the naturally occurring IAA conjugates in tobacco leaves. Accordingly the sucrose-stimulated ethylene production in tobacco leaves can be ascribed mainly to the sucrose-stimulated hydrolysis of the esteric IAA conjugate.     

The role of phytohormones ethylene and auxin in plant-nematode interactions

The phytohormones ethylene and auxin regulate many important processes in plants, including cell differentiation, cell expansion, and responses to abiotic stresses. These hormones also play important roles in many plant-pathogen interactions, including regulation of plant defense responses and symptom development. Sedentary plant-parasitic nematodes, which require the formation of a complex feeding site within the host root, are among the world’s most destructive plant pathogens. Nematode-induced feeding sites show dramatic changes in host cell morphology and gene expression. These changes are likely mediated, at least in part, by phytohormones. In the present review, current knowledge of the roles of ethylene and auxin will be explored in two main areas: the specific role of phytohormones in mediating feeding site development by plant-parasitic nematodes and the general role of phytohormones in affecting the ability of parasitic nematodes to cause disease. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 1, pp. 3–7. This article was presented in original.     

Effect of calcium, (2-chloroethyl) phosphonic acid and ethylene on bean leaf abscission

Summary The effects of CaCl₂, (2-chloroethyl) phosphonic acid (Ethephon) and ethylene on leaf abscission of debladed and intact bean plants (Phaseolus vulgaris L.) were studied. Ethephon (1000 g/l) and ethylene (8 l/l) induced abscission in debladed and intact plants in 24–72 h whereas IAA (10^-5M), cycloheximide (10^-5M) and CaCl₂ (0.068M) delayed abscission in debladed plants. CaCl₂ completely inhibited the abscission-enhancing effect of Ethephon in intact bean leaves. When CaCl₂ and Ethephon were applied simultaneously to separate halves of the leaf blade, leaves with Ethephon applied closest to the pulvinus abscised rapidly; when CaCl₂ was applied closest to the pulvinus, abscission was prevented. Calcium pre-treatment prior to ethylene (8 l/l) treatment of debladed plants delayed abscission as compared to those treated with ethylene alone.Michigan Agricultural Experiment Station Journal Article No. 6299.     

A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis

Although auxin response factors (ARFs) are the first well-characterized proteins that bind to the auxin response elements, elucidation of the roles of each ARF gene in auxin responses and plant development has been challenging. Here we show that ARF19 and ARF7 not only participate in auxin signaling, but also play a critical role in ethylene responses in Arabidopsis (Arabidopsis thaliana) roots, indicating that the ARFs serve as a cross talk point between the two hormones. Both arf19 and arf7 mutants isolated from our forward genetic screens are auxin resistant and the arf19arf7 double mutant had stronger auxin resistance than the single mutants and displayed phenotypes not seen in the single mutants. Furthermore, we show that a genomic fragment of ARF19 not only complements arf19, but also rescues arf7. We conclude that ARF19 complements ARF7 at the protein level and that the ARF7 target sequences are also recognized by ARF19. Therefore, it is the differences in expression level/pattern and not the differences in protein sequences between the two ARFs that determines the relative contribution of the two ARFs in auxin signaling and plant development. In addition to being auxin resistant, arf19 has also ethylene-insensitive roots and ARF19 expression is induced by ethylene treatment. This work provides a sensitive genetic screen for uncovering auxin-resistant mutants including the described arf mutants. This study also provides a likely mechanism for coordination and integration of hormonal signals to regulate plant growth and development.     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

The role of endogenous gibberellin-like substances and inhibitors in the growth of pea internodes

Third internodes or whole stems of 7-days old etiolated pea plants were extracted and the content of gibberellin-like substances and inhibitors has been determined. Extracts were found to contain four or five different gibberellin-like substances, some of which are chromatographically similar to GA₃. The content of gibberellins has been high in young internodes and decreased along with the internodes elongation. Brief red light irradiation brings about quantitative changes in gibberellin content, depending also on the length of internodes. The extracts contain acidic and neutral inhibitors which interfere with the response to GA₃. The content of the inhibitors does not seem to be affected by the ageing of internodes or by the light treatment.     

Abscission: the role of ethylene modification of auxin transport

The role of ethylene-mediated reduction of auxin transport in natural and ethylene-induced leaf abscission was studied in the cotton (Gossypium hirsutum L., cv. Stoneville 213) cotyledonary leaf system. The threshold level of ethylene required to cause abscission of intact leaves was between 0.08 and 1 μl/l with abscission generally occurring 12 to 24 hours following ethylene fumigation. The threshold level of ethylene required to reduce the auxin transport capacity in the cotyle-donary petiole paralleled that required for stimulation of abscission. In plants where cotyledons are allowed to senesce naturally there is a decline in auxin transport capacity of petioles and increase in ethylene synthesis of cotyledons. The visible senescence process which precedes abscission requires up to 11 days, and increases in ethylene production rates and internal levels were detected well before abscission. Ethylene production rates for entire cotyledons rose to 2.5 mμ1 g⁻¹ hr⁻¹ and internal levels of 0.7 μl/l were observed. These levels appear to be high enough to cause the observed decline in auxin transport capacity. These findings, along with those of others, indicate that ethylene has several roles in abscission control (e.g., transport modification, enzyme induction, enzyme secretion). The data indicate that ethylene modification of auxin transport participates in both natural abscission and abscission hastened by exogenous ethylene.     

Chilling-induced leaf abscission of Ixora coccinea plants. I. Induction by oxidative stress via increased sensitivity to ethylene

Exposing ixora ( Ixora coccinea ) plants to chilling temperatures (3–9°C for 3 days) resulted in increased leaf abscission, initiated 3 days after transfer to 20°C. Exposure to chilling also induced a 7-fold increase in ethylene production rates of abscission zone (AZ) tissue during the initial 5 h after chilling. The ethylene burst resulted from the high levels of 1-aminocyclopropane-1-carboxylic acid (ACC) accumulated in the AZ during the chilling period. ACC levels following chilling decreased also due to enhanced conjugation to 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC). Treating plants prior to chilling with antioxidants, such as butylated hydroxyanisole (BHA), n -propyl gallate (PG), and vitamin E, significantly reduced chilling-induced leaf abscission. This effect was obtained despite the fact that ethylene production in the treated plants resembled that of chilled plants receiving no BHA. In addition, exposure of plants to ethylene (0.5–10 μl l⁻¹) for 1–3 days significantly enhanced leaf abscission only when they had been pre-chilled. These data imply that chilling-induced leaf abscission was closely correlated with increased sensitivity of the AZ to ethylene rather than with the chilling-induced ethylene burst. Based on the findings that the ethylene action inhibitor, 1-methylcyclopropene (1-MCP), and the antioxidant BHA inhibited both the chilling-induced and the ethylene-enhanced leaf abscission, it is concluded that: (1) although ethylene is essential for chilling-induced abscission, it is not the triggering factor; (2) oxidative processes derived from the chilling stress seem to be the trigger of chilling-induced leaf abscission, operating via increased sensitivity to ethylene.