EFEITO DO FRIO NA BROTAÇÃO DE GEMAS DE PEREIRA (Pyrus communis L.) cv. Carrick, EM PELOTAS, RS

Objetivou-se, no presente trabalho, identificar a profundidade de dormência e a velocidade de brotação em gemas de pereira, submetidas a diferentes períodos de frio à temperatura de 4ºC ±1. O experimento foi conduzido na Embrapa-Clima Temperado, em Pelotas, em 1999. Em 1º de junho, foram coletados 50 ramos, na cultivar Carrick, com aproximadamente 30 cm de comprimento. Após, foram divididos em 5 lotes de 10 ramos, sendo 4 mantidos a 4ºC± 1, e um em condições ambiente, constituindo, assim, 5 tratamentos: 0 (Testemunha); 272; 544; 816 e 1088 horas de frio (HF). No final de cada tratamento, os ramos foram divididos em pequenas estacas, contendo apenas uma única gema, sendo, após, armazenados em câmara climática a 25ºC ± 1. Avaliou-se a brotação, considerando-se o estádio de ponta verde. A partir destes dados, calculou-se o tempo médio de brotação (TMB), bem como a percentagem de gemas brotadas, em cada um dos tratamentos. Utilizou-se o índice de velocidade de brotação (IVB), para determinar a eficiência da temperatura na brotação das gemas. A profundidade de dormência, das gemas terminais, diminuiu à medida que se aumentou o período de frio. As gemas axilares não foram influenciadas pelo tempo de exposição ao frio. Com base nos dados do IVB e dos coeficientes angulares, as gemas terminais da cv. Carrick necessitam de 800 horas de frio para completar a brotação, nas condições que foram conduzidos os experimentos.

Qualidade pós-colheita da pêra (Pyrus communis L.) cultivar Carrick submetida a diferentes condições de armazenamento

As pêras européias não alcançam a maturidade de consumo na planta, sendo amadurecidas após a colheita, mediante armazenamento. O presente trabalho objetivou avaliar a qualidade pós-colheita de pêras cv. Carrick sob diferentes condições de armazenamento. As frutas foram armazenadas à temperatura de 0±0,5ºC e umidade relativa (UR) de 90-95% e, para simulação da comercialização, temperatura de 20º±1ºC e UR de 75-80%. As frutas foram submetidas aos tratamentos: T1) 30 dias a 0 ºC; T2) 28 dias a 0ºC + 2 dias a 20ºC; T3) 26 dias a 0ºC + 4 dias a 20ºC; T4) 24 dias a 0ºC + 6 dias a 20ºC. Após 30 dias, foram analisadas as seguintes variáveis: sólidos solúveis totais (SST); acidez titulável (AT); relação SST/AT; cor; firmeza de polpa e ocorrência de podridão. Foram avaliadas, ainda, as características sensoriais de sabor e textura, com equipe treinada. Os valores de firmeza da polpa variaram de 15,51 a 3,70 libras. As cores amarelo e vermelho da epiderme tornaram-se mais intensas nos tratamentos T3 e T4; a AT e os SST variaram de 0,39 a 0,32% e 13,4 a 13,87°Brix, respectivamente. Os tratamentos T3 e T4 apresentaram melhores características de sabor e qualidade geral, sendo as frutas classificadas de boa a ótima qualidade. As frutas armazenadas sob refrigeração por 24 e 26 dias, e transferidas por 4 a 6 dias para temperatura de 20°C apresentaram melhor qualidade na comercialização e consumo.

Comportamiento fisiológico de la pera variedad Triunfo de Viena (Pyrus communis L.) durante el período poscosecha

La pera variedad Triunfo de Viena es una fruta producida en Colombia y actualmente no se dispone de una red de frío para su manejo poscosecha. El objetivo de este trabajo fue establecer el comportamiento fisiológico de la pera variedad Triunfo de Viena en tres condiciones de temperatura y humedad relativa: 3ºC y 85%, 11ºC y 80%, 18ºC y 75%. La pera variedad Triunfo de Viena es un fruto climatérico, cuyo punto de máxima intensidad respiratoria puede desplazarse en el tiempo dependiendo de las condiciones de almacenamiento. Para las condiciones estudiadas, el climaterio se presentó en el día 22 (35,87 mg CO2 kg-1 h-1), en el día 36 (28,59 mg CO2 kg-1 h-1) y en el día 93 (15,72 mg CO2 kg-1 h-1) del almacenamiento, para las temperaturas de 18ºC, 11ºC y 3ºC respectivamente. La pérdida de peso presentó una relación lineal y directa respecto al tiempo de almacenamiento, siendo mayor con temperaturas de 18ºC y 11ºC. La acidez titulable y el pH no presentan grandes variaciones durante el almacenamiento. La intensidad respiratoria, la pérdida de peso, la relación SST/AT y la firmeza de la pulpa, son las características que representan mejor la evolución de la madurez de las peras variedad Triunfo de Viena.

Anelamento e Paclobutrazol na produção e absorção de nutrientes em Pereira (Pyrus communis L.) cultivar Packham´s Triumph

Foram conduzidos dois experimentos para estudar o efeito do anelamento e do paclobutrazol na produção e absorção de macronutrientes em pereira cv Packham´s Triumph, no munícipio de São Joaquim-SC, localizado a 1.400 m de altitude, em pomar comercial, utilizando plantas adultas.O delineamento utilizado no primeiro e segundo foi o de blocos casualizados. No primeiro estudo, foram utilizados sete tratamentos: 1)testemunha; 2) 1,5 g /planta do paclobutrazol, aplicados no solo; 3) 3,0 g/planta de paclobutrazol, aplicados no solo; 4 ) 4,5 g/planta de paclobutrazol, aplicados no solo; 5) anelamento simples (1 anel); 6) anelamento duplo realizado (dois anéis), e 7 ) anelamento pleno (um anelamento seguido de outro anelamento no mesmo local após a cicatrização do primeiro). No segundo experimento, os tratamentos foram: 1)-testemunha; 2) 2 g/planta de paclobutrazol, aplicados o solo; 3 ) 4 g/planta de paclobutrazol, aplicados no solo; 4 )-1.000 ppm de paclobutrazol em aplicação foliar; 5 ) 2.000 ppm de paclobutrazol em aplicação foliar; 6 ) anelamento simples, e 7) anelamento duplo. A maior produção foi observada no anelamento pleno e no anelamento duplo. O anelamento teve maior efeito na produção do que o paclobutrazol, e este via solo teve maior efeito do que a aplicação foliar. Os tratamentos não influenciaram nos níveis de nutrientes no fruto, com exceção do potássio. O maior teor de potássio foi encontrado na testemunha que apresentou o maior tamanho de frutos.

Studio molecolare dei meccanismi dell'autoincompatibilità gametofitica in pero europeo (Pyrus communis)

Self-incompatibility (SI) systems have evolved in many flowering plants to prevent self-fertilization and thus promote outbreeding. Pear and apple, as many of the species belonging to the Rosaceae, exhibit RNase-mediated gametophytic self-incompatibility, a widespread system carried also by the Solanaceae and Plantaginaceae. Pear orchards must for this reason contain at least two different cultivars that pollenize each other; to guarantee an efficient cross-pollination, they should have overlapping flowering periods and must be genetically compatible. This compatibility is determined by the S-locus, containing at least two genes encoding for a female (pistil) and a male (pollen) determinant. The female determinant in the Rosaceae, Solanaceae and Plantaginaceae system is a stylar glycoprotein with ribonuclease activity (S-RNase), that acts as a specific cytotoxin in incompatible pollen tubes degrading cellular RNAs. Since its identification, the S-RNase gene has been intensively studied and the sequences of a large number of alleles are available in online databases. On the contrary, the male determinant has been only recently identified as a pollen-expressed protein containing a F-box motif, called S-Locus F-box (abbreviated SLF or SFB). Since F-box proteins are best known for their participation to the SCF (Skp1 - Cullin - F-box) E3 ubiquitine ligase enzymatic complex, that is involved in protein degradation through the 26S proteasome pathway, the male determinant is supposed to act mediating the ubiquitination of the S-RNases, targeting them for the degradation in compatible pollen tubes. Attempts to clone SLF/SFB genes in the Pyrinae produced no results until very recently; in apple, the use of genomic libraries allowed the detection of two F-box genes linked to each S haplotype, called SFBB (S-locus F-Box Brothers). In Japanese pear, three SFBB genes linked to each haplotype were cloned from pollen cDNA. The SFBB genes exhibit S haplotype-specific sequence divergence and pollen-specific expression; their multiplicity is a feature whose interpretation is unclear: it has been hypothesized that all of them participate in the S-specific interaction with the RNase, but it is also possible that only one of them is involved in this function. Moreover, even if the S locus male and female determinants are the only responsible for the specificity of the pollen-pistil recognition, many other factors are supposed to play a role in GSI; these are not linked to the S locus and act in a S-haplotype independent manner. They can have a function in regulating the expression of S determinants (group 1 factors), modulating their activity (group 2) or acting downstream, in the accomplishment of the reaction of acceptance or rejection of the pollen tube (group 3). This study was aimed to the elucidation of the molecular mechanism of GSI in European pear (Pyrus communis) as well as in the other Pyrinae; it was divided in two parts, the first focusing on the characterization of male determinants, and the second on factors external to the S locus. The research of S locus F-box genes was primarily aimed to the identification of such genes in European pear, for which sequence data are still not available; moreover, it allowed also to investigate about the S locus structure in the Pyrinae. The analysis was carried out on a pool of varieties of the three species Pyrus communis (European pear), Pyrus pyrifolia (Japanese pear), and Malus × domestica (apple); varieties carrying S haplotypes whose RNases are highly similar were chosen, in order to check whether or not the same level of similarity is maintained also between the male determinants. A total of 82 sequences was obtained, 47 of which represent the first S-locus F-box genes sequenced from European pear. The sequence data strongly support the hypothesis that the S locus structure is conserved among the three species, and presumably among all the Pyrinae; at least five genes have homologs in the analysed S haplotypes, but the number of F-box genes surrounding the S-RNase could be even greater. The high level of sequence divergence and the similarity between alleles linked to highly conserved RNases, suggest a shared ancestral polymorphism also for the F-box genes. The F-box genes identified in European pear were mapped on a segregating population of 91 individuals from the cross 'Abbé Fétel' × 'Max Red Bartlett'. All the genes were placed on the linkage group 17, where the S locus has been placed both in pear and apple maps, and resulted strongly associated to the S-RNase gene. The linkage with the RNase was perfect for some of the F-box genes, while for others very rare single recombination events were identified. The second part of this study was focused on the research of other genes involved in the SI response in pear; it was aimed on one side to the identification of genes differentially expressed in compatible and incompatible crosses, and on the other to the cloning and characterization of the transglutaminase (TGase) gene, whose role may be crucial in pollen rejection. For the identification of differentially expressed genes, controlled pollinations were carried out in four combinations (self pollination, incompatible, half-compatible and fully compatible cross-pollination); expression profiles were compared through cDNA-AFLP. 28 fragments displaying an expression pattern related to compatibility or incompatibility were identified, cloned and sequenced; the sequence analysis allowed to assign a putative annotation to a part of them. The identified genes are involved in very different cellular processes or in defense mechanisms, suggesting a very complex change in gene expression following the pollen/pistil recognition. The pool of genes identified with this technique offers a good basis for further study toward a better understanding of how the SI response is carried out. Among the factors involved in SI response, moreover, an important role may be played by transglutaminase (TGase), an enzyme involved both in post-translational protein modification and in protein cross-linking. The TGase activity detected in pear styles was significantly higher when pollinated in incompatible combinations than in compatible ones, suggesting a role of this enzyme in the abnormal cytoskeletal reorganization observed during pollen rejection reaction. The aim of this part of the work was thus to identify and clone the pear TGase gene; the PCR amplification of fragments of this gene was achieved using primers realized on the alignment between the Arabidopsis TGase gene sequence and several apple EST fragments; the full-length coding sequence of the pear TGase gene was then cloned from cDNA, and provided a precious tool for further study of the in vitro and in vivo action of this enzyme.

Evaluación de las características físico-químicas y biológicas en dos suelos superficiales cultivados con pera (Pyrus communis L.) cv. Williams bajo manejo convencional

El objetivo del trabajo fue identificar las características físico-químicas y biológicas en dos suelos superficiales fertilizados con nitrógeno y enmienda orgánica en el Alto Valle de Río Negro (huertos H1 y H2). En ambos huertos se aplicó fertilizante nitrogenado durante las temporadas 2008-2009 y 2009-2010 y en H2 se aplicó estiércol de pollo. Se extrajeron muestras de suelos en primavera y otoño y se determinó: carbono orgánico total, conductividad eléctrica, cationes de intercambio, relación de adsorción de sodio y nitratos, respiración microbiana, carbono de la biomasa microbiana, actividad de la deshidrogenasa y el índice de mineralización. La concentración de carbono orgánico total, potasio y nitrógeno fueron adecuadas para la producción de pera. El comportamiento de las variables biológicas fue diferente en los huertos. En H1 fueron mayores en primavera, en ambas temporadas y el índice de mineralización fue ligeramente superior a 1 en otoño, indicando equilibrio entre la mineralización y la humificación del carbono. En H2 las mediciones biológicas fueron similares entre las estaciones como consecuencia de realizar fertilización nitrogenada (N) en primavera y en otoño. La enmienda orgánica no reflejó un aumento de la actividad biológica en primavera. La actividad microbiana y enzimática en H1 y H2 fue sensible a los cambios que ocurrieron en los suelos.

Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells

Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development. We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor. However, paradoxically, both AGPs were buffer soluble rather than membrane associated. We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs. This GPI anchor has the minimal core oligosaccharide structure, d-Manα(1–2)-d-Manα(1–6)-d-Manα(1–4)-d-GlcN-inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial β(1–4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid. The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D. The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.

A transcriptome approach towards understanding the development of ripening capacity in 'Bartlett' pears (Pyrus communis L.)

Background: The capacity of European pear fruit (Pyrus communis L.) to ripen after harvest develops during the final stages of growth on the tree. The objective of this study was to characterize changes in 'Bartlett' pear fruit physico-chemical properties and transcription profiles during fruit maturation leading to attainment of ripening capacity. Results: The softening response of pear fruit held for 14days at 20°C after harvest depended on their maturity. We identified four maturity stages: S1-failed to soften and S2- displayed partial softening (with or without ET-ethylene treatment); S3 - able to soften following ET; and S4 - able to soften without ET. Illumina sequencing and Trinity assembly generated 68,010 unigenes (mean length of 911bp), of which 32.8% were annotated to the RefSeq plant database. Higher numbers of differentially expressed transcripts were recorded in the S3-S4 and S1-S2 transitions (2805 and 2505 unigenes, respectively) than in the S2-S3 transition (2037 unigenes). High expression of genes putatively encoding pectin degradation enzymes in the S1-S2 transition suggests pectic oligomers may be involved as early signals triggering the transition to responsiveness to ethylene in pear fruit. Moreover, the co-expression of these genes with Exps (Expansins) suggests their collaboration in modifying cell wall polysaccharide networks that are required for fruit growth. K-means cluster analysis revealed that auxin signaling associated transcripts were enriched in cluster K6 that showed the highest gene expression at S3. AP2/EREBP (APETALA 2/ethylene response element binding protein) and bHLH (basic helix-loop-helix) transcripts were enriched in all three transition S1-S2, S2-S3, and S3-S4. Several members of Aux/IAA (Auxin/indole-3-acetic acid), ARF (Auxin response factors), and WRKY appeared to play an important role in orchestrating the S2-S3 transition. Conclusions: We identified maturity stages associated with the development of ripening capacity in 'Bartlett' pear, and described the transcription profile of fruit at these stages. Our findings suggest that auxin is essential in regulating the transition of pear fruit from being ethylene-unresponsive (S2) to ethylene-responsive (S3), resulting in fruit softening. The transcriptome will be helpful for future studies about specific developmental pathways regulating the transition to ripening. © 2015 Nham et al.

Red flesh fruit in european pear (Pyrus communis): genetic sources, QTLs, candidate genes and tools for breeding

Red flesh fruit is a character which interest is increasing in several commercial species. Following a review of the research on the biosynthesis and accumulation of anthocyanin in pears (Chapter 1) the general aim of the project is reported in Chapter 2. Chapter 3 reports the results of a molecular analysis of 33 red-fleshed pear accessions, genotyped with 18 SSR markers with the aim of improving germplasm conservation strategies to support ongoing breeding programs. The molecular profiles revealed both cases of synonymy and homonymy and 6 unique genotypes were identified. The S-allele were established to highlight the genetic relationships among these landraces. Four of the unique genotypes have been clustered based on pomological data. In the Chapter 4, the work was directed to identify the putative genomic regions involved in the appearance of this character in pear fruit. A crossing population (‘Carmen’ x ‘Cocomerina Precoce’) segregating for the trait was phenotyped for 2 consecutive years and used for QTL analysis. A strong QTL was identified in a small genomic region related to the red flesh fruit trait at 27 Mb from the start of LG5. Two candidate genes were detected in this genomic region: ‘PcMYB114’ and ‘PcABCC2’. SSR marker SSR114 was found able to detect the red flesh phenotype segregation in all the red-fleshed pear accessions and segregating progenies tested. Chapter 5 focuses on examining the trend of anthocyanin synthesis and accumulation during the fruit development, from fruit set to ripening time. Three different trials were planned: qPCR and HPLC methods were performed to correlate the genes expression with the anthocyanin accumulation in ‘Cocomerina Precoce’ and six progenies. Total transcriptome sequencing was used to compare the differential genes expression between red and white-fleshed fruit. Chapter 6 reviews and analyses all the earlier study findings while providing new potential future perspectives.

Fertilizantes foliares em culturas perenes: pereira japonesa (Pyrus pyrifolia var. Culta), pinheira (Annona squamosa L.) e videira (Vitis labrusca L.)

Pós-graduação em Agronomia - FEIS

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptI1 or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease. (C) 2009 Elsevier B.V. All rights reserved.

Diversity of the bacterial community in the surface soil of a pear orchard based on 16S rRNA gene analysis

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes was used to characterize bacterial populations in the surface soil of a commercial pear orchard consisting of different pear cultivars during two consecutive growing seasons. Pyrus communis L. cvs Blanquilla, Conference, and Williams are among the most widely cultivated cultivars in Europe and account for the majority of pear production in Northeastern Spain. To assess the heterogeneity of the community structure in response to environmental variables and tree phenology, bacterial populations were examined using PCR-denaturing gradient gel electrophoresis (DGGE) followed by cluster analysis of the 16S ribosomal DNA profiles by means of the unweighted pair group method with arithmetic means. Similarity analysis of the band patterns failed to identify characteristic fingerprints associated with the pear cultivars. Both environmentally and biologically based principal-component analyses showed that the microbial communities changed significantly throughout the year depending on temperature and, to a lesser extent, on tree phenology and rainfall. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant bacterial populations. Most DGGE band sequences were related to bacterial phyla, such as Bacteroidetes, Cyanobacteria, Acidobacteria, Proteobacteria, Nitrospirae, and Gemmatimonadetes, previously associated with typical agronomic crop environments

Efeito do ethephon e da frigoconservação na maturação de pêras cv. Packham's Triumph

Este trabalho, realizado em 1996 na Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul, em Eldorado do Sul, RS, e na Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado (CPACT), em Pelotas, RS, objetivou avaliar o efeito do ethephon aplicado em pré-colheita e da frigoconservação na maturação da pêra (Pyrus communis L.) cv. Packham's Triumph. O ethephon foi pulverizado nas concentrações de 0, 12,5, 25, 50 e 100 ppm, e as pêras frigorificadas a -1,0ºC e 92-96% de umidade relativa por 0, 10, 20, 40 e 80 dias. Após os períodos de armazenamento refrigerado, os frutos foram conservados em temperatura ambiente por dois ou oito dias. A firmeza da polpa apresentou uma tendência de diminuição com o aumento das concentrações de ethephon, do período de frigorificação e dos dias à temperatura ambiente. O comportamento da relação sólidos solúveis totais (SST)/acidez titulável (AT) variou mais em função da AT do que dos valores de SST. A relação SST/AT aumentou desde a aplicação dos tratamentos até a colheita. A produção de etileno aumentou com o aumento do período de frigorificação e do número de dias em temperatura ambiente; mas não foi influenciada pela concentração de ethephon.

Polimorfismo enzimático nos tecidos de pereira

O trabalho foi realizado com o objetivo de avaliar o polimorfismo enzimático em diferentes tecidos de oito cultivares de pereira Pyrus communis L. Os genótipos utilizados fazem parte da coleção de plantas disponíveis na Universidade de Estudos de Bolonha. Para as análises isoenzimáticas foram utilizadas gemas floríferas dormentes no inverno, casca de ramos de um ano obtida de plantas em pleno desenvolvimento, folhas obtidas no início da primavera e folhas de plantas mantidas in vitro. A corrida eletroforética foi realizada em gel de poliacrilamida a gradiente com (5% a 12,5%). Os resultados obtidos com os genótipos utilizados indicaram que o sistema enzimático beta-glucosidase (E.C.3.2.1.21) apresentou atividade apenas nas folhas das plantas in vitro, com uma banda na mesma posição para todas as cultivares, ao passo que os sistemas para as enzimas esterase (E.C.3.1.1.2) e peroxidase (E.C.1.11.1.7) apresentaram elevado polimorfismo. Nas gemas dormentes analisadas, o sistema peroxidase permitiu diferenciar todos os genótipos. As formas isoenzimáticas da esterase permitiram separar todos os genótipos independentemente dos tecidos utilizados.