Dissertation (PhD) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: In most plants the efficiency of sucrose production and the systemic distribution
thereof are the major determinants of growth, development and yield. The factors
governing sugar partitioning co-ordinate its distribution in response to intrinsic and
environmental signals. These factors include sugar transporters and invertases as
well as metabolites, including sucrose and glucose, which function as signalling
molecules to modulate gene expression.
The genetic transformation of plants and the subsequent development of
transgenic lines with disturbed sugar metabolism have made an unprecedented
impact on the study of sugar translocation and -partitioning. For instance, the
transformation of plants with a yeast-derived invertase targeted to different
subcellular compartments has led to the elucidation of several key aspects of sugar
metabolism, including phloem loading mechanisms, the regulation of photosynthesis
by sugars, the importance of sugar-metabolism compartmentation with regards to
sucrose biosynthesis, storage and distribution, as well as the role of cell-wall
invertase in phloem unloading and sink strength.
In this study, a similar strategy of transgenic plant analysis was employed to
expand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2
gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol,
vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayed
varying increases in invertase activity, altered sugar levels and consequently
disturbed sink-source interactions and sugar partitioning. Transgenic lines
overproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast
(Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink and
source organs on the expression of sugar transporters, as well as the endogenous
cell wall invertase and inhibitors in these plants.
Transcript levels of the sucrose transporter NtSUT1 and hexose transporter
NtMST1 encoding genes increased significantly in the source leaves and roots of
Vac-Inv lines, whereas increased NtMst1 transcript levels were also detected in the
roots of Apo-Inv lines. The increased mRNA levels could be correlated to the altered
invertase activities and sugar levels in these tissues. It is concluded that NtSUT1 and
NtMST1 are differentially regulated by sucrose and/or hexose content on a
transcriptional level. Furthermore, the regulatory effect of the altered sugar levels on
transporter expression depended on the subcellular compartment in which the yeast
invertase was expressed. It would seem that the subcellular compartmentation of
sugar metabolism is also fundamental to the regulation of sugar partitioning.
The transcription levels of the endogenous cell wall invertase (CWt) and cell
wall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues of
Apo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. In
comparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-source
status and growth stage. However, no obvious correlation between the Cwi and
Cwi-Inh expression levels and soluble sugar content of these tissues was found. It is
suggested that the post-transcriptional and post-translation control of these proteins
by sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnh
mRNA ratio and growth observations of the various tissues of control as well as
Apo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurate
indicator of the sink strength of sink organs.
In addition, the influence of sink-source interactions on sugar partitioning was
investigated. Reciprocal grafting between Apo-Inv and control lines resulted in scions
with an altered sucrose metabolism in either the sink or source organs. These scions
were subjected to biomass distribution, soluble sugar quantification and C4C]-
radiolabelling experiments. The latter revealed an unaltered state of sugar
partitioning from the above-ground tissues of the Apo/GUS scions and a significant
shift in sugar partitioning towards the roots of the GUS/Apo scions in comparison to
the control GUS/GUS scions. Phenotypic changes, opposite to those observed in
Apo-Inv lines expressing the heterologous invertase in both sink and source organs,
could initially be observed in the GUS/Apo and Apo/GUS scions. However, no
significant differences in phenotype or biomass distribution could be observed
between the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting.
This inconsistency between phenotype and sugar partitioning might be
explained by an increase in the respiration rate of the tissues as supported by the
soluble sugar content. These results highlight the complexity and adaptability of
sucrose metabolism and sugar partitioning. In addition, it confirms that sugar
partitioning can be modulated by sink-source interactions and emphasise the
importance of invertases in the regulation of sugar partitioning through its ability to
alter sink strength.
This study forms part of the rapidly expanding initiative to unravel the control
mechanisms of sugar partitioning. The results obtained in this study confirmed again
that the introduction and expression of a single heterologous gene in transgenic
plants could provide significant insight into the regulation of this process. It was
shown here that the expression of sugar transporters is closely regulated by sugar
levels and therefore fulfils a vital function in sugar sensing and consequently the
regulation of sugar partitioning. The data presented in this study also demonstrated
the intricate and flexible nature of the relationship that exists between sugar
metabolism, partitioning and growth phenomena. / AFRIKAANSE OPSOMMING: Die doeltreffendheid van sukroseproduksie, tesame met die sistemiese verspreiding
daarvan, is die vernaamste faktore wat die groei, ontwikkeling en opbrengsvermoë
van die meeste plante bepaal. Die faktore wat suikerverdeling beheer, funksioneer
om suikerverspreiding te koordineer in reaksie op beide inherente- en
omgewingsseine. Hierdie faktore sluit suikertransporters en invertases in, asook
metaboliete soos sukrose en glukose wat funksioneer as seinmolekule in die
modulering van geenuitdrukking.
Die genetiese transformasie van plante en die gevolglike daarstelling van
transgeniese lyne met veranderde suikermetabolismes het 'n beduidende inwerking
op die bestudering van suikervervoer en -verdeling gehad. Byvoorbeeld, die
transformasie van plante met 'n gis-invertase geteiken na verskillende sub-sellulêre
kompartemente, het tot die toeligting van verskeie aspekte van suikermetabolisme
gelei, insluitende dié van floëemladingsmeganismes, die regulering van fotosintese
deur suikers, die belang van kompartementalisering ten opsigte van
sukrosebiosintese, -opberging en -verspreiding, en die rol van selwand-invertases in
floëemontlaaiing en swelgpuntkrag.
In hierdie studie is van soortgelyke transgeniese plantontledings gebruik gemaak
om 'n dieper insig tot die regulering van suikerverdeling te verkry. Die gis-invertase
Suc2 geen, afkomstig van Saccharomyces cerevisiae, is ooruitgedruk in óf die
sitosol, vakuool óf apoplastiese ruimte van transgeniese tabakplante. Hierdie
transgeniese lyne het wisselende toenames in invertase-aktiwiteite en veranderde
suikervlakke getoon, asook gevolglike versteurde bron-swelgpunt interaksies en
suikerverdeling. Transgeniese lyne met ooruitdrukking van die gis-invertase in óf die
vakuool (Vac-Inv) óf die apoplast (Apo-Inv) is gebruik om die gevolg van die
veranderde suikervlakke in bron- en swelgpuntorgane op die uitdrukking van
suikertransporters, asook die endogene selwand-invertase en invertase-inhibitor in
hierdie plante te bepaal.
Transkripsievlakke van die sukrosetransporter NtSut1 en die heksosetransporter,
NtMst1, het beduidend toegeneem in die bron-blare en wortels van die
Vac-Inv lyne; 'n toename in NtMst1 transkripsievlakke is ook in die wortels van
Apo-Inv lyne bevestig. Die toenames in boodskapper RNA kon gekorreleer word met
die veranderde invertase-aktiwiteite en suikervlakke in hierdie weefsels. Die
gevolgtrekking word gemaak dat NtSUT1 en NtMST1 differensieël gereguleer word
op transkripsionele vlak deur die sukrose en/of heksose inhoud van weefsels. Meer
nog, die regulerende effek van die veranderde suikervlakke op transporteruitdrukking
het afgehang van die subsellulêre kompartement waarin die gis-invertase
uitgedruk is. Dit wil dus voorkom dat die subsellulêre kompartementalisering van
suikermetabolisme fundamenteel tot die deurgee en waarneming van suikerseine is,
met In gevolglike eweneens belangrike rol in die regulering van suikerverdeling. Die transkripsievlakke van beide die endogene selwand-invertase (CWI) en
die selwand-invertase-inhibitor (CWI-Inh) enkoderende gene is in verskeie weefsels
van die Apo-Inv en Vac-Inv lyne, tydens beide die vegetatiewe- en blomstadia,
bestudeer. Die onderskeie weefsels van die Apo-Inv en Vac-Inv lyne het, in
vergelyking met die kontrole lyne, veranderde Cwi en Cwi-inh transkripsievlakke
getoon wat bepaal is deur bron-swelgpunt status en groeistadium. Geen duidelike
korrelasie kon tussen beide Cwi en Cwi-inh uitdrukkingsvlakke en oplosbare suiker
inhoud gevind word nie. Daar word voorgestel dat post-transkripsionele en posttranslasionele
beheer deur suikers 'n belangrike rol in die regulering van hierdie
proteïne speel. Bestudering van die Cwi:Cwi-lnh mRNA verhouding, asook groei
verskynsels van die onderskeie weefsels van kontrole en Apo-Inv en Vac-Inv lyne,
dui daarop dat hierdie transkripsievlak-verhouding moontlik 'n akkurate aanwyser van
die swelgpuntkrag van 'n swelgpuntorgaan kan wees.
Voorts is die invloed van bron-swelgpuntorgaan interaksies op suikerverdeling
ondersoek. Omgekeerde enting tussen Apo-Inv en kontrole lyne het entlote met
gemodifiseerde suikermetabolisme in óf hul bron- óf hul swelgpuntorgane tot gevolg
gehad. Hierdie entlote is aan biomassaverspreidings-, oplosbare suiker kwantifisering
en C4C]-radiomerking eksperimente onderwerp. Hierdie resultate het gewys dat, in
vergelyking met die kontrole (GUS/GUS) ente, daar geen verandering in die status
van suikerverdeling vanaf die bogrondse plantdele in die Apo/GUS ente is nie, maar
wel 'n beduidende verskuiwing in suikerverdeling na die wortels van die GUS/Apo
ente. Fenotipiese veranderinge, wat teenoorgesteld van dié teenwoordig in die Apo-
Inv lyne waar die heteroloë invertase in beide bron en swelgpuntorgane uitgedruk
word, is aanvanklik in die GUS/Apo en Apo/GUS ente waargeneem. Geen verskille in
fenotipe of biomassa-verspreiding kon egter sewe weke na die entings prosedures
tussen die GUS/Apo, Apo/GUS and GUS/GUS ente gevind word nie. Dit mag
verduidelik word deur 'n moontlike toename in respirasietempo in die betrokke
weefsels; die oplosbare suikervlakke wat in die verskillende ente aangeteken is
ondersteun dié moontlikheid. Hierdie resultate as geheelonderstreep die
kompleksiteit en aanpasbaarheid van suikermetabolisme en -verdeling. Verder
bevestig dit dat suikerverdeling beïnvloed kan word deur bron-swelgpunt interaksies,
asook die belang van invertases in die regulering van suikerverdeling gegewe die
vermoë om swelgpuntkrag te verander.
Hierdie studie vorm deel van 'n vinnig groeiende inisiatief om die beheermeganismes
van suikerverdeling te ontrafel. Die resultate verkry in hierdie studie
bekragtig die belang van rekombinante DNA tegnologie in die bestudering van
fundamentele plantprosesse. Die invoeging en uitdrukking van 'n geteikende gisinvertase
in transgeniese plante het gelei tot veranderde suikervlakke en bronswelgpunt
interaksies in hierdie lyne met die gevolglike ontginning van waardevolle
inligting ten opsigte van die regulering van suikerverdeling in reaksie tot interne
seine. Daar is aangetoon dat suikertransporters onlosmaakbaar gekoppel is aan die
deurgee en waarneming van suikerseine, spesifiek op die vlak van transkripsionele regulering, en dus ook die regulering van suikerverdeling. Voorts wys die resultate op
die komplekse en aanpasbare aard van die verhouding wat bestaan tussen
suikermetabolisme, -verdeling en groeiverskynsels.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/49854 |
| Date | 12 1900 |
| Creators | Champanis, Reinette |
| Contributors | Vivier, M. A., Botha, F. C., Pretorius, I. S., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 143 p. : col. ill. |
| Rights | Stellenbosch University |
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