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

[摘要] ENGLISH ABSTRACT: The goals of this project were firstly to develop the tissue preparation and in situhybridisation protocols for sugarcane culm tissue, and secondly to use the developedtechniques to examine the expression patterns of three invertase isoforms in sugarcaneinternodes of various developmental stages. Sugarcane invertases have been the focus ofintense research for many years, yet almost nothing is known of their tissue-specificdistribution. It was thought that by characterising their expression patterns using in situhybridisation, more knowledge of their functions and involvement in sucroseaccumulation 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 bycomparing different tissue preparation methods, as well as by systematically testing thevarious parameters pertaining to each method. The in situ hybridization technique wasalso developed by testing and comparing a number of key parameters. It was found thatfixing whole mount tissue for 48 h preserved sugarcane tissue adequately. Highhybridization temperatures and probe concentrations provided the best signal, andincluding pre-treatment with HCl and Pronase was essential in sensitizing the tissue tothe probe. A less viscous detection buffer reduced both osmotic effects and time requiredfor signal detection.In the second part of this study, the developed method was used to examine theexpression patterns of the three invertase isoforms in young, maturing and matureinternodes 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 inthe phloem tissue. Transcript levels of all three isoforms declined in maturing tissue, withsoluble acid invertase declining sharply and dropping below detection in maturing andmature tissue. Transcript levels of cell wall invertase and neutral invertase declined onlygradually, and appreciable levels of both were still present in mature tissue. Acid invertase is suggested to be mainly involved in internode elongation, while cell wallinvertase 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 ofhexose pools, however the function of this enzyme remains unclear.This study has demonstrated the value of in situ hybridization, yet at the same time hasshown its limitations, especially when more traditional biochemical techniques are notemployed to complement the results. Although the precise functions of the invertaseisoforms in sugarcane remain inconclusive, this study has opened up the way for tissuespecificpromoter design and future in situ studies of sugarcane invertases

[发布机构] Stellenbosch University