Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The goals of this project were firstly to develop the tissue preparation and in situ
hybridisation protocols for sugarcane culm tissue, and secondly to use the developed
techniques to examine the expression patterns of three invertase isoforms in sugarcane
internodes of various developmental stages. Sugarcane invertases have been the focus of
intense research for many years, yet almost nothing is known of their tissue-specific
distribution. It was thought that by characterising their expression patterns using in situ
hybridisation, more knowledge of their functions and involvement in sucrose
accumulation would be gained.
Although in situ hybridisation is now regularly used to study gene expression in plants,
there is to date only a single publication describing its use on immature sugarcane tissue.
Therefore this technique needed further development, and this was achieved by
comparing different tissue preparation methods, as well as by systematically testing the
various parameters pertaining to each method. The in situ hybridization technique was
also developed by testing and comparing a number of key parameters. It was found that
fixing whole mount tissue for 48 h preserved sugarcane tissue adequately. High
hybridization temperatures and probe concentrations provided the best signal, and
including pre-treatment with HCl and Pronase was essential in sensitizing the tissue to
the probe. A less viscous detection buffer reduced both osmotic effects and time required
for signal detection.
In the second part of this study, the developed method was used to examine the
expression patterns of the three invertase isoforms in young, maturing and mature
internodes of sugarcane, and the results were complemented with Northern blot analysis.
Transcript of all three isoforms was found to be present in the storage parenchyma and in
the phloem tissue. Transcript levels of all three isoforms declined in maturing tissue, with
soluble acid invertase declining sharply and dropping below detection in maturing and
mature tissue. Transcript levels of cell wall invertase and neutral invertase declined only
gradually, and appreciable levels of both were still present in mature tissue. Acid invertase is suggested to be mainly involved in internode elongation, while cell wall
invertase would appear to play important roles in phloem unloading and turgor control.
Neutral invertase is suggested to be involved in either sucrose cycling or maintenance of
hexose pools, however the function of this enzyme remains unclear.
This study has demonstrated the value of in situ hybridization, yet at the same time has
shown its limitations, especially when more traditional biochemical techniques are not
employed to complement the results. Although the precise functions of the invertase
isoforms in sugarcane remain inconclusive, this study has opened up the way for tissuespecific
promoter design and future in situ studies of sugarcane invertases / AFRIKAANSE OPSOMMING: Die doel van hierdie projek was tweeledig: eerstens om weefselvoorbereiding en in situhibridisasie-
protokolle vir die stingelweefsel van suikerriet te ontwikkel; en tweedens om
die ontwikkelde tegnieke te gebruik om die uitdrukkingspatrone van drie invertaseisovorme
in die suikerriet-internodes van verskeie ontwikkelingstadia te ondersoek.
Suikerriet-invertases is al vir jare lank die fokus van intense navorsing, maar baie min is
bekend oor hulle weefselspesifieke verspreiding. Die idee was om meer kennis oor
suikerriet-invertases se funksies en betrokkenheid by sukrose-akkumulasie te verkry deur
in situ-hibridisasie te gebruik om hulle uitdrukkingspatrone te karakteriseer.
Alhoewel in situ-hibridisasie deesdae gereeld gebruik word om geenuitdrukking in plante
te bestudeer, is daar tot op datum slegs een publikasie wat die gebruik daarvan in
onvolwasse suikerrietweefsel beskryf. Hierdie tegniek moes dus verder ontwikkel word,
en dit is gedoen deur verskillende weefselvoorbereidingsmetodes te vergelyk en
sistematies die verskillende parameters wat op elke metode van toepassing is te toets.
Die in situ-hibridisasie-tegniek is ook ontwikkel deur die toetsing en vergelyking van 'n
aantal sleutelparameters. Daar is gevind dat suikerrietweefsel voldoende gepreserveer
word deur die intakte gemonteerde weefsel vir 48 uur te fikseer. Hoë hibridisasietemperature
en hoë peilerkonsentrasies het die beste sein gegee; die insluiting van voorbehandeling
met HCl en Pronase was noodsaaklik om die weefsel meer gevoelig vir die
peiler te maak. Osmotiese invloede en die tyd nodig vir seindeteksie is verminder deur
die viskositeit van die buffer te verminder.
In die tweede deel van die studie is die ontwikkelde metode gebruik om die
uitdrukkingspatrone van die drie invertase-isovorme in jong, ontwikkelende en volwasse
internodes te ondersoek en die resultate is deur 'n noordelike oordraganalise
gekomplementeer. Transkripte van al drie isovorme is in die stoorparenchiem en
floëemweefsel gevind. Transkripvlakke van al drie isovorme het afgeneem in
ontwikkelende weefsel, met oplosbare suurinvertase wat skerp afgeneem en tot onder die deteksie-limiet gedaal het in ontwikkelende en volwasse weefsel. Transkripvlakke van
selwandinvertase en neutrale invertase het slegs geleidelik afgeneem en merkbare vlakke
van albei was teenwoording in ontwikkelende en volwasse weefsel. Daar word
voorgestel dat suurinvertase hoofsaaklik betrokke is by internodeverlenging, terwyl
selwandinvertase skynbaar 'n belangrike rol in floëem-ontlading en turgor-beheer speel.
Daar word voorgestel dat neutrale invertase betrokke is óf by die sukrose-sirkulering óf
by die onderhoud van heksose-poele; die funksie van hierdie ensiem is egter steeds nie
duidelik nie.
Hierdie studie het die waarde van in situ-hibridisasie gedemonstreer maar terselfdetyd
ook die beperkinge daarvan uitgewys, veral as meer tradisionele biochemiese tegnieke
nie gebruik word om die resultate aan te vul nie. Alhoewel daar onsekerheid is oor die
presiese funksies van die invertase-isovorme in suikerriet, het die studie die weg gebaan
vir weefselspesifieke promotorontwerp en toekomstige in situ-studies van
suikerrietinvertases.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16621 |
| Date | 04 1900 |
| Creators | Turner, Gabrielle M. |
| Contributors | Cramer, M. D., Botha, F. C., University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB). |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xiv, 77 leaves : ill. |
| Rights | University of Stellenbosch |
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