Cellulase and β-galactosidase activities in 'golden' and 'gran golden' papaya softening

O objetivo desse trabalho foi avaliar a ação das enzimas celulase e β-galactosidase em relação à perda de firmeza dessas cultivares de mamões 'Gran Golden' e 'Golden' devido a relatos de uma perda de firmeza diferenciada entre as cvs. Os frutos foram armazenados a 25ºC e analisados diariamente quanto à firmeza da polpa e à atividade enzimática da celulase e β-galactosidase durante 8 dias. Os resultados de firmeza da polpa e atividade enzimática foram submetidos às análises de correlação e regressão. No 4º dia pós-colheita os mamões 'Golden' apresentaram firmeza média de 60,6 N e os 'Gran Golden' 31,1 N e a um aumento da atividade da celulase e da β-galactosidase. Os dados gerados neste trabalho sugerem que as enzimas celulase e β-galactosidase atuam diferentemente no processo de perda de firmeza dos frutos das cultivares Goldene Gran Golden. Aantecipaçãonaperdade firmezade 'Gran Golden' pode estar relacionada com a maior atividade dessas enzimas.It has been reported by orchards from the north of Espírito Santo state that 'Gran Golden' papaya loses firmness faster than 'Golden'. The goal of this work was to evaluate the action of cellulase and β-galactosidase related to the softening on papaya. The fruits have been stored at 25ºC and firmness and enzymes activities were daily analyzed during 8 days. The results were submitted to correlation and regression analysis. The activity of cellulase and β-galactosidase had increased for both cultivars. The 4th postharvest day showed that 'Golden' firmness was 6.18 while 'Gran Golden' was 31.1 N. Fruit softening in 'Gran Golden' was intense and the fruit was very soft at ripe stage. These works show that hydrolytic enzymes cellulase and β-galactosidase act differently in the softening process in 'Golden'and 'Gran Golden'papaya. The flesh firmness on 'Gran Golden' is related to the increased activity of these enzymes. These results can help to choose which cultivar to produce in relation to shelf-life and fruit quality at commercialization places.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Banco do NordesteBrape

Different planting spacings and fertilization levels on the activity of nitrate reductase in leaves of papaya hybrid UENF/CALIMAN-01

O objetivo deste trabalho foi avaliar o efeito de diferentes espaçamentos de plantio e de níveis de adubação NPK sobre a atividade da redutase do nitrato (RN) nas folhas do híbrido de mamoeiro UENF/CALIMAN-01 , visando a sugerir possível ajuste em seu manejo de adubação nitrogenada, no sentido de maximizar a eficiência do uso do nitrogênio. O experimento foi conduzido na fazenda Caliman Agrícola S.A., no município de Linhares - ES. Utilizou-se o delineamento estatístico experimental em blocos casualizados, com esquema fatorial, com três espaçamentos de plantio entre plantas (E1 = 1,8 m; E2 = 2,25 m, e E3 = 2,7 m), cinco níveis de adubação NPK convencional (A1 = 80% do padrão; A2 = 100% padrão da empresa; A3 = 120% do padrão; A4 = 140% do padrão, e A5 = 160% do padrão) e cinco períodos de avaliação (meses de março a julho). O padrão de adubação NPK da empresa consiste em 350; 105 e 660 kg ha-1ano-1 de sulfato de amônio (20% de N), superfosfato simples (18% de P) e cloreto de potássio (60% de K), respectivamente. Os dados obtidos para a atividade da RN foram submetidos a uma análise de variância e teste de médias. Dentre os tratamentos testados, o nível A1 (80% do padrão), independentemente do espaçamento, poderia ser indicado no manejo do híbrido de mamoeiro UENF/CALIMAN-01, pois em todos eles a atividade da redutase do nitrato, em praticamente todos os períodos avaliados, apresentou valores adequados, ou até mesmo superiores aos encontrados na literatura em cultivares de mamoeiro. A redução da adubação NPK pôde ser justificada, uma vez que não houve diferença na produtividade das plantas entre os tratamentos avaliados.The objective of this work was to evaluate the effect of different planting spacings and levels of NPK manuring on the activity of the nitrate reductase (NR) in the leaves of the papaya hybrid UENF/CALIMAN-01, aiming to suggest possible adjustment in the handling of nitrogen fertilization, in the sense of maximizing the efficiency of the use of the nitrogen. The experiment was driven in the Caliman Agrícola S.A. farm, in the municipal district of Linhares- ES. A complete block design, factorial, with three planting spacings among plants (E1 = 1.8 m, E2 = 2.25 m and E3 = 2.7 m), five levels of NPK conventional manuring (A1 = 80% of the standard, A2 = 100% of the company standard, A3 = 120% of the standard, A4 = 140% of the standard and A5 = 160% of the standard) and five evaluation periods ( from March to July) was used. The standard of the company NPK manuring consists of 350, 105 and 660 Kg ha-1year -1 of sulfate of ammonium (20% of N), simple superphosphate (18% of P) and potassium chloride (60% of K), respectively. The data obtained for the activity of NR were submitted to a variance analysis and average test. Among the tested treatments, A1 (80% of standard), independent of the spacing, could be indicated in the handling of the hybrid UENF/CALIMAN-01, because in all of them the activity of the nitrate reductase in, practically, all of the appraised periods, presented appropriate values, or even, superiors to the ones found in the literature for the papaya tree. The reduction of NPK fertilization could be justified, once that did not have difference in the productivity of the plants among the evaluated treatments.FINEPCNPq(FAPERJ) Fundacao de Amparo a Pesquisa do Estado do Rio de Janeir

Cloning and expression analysis of papaya genes encoding proteins with inhibitory activity against fungal polygalacturonases

As proteínas inibidoras de poligalacturonases (PGIPs) presentes na parede celular são capazes de limitar o potencial destrutivo da poligalacturonase (PG) fúngica e, assim, constituem um tipo importante dentre os diversos sistemas de defesa do tecido vegetal frente à infecção fúngica. No mamão, o ataque fitopatogênico é o principal causador de danos pós-colheita, e sua alta susceptibilidade pode estar relacionada com a baixa eficácia ou pouca abundância dos meios de defesa anti-fitopatogênica. Uma vez que isso pode estar relacionado com as PGIPs e nada se conhece sobre o papel dessas proteínas nesse fruto, o objetivo do trabalho foi clonar os genes das PGIPs de mamoeiro e definir seu padrão de expressão em diferentes órgãos e tecidos e ao longo do amadurecimento. Para tanto, foram identificadas no genoma do mamoeiro, a partir de critérios que definem a identidade de uma PGIP, duas prováveis sequências dentre 13 candidatas iniciais. Ambas foram clonadas a partir das sequências genômicas e de cDNA, sequenciadas e sua identidade confirmada, sendo denominadas Cppgip4 e Cppgip6. As análises de expressão relativa em diversos tecidos e idades fisiológicas do mamoeiro demonstraram que os dois genes apresentaram diminuição da expressão com o desenvolvimento dos frutos, sendo que com a polpa apresentou redução dos níveis de expressão relativa de Cppgip4 em até 18 vezes dos 30 dias pós-antese (DPA) ao 9 dias pós-colheita (DPC). Na casca também houve redução significativa da expressão com o desenvolvimento. Para a expressão absoluta, nos frutos, sementes, caules, raízes e folhas, o número de cópias de ambos os transcritos decresceu com o desenvolvimento, sendo cerca de cem mil vezes mais abundante para Cppgip6 que para Cppgip4. As tentativas de expressão de proteínas recombinantes em Pichia pastoris não geraram resultado positivo, provavelmente em virtude das condições ideais de indução ainda não terem sido estabelecidas corretamente para o ensaio. A atividade de PGIPs extraídas diretamente do tecido foi medida por análise de difusão em ágar empregando pectinase de Aspergillus niger e revelou uma tendência à diminuição da porcentagem de inibição à medida que os frutos se desenvolveram, em concordância com os resultados da análise por qPCR. O conjunto de resultados sugere que a expressão varia com o estádio de desenvolvimento do fruto e é tecido-específica, possivelmente em resposta à diferente susceptibilidade dos tecidos ao ataque fitopatogênico, indicando que menores níveis de transcritos e atividade no amadurecimento, período de maior susceptibilidade, poderiam sinalizar para a regulação do processo degradativo marcando o início da senescência.Polygalacturonase inhibiting proteins (PGIPs) present in plant cell walls are able to inhibit the destructive action of fungal polygalacturonase (PG). In this way, they constitute an important type of plant defense system against fungal infections. In papaya fruit, the pathogenic attack is the main cause of post harvesting loss, and its high susceptibility may be related to the low efficiency or low abundance of anti-phytopathogenic defense. Since this fact could be related to PGIPs expression and little is known about the response of these proteins in the fruit, the aim of the present work was to clone the genes of PGIPs papaya fruit and set their expression pattern in different organs and tissues throughout fruit ripening. Thus, two probable PGIP sequences among 13 initial candidates were identified in the papaya genome by using specific criteria. Both sequences were cloned from cDNA and genomic samples, sequenced and confirmed its identity, and then being named Cppgip4 and Cppgip6. Analysis of relative expression in various tissues at different physiological stages demonstrated that both genes were down regulated during fruit development. The relative expression levels of Cppgip4 in papaya pulp was reduced by 18 times from the 30 days post-anthesis (DPA) to the 9 days post-harvest (DPH). Similarly, gene expression in papaya peel was significant down regulated during fruit development. Absolute expression analysis revealed gene expressions in the fruit pulp, seed, stem, root and leaf were also down regulated within development. Moreover, Cppgip6 gene expression was a hundred thousand times more abundant than Cppgip4. The recombinant protein expression in Pichia pastoris did not result positive, probably because of the ideal conditions of induction have not been properly established the yet. The activity of PGIPs extracted directly from the tissue was measured by the agar diffusion assay using pectinase from Aspergillus niger and showed decrease of inhibition during fruit developed in accordance with the results of the qPCR analysis. Based on the results it is possible to suggest the expression of these genes varies temporally with the developmental stage of the fruit and is tissue-specific, possibly in response to the different susceptibility of tissues to pathogenic attack. In addition, the lowest levels of PGIP expression were achieved at the fruit ripening, when the susceptibility to fungal infection is high and could signal for regulating the degradation process characterized by the onset of senescence

Cloning and expression analysis of papaya genes encoding proteins with inhibitory activity against fungal polygalacturonases

As proteínas inibidoras de poligalacturonases (PGIPs) presentes na parede celular são capazes de limitar o potencial destrutivo da poligalacturonase (PG) fúngica e, assim, constituem um tipo importante dentre os diversos sistemas de defesa do tecido vegetal frente à infecção fúngica. No mamão, o ataque fitopatogênico é o principal causador de danos pós-colheita, e sua alta susceptibilidade pode estar relacionada com a baixa eficácia ou pouca abundância dos meios de defesa anti-fitopatogênica. Uma vez que isso pode estar relacionado com as PGIPs e nada se conhece sobre o papel dessas proteínas nesse fruto, o objetivo do trabalho foi clonar os genes das PGIPs de mamoeiro e definir seu padrão de expressão em diferentes órgãos e tecidos e ao longo do amadurecimento. Para tanto, foram identificadas no genoma do mamoeiro, a partir de critérios que definem a identidade de uma PGIP, duas prováveis sequências dentre 13 candidatas iniciais. Ambas foram clonadas a partir das sequências genômicas e de cDNA, sequenciadas e sua identidade confirmada, sendo denominadas Cppgip4 e Cppgip6. As análises de expressão relativa em diversos tecidos e idades fisiológicas do mamoeiro demonstraram que os dois genes apresentaram diminuição da expressão com o desenvolvimento dos frutos, sendo que com a polpa apresentou redução dos níveis de expressão relativa de Cppgip4 em até 18 vezes dos 30 dias pós-antese (DPA) ao 9 dias pós-colheita (DPC). Na casca também houve redução significativa da expressão com o desenvolvimento. Para a expressão absoluta, nos frutos, sementes, caules, raízes e folhas, o número de cópias de ambos os transcritos decresceu com o desenvolvimento, sendo cerca de cem mil vezes mais abundante para Cppgip6 que para Cppgip4. As tentativas de expressão de proteínas recombinantes em Pichia pastoris não geraram resultado positivo, provavelmente em virtude das condições ideais de indução ainda não terem sido estabelecidas corretamente para o ensaio. A atividade de PGIPs extraídas diretamente do tecido foi medida por análise de difusão em ágar empregando pectinase de Aspergillus niger e revelou uma tendência à diminuição da porcentagem de inibição à medida que os frutos se desenvolveram, em concordância com os resultados da análise por qPCR. O conjunto de resultados sugere que a expressão varia com o estádio de desenvolvimento do fruto e é tecido-específica, possivelmente em resposta à diferente susceptibilidade dos tecidos ao ataque fitopatogênico, indicando que menores níveis de transcritos e atividade no amadurecimento, período de maior susceptibilidade, poderiam sinalizar para a regulação do processo degradativo marcando o início da senescência.Polygalacturonase inhibiting proteins (PGIPs) present in plant cell walls are able to inhibit the destructive action of fungal polygalacturonase (PG). In this way, they constitute an important type of plant defense system against fungal infections. In papaya fruit, the pathogenic attack is the main cause of post harvesting loss, and its high susceptibility may be related to the low efficiency or low abundance of anti-phytopathogenic defense. Since this fact could be related to PGIPs expression and little is known about the response of these proteins in the fruit, the aim of the present work was to clone the genes of PGIPs papaya fruit and set their expression pattern in different organs and tissues throughout fruit ripening. Thus, two probable PGIP sequences among 13 initial candidates were identified in the papaya genome by using specific criteria. Both sequences were cloned from cDNA and genomic samples, sequenced and confirmed its identity, and then being named Cppgip4 and Cppgip6. Analysis of relative expression in various tissues at different physiological stages demonstrated that both genes were down regulated during fruit development. The relative expression levels of Cppgip4 in papaya pulp was reduced by 18 times from the 30 days post-anthesis (DPA) to the 9 days post-harvest (DPH). Similarly, gene expression in papaya peel was significant down regulated during fruit development. Absolute expression analysis revealed gene expressions in the fruit pulp, seed, stem, root and leaf were also down regulated within development. Moreover, Cppgip6 gene expression was a hundred thousand times more abundant than Cppgip4. The recombinant protein expression in Pichia pastoris did not result positive, probably because of the ideal conditions of induction have not been properly established the yet. The activity of PGIPs extracted directly from the tissue was measured by the agar diffusion assay using pectinase from Aspergillus niger and showed decrease of inhibition during fruit developed in accordance with the results of the qPCR analysis. Based on the results it is possible to suggest the expression of these genes varies temporally with the developmental stage of the fruit and is tissue-specific, possibly in response to the different susceptibility of tissues to pathogenic attack. In addition, the lowest levels of PGIP expression were achieved at the fruit ripening, when the susceptibility to fungal infection is high and could signal for regulating the degradation process characterized by the onset of senescence

Vegetative and fruit characteristics of papaya trees obtained by mass selection

O presente trabalho teve como objetivo monitorar características vegetativas e dos frutos de mamoeiro (Carica papaya L.), obtidos por seleção massal de plantas da cv. Golden, nos primeiros meses de produção. As amostragens foram realizadas em uma lavoura comercial aos 0, 20, 40, 70, 130, 180, 230, 260, 280, 310 e 340 dias após o plantio (DAP) e os primeiros frutos foram colhidos 230 DAP. Os resultados obtidos evidenciaram baixa altura das plantas (199 cm em 340 DAP) e baixa altura da primeira floração (71 cm), aspectos que facilitam a colheita. As plantas apresentaram boa produtividade, com elevado número de folhas (ampla área de recobrimento dos frutos) e cerca de 60 frutos por planta. Os frutos mantiveram características semelhantes aos da cv. Golden. A massa fresca dos frutos variou de 302,4 a 467,5g, encontrando-se dentro da faixa recomendada para comércio interno. A média da espessura da polpa foi de 2,3 cm, atributo de grande interesse econômico. A firmeza da polpa mostrou uma estreita relação com os fatores climáticos, onde grandes variações de temperatura e pluviosidade aceleraram a perda de firmeza.The present work had as purpose to evaluate some characteristics of papaya trees (Carica papaya L.), Golden cultivar, obtained trough plant mass selection, regarding plant and fruit quality in the first months of production. The samples were evaluated in a commercial crop at: 0, 20, 40, 70, 130, 180, 230, 260, 280, 310 and 340 days after the planting (DAP) and the first fruits were harvested at 230 DAP. The results showed the low height (199cm in 340 DAP) and low first flowering’s heigth (71cm), which is important to facilitate the harvest process. The plants presented good yield with high number of leafs (allowing a great area of fruit cover) and about 60 fruits per plant. The fruits kept similar features to cv. Golden. The fruit’s fresh weight ranged from 302.4 to 467.5g, which is in the range of the Brazilian market. The pulp thickness was 2.35cm, which is a feature of great economic interest. The pulp thickness showed close relation with climatic factors, and great variations of temperature and precipitation accelerated the pulp loss of thickness

Physiological Degradation of Pectin in Papaya Cell Walls: Release of Long Chains Galacturonans Derived from Insoluble Fractions During Postharvest Fruit Ripening

Papaya (Carica papaya L.) is a fleshy fruit that presents a rapid pulp softening during ripening. However, the timeline on how papaya pectinases act in polysaccharide solubilization and the consequent modification of the cell wall fractions during ripening is still not clear. In this work, the gene expression correlations between, on one hand, 16 enzymes potentially acting during papaya cell wall disassembling and, on the other hand, the monosaccharide composition of cell wall fractions during papaya ripening were evaluated. In order to explain differences in the ripening of papaya samplings, the molecular mass distribution of polysaccharides from water-soluble and oxalate-soluble fractions (WSF and OSF, respectively), as well as the oligosaccharide profiling from the WSF fraction, were evaluated by high performance size exclusion chromatography coupled to a refractive index detector (HPSEC-RID) and high performance anion-exchange chromatography coupled to pulse amperometric detection (HPAEC-PAD) analyses, respectively. Results showed that up-regulated polygalacturonase and β-galactosidase genes were positively correlated with some monosaccharide profiles. In addition, an overall increase in the retention time of high molecular weight (HMW) and low molecular weight (LMW) polysaccharides in WSF and OSF was shown. The apparent disappearance of one HMW peak of the OSF may result from the conversion of pectin that were crosslinked with calcium into more soluble forms through the action of PGs, which would increase the solubilization of polysaccharides by lowering their molecular weight. Thus, the results allowed us to propose a detailed process of papaya cell wall disassembling that would affect sensorial properties and post-harvesting losses of this commercially important fruit

Vegetative and fruit characteristics of papaya trees obtained by mass selection

The present work had as purpose to evaluate some characteristics of papaya trees (Carica papaya L.), Golden cultivar, obtained trough plant mass selection, regarding plant and fruit quality in the first months of production. The samples were evaluated in a commercial crop at: 0, 20, 40, 70, 130, 180, 230, 260, 280, 310 and 340 days after the planting (DAP) and the first fruits were harvested at 230 DAP. The results showed the low height (199cm in 340 DAP) and low first flowering`s heigth (71cm), which is important to facilitate the harvest process. The plants presented good yield with high number of leafs (allowing a great area of fruit cover) and about 60 fruits per plant. The fruits kept similar features to cv. Golden. The fruit`s fresh weight ranged from 302.4 to 467.5g, which is in the range of the Brazilian market. The pulp thickness was 2.35cm, which is a feature of great economic interest. The pulp thickness showed close relation with climatic factors, and great variations of temperature and precipitation accelerated the pulp loss of thickness

Analysis of Papaya Cell Wall-Related Genes during Fruit Ripening Indicates a Central Role of Polygalacturonases during Pulp Softening

<div><p>Papaya (<i>Carica papaya</i> L.) is a climacteric fleshy fruit that undergoes dramatic changes during ripening, most noticeably a severe pulp softening. However, little is known regarding the genetics of the cell wall metabolism in papayas. The present work describes the identification and characterization of genes related to pulp softening. We used gene expression profiling to analyze the correlations and co-expression networks of cell wall-related genes, and the results suggest that papaya pulp softening is accomplished by the interactions of multiple glycoside hydrolases. The polygalacturonase <i>cpPG1</i> appeared to play a central role in the network and was further studied. The transient expression of <i>cpPG1</i> in papaya results in pulp softening and leaf necrosis in the absence of ethylene action and confirms its role in papaya fruit ripening.</p></div

Transient expression of the <i>cpPG1</i> gene in papaya leaves.

<p>Constructs carrying <i>GFP</i> or <i>cpPG1-GFP</i> genes were agroinfiltrated into papaya leaves, and the GFP expression in 20-day-old leaves was observed under UV light excitation (<b>Figure A</b>). The images are representative of triplicate experiments that were performed on each agroinfiltrated leaf (<i>n</i> = 3). The absolute quantification of the <i>cpPG1</i> mRNA levels in <b>Figure B</b> demonstrate that <i>cpPG1</i> (gray bar) is transiently expressed. The light gray bar indicates data from the transient expression of <i>cpPG1</i>, the gray bars indicate data from endogenous <i>cpPG1</i> expression, and the black bars indicate the threshold cycle values (Ct) for the two genes used as internal controls (<i>cpACT</i> and <i>cp_EF1</i>). The images are representative of triplicate experiments that were performed on each agroinfiltrated leaf (<i>n</i> = 3). The figure bars are scaled to 1 cm.</p