Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and
accumulation in sugarcane is poorly understood. The present study was therefore aimed at the
development of an in situ hybridisation (ISH) technique to study differential gene expression
among the various cell types of the sugarcane culm. This technique in conjunction with
northern and western blotting was then used to determine the sites of cellular and tissue
specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate
dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose
metabolism.
This study revealed that the determination of the influencing parameters associated with the
development of an ISH protocol was essential for the successful detection of the endogenous
RNA sequences in sugarcane internodal tissues. The parameters that were investigated
included the type of embedding medium, duration of fixation period, pre-treatment procedures
and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed
for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the
analysis of cytological gene expression at all stages of sugarcane development.
The second part of this study revealed very localised transcript expression for UDP-Glc DH,
PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and
UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed
the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all
expressed in the parenchyma cells that were associated with the vascular bundles and the stem
storage compartment, suggesting that the parenchyma cells distributed throughout the stem in
the different tissue types complement each other in function for the purposes of phloem
loading, unloading and assimilate transport processes.
Complimentary northern and western hybridisations demonstrated that internode 7 represents
a shift in the sink from utilisation to storage. This is evident by the observed decline in both
the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage
of development. The relative mRNA and protein abundances for the three enzymes showed a
similar trend. Higher levels of the gene transcripts and translated products were observed in
the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At
a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase
expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was
expressed exclusively in parenchyma cells that were associated with the vascular bundles and
those serving a storage function in the stem pith and UGPase was found to be localised in the
phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such
findings have demonstrated an increase in resolution with which gene expression can be
examined at a cellular level. Hence, the results from this study have demonstrated that the
knowledge of metabolic compartmentation between different tissue and cell types is a
requisite to understanding the function(s) of individual enzymes within complex structures
such as the sugarcane culm. / AFRIKAANSE OPSOMMING: Die sellulêre lokalisering van die ensieme wat geïmpliseer word in die regulering van sukrose
metabolisme is onbekend. Met dit in gedagte, was hierdie studie gefokus op die ontwikkeling
van 'n in situ hibridisasie (ISH) tegniek om differensiële geenuitdrukking in die verskillende
seltipes van die suikerrietstingel te ondersoek. Hierdie tegniek, tesame met RNA-en proteïen
gel blots, is volgens aangewend om die areas van sellulêre-en weefselspesifieke uitdrukking
van die sitosoliese ensieme UDP-glukose dehydrogenase, pirofosfaat-afhanklike
fosfofruktokinase en UDP-glukose pirofosforilase, wat almal betrokke is by
sukrosemetabolisme, te bepaal.
Dit het duidelik geword gedurende die studie dat die bepaling van die optimale parameters
van die ISH protokol vir suikerriet van deurslaggewende belang sou wees vir die opsporing
van endogene RNA volgordes. Die parameters wat ondersoek is het ingesluit die tipe
inbeddingsmedium, die tydsduur van fiksering, vooratbehandelings- en hibridisasiemetodes.
Dit het duidelik geword dat vars internodale weefselsnitte wat vir 24 h gefikseer is en daarna
voorafbehandeling en hibridisasie ondergaan het, die bepaling van geenuitdrukking tydens
alle fases van suikkerrietontwikkeling moontlik gemaak het.
Die tweede fase van hierdie studie het aangetoon dat al drie ensieme spesifiek gelokaliseerde
uitdrukkingspatrone gehad het in verskillende internodale weefsels en seltipes. Al drie gene is
konstitutief uitgedruk in internodes. Die UDP-glukose dehydrogenase en UDP-glukose
pirofosforilase transkripte is gelokaliseer na die floeëm elemente, terwyl xileem slegs die
UDP-glukose dehydrogenase transkripte bevat het. Al die gene is in die parenchiemselle
uitgedruk wat geassosieer is met die vaatbondels en die stingel stoorkompartement, wat
moontlik beteken dat die parenchiem selle wat deur die stingel versprei is 'n sentrale netwerk
vorm wat direk of indirek koolstofassimileringsprosesse beïnvloed.
RNA-en proteïen gel blots op dieselfde internodes het gewys dat internode sewe 'n
verskuiwing, van koolstofverbruik na berging, verteenwoordig. Dit word gerllustreer deur die
afname in beide transkrip en proteïen vlakke van die drie ensiem in hierdie stadium van
ontwikkeling. Alhoewel beide mRNA en proteïen vlakke vir al die ensieme 'n soortgelyke
tendens getoon het, het die sellulêre uitdrukking van die ensieme volgens ISH verskil, wat die
krag van die tegniek illustreer. Die resultate van hierdie studie het gedemonstreer dat begrip
van die kompartementalisasie van metabolisme tussen verskillende weefsel-en seltipes 'n voorvereiste is om die funksie/s van individuele ensieme in komplekse strukture soos die
suikerrietstingel te bepaal.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/49795 |
| Date | 12 1900 |
| Creators | Ramoutar, Rakeshnie |
| Contributors | Cramer, M. D., Botha, F. C., Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xi, 67 p. : ill. |
| Rights | Stellenbosch University |
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