Methods for the quantitation of abscisic acid and its precursors from plant tissues

Methods are given for the quantitation of the plant stress hormone, abscisic acid (ABA), and its two metabolic precursors, ketone and enolate, that are applicable to all species tested so far. The plant extract is homogenized at neutral pH, hexane-soluble neutrals are extracted and discarded, and then the free ABA and other organic acids are extracted as ion pairs. The remaining aqueous phase is acidified, allowed to stand, neutralized, and extracted to give the ABA ex ketone fraction and then the aqueous phase is treated with base and again extracted to give the ABA ex enolate fraction. Each of these three fractions, free ABA, ABA ex ketone, and ABA ex enolate, along with a deuteriated internal standard, [side-chain-(2)H(4)]ABA, is then derivatized with pentafluorobenzyl bromide and purified on an automated sample preparation system. The resulting pentafluorobenzyl abscisate samples are then quantified using electron capture negative ionization mass spectrometry with methane as the reagent gas. Using these procedures free ABA, and ABA from its precursors, can be quantified at the level of 100 fg on column. If a large volume injector is used so that the total sample is injected it should be possible to quantify ABA and its precursors in the parts per billion range on a few milligrams of plant tissue.     

Role of the mitochondria in the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene in plant tissues

Intact plant mitochondria, isolated from climacteric (Lycopersicon esculentum, Mill., tomato) or non-climacteric (Solanum tuberosum, L., potato) tissues, and purified on Percoll density gradients, were unable to convert 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene. Energization or sonication did not enhance ethylene production. For both tissues, the low activity of ACC conversion found in crude mitochondrial fractions from both tissues was increased by sonication. After mitochondrial purification, this activity was located on top of the gradient together with the microsomal membrane fraction containing a high lipoxygenase activity. Addition of exogenous lipoxygenase and linoleic acid to isolated tomato or potato mitochondria greatly enhanced ACC conversion (to approx. 300 pmol h⁻¹ mg⁻¹ protein). Direct measurements of ACC uptake by mitochondria indicated that ACC uptake is not dependent on energization.     

The importance of linoleic acid and linolenic acid as precursors of hexanal and trans-2-hexenal in black tea

During tea fermentation, linoleic acid in the neutral fat fraction,and linolenic acid in both the neutral fat and phospholipidfractions from leaves decreased. The addition of linoleic orlinolenic acid to leaf macerates during fermentation resultedin an increase in hexanal or trans-2-hexenal in the volatilefraction. Tracer experiments showed the direct conversion oflinoleic-U-¹⁴C and linolenic-U-¹⁴C acids to labeled hexanaland trans-2-hexenal, respectively, which were identified as2,4-DNPH derivatives. Further conversion of hexanal and trans-2-hexenal into hexanoicand trans-2-hexenoic acids during tea fermentation was suggestedby the increases in these compounds after the addition of hexanaland trans-2-hexenal to leaf macerates. (Received December 21, 1971; )     

Cyanide metabolism in relation to ethylene production in plant tissues

HCN is the putative product of C-1 and amino moieties of 1-aminocyclopropane-1-carboxylic acid (ACC) during its conversion to ethylene. In apple (Malus sylvestrus Mill.) slices or auxin-treated mungbean (Vigna radiata L.) hypocotyls, which produced ethylene at high rates, the steady state concentration of HCN was found to be no higher than 0.2 micromolar, which was too low to inhibit respiration (reported Ki for HCN to inhibit respiration was 10-20 micromolar). However, these tissues became cyanogenic when treated with ACC, the precursor of ethylene, and with 2-aminoxyacetic acid, which inhibits β-cyanoalanine synthase, the main enzyme to detoxify HCN; the HCN levels in these tissues went up to 1.7 and 8.1 micromolar, respectively. Although ethylene production by avocado (Persea gratissima) and apple fruits increased several hundred-fold during ripening, β-cyanoalanine synthase activity increased only one- to two-fold. These findings support the notion that HCN is a co-product of ethylene biosynthesis and that the plant tissues possess ample capacity to detoxify HCN formed during ethylene biosynthesis so that the concentration of HCN in plant tissues is kept at a low level.     

Metabolism of unsaturated fatty acids in tomato fruit: linoleic and linolenic acid as precursors of hexanal

Incubation of linoleic or linolenic acid with tissue slicesas well as cell-free extracts of tomato fruits produced hexanal.Biogenesis of hexanal from these fatty acids was further substantiatedby the use of uniformally labelled ¹⁴C substrates. Based onthe fact that hydrogen peroxide inhibited oxygen uptake andalso production of carbonyls, it is apparent that lipoxidaseis involved in these reactions. The activity of the crude solubleextract was increased by dialysis and ammonium sulphate fractionation. In general, ripe fruits contained greater enzymatic activitiesbut smaller amounts of linoleic and linolenic acid than greenfruits. The enzymatic activity was enhanced by metal ions andcompounds containing free -SH groups. (Received December 27, 1971; )     

Relationship between the presence of linolenic acid and the ability to form respiration-deficient mutants in yeast

The relationship between the presence of linolenic acid and the ability to form respirationdeficient mutants induced by acriflavin has been established. The metabolic coefficients and the composition of fatty acids in samples metabolizing lactose are presented.     

Salicylic acid inhibition of ethylene production by apple discs and other plant tissues

Ethylene production by apple discs is effectively inhibited by salicylic acid. Inhibition is pH dependent, being greatest from pH 3.5–4.5 and minimal at pH 6.5 and above. With 100 M salicylic acid maximal inhibition, approximately 90%, is achieved in 3 h with an apparent K_i of 40 M. At somewhat higher concentrations salicylic acid also inhibits the conversion of 1-aminocyclo-propane-1-carboxylic acid to ethylene by pear discs and mung bean hypocotyls. Salicylic acid interferes with action of the putative ethylene-forming enzyme and in this respect is somewhat more effective than cobalt ion. The inhibitory effects of salicylic acid and cobalt ion are not additive. Implications for the limits and locus of salicylic acid inhibition are discussed.     

Surfactants identified in lung lymph and their ability to act as abhesives

Phospholipid has been extracted from pulmonary lymph collected from 10 dogs. Thin-layer chromatography was used to identify phosphatidylcholine (PC) 55.6 +/- 2.9%, sphingomyelin 21.3 +/- 1.7%, phosphatidylethanolamine 11.2 +/- 4.9%, and lysophosphatidylcholine 5.9 +/- 0.8%. All extracts proved highly surface active, reducing the surface tension of saline to 27.7 +/- 0.7 dyn/cm upon 80% film compression and increasing the maximum contact angle on glass (theta) from 7 +/- 1 to 47.4 +/- 1.4 degrees. The hydrophobic properties induced on glass were further demonstrated by the ability to cause saline to withdraw and expose a dry surface. A standard adhesion test was used to measure the "tack" produced by the major proteins in lymph. However, when the surface energy of the hydrophilic glass surfaces was reduced by a monolayer of lymph phospholipid extract or an equivalent mixture of synthetic surfactants, the adhesive force was reduced by 79 +/- 4% for albumin and 55 +/- 4% for globulin. As a 0.1% liposomal suspension, PC gave 55% release with albumin. Reversible bonding of the lumen of lymph vessels by the "tacky" proteins present is discussed as a possible factor contributing to the large changes in flow resistance known to occur in the pulmonary lymphatic system.     

Ozone-induced ethylene production is dependent on salicylic acid,and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death

Ethylene is known to influence plant defense responses including cell death in response to both biotic and abiotic stress factors. However, whether ethylene acts alone or in conjunction with other signaling pathways is not clearly understood. Ethylene overproducer mutants, eto1 and eto3, produced high levels of ethylene and developed necrotic lesions in response to an acute O3 exposure that does not induce lesions in O3-tolerant wild-type Col-0 plants. Treatment of plants with ethylene inhibitors completely blocked O3-induced ethylene production and partially attenuated O3-induced cell death. Analyses of the responses of molecular markers of specific signaling pathways indicated a relationship between salicylic acid (SA)- and ethylene-signaling pathways and O3 sensitivity. Both eto1 and eto3 plants constitutively accumulated threefold higher levels of total SA and exhibited a rapid increase in free SA and ethylene levels prior to lesion formation in response to O3 exposure. SA pre-treatments increased O3 sensitivity of Col-0, suggesting that constitutive high SA levels prime leaf tissue to exhibit increased magnitude of O3-induced cell death. NahG and npr1 plants compromised in SA signaling failed to produce ethylene in response to O3 and other stress factors suggesting that SA is required for stress-induced ethylene production. Furthermore, NahG expression in the dominant eto3 mutant attenuated ethylene-dependent PR4 expression and rescued the O3-induced HR (hypersensitive response) cell death phenotype exhibited by eto3 plants. Our results suggest that both SA and ethylene act in concert to influence cell death in O3-sensitive genotypes, and that O3-induced ethylene production is dependent on SA.     

A comparative study of the effects of abscisic acid and new terpenoid abscisic acid analogues on plant physiological processes

The effects of new terpenoid abscisic acid analogues in comparison to abscisic acid (ABA) on some physiological and biochemical processes of various plant species have been investigated. The analogues exhibited ABA-like effects by inhibiting cell elongation and germination, and by promoting abscission, as well as the accumulation of proline and induction of stomatal closure coupled by a reduction in transpiration. In response to low temperature, the analogue-treated plants showed improved resistance to cold accompanied by a decrease in electrolyte leakage.     

A comparative study of the effects of abscisic acid and new terpenoid abscisic acid analogues on plant physiological processes

The effects of new terpenoid abscisic acid analogues in comparison to abscisic acid (ABA) on some physiological and biochemical processes of various plant species have been investigated. The analogues exhibited ABA-like effects by inhibiting cell elongation and germination, and by promoting abscission, as well as the accumulation of proline and induction of stomatal closure coupled by a reduction in transpiration. In response to low temperature, the analogue-treated plants showed improved resistance to cold accompanied by a decrease in electrolyte leakage.     

Selective enhancement of alternative path capacity in plant storage organs in response to ethylene plus oxygen: a comparative study

The respiration rise in bulky storage organs induced by ethylene plus pure O₂ is accompanied by an increase in the CN-resistant respiration, or alternative path. Whereas a lesser respiratory stimulation in response to ethylene is demonstrable in air and increased by peeling, ethylene-induced alternative path development depends on the synergistic effect of pure O₂, with or without peeling. The effect of ethylene plus O₂ is evident, whether untreated parent organs yield CN-sensitive or CN-resistant fresh slices. Alternative path capacity and maximal cytochrome oxidase-mediated electron transport have been separately estimated. Ethylene plus O₂ selectively enhances the alternative path. It is proposed that the gross rise in respiration evoked by ethylene is implemented by a system with an O₂ requirement much higher than that of cytochrome oxidase, while the ethylene-induced development of the alternative path depends on a system of still higher O₂ requirement.     

A comparative study of ethylene oxidation inVicia faba andMycobacterium paraffinicum

Vicia faba L. ‘Herz Freya’ (fababean) cotyledons andMycobacterium paraffinicum Bardane strain (MPB) cells were studied to describe and compare physiological and biochemical factors regulating ethylene oxidation. Both organisms demonstrated a linear rate of ethylene uptake as a function of concentration from 1 ppm to 1,000 ppm. CO₂ did not influence ethylene oxidation by either organism. Zero degree temperatures and CO inhibited ethylene oxidation by fababeans but not by MPB. An N₂ gas phase blocked ethylene consumption by fababeans. In contrast, MPB continued to consume ethylene at a reduced rate under anaerobic conditions. Hydrocarbon oxidation was limited to alkenes. Alkanes were not oxidized by either organism. Both organisms were sensitive to diethyldithiocarbamic acid, o-phenanthroline, carbonyl cyanidem-chlorophenyl hydrazone, and CS₂. The possibility that CS₂ acted as a suicide substrate is discussed. Evidence is presented that hydrocarbon gas oxidation by fababeans is not a part of, or reflection of, the way ethylene acts as a hormone.     

E. coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide

The monomeric peptide methionine sulfoxide reductase (MsrA) catalyzes the irreversible thioredoxin-dependent reduction of methionine sulfoxide. The crystal structure of MsrAs from Escherichia coli and Bos taurus can be described as a central core of about 140 amino acids that contains the active site. The core is wrapped by two long N- and C-terminal extended chains. The catalytic mechanism of the E. coli enzyme has been recently postulated to take place through formation of a sulfenic acid intermediate, followed by reduction of the intermediate via intrathiol-disulfide exchanges and thioredoxin oxidation. In the present work, truncated MsrAs at the N- or C-terminal end or at both were produced as folded entities. All forms are able to reduce methionine sulfoxide in the presence of dithiothreitol. However, only the N-terminal truncated form, which possesses the two cysteines located at the C-terminus, reduces the sulfenic acid intermediate in a thioredoxin-dependent manner. The wild type displays a ping-pong mechanism with either thioredoxin or dithiothreitol as reductant. Kinetic saturation is only observed with thioredoxin with a low K(M) value of 10 microM. Thus, thioredoxin is likely the reductant in vivo. Truncations do not significantly modify the kinetic properties, except for the double truncated form, which displays a 17-fold decrease in k(cat)/K(MetSO). Alternative mechanisms for sulfenic acid reduction are also presented based on analysis of available MsrA sequences.