Dissertations / Theses on the topic 'Raffinose oligosaccharides'

The raffinose family oligosaccharides (RFO) are alpha-galactosyl derivatives of sucrose, and the most common are raffinose, stachyose and verbascose. Raffinose is found exclusively in the embryo. Embryos and endosperms of barley (Hordeum vulgare. Cv. Golden Promise) were studied throughout their development and into maturity to investigate the 'relationships among a-amylase, carbohydrate, abscisic acid (ABA), raffinose synthesis pathway and the onset of desiccation tolerance. The onset of desiccation tolerance corresponds to an increase in starch, a-amylase, raffinose and a decrease in monosaccharides, stachyose and sucrose in the embryo of immature grains. A 5 fold increase was observed between 3-4 W.P.A of raffinose. 4-5 W.P.A, in the embryo a-galactosidase increases from 0.125 - 0.54 U/dry wt. The increase of raffinose precedes by a week the rise in a.:.galactosidase. In immature embryos, compared to the control without ABA, raffinose and galctose slightly increaSed and myo-inositol and glycerol decreased. ABA induced the down regulation of photochrome Band glutathione S-transferases. During imbition of mature grains in the embryo raffinose, melibiose and stachyose decreased to undetectable by 28, 17 and 17 h respectivlty however, sucrose increased. In the dormant samples metabolism fluctuated only slightly'. Galactose and raffinose was only present in dormant grain. Germination percentage was adversely influenced by salt and osmotic stress (PEG). Expression of raffinose synthase increased after 5 and 48 h of NaCI treatment and at 30°C during dehydration and re-hydration. The gene was also detected throughout 5 days of germination. In summary, water deficit stress caused pronounced effects on carbohydrate metabolism in barley embryos.RFO,s and glycerol were generally depressed by all stress treatments, which suggests RFO's may not playa direct role in inducing tolerance in embryo tissues.

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.

The raffinose family of oligosaccharides (RFO) function as transport carbohydrates in the phloem, storage compounds in sink tissues, and as putative metabolic agents that combat plant stresses. Research on the RFO pathway has focused on seed biology and plants that transport raffinose as their primary photoassimilate. In contrast, few studies have explored this pathway in woody species. As such, this thesis investigated the fundamental function of the RFO enzymes in hybrid poplar, with emphasis on galactinol synthase (GolS), an enzyme key to the pathway. Phylogenetic comparisons of Populus Go1S with other known GolS suggest a putative role for these enzymes in stress response. Protein analysis of two heterologously expressed isoforms demonstrated that they are true Go1S; with Pa×gGolSI possessing a broader pH and temperature range than Pa×gGolSII. Expression patterns also revealed that the Pa×gGolSII transcript abundance varied seasonally. Together, the results suggest that Pa×gGolSI may be involved in basic metabolic activities, while Pa×gGolSII is likely involved in seasonal mobilization of carbohydrates. To further elucidate the in-planta Go1S function, transgenic trees with mis-regulated GolS were generated. Two AtGolS3 over-expression (OE) transgenic lines showed effects on growth, while other lines appeared normal and possessed marginally modified cell wall characteristics. The extreme over-expressers were severely stunted and had cell wall traits characteristic of tension wood. AtGolS3-OE lines showed reduction in the microfibril angle, increase in cell wall crystallinity and possessed higher cellulose, and lower mannose and lignin contents. Interestingly, although galactinol and raffinose contents increased dramatically, they were not more tolerant to abiotic stress under the conditions tested. These results suggest that the over-expression of GolS and its product galactinol may serve as a molecular signal that initiates different metabolic changes for combating stress, culminating in the formation of tension wood. Additionally, over expression of raffinose synthase (RFS) resulted in increased biomass and total cellulose content. However, it does not appear to have a similar signalling role. Collectively, this research opens new insight about functions of the RFO in poplar, with the participation of GolS in stress signalling and consequent tension wood formation, and the importance of RFS to carbon allocation and growth.

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’.

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

Les α-galactosides sont des glucides non digestibles constitués d'unités galactose liées en α(1,6). Les α-galactosides de la famille du raffinose (RFO) sont, avec le saccharose, les principaux oligosaccharides des légumineuses. Cependant, aucune activité α(1,6)-galactosidase n'est retrouvée au niveau de l'épithélium intestinal humain, les RFO sont donc exclusivement fermentés par les enzymes microbiennes. Ces travaux introduisent une enzyme bifonctionnelle de Ruminococcus gnavus E1, un membre majoritaire du microbiote intestinal humain, présentant une activité α(1,6)-galactosidase/ saccharose kinase (AgaSK). L'analyse de la séquence peptidique montre qu'AgaSK présente deux domaines : un domaine homologue aux α-galactosidases GH36, et un autre contenant un motif de fixation des nucléotides (motif A de Walker). La caractérisation des paramètres biochimiques d'AgaSK met en évidence cette bifonctionnalité puisqu'elle est capable d'hydrolyser les α(1,6)-galactosides solubles, et parallèlement en présence d'ATP de phosphoryler le saccharose spécifiquement sur la position C6 du glucose. La production directe de saccharose-6-phosphate à partir de l'hydrolyse du raffinose constitue une voie métabolique jamais décrite chez les bactéries. L'analyse de chacun des domaines montre que les domaines isolés d'AgaSK sont actifs mais la comparaison de leurs paramètres cinétiques montre qu'il y a des différences entre la protéine entière et les domaines isolés. La résolution de la structure du domaine α-galactosidase en complexe avec le galactose démontre que l'état oligomérique est nécessaire pour le bon repliement de la protéine et pour une fixation efficace du substrat
Α-galactosides are non digestible carbohydrates present in many leguminous plants. Soluble α-galactosides consist of galactose units α(1,6) linked to different carbohydrates. Among these, the raffinose family oligosaccharides (RFO) and sucrose, are the most abundant oligosaccharides found in legumes. However, no α(1,6)galactosidase activity exists in the human intestine mucosa and α-galactosides are exclusively fermented by microbial α(1,6)galactosidases (EC3.2.1.22). Here we introduce a bifunctional enzyme, the α(1,6)galactosidase/sucrose kinase (AgaSK) whose gene is highly transcribed in vivo by Ruminococcus gnavus E1, a major member of human dominant intestinal microbiota. Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide binding motif (Walker A motif). Its biochemical characterization showed that AgaSK is able to hydrolyze efficiently soluble α-galacosides. Furthermore, AgaSK it is able to bind nucleotide to phosphorylate specifically on the C6 position of glucose sucrose. The production of sucrose-6-P directly from raffinose brings out a glycolytic pathway in bacteria, not described so far. In addition, AgaSK isolated domains are active but the biochemical characterization has shown that there are differences in the activities between the whole protein and isolated domains. The crystal structures of the galactosidase domain in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding

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.

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.

Soybean [Glycine max (L.) Merr] is one of the U.S.'s most economically important crops due to the protein and oil content of seeds. The major storage form of phosphorus in soybean seeds is found in the form of phytate, but because of its negative nutritional and environmental impacts, seed phytate and raffinosaccharide content have been a recent focus of breeders and molecular geneticists. The soybean line CX1834 is a low phytate mutant known to have two low phytate QTLs on linkage groups (LGs) L and N. The first objective of this research was to determine the genetic basis of the low phytate trait in CX1834. By using the whole genome sequence, we identified two candidate multidrug resistance-associated (MRP) ABC transporter genes. Sequencing the genes from CX1834 and comparing them to the reference genome sequence revealed a single nucleotide polymorphism (SNP) in the MRP gene located on LG N (causing a stop codon), and a SNP mutation in the MRP gene located on LG L (causing an amino acid change from arginine to lysine). One major concern with low phytate soybeans is the low seedling emergence. The second objective was to undertake a population-wide study of emergence in the recombinant inbred population CX1834 x V99-3337, over two years and two locations. We found a positive correlation between phytate level and emergence, and that variation among year, location, genotypic class, year x genotypic class, and year x location interactions were significantly affecting emergence. V99-5089, in addition to being low phytate, has high sucrose and low raffinosaccharide content. This phenotype of V99-5089 has been previously determined to be due to a SNP mutation in its myo-inositol phosphate synthase (MIPS) gene located on LG B1. The third objective was to use the recombinant inbred population derived from CX1834 x V99-5089 to observe the combinations of all three mutations to see how the different alleles impact phytate and raffinosaccharide content. The individuals with all three mutations, as well as those with the two MRP mutations together had lower phytate than the other genotypic classes. However, these lines (all three mutations) had unexpectedly high stachyose.
Ph. D.

La relation entre l'acquisition de la tolérance au gel et les teneurs en sucres solubles, l'amidon, les acides organiques et la proline a été étudiée chez deux variétés de pois (Pisum sativum L. ): Champagne, tolérant au gel et Térèse, sensible au gel. Le contenu en saccharose et raffinose dans l'appareil aérien et racinaire de Champagne augmente significativement en réponse à l'acclimatation au froid. Cependant chez les 59 lignées recombinantes, issues du croisement Champagne et Térèse cultivées en champ, il semble que l'accumulation des sucres solubles ne garantit pas l'acquisition de la tolérance au gel. L'analyse du profil d'expression des protéines de bourgeon, feuille, tige et racine de Champagne et Térèse au cours de l'acclimatation au froid, a permis de mettre en évidence l'expression spécifique de protéines de l'antenne collectrice de lumière et de la chaîne de transport d'électrons de l'appareil photosynthétique et impliquées dans l'inhibition du stress oxydatif chez Champagne.

To increase the global acceptability of chickpea by improving its nutritional quality, seed RFO (Raffinose Family Oligosaccharides) concentration needs to be reduced without affecting their role during seed development and positive impact on human health. To achieve this objective, the key regulating step(s) of RFO biosynthesis needs to be identified. The three main objectives of the thesis were: (1) to optimize an analytical method to determine soluble sugars concentration in chickpea seeds including RFO, (2) to determine chickpea genotypes with contrasting seed RFO concentration, and (3) to optimize and validate RFO biosynthetic enzyme activity assays. These three objectives of the thesis provided basis of the fourth objective. For the first objective, a modified HPAEC-PAD (High performance anion exchange chromatography with pulsed amperometric detector) based gradient approach was optimized to study the concentration and composition of soluble sugars in chickpea seeds. The optimized method separated all the soluble sugars within 20 min of run time with higher accuracy, sensitivity and precision compared to previously reported methods. Therefore, the optimized method was utilized to study the natural variation in RFO concentration of 171 chickpea genotypes. Sucrose (0.60 - 3.59 g/100 g) and stachyose (0.18 − 2.38 g/100 g) were predominant among soluble sugars and RFO, respectively. Analysis of variance revealed a significant impact (P ≤ 0.001) of genotype (G), environment (E), and their interaction (G×E) on seed RFO concentration in chickpea. A significant positive correlation was observed between substrate and product concentration in RFO biosynthesis. Raffinose, stachyose and verbascose showed moderate broad sense heritability (0.25 − 0.56) suggesting the quantitative nature of the RFO trait in chickpea seeds. Desi (ICC 1163, ICC 1471, ICC 9562, ICCV 07115, ICCV 07116 and ICCV 07117) and kabuli (ICC 5270, ICC 10674, ICC 16216, ICC 16528, ICCV 3 and ICCV 91302) chickpea genotypes with high and low RFO concentrations (high RFO genotypes are underlined) were identified. RFO biosynthetic enzymes activities were optimized for substrate and protein concentration, temperature (25 °C), time (10 min for galactinol synthase and 60 min for other biosynthetic enzymes) and pH (7.0). These assays were validated at different seed developmental stages of two released varieties: CDC Vanguard and CDC Frontier. Simultaneously, RFO accumulation at different seed developmental stages was also studied. During 18 - 38 DAF (days after flowering), about a 75 % decrease in seed moisture was observed coinciding with the accumulation of RFO providing desiccation tolerance to maturing seeds. The initial substrates viz. myo-inositol and sucrose were observed throughout seed development process having maximum accumulation at 18 - 20 (0.50 – 0.57 g/100 g) and 20 - 22 (9.94 - 11.17 g/100 g) DAF that decreased afterwards supporting the biosynthesis of galactinol and raffinose, respectively. Galactinol is considered as the universal galactosyl donor, it showed the highest concentration at 30 DAF and this was later utilized for increased RFO accumulation till 36 DAF. Activity of RFO biosynthetic enzymes was observed 2 - 6 days prior to first detection of their corresponding products whereas the highest activities were determined 2 - 4 days prior to maximum accumulation of RFO. However, maximum GS (galactinol synthase) activity was observed at 36 DAF but this did not correspond to amount of galactinol accumulation in seeds. This indicated that galactinol was synthesized in higher amount even after 30 DAF but directed towards RFO biosynthesis thus could not necessarily accumulate in seeds. A galactinol independent pathway was also found operative in chickpea seeds. These results suggested that substrate concentration and GS activity might be the possible factors regulating seed RFO concentration in chickpea. The fourth objective utilized the information, material and methods from the previous three objectives. Chickpea genotypes with contrasting RFO concentration were compared for seed size and weight, germination capacity and RFO biosynthesis (accumulation and biosynthetic enzymes activities during seed development). Sucrose concentration showed a significant positive (r = 0.728, P ≤ 0.05) correlation with seed size/weight. RFO concentration was a facilitator of seed germination rather than regulating factor. Higher accumulation of myo-inositol and sucrose in high RFO genotypes during early seed developmental stages suggested that initial substrates concentrations may influence seed RFO concentration. High RFO genotypes expressed about 2 - 3 fold higher activity for all RFO biosynthetic enzymes compared to those with low RFO concentration. The enzyme activity data corresponded with the accumulation of individual RFO during chickpea seed development. In conclusion, regulating galactinol synthase activity is a potential strategy to reduce seed RFO concentration in chickpea. The present study can be extended to study RFO biosynthesis at the transcript level and the influence of RFO biosynthetic enzymes on seed size and weight, germination, RFO concentration, yield, and stress tolerance.