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’.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/98052 |
| Date | 12 1900 |
| Creators | Loedolff, Bianke |
| Contributors | Peters, S., Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB) |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 143 pages : illustrations |
| Rights | Stellenbosch University |
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