The development of an in situ hybridisation technique to determine the gene expression patterns of UDP-Glucose dehydrogenase, pyrophosphate-dependent phosphofructokinase and UDP-Glucose pyrophosphorylase in sugarcane internodal tissues

[摘要] ENGLISH ABSTRACT:The cellular expression of the enzymes implicated in regulating sucrose metabolism andaccumulation in sugarcane is poorly understood. The present study was therefore aimed at thedevelopment of an in situ hybridisation (ISH) technique to study differential gene expressionamong the various cell types of the sugarcane culm. This technique in conjunction withnorthern and western blotting was then used to determine the sites of cellular and tissuespecific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphatedependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrosemetabolism.This study revealed that the determination of the influencing parameters associated with thedevelopment of an ISH protocol was essential for the successful detection of the endogenousRNA sequences in sugarcane internodal tissues. The parameters that were investigatedincluded the type of embedding medium, duration of fixation period, pre-treatment proceduresand hybridisation temperature. It further revealed that fresh internodal tissue sections, fixedfor a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated theanalysis 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 andUGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressedthe UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were allexpressed in the parenchyma cells that were associated with the vascular bundles and the stemstorage compartment, suggesting that the parenchyma cells distributed throughout the stem inthe different tissue types complement each other in function for the purposes of phloemloading, unloading and assimilate transport processes.Complimentary northern and western hybridisations demonstrated that internode 7 representsa shift in the sink from utilisation to storage. This is evident by the observed decline in boththe relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stageof development. The relative mRNA and protein abundances for the three enzymes showed asimilar trend. Higher levels of the gene transcripts and translated products were observed inthe younger sucrose importing tissues, than in the older sucrose accumulating internodes. Ata cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPaseexpression 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 wasexpressed exclusively in parenchyma cells that were associated with the vascular bundles andthose serving a storage function in the stem pith and UGPase was found to be localised in thephloem and parenchyma of the vascular bundles and the storage parenchyma cells. Suchfindings have demonstrated an increase in resolution with which gene expression can beexamined at a cellular level. Hence, the results from this study have demonstrated that theknowledge of metabolic compartmentation between different tissue and cell types is arequisite to understanding the function(s) of individual enzymes within complex structuressuch as the sugarcane culm.

[发布机构] Stellenbosch University