Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase

 Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination. Received: 28 September 1999 / Accepted: 27 December 1999     

Ameliorative effect of brassinosteroid and ethylene on germination of cucumber seeds in the presence of sodium chloride

Brassinosteroids are a class of plant polyhydroxysteroids with a diverse of functions in plant growth and development, while ethylene is a gaseous hormone involved in regulation of numerous physiological processes. To evaluate the roles of BR and ethylene in seed germination under conditions of salt stress, effects of 24-Epibrassinolide (EBR) and 1-aminocyclopropane-1-carboxylic acid (ACC) on seed germination of cucumber (Cucumis sativus) seeds in the presence of 250 mM NaCl were investigated. Seed germination was significantly inhibited by the presence of NaCl in the incubation medium, and the inhibitory effect was significantly alleviated by addition of EBR and ACC to the incubation medium containing NaCl. There was an increase in ethylene evolution during seed germination and this increase was suppressed by salt stress. The reduction in ethylene evolution from imbibed seeds by salt stress was attenuated by EBR. Salt stress inhibited ACC oxidase (ACO) activity and EBR reversed the salt stress-induced decrease in ACO activity. Salt stress reduced expression of gene encoding ACO (CsACO2), and EBR reversed the salt stress-induced down-regulation of CsACO2. The alleviative effect of EBR on seed germination in the presence of NaCl was diminished by antagonist of ethylene synthesis, aminoethoxyvinylglycine. These results indicate that both ethylene and BR are likely to be associated with suppression of seed germination under salt stress and that the mitigating effect of BR on salt stress-induced inhibition of seed germination may occur through its interaction with ethylene synthesis.     

Manipulation of ethylene biosynthesis

The plant hormone ethylene is involved in many plant processes ranging from seed germination to leaf and flower senescence and fruit ripening. Ethylene is synthesized from methionine, via S-adenosyl-L-methionine (SAM) and 1-amino-cyclopropane-1-carboxylic acid (ACC). The key ethylene biosynthetic enzymes are ACC synthase (ACS) and ACC oxidase (ACO). Manipulation of ethylene biosynthesis by chemicals and gene technology is discussed. Biotechnological modification of ethylene synthesis is a promising method to prevent spoilage of agricultural and horticultural products.     

Germination of salt-stressed seeds as related to the ethylene biosynthesis ability in three Stylosanthes species

Stylosanthes, a genus of tropical forage legume, is known to exhibit good persistence in saline soils, yet mechanisms for regulation of seed germination under salt stress are poorly understood. This study was carried out to evaluate the mode of action of salt stress on seed germination of Stylosanthes. 1-Aminocyclopropane-1-carboxylic acid (ACC) increased ethylene biosynthesis and germination of NaCl-inhibited seeds in a dose-dependent manner. Contents of ACC and germination of Stylosanthes humilis seeds increased following transfer from NaCl solution to deionised water, but not after transfer to l-α-(2-aminoethoxyvinyl)-glycine (AVG) solution, an inhibitor of ethylene biosynthesis. Ethylene biosynthesis was much larger in NaCl-treated seeds of Stylosanthes guianensis than in seeds of S. humilis and Stylosanthes capitata, a fact which was reflected in higher germination rates. S. guianensis seedlings also displayed higher growth and survival rates than S. humilis and S. capitata under salt stress. Moreover, smaller ACC levels, as well as reduced ethylene biosynthesis of S. capitata seeds were accompanied by lower germination under salt stress. In addition, S. capitata seedlings treated with NaCl solutions exhibited relatively lower growth and survival rates in comparison with S. humilis and S. guianensis. Thus, different abilities to synthesize ethylene by S. guianensis, S. humilis and S. capitata seeds explain the differences in tolerance to salt stress of the three species.     

Ethylene biosynthesis in Amaranthus caudatus seeds in response to methyl jasmonate

Methyl jasmonate (JA-Me) at 10^–3 M completely inhibited Amaranthus caudatus seed germination. Exogenous ethylene could totally reverse this inhibition. The inhibitor of ethylene action, 2,5-norbornadiene (NBD), increased the sensitivity of seeds to JA-Me. Methyl jasmonate inhibited ethylene production and also decreased both 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl ACC (MACC) content. Likewise, ACC oxidase activity in vivo was decreased by jasmonate. Similarly ACC oxidase activity in vitro isolated from seeds incubated in the presence of JA-Me was lower than that isolated from untreated seeds.The inhibitory JA-Me action on seed germination seems to be mainly associated with the inhibition of ethylene biosynthesis. Both inhibition of ACC synthase and ACC oxidase activity and/or synthesis can be involved.     

The last step of the ethylene biosynthesis pathway in turnip tops (Brassica rapa) seeds: Alterations related to development and germination and its inhibition during desiccation

The involvement of ethylene in zygotic embryogenesis is a little known aspect of the growth and development in higher plants. In the present work, we study the alterations of the last step of the ethylene biosynthesis pathway during the formation period of turnip tops ( Brassica rapa cv. Rapa) seeds and its repercussions in the germination process and post-germinative growth. For this, we chose 11 different phases of silique development, the first being the recently fertilized pistil and the last being the silique just prior to its dehiscence (ca. 2 months post-anthesis). In the 11 phases, ethylene production was detected in both whole silique (with or without seeds) and in the seeds enclosed by the silique wall. The levels of ACC, ACO and ethylene production proved high in seeds belonging to: (1) the pod in the very early phases, when the seeds were growing but without photosynthetic competence; (2) the silique at maximum growth, in which the seeds will initiate desiccation and loss of photosynthetic activity. During the phases prior to dehiscence, there was a marked inhibition in the last step of the ethylene biosynthesis pathway. In viable dry seeds, no ACO activity was detected and the ACC levels were 4-fold lower than at the onset of the silique senescence. Germination brings about a net synthesis of ACC with respect of the stores dry seed. This fact, together with other results presented in this work, point towards, as in other seeds, a dependence of ethylene synthesis for radicle emergence. The possible role played by the silique wall in the control of ethylene biosynthesis during zygotic embryogenesis, as well as the participation of ethylene as a hormonal signal in the triggering of seed desiccation in Brassica rapa cv. Rapa, are discussed in depth.     

Ethylene in seed dormancy and germination

The role of ethylene in the release of primary and secondary dormancy and the germination of non-dormant seeds under normal and stressed conditions is considered. In many species, exogenous ethylene, or ethephon – an ethylene-releasing compound - stimulates seed germination that may be inhibited because of embryo or coat dormancy, adverse environmental conditions or inhibitors (e.g. abscisic acid, jasmonate). Ethylene can either act alone, or synergistically or additively with other factors. The immediate precursor of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC), may also improve seed germination, but usually less effectively. Dormant or non-dormant inhibited seeds have a lower ethylene production ability, and ACC and ACC oxidase activity than non-dormant, uninhibited seeds. Aminoethoxyvinyl-glycine (AVG) partially or markedly inhibits ethylene biosynthesis in dormant or non-dormant seeds, but does not affect seed germination. Ethylene binding is required in seeds of many species for dormancy release or germination under optimal or adverse conditions. There are examples where induction of seed germination by some stimulators requires ethylene action. However, the mechanism of ethylene action is almost unknown.
The evidence presented here shows that ethylene performs a relatively vital role in dormancy release and seed germination of most plant species studied.     

Action of ferric and aluminium ions on the dormancy breakage of <Emphasis Type="Italic">Stylosanthes humilis</Emphasis> seeds

Dormancy of scarified seeds of Stylosanthes humilis was broken by acidic Al³⁺ and Fe³⁺ solutions. Fe⁺³-stimulated seeds exhibited a high activity of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and produced great amounts of ethylene, which showed correlated with the germination process. In addition, specific inhibitors of ethylene biosynthesis and action largely depressed the Fe³⁺-stimulated germination, leading to the conclusion that the ion broke dormancy by triggering ethylene production by the seeds. By contrast, inhibitors of ethylene biosynthesis and action did not impair germination of Al³⁺-stimulated dormant seeds. Moreover, ethylene production and activity of ACC oxidase of Al³⁺-treated seeds was substantially decreased by inhibitors of ethylene biosynthesis, but germination kept large. Together these data suggest that ethylene biosynthesis was not required in the chain of events triggered by Al³⁺ leading to dormancy breakage. Methyl viologen (MV), a reactive oxygen species-generating compound, broke dormancy of seeds to the same extent as Al³⁺ did. Germination of both Al³⁺- and MV-stimulated dormant seeds was inhibited by sodium selenate, an antioxidant compound; selenate, however had no effect on germination of Fe³⁺-stimulated seeds. Together these data indicate that the mechanisms underlying the germination of Al³⁺- and Fe³⁺-treated seeds are not the same.     

Inhibitory Effects of Methyl Jasmonate on the Germination and Ethylene Production in Cocklebur Seeds

Methyl jasmonate (JA-Me) inhibited the germination of cocklebur (Xanthium pennsylvanicum Wallr.) seeds. The inhibition of the germination of cocklebur seeds treated with JA-Me at concentrations less than 300 μm was nullified by ethylene applied exogenously, although the inhibitory effect of 1,000 μm JA-Me was not recovered completely even by high concentrations of ethylene (10,000 μL/liter). JA-Me inhibited ethylene production before seed germination. The level of 1-aminocyclopropane-1-carboxylic acid (ACC) in the cotyledonary tissues treated with JA-Me decreased but not the level of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC). JA-Me inhibited the conversion of ACC to ethylene in the tissues. These results suggested that JA-Me inhibits ethylene production by prevention of ACC oxidation in addition to ACC synthesis. We believe that the inhibition of ethylene production by JA-Me results in the retardation of the germination of cocklebur seeds. Received June 4, 1997; accepted October 23, 1997     

Effects of matriconditioning on onion seed germination, seedling emergence and associated physical and metabolic events

The effect of matriconditioning, the physiological presowing seed technique, using Micro-Cel E on Allium cepa L. cv. Czerniakowska seed quality was studied. Several ratios of seeds, carrier, water and time of priming were tested. The most effective treatment for improving onion seed germination at most tested temperatures was priming to a ratio of 2 g seed:1 g Micro-Cel:3 g water for 5 days in light at 15 °C. Matriconditioning greatly improved the germination and emergence percentage, seedling fresh and dry weight and reduced electrolyte leakage compared to that of untreated seeds; this beneficial effect was especially evident at suboptimal temperatures. Matriconditioning improved the germinability of aged seeds, the effect being more pronounced in the more aged seeds. No significant differences in ethylene production by primed and non-primed seeds were observed in the absence of its precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), but its presence during imbibition caused an increase in ethylene production; an enhanced activity of in vivo ACC oxidase in Allium cepa matriconditioned seeds in comparison to untreated seeds, indicates that the endogenous level of ACC is a limiting factor of ethylene production. Likewise, the activity of ACC oxidase isolated from matriconditioned seeds was higher than that from untreated seeds. Higher endo--mannanase and total dehydrogenase activities were observed in primed air-dried seeds in comparing to non-primed seeds.     

Evidence that ethylene, light and abscisic acid interact to inhibit germination in the recalcitrant seeds of Quercus robur L.

The effects of ethylene, light and abscisic acid (ABA) on thegermination of prematurely-harvested and mature Q. robur seedswere studied. There was evidence of active biosynthesis of ethylene,but not of ABA in seeds from both harvests. Addition of ABA,the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid(ACC) or the ethylene-releasing compound Ethephon (E) to germinationmedia and exposure to light individually inhibited germinationand their effects were additive. The ethylene biosynthesis inhibitoraminoethoxyvinylglycine (AVG) tended to promote germinationof premature seeds only. Key words: Quercus robur, germination, ethylene, light, abscisic acid, recalcitrant seed     

The effect of plant growth regulators, nitric oxide, nitrate, nitrite and light on the germination of dimorphic seeds of Suaeda salsa under saline conditions

Suaeda salsa, a leaf succulent shrub in the family Chenopodiaceae, is one of the most important halophytes in China. Suaeda salsa produces dimorphic seeds (soft brown seeds and hard black seeds). Seeds of S. salsa were collected from the coastal salt flats near Huanghua City, China. Experiments were conducted to determine the salinity-alleviating effect of plant growth regulators, nitric oxide, nitrate, nitrite and light on the germination of dimorphic seeds of S. salsa. Brown seeds had a higher germination rate than black seeds in all experiments. Black seeds were more sensitive to salt in the absence of light in comparison to brown seeds. Brown seeds absorbed water more quickly in comparison to black seeds and were found to be more tolerant of salt stress. Our results showed that 1-aminocyclopropane-1-carboxylate (ACC, the immediate precursor of ethylene), nitrite, GA₄ and BA improved seed germination in the presence of salt. However, nitrate, GA₁, GA₃ failed to alleviate salt stress. ABA inhibited seed germination and seedling growth. Possible mechanisms involved in the alleviation of salt stress in S. salsa seeds and the ecological adaptation of the seeds to the environment are discussed.     

Dormancy removal in apple embryos by nitric oxide or cyanide involves modifications in ethylene biosynthetic pathway

The connection between classical phytohormone-ethylene and two signaling molecules, nitric oxide (NO) and hydrogen cyanide (HCN), was investigated in dormancy removal and germination “sensu stricto” of apple (Malus domestica Borkh.) embryos. Deep dormancy of apple embryos was removed by short-term (3–6 h) pre-treatment with NO or HCN. NO- or HCN-mediated stimulation of germination was associated with enhanced emission of ethylene by the embryos, coupled with transient increase in ROS concentration in embryos. Ethylene vapors stimulated germination of dormant apple embryos and eliminated morphological anomalies characteristic for young seedlings developed from dormant embryos. Inhibitors of ethylene receptors completely impeded beneficial effect of NO and HCN on embryo germination. NO- and HCN-induced ethylene emission by apple embryo was only slightly reduced by inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity during first 4 days of germination. Short-term pre-treatment of the embryos with NO and HCN modified activity of both key enzymes of ethylene biosynthetic pathway: ACC synthase and ACC oxidase. Activity of ACC synthase declined during first 4 days of germination, while activity of ACC oxidase increased markedly at that time. Additional experiments point to non-enzymatic conversion of ACC to ethylene in the presence of ROS (H₂O₂). The results indicate that NO and HCN may alleviate dormancy of apple embryos “via” transient accumulation of ROS, leading to enhanced ethylene emission which is required to terminate germination “sensu stricto”. Therefore, ethylene seems to be a trigger factor in control of apple embryo dormancy removal and germination.     

Developing tools for investigating the multiple roles of ethylene: identification and mapping genes for ethylene biosynthesis and reception in barley

The plant hormone ethylene is important to many plant processes from germination through senescence, including responses to in vitro growth and plant regeneration. Knowledge of the number and function of genes that are involved in ethylene biosynthesis and reception is necessary to determine the role of specific genes within gene families known to influence ethylene biosynthesis and other aspects of ethylene function in plants. Our objective was built on previous studies that have established the critical role of ethylene in the in vitro response of barley (Hordeum vulgare L.), and that have identified ethylene-related QTL in the barley genome. In this study, we have identified the locations of genes in the barley 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO), and ethylene receptor (ETR) gene families. Specific primers for PCR amplification of each gene were developed and used to map these genes in the Oregon Wolf Barley mapping population. Five ACS, 8 ACO, and 7 ETR genes were identified and mapped to six of the barley chromosomes. Gene locations were syntenous to the orthologs in rice except for two that mapped to chromosome 6H. Gene duplication was evident for ACO genes on chromosomes 5H and 6H. Gene-specific primers will be useful for determining expression of each gene under various environmental conditions, including in vitro environments, to better understand the role of ethylene. Of the six known QTL for green plant regeneration in barley, three were located near the genes mapped in this study.     

ACC conversion to ethylene by sunflower seeds in relation to maturation,germination and thermodormancy

Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene was studied in sunflower (Helianthus annuus L., cv. Mirasol) seeds in relation to germinability. Ethylene production from ACC decreased during seed maturation, and non-dormant mature seeds were practically unable to synthesize ethylene until germination and growth occurred, indicating that ethylene forming enzyme (EFE) activity developed during tissue imbibition and growth. ACC conversion to ethylene was reduced by the presence of pericarp, and in young seedlings it was less in cotyledons than in growing axes.ACC conversion to ethylene by cotyledons from young seedlings was optimal at c. 30°C, and was strongly inhibited at 45°C. Pretreatment of imbibed seeds at high temperature (45°C) induced a thermodormancy and a progressive decrease in EFE activity.Abscisic acid and methyl-jasmonate, two growth regulators which inhibit seed germination and seedling growth, and cycloheximide were also shown to inhibit ACC conversion to ethylene by cotyledons of 3-day-old seedlings and by inbibed seeds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - CH cycloheximide - EFE ethylene forming enzyme - IAA indole-3-acetic acid - Me-Ja methyl-jasmonate     

No difference in the regulation pattern of calcium on ethylene biosynthesis between wild-type and never-ripe tomato fruit at mature green stage

This work investigated how calcium regulates the ethylene biosynthesis in the fruits of wild-type tomato (Lycopersicon esculentum L.) and their ethylene receptor never-ripe (Nr) mutants. In Nr tomato, the ethylene perception was blocked. When both materials were treated with calcium, the content of 1-aminocyclopropane-1-carboxylic acid (ACC)/malonyl-ACC and the activity of ACC oxidase (ACO) in tomato fruit discs increased, whereas the production of ethylene, content of malondialdehyde, and membrane permeability decreased. Calcium treatment did not affect the activity of ACC synthase, which is the first committed step in the ethylene biosynthesis pathway. The expression of LeACO1 in mature green fruit was inhibited significantly by calcium treatment in wild-type and Nr tomatoes, but the expression of LeACS2, the key ACC synthase gene in ethylene synthesis during tomato fruit maturing, was not affected. These results revealed that the effect of calcium on ethylene biosynthesis in tomato mature green fruit was independent of ethylene perception. The results also revealed that the targeting step of calcium preventing ethylene production was located at the ACC conversion to ethylene, by means of inhibiting ACC availability for ACO through enhancing cell membrane integrity and by means of preventing LeACO1 gene expression. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 60–67. The text was submitted by the authors in English.