cellulase and ethylene

Plant-pathogenic fungi produce cellulases. However, little information is available on cellulase as an elicitor in plant-pathogen interactions. Here, an endocellulase (EG1) was isolated from Rhizoctonia solani. It contains a putative protein of 227 amino acids with a signal peptide and a family-45 glycosyl hydrolase domain. Its aspartic acid (Asp) residue at position 32 was changed to alanine (Ala), resulting in full loss of its catalytic activity. Wild-type and mutated forms of the endoglucanase were expressed in yeast and purified to homogeneity. The purified wild-type and mutant forms induced cell death in maize, tobacco and Arabidopsis leaves, and the transcription of three defence marker genes in maize and tobacco and 10 genes related to defence responses in maize. Moreover, they also induced the accumulation of reactive oxygen species (ROS), medium alkalinization, Ca(2+) accumulation and ethylene biosynthesis of suspension-cultured tobacco cells. Similarly, production of the EG1 wild-type and mutated forms in tobacco induced cell death using the Potato virus X (PVX) expression system. In vivo, expression of EG1 was also related to cell death during infection of maize by R. solani. These results provide direct evidence that the endoglucanase is an elicitor, but its enzymatic activity is not required for its elicitor activity.

Topics: Calcium; Cell Death; Cellulase; Cloning, Molecular; Cytosol; Electrophoresis, Polyacrylamide Gel; Ethylenes; Gene Expression Regulation, Plant; Genes, Fungal; Molecular Sequence Data; Mutant Proteins; Nicotiana; Pichia; Plant Diseases; Plant Leaves; Potexvirus; Reactive Oxygen Species; Rhizoctonia; Sequence Analysis, DNA; Zea mays

Papaya fruits (Carica papaya L. cv 'Sui you 2') harvested with < 5% yellow surface at the blossom end were fumigated with 60 microL/L of nitric oxide for 3 h and then stored at 20 degrees C with 85% relative humility for 20 days. The effects of nitric oxide treatment on ethylene production rate, the activities of cell wall softening related enzymes including polygalacturonase, pectin methyl esterase, pectate lyase and cellulase and the levels of hormones including indole acetic acid, abscisic acid, gibberellin and zeatin riboside were examined. The results showed that papaya fruits treated with nitric oxide had a significantly lower rate of ethylene production and a lesser loss of firmness during storage. A decrease in polygalacturonase, pectin methyl esterase, pectate lyase and cellulase activities was observed in nitric oxide treated fruit. In addition, the contents of indole acetic acid, abscisic acid and zeatin riboside were reduced in nitric oxide treated fruit, but no significant reduction in the level of gibberellin was found. These results indicate that nitric oxide treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of cell wall softening related enzymes and certain hormones.

Topics: Abscisic Acid; Carboxylic Ester Hydrolases; Carica; Cell Wall; Cellulase; Ethylenes; Food Storage; Free Radical Scavengers; Gibberellins; Indoleacetic Acids; Isopentenyladenosine; Nitric Oxide; Plant Growth Regulators; Polygalacturonase; Polysaccharide-Lyases

Although auxin and ethylene play pivotal roles in leaf abscission, the subsequent signaling molecules are poorly understood. This is mainly because it is difficult to effectively treat the intact abscission zone (AZ) with pharmacological reagents. We developed an in vitro experimental system that reproduces stress-induced leaf abscission in planta. In this system, 1-mm-thick petiole strips, encompassing the AZ, were separated within 4 days of abscission at the AZ through cell wall degradation in an auxin depletion- and ethylene-dependent manner. The system allowed us to show that hydrogen peroxide (H(2)O(2)) is involved in abscission signaling. Microscopic analyses revealed continuous H(2)O(2) production by AZ cells. H(2)O(2) scavengers and diphenylene iodonium, an inhibitor of NADPH oxidase, suppressed in vitro abscission and cellulase expression. Conversely, the application of H(2)O(2) promoted in vitro abscission and expression of cellulase. Ethephon-induced abscission was suppressed by inhibitors of H(2)O(2) production, whereas the expression of ethylene-responsive genes was unaffected by both H(2)O(2) and an H(2)O(2) inhibitor. These results indicated that H(2)O(2) acts downstream from ethylene in in vitro abscission signaling. In planta, salinity stress induced the expression of genes that respond to ethylene and reactive oxygen species, and also induced H(2)O(2) production at the AZ, which preceded leaf abscission. These results indicate that H(2)O(2) has roles in leaf abscission associated with ethylene both in vitro and in planta.

Topics: Capsicum; Cellulase; Ethylenes; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Hydrogen Peroxide; Indoleacetic Acids; Microscopy, Fluorescence; Onium Compounds; Organophosphorus Compounds; Plant Growth Regulators; Plant Leaves; RNA, Messenger; RNA, Plant; Sodium Chloride

Ethylene induced cotton (Gossypium hirsutum var RST-39) leaf abscission has been characterized by measuring the activities of ACC synthase (ACS, E.C. 4.4.1.14), ACC oxidase (ACO, E.C. 1.14.17.4) and cellulase (E.C. 3.2.1.4). In addition, a leaf abscission specific cDNA (GhCel1) has been cloned from cotton, which belongs to the alpha(2) subgroup of cellulases that possess a C-terminus carbohydrate-binding domain. Measurement of enzyme activity in the abscission zones of cotton leaf explants exposed to ethylene for 48h compared to non-treated controls indicated a more than 5-fold increase in the activity of ACS, 1.2-fold increase in the activity of ACO and about 2.7-fold increase in the activity of cellulase in the ethylene treated explants. This increase was accompanied by a substantial decrease in the force required to separate the petiole from the stem (break strength) and an increased accumulation of cellulase transcript in the abscission zone. Treatment of explants with 1-Methylcyclopropene (1-MCP) prior to ethylene resulted in significant inhibition of enzyme activities and transcript accumulation. It is concluded that ethylene response of cotton leaf abscission leads to higher cellulase expression and increased activities of ethylene biosynthesis enzymes in the abscission zone.

Topics: Abscisic Acid; Amino Acid Oxidoreductases; Cellulase; Cloning, Molecular; Cyclopropanes; Ethylenes; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gossypium; Lyases; Plant Growth Regulators; Plant Leaves; Plant Proteins; Protein Structure, Tertiary; Time Factors

Two aspects of the competence of abscission zone cells as a specific class of hormone target cell are examined. The first is the competence of these target cells to respond to a remote stele-generated signal, and whether ethylene acts in concert with this signal to initiate abscission of the primary leaf in Phaseolus vulgaris. The second is to extend the concept of dual control of abscission cell competence. Can the concept of developmental memory that is retained by abscission cell of Phaseolus vulgaris post-separation in terms of the inductive/repressive control of beta-1,4-glucan endohydrolase (cellulase) activity exerted by ethylene/auxin be extended to the rachis abscission zone cells of Sambucus nigra?. Abscission assays were performed using the leaf petiole-pulvinus explants of P. vulgaris with the distal pulvinus stele removed. These (-stele) explants do not separate when treated with ethylene and require a stele-generated signal from the distal pulvinus for separation at the leaf petiole-pulvinis abscission zone. Using these explants, the role of ethylene was examined, using the ethylene action blocker, 1-methyl cyclopropene, as well as the significance of the tissue from which the stele signal originates. Further, leaf rachis abscission explants were excised from the compound leaves of S. nigra, and changes in the activity of cellulase in response to added ethylene and auxin post-separation was examined.. The use of (-stele) explants has confirmed that ethylene, with the stele-generated signal, is essential for abscission. Neither ethylene alone nor the stelar signal alone is sufficient. Further, in addition to the leaf pulvinus distal to the abscission zone, mid-rib tissue that is excised from senescent or green mid-rib tissue can also generate a competent stelar signal. Experiments with rachis abscission explants of S. nigra have shown that auxin, when added to cells post-separation can retard cellulase activity, with activity re-established with subsequent ethylene treatment.. The triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues, in concert with ethylene, a requisite part of the process. Once evoked, abscission cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.

Topics: Biological Assay; Cell Extracts; Cellulase; Ethylenes; Glycoside Hydrolases; Indoleacetic Acids; Phaseolus; Pulvinus; Sambucus nigra; Signal Transduction

In peach (Prunus persica L. Batsch.) the degradation of the pectic compounds of the cell wall is considered to be the principal component responsible for fruit softening. Many genes encoding enzymes acting on the different polymers of the pectic matrix have been shown to be highly expressed during the late phases of softening, with polygalacturonase being the most important. Nevertheless, it is known that softening starts well before the ethylene climacteric rise which occurs concomitant with the maximal expression of the pectolytic enzymes. The cloning and characterization of PpEG4, an endo-beta-1,4-glucanase (EGase) gene preferentially expressed in preclimacteric fruits, are presented here. PpEG4 belongs to the group of EGases containing, at their carboxy-terminus, a peptide similar to the cellulose binding domain of microbial origin. This EGase is also expressed during abscission of both leaves and fruits. The effect of exogenous ethylene treatments on PpEG4 transcription is null in young fruits and negative in preclimacteric ones, while it is positive in abscission zones. Thus, the expression of PpEG4 seems to be more dependent on the type of separation process rather than being influenced by a direct hormone action. The ability of the PpEG4 regulatory sequences to drive transcription in cells undergoing separation events is also maintained in tomato, where about 3 kb of the gene promoter could drive the expression of gusA in preclimacteric fruits and in the fruit abscission zones.

Topics: Amino Acid Sequence; Base Sequence; Cellulase; Cloning, Molecular; Ethylenes; Fruit; Gene Expression Regulation, Plant; Genes, Plant; Genes, Reporter; Molecular Sequence Data; Naphthaleneacetic Acids; Plant Proteins; Plants, Genetically Modified; Promoter Regions, Genetic; Protein Structure, Tertiary; Prunus; Recombinant Fusion Proteins; Reproduction; Restriction Mapping; Sequence Alignment; Solanum lycopersicum

The sloughing of root cap cells from the root tip is important because it assists the growing root in penetrating the soil. Using a promoter-reporter (GUS) and RT-PCR analysis, we identified an endo-beta-1,4-glucanase (AtCel5) of Arabidopsis thaliana that is expressed exclusively in root cap cells of both primary and secondary roots. Expression is inhibited by high concentrations of IAA, both exogenous and internal, as well as by ABA. AtCel5 expression begins once the mature tissue pattern is established and continues for 3 weeks. GUS staining is observed in both root cap cells that are still attached and cells that have already been shed. Using AtCel5-GUS as a marker, we observed that the root cap cells begin to separate at the sides of the tip while the cells of the central region of the tip separate last. Separation involves sequential tiers of intact cells that separate from the periphery of the root tip. A homozygous T-DNA insertion mutant that does not express AtCel5 forms the root cap and sheds root cap cells but sloughing is less efficient compared to wild type. The reduction in sloughing in the mutant does not affect the overall growth performance of the plant in loose media. The modest effect of abolishing AtCel5 expression suggests that there are multiple redundant genes regulating the process of sloughing of the root cap, including AtCel3/At1g71380, the paralog of the AtCel5 gene that is also expressed in the root cap cells. Thus, these two endo-1,4-beta-D-glucanases may have a role in the sloughing of border cells from the root tip. We propose that AtCel5, provides a new molecular marker to further analyze the process of root cap cell separation and a root cap specific promoter for targeting to the environment genes with beneficial properties for plant growth.

Topics: Abscisic Acid; Amino Acid Sequence; Arabidopsis; Cellulase; DNA, Bacterial; Ethylenes; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genetic Markers; Glucuronidase; Indoleacetic Acids; Isoenzymes; Meristem; Microscopy, Electron, Scanning; Molecular Sequence Data; Mutagenesis, Insertional; Mutation; Phthalimides; Plant Growth Regulators; Plant Roots; Plants, Genetically Modified; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Homology, Amino Acid

In order to investigate the physiological role of ethylene in the initiation and subsequent progression of softening, pear fruit were treated with propylene, an analogue of ethylene or 1-methylcyclopropene (1-MCP), a gaseous inhibitor of ethylene action at the preclimacteric or ripening stages. The propylene treatment at the pre-ripe stage stimulated ethylene production and flesh softening while the 1-MCP treatment at the same stage markedly retarded the initiation of the ripening-related events. Moreover, 1-MCP treatment after the initiation of ripening markedly suppressed the subsequent flesh softening and ethylene production. These results clearly indicate that ethylene is not merely a by-product, but plays a crucial role in both the initiation and maintenance of regulating the softening process during ripening. The observations also suggest that ethylene in ripening is regulated entirely in an autocatalytic manner. The mRNA accumulation of pear polygalacturonases (PG) genes, PC-PG1 and PC-PG2, was in parallel with the pattern of fruit softening in both propylene and 1-MCP treatments. However, the expression pattern of pear endo-1,4-beta-D-glucanases (EGase) genes, PC-EG1 and PC-EG2, was not affected in both treatments. The results suggest that ethylene is required for PGs expression even in the late ripening stage, but not for EGases.

Topics: Alkenes; Cellulase; Cloning, Molecular; Cyclopropanes; Ethylenes; Fruit; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Isoenzymes; Molecular Sequence Data; Phylogeny; Plant Proteins; Polygalacturonase; Pyrus; RNA, Messenger

Site-directed mutagenesis was used to identify cis-acting elements that control hormonal and abscission-specific expression of the bean (Phaseolus vulgaris) abscission cellulase (BAC) promoter. Auxin inhibition of BAC promoter expression is at least in part controlled by a negatively regulated element and ethylene induction by a positively regulated element. One of a series of 15 different 10-bp mutations created in a 2.9-kb BAC promoter reduced reporter gene expression by 60%. The native sequence for this 10-bp mutation includes a TGA-type basic leucine zipper (bZIP) motif. Tandem ligation of three 18-bp BAC elements (Z-BAC), which includes the bZIP motif to a minimal -50 35S cauliflower mosaic virus promoter, enhanced expression in abscission zones (AZs) 13-fold over that of the minimal promoter alone. The native forward orientation of the Z-BAC elements was essential for high expression levels. Expression of the Z-BAC minimal construct was 3-fold greater in AZ than stems when compared with the expression levels of an internal control with an enhanced 35S cauliflower mosaic virus promoter. Polymerase chain reaction was used to identify three TGA-type bZIP transcription factors in an AZ cDNA library. One of these factors was of the class I type and two of the class II type. RNA-blot analysis was completed for these genes and electrophoretic mobility shift assays used to confirm their binding to the Z-BAC element. Electrophoretic mobility shift assay-binding affinity was greatest for the class I TGA-type bZIP factor. The results indicate a complex interaction of negative and positive regulating transcription factors that control BAC gene expression.

Topics: Base Sequence; Binding Sites; Binding, Competitive; Cellulase; DNA, Complementary; Ethylenes; Gene Expression Regulation, Plant; Leucine Zippers; Molecular Sequence Data; Mutagenesis, Site-Directed; Phaseolus; Promoter Regions, Genetic; Protein Binding; Regulatory Sequences, Nucleic Acid; Soybean Proteins; Transcription Factors

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease.

Topics: Adaptation, Physiological; Cellulase; Chitinases; Cotyledon; Cucurbitaceae; Ethylenes; Hot Temperature; Peroxidase; Plant Diseases; Protein Denaturation; Respiratory Burst; Salicylic Acid; Signal Transduction; Trichoderma

Treatment with ethylene accelerated the abscission of branches of Azolla filiculoides plants. An Azolla plantlet treated with ethylene at 10 microl liter(-1) divided into 4-5 fragments after a lag period of 6-8 h. Ethylene-induced abscission was effectively inhibited by cycloheximide and was associated with an increase in the activities of cellulase and polygalacturonase. At the fracture surface abscised after treatment with ethylene, dissolution of the primary walls of the abscission zone cells was apparent. However, the middle lamella between abscission zone cells was still present. Immunoelectron microscopy using anti-unesterified pectin (JIM5) and anti-methylesterified pectin (JIM7) monoclonal antibodies revealed the presence of both JIM5 and JIM7 epitopes in the wall between abscission zone cells of branches before abscission occurred. In the middle lamella remaining after ethylene-induced abscission, only JIM7 epitopes were observed. The features of ethylene-induced abscission described herein were different from those of the rapid abscission induced by sodium azide, which implies that they are mediated by different mechanisms. The possible mechanisms are discussed.

Topics: Cellulase; Cycloheximide; Dactinomycin; Ethylenes; Microscopy, Electron; Plant Development; Plants; Polygalacturonase; Sodium Azide

Plants of tomato (Lycopersicon esculentum Mill. cv. T5) were transformed with an antisense endo-1,4-beta-glucanase (cellulase, EC 3.2.1.4) Cel2 transgene under the control of the constitutive cauliflower mosaic virus 35S promoter in order to suppress mRNA accumulation of Cel2. In two independent transgenic lines, Cel2 mRNA abundance was reduced by >95% in ripe fruit pericarp and ca. 80% in fruit abscission zones relative to non-transgenic controls. In both transgenic lines the softening of antisense Cel2 fruit pericarp measured using stress-relaxation analysis was indistinguishable from control fruit. No differences in ethylene evolution were observed between fruit of control and antisense Cel2 genotypes. However, in fruit abscission zones the suppression of Cel2 mRNA accumulation caused a significant (P<0.001) increase in the force required to cause breakage of the abscission zone at 4 days post breaker, an increase of 27% in one transgenic line and of 46% in the other transgenic line. Thus the Cel2 gene product contributes to cell wall disassembly occurring in cell separation during fruit abscission, but its role, if any, in softening or textural changes occurring in fruit pericarp during ripening was not revealed by suppression of Cel2 gene expression.

Topics: Cellulase; Ethylenes; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genotype; Plants, Genetically Modified; RNA, Antisense; RNA, Messenger; Solanum lycopersicum

The endo-beta-1,4-glucanases, or cellulases, of higher plants are cell wall-associated enzymes believed to function in cell wall changes associated with the diverse processes of fruit ripening, organ abscission and cell elongation. We have isolated and characterized cDNA and genomic clones encoding a cellulase, PCEL1, which is abundant in ripening pepper fruit. Genomic analysis indicates that PCEL1 is encoded by a single gene, PCEL1, which belongs to a small, structurally divergent gene family. In ripening fruit, PCEL1 transcription is initiated at two distinct sites which yields overlapping mRNA species of 1.7 and 2.1 kb. High-level accumulation of both transcripts occurs in red fruit, while the 1.7 kb transcript is detected at a much lower level in stem and petiolar tissue. The increase in cellulase activity which is measured during fruit ripening is the product of PCEL1 expression and is tightly coupled to fruit reddening. High-level applications of ethylene serve to enhance the rate of ripening and the accumulation of PCEL1 mRNA. A direct role for ethylene in regulating PCEL1 expression is shown by the exclusive induction, in immature green fruit, of the 1.7 kb transcript in response to prolonged high-level exposure to ethylene--a pattern of expression not observed in fruit development on the vine.

Topics: Amino Acid Sequence; Base Sequence; Capsicum; Cellulase; DNA Primers; Ethylenes; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Genomic Library; Molecular Sequence Data; Oligodeoxyribonucleotides; Plant Stems; Plants, Medicinal; RNA, Messenger; Seeds; Transcription, Genetic

Cellulysin, a crude cellulase from the plant parasitic fungus Trichoderma viride, induces the biosynthesis of volatiles in higher plants (Nicotiana plumbaginifolia, Phaseolus lunatus, and Zea mays) when applied to cut petioles by the transpiration stream. The pattern of the emitted volatiles largely resembles that from a herbivore damage or treatment of the plants with jasmonic acid (JA) indicating that cellulysin acts via activation of the octadecanoid signalling pathway. The treatment with cellulysin raises the level of endogenous JA after 30 min and is followed by a transient emission of ethylene after 2-3 h. Volatile production becomes significant after 12-24 h. Inhibitors of the JA pathway effectively block the cellulysin-dependent volatile biosynthesis.

Topics: Cellulase; Chromatography, Gas; Cyclopentanes; Ethylenes; Fabaceae; Kinetics; Nicotiana; Oxylipins; Plants; Plants, Medicinal; Plants, Toxic; Signal Transduction; Stearic Acids; Trichoderma; Zea mays

Six cellulase genes were isolated from total RNA of the ethylene-treated tomato (Lycopersicon esculentum Mill.) flower abscission zone by reverse-transcription polymerase chain reaction using degenerate primers to conserved amino acid sequences from known plant cellulases. Four of the gene fragments are homologous to fruit pericarp cellulases. The other two are novel cellulase genes, referred to as Cel5 and Cel6. Breakstrength and cellulase gene expression were then analyzed in naturally abscising flowers and flower explants. In both naturally abscising flowers and flower explants induced to abscise in air or ethylene, both new cellulase mRNAs were correlated with flower shedding. Whereas the Cel5 mRNA increased in later stages of abscission, the Cel6 mRNA was present in nonabscising flowers and then decreased in the final stage of abscission. A third cellulase, Cel1, increased during the final stage of abscission in flower explants and yet did not increase during shedding in planta, although it was detectable at low levels in all abscission stages. Cel1 and Cel5 mRNA decreased 99% when indole-3-acetic acid was added during ethylene treatment, consistent with low levels of abscission (3%). In contrast, Cel6 mRNA increased slightly when indole-3-acetic acid was added. These results suggest that abscission is a multistep process involving both activated and repressed cellulase genes and that the relative importance of each cellulase in the process depends on the physiological conditions under which abscission takes place.

Topics: Amino Acid Sequence; Base Sequence; Cellulase; DNA Primers; Ethylenes; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Molecular Sequence Data; Polymerase Chain Reaction; Sequence Homology, Amino Acid; Solanum lycopersicum

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

Topics: Base Sequence; Cellulase; Conserved Sequence; Ethylenes; Fabaceae; Genes, Reporter; Glycine max; Indoleacetic Acids; Luciferases; Molecular Sequence Data; Plants, Genetically Modified; Plants, Medicinal; Polymerase Chain Reaction; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Messenger; Sequence Homology, Nucleic Acid; Solanum lycopersicum; Soybean Proteins; Thiosulfates; Transcription, Genetic

Ethylene promotes the abscission of leaves and the ripening of fruits in pepper plants, and in both events an increase in cellulase activity is observed. However, two enzyme isoforms (pI 7.2 and 8.5, respectively) are differentially involved in the two physiological phenomena. The pI 8.5 form has been purified from ripe fruits. It is a glycoprotein with an apparent molecular mass of 54 kDa. Two short peptides were sequenced and a very high homology to a tomato cellulase was observed. Polyclonal antibodies, raised against the purified enzyme, have allowed us to demonstrate that the observed ethylene-induced increase in cellulase activity is paralleled by de novo synthesis of protein. Three cDNAs (CX1, CX2 and CX3), encoding different cellulases, were obtained and characterized and their expression investigated. Accumulation of all three mRNAs is induced by ethylene treatment, though to different levels. CX1 is mainly expressed in ripe fruits while CX2 is especially found in abscission zones. CX3 accumulates at very low levels in activated abscission zones. Comparisons with other known cellulases demonstrate clear heterogeneity within the higher plant cellulases. Differences in ethylene inducibility and molecular structure suggest different physiological roles for cellulase in pepper plants.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Northern; Capsicum; Cellulase; Cloning, Molecular; Cross Reactions; Ethylenes; Gene Expression Regulation, Plant; Immunoblotting; Isoenzymes; Molecular Sequence Data; Plant Growth Regulators; Plant Leaves; Plant Stems; Plants, Medicinal; Polymerase Chain Reaction; Sequence Analysis; Sequence Homology, Amino Acid

Two structurally divergent endo-beta-1,4-glucanase (EGase) cDNAs were cloned from tomato. Although both cDNAs (Cel1 and Cel2) encode potentially glycosylated, basic proteins of 51 to 53 kD and possess multiple amino acid domains conserved in both plant and microbial EGases, Cel1 and Cel2 exhibit only 50% amino acid identity at the overall sequence level. Amino acid sequence comparisons to other plant EGases indicate that tomato Cel1 is most similar to bean abscission zone EGase (68%), whereas Cel2 exhibits greatest sequence identity to avocado fruit EGase (57%). Sequence comparisons suggest the presence of at least two structurally divergent EGase families in plants. Unlike ripening avocado fruit and bean abscission zones in which a single EGase mRNA predominates, EGase expression in tomato reflects the overlapping accumulation of both Cel1 and Cel2 transcripts in ripening fruit and in plant organs undergoing cell separation. Cel1 mRNA contributes significantly to total EGase mRNA accumulation within plant organs undergoing cell separation (abscission zones and mature anthers), whereas Cel2 mRNA is most abundant in ripening fruit. The overlapping expression of divergent EGase genes within a single species may suggest that multiple activities are required for the cooperative disassembly of cell wall components during fruit ripening, floral abscission, and anther dehiscence.

Topics: Amino Acid Sequence; Base Sequence; Cellulase; Ethylenes; Fruit; Gene Expression Regulation, Plant; Genes, Plant; Molecular Sequence Data; Plant Growth Regulators; RNA, Messenger; Sequence Homology, Amino Acid; Solanum lycopersicum; Tissue Distribution

Ethylene-induced abscission in leaf and fruit explants of peach involves different enzymes. In leaves abscission is accompanied by increased occurrence of cellulase forms differing in isoelectric point (pI 6.5 and 9.5). A polypeptide with a molecular mass of 51 kDa gives in a western blot a strong cross-reaction with an antibody raised against a maturation cellulase from avocado fruit. Cellulase activity is also found in abscising fruit explants but the amount is very low compared to that of the leaf explants. A northern analysis with a cellulase clone from avocado reveals the presence of two hybridizing mRNAs with a size of 2.2 kb and 1.8 kb, respectively. The steady-state level of the 2.2 kb mRNA is significantly increased by treatment with ethylene. Polygalacturonases are not detected in abscising leaves, but are strongly induced by ethylene in fruit explants. Of the three forms found, two are exopolygalacturonases while the third is an endoenzyme. Ethylene activates preferentially the endoenzyme and the basic exoenzyme but depresses the acid exopolygalacturonases. A northern analysis carried out with a cDNA coding for tomato endopolygalacturonase shows hybridization only with one endopolygalacturonase mRNA form in the fruit abscission zone. Treatment with ethylene causes an increase in the steady-state level of this mRNA. The differences in the enzyme patterns observed in fruit and leaf abscission zones and a differential enzyme induction suggest the feasibility to regulate fruit abscission in peach with the aid of antisense RNA genes.

Topics: Blotting, Northern; Blotting, Western; Cellulase; Ethylenes; Fruit; Isoelectric Focusing; Kinetics; Molecular Weight; Plant Physiological Phenomena; Plants; Poly A; Polygalacturonase; RNA; RNA, Messenger

We determined the primary structure of a tobacco beta-1,3-glucanase gene. The beta-1,3-glucanase gene has a single large intron, and the intron separates coding regions of the signal peptide and the mature enzyme. Analysis of the 5'-flanking region sequence revealed an 11 bp GC-rich element with perfect homology to the putative regulatory sequence of tobacco chitinase genes. RNA blot analysis showed that levels of mRNAs of beta-1,3-glucanase and chitinase are coordinately increased in response to ethylene and salicylic acid. Accumulation of beta-1,3-glucanase mRNA in suspension-cultured cells is rapidly induced at late logarithmic growth phase. Members of the tobacco beta-1,3-glucanase gene families are classified into two subfamilies. One of the subfamilies appeared to be transcriptionally inactive.

Topics: Amino Acid Sequence; Base Sequence; Cellulase; Chitinases; Ethylenes; Gene Expression Regulation; Genes, Plant; Molecular Sequence Data; Multigene Family; Nicotiana; Plant Proteins; Plants, Toxic; Salicylates; Salicylic Acid; Sequence Homology, Nucleic Acid; Transcription, Genetic