RESUMEN
Lipoxygenases (LOXs) are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones, CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino) cultivar "Yumeiren", encode 902 and 906 amino acids, respectively. Bioinformatics analysis showed that CmLOX10 and CmLOX13 included all of the typical LOX domains and shared 58.11% identity at the amino acid level with each other. The phylogenetic analysis revealed that CmLOX10 and CmLOX13 were members of the type 2 13-LOX subgroup which are known to be involved in biotic and abiotic stress. Heterologous expression of the full-length CmLOX10 and truncated CmLOX13 in Escherichia coli revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the lipoxygenase activities. The purified CmLOX10 and CmLOX13 recombinant enzymes exhibited maximum activity at different temperature and pH and both had higher affinity for linoleic acid than linolenic acid. Chromatogram analysis of reaction products from the CmLOX10 and CmLOX13 enzyme reaction revealed that both enzymes produced 13S-hydroperoxides when linoleic acid was used as substrate. Furthermore, the subcellular localization analysis by transient expression of the two LOX fusion proteins in tobacco leaves showed that CmLOX10 and CmLOX13 proteins were located in plasma membrane and chloroplasts respectively. We propose that the two lipoxygenases may play different functions in oriental melon during plant growth and development.
Asunto(s)
Cucumis/enzimología , Lipooxigenasa/metabolismo , Secuencia de Aminoácidos , ADN Complementario , Lipooxigenasa/química , Lipooxigenasa/genética , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/enzimologíaRESUMEN
Cucumber (Cucumis sativus L.), a kind of fruit usually harvested at the immature green stage, belongs to non-climacteric fruit. To investigate the contribution of abscisic acid (ABA) to cucumber fruit development and ripening, variation in ABA level was investigated and a peak in ABA level was found in pulp before fruit get fully ripe. To clarify this point further, exogenous ABA was applied to cucumber fruits at two different development stages. Results showed that ABA application at the turning stage promotes cucumber fruit ripening, while application at the immature green stage had inconspicuous effects. In addition, with the purpose of understanding the transcriptional regulation of ABA, two partial cDNAs of CsNCED1 and CsNCED2 encoding 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthetic pathway; one partial cDNA of CsCYP707A1 for 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA and two partial cDNAs of CsBG1 and CsBG2 for ß-glucosidase (BG) that hydrolyzes ABA glucose ester (ABA-GE) to release active ABA were cloned from cucumber. The DNA and deduced amino acid sequences of these obtained genes respectively showed high similarities to their homologous genes in other plants. Real-time PCR analysis revealed that ABA content may be regulated by its biosynthesis (CsNCEDs), catabolism (CsCYP707A1) and reactivation genes (CsBGs) at the transcriptional level during cucumber fruit development and ripening, in response to ABA application, dehydration and pollination, among which CsNCED1, CsCYP707A1 and CsBG1 were highly expressed in pulp and may play more important roles in regulating ABA metabolism.
Asunto(s)
Ácido Abscísico/metabolismo , Cucumis/metabolismo , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas de Plantas/genética , Transcripción Genética , Ácido Abscísico/genética , Ácido Abscísico/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cucumis/enzimología , Cucumis/genética , Cucumis/crecimiento & desarrollo , ADN Complementario , ADN de Plantas , Deshidratación , Frutas/enzimología , Frutas/crecimiento & desarrollo , Datos de Secuencia Molecular , Reguladores del Crecimiento de las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Polinización , Homología de SecuenciaRESUMEN
Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Clorofila/biosíntesis , Cucumis/enzimología , Oryza/enzimología , Oxidorreductasas/metabolismo , Zea mays/enzimología , Secuencia de Aminoácidos , Arabidopsis/genética , Clonación Molecular , Cucumis/genética , Activación Enzimática , Pruebas de Enzimas , Genes de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Sistemas de Lectura Abierta , Oryza/genética , Protoclorofilida/metabolismo , Protoporfirinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por Sustrato , Zea mays/genéticaRESUMEN
Oilseed germination is characterized by the mobilization of storage lipids as a carbon and energy source for embryonic growth. In addition to storage lipid degradation in germinating oilseeds via the direct action of a triacylglycerol lipase (TGL) on the storage lipids, a second degradation pathway that is dependent on a specific lipid body trilinoleate 13-lipoxygenase (13-LOX) has been proposed in several plant species. The activity of this specific 13-LOX leads first to the formation of ester lipid hydroperoxides. These hydroperoxy fatty acids are then preferentially cleaved off by a TGL and serve as a substrate for glyoxysomal ß-oxidation. As a prerequisite for triacylglycerol (TAG) mobilization, a partial degradation of the phospholipid monolayer and/or membrane proteins of the oil body has been discussed. Evidence has now been found for both processes: partial degradation of the proteins caleosin and oleosin was observed and simultaneously a patatin-like protein together with transient phospholipase (PLase) activity could be detected at the oil body membranes during germination. Moreover, in vitro experiments with isolated oil bodies from mature seeds revealed that the formation of 13-LOX-derived lipid peroxides in lipid body membranes is increased after incubation with the purified recombinant patatin-like protein. These experiments suggest that in vivo the degradation of storage lipids in cucumber cotyledons is promoted by the activity of a specific oil body PLase, which leads to an increased decomposition of the oil body membrane by the 13-LOX and thereby TAGs may be better accessible to LOX and TGL.
Asunto(s)
Cotiledón/enzimología , Cucumis/enzimología , Membranas Intracelulares/enzimología , Lipooxigenasa/metabolismo , Orgánulos/enzimología , Fosfolipasas/metabolismo , Proteínas de Plantas/metabolismo , Cotiledón/metabolismo , Cucumis/embriología , Cucumis/genética , Cucumis/metabolismo , Membranas Intracelulares/metabolismo , Metabolismo de los Lípidos , Lipooxigenasa/genética , Orgánulos/metabolismo , Fosfolipasas/genética , Proteínas de Plantas/genética , SemillasRESUMEN
In this research, biological function of CsNMAPK, encoding a mitogen-activated protein kinase of cucumber, was investigated under salt and osmotic stresses. Northern blot analysis showed that the expression of CsNMAPK was induced by salt and osmotic stresses in the cucumber root. In order to determine whether CsNMAPK was involved in plant tolerance to salt and osmotic stresses, transgenic tobacco plants constitutively overexpressing CsNMAPK were generated. Northern and Western blot analysis showed that strong signals were detected in the RNA and protein samples extracted from transgenic lines, whereas no signal was detected in the wild type tobacco, indicating that CsNMAPK was successfully transferred into tobacco genome and overexpressed. The results of seed germination showed that germination rates of transgenic lines were significantly higher than wild type under high salt and osmotic stresses. In addition, seed growth of transgenic lines was much better than wild type under salt and osmotic stresses. These results indicated that overexpression of CsNMAPK positively regulated plant tolerance to salt and osmotic stresses.
Asunto(s)
Cucumis/enzimología , Germinación/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nicotiana/metabolismo , Semillas/metabolismo , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Northern Blotting , Western Blotting , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Manitol/farmacología , Proteínas Quinasas Activadas por Mitógenos/genética , Ósmosis/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Semillas/efectos de los fármacos , Nicotiana/efectos de los fármacos , Nicotiana/genética , Nicotiana/crecimiento & desarrolloRESUMEN
Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of Cucumis sativus (cucumber) and Cucumis melo (melon) that have diverged over 40 My ago. The isolation of the genes for andromonoecy in Cucumis species provides a molecular basis for understanding how sexual systems arise and are maintained within and between species.
Asunto(s)
Cucumis/fisiología , Etilenos/biosíntesis , Liasas/metabolismo , Secuencia de Aminoácidos , Cucumis/enzimología , Cucumis/genética , Liasas/química , Liasas/genética , Datos de Secuencia Molecular , Reproducción , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Free as well as alginate immobilized urease was utilized for detection and quantitation of cadmium (Cd2+) in aqueous samples. Urease from the seeds of pumpkin (Cucumis melo), being a vegetable waste, was extracted and purified to apparent homogeneity (Sp. Activity 353 U/mg protein; A280/A260=1.12) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization and no leaching of the enzyme was found over a period of 15 days at 4 degrees C. Urease catalyzed urea hydrolysis by both soluble and immobilized enzyme revealed a clear dependence on the concentration of Cd2+. The inhibition caused by Cd2+ was non-competitive (Ki=1.41 x 10(-5) M). The time dependent inhibition both in the presence and in absence of Cd2+ ion revealed a biphasic inhibition in the activity. A Response Surface Methodology (RSM) for the parametric optimization of this process was performed using two-level-two-full factorial (2(2)), central composite design (CCD). The regression coefficient, regression equation and analysis of variance (ANOVA) was obtained using MINITAB 15 software. The predicted values thus obtained were closed to the experimental value indicating suitability of the model. In addition to this 3D response surface plot and isoresponse contour plot were helpful to predict the results by performing only limited set of experiments.
Asunto(s)
Cadmio/análisis , Proyectos de Investigación , Ureasa/química , Verduras/química , Agua/química , Alginatos/química , Cadmio/química , Cucumis/enzimología , Interpretación Estadística de Datos , Enzimas Inmovilizadas , Análisis Factorial , Hidrólisis , Cinética , Semillas/enzimología , Solubilidad , Soluciones , Propiedades de Superficie , Temperatura , Factores de Tiempo , Urea/metabolismo , Ureasa/antagonistas & inhibidoresRESUMEN
Kachri fruit, Cucumis trigonus Roxburghi, contains high protease activity and has been used as meat tenderizer in the Indian subcontinent. A 67 kDa serine protease from Kachri fruit was purified by DEAE-Sepharose and CM-Sepharose chromatography, whose optimum activity was at pH 11 and 70 degrees C. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The substrate specificity of the purified protease towards synthetic peptides was comparable to cucumisin, the first characterized subtilisin class plant protease from the sarcocarp of melon fruit (Cucumis melo). These characteristics, along with the N-terminal amino acid sequence, indicated that the isolated protease from Cucumis trigonus Roxburghi is a cucumisin homologue, which belongs to the serine protease family.
Asunto(s)
Cucumis/enzimología , Serina Endopeptidasas/química , Serina Endopeptidasas/aislamiento & purificación , Secuencia de Aminoácidos , Cucumis/química , Frutas/química , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Extractos Vegetales/química , Subtilisinas/químicaRESUMEN
Previous studies show that low temperature strongly induces suberin layers in the roots of chilling-sensitive cucumber plants, while in contrast, low temperature produces a much weaker induction of suberin layers in the roots of the chilling-tolerant figleaf gourd [S.H. Lee, G.C. Chung, S. Steudle, Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and -tolerant figleaf gourd, J. Exp. Bot. 56 (2005) 985-995; S.H. Lee, G.C. Chung, E. Steudle, Low temperature and mechanical stresses differently gate aquaporins of root cortical cells of chilling-sensitive cucumber and figleaf gourd, Plant Cell Environ. (2005) in press; S.J. Ahn, Y.J. Im, G.C. Chung, B.H. Cho, S.R. Suh, Physiological responses of grafted-cucumber leaves and rootstock roots affected by low root temperature, Scientia Hort. 81 (1999) 397-408]. Here, the effect of low temperature on fatty acid unsaturation and lipoxygenase activity was examined in cucumber and figleaf gourd. The double bond index demonstrated that membrane lipid unsaturation shows hyperbolic saturation curve in figleaf gourd roots while a biphasic response in cucumber roots to low temperature. In figleaf gourd, the hyperbolic response in the double bond index was primarily due to accumulation of linolenic acid. Chilling stress also significantly induced lipoxygenase activity in figleaf gourd roots. These results suggest that the degree of unsaturation of root plasma membrane lipids correlates positively with chilling-tolerance. Therefore, studies that compare the effects of chilling on cucumber and figleaf gourd may provide broad insight into stress response mechanisms in chilling-sensitive and chilling-tolerant plants. Furthermore, these studies may provide important information regarding the relationship between lipid unsaturation and lipoxygenase function/activity, and between lipoxygenase activity and water channeling during the response to chilling stress. The possible roles of these processes in chilling tolerance are discussed.
Asunto(s)
Frío , Cucumis/enzimología , Cucurbita/enzimología , Ácidos Grasos Insaturados/metabolismo , Lipooxigenasa/metabolismo , Raíces de Plantas/enzimología , Membrana Celular/enzimología , Activación EnzimáticaRESUMEN
A cucumber cDNA designated CsCPK5 and encoding a calcium-dependent protein kinase (CsCDPK5) was isolated and characterized. An open reading frame of 1542 bp was detected that could encode a protein of 514 amino acid residues with a calculated molecular mass of 56.5kDa. Comparison of the deduced amino acid sequence of CsCDPK5 with sequences of other CDPKs revealed the highest similarity (85%) to AtCDPK6. As described for other CDPKs, CsCDPK5 has a long variable domain preceding a catalytic domain, an autoinhibitory function domain, and a C-terminal calmodulin-domain containing 4 EF-hand calcium-binding motifs. The N-terminal long variable domain of CsCDPK5 does not contain the N-myristoylation motif, which is found in many CDPKs. The relative expression level of the CsCPK genes in various organs of cucumber plants and seedlings and in etiolated, excised cotyledons and hypocotyls following treatments with light and/or benzyladenine (BA), abscisic acid (ABA), gibberellic acid (GA) or indole acetic acid (IAA) was determined by northern analysis using the CsCPK5 cDNA probe. The CsCPK transcripts are most abundant in cucumber plant Leaves with less accumulation in cucumber seedling roots and hypocotyls and lowest Levels in cucumber plant flowers and seedling hooks and cotyledons. All phytohormones tested enhanced the accumulation of the transcripts 2-3-fold in etiolated cotyledons. On the other hand, levels of the transcripts increased to a lesser extent in both light and BA- or IAA-treated cotyledons and no effect was noted in response to light treatment with GA. In hypocotyls, no major changes in the relative levels of CsCPK transcripts were observed in the phytohormone-treated etiolated and light-exposed tissues, except an up-regulatory effect with IAA treatment in the etiolated and IAA, ABA and GA treatments in light-exposed hypocotyls. These observations suggest that exogenous phytohormones can up-regulate the CsCPK transcript levels in tissue-specific, and light-dependent and independent manners.
Asunto(s)
Cucumis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Quinasas/genética , ARN de Planta/genética , Secuencia de Aminoácidos , Clonación Molecular , Cucumis/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Biblioteca de Genes , Luz , Datos de Secuencia Molecular , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas Quinasas/biosíntesis , ARN de Planta/biosíntesis , Plantones/enzimología , Plantones/genética , Alineación de SecuenciaRESUMEN
Raffinose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage. These sugars are initially metabolised by alpha-galactosidases (alpha-gal). We report the cloning and functional expression of the first genes, CmAGA1 and CmAGA2, encoding for plant alpha-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raffinose and stachyose translocating species. The alkaline alpha-gal genes show very high sequence homology with a family of undefined 'seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to confirm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline alpha-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline alpha-gals shares little homology with the known prokaryotic and eukaryotic alpha-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step. This previously uncharacterised, plant-specific alpha-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate.
Asunto(s)
Cucumis/genética , alfa-Galactosidasa/genética , Secuencia de Aminoácidos , Clonación Molecular , Cucumis/clasificación , Cucumis/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Concentración de Iones de Hidrógeno , Cinética , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , alfa-Galactosidasa/química , alfa-Galactosidasa/metabolismoRESUMEN
The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.
Asunto(s)
Antioxidantes/metabolismo , Cucumis/anatomía & histología , Cucumis/metabolismo , Cucurbita/anatomía & histología , Cucurbita/metabolismo , Estrés Oxidativo , Secuencia de Aminoácidos , Transporte Biológico , Cucumis/enzimología , Cucurbita/enzimología , Bases de Datos Genéticas , Desastres , Enzimas/química , Enzimas/aislamiento & purificación , Enzimas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Análisis de Secuencia de Proteína , Agua/metabolismoRESUMEN
Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kb cucumisin promoter fused to a beta-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides -234 and -214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with the cucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possible cis-elements, an I-box-like sequence (AGATATGATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions -254 and -215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.