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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.

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.

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.

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’.

Raffinose family oligosaccharides (RFO) are major soluble carbohydrates of lentil (Lens culinaris Medik) seeds. When consumed by humans, RFO pass indigested through upper digestive tract as ¦Á-galactosidase enzyme required for RFO breakdown is not produced in humans. Consumption of lentils with high concentrations of RFO result in stomach discomfort, bloating and diarrhea due to bacterial fermentation of RFO in large intestine. This has lead to a relatively low use of lentils for human consumption. RFO are therefore considered anti-nutritional factors and development of lentil cultivars with reduced RFO concentrations is desired to improve lentil quality and consumption. To explore the possibility to develop lentil cultivars for low-RFO concentration, heritability of RFO trait and influence of environmental conditions on RFO must be known. In addition, RFO biosynthesis and accumulation in lentil seeds must be understood. However, very limited information is available on the above mentioned aspects of RFO in general and in lentil in particular. Therefore, the objectives of this study were: (1) to evaluate natural variation in RFO concentration and composition in commonly grown lentil cultivars and to determine the correlation between RFO concentration and other important seed constituents, (2) to investigate heritability and effect of environment on concentration and composition of lentil seed soluble carbohydrates, (3) to assess natural variation and diversity in RFO concentration in the genus Lens, (4) and to evaluate the association between galactinol synthase activity and accumulation of RFO in lentil seeds. Analysis of 22 lentil genotypes revealed significant (P ¡Ü 0.05) variation in total starch, amyolse, protein, total RFO and seed weight and seed colour. Stachyose was the major RFO in all lentil genotypes followed by raffinose and verbascose. A significant (P ¡Ü 0.05) inverse correlation was found between RFO and amylose concentration (r = -0.34); whereas RFO concentration and thousand seed weight correlated positively (r = 0.35). The analyses of variance of eleven cultivars grown at ten different environments showed that cultivar, environment and their interaction had significant effects on sugar concentration in lentil seeds. The high broad sense heritability of RFO (h2= 0.85) indicated that RFO concentration in lentil seeds is highly heritable and thus amenable to genetic improvement. An extensive evaluation of domesticated and wild species and subspecies of the genus Lens revealed significant (P ¡Ü 0.05) variation and diversity in RFO concentration and composition of individual oligosaccharides. Higher Shannon-Weaver diversity indices (SDI) for total RFO, raffinose and verbascose traits were observed in wild lentils compared to domesticated genotypes. Lens ervoides genotypes and some wild genotypes contained almost half the RFO concentration of cultivated lentils and therefore, wild genotypes may be useful for developing low-RFO lines. Higher verbascose and lower stachyose concentration was found in Lens ervoides genotypes, whereas higher raffinose and lower verbascose concentration is found in Lens nigricans genotypes. Study of galactinol synthase activity in developing seeds with varying RFO concentration showed no clear association between galactinol synthase activity and RFO concentration. The sucrose and galactinol concentration of developing seeds were also not associated with total RFO concentration of lentil seeds. This finding suggests a non-regulatory role of galactinol synthase in RFO biosynthetic pathway in lentil seeds. Together, all these findings are not only significant to devise strategies to develop lentil cultivars with reduced RFO concentration but also for understanding RFO biosynthesis in lentil seeds.

Raffinose family oligosaccharides (RFO) are major soluble carbohydrates of lentil (Lens culinaris Medik) seeds. When consumed by humans, RFO pass indigested through upper digestive tract as ¦Á-galactosidase enzyme required for RFO breakdown is not produced in humans. Consumption of lentils with high concentrations of RFO result in stomach discomfort, bloating and diarrhea due to bacterial fermentation of RFO in large intestine. This has lead to a relatively low use of lentils for human consumption. RFO are therefore considered anti-nutritional factors and development of lentil cultivars with reduced RFO concentrations is desired to improve lentil quality and consumption. To explore the possibility to develop lentil cultivars for low-RFO concentration, heritability of RFO trait and influence of environmental conditions on RFO must be known. In addition, RFO biosynthesis and accumulation in lentil seeds must be understood. However, very limited information is available on the above mentioned aspects of RFO in general and in lentil in particular. Therefore, the objectives of this study were: (1) to evaluate natural variation in RFO concentration and composition in commonly grown lentil cultivars and to determine the correlation between RFO concentration and other important seed constituents, (2) to investigate heritability and effect of environment on concentration and composition of lentil seed soluble carbohydrates, (3) to assess natural variation and diversity in RFO concentration in the genus Lens, (4) and to evaluate the association between galactinol synthase activity and accumulation of RFO in lentil seeds. Analysis of 22 lentil genotypes revealed significant (P ¡Ü 0.05) variation in total starch, amyolse, protein, total RFO and seed weight and seed colour. Stachyose was the major RFO in all lentil genotypes followed by raffinose and verbascose. A significant (P ¡Ü 0.05) inverse correlation was found between RFO and amylose concentration (r = -0.34); whereas RFO concentration and thousand seed weight correlated positively (r = 0.35). The analyses of variance of eleven cultivars grown at ten different environments showed that cultivar, environment and their interaction had significant effects on sugar concentration in lentil seeds. The high broad sense heritability of RFO (h2= 0.85) indicated that RFO concentration in lentil seeds is highly heritable and thus amenable to genetic improvement. An extensive evaluation of domesticated and wild species and subspecies of the genus Lens revealed significant (P ¡Ü 0.05) variation and diversity in RFO concentration and composition of individual oligosaccharides. Higher Shannon-Weaver diversity indices (SDI) for total RFO, raffinose and verbascose traits were observed in wild lentils compared to domesticated genotypes. Lens ervoides genotypes and some wild genotypes contained almost half the RFO concentration of cultivated lentils and therefore, wild genotypes may be useful for developing low-RFO lines. Higher verbascose and lower stachyose concentration was found in Lens ervoides genotypes, whereas higher raffinose and lower verbascose concentration is found in Lens nigricans genotypes. Study of galactinol synthase activity in developing seeds with varying RFO concentration showed no clear association between galactinol synthase activity and RFO concentration. The sucrose and galactinol concentration of developing seeds were also not associated with total RFO concentration of lentil seeds. This finding suggests a non-regulatory role of galactinol synthase in RFO biosynthetic pathway in lentil seeds. Together, all these findings are not only significant to devise strategies to develop lentil cultivars with reduced RFO concentration but also for understanding RFO biosynthesis in lentil seeds.

<p><strong>Apstrakt je obrađen tehnologijama za optičko prepoznavanje teksta (OCR).</strong></p><p>Analizom podloge iz standardizovanog fiziolo&scaron;kog testa fermentacije rafinoze<br />primenom HPTLC metode, utvrđeno je da se u podlozi javlja jedinjenje sa<br />retencijom većom od rafinoze, dok svi konstituenti rafinoze imaju manju retenciju.<br />Za nastajanje takvog jedinjenja tokom testa fermentacije rafinoze i njenih<br />konstituenata kvascima nije bilo literatume potvrde.<br />Ispitivano je kod kojih kvasaca i kod fermentacije kojih ugljenih hidrata se u<br />podlozi javlja ispitivano jedinjenje. Zatim je ispitivana veza određenih parametara<br />procesa fermentacije sa nastajanjem zapaženog jedinjenja. Pored toga, ispitivanom<br />jedinjenju su, nakon izolovanja iz fermentacione podloge i preči&scaron;ćavanja radi<br />dobijanja čiste supstance, određeni neki elementi strukture.<br />Utvrđeno je da se samo kod kvasaca koji imaju fiziolo&scaron;ko svojstvo fermentacije<br />celog molekula rafinoze i svojstvo fermentacije melibioze tokom procesa<br />javlja ispitivano jedinjenje i da je ono intermedijer metabolizma. Činioci značajni<br />za proces fermentacije - inokulum (količina i podloge za njegovu pripremu),<br />sastav podloge (sadržaj rafinoze i melibioze, kao i različiti izvori azota) i uslovi<br />gajenja, varirani u odnosu na standardizovani fiziolo&scaron;ki test fermentacije, ne<br />prouzrokuju promenu kvalitativnog sastava jedinjenja u podlozi tokom fermentacije<br />rafinoze i melibioze. Dakle, ispitani intermedijer je uvek prisutan u podlozi.<br />Na povećanje njegovog sadržaja u podlozi utiče dodatak galaktoze tokom fermentacije<br />rafinoze. Najveća koncentracija ispitivanog intermedijera (0,87 mg/ml) je<br />trećeg dana fermentacije melibioze, odnosno drugog dana fermentacije rafinoze<br />(1,48 mg/ml). Sadržaj ispitivanog intermedijera na kraju procesa fermentacije<br />rafinoze je 6 puta veći nego kod melibioze (1,04 prema 0,17 mg/ml). Ispitivani<br />intermedijer je redukujući ugljeni hidrat molekulske mase 504 &scaron;to odgovara<br />trisaharidima. Konstituenti ovog jedinjenja su glukoza i galaktoza koje su povezane<br />najverovatnije a-tipom veze.</p> / <p><strong>Abstract was processed by technology for Optical character recognition (OCR).</strong></p><p>Analyzing the media from standardized physiological test for raffinose fermentation<br />by means of HPTLC method, the appearance of a compound was registered<br />with retention higher than that of raffinose or its constituents indicating also<br />smaller retentions. No literature data were found for the formation of such a<br />compound during fermentation tests for raffinose and its constituents.<br />It was investigated which yeast will indicate formation of the investigated<br />compound in the course of fermentation as well as which carbohydrate during its<br />fermentation will indicate the presence of this compound. Further investigation<br />included correlation of particular parameters known as significant for the process<br />of fermentation, with the appearance of the noticed compound. Moreover, some<br />structural elements were also determined for the investigated compound, after<br />isolation from fermentation media and purification in order to obtain pure<br />substance.<br />It was proved that the investigated compound appeared just with the yeasts<br />physiologically characterized by fermentation of entire molecule of raffinose as<br />well as melibiose in the course of fermentation. Ibis compound proved to be an<br />intermediate compound of metabolism. Parameters significant for the process of<br />fermentation - inoculum (size and media for its growth), media composition<br />(content of raffinose and melibiose as different nitrogen source) and the condition<br />of incubation, exchanged from the standardized physiological test of fermentation,<br />didn&#39;t change qualitatively the compound composition in the media during<br />raffinose and melibiose fermentation. Therefore, it is obvious that the investigated<br />intermediate compound was present constantly in the media. Its content is<br />effected by the addition of galactose in the course of raffinose fermentation. The<br />highest concentration (0.87 mg/ml) of the investigated compound was registered<br />on the third day of melibiose fermentation, i.e. the second day of raffinose<br />fermentation (1.48 mg/ml). Content of the investigated intermediate compound<br />at the end of raffinose fermentation was six limes higher than of melibiose (1.04<br />in relation to 0.17 mg/ml). The investigated intermediate compound proved to be<br />the reduced carbohydrate with molecular mass of 504 corresponding to trisaccharides.<br />Constituents of this compound, glucose and galactose are probably connected<br />by a-type of bonds.</p>

Orientador: Edvaldo Aparecido Amaral da Silva / Resumo: Os oligossacarídeos da família rafinose (OFR), presentes nas sementes/grãos de soja, são açúcares solúveis considerados fatores antinutricionais para humanos e animais monogástricos, e por isso, limitam o uso da cultura para estes. Entretanto, estes compostos aparentam exercer um papel fundamental na aquisição da qualidade fisiológica das sementes de soja. Diante disso, genótipos mutantes que expressam o fenótipo de baixo ou ultrabaixo teor de OFR têm sido estudados visando à eficiência da alimentação humana e animal. Portanto, é preciso elucidar as funções dos OFR sobre as propriedades da qualidade fisiológica de sementes, a fim de preconizar materiais genéticos de alta qualidade, com capacidade de armazenamento e propagação. Todavia, a qualidade fisiológica e os teores de açúcares solúveis podem ser alterados em função da genética do genótipo ou do ambiente. Por isso, genótipos mutantes em OFR podem apresentar a aquisição da qualidade fisiológica distinta quando produzidos em diferentes ambientes. Objetivou-se com este trabalho elucidar a contribuição dos OFR na aquisição da qualidade fisiológica de sementes de soja em genótipos que expressam teores contrastantes destes. Para tal, foram utilizadas sementes com fenótipos para baixo e ultrabaixo teor de OFR, mutantes nas enzimas rafinose sintase (rs) 2 e rs2 rs3, respectivamente, em comparação a genótipos com alelos funcionais para RS2 (teores normais de OFR), produzidos em três safras. As sementes foram colhidas em diferente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The raffinose family of oligosaccharides (OFR), present in soybean seeds/grains, are soluble sugars considered as antinutritional factors for humans and monogastric animals, and therefore, they limit the use of the crop. However, these compounds seem to play a fundamental role in the acquisition of physiological quality of soybean seeds. Thus, mutant genotypes expressing phenotype of low or ultralow content of OFR have been studied aiming at the efficiency of human and animal feeding. Therefore, it is necessary to elucidate the functions of OFR on the properties of physiological seed quality, in order to recommend materials of high-quality genetic material with storability and propagation capacity. The physiological quality and soluble sugar content may be altered in genetic or environmental function. Therefore, mutant OFR genotypes may acquire distinct physiological quality when grown in different environments. The aim of this research was to elucidate the contribution of OFR in the acquisition of the physiological quality of soybean seeds in genotypes that express contrasting contents for these in three experimental crops. For this, seeds with low and ultralow OFR phenotypes, mutants in raffinose synthase (rs) 2 and rs2 rs3, respectively, were used in comparison to genotypes with RS2 functional alleles (normal OFR levels). The seeds were harvested at different phenological stages during their maturation. Subsequently, it was evaluated for all genotypes studied in all harves... (Complete abstract click electronic access below) / Mestre

Carbohydrate content sucrose, raffinose, and stachyose are one of the important seed quality traits in soybean. However, little is known about the genetics of these quantitative traits. Recombinant inbred lines (RIL) were developed from the interspecific hybridization between a Glycine max breeding line (V71-370) and a Glycine soja plant introduction (PI407162). The 308 RILs, each parent, and one cultivar were arranged in a randomized complete block design with two replications and planted at two locations in Virginia. The main objective of the first part of this research was to devise a quick, economical, and reliable HPLC methodology to determine the amount of sucrose, raffinose, and stachyose in soybean seeds. Concentration of sucrose, raffinose and stachyose are quantitative traits, which are hard to manipulate genetically due to the influence of genotype, environment, and genotype by environment interactions on seed chemical composition. The objectives of the second study were to evaluate agronomic and quality traits over locations and to study correlations among traits. The agronomic traits analyzed in this study included; maturity, plant canopy height, canopy spread, leaflet length, leaflet width, yield, and seed size. Seed quality traits studied were sucrose, raffinose, and stachyose content. Although some correlation coefficients were statistically significant at P<0.001, many were not large enough to be of practical value. A positive correlation was observed between all three sugars. Significant variation was observed among RILs and locations for all traits studied. Genotype by environment interaction was significant for all of the agronomic traits, but was not significant for seed sucrose, stachyose, or raffinose. Maturity, seed size, and sucrose content were highly heritable traits, whereas plant height, canopy spread, yield, leaf length, leaf width, stachyose content, and raffinose content had relatively low broad-sense heritabilities. The RIL population was used to investigate the genetic basis for these agronomic and seed quality traits. Seven out of twenty soybean molecular linkage groups (MLG), A1, A2, E, F, G, I, and M, were selected on the basis of previous research and mapped in this population with restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers. Five QTL for seed sucrose content, one QTL each for raffinose and stachyose, and one for each agronomic trait except yield and leaflet width, and two QTL each for yield and leaflet width were detected using the Blacksburg data. Four QTL for seed sucrose content, one QTL for raffinose, two QTL for stachyose, one QTL each for plot width and yield and two QTL for leaflet width were detected using the Warsaw data. Several QTL affecting different agronomic traits shared common genomic regions suggesting pleiotropy at some loci. The majority of the seed quality QTL was stable at both locations. Agronomic traits were more environmentally sensitive and no QTL were common to both locations. Epistasis analysis showed interactions between QTL that detected new genomic regions associated with raffinose content. These results suggest that these potential QTL are definitely on the genomic regions of interest and would be more powerful in marker-assisted selection when we find closer markers to each QTL. / Ph. D.