Development of in situ hybridisation to examine tissue-specific expression patterns of the invertase genes in sugarcane culm

Turner, Gabrielle M. (2005-04)

Thesis (MSc)--University of Stellenbosch, 2005.

Thesis

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.

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