Trehalose and carbon partitioning in sugarcane

[摘要] The current understanding of the regulation of sucrose accumulation is still incomplete eventhough many scientists have investigated this subject. Components of trehalose metabolism havebeen implicated in the regulation of carbon flux in bacteria, yeast and more recently in plants. Witha view to placing trehalose metabolism in the context of cytosolic sugarcane sucrose metabolismand carbon partitioning we have investigated the metabolites, transcripts and enzymes involved inthis branch of carbohydrate metabolism in sugarcane internodal tissues.Sugarcane internodal trehalose levels varied between 0.31 ± 0.09 and 3.91 ± 0.99 nmol.g-1 freshweight (FW). From statistical analysis of the metabolite profile it would appear that trehalose doesnot directly affect sucrose accumulation, although this does not preclude involvement of trehalose-6-phosphate in the regulation of carbon partitioning. The metabolite data generated in this studydemanded further investigation into the enzymes (and their transcripts) responsible for trehalosemetabolism.Trehalose is synthesised in a two step process by the enzymes trehalose-6-phosphate synthase(EC 2.4.1.15, TPS) and trehalose-6-phosphate phosphatase (EC 3.1.3.12, TPP), and degradedby trehalase (EC 3.2.1.28). Two novel sugarcane partial cDNAs that coded for trehalase (tre) andactin (required for normalisation in profiling experiments) were isolated and used along with partialtranscripts for TPS and TPP to determine transcript levels in different tissue- and genotypes. Aputative full-length SugTPS cDNA was isolated and characterised. Enzyme activities for TPS, TPPand trehalase were measured at levels of 2.7 nmol.min-1.mg-1protein, 8.5 nmol.min-1.mg-1proteinand 6.2 nmol.min-1.mg-1protein respectively, from young internodal protein extracts of sugarcane,variety N19. TPP enzyme activity and transcript levels were higher in S. spontaneum thanSaccharum interspecific hybrids.Kinetic analysis of TPP and trehalase activities were performed with the purpose of providingparameters for an in silico kinetic model of trehalose and sucrose metabolism. Three isoforms ofTPP were identified and desingated TPPAI, TPPAII and TPPB. Both TPPA isoforms had pHoptima of 6.0, and TPPB of pH 6.5. Apparent Km values were determined as 0.447 ± 0.007 mM forTPPAI, 13.82 ± 1.98 mM for TPPAII and 1.387 ± 0.18 mM for TPPB. Partial purification andcharacterisation of trehalase demonstrated dual pH optima of 3.5 and 6.0, with Km values between0.345 and 0.375 mM. These data were used as the basis for a kinetic model of trehalosemetabolism.A previously described kinetic model of cytosolic sucrose metabolism has been expanded toinclude the trehalose pathway (TPS, TPP and trehalase). The aim was to supplement theavailable information on cytosolic metabolism in sugarcane storage parenchyma, identify points ofcontrol between sucrose and trehalose metabolism, and provide a platform from which furtherexperimental and in silico modelling can be launched. The model predicted trehalose in the sameorder of magnitude as those determined in the metabolite profiling experiments. The majority ofcontrol of flux over the trehalose pathway resided in the TPS step, with flux control coefficients >70% of the total pathway. Incorporation of the trehalose branch into the original sucrose modelshowed that reactions from the original model significantly affected the steady-state attributes ofthe trehalose pathway.Due to the relatively low flux through the trehalose branch of the expanded model, completerecycling of trehalose, and the lack of allosteric regulation by trehalose-6-phosphate or trehaloseon any of the reactions from the original sucrose model, incorporation of the trehalose branch hadno significant effect on either steady-state cytosolic sucrose concentration or flux of sucrose intothe vacuole. The expanded model affords a basis from which to further investigate trehalosemetabolism in the context of plant sucrose accumulation.

[发布机构] Stellenbosch University