RESUMEN
The avocado fruit (Persea americana) has become a significant fruit in the human diet for its nutritional properties. However, the seed is a source of bioactive molecules and has been poorly utilized. Previously, we reported that the PaSn gene is expressed in the avocado seeds, a cysteine-rich antimicrobial peptide (GASA/Snakin), and demonstrated its antibacterial activity. In this work, we report the recombinant production of PaSn in the Escherichia coli system and evaluate its antifungal activity against plant and human pathogen fungi. The recombinant peptide showed antifungal activity at 200 µg/mL against phytopathogens Colletotrichum gloeosporioides and Fusarium oxysporum and human pathogens Candida albicans and C. glabrata. Our results demonstrate the usefulness of a prokaryotic expression system for avocado antimicrobial peptide production. In conclusion, the snakin PaSn could be helpful in the control of postharvest avocado and other fruits' fungal diseases and human fungal pathogens.
RESUMEN
Blackberry is an economically important crop in Mexico, and its yield is substantially reduced by gray mold, a disease caused by Botrytis cinerea. One of the means to obtain B. cinerea-resistant plants is gamma irradiation. Shoot tips of in vitro-micropropagated blackberry plants (Rubus fruticosus 'Tupy') were irradiated with five doses of Cobalt-60 gamma radiation (0, 15, 30, 45, and 60 Gy) and cultured on Murashige and Skoog basal medium containing 1.0 mg l-1 benzylaminopurine and 0.06 mg l-1 indole-3-butyric acid (MSB medium). After 28 days of culture, survival was evaluated to determine mean lethal dose (LD50), and 200 shoots were further irradiated at the determined LD50 (30.8 Gy). After 28 days, the surviving shoots were micropropagated on MSB medium for 60 days. Non-irradiated shoots were screened for the in vitro selection of resistant B. cinerea, exposing them to different concentrations of sterile culture filtrate of B. cinerea (0, 2, 4, 6, 8, and 10 g l-1) for 28 days to determine mean lethal concentration (LC50), and the irradiated surviving shoots were further exposed to the determined LC50 (4.6 g l-1). Three surviving lines (rfgum5, rfgum6, and rfgum17) that did not present changes compared with the control shoots were micropropagated to obtain plantlets, which were further subjected to in vitro resistance assays using detached leaves inoculated with B. cinerea (1×103 spores ml-1). Plants of rfgum5 and rfgum6 mutant lines were highly resistant and presented similar growth to control plants. Therefore, this methodology is useful to obtain B. cinerea-resistant blackberry plants.
RESUMEN
In recent years, it has been recognized that epigenetic alterations play an important role in the development and maintenance of cancer, including leukemias. Furthermore, it is known that these alterations are involved in the emergence of resistance to conventional chemotherapeutics. Consequently, molecules with an anticancer activity whose activity is ruled by epigenetic modifications are attractive to search for new therapies against cancer. The plant antimicrobial peptides have been widely evaluated as molecules with anticancer activity; however, the analysis of the epigenetic regulation induced by these molecules associated with this activity is scarce and still is an unexplored field. In this work, we show that the PaDef defensin, a plant antimicrobial peptide from Mexican avocado fruit (Persea americana var. drymifolia) is cytotoxic for Jurkat cell line from acute lymphoid leukemia cells, through an apoptotic process. PaDef inhibited cell viability in a concentration-dependent manner, with an IC50 = 47.3 µM. Treatment of Jurkat cells with PaDef (IC50) induced cell death by apoptosis dependent on caspases 8 and 9; besides, it was related to an increase in the production of reactive oxygen species and the loss of mitochondrial membrane potential. Interestingly, the inhibition of caspase activation by inhibitors of caspases 8 and 9 does not revert the reduction in viability, suggesting that other mechanisms, in addition to caspase activity, could be participating in the PaDef cytotoxic effect. Also, the modifications in the histone 3 tails induced by PaDef in Jurkat cells were evaluated, specifically acetylation and methylation. PaDef increased global histone 3 acetylation and lysine 9 specific marks (2-fold and up to 4-fold, respectively). These effects correlated with the reduction of the Histone Deacetylase activity (HDAC, â¼50%). Based on methylation marks, PaDef treatment increased lysine 9 di- and tri-methylation tags (2-fold in both cases). The epigenetic modulation induced by PaDef on Jurkat cells could be related to the chromatin compaction-decompaction promoting gene expression or repression; however, further studies are necessary to correlate these marks with the transcription of specific genes. Therefore, the study of new molecules that may have anticancer activity through epigenetic modulation is interesting.
RESUMEN
Avocado (Persea americana Mill.) is a tree native from central and eastern México that belongs to the Lauraceae family. Avocado has three botanical varieties known as Mexican (P. americana var. drymifolia), West Indian (P. americana var. americana), and Guatemalan (P. americana var. guatemalensis). It is an oil-rich fruit appreciated worldwide because of its nutritional value and the content of bioactive molecules. Several avocado molecules show attractive activities of interest in medicine. Avocado fatty acids have beneficial effects on cardiovascular disease risk factors. Besides, this fruit possesses a high content of carotenoids and phenolic compounds with possible antifungal, anti-cancer and antioxidant activities. Moreover, several metabolites have been reported with anti-inflammatory effects. Also, an unsaponifiable fraction of avocado in combination with soybean oil is used for the treatment of osteoarthritis. The Mexican variety is native from México and is characterized by the anise aroma in leaves and by small thin-skinned fruits of rich flavor and excellent quality. However, the study of the bioactive molecules of the fruit has not been addressed in detail. In this work, we achieved a literature review on the inflammatory, immunomodulatory and cytotoxic properties of long-chain fatty acids and derivatives from Mexican avocado seed. Also, the antioxidant and anti-inflammatory properties of the oil extracted from the avocado seed are referred. Finally, the antimicrobial, immunomodulatory, and cytotoxic activities of some antimicrobial peptides expressed in the fruit are reviewed.
Asunto(s)
Antiinfecciosos , Persea , Antiinfecciosos/farmacología , Frutas , México , SemillasRESUMEN
Intracellular lipid droplets (LD) provide the oil storage mechanism of plants. They are found within seeds as individual structures, even under conditions of cold stress and dehydration, due to the protein that covers them. This protein, called oleosin, is found exclusively in plants and has been widely studied in seeds. Avocado fruits (Persea americana Mill.) are rich in oil, which is stored in the mesocarp, not in the seeds. The presence of oleosin in the mesocarp tissue of avocadoes has been reported, but its physiological role is still unknown. In this study, we identify two genes that code for oleosin in the mesocarp of the native Mexican avocado. These sequences are very different from those of seed oleosins. Both genes are expressed during fruit ripening, while one, PaOle1, has the highest expression in the green fruit stage. The protein of PaOle1 is stable during the fruit ripening process and covers all the mesocarp LDs. The expression of PaOle1 gene and protein is organ specific to avocado mesocarp. Among avocadoes varieties oleosin abundance is directly related to oil content.
Asunto(s)
Persea , Frutas/genética , Persea/genética , Plantas , Semillas/genéticaRESUMEN
Studies on avocado oil have focused on the most common commercial cultivars, Hass, Fuerte, and Bacon, rather than the less common varieties, P. americana var. drymifolia and P. americana var. americana, even though the drymifolia variety has a higher oil content and the americana variety is the most common avocado grown in the tropics. The most abundant storage structures for plant oils are the oleosomes, and the aim of this study was to determine the oleosome size, oil yield, and fatty acid composition of the americana and drymifolia varieties, using the Hass cultivar as a reference. Differences were found between the three avocado types for 1) oil yield, with drymifolia having higher and americana lower oil content (p < 0.05%), 2) oleosome size, with Hass having a larger (41.53 µm) and americana a smaller (11.96 µm) size, and 3) fatty acid composition, with the americana and drymifolia varieties showing less monounsaturated fatty acids (oleic) and more polyunsaturated fatty acids (linoleic) and saturated fatty acids (palmitic); while Hass had a high level (60%) of monounsaturated fatty acids. Small but significant differences were also found between oleosome and mesocarp oils isolated from the drymifolia and Hass types.
Asunto(s)
Ácidos Grasos Insaturados/análisis , Gotas Lipídicas/química , Persea/química , Aceites de Plantas/análisis , Ácidos Grasos Insaturados/aislamiento & purificación , Persea/clasificación , Aceites de Plantas/aislamiento & purificación , Triglicéridos/análisis , Triglicéridos/aislamiento & purificaciónRESUMEN
Plant defensins, a group of antimicrobial peptides, show selective cytotoxicity toward cancer cells. However, their mechanisms of action remain poorly understood. Here, we evaluated the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on K562 chronic myeloid leukemia cells and analyzed the pathway involved in the induction of cell death. The defensin PaDef was not cytotoxic against human PBMCs; however, it was cytotoxic for K562 cell line (IC50â¯=â¯97.3⯵g/ml) activating apoptosis at 12â¯h. PaDef did not affect the mitochondrial membrane potential (ΔΨm), neither the transmembranal potential or the release of intracellular calcium. Also, PaDef induced gene expression of caspase 8 (â¼2 fold), TNF-α (â¼4 fold) and TNFR1 (â¼10 fold). In addition, the activation of caspase 8 was detected at 24â¯h, whereas caspase 9 activity was not modified, suggesting that the extrinsic apoptosis pathway could be activated. In conclusion, PaDef induces apoptosis on K562 cells, which is related to the activation of caspase 8 and involves the participation of TNF-α, which is a novel property for a plant defensin.
Asunto(s)
Antiinfecciosos/farmacología , Apoptosis/efectos de los fármacos , Defensinas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Persea/química , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Células Tumorales CultivadasRESUMEN
Antimicrobial peptides (AMPs) are cytotoxic to cancer cells; however, mainly the effects of AMPs from animals have been evaluated. In this work, we assessed the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on the MCF-7 cancer cell line (a breast cancer cell line) and evaluated its mechanism of action. PaDef inhibited the viability of MCF-7 cells in a concentration-dependent manner, with an IC50=141.62µg/ml. The viability of normal peripheral blood mononuclear cells was unaffected by this AMP. Additionally, PaDef induced apoptosis in MCF-7 cells in a time-dependent manner, but did not affect the membrane potential or calcium flow. In addition, PaDef IC50 induced the expression of cytochrome c, Apaf-1, and the caspase 7 and 9 genes. Likewise, this defensin induced the loss of mitochondrial Δψm and increased the phosphorylation of MAPK p38, which may lead to MCF-7 apoptosis by the intrinsic pathway. This is the first report of an avocado defensin inducing intrinsic apoptosis in cancer cells, which suggests that it could be a potential therapeutic molecule in the treatment of cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Defensinas/farmacología , Persea/química , Proteínas de Plantas/farmacología , Apoptosis/genética , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Membrana Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Avocado (Persea americana) is an economically important tropical fruit considered to be a good source of fatty acids. Despite its importance, the molecular and cellular characterization of biochemical and developmental processes in avocado is limited due to the lack of transcriptome and genomic information. RESULTS: The transcriptomes of seeds, roots, stems, leaves, aerial buds and flowers were determined using different sequencing platforms. Additionally, the transcriptomes of three different stages of fruit ripening (pre-climacteric, climacteric and post-climacteric) were also analyzed. The analysis of the RNAseqatlas presented here reveals strong differences in gene expression patterns between different organs, especially between root and flower, but also reveals similarities among the gene expression patterns in other organs, such as stem, leaves and aerial buds (vegetative organs) or seed and fruit (storage organs). Important regulators, functional categories, and differentially expressed genes involved in avocado fruit ripening were identified. Additionally, to demonstrate the utility of the avocado gene expression atlas, we investigated the expression patterns of genes implicated in fatty acid metabolism and fruit ripening. CONCLUSIONS: A description of transcriptomic changes occurring during fruit ripening was obtained in Mexican avocado, contributing to a dynamic view of the expression patterns of genes involved in fatty acid biosynthesis and the fruit ripening process.
Asunto(s)
Ácidos Grasos/metabolismo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Persea/genética , Proteínas de Plantas/genética , Análisis de Secuencia de ARN/métodos , Flores/genética , Flores/crecimiento & desarrollo , Frutas/genética , Frutas/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Persea/química , Persea/crecimiento & desarrollo , Persea/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms and are promising candidates to treat infections caused by pathogenic bacteria to animals and humans. AMPs also display anticancer activities because of their ability to inactivate a wide range of cancer cells. Cancer remains a cause of high morbidity and mortality worldwide. Therefore, the development of methods for its control is desirable. Attractive alternatives include plant AMP thionins, defensins, and cyclotides, which have anticancer activities. Here, we provide an overview of plant AMPs anticancer activities, with an emphasis on their mode of action, their selectivity, and their efficacy.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/uso terapéutico , Antineoplásicos/uso terapéutico , Inmunidad Innata/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Antiinfecciosos/uso terapéutico , Ciclotidas/uso terapéutico , Defensinas/uso terapéutico , Humanos , Inmunidad Innata/inmunología , Neoplasias/patología , Plantas/química , Tioninas/uso terapéuticoRESUMEN
Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP) represent attractive control agents. Mexican avocado (Persea americana var. drymifolia) is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp) encoding a protein (78 aa) homologous with plant defensins (>80%). We expressed the defensin PaDef cDNA (pBME3) in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 µg/mL of total protein from clones (>55%). Also, S. aureus viability was inhibited from 50 µg/mL total protein (27-38%) but was more evident at 100 µg/mL (52-65%). This inhibition was higher than the effect showed by polyclonal population (~23%). Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Defensinas/farmacología , Escherichia coli/efectos de los fármacos , Proteínas de Plantas/farmacología , Staphylococcus aureus/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Secuencia de Bases , Candida albicans/efectos de los fármacos , Bovinos , Línea Celular , ADN de Plantas/genética , Defensinas/química , Defensinas/genética , Células Endoteliales/metabolismo , Expresión Génica , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Persea/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Medicinales/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Avocado is one of the most important fruits in the world. Avocado "native mexicano" (Persea americana var. drymifolia) seeds are widely used in the propagation of this plant and are the primary source of rootstocks globally for a variety of avocado cultivars, such as the Hass avocado. Here, we report the isolation of 5005 ESTs from the 5' ends of P. americana var. drymifolia seed cDNA clones representing 1584 possible unigenes. These avocado seed ESTs were compared with the avocado flower EST library, and we detected several genes that are expressed either in both tissues or only in the seed. The snakin gene, which encodes an element of the innate immune response in plants, was one of those most frequently found among the seed ESTs, and this suggests that it is abundantly expressed in the avocado seed. We expressed the snakin gene in a heterologous system, namely the bovine endothelial cell line BVE-E6E7. Conditioned media from transfected BVE-E6E7 cells showed antimicrobial activity against strains of Escherichia coli and Staphylococcus aureus. This is the first study of the function of the snakin gene in plant seed tissue, and our observations suggest that this gene might play a protective role in the avocado seed.
Asunto(s)
Etiquetas de Secuencia Expresada/metabolismo , Genes de Plantas , Persea/genética , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Proteínas de Plantas/genética , Semillas/metabolismo , Adaptación Fisiológica/genética , Animales , Antiinfecciosos/metabolismo , Bovinos , Línea Celular , ADN Complementario , Escherichia coli , Flores/metabolismo , Expresión Génica , Péptidos/genética , Péptidos/metabolismo , Persea/metabolismo , Proteínas de Plantas/metabolismo , StaphylococcusRESUMEN
BACKGROUND: Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a ß-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel ß-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides.The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively. RESULTS: Here we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides. CONCLUSIONS: The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin lyases. A time-course analysis revealed significant differences between the two fungal races in terms of the expression of Clpnl2 encoding for pectin lyase 2. According to the results, pectin lyases from bacteria and fungi separated early during evolution. Likewise, the enzymes from fungi and oomycetes diverged in accordance with their differing lifestyles. It is possible that the diversity and nature of the assimilatory carbon substrates processed by these organisms played a determinant role in this phenomenon.
Asunto(s)
Colletotrichum/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Polisacárido Liasas/genética , Clonación Molecular , Colletotrichum/genética , ADN Complementario , Biblioteca de Genes , Datos de Secuencia Molecular , Filogenia , Polisacárido Liasas/química , Polisacárido Liasas/metabolismo , Análisis de Secuencia de ADNRESUMEN
Transgenic plants are able to express molecules with antigenic properties. In recent years, this has led the pharmaceutical industry to use plants as alternative systems for the production of recombinant proteins. Plant-produced recombinant proteins can have important applications in therapeutics, such as in the treatment of rheumatoid arthritis (RA). In this study, the mycobacterial HSP65 protein expressed in tobacco plants was found to be effective as a treatment for adjuvant-induced arthritis (AIA). We cloned the hsp65 gene from Mycobacterium leprae into plasmid pCAMBIA 2301 under the control of the double 35S promoter from cauliflower mosaic virus. Agrobacterium tumefaciens bearing the pChsp65 plasmid was used to transform tobacco plants. Incorporation of the hsp65 gene was confirmed by PCR, reverse transcription-PCR, histochemistry, and western blot analyses in several transgenic lines of tobacco plants. Oral treatment of AIA rats with the HSP65 protein allowed them to recover body weight and joint inflammation was reduced. Our results suggest a synergistic effect between the HSP65 expressed protein and metabolites presents in tobacco plants.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Proteínas Bacterianas/uso terapéutico , Chaperonina 60/uso terapéutico , Nicotiana/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Administración Oral , Agrobacterium tumefaciens/genética , Animales , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Chaperonina 60/administración & dosificación , Chaperonina 60/genética , Chaperonina 60/metabolismo , Humanos , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Plantas Modificadas Genéticamente/genética , Plásmidos , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/genética , Resultado del TratamientoRESUMEN
Papaya (Carica papaya) is a very important crop in many tropical countries but it is highly susceptible to parasitic diseases, physiological disorders, mechanical damage and fruit overripening. Here we report a study on ACC oxidase cosuppression and its effects on papaya fruit ripening. Papaya ACC oxidase was isolated using PCR and embriogenic cells transformed by biolistic using the CaMV 35S promoter to drive the expression of the PCR fragment in sense orientation. Fifty transgenic lines were recovered and 20 of those were grown under field conditions. Southern analysis showed incorporation of the transgene in different copy numbers in the papaya genome. Fruits were evaluated in terms of texture (firmness), colour development, respiration and ethylene production. A sharp reduction in ethylene and CO2 production was detected, whereas softening and colour development of the peel were also altered. Overall, transgenic fruits showed a delay in ripening rate. A reduction in mRNA level for ACC oxidase in transgenic fruit was clearly detectable by northern blot. More studies are necessary before this technology can be used to extend the shelf life of papaya fruit.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Carica/enzimología , Carica/crecimiento & desarrollo , Etilenos/metabolismo , Frutas/enzimología , Frutas/crecimiento & desarrollo , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Aminoácido Oxidorreductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Dióxido de Carbono/metabolismo , Carica/genética , Respiración de la Célula , Frutas/genética , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Interferencia de ARNRESUMEN
In this paper are presented structural analysis and expression studies of one genomic clone encoding a 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) from papaya. Using RT-PCR amplification of ACC oxidase cDNAs from ripe papaya, a product of 800 bp was obtained, which after sequence analysis was found to code for a protein highly homologous to ACC oxidase proteins. This PCR product was used as a probe for screening a genomic library, and two different groups of clones were obtained as indicated by restriction mapping. One clone (CPACCO-1) was selected for further study and fully sequenced. Comparison of this sequence with the PCR product and other cloned ACC oxidase genes revealed that CPACCO-1 encoded the transcript in four exons interrupted by three introns. Southern blot analysis showed one or two major bands hybridized to the PCR probe, suggesting that the ACC oxidase gene is present in one or two copies in the papaya genome. By northern blot analysis it was found that the ACC oxidase transcripts appear in the pulp earlier than in the peel, suggesting a developmental regulation. A wounding experiment revealed the highest expression of this gene by 2 h. Transcriptional regulation by ethylene could be due to the presence of a putative GCC box in the promoter region.