Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Plants are constantly exposed to biotic and abiotic stress inducing factors that threaten their existence. Biotic factors such as pathogens are the cause of huge yield losses to crop plants worldwide with fungal pathogens debatably constituting the worst damage. Fungal pathogens such as Botrytis cinerea, which has a wide host range, release cell wall degrading enzymes called endopolygalacturonases (ePGs) during plant infection. These ePGs break down the pectin component of the cell wall, thus providing an entry route, as well as nutrients for the fungus. Plants have evolved mechanisms to counteract and suppress the action of the ePGs. This is achieved through the action of cell wall associated proteins called polygalacturonaseinhibiting proteins, PGIPs. PGIPs directly inhibit ePGs and their inhibitory action also prolongs the existence of longer chain oligogalacturonide residues which are believed to elicit a cascade of defence responses. In grapevine, a PGIP encoding gene, VvPGIP1, was previously isolated and characterised. VvPGIP1, as well as nine non-vinifera grapevine PGIPs have been expressed in tobacco and shown to be potent antifungal proteins that caused the transgenic tobacco to have strong resistance phenotypes against Botrytis in whole plant infection assays. Following on the tobacco study, two of the non-vinifera PGIPs were expressed in cultivars of the susceptible Vitis vinifera. Characterisation of the putative transgenic population showed that transgene integration was successful, the transgenes were being expressed and there were at least 29 transgenic lines with independent integration events. The transgenic lines were confirmed to have active PGIPs (transgene-derived) in their leaves. Crude protein extracts from 22 lines exhibited 100% inhibition against crude B. cinerea PGs (BcPGs). The plant lines with positive transgene integration, expression, independent integration events and exhibiting 100% transgene-derived PGIP activity were further selected for whole plant and detached leaf antifungal assays where they were challenged with B. cinerea. The whole plant infection assay showed that expression of the non-vinifera PGIPs in V. vinifera promotes susceptibility to B. cinerea, not resistance. This surprising result could perhaps be explained by a quicker and stronger recognition between the pathogen and the host and the stronger activation of defence responses in the host. A more active hypersensitive response in the host would benefit Botrytis being a necrotroph. The type of lesions and the onset and speed of lesion development observed on the transgenics lines versus the wild type support this possibility. Knowledge gaps with regards to the efficiency of the ePG inhibition by the nonvinifera PGIPs during infection of grapevine tissue; the potential changes that might be caused by expressing PGIPs in a grapevine host with a native PGIP with high homology to the transgenes (including potential gene silencing) and the potential impact on defence signalling and defence responses all provides further avenues of study to elucidate this very interesting phenotype further. Overall, this study provides a comprehensively characterised population of transgenic plants that provides useful resources for in vivo analysis of PGIP function in defence, where the host plant harbours a native copy of the PGIP encoding gene. / AFRIKAANSE OPSOMMING: Plante word voortdurend blootgestel aan biotiese en abiotiese faktore, wat stres veroorsaak en hul bestaan bedreig. Biotiese faktore, soos patogene, veroorsaak groot verliese in wêreldwye gewasopbrengste, met swampatogene wat moontlik die grootste skade veroorsaak. Swampatogene, soos Botrytis cinerea, wat ‘n wye reeks gasheerplante kan infekteer, stel selwand-afbrekende ensieme tydens plantinfeksie vry, wat as endo-poligalakturonases (ePG’s). bekend staan. Hierdie ePG’s breek die pektienkomponent van die selwand af, wat gevolglik as ‘n ingangspunt dien,asook voedingstowwe vir die swam verskaf. Plante het meganismes ontwikkel om die aktiwiteit van hierdie ePG’s te bekamp en te onderdruk. Die aktiwiteit van die selwand-geassosieërde proteïene, genaamd poligalakturonase-inhiberende proteïene (PGIP’s), speel hier ‘n rol. PGIP’s inhibeer ePG’s direk en hul inhiberende aktiwiteit verleng ook die bestaan van langketting oligogalakturoniedresidu’s, wat blykbaar ‘n kaskade van weerstandsreaksies kan inisieer. ‘n PGIP-koderende geen, VvPGIP1, is voorheen uit wingerd geïsoleer en gekarakteriseer. VvPGIP1, asook nege nie-vinifera wingerd-PGIP’s is voorheen in tabak uitgedruk en bevestig as proteïene met sterk anti-swamaktiwiteit, soos bevestig deur die bevinding dat die transgeniese tabak ‘n weerstandsfenotipe teen Botrytis in heelplant-infeksietoetse het. Ná die tabakstudie is twee van die nie-vinifera PGIP’s uitgedruk in vatbare V. vinifera-kultivars. Karakterisering van die vermeende transgeniese bevolking het getoon dat die transgeen-integrasie suksesvol was, dat die transgeen uitgedruk word en dat daar ten minste 29 transgeniese lyne met onafhanklike integrasie gebeurtenisse geskep is. Daar is verder bevestig dat die transgeniese lyne aktiewe PGIP’s (transgeen-afkomstig) in hul blare het. Ongesuiwerde proteïenekstrakte van 22 lyne het 100% inhibisie teen ‘n mengsel van ongesuiwerde B. cinerea PGs (BcPGs) getoon. Die plantlyne met positiewe transgeenintegrasie en -uitdrukking, asook onafhanklike integrasiegebeure en wat 100% transgeen-afkomstige PGIP-aktiwiteit getoon het, is verder aan heel-plant en verwyderde blaarswaminfeksies met B cinerea onderwerp. Die heelplantinfeksietoetse het getoon dat uitdrukking van nie-vinifera PGIP’s in V. vinifera ‘n toename, in plaas van ‘n afname, in vatbaarheid teen B. cinerea veroorsaak. Hierdie verbasende resultaat kan moontlik toegeskryf word aan ‘n vinniger en sterker herkenningsreaksie tussen patogeen en gasheer en die moontlike sterker stimulering van weerstandsreaksies in die gasheer. ‘n Meer aktiewe hipersensitiewe reaksie in die gasheer sal tot die voordeel van Botrytis, wat ‘n nektrotroof is, wees. Die tipe letsel, asook die aanvang en spoed van letselontwikkeling wat waargeneem is in transgeniese lyne teenoor die wilde-tipe ondersteun hierdie moontlikheid. Gapings in kennis ten opsigte van die doeltreffendheid van die ePG-inhibisie deur die nievinifera PGIP’s tydens infeksie van wingerdweefsel, die moontlike veranderinge (insluitend ‘n moontlike geenuitdowingseffek) wat veroorsaak kan word deur die uitdrukking van PGIP-gene in ‘n kultivar met ‘n inheemse en baie homoloë PGIP-geen, kon ‘n invloed op weerstandseine en weerstandsreaksies gehad het. Hierdie aspekte lewer verdere studiemoontlikhede om hierdie interessante fenotipe verder te verklaar.Algeheel lewer hierdie studie ‘n breedvoeriggekarakteriseerde bevolking trangeniese plante, wat dien as nuttige hulpbronne vir in vivoanalise van PGIP se funksie in siekteweerstandbiedendheid, veral waar die gasheerplant ‘n inheemse kopie van die PGIP-koderende geen huisves.

Orientador: Paulo Mazzafera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T10:18:49Z (GMT). No. of bitstreams: 1 Melo_GeraldoAclecio_D.pdf: 1320706 bytes, checksum: 5f501b7dd60a44ae72341950498d75f7 (MD5) Previous issue date: 2005 / Resumo: Polifenoloxidase - PFO (EC 1.14.18.1 ou EC 1.10.3.2) é uma enzima de ampla distribuição entre as plantas e catalisa a hidroxilação de monofenóis a o-difenóis e a oxidação destes para o-diquinonas. Sua função em plantas tem sido relacionada a mecanismos de defesa contra patógenos e pragas. Em cafeeiro, o ácido 5-cafeoilquínico, também conhecido como ácido clorogênico (CGA) é o principal substrato da PFO e ambos, enzima e substrato, estão presentes em quantidades expressivas nos frutos e nas folhas desta planta. O CGA também está relacionado com mecanismos de defesas das plantas e como tal é considerando importante substrato em reações de oxidação, principalmente aquelas mediadas pela PFO. No presente estudo, com objetivo de conhecer características da PFO de folhas do cafeeiro, de averiguar sua ação em mecanismos de defesa nessa planta e de entender fatores ligados à síntese e ao acúmulo de seu principal substrato foram feitas a purificação e caracterização dessa enzima, estudos da expressão de sua atividade, bem como estudos de expressão de enzimas da via de síntese do CGA em cafeeiro. Com o uso de técnicas de precipitação com sulfato de amônio, cromatografias de troca iônica, interação hidrofóbica e exclusão molecular foi possível obter a PFO com alto grau de pureza. A enzima apresentou massa molecular de 40,5 Kda e preferência pelo ácido 5-cafeoilquínico como substrato. Seqüências de peptídeos obtidas após digestão da proteína e análise por espectrometria de massas mostraram-se homólogas a seqüências de PFO de várias outras plantas. O nível constitutivo de atividade da PFO observado para quinze genótipos de café variou de 3,8 a 88,0 unidades de atividade/mg de proteína, entretanto não teve relação direta com resistência a pragas e doenças nessa planta. A resistência ao bicho mineiro foi significativamente relacionada ao nível de compostos fenólicos, entretanto, ácido 5-cafeoilquínico, o principal substrato da PFO em café, não teve relação com essa resistência, sugerindo a importância de outros compostos fenólicos como substratos da PFO. Dano mecânico, tratamento com ácido metiljasmônico, inoculação com esporos do fungo Hemileia vastatrix e a infestação com ovos do inseto Perileucoptera coffeella levaram a respostas variadas nos níveis de atividade de PFO nos genótipos avaliados. Baseando-se nesses resultados, conclui-se que a ação da PFO na resistência do cafeeiro a pragas e doenças pode estar relacionada ao potencial oxidativo do tecido e não simplesmente uma maior atividade; que o tipo e quantidade de substrato encontrado no tecido podem ser importantes na resistência do cafeeiro e que entre os genótipos pode existir a especialização de mecanismos de resistência envolvendo a ação da PFO. Estudos de expressão por RT-PCR de fenilalanina amônia-liase (PAL), cinamato 4-hiroxilase (C4H), coumarato 3-hidroxilase (C3H), hidroxicinamoil-CoA ligase (4CL) e hidroxicinamoil-CoA:D-quinato hidroxicinamoil transferase (CQT), enzimas da via de síntese do CGA, tiveram sua expressão reduzida à medida que o tecido envelhece. No endosperma foi observado um decréscimo acentuado de expressão no final da maturação dos frutos. Plântulas estioladas obtidas pela germinação de sementes no escuro e transferidas para luz mostraram aumentos significativos no conteúdo de CGA após 24 horas. Esses aumentos foram transientes e coincidiram com a expressão da PAL, C4H, C3H, 4CL e CQT. Os resultados indicam existência de controle da síntese de CGA e a existência de mecanismos de controle da expressão em comum para as cinco enzimas estudadas / Abstract: Polyphenoloxidase - PPO (EC 1.14.18.1 ou EC 1.10.3.2) is an enzyme with broad distribution among plants and catalyzes the hydroxylation of monophenols to o-diphenols and the oxidation of these to o-diquinones. Its function on plants has been related to defense mechanisms against pathogens and plagues. 5-Caffeoylquinic acid, also known as chlorogenic acid (CGA), is the main PPO substrate in coffee tissues and both, enzyme and substrate are present on substantial quantities in fruits and leaves. CGA is also referred to having connection with plants defense mechanisms and it is also an important substrate on oxidation reactions, mainly those mediated by PPO. Therefore, in order to increase our knowledge on the coffee PPO characteristics, to verify its role in defense mechanisms and also to understand the factors connected to the synthesis of CGA, coffee leaf PPO was purified and characterized regarding kinetic parameters and its activity in leaves of several coffee species exposed or not to pest (leaf miner) and disease (leaf rust). Also studies on the expression of the enzymes of CGA synthesis were carried out. By using ammonium sulfate precipitation followed by chromatographic steps on ionic exchange, hydrophobic interaction and molecular exclusion resins it was possible to purify PPO to homogeneity. The enzyme presented a molecular mass of 40,5 Kda and used 5-cafeoylquinic acid as the preferred substrate. Peptide sequences obtained after digestion of the purified PPO and analysis through mass spectrometry were homologous to PPO sequences of several other plants. The constitutive level of PPO activity observed for 15 coffee genotypes varied from 3,8 to 88,0 units of activity/mg of protein, but did not have a direct relationship with resistance to plagues in this plant. Resistance to leaf miner was significantly related to the level of phenolic compounds. However, 5-caffeoylquínic acid, the main substrate of PPO on coffee, was not related with resistance, suggesting the importance of other phenolic compounds as PPO substrates. Mechanical damage, treatment with methyljasmonic acid, inoculation with spores from Hemileia vastatrix and the infestation with the insect Perileucoptera coffeella led to varied results of the PPO activity in the evaluated genotypes. Based on these results, we conclude that the PPO role in the coffee resistance to plagues and diseases might be related to the oxidative potential of the tissue and not only on the PPO activity; that the kind and quantity of PPO substrate found in the tissue might be important for the resistance of the coffee tree and that there may be specific mechanisms of resistance involving PPO action among the genotypes. RT-PCR studies of the expression of phenylalanine ammonia-lyase (PAL), cinnamate 4-hyroxylase (C4H), coumarate 3-hydroxylase (C3H), hydroxycinnamoyl-CoA ligase (4CL) and hydroxycinnamoyl-CoA:D-quinate hydroxycinnamoyl transferase (CQT), which code for enzymes of the CGA biosynthetic pathway, showed that the expression of these enzymes decrease with tissue aging. In the endosperm, an evident decrease on the expression was observed in the end of the fruit ripening. Etiolated seedlings obtained by germination of coffee seeds in the dark and transferred into light showed significant increasing on the CGA content after 24 hours. The increase was transient and followed the expression pattern of PAL, C4H, C3H, 4CL and CQT. The results indicate that CGA biosynthesis is coordinately regulated by the expression of the five enzymes / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal