The expression, accumulation and kinetic properties of 1 -aminocyclopropane- 1 -carboxylic acid (ACC) oxidase (ACO), the enzyme which catalyses the final step in the ACC-dependent pathway of ethylene biosynthesis in plants, is examined. The investigation is divided into three sections: (i) identification of two ACO genes in apple leaf tissue, designated MD-ACO2 and MD-ACO3, (ii) kinetic analyses of each of the three isoforms of ACO in apple (MD-ACO1, MD-ACO2 and MD-ACO3) expressed in E. coli, and (iii) temporal and developmental expression in vivo of each of the ACO genes and accumulation of the corresponding gene products in leaf and fruit tissue. The coding regions of putative ACO gene transcripts were generated from leaf tissue using RT-PCR. Sequence alignments and interrogation of the expressed sequence tags (ESTs) database indicated that the entire open reading frame (ORF) sequences were encoded by two distinct genes, and these are designated MD-ACO2 and MD-ACO3. A third ACO gene had been identified in apple by other research workers previously, and designated MD-ACO1. Differences are obvious in the number of base-pairs (bp) constituting the entire ORF of MD-ACO1 (942 bp), MD-ACO2 (990 bp) and MD-ACO3 (966 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9 % in the ORF but diverge in the 3' untranslated regions (3' -UTR) (69.5 %). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5 %) and MD-ACO2 (77.8 %) in the ORF, and in the 3'-UTR (MD-ACO1, 68.4 %; MD-ACO2, 71 %). A comparison of the gene structures show that the endonuclease restriction sites are unique to each individual MD-ACO sequence. Genomic Southern analysis, using probes spanning the 3'-UTR and the 3'-end of the coding region confirmed that MD-ACO3 is encoded by a distinct gene. However, while the distinction between MD-ACO1 and MD-ACO2 is not as definitive, different gene expression patterns adds credence to their distinctiveness. Each of the three deduced amino acid sequences contain all of the residues hitherto reported to be necessary for maximal ACO activity. Expression of MD-ACO1, MD-ACO2 and MD-ACO3 as fusion proteins in E. coli was induced using isopropy1-β-D-thiogalactopyranoside (IPTG), the recombinant proteins purified by nickel-nitrilotriacetic acid (NiNTA) affinity chromatography and the products had predicted masses determined from the nucleotide sequences, including the His-tag of 38.53 kDa (MD-ACO1), 40.39 kDa (MD-ACO2) and 39.3 kDa (MD-ACO3). Polyclonal antibodies were raised against the MD-ACO3 fusion in rabbit for western blot analysis. Antibodies had been raised previously against recombinant MD-ACO1, and while it was considered likely the MD-ACO2 would be recognized by the MD-ACO1 antibodies, MD-ACO2 does not appear to be recognized in vivo by the antibody. Analyses of the kinetic properties of the three apple ACOs was undertaken. Apparent Michaelis constants (Km) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) have been determined which suggests differences in the affinity of each enzyme for the substrate ACC. Maximum velocity (Vmax) was determined for MD-ACO1 (15.15 nmol), MD-ACO2 (12.94 nmol) and MD-ACO3 (18.94 nmol). The catalytic constant (Kcat) was determined for MD-ACO1 (6.6 x 10-2), MD-ACO2 (3.44 x 10-2) and for MD-ACO3 (9.14 x 10-2), with kcat/Km(μM s-1) values of 7.38 x 10-4 μM s-1 (MD-ACO1), 0.86 x 10-4 M s-1 (MD-ACO2) and 3.8 x 10-4 μM s-1 (MD-ACO3). The optimal pH for MD-ACO1 was 7.0 - 7.5, MD-ACO2 7.5 - 8.0 and MD-ACO3 7.0 - 8.0. All three isoforms exhibited absolute requirements for the co-substrate ascorbate in vitro with optimal activity at 30 mM. Similarly, ferrous iron (FeSO4.7H20; of 15 - 25 μM) and sodium bicarbonate (NaHCO3; of 30 mM) were required for optimal activity, and were the same for all isoforms. No significant difference in thermostability was found in this study between the MD-ACO isoforms at the P = 0.05 level. However, the activities of the enzyme differed significantly between temperatures over time. In vivo expression of each of the ACO genes in leaf tissue determined using RT-PCR and cDNA Southern analysis reveal that both MD-ACO2 and MD-ACO3 are expressed in young leaf tissue and in mature leaf tissue. While MD-ACO3 is expressed predominantly in young leaf tissue, MD-ACO2 (in common with MD-ACO1) is expressed predominantly in mature fruit tissue. None of the MD-ACOs were observed to be senescence associated genes (SAG). MD-ACO3 protein accumulated predominantly in young leaf tissue and less intensely in both mature leaf tissue and young fruit tissue, while MD-ACO1 accumulated only in mature fruit tissue. For the developmental studies, samples were collected at approximately 11 am in this study. MD-ACO2 and MD-ACO3 were also expressed in leaf tissue collected over a 24 h period in the spring and also in the autumn. For both genes transcripts accumulated in the presence of fruit but tended to disappear in the absence of fruit. These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed, and that MD-ACO3 is encoded by a gene distinct from MD-ACO1 and MD-ACO2. MD-ACO1 and MD-ACO2 are either allelic forms of the same gene or closely clustered. Although there is some variation in kinetic properties which may reflect different physiological environments, they do not vary greatly.

Made available in DSpace on 2016-12-08T16:44:53Z (GMT). No. of bitstreams: 1 PGPV07MA016.pdf: 1679932 bytes, checksum: ff6b141d76da54e056823ae64536de56 (MD5) Previous issue date: 2007-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The first cultivations of apple in Brazil was initiated in the end of the sixty s and beginning of the seventy decade and the quality of the available propagative materials used to be very low for plantation. Europeu and United State were the main producers of this fruit and were the forst ones to work with clonal cleaning, but allowing many infected propagative materials with vírus and similars were introduced in Brazil. In the last years the Brazilian apple culture increased its social-economic importance, especially in the Rio Grande do Sul and Santa Catarina State. However the quality of the imported and produced seedling in Brazil still is not satisfactory. Recent introductions of propagative materials from United States, Europe and Japan, revealed partially infected with latente viroses, according with the Virology Laboratory of the EMBRAPA GRAPE and WINE. The culture of the dtrawberry also is distinguished in the national scene of production of fruits of tempered climate, assuming reat relevance due its exploration in properties of familiar base. The plants used for the producers are deriving mainly of Chile, Argentina ando f the United States, that after enter in the country controls of little severity Viruses are responsible for injuries in all stages of apple development by interference in physiological processes which are necessary for the metabolismo and normal growth of the plant. The detention and elimination of thesepathogens is fundamental for clonal cleaning programs. Therefore, the characterization and utilization of new techniques in detection of these pathogens are very importante in search higher sensitivity, security and less costs for obtainment and production of free vírus and viroid propagative materials. The objective of this work was: a) develop a method of multiple biological indexing in substitution of individual biological indexing in detection of apple latente vírus; b) molecular characterization by analysis of nucleoitides and amino acids sequence of the capsidial protein of the isolate BR1 of apple chlorotic leaf spot vírus (ACLSV); and c) detection and partial characterization of the coat protein gene of Strawberry mild yellow edge vírus (SMYEV) in strawberry cv camarosa . The multiple biological indexing in which all indicator plantare grafting over the same root-stock, showed a substantial economy of space and costs due to reduction of the plant numbers in approximately 60 to 80%. This method showed to be reliable with a satisfactory sprouting and a good exteriorization of classic symptoms in the biological detection and characterization of Apple chlorotic leaf spot vírus (ACLSV), Apple stem grooving vírus (ASGV) and Apple vírus stem pitting (ASPV). The molecular characterization of the ACLSV isolate BR1 showed high homology when compared with the Japanese isolate MO-31 which cause latente infection in Malus prunifolia Malus Borkh var. ringo (= Maruba-Kaido). The Japanese isolate MO-31 is a latente vírus in Maruba-Kaido and is the more importante ACLSV biotipe. In nursery and orchards eith Maruba-Kaido root-stock, the symptoms of the ACLSV infection is not visible, allowing the maintenance of infected plants. The Strawberry mild yellow edge vírus was detected by biological indexing and the analysis of nucleotides and aminoavids sequence of the Brasilian isolates (BR1) showed, when compared with others isolates, a 88 to 92% between nucleotides and 98,5 to 100% between aminoacids / Os primeiros cultivos de macieira no Brasil iniciaram-se no final da década de 60 e início da década de 70 do século passado. A qualidade do material disponível para o plantio naquela época era baixa, visto que os principais produtores desta fruta, como a Europa e os Estados Unidos apenas iniciavam programas de limpeza clonal, e com isso ocorreram introduções de materiais infectados por vírus e similares. Nos últimos anos a cultura da macieira assumiu grande importância sócio-econômica no Brasil, especialmente no Rio Grande do Sul e Santa Catarina. Entretanto a qualidade das mudas importadas e produzidas no país ainda não é satisfatória. Introduções recentes de materiais propagativos provenientes dos Estados Unidos, Europa e Japão, mostraram-se parcialmente infectadas por vírus latentes, conforme análises do Laboratório de Virologia da Embrapa Uva e Vinho. A cultura do morango também se destaca no cenário nacional de produção de frutos de clima temperado, assumindo grande relevância devido a sua exploração em propriedades de base familiar. As mudas utilizadas pelos produtores são oriundas principalmente do Chile, Argentina e dos Estados Unidos, que entram no país sem controle quanto a presença de vírus. Os Vírus são responsáveis por danos em todas as fases de desenvolvimento da macieira e do morangueiro, pois interferem nos processos fisiológicos necessários para o metabolismo e crescimento normal da planta. A detecção e eliminação de vírus são componentes fundamentais em programas de limpeza clonal. Por isso, o desenvolvimento e a utilização de novas técnicas de detecção são relevantes na busca de maior sensibilidade, segurança e menor custo na obtenção e produção de materiais propagativos livres de vírus. Os objetivos deste trabalho foram: a) desenvolver um método de indexação biológica múltipla como alternativa a indexação biológica individual na detecção de vírus latentes em macieira; b) a caracterização molecular pela análise da seqüência de nucleotídeos e aminoácidos do gene da proteína capsidial do isolado BR1 de Apple chlorotic leaf spot virus (ACLSV); e c) detecção e caracterização parcial do gene da proteína capsidial de Strawberry mild yellow edge virus SMYEV em morangueiro cultivar Camarosa. A indexação biológica múltipla, na qual todas as indicadoras são enxertadas sobre o mesmo porta-enxerto, permitiu substancial economia de espaço e custos, devido à redução do número de plantas em cerca de 60 a 80%. Este método conferiu resultados confiáveis, como uma brotação satisfatória e a expressão de sintomas clássicos, na detecção e caracterização biológica de ACLSV, Apple stem grooving virus (ASGV) e Apple stem pitting virus (ASPV). A caracterização molecular do isolado BR1, demonstrou a maior homologia deste isolado com o isolado japonês MO-31, o qual causa infecção latente em Malus prunifolia Borkh var. ringo (=Maruba-Kaido), e o ACLSV não sendo visível, pode ser propagado em plantas infectadas. SMYEV foi detectado por indexação biológica e molecular e a análise da seqüência de nucleotídeos e aminoácidos deduzidos do isolado brasileiro (BR1) de SMYEV, demonstrou comparativamente com outros isolados, homologia de 88 a 92% entre nucleotídeos e 98,5 a 100% entre aminoácidos deduzidos