Sucrose accumulation and the expression of neutral invertase in sugarcane

[摘要] ENGLISH ABSTRACT:The goals of this project were to (i) determine maximum extractable neutral invertase (NI)activity in the sugarcane culm, (ii) sequence a cDNA encoding for the sugarcane NI (SNI),(iii) determine SNI copy number in the genome, (iv) describe SNI transcript and proteinexpression patterns throughout the plant, and (v) attempt to determine the contribution ofhydrolysis to sucrose accumulation.SNI and sugars were extracted from the developing culm tissues of sugarcane,commercial variety N19. Tissues were divided according to developmental stage(internodes 3, 6 and 9) and anatomical differentiation (enriching for elongating, vascular orstorage tissues). The lowest sucrose content was found in the core of the bottom of eachof the internodes. The ratio between hexoses and sucrose was highest in the younginternodes. In these internodes hexose content was higher in the bottom than the top.There was a significant correlation between sucrose content and NI. Fluxes involved insucrose synthesis and hydrolysis were investigated. The hexoses glucose and fructosewere supplied as a carbon source for tissue discs of young and maturing internodal tissuesof sugarcane, varieties N19 and US6656-15. Sucrose content was 10-fold higher inmaturing internodes of N19 than US6656-15. Calculated sucrose hydrolysis rates viainvertase were higher in maturing internodes of US6656-15 than N19. Taking metaboliccompartmentation into account, hydrolysis of sucrose via invertase made a significantcontribution to the net turnover of sucrose. Along with this, it would appear that the abilityto partition sucrose between the vacuole and cytosol causes a significant difference insucrose content between varieties.A full-length cDNA for SNI was sequenced. This expressed gene showed significanthomology to known NI sequences on both nucleic and amino acid levels. The SNIsequence did not contain the putative invertase catalytic amino acid sequence, suggestingit developed separately from the other classes of invertases. Approximately 1.8 kb of theSNI cDNA was incorporated into a vector suitable for direct bombardment into sugarcanetissue. Southern blot analysis showed the enzyme has a low copy number. SNI transcriptexpression was observed in all tissues of the sugarcane plant: roots, internodes, leaf rolland leaves. In culm tissues where sucrose content was low and hexose contents werehigh, SNI transcript and protein levels were high. This suggests that SNI is involved ingrowth metabolism.

[发布机构] Stellenbosch University