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’.

Phloem transport is along hydrostatic pressure gradients generated by differences in solute concentration between source and sink tissues. Numerous species accumulate raffinose-family oligosaccharides (RFOs) in the phloem of mature leaves to accentuate the pressure gradient between source and sinks. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which transports predominantly sucrose. To do this, three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Sugars were extracted and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and 21-day old plants of both lines had levels of galactinol, raffinose, and stachyose approaching 50% of total soluble sugar. All three exotic sugars were also identified in phloem exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type leaves. Differences in starch accumulation or degradation between wild type and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between wild type and engineered plants, but engineered plants flowered earlier. Finally, since the sugar composition of the phloem translocation stream is altered in these plants, we tested for aphid feeding. When green peach aphids were given a choice between WT and transgenic plants, WT plants were preferred. When aphids were reared on only WT or only transgenic plants, aphid fecundity was reduced on the transgenic plants. When aphids were fed on artificial media with and without RFOs, aphid reproduction did not show differences, suggesting the aphid resistance is not a direct effect of the exotic sugars.

No description available.

Orientador: Prof. Dr. Danilo da Cruz Centeno / Coorientadora: Profª. Drª. Hana Paula Masuda / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, São Bernardo do Campo, 2018. / A busca por fontes renováveis de energia tem aumentado devido à preocupação mundial com o uso excessivo de combustíveis fósseis, destacando-se como alternativa o etanol produzido a partir da cana-de-açúcar. O plantio de cana é intenso no Brasil, por ser uma cultura relativamente bem adaptada às distintas condições locais. No entanto, condições de estresse biótico e abiótico podem reduzir a produtividade e gerar grandes prejuízos, sobretudo em períodos de seca. Nesta perspectiva, o estudo de características agronomicamente desejáveis, como a tolerância ao déficit hídrico, em diferentes variedades é fundamental para a seleção de cultivares apropriadas e o desenvolvimento de cultivares mais produtivas, que elevem a produção de etanol, sem que haja uma expansão demasiada das áreas de cultivo de cana. Neste trabalho, indivíduos de cana-de-açúcar SP80-3280, uma das cultivares mais produzidas no Estado de São Paulo, foram cultivados em condições hídricas distintas e tiveram suas folhas mais jovens (folha +1) analisadas em diferentes aspectos. Os dados fisiológicos foram medidos no Infra-Red GAS Analyzer (IRGA). A quantificação relativa de transcritos foi feita através do método de PCR em tempo real (qPCR), para galactinol sintase (ScgolS), rafinose sintase (ScrafS) e estaquiose sintase (ScstaS), genes-chave na via dos Oligossacarídeos da Série da Rafinose (OSR), envolvida na tolerância à seca. As sequências codificantes de ScgolS, ScrafS e ScstaS, foram obtidas através de análises fenéticas, realizadas com as regiões codificantes de homólogos em milho, sorgo e cana. A quantificação dos metabólitos primários foi realizada através da técnica de cromatografia gasosa acoplada à espectrometria de massas (GC-MS). Plantas que permaneceram sob déficit hídrico por 7 dias (DH - 7 dias) apresentaram diminuição em todos os parâmetros ecofisiológicos, e aumento nos níveis de mio-inositol, manitol, glicose, frutose, ácido quínico e ácido chiquímico. Já plantas com 20 dias de déficit hídrico (DH - 20 dias) mostraram diminuição na taxa de crescimento, no número de folhas verdes e na taxa fotossintética, mesmo com aumento nos níveis de condutância estomática e na concentração interna de CO2. Também apresentaram aumento nos níveis de frutose, galactose, ácido quínico e ácido chiquímico, e redução nos níveis de manitol. As plantas Reidratadas sobreviverem à reidratação e voltaram à crescer após o reestabelecimento da água, apresentando aumento na taxa fotossintética, mesmo com redução nos níveis de condutância estomática, na concentração interna de CO2 e na transpiração. Apresentaram também, aumento nos níveis de mio-inositol, manitol, frutose, ácidos orgânicos e ácidos graxos, e na quantidade relativa de transcritos de ScstaS. Em conclusão, plantas que passaram pelo déficit hídrico apresentaram redução da taxa de crescimento, maior quantidade de compostos relacionados ao ajuste osmótico, e não foi observada indução elevada dos genes-chave da via dos OSR. A sensibilidade de SP80-3280 ao déficit hídrico foi reafirmada, mas foi surpreendente a sobrevivência e reestabelecimento após a reidratação. Aparentemente, SP80-3280 induz mecanismos de prevenção ao déficit hídrico, que possibilitam sua sobrevivência, mas não consegue induzir suficientemente mecanismos de tolerância que garantam à planta melhores condições de crescimento diante do estresse. / The search for renewable sources of energy has increased due to the worldwide preoccupation with the excessive use of fossil fuels, highlighting as alternative the ethanol produced from sugarcane. Cane planting is intense in Brazil, because it is a relatively well adapted crop to the different local conditions. However, biotic and abiotic stress can reduce productivity and generate great losses, especially in periods of drought. In this perspective, the study of agronomically desirable characteristics, such as tolerance to water deficit, in different varieties is fundamental for the selection of appropriate cultivars and the development of more productive cultivars, increasing the ethanol production, without extensive expansion of sugarcane planting areas. In this work, individuals of cultivar SP80-3280, one of the most produced in the State of São Paulo, were cultivated under different water conditions and had their youngest leaves (leaf +1) analyzed in different aspects. Physiological data were measured in the Infra-Red GAS Analyzer (IRGA). The relative quantification of transcripts was done by real-time PCR method (qPCR) for galactinol synthase (ScgolS), raffinose synthase (ScrafS) and stachyose synthase (ScstaS), keygenes in the Raffinose Family Oligosaccharides (RFO), involved in drought tolerance. The coding sequences of ScgolS, ScrafS and ScstaS were obtained through phenetic analyzes performed with the homologous coding regions of maize, sorghum and cane. The primary metabolites were quantified by gas chromatography coupled to mass spectrometry (GC-MS). The plants that remained under water deficit for 7 days (DH - 7 days) presented a decrease in all the ecophysiological parameters, and increased levels of myo-inositol, mannitol, glucose, fructose, quinic acid and shikimic acid. Plants with 20 days of water deficit (DH - 20 days) showed a decrease in the growth rate, in the number of green leaves and in the photosynthetic rate, even with an increase in stomatal conductance and in the internal CO2 concentration. There was also an increase in the levels of fructose, galactose, quinic acid and shikimic acid, and reduction in mannitol levels. The rehydrated plants survived rehydration and returned to grow after water reestablishment, with an increasing in the photosynthetic rate, even with a reduction in stomatal conductance, internal CO2 concentration and transpiration levels. They also showed increased levels of myo-inositol, mannitol, fructose, organic acids and fatty acids, and at the relative amount of ScstaS transcripts. In conclusion, plants that underwent water deficit had a reduction in the growth rate, a greater number of compounds related to the osmotic adjustment, and no high induction of the key genes of the RFO pathway was observed. The sensitivity of SP80-3280 to water deficit was reaffirmed, but survival and reestablishment after rehydration was surprising. Apparently, SP80-3280 induces prevention mechanisms that allow its survival, but it can not sufficiently induce tolerance mechanisms that guarantee the plant better growth conditions in the face of stress.

No description available.

Les agrobactéries établissent des relations à long terme avec les plantes et ce, dans deux styles de vie différents, rhizosphérique et pathogène (galle du collet). Dans ce mode de vie, les bactéries modifient génétiquement leur hôte et se créent ainsi une niche écologique spécifique (tumeur). La transition entre les deux styles de vie est déclenchée par la perception de signaux végétaux, parmi lesquels des acides hydroxycinnamiques (HCAs) comme l’acide férulique. Or dans l’espèce Agrobacterium fabrum, des gènes spécifiques permettent la dégradation des HCAs. Nous avons émis l’hypothèse que cette dégradation était un signal de proximité de la plante et influençait alors des fonctions importantes pour l’interaction avec celle-ci. Nous avons caractérisé la régulation de la dégradation des HCAs, évalué son rôle dans la valeur sélective d’A. fabrum, et suggéré son importance dans la transition entre les styles de vie rhizosphérique et pathogène. Nous avons montré que la dégradation des HCAs module le métabolisme carboné bactérien, notamment l’utilisation d’acide aminés et d’oligosaccharides de la famille du raffinose. Nous avons caractérisé la protéine MelB qui permet l’import de ces sucres, du mélibiose et du galactinol. Leur utilisation est importante pour la colonisation des plantes dès la germination. L’analyse de l’expression des gènes et du métabolisme bactérien en présence d'un composé signal de la plante, nous a révélé de nouveaux déterminants importants pour l’écologie de ce phytopathogène, notamment des facteurs de transcription. En outre, cette analyse a confirmé l’importance des échanges cellulaires et de déterminants impliqués dans la compétition bactérienne / Agrobacterium establish long term interactions with plants, either in a rhizosphere or pathogenic lifestyle. Pathogenic agrobacteria are causing the crown gall disease by genetically modifying the plant cells host, thus creating a specific ecological niche (tumor). The transition from the rhizosphere to the pathogenic lifestyle is triggered by bacterial perception of plant-derived signals, including hydroxycinnamic acids (HCAs) such as ferulic acid. However, A. fabrum strains have species-specific genes that allow HCAs degradation.We hypothesized that in A. fabrum, the degradation of the HCAs is perceived as a plant signal which influences important functions involved in the interaction with plants. We characterized the regulation of HCAs degradation, evaluated its role in the fitness of A. fabrum, and suggested its importance for the transition between the rhizosphere and pathogenic lifestyles. Then, we showed that the degradation of HCAs modulates carbon metabolism, such as the use of amino acids and sugars belonging to the raffinose family oligosaccharides (RFO). We have demonstrated that besides these sugars, the MelB protein allows the import melibiose and galactinol. Their use is important for plant colonization, since seed germination. The analyzes of gene expression and bacterial metabolism in the presence of a plant signal compound, revealed new determinants important for A. fabrum ecology, including transcription factors. In addition, it confirmed the importance of cellular exchanges and bacterial competition for Agrobacterium fitness in planta