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1.
1-aminocyclopropane-1-carboxylate (ACC) synthase is a principal enzyme that catalyses the committed step in phytohormone ethylene biosynthesis. Previous evidence indicates that the hypervariable C-terminus of ACC synthase is most likely to be processed proteolytically in vivo. However, the protease responsible has not been identified thus far. In the present study, we detected proteolytic activity against ACC synthase (LeACS2) in tomato (Lycopersicon esculentum Mill.) fruit extract based on a newly established in vitro assay system. Purification of the protease through DEAE, gel filtration and MonoQ chromatography resulted in considerable enrichment of a 64-kDa protein species. Subsequent biochemical analysis of the purified tomato protease revealed that the optimal conditions for its proteolytic activity were at pH 8.0 and at 37 ℃. In addition, the protease activity was blocked completely by the metalloprotease inhibitor 1,10-phenanthroline. The present study represents the first report on the isolation of an ACC synthaseprocessing protease from plant tissues. 相似文献
2.
Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC
1-aminocyclopropane-1-carboxylic acid 相似文献
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6.
A partially purified preparation of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) from tomato (Lycopersicon esculentum (Mill.) fruit tissue was used to generate monoclonal antibodies (MAb) specific for the two different MAbs yielded a 50-kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An enzyme-linked immunosorbent assay (ELISA) capable of detecting <1 ng of antigen was developed. The ELISA system was used to demonstrate that two of the MAbs recognized different epitopes on the ACC-synthase protein. Wound-induced increases in ACC-synthase activity in tomato fruit tissue were correlated with changes in ELISA-detectable protein. In-vivo labeling of wounded tissue with [35S]methionine followed by extraction and immunopurification in the presence of various protease inhibitors yielded one major radioactive band of 50 kDa molecular mass. Pulse labeling with [35S]methionine at various times after wounding indicated that the wound-induced increase in ACC-synthase activity involved de-novo synthesis of a rapidly turning over 50-kDa polypeptide.Abbreviations ACC
1-aminocyclopropane-1-carboxylic acid
- ELISA
enzyme-linked immunosorbent assay
- MAb
monoclonal antibody
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
7.
Properties and Partial Purification of 1-Aminocyclopropane-1-carboxylate Synthase 总被引:2,自引:2,他引:0
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We studied the regulation of 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in tomato (Lycopersicon esculentum Mill.) fruit tissue and attempted the purification of this enzyme. The increase of ACC synthase activity in wounded tomato pericarp was inhibited by cordycepin and cycloheximide. Density labeling studies showed a 0.75% increase in the buoyant density of ACC synthase isolated from tomato pericarp tissue that had been incubated on 2H2O as compared to ACC synthase from H2O-treated tissue. These data are consistent with the hypothesis that ACC synthase is synthesized de novo following wounding of tomato pericarp tissue. SDS-gel electrophoresis and fluorography showed that the pattern of incorporation of l-[35S]methionine into protein changed with time after wounding of the tissue. Radioactive protein bands that were not detected 1 hour after wounding, became apparent 2 to 3 hours after wounding. 相似文献
8.
The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding, and beyond
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A pyridoxal 5'-phosphate (PLP)-dependent enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-Met methylthioadenosine-lyase, EC 4.4.1.14), catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC. A tomato ACC synthase isozyme (LE-ACS2) with a deletion of 46 amino acids at the C terminus was chosen as the control enzyme for the study of the function of R286 in ACC synthase. R286 of the tomato ACC synthase was mutated to a leucine via site-directed mutagenesis. The ACC synthase mutant R286L was purified using a simplified two-step purification protocol. Circular dichroism (CD) analysis indicated that the overall three-dimensional structure of the mutant was indistinguishable from that of the control enzyme. Fluorescence spectroscopy revealed that the binding affinity of R286L ACC synthase for its cofactor PLP was reduced 20- to 25-fold compared with control. Kinetic analysis of R286L showed that this mutant ACC synthase had a significantly reduced turnover number (k(cat)) of 8.2 x 10(-3) s(-1) and an increased K(m) of 730 microM for AdoMet, leading to an 8,000-fold decrease in overall catalytic efficiency compared with the control enzyme. Thus, R286 of tomato ACC synthase is involved in binding both PLP and AdoMet. 相似文献
9.
《植物学报(英文版)》2005,47(11)
l-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS (LeACS2) into 52-, 50- and 49-kDa truncated isoforms in ripening tomato (Lycopersicon esculentum Mill. cv.Cooperation 903) fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys438, Glu447, Lys448, Asn456, Ser460, Ser462, Lys463, and Leu474, but does not cleave the Nterminus of LeACS2. Four C-terminus-deleted (26-50 amino acids) LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain (H14) and Ser460 for this metalloprotease.Furhermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo. 相似文献
10.
Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [35S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins. 相似文献
11.
Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits 总被引:11,自引:0,他引:11
Biggs, M. S., Woodson, W. R. and Handa, A. K. 1988. Biochemical basis of high-temperature inhibition of ethylene biosynthesis in ripening tomato fruits. Physiol. Plant. 72: 572578
Incubation of fruits of tomato ( Lycopersicon esculentum Mill. cv. Rutgers) at 34°C or above resulted in a marked decrease in ripening-associated ethylene production. High temperature inhibition of ethylene biosynthesis was not associated with permanent tissue damage, since ethylene production recovered following transfer of fruits to a permissive temperature. Determination of pericarp enzyme activities involved in ethylene biosynthesis following transfer of fruits from 25°C to 35 or 40°C revealed that 1-aminocyclopropane-l-carboxylic acid (ACC) synthase (EC 4.4.1.14) activity declined rapidly while ethylene forming enzyme (EFE) activity declined slowly. Removal of high temperature stress resulted in more rapid recovery of ACC synthase activity relative to EFE activity. Levels of ACC in pericarp tissue reflected the activity of ACC synthase before, during, and after heat stress. Recovery of ethylene production following transfer of pericarp discs from high to permissive temperature was inhibited in the presence of cycloheximide, indicating the necessity for protein synthesis. Ethylene production by wounded tomato pericarp tissue was not as inhibited by high temperature as ripening-associated ethylene production by whole fruits. 相似文献
Incubation of fruits of tomato ( Lycopersicon esculentum Mill. cv. Rutgers) at 34°C or above resulted in a marked decrease in ripening-associated ethylene production. High temperature inhibition of ethylene biosynthesis was not associated with permanent tissue damage, since ethylene production recovered following transfer of fruits to a permissive temperature. Determination of pericarp enzyme activities involved in ethylene biosynthesis following transfer of fruits from 25°C to 35 or 40°C revealed that 1-aminocyclopropane-l-carboxylic acid (ACC) synthase (EC 4.4.1.14) activity declined rapidly while ethylene forming enzyme (EFE) activity declined slowly. Removal of high temperature stress resulted in more rapid recovery of ACC synthase activity relative to EFE activity. Levels of ACC in pericarp tissue reflected the activity of ACC synthase before, during, and after heat stress. Recovery of ethylene production following transfer of pericarp discs from high to permissive temperature was inhibited in the presence of cycloheximide, indicating the necessity for protein synthesis. Ethylene production by wounded tomato pericarp tissue was not as inhibited by high temperature as ripening-associated ethylene production by whole fruits. 相似文献
12.
Moeder W Barry CS Tauriainen AA Betz C Tuomainen J Utriainen M Grierson D Sandermann H Langebartels C Kangasjärvi J 《Plant physiology》2002,130(4):1918-1926
We show that above a certain threshold concentration, ozone leads to leaf injury in tomato (Lycopersicon esculentum). Ozone-induced leaf damage was preceded by a rapid increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, ACC content, and ethylene emission. Changes in mRNA levels of specific ACC synthase, ACC oxidase, and ethylene receptor genes occurred within 1 to 5 h. Expression of the genes encoding components of ethylene biosynthesis and perception, and biochemistry of ethylene synthesis suggested that ozone-induced ethylene synthesis in tomato is under biphasic control. In transgenic plants containing an LE-ACO1 promoter-beta-glucuronidase fusion construct, beta-glucuronidase activity increased rapidly at the beginning of the O(3) exposure and had a spatial distribution resembling the pattern of extracellular H(2)O(2) production at 7 h, which coincided with the cell death pattern after 24 h. Ethylene synthesis and perception were required for active H(2)O(2) production and cell death resulting in visible tissue damage. The results demonstrate a selective ozone response of ethylene biosynthetic genes and suggest a role for ethylene, in combination with the burst of H(2)O(2) production, in regulating the spread of cell death. 相似文献
13.
Jian-Feng LI Robert QI Liang-Hu QU Autar K Mattoo Ning LI 《植物学报(英文版)》2005,47(11):1352-1363
1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS (LeACS2) into 52-, 50- and 49-kDa truncated isoforms in ripening tomato (Lycopersicon esculentum Mill. cv. Cooperation 903) fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted (26-50 amino acids) LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain (H14) and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo. 相似文献
14.
Shigeru Satoh Naoko Oyamada Toshihito Yoshioka Naoki Midoh 《Plant Growth Regulation》1997,23(3):191-193
The effects of 1,1-dimethyl-4-(phenylsulfonyl)semicarbazide (DPSS) on the in vitro activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase isolated from senescing carnation petals were investigated. In contrast to a previous proposal, DPSS at 1 mM did not inhibit the in vitro activity of ACC oxidase. It was confirmed that DPSS does not inhibit ACC synthase activity. DPSS probably does not exert its inhibitory action on ethylene production by a direct action on ACC oxidase and ACC synthase, but by some unknown action. 相似文献
15.
Capitani G McCarthy DL Gut H Grütter MG Kirsch JF 《The Journal of biological chemistry》2002,277(51):49735-49742
The 1.6-A crystal structure of the covalent ketimine complex of apple 1-aminocyclopropane-1-carboxylate (ACC) synthase with the potent inhibitor l-aminoethoxyvinylglycine (AVG) is described. ACC synthase catalyzes the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. AVG is widely used in plant physiology studies to inhibit the activity of ACC synthase. The structural assignment is supported by the fact that the complex absorbs maximally at 341 nm. These results are not in accord with the recently reported crystal structure of the tomato ACC synthase AVG complex, which claims that the inhibitor only associates noncovalently. The rate constant for the association of AVG with apple ACC synthase was determined by stopped-flow spectrophotometry (2.1 x 10(5) m(-1) s(-1)) and by the rate of loss of enzyme activity (1.1 x 10(5) m(-1) s(-1)). The dissociation rate constant determined by activity recovery is 2.4 x 10(-6) s(-1). Thus, the calculated K(d) value is 10-20 pm. 相似文献
16.
Immunochemical cross-reactivity of wound- and auxin-induced1-aminocyclopropane-1-carboxylate (ACC) synthase was examinedwith the antibody against wound-induced ACC synthase purifiedfrom mesocarp of winter squash (Cucurbita maxima Duch.). Theantibody recognized ACC synthase from wounded hypocotyls ofwinter squash and from wounded pericarp of tomato fruits, butnot the enzyme from IAA-treated hypocotyls of winter squash,tomato and mung bean. These results indicate that the primarystructure of the wound-induced enzyme is different from thatof the auxin-induced enzyme in the same species, and impliesthat there are two different genes for ACC synthase, one forwound induction and the other for auxin induction. (Received June 14, 1988; Accepted July 20, 1988) 相似文献
17.
1-Aminocyclopropane-1-carboxylate (ACC) synthase, which catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC, is irreversibly inactivated by its substrate AdoMet. AdoMet has two diastereomers with respect to its sulfonium center, (-)-AdoMet and (+)-AdoMet. We prepared (+)- and (-)-AdoMet from a commercial source, and compared their activities as a substrate and as an inactivator of ACC synthase isolated from tomato (Lycopersicon esculentum Mill). fruits. Only (-)-AdoMet produced ACC, whereas both (-)- and (+)-AdoMet inactivated ACC synthase; (+)-AdoMet inactivated the enzyme three times faster than (-)-AdoMet. We have previously shown that ACC synthase was specifically radiolabeled when the enzyme was incubated with S-adenosyl-L-[3,4-14C]methionine. The present results further indicate that S-adenosyl-L-[carboxyl-14C]methionine, but not S-adenosyl-L-[methyl-14C]methionine, radiolabeled the enzyme. These data suggest that the 2-aminobutyric acid portion of AdoMet is linked to ACC synthase during the autoinactivation process. A possible mechanism for ACC synthase inactivation by AdoMet is discussed. 相似文献
18.
Inactivation of stress induced 1-aminocyclopropane carboxylate synthase in vivo differs from substrate-dependent inactivation in vitro 总被引:4,自引:1,他引:3
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The activity of 1-aminocyclopropane carboxylate (ACC) synthase increased rapidly in tomato (Lycopersicon esculentum Mill.) leaf discs after vacuum infiltration, reached a maximum after about 30 minutes, and subsequently decayed with an apparent half-life of about 20 minutes. Aminoethoxyvinylglycine, a known inhibitor of ACC synthase, did not alter the apparent turnover of ACC synthase in vivo although it efficiently blocked inactivation of the enzyme by its substrate S-adenosylmethionine in vitro. Similar results were obtained, using a novel assay with permeabilized cells, for ACC synthase in tomato cell cultures treated with a fungal elicitor. The results indicate that inactivation of ACC synthase in vivo differs from substrate-dependent inactivation in vitro. 相似文献
19.
We determined the time course of increases in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in ripening tomato
(Lycopersicon esculentum (L.) Mill.) pericarp discs following wounding and treatment with 75 mM LiCl. Over the course of 24 h, we detected oscillations
in the amount of enzyme activity from an initial peak at 6 h to a subsequent, even higher level at 18 h. In-vitro translation
products derived from poly(A)+ RNAs isolated at various times of treatment and in-vivo-labeled proteins were immunoprecipitated using antibodies specific
for ACC synthase. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that wounding
and treatment with LiCl induced an accumulation of translatable ACC-synthase-specific mRNAs. In addition, single, prominent
bands were apparent for both in-vivo and in-vitro samples but their molecular masses differed. It appears that the in-vitro
translation product is a polypeptide of 56 kDa while the in-vivo-labeled enzyme has a molecular mass of 47 kDa.
The authors greatly appreciate the skilled technical assistance of Renate deZacks and Gail Robinson. This research was supported
by the National Science Foundation through Grant No. DCB-8718873 and by the Department of Energy through Contract No. DE-AC02-76ER-01338. 相似文献