Cross species identification of genetic factors that determine fruit surface characteristics

Thesis (PhD)--Stellenbosch University, 2015.ENGLISH ABSTRACT: The plant cuticle is a typically waxy layer that covers the entire above ground part of higher plants and performs a number of important roles in vegetative organs and during fruit development and ripening, including protection from a range of abiotic and biotic stresses. This complex hydrophobic layer consists of a cutin matrix of predominantly fatty acids embedded with long chain waxes, synthesized in (and secreted from) the epidermal cells. Suberin, an aliphatic polymer which is higher in phenolic content than cutin and less elastic, may be formed due to wounding and/or cuticle damage. While the synthesis and transport of cutin and wax have been investigated for a number of decades, there are still steps in these pathways that are yet to be elucidated. In the case of suberin biosynthesis, much less has been described. The understanding of the regulatory mechanisms for these processes has only recently received attention, and has already proven to be an area of significant interest for plant scientists, particularly with regard to the interaction between the regulation of epidermal cell identity and cuticle development. In terms of fruit production the cuticle can be linked to many quality parameters, especially postharvest storage. This is particularly true for apple, a crop dependent on a long shelf life. Apple is also susceptible to the formation of a cuticle failure disorder known as russet. Apple russet results from micro-cracking of the cuticle and the formation of a corky suberized layer. During normal growth suberin can be found in potato tubers, roots, bark, and seed coat. However, on most fruit, suberin is typically an undesirable consumer trait, and in the case of apples can have a negative impact on the postharvest storage. In this work, the current understanding of plant cuticles is built upon with particular emphasis on fleshy fruit cuticle assembly and the regulation of the biosynthetic pathways generating its constituents. Research goals focused on identifying and characterizing genetic factors involved in fruit surface formation, particularly with regard to cuticle biosynthesis. Further attention is paid to understanding the regulatory mechanisms of cuticular pathways, including the emerging concept of co-regulation of epidermal cell differentiation and cuticle biosynthesis. Tomato and apple were the species in which these investigations were focused. This was due to the fact that tomato is seen as a model species for fruit surface biology research, while the applied aspects of apple surface research have far reaching impact. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface the tomato SlMIXTA-like gene was investigated. MIXTA/MIXTA-like proteins, initially described in snapdragon petals, are regulators of epidermal cell differentiation. In an effort to understand these processes in fruit, tomato was transgenically silenced for SlMIXTA-like expression. Plants displayed defects in the patterning of conical epidermal cells of fruit, and also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling, coupled with comprehensive microscopy, revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes acting downstream of SlMIXTA-like included oxidases, transferases and transporters involved in cutin synthesis and assembly. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, it was found that SlMIXTA-like acts downstream of the cutin biosynthesis regulator SlSHN3 and possibly co-operates with homeodomain-leucine zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly and epidermal cell patterning. While russeting may occur in apples after cuticle damage, it is also a heritable trait, and therefore is to some extent under genetic control. In order to identify genetic factors controlling cuticle biosynthesis in apple (and thus preventing russet), a QTLmapping survey was performed on a full-sib population. Two genomic regions located on chromosome 2 and 15 that could be associated with russeting were identified. Apples with compromised cuticles were identified through a novel and high throughput tensile analysis of the skin, while histological analysis confirmed cuticle failure in a subset of the progeny. Additional genomic investigation of the determined QTL regions identified a set of underlying genes involved in cuticle biosynthesis. Candidate gene expression profiling by qRT-PCR on a subset of the progeny highlighted the specific expression pattern of a SHN1/WIN1 transcription factor (termed MdSHN3) on chromosome 15. The MdSHN3 transcription factor displayed extremely low expression in lines with improper cuticle formation suggesting it to be a fundamental regulator of cuticle biosynthesis in apple fruit, and thus necessary for the prevention of suberized fruit surfaces (russet). In an effort to gain a greater understanding of the mechanisms underlying suberin biosynthesis in fruit, and in plants in general, transgenic tomato were generated with compromised cutin formation. This was achieved via the transcriptional silencing of SlDCR, an orthologue to AtDCR which was previously identified as a key step in cutin biosynthesis in Arabidopsis. Silencing of this BAHD acyltransferase resulted in an almost total elimination of the major monomer from the fruit cutin (C16-9,10-dihydroxy fatty acid), and the plants developed fruit with a suberized surface. This provided an excellent opportunity for transcriptome and chemical characterization of the suberization process in fleshy fruit. In parallel an apple clone that developed a russeted fruit surface was identified, and characterized. A large scale comparative transcriptomic analysis of these tomato and apple mutants was performed, generating a list of candidate genes for suberin deposition. Increasing the comparison to include data mined from literature resulted in the elucidation of a multi-species gene expression signature for suberin biosynthesis, and allowed for the identification and characterization of novel genetic elements, including those involved in the regulation of suberin formation and its deposition. Of these genetic elements, MYB107, was demonstrated to be a positive regulator of suberin accumulation in Arabidopsis seed coat. In totality this study has produced a greater understanding of genetic mechanisms governing cuticle biosynthesis, particularly in developing fruit. This was achieved primarily through the functional characterization of regulatory elements (MIXTA-like, SHN3 and MYB107 transcription factors) controlling the synthesis of cutin and suberin matrices. Additionally the DCR enzyme was demonstrated to be a crucial step in fruit cutin biosynthesis. Finally the intricate relationship between epidermal cell development and cuticle biosynthesis has been further highlighted.AFRIKAANSE OPSOMMING: Die plant kutikula is 'n tipiese wasagtige laag wat die hele bogrondse deel van hoër plante dek en voer 'n aantal belangrike rolle in vegetatiewe organe en tydens die ontwikkeling en rypwording vrugte, insluitend die beskerming van 'n verskeidenheid van abiotiese en biotiese stres. Hierdie komplekse hidrofobiese laag bestaan uit 'n cutin matriks van oorwegend vetsure ingesluit met lang ketting wasse, gesintetiseer in (en afgeskei van) die epidermale selle. Suberien, 'n alifatiese polimeer wat hoër in fenoliese inhoud as cutin en minder elasties, kan gevorm word as gevolg van gewond en/of skade kutikula. Terwyl die sintese en vervoer van cutin en was reeds ondersoek vir 'n aantal dekades, is daar nog stappe in hierdie paaie wat nog beantwoord word. In die geval van suberien biosintese, veel minder is al beskryf. Die begrip van die regulerende meganismes vir hierdie prosesse het eers onlangs ontvang aandag en het reeds bewys dat 'n oppervlakte van beduidende belang vir plant wetenskaplikes, veral met betrekking tot die interaksie tussen die regulering van epidermale sel identiteit en kutikula ontwikkeling. In terme van die vrugte produksie kan die kutikula gekoppel word aan baie kwaliteit parameters, veral na-oes stoor. Dit is veral waar vir appel, 'n gewas afhanklik van 'n lang raklewe. Apple is ook vatbaar vir die vorming van 'n kutikula mislukking siekte bekend as rooibruin. Apple rooibruin resultate van mikro-krake van die kutikula en die vorming van 'n Corky suberized laag. Gedurende normale groei suberien kan gevind word in moere, wortels, bas, en saadhuid. Maar op die meeste vrugte, suberien is tipies 'n ongewenste eienskap verbruiker, en in die geval van appels kan 'n negatiewe impak op die oes stoor het. In hierdie werk, is die huidige begrip van plant cuticles gebou op met spesifieke klem op vlesige vrugte kutikula vergadering en die regulering van die biosintetiese weë genereer sy bestanddele. Navorsing doelwitte gefokus op die identifisering en karakterisering van genetiese faktore wat betrokke is in die vorming vrugte oppervlak, veral met betrekking tot kutikula biosintese. Verdere aandag word aan die begrip van die regulerende meganismes van kutikulêre paaie, insluitend die opkomende konsep van mede-regulering van epidermale seldifferensiasie en kutikula biosintese. Tamatie en appel was die spesies in wat hierdie ondersoeke is gefokus. Dit was as gevolg van die feit dat tamatie is gesien as 'n model vir spesie vrugte oppervlak biologie navorsing, terwyl die toegepaste aspekte van appel oppervlak navorsing verreikende impak. Om die verhouding tussen epidermale sel ontwikkeling en kutikula vergadering in die konteks van die vrugte te ondersoek oppervlak die tamatie SlMIXTA-like gene ondersoek. MIXTA/MIXTA-like proteïene, aanvanklik beskryf in leeubekkies kroonblare, is reguleerders van epidermale seldifferensiasie. In 'n poging om hierdie prosesse in vrugte te verstaan, was tamatie transgenically stilgemaak vir SlMIXTA-like uitdrukking. Plante vertoon gebreke in die patroon van koniese epidermisselle van die vrugte, en het ook getoon veranderde oes water verlies en weerstand teen patogene. Transkriptoom en kutikulêre lipiede profilering, tesame met 'n omvattende mikroskopie, geopenbaar beduidende veranderinge aan die gemeente kutikula en voorgestel SlMIXTA-like om cutin biosintese reguleer. Kandidaat gene optree stroomaf van SlMIXTA-like ingesluit oxidases, transferases en karweiers betrokke in cutin sintese en die gemeente. As deel van 'n groter netwerk van regulerende epidermale sel patrone en L1-laag identiteit, is daar gevind dat SlMIXTA-like handelinge stroomaf van die cutin biosintese reguleerder SlSHN3 en moontlik werk saam met homeodomain-leucine rits IV transkripsie faktore. Dus, SlMIXTA-like is 'n positiewe reguleerder van beide kutikula en koniese epidermale sel vorming in tamatie vrugte, wat as 'n bemiddelaar van die stywe assosiasie tussen vrugte cutin polimeer vorming, kutikula vergadering en epidermale sel patrone. Terwyl skilverruwing mag voorkom in appels na kutikula skade, is dit ook 'n oorerflike eienskap, en daarom is tot 'n mate onder genetiese beheer. Ten einde genetiese faktore beheer kutikula biosintese in die appel (en dus die voorkoming van rooibruin) identifiseer, is 'n QTL-kartering opname uitgevoer op 'n full-sib bevolking. Twee genoom gebiede geleë op chromosoom 2 en 15 wat kan geassosieer word met skilverruwing is geïdentifiseer. Appels met verswakte cuticles is geïdentifiseer deur 'n roman en 'n hoë deurset trek ontleding van die vel, terwyl histologiese analise bevestig kutikula mislukking in 'n subset van die nageslag. Bykomende genomiese ondersoek van die bepaal QTL streke geïdentifiseer is 'n stel van onderliggende gene betrokke by kutikula biosintese. Kandidaat geenuitdrukking profiele deur QRT-PCR op 'n subset van die nageslag beklemtoon die spesifieke uitdrukking patroon van 'n SHN1/WIN1 transkripsie faktor (genoem MdSHN3) op chromosoom 15. Die MdSHN3 transkripsie faktor vertoon uiters lae uitdrukking in lyne met onbehoorlike kutikula vorming daarop dui dit 'n fundamentele reguleerder van kutikula biosintese in die appel vrugte, en dus nodig is vir die voorkoming van suberized vrugte oppervlaktes (rooibruin) wees. In 'n poging om 'n beter begrip van die meganismes onderliggend suberien biosintese in vrugte te kry, en in plante in die algemeen, is transgeniese tamatie gegenereer met verswakte cutin vorming. Dit is bereik deur middel van die transkripsionele swye van SlDCR, 'n orthologue om AtDCR wat voorheen geïdentifiseer as 'n belangrike stap in cutin biosintese in Arabidopsis. Swye van hierdie BAHD acyltransferase gelei tot 'n byna totale uitwissing van die groot monomeer van die vrugte cutin (C16-9,10-dihydroxy vetsuur), en die plante ontwikkel vrugte met 'n suberized oppervlak. Dit verskaf 'n uitstekende geleentheid vir transkriptoom en chemiese karakterisering van die suberization proses vlesige vrugte. In parallel 'n appel kloon dat 'n russeted vrugte oppervlak ontwikkel is geïdentifiseer, en wat gekenmerk word. A groot skaal vergelykende transcriptomic ontleding van hierdie tamatie en appel mutante is uitgevoer, wat 'n lys van kandidaat gene vir suberien afsetting. Die verhoging van die vergelyking in te sluit data ontgin uit die literatuur het gelei tot die toeligting van 'n multi-spesie geenuitdrukking handtekening vir suberien biosintese en toegelaat word vir die identifisering en karakterisering van nuwe genetiese elemente, insluitend diegene wat betrokke is by die regulering van suberien vorming en sy afsetting. Van hierdie genetiese elemente, MYB107, is gedemonstreer om 'n positiewe reguleerder van suberien opeenhoping in Arabidopsis saadhuid wees. In totaliteit het hierdie studie 'n groter begrip van genetiese meganismes regerende kutikula biosintese, veral in ontwikkelende vrugte geproduseer. Dit is bereik deur middel van die funksionele hoofsaaklik karakterisering van regulatoriese elemente (MIXTA-like, SHN3 en MYB107 transkripsie faktore) die beheer van die sintese van cutin en suberien matrikse. Daarbenewens is die DCR ensiem gedemonstreer belangrike stap in vrugte cutin biosintese wees. Ten slotte word die ingewikkelde verhouding tussen epidermale sel ontwikkeling en kutikula biosintese is verder uitgelig