Identification and characterisation of a drought-responsive galactinol synthase in wild rocket (Diplotaxis tenuifolia)

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dc.contributor.advisorPeters, Shaun W.en_ZA
dc.contributor.advisorLoedolff, Biankeen_ZA
dc.contributor.authorXonti, Alukhanyoen_ZA
dc.contributor.otherStellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.en_ZA
dc.date.accessioned2023-12-01T09:48:45Z
dc.date.accessioned2024-01-08T17:16:24Z
dc.date.available2023-12-01T09:48:45Z
dc.date.available2024-01-08T17:16:24Z
dc.date.issued2023-12
dc.descriptionThesis (MScAgric)--Stellenbosch University, 2023. en_ZA
dc.description.abstractENGLISH ABSTRACT: The raffinose family of oligosaccharides (RFOs) are sucrosyl-galactosides that occur uniquely in the plant kingdom. The elucidated functional roles attributed to RFOs include serving as carbohydrate transporters, storage reserves and acting as protective agents against biotic and abiotic stress effects. Galactinol synthase (GolS; EC 2.4.1.123) is the key biocatalyst of the RFO biosynthetic pathway and initiates the synthesis of the precursor galactinol (Gol) with the transfer of a galactosyl moiety from UDP-galactose (UDP-Gal) to myo-inositol (Ino). RFO biosynthesis involves the sequential addition of activated galactosyl moieties from Gol to sucrose (Suc) with the enzymes raffinose synthase (RafS; EC 2.4.1.82) and stachyose synthase (StaS; EC 2.4.1.67) to produce raffinose (Raf) and stachyose (Sta) plus higher order RFOs respectively. The coupled transcriptional upregulation of GolS and subsequent RFO accumulation during water deficit stress has been well-studied in several plants; however, these functional dynamics have not been explored in wild rocket (Diplotaxis tenuifolia). The focus of the current study was to isolate and functionally characterise a putative DtGolS1 and investigate its transcriptional regulation and subsequent water-soluble carbohydrates (WSC) profile change in D. tenuifolia leaves under mild water deficit stress. With the heterologous expression of DtGolS1 in E. coli (DH5α), it was demonstrated that the extracts synthesized galactinol in vitro. The predicted DtGolS1 amino acid sequence exhibited GolS hallmarks from other plant species including the C-terminal APSAA pentapeptide and a serine phosphorylating site. Transcriptional analyses also indicated that DtGolS1 is sensitive to water deficit stress as transcript levels observed an upregulation for the most stressed rocket plants. Based on the current findings, the identification of a promising DtGolS1 candidate gene for sugar metabolic manipulation in improving water stress tolerance in wild rocket was revealed. In addition, owing to the prebiotic properties of RFOs, a greater understanding of RFO metabolism particularly in salad greens such as wild rocket could provide insight into biofortification strategies leading to the production of sustainable salad crops.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Die raffinose familie van oligosakkariede (RFO's) is sukrosiel-galaktosiede wat uniek in die planteryk voorkom. Die toegeligte funksionele rolle wat aan RFO's toegeskryf word, sluit onder andere die volgende in: dien as koolhidraatvervoerders, bergingsreserwes en dien as beskermende middels teen biotiese en abiotiese stres effekte. Galaktinol sintase (GolS; EC 2.4.1.123) is die sleutel biokatalisator van die RFO biosintetiese pad en inisieer die sintese van die voorloper galaktinol (Gol) met die oordrag van 'n galaktosiel deel van UDP-galaktose (UDP- Gal) na mio-inositol (Ino). RFO-biosintese behels die opeenvolgende toevoeging van geaktiveerde galaktosiede vanaf Gol tot sukrose (Suc) met die ensieme raffinose-sintase (RafS; EC 2.4.1.82) en stagiose-sintase (StaS; EC 2.4.1.67) om raffinose (Raf) en stagiose (Sta) te produseer plus hoër orde RFO's onderskeidelik. Die gekoppelde transkripsionele opregulering van GolS en daaropeenvolgende RFO-akkumulasie tydens watertekortstres is goed bestudeer in verskeie plante; hierdie funksionele dinamika is egter nie in wilde vuurpyl (Diplotaxis tenuifolia) ondersoek nie. Die fokus van die huidige studie was om 'n vermoedelike DtGolS1 te isoleer en funksioneel te karakteriseer en die transkripsionele regulering daarvan en daaropvolgende wateroplosbare koolhidrate (WSC's) profiel-verandering in D. tenuifolia blare onder ligte watertekortstres te ondersoek. Met die heteroloë uitdrukking van DtGolS1 in E. coli (DH5α), is dit gedemonstreer dat die ekstrakte galaktinol in vitro gesintetiseer het. Die voorspelde DtGolS1-aminosuurvolgorde het GolS-kenmerke van ander plantspesies getoon, insluitend die C-terminale APSAA-pentapeptied en 'n serien-fosforileringsplek. Transkripsionele ontledings het ook aangedui dat DtGolS1 sensitief is vir watertekortstres aangesien transkripsiesvlak 'n 9.6-voudige verandering waargeneem het vir die plante wat die meeste gestres het. Gebaseer op die huidige bevindinge, is die identifikasie van 'n belowende DtGolS1-kandidaatgeen vir suikermetaboliese manipulasie in die verbetering van waterstres- verdraagsaamheid in wilde vuurpyl aan die lig gebring en as gevolg van die prebiotiese eienskappe van RFO's, 'n groter begrip van RFO-metabolisme veral in groenteslaai soos wilde vuurpyl kan insig gee in biofortifikasiestrategieë wat lei tot die produksie van volhoubare slaaigewasse.af_ZA
dc.description.abstractISISHWANKATHELO NGESIXHOSA: Intsapho yee-raffinose yee-oligosaccharides (RFOs) zi-sucrosyl-galactosides ezenzeka ngokukodwa kwizityalo. Iindima ezicacileyo zokusebenza ezibalelwa kwii-RFO ziquka ukusebenza njengezithuthi zeswekile, oovimba bokugcina kunye nokusebenza njengee- arhente ezikhuselayo kwimiphumo yoxinzelelo yeziphilayo nezingaphiliyo. I-Galactinol synthase (GolS; EC 2.4.1.123) yi-ikhompawundi yemichiza ehamba phambili ekuqaliseni ukusabela ekwenzeni kwee-RFOs kwaye iqalisa ukuhlanganiswa kwe-galactinol (Gol) ngokudluliswa kwe-galactosyl ukusuka kwi-UDP-galactose (UDP-Gal) ukuya kwi-myo-inositol (Ino). Indela yokwenziwa kweeRFOs ibandakanya ukongezwa okulandelelanayo kwe- galactosyl ukusuka kwi-Gol ukuya kwi-sucrose (Suc) nge-raffinose synthase (RafS; EC 2.4.1.82) kunye ne-stachyose synthase (StaS; EC 2.4.1.67) ukuvelisa i-raffinose (Raf), i-stachyose (Sta) kunye nee-RFO ezikumgangatho ophezulu ngokulandelelanayo. Ukonyuka kwenguqulo lwe- GolS kunye nokuqokelela kwee-RFOs ngexesha loxinzelelo lwentsilelo yamanzi ifundwe kakuhle kwizityalo ezininzi; nangona kunjalo, indlela ezisebenza ngazo azikhange zihlolwe kwi mfino yasendle (Diplotaxis tenuifolia). Ingqwalasela yophononongo ibikukwahlula nokuphanda iimpawu zokusebenza lwe-DtGolS1 ecingelwayo kunye nokuphanda ummiselo kwenguqulo lwayo notshintsho lweprofayile kwee-water-soluble carbohydrates (WSCs) kwamagqabi e-D. tenuifolia phantsi koxinzelelo lwentsilelo yamanzi. Ngokubonakaliswa kwe- DtGolS1 kwi-E. coli (DH5α), kuboniswe ukuba izicatshulwa zenze i-GolS kwityhubhu yovavanyo. Ulandelelwano oluqikelelweyo lwe-amino acid ye-DtGolS1, lubonise iimpawu ze- GolS ezivela kwezinye iintlobo zezityalo eziquka impawu ye-APSAA ekugqibeleni kunye ne- serine. Uhlalutyo lukwabonise ukuba i-DtGolS1 inovelwano kuxinzelelo lwentsilelo yamanzi njengoko inqanaba loshicilelo libone utshintsho oluphindwe kayi-9.6 kwezona zityalo zixinzelelwe kakhulu. Ngokusekwe kwiziphumo zangoku, ukuchongwa kwejini elithembisayo le-DtGolS1 lokuguqula iswekile ekuphuculeni ukunyamezela uxinzelelo lwamanzi kwimfino yasendle ityhiliwe kwaye ngenxa yeempawu zee-RFO njengee-arhente ezikhuthaza ukukhula kwee-bhaktheriya eziluncedo zamathumbu, ukuqonda ngakumbi inkqubo zee-RFO ibalulekile. Eziziphumo zibonisa indlela ezinokwenziwa ngayo izityalo zokutya eziphuculweyo ngendlela ezinzileyo.xh_ZA
dc.description.versionMastersen_ZA
dc.format.extentxii, 63 pages : illustrations (some color)en_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/128959
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshRaffinoseen_ZA
dc.subject.lcshDiplotaxis tenuifolia -- Morphologyen_ZA
dc.subject.lcshWild rocket (Diplotaxis tenuifolia) -- Effect of water levels onen_ZA
dc.subject.lcshDiplotaxis tenuifolia -- Effect of stress onen_ZA
dc.subject.lcshDrought-tolerant plantsen_ZA
dc.subject.lcshRaffinose family of oligosaccharides -- Synthesisen_ZA
dc.subject.nameUCTDen_ZA
dc.titleIdentification and characterisation of a drought-responsive galactinol synthase in wild rocket (Diplotaxis tenuifolia)en_ZA
dc.typeThesisen_ZA
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