Changes in free polyamine concentration induced by salt stress in seedlings of different species

Growth rate, mineral composition and changes in polyamine concentration induced in response to salinity were studied in six crop species: spinach, lettuce, bean, pepper, beetroot and tomato. Salinity decreased growth rate, but sensitivity differed amongst the species: pepper being the most sensitive, followed by bean, tomato, lettuce and spinach, with beetroot being the most tolerant. The increase of Na⁺ and total cation with salinity in shoots was the highest in spinach and beetroot, the most tolerant species, while in pepper it was the lowest. Changes in putrescine (Put) concentration in shoots were related to salinity tolerance (increased in the most sensitive), while changes in spermidine (Spd; decreases) and spermine (Spm; increases) were similar with most species, except for pepper in which salinity strongly increased Put, Spd and Spm. Therefore, total polyamine concentration increased in pepper shoot, while it decreased in the other species. Thus, results show that Put accumulation was a consequence of salt stress in the most sensitive species, while salt tolerant species (beetroot) showed little change in polyamine concentration, and higher concentration in both Na⁺ and total cations. The role of polyamines or cation increased concentration after saline treatment in species with different salt tolerance is discussed.     

Agrobacterium tumefaciens-mediated transformation to alter ethylene and cytokinin biosynthesis in broccoli

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. Postharvest treatment of broccoli with 6-benzylaminopurine delays senescence, whilst exogenous ethylene has been shown to accelerate this process following harvest. To alter ethylene biosynthesis, broccoli was transformed, using Agrobacterium tumefaciens-mediated transformation, with an antisense ACC oxidase gene from broccoli driven by the asparagine synthetase promoter from asparagus. In addition, broccoli was transformed with the chimeric gene construct SAG12-IPT to alter cytokinin biosynthesis during harvest-induced senescence. Transformation was achieved using both hypocotyl and cotyledonary petiole explants. The presence of an antisense ACC oxidase gene enhanced transformation efficiency, but Ag⁺ incorporated into the medium did not. The transgenic nature of these plants was confirmed by PCR and Southern analyses.     

Xylem Transport of 1-Aminocyclopropane-1-carboxylic Acid, an Ethylene Precursor, in Waterlogged Tomato Plants

Waterlogging is known to cause an increase in ethylene synthesis in the shoot which results in petiole epinasty. Evidence has suggested that a signal is synthesized in the anaerobic roots and transported to the shoot where it stimulates ethylene synthesis. Experimental data are presented showing that 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, serves as the signal. Xylem sap was collected from detopped tomato plants (Lycopersicon esculentum Mill. cv. VFN8). ACC in the sap was quantitated by a sensitive and specific assay, and its tentative chemical identity verified by paper chromatography. ACC levels in both roots and xylem sap increased markedly in response to waterlogging or root anaerobiosis. The appearance of ACC in the xylem sap of flooded plants preceded both the increase in ethylene production and epinastic growth, which were closely correlated. Plants flooded and then drained showed a rapid, simultaneous drop in ACC flux and ethylene synthesis rate. ACC supplied through the cut stem of tomato shoots at concentrations comparable to those found in xylem sap caused epinasty and increased ethylene production. These data indicate that ACC is synthesized in the anaerobic root and transported to the shoot where it is readily converted to ethylene.     

Broccoli growth parameters and level of head infestations in simple and mixed plantings: Impact of increased flora diversification

The impacts of interplanting broccoli, Brassica oleracea var. italica L., with chili pepper, Capsicum annuum L., or yellow sweetclover (YSC), Melilotus officinalis L., on broccoli growth parameters and levels of lepidopteran head infestations were studied during two field seasons (winter & summer). During both seasons, several broccoli growth characteristics (e.g. plant vertical area, whole plant biomass, etc.) were significantly reduced in broccoli‐YSC habitats. However, broccoli heads in broccoli‐YSC habitats did not differ significantly in size from other treatment habitats. Additionally, these heads contained the lowest mean lepidopteran contaminants per broccoli crown during the winter season. During this period, the average number of lepidopteran contaminants per broccoli head was more than twice that in monoculture and pepper intercropped broccoli than in broccoli‐YSC habitats. During the summer experiment, the average number of lepidopteran contaminants per broccoli head was greater in pepper intercropped broccoli than in both monoculture and YSC interplanted broccoli. Potential causes of broccoli growth differences and their relationship to lepidopteran ovipositional behavior are discussed.     

The effects of the herbicide quinclorac on shoot growth in tomato is alleviated by inhibitors of ethylene biosynthesis and by the presence of an antisense construct to the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene in transgenic plants

Reduction of shoot growth, leaf epinasty and chlorosis in young tomato plants (Lycopersicon esculentum Mill. cv. Hellfrucht/Frühstamm) treated hydroponically with 10^-7 M of the herbicide quinclorac were partially compensated when the plants were simultaneously sprayed with salicyclic acid or the oxime ether derivative PACME. Since salicyclic acid and PACME are known inhibitors of ethylene biosynthesis, it is suggested that this pathway is implicated in quinclorac action. Further support for this hypothesis was obtained in experiments with transgenic tomato plants containing an antisense gene to 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in ethylene biosynthesis. When quinclorac was applied via the root antisense plants showed reduced phenotypical alterations compared to those of wild-type plants.     

Response of tomato cultivars to salinity

The responses of five tomato cultivars (L. esculentum Mill) of different degrees of salt tolerance were examined over a range of 0 to 140 mM NaCl applied for 3 and 10 weeks. Judged by both Na and Cl accumulations and maintenance of K, Ca and Mg contents with increasing salinity, the most tolerant cultivars (Pera and GC-72) showed different responses. The greater salt tolerance of cv Pera was associated with a higher Cl and Na accumulation and a lower K content in the shoot than those found in the other cultivars, typical of a halophytic response to salinity. However, the greater salt tolerance of cv GC-72 was associated with a retention of Na and Cl in the root, restriction of their translocation to the shoot and maintenance of potassium selectivity under saline conditions. The salt tolerance mechanisms that operated in the remaining cultivars were similar to that of cv GC-72, as at first they excluded Na and Cl from the shoots, accumulating them in the roots; with longer treatment, the ability to regulate Na and Cl concentrations in the plant was lost only in the most salt sensitive cultivar (Volgogradskij), resulting in a massive influx of both ions into the shoot.The salt sensitivity of some tomato cultivars to salinity could be due to both the toxic effect of Na and Cl ions and nutritional imbalance induced by salinity, as plant growth was inversely correlated with Na and Cl contents and directly correlated with K and Ca contents. This study displays that there is not a single salt tolerance mechanism, since different physiological responses among tomato cultivars have been found.     

Ethylene inhibitors enhance in vitro root formation on faba bean shoots regenerated on medium containing thidiazuron

The possible involvement of ethylene in in vitrorooting of faba bean (Vicia faba L.) shootsregenerated on medium containing thidiazuron wasinvestigated. The effects of the ethylene precursor1-aminocyclopropane-1-carboxylic acid (ACC) and threeethylene inhibitors, silver nitrate (AgNO₃),acetyl salicylic acid (ASA) and cobalt chloride(CoCl₂) on root formation were tested in vitrousing TDZ-induced shoots of faba bean accession 760.ACC inhibited root formation. In contrast, ethyleneinhibitors promoted root formation, AgNO₃ at theappropriate concentrations enhanced root emergence andincreased root number per shoot, root growth rate, androot length. Both CoCl₂ and ASA at theappropriate concentrations increased rootingefficiency. These promotive effects may result from areduction in ethylene concentration or inhibition ofethylene action. The results offer a new approach toimprove the rooting efficiency of TDZ-induced shootsof faba bean and possibly of other plant species.     

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.     

A role for jasmonates in climacteric fruit ripening

Jasmonates are a class of oxylipins that induce a wide variety of higher-plant responses. To determine if jasmonates play a role in the regulation of climacteric fruit ripening, the effects of exogenous jasmonates on ethylene biosynthesis and color, as well as the endogenous concentrations of jasmonates were determined during the onset of ripening of apple (Malus domestica Borkh. cv. Golden Delicious) and tomato (Lycopersicon esculentum Mill. cv. Cobra) fruit. Transient (12 h) treatment of pre-climacteric fruit discs with exogenous jasmonates at low concentration (1 or 10 μM) promoted ethylene biosynthesis and color change in a concentration-dependent fashion. Activities of both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC synthase were stimulated by jasmonate treatments in this concentration range. The endogenous concentration of jasmonates increased transiently prior to the climacteric increase in ethylene biosynthesis during the onset of ripening of both apple and tomato fruit. The onset of tomato fruit ripening was also preceded by an increase in the percentage of the cis-isomer of jasmonic acid. Inhibition of ethylene action by diazocyclopentadiene negated the jasmonate-induced stimulation of ethylene biosynthesis, indicating jasmonates act at least in part via ethylene action. These results suggest jasmonates may play a role together with ethylene in regulating the early steps of climacteric fruit ripening. Received: 14 August 1997 / Accepted: 4 October 1997     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

Stereospecific conversion of 1-aminocyclopropanecarboxylic Acid to ethylene by plant tissues : conversion of stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic Acid to 1-butene

Inasmuch as the molecule of 1-aminocyclopropanecarboxylic acid (ACC) possesses reflective symmetry but lacks rotational symmetry, the two chemically alike methylene groups can be distinguished by a stereospecific enzyme. To determine whether ACC conversion to ethylene by plant tissues proceeds in a stereospecific fashion, the four stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid (AEC) were administered to postclimacteric apple (Malus sylvestris Mill., var. Golden Delicious), excised preclimacteric cantaloupe (Cucumis melo L., var. reticulatis Naud cv. PMR-45), and etiolated mung bean (Vigna radiata L., Wilczek, var. Berken) hypocotyls. In each case (1R,2S)-AEC was the preferred substrate yielding 1-butene. In contrast, all AEC isomers were converted equally well to butene by chemical oxidation using NaOCl. Both ACC and AEC appear to be substrates for the same enzyme since both reactions are inhibited in parallel by N₂ or Co²⁺, both reactions are induced in parallel by excision, and when both substrates are present simultaneously each will act as an inhibitor with respect to the other. The aforementioned observations indicate that ACC is stereospecifically converted to ethylene. For AEC to be the most active precursor of 1-butene, the ethyl substituent should be trans to the carboxyl group and the pro-(S) methylene group should be unsubstituted. This observation leads to the suggestion that the enzyme interacts with amino, carboxyl, and pro-(S) methylene groups, a configuration corresponding to a l-amino acid. This view is consistent with the observation that the l-forms of alanine and methionine inhibit the conversion of ACC to ethylene more than the corresponding d-amino acids in the mung bean hypocotyl system.     

Growth and nutrient concentrations of alfalfa and common bean as influenced by soil acidity

Growth and nutrient utilization of alfalfa (Medicago sativa L. cv. Arc) and common bean (Phaseolus vulgaris L. cv. Carioca) were studied in an acid soil adjusted to eight levels of soil acidity by lime addition. Application of lime significantly (P<0.05) increased shoot and root growth for both species. However, common bean was far less sensitive to soil acidity than alfalfa. Maximum alfalfa growth was obtained at a soil pH of 5.8 and maximum bean growth was achieved at pH 5.0. Root and shoot growth of both legumes was positively correlated (P<0.01) with soil pH, exchangeable Ca and exchangeable Mg and negatively correlated (P<0.01) with soil exchangeable Al. Common bean had a lower internal P requirement for maximum growth and was more efficient than alfalfa in taking up Ca and Mg. These characteristics would contribute to the favorable growth of common bean in acid-infertile soils.     

Phytochrome-controlled ethylene biosynthesis of intact etiolated bean seedlings

Intact etiolated bean (Phaseolus vulgaris L. cv. Limburgse vroege) seedlings were illuminated with red light (10.5 W·m^-2) for 10 min. After different time intervals ethylene production, and contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-1-carboxylic acid were measured. The red-light-induced decrease of ethylene production in 8-d-old intact etiolated bean seedlings was fast, strong and long-lasting ad was mediated through the phytochrome system. This effect appeared to be strictly age-dependent, as it could not be detected in plants younger than 6 d or older than 11 d.The capacity for the conversion of ACC to ethylene was not affected by red light. The inhibitory effect of the light treatment on ethylene production could be related to a reduced free-ACC content. This reduction was a consequence of a temporary non-reversible increase of ACC malonylation and a long-lasting, for a certain time reversible, inhibition of ACC synthesis. The effect of a brief irradiation with red light on the decrease of ethylene production and free-ACC content was completed after about 2 h. Reversibility by far-red, however, persisted for at least 3 h, and was lost between 3 and 6 h.Abbrevation ACC 1-aminocyclopropane-1-carboxylic acid - M-ACC 1-(malonylamino)cyclopropane-1-carboxylic acid     

Interactive effects of boron and salinity stress on the growth,membrane permeability and mineral composition of tomato and cucumber plants

A greenhouse study was conducted in order to determine interactive effects of NaCl salinity and B on the growth, sodium (Na), chloride (Cl), boron (B), potassium (K) concentrations and membrane permeability of salt resistant Tomato (Lycopersicon esculentum L. cv. Lale F₁) and salt sensitive cucumber (Cucumis sativus L. cv. Santana F₁) plants. Plants were grown in a factorial combination of NaCl (0 and 30 mM for cucumber and 0 and 40 mM for tomato) and B (0, 5, 10 and 20 mg kg^–1 soil). Boron toxicity symptoms appeared at 5 mg kg^–1 B treatments in both plants. Salinity caused an increase in leaf injury due to B toxicity, but it was more severe in cucumber. Dry weights of the plants decreased with the increasing levels of applied B in nonsaline conditions, but the decrease in dry weights due to B toxicity was more pronounced in saline conditions especially in cucumber. Salinity × B interaction on the concentration of B in both plants was found significant. However, increase in B concentrations of tomato decreased under saline conditions when compared to nonsaline conditions. Contrary to this, B concentration of cucumber increased as a result of increasing levels of applied B and salinity. Salinity increased Na and Cl concentrations of both plants.Potassium concentration of tomato was not affected by salinity and B treatments, but K concentration of cucumber was decreased by salinity. Membrane permeability of the plants was increased by salinity while toxic levels of B had no effect on membrane permeability in nonsaline conditions. Membrane permeability was significantly increased in the presence of salinity by the increasing levels of applied B.     

Inhibition of ethylene synthesis in tomato plants subjected to anaerobic root stress

Enhanced ethylene production and leaf epinasty are characteristic responses of tomato (Lycopersicon esculentum Mill.) to waterlogging. It has been proposed (Bradford, Yang 1980 Plant Physiol 65: 322-326) that this results from the synthesis of the immediate precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), in the waterlogged roots, its export in the transpiration stream to the shoot, and its rapid conversion to ethylene. Inhibitors of the ethylene biosynthetic pathway are available for further testing of this ACC transport hypothesis: aminooxyacetic acid (AOA) or aminoethoxyvinylglycine (AVG) block the synthesis of ACC, whereas CO²⁺ prevents its conversion to ethylene. AOA and AVG, supplied in the nutrient solution, were found to inhibit the synthesis and export of ACC from anaerobic roots, whereas Co²⁺ had no effect, as predicted from their respective sites of action. Transport of the inhibitors to the shoot was demonstrated by their ability to block wound ethylene synthesis in excised petioles. All three inhibitors reduced petiolar ethylene production and epinasty in anaerobically stressed tomato plants. With AOA and AVG, this was due to the prevention of ACC import from the roots as well as inhibition of ACC synthesis in the petioles. With Co²⁺, conversion of both root- and petiole-synthesized ACC to ethylene was blocked. Collectively, these data support the hypothesis that the export of ACC from low O₂ roots to the shoot is an important factor in the ethylene physiology of waterlogged tomato plants.     

Ethylene and in vitro rooting of rose shoots

Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene biosynthesis inhibitor, (CoCl₂), and inhibitor of ethylene binding to receptors, 1-methylcyclopropene (1-MCP), on ethylene production and rooting in shoot culture of Rosa hybrida L. cv. Alba meidiland were studied. Additionally, effect of ethylene removal by KMnO₄ and HgClO₄ on rooting was tested. ACC increased ethylene production and delayed root formation, decreased the number of roots per shoot and inhibited root growth. In contrast, inhibition of ethylene production by CoCl₂ accelerated root emergence, and increased the number of roots per shoot. Likewise, removing ethylene from the ambient atmosphere improved root emergence and, increased root number of per shoot and markedly inhibited root growth. Blocking the ethylene receptors by 1-MCP increased ethylene level in the ambient atmosphere and increased both emergence and root formation. Both ethylene biosynthesis and action are involved in the control of rooting. Ethylene concentration in glass jars was too high for root emergence and formation, but was appropriate for root growth. CoCl₂ or 1-MPC can be recommended for regulation of rooting in rose shoot culture, since both emergence and number of roots were improved but root growth was not inhibited.     

Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro

Summary The role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20–30 M aminoethoxyvinylglycine (AVG) or AgNO₃] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N⁶-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO₃ in combination with AVG. Culture medium solidified with agarose in the presence of AgNO₃ but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10–25 mM putrescine in combination with 30 M AgNO₃ or AVG. Explants with high regenerability grown in the presence of AgNO₃ or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1–5 mg/l were more effective than NAA.Abbreviations ACC 1-aminocyclopropane-1-carboxylate - AVG aminoethoxyvinylglycine - BA N⁶-benzyladenine - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAs polyamines - SAM S-adenosyl-L-methionine     

Regulation of Harvest-induced Senescence in Broccoli (Brassica oleracea var. italica) by Cytokinin, Ethylene, and Sucrose

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene by accelerating the process, and cytokinin by delaying it. To determine the level at which these hormones influenced senescence, we isolated and monitored the expression of genes normally associated with senescence in broccoli florets treated with exogenous 6-benzyl aminopurine (6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC), a combination of 6-BAP and ACC, and sucrose, in the five days following harvest. Exogenous 6-BAP caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression. The expression of genes used as senescence markers, BoCP5 and BoMT1, was reduced, whereas BoCAB1 levels were maintained after harvest in response to exogenous 6-BAP. In addition, the expression of genes encoding sucrose transporters (BoSUC1 and BoSUC2) and carbohydrate metabolizing enzymes (BoINV1 and BoHK1) was also reduced upon 6-BAP feeding. Interestingly, the addition of ACC prevented the 6-BAP-induced increase in expression of BoACS, but 6-BAP negated the ACC-induced increase in expression of BoACO. The culmination of these results indicates a significant role for cytokinin in the delay of senescence. The implication that cytokinin regulates postharvest senescence in broccoli by inhibiting ethylene perception and/or biosynthesis, thus regulating carbohydrate transport and metabolism, as well as senescence-associated gene expression, is discussed and a model presented.