Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The raffinose family of oligosaccharides (RFOs) are α1,6-galactosyl extensions of sucrose (Suc-Galn) unique to the plant kingdom. Their biosynthesis is mediated via α1,6-galactosyltransferases which catalyse the formation of raffinose (Raf, Suc-Gal1), stachyose (Sta, Suc-Gal2) and higher oligomers (Suc-Galn, n ≥ 13) in a stepwise manner. RFOs are well known for their historical roles as phloem translocates and general carbon storage reserves. In recent years their physiological roles have expanded to include potential functions in global plant stress-responses, where correlative mass increases are associated with abiotic stresses such as desiccation, salinity and low temperatures and, to a lesser extent biotic stress (pathogen infection). This study focused on (i) the functional characterisation of a putatively annotated stachyose synthase from Arabidopsis seeds (RS4, At4g01970), (ii) dissection of the proposed functional role of the RFO precursor galactinol in biotic stress tolerance using the Arabidopsis/Botrytis cinerea pathosystem and, (iii) an attempt to engineer long-chain RFOs into Arabidopsis by constitutive over-expression of the unique RFO chain elongation enzyme galactan:galactan galactosyltransferase (ArGGT) from Ajuga reptans. In Arabidopsis Raf is the only RFO known to accumulate in leaves, strictly during conditions of abiotic stress. However, seeds accumulate substantial amounts of both Raf and Sta. While RFO physiology in Arabidopsis leaves and roots is quite well characterised, little is known about the RFO physiology in the seeds. Apart from a single enzyme being described to partially contribute to seed Raf accumulation (RS5, At5g40390), no other RFO biosynthetic genes are known. In this work we functionally characterised an α1,6-galactosyltransferase putatively annotated as a stachyose synthase (RS4, At4g01970) in the Arabidopsis database. Using two insertion mutants (atrs4-1 and 4-2) we demonstrated Sta deficiency in mature seeds. A double mutant with the recently characterised RS5, shown to partially be responsible for Raf accumulation in mature seeds was completely deficient in seed RFOs. This provided the first hint that RS4 could potentially also be involved in Raf biosynthesis. Seed specific expression of RS4 was deregulated by constitutive over-expression in wild-type (Col-0) and the atrs5 mutant background (RS and Raf deficient). Both Raf and Sta unusually accumulated in Col-0 leaves over-expressing RS4, under normal growth conditions. Further, leaf crude extracts from atrs5 insertion mutants (RS and Raf deficient) over-expressing RS4 showed enzyme activities for both RS and SS, in vitro. Collectively our findings have physiologically characterised RS4 as a RFO synthase responsible for Sta and, partially Raf (along with RS5) accumulation during Arabidopsis seed development. The galactosyl donor in RFO biosynthesis, galactinol (Gol) has recently been implicated in biotic stress signalling (pathogen response) in cucumber, tobacco and Arabidopsis. Those studies focused exclusively on Gol in their experimental approaches using both over-expression (tobacco, Arabidopsis) and loss-of-function (Arabidopsis) strategies. However, they did not address the invariable accumulation of Raf that is routinely obtained from such over-expression strategies. We therefore investigated if Raf could play a functional role in induced systemic resistance (ISR), a well-studied mechanism employed by plants to combat necrotrophic pathogens such as Botrytis cinerea. To this end we looked to the RS5 mutant backgrounds (Raf deficient but Gol hyper-accumulating) reasoning that the Gol accumulating mutants should be resistant to B. cinerea (as previously described for transgenic over-expression of GolS1 isoforms in tobacco and Arabidopsis). Such findings would then preclude a role for Raf, since the system would be Raf deficient. Surprisingly, two independent T-DNA insertion mutants for RS5 (atrs5-1 and 5-2) were equally hypersensitive to B. cinerea infection as two independent T-DNA insertion mutants for GolS1 (atgols1-1 and 1-2). The hyper-sensitivity of the GolS1 mutant background has previously been demonstrated. The RS5 mutant backgrounds accumulate substantial amounts of Gol, comparable to those reported for transgenic plants (tobacco and Arabidopsis) where pathogen resistance was reported. Further, during the course of our investigations we discovered that both AtGolS1 mutants also accumulated substantial amounts of both Gol and Raf under normal growing conditions. This was not reported in previous studies. Collectively our findings argue against a role for either Gol or Raf being responsible for the induction/signalling of ISR. However, we do not preclude that the RFO pathway is somehow involved, given the previous reports citing pathogen resistance when GolS1 genes are over-expressed. We are further investigating a potential role for the GolS transcript and/or protein being the component of the suggested signalling function in ISR. The unique enzyme from A. reptans (galactan:galactan galactosyltransferase, ArGGT) is able to catalyse the formation of higher oligomers in the RFO pathway without the use of Gol as a galactosyl donor but rather, using RFOs themselves as galactose donors and acceptors (Gol-independent biosynthesis). We constitutively over-expressed ArGGT in Arabidopsis as a way to engineer long-chain RFO accumulation to further dissect a role for them in improving freezing tolerance. To this end we have been unsuccessful in obtaining RFOs higher than Sta (which occurred in extremely low abundance) in the leaves. Since ArGGT would appear to show substrate preference for Sta, and Arabidopsis seeds accumulate substantial quantities of Sta, we further analysed the seed water soluble carbohydrate (WSC) profiles of three independent transgenic lines but detected no additional RFO oligomers beyond the normally accumulating Raf and Sta. We suggest further strategies to improve this approach (Chapter 4). Collectively this work represents case studies of RFOs in seed physiology, their abilities/requirement in biotic stress and the use of unique enzymes to engineer long-chain RFO accumulation using the Arabidopsis model. At the time of submission of this dissertation the following contributions have been made to the general scientific community: (i) Presentation of chapter 2 at the 26th International Conference for Arabidopsis Research (26th ICAR, 2015, Paris, France) and, (ii) Submission of chapter 2 as a manuscript presently under peer review for possible publication in Plant and Cell Physiology. / AFRIKAANSE OPSOMMING: Die raffinose familie van oligosakkariede (RFO) is α1,6-galactosyl uitbreidings van sukrose (Suc-Galn) uniek aan die plante koningryk. Hul biosintese word bemiddel deur α1,6-galactosyltransferases wat in 'n stapsgewyse manier die vorming van raffinose (Raf, Suc-Gal1), stachyose (Sta, Suc-Gal2) en hoër oligomere (Suc-Galn, n ≥ 13) kataliseer. RFOs is bekend vir hul historiese rol as floëem translokate en algemene koolstof reserwes. Meer onlangs was hul fisiologiese rolle uitgebrei om potensiële funksies te vervul in globale plant stres-reaksies, waar korrelatiewe massa toenames geassosieer word met abiotiese stresfaktore soos uitdroging, soutgehalte en lae temperature en tot 'n mindere mate biotiese stres (patogeen infeksie). Hierdie studie fokus op (i) die funksionele karakterisering van 'n tentatief ge-annoteerde stachyose sintase van Arabidopsis sade (RS4, At4g01970), (ii) disseksie van die voorgestelde funksionele rol van die RFO voorloper galactinol in biotiese stres verdraagsaamheid, met behulp van die Arabidopsis/Botrytis cinerea patogeen sisteem en (iii) 'n poging om 'n lang-ketting RFOs in Arabidopsis te inisieer deur konstitutiewe oor-uitdrukking van die unieke RFO ketting-verlengings ensiem galactan:galactan galactosyltransferase (ArGGT) afkomstig van Ajuga reptans. In Arabidopsis is Raf die enigste RFO bekend daarvoor om te versamel in die blare, ekslusief tydens toestande van abiotiese stres. Maar, sade versamel aansienlike konsentrasies van beide Raf en Sta. Terwyl RFO fisiologie in Arabidopsis (blare en wortels) baie goed gekenmerk is, is min bekend oor die RFO fisiologie in die saad. Afgesien van 'n enkele ensiem wat beskryf word om gedeeltelik by te dra tot Raf versameling (RS5, At5g40390), is geen ander RFO biosintetiese gene bekend in saad nie. In hierdie werk beskryf ons die funksionele karakterisering van ‘n α1,6-galactosyltransferase wat tenetatief ge-annoteer word as 'n stachyose sintase (RS4, At4g01970) in die Arabidopsis databasis. Met die gebruik van twee invoegings mutante (atrs4-1 en 4-2) het ons die verlies van Sta in volwasse sade gedemonstreer. RFOs was heeltemal absent in sade van 'n dubbele mutant met die onlangs gekarakteriseerde RS5 (verantwoordelik vir gedeeltelike Raf versameling in volwasse sade). Dit het die eerste aanduiding daargestel dat RS4 potensieel ook betrokke kan wees in Raf biosintese. Saad-spesifieke uitdrukking van RS4 was gedereguleer deur konstitutiewe oor-uitdrukking in wilde-tipe (Col-0) en die atrs5 mutant agtergrond (RS en Raf gebrekkig). Oor-uitdrukking van RS4 in Col-0 blare het gelei tot beide buitengewone Raf en Sta konsentrasies, onder normale groeitoestande. Verder, oor-uitdrukkingvan RS4 in atrs5 invoeg mutante (waar beide RS en Raf absent is) het in vitro ensiemaktiwiteite vir beide RS en SS getoon. Gesamentlik beskryf ons bevindinge die fisiologies karakterisering van RS4 as 'n RFO sintase, verantwoordelik vir Sta en gedeeltelik Raf (saam met RS5) sintese tydens Arabidopsis saad ontwikkeling. Die galactosyl skenker in RFO biosintese, galactinol (Gol), was onlangs beskryf om ‘n rol te speel in biotiese stres (patogeen reaksie) in komkommer, tabak en Arabidopsis. Daardie studies het uitsluitlik gefokus op Gol in hul eksperimentele benaderings deur die gebruik van beide oor-uitdrukking (tabak, Arabidopsis) en die verlies-van-funksie (Arabidopsis) strategieë. Maar hulle het nie die onveranderlike opeenhoping van Raf, wat gereeld verky word uit sulke oor-uitdrukking strategieë, aangespreek nie. Ons het dus ondersoek of daar 'n funksionele rol vir Raf in geïnduseerde sistemiese weerstand (ISR) kan wees. ISR is 'n goed-bestudeerde meganisme wat deur plante ge-implementeer word om nekrotrofiese patogene soos Botrytis cinerea te beveg. Vir hierdie doel het ons gekyk na die RS5 mutant agtergronde (absent in Raf, maar hiper-akkumulasie van Gol) met die redenasie dat die Gol akkumulerende mutante weerstandbiedig teen B. cinerea moet wees (soos voorheen beskryf vir transgeniese oor-uitdrukking van GolS1 in tabak en Arabidopsis). Sulke bevindings verhinder dan 'n rol vir Raf, aangesien die stelsel geen Raf akkumuleer nie. Verbasend, twee onafhanklike T-DNA invoeg mutante vir RS5 (atrs5-1 en 5-2) was ewe hiper-sensitief vir B. cinerea infeksie as twee onafhanklike T-DNA invoeg mutante vir GolS1 (atgols1-1 en 1-2). Die hiper-sensitiwiteit van die GolS1 mutant agtergrond was reeds voorheen gedemonstreer. Die RS5 mutant agtergronde versamel aansienlike konsentrasies van Gol, vergelykbaar met dié berig vir transgeniese plante (tabak en Arabidopsis) waar patogeen-weerstandbiedigheid aangemeld is. Verder, in die loop van ons ondersoeke het ons ontdek dat beide AtGolS1 mutante ook aansienlike konsentrasies van beide Gol en Raf onder normale groei-toestande akkumuleer. Dit was nie aangemeld in die vorige studies nie. Gesamentlik argumenteer ons bevindinge teen 'n rol vir óf Gol, of Raf, tydens die induksie van ISR. Alhoewel, ons elimineer nie ‘n rol vir die RFO padweg nie, gegewe dat oor-uitdrukking van GolS1 gene tydens patogeen-weerstandbiedigheid in vorige verslae verwysig was. Ons ondersoek verder 'n moontlike rol vir die aanwesigheid van die GolS transkrip en/of proteïen as ‘n moontlike komponent van die voorgestelde funksie in ISR. Die unieke ensiem van A. reptans (galactan:galactan galactosyltransferase, ArGGT) is in staat om die vorming van hoër oligomere in die RFO pad te kataliseer sonder die gebruik van Gol as 'n skenker galactosyl, maar eerder, met behulp van die RFO's hulself as galaktose skenkers en aanvaarders (Gol-onafhanklike biosintese). Ons het ArGGT konstitutief ooruitgedruk in Arabidopsis as 'n manier om 'n lang-ketting RFO akkumulasie daar te stel met die doel om 'n rol vir hulle in die verbetering van vriestoleransie verder te ontleed. Ons was tot dusver onsuksesvol in die verkryging van RFOs hoër as Sta in die blare (wat akkumuleer het in 'n baie lae konsentrasie). Sedert ArGGT ‘n affiniteit vir Sta as substraat toon, en Arabidopsis sade versamel aansienlike hoeveelhede Sta, het ons verder die saad water oplosbare koolhidraat (WSC) profiele van drie onafhanklike transgeniese lyne ontleed, maar bespeur geen bykomende RFO oligomere buite die normale Raf en Sta konsentrasie nie. Ons stel verdere strategieë voor om hierdie benadering (Hoofstuk 4) te verbeter. Gesamentlik verteenwoordig hierdie werk gevallestudies van RFOs in saadfisiologie, hul vermoëns/vereiste in biotiese stres en die gebruik van unieke ensieme om lang-ketting RFO akkumulasie daar te stel met behulp van die Arabidopsis model. Teen die tyd van die indiening van hierdie tesis was die volgende bydraes gemaak aan die algemene wetenskaplike gemeenskap: (i) Aanbieding van hoofstuk 2 op die 26ste Internasionale Konferensie vir Arabidopsis Navorsing (26ste ICAR, 2015, Parys, Frankryk), en (ii) indiening van hoofstuk 2 as 'n manuskrip tans onder nasiening vir moontlike publikasie in die joernaal ‘Plant and Cell Physiology’.

Orientadora: Profª. Drª. Hana Paula Masuda / Coorientador: Prof. Dr. Danilo da Cruz Centeno / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, São Bernardo do Campo, 2018. / Os oligossacarídeos da série da rafinose (OSRs) são carboidratos formados pela adição sequencial de um grupo galactosil, geralmente doado por uma molécula de galactinol, à molécula de sacarose. Essa via é regulada principalmente por três enzimas. A galactinol sintase (GOLS) que é responsável pela síntese de galactinol. A rafinose sintase (RAFS) que transfere o resíduo galactosil do galactinol à molécula de sacarose dando origem a rafinose. E a estaquiose sintase (STS) que é responsável pela transferência de galactosil para a rafinose, dando origem a estaquiose. Esses açúcares desempenham importantes papéis fisiológicos nas células vegetais e têm sido considerados como moléculas chave na resposta ao estresse abiótico. Cada enzima envolvida no metabolismo dos OSRs é codificada por uma família de gênica. No entanto, ainda são escassos os trabalhos que apresentem descrições sistemáticas dos genes e suas relações evolutivas nas espécies vegetais. Os poucos trabalhos disponíveis focam nos genes que codificam GOLS, frequentemente considerada a enzima-chave da via. O objetivo deste trabalho foi estudar a diversidade e evolução dos genes rafs e sts em monocotiledôneas, para ampliar o conhecimento sobre os genes nessas espécies. Foram investigados genes rafs e sts em oito espécies vegetais, seis monocotiledôneas e duas dicotiledôneas. Também foram produzidas análises filogenéticas, de ortologia e a caracterização dos domínios proteicos nos genes identificados. Os resultados mostraram que RAFS e STS existem em grande diversidade e que são codificadas por vários genes putativos. As árvores filogenéticas permitiram diferenciar rafs de sts, sugerir relações evolutivas entre os genes e identificar diferentes grupos nessa família gênica. Análises de sintenia indicam a existência de genes ortólogos e duplicações in tandem. Por fim, a análise dos domínios proteicos confirmou a similaridade entre rafs e sts. Como conclusão, essa dissertação expande o conhecimento a respeito dos genes codificadores da via do OSRs, fornece informações para futuros trabalhos com foco em biotecnologia e contribui com a descrição das informações genômicas obtidas nos projetos de sequenciamento genético de espécies vegetais. / The raffinose series oligosaccharides (RFOs) are small carbohydrates synthetized by the sequential addition of a galactosil group, usually donated by a galactinol to sucrose. This metabolic pathway is regulated, among others, by the galactinol synthase (GOLS) enzyme, responsible for the synthesis of galactinol; the raffinose synthase (RAFS), responsible for the transfer of a galactosil group to sucrose, synthetizing rafinose, and; stachyose synthase (STS), responsible for the transfer of another galactosil group to raffinose, thus producing stachyose. These sugars play important physiological roles on plant cells and are considered key molecules in the response to abiotic stress. The enzymes involved on the RFOs metabolism exhibit a large number of functional genes. However, few studies present systematic descriptions of these genes and their evolutionary relationships on plant species. The few available studies focused on the genes that code for GOLS, frequently considered the key enzyme of RFOs metabolic pathway. The objective of this study was to understand the diversity and evolution of the rafs and sts genes in monocot species, to extend the knowledge on these plant genes. Rafs and sts genes were surveyed in eight plant species, six monocot and two dicot species. Phylogenetic and synteny analyses were performed, as well as, the characterization of the protein domains. The results showed that a large number of putative genes codifies both RAFS and STS, indicating that this gene family have a high diversity in plant genomes. The phylogenetic trees allowed proposing the evolutionary relationships between those genes and suggested the existence of different sequence groups. Synteny analyses showed groups of orthologue genes and in tandem gene duplications. Finally, the protein domain analyses corroborated the high similarity between rafs and sts. In conclusion, this work expands the knowledge about RFOs metabolism genes, provided information for further biotechnology studies and contributes to the description of sequence data from genomics projects.