Aspects of sucrose metabolism in transgenic tobacco

[摘要] ENGLISH ABSTRACT:In most plants the efficiency of sucrose production and the systemic distributionthereof are the major determinants of growth, development and yield. The factorsgoverning sugar partitioning co-ordinate its distribution in response to intrinsic andenvironmental signals. These factors include sugar transporters and invertases aswell as metabolites, including sucrose and glucose, which function as signallingmolecules to modulate gene expression.The genetic transformation of plants and the subsequent development oftransgenic lines with disturbed sugar metabolism have made an unprecedentedimpact on the study of sugar translocation and -partitioning. For instance, thetransformation of plants with a yeast-derived invertase targeted to differentsubcellular compartments has led to the elucidation of several key aspects of sugarmetabolism, including phloem loading mechanisms, the regulation of photosynthesisby sugars, the importance of sugar-metabolism compartmentation with regards tosucrose biosynthesis, storage and distribution, as well as the role of cell-wallinvertase in phloem unloading and sink strength.In this study, a similar strategy of transgenic plant analysis was employed toexpand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol,vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayedvarying increases in invertase activity, altered sugar levels and consequentlydisturbed sink-source interactions and sugar partitioning. Transgenic linesoverproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast(Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink andsource organs on the expression of sugar transporters, as well as the endogenouscell wall invertase and inhibitors in these plants.Transcript levels of the sucrose transporter NtSUT1 and hexose transporterNtMST1 encoding genes increased significantly in the source leaves and roots ofVac-Inv lines, whereas increased NtMst1 transcript levels were also detected in theroots of Apo-Inv lines. The increased mRNA levels could be correlated to the alteredinvertase activities and sugar levels in these tissues. It is concluded that NtSUT1 andNtMST1 are differentially regulated by sucrose and/or hexose content on atranscriptional level. Furthermore, the regulatory effect of the altered sugar levels ontransporter expression depended on the subcellular compartment in which the yeastinvertase was expressed. It would seem that the subcellular compartmentation ofsugar metabolism is also fundamental to the regulation of sugar partitioning.The transcription levels of the endogenous cell wall invertase (CWt) and cellwall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues ofApo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. Incomparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-sourcestatus and growth stage. However, no obvious correlation between the Cwi andCwi-Inh expression levels and soluble sugar content of these tissues was found. It issuggested that the post-transcriptional and post-translation control of these proteinsby sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnhmRNA ratio and growth observations of the various tissues of control as well asApo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurateindicator of the sink strength of sink organs.In addition, the influence of sink-source interactions on sugar partitioning wasinvestigated. Reciprocal grafting between Apo-Inv and control lines resulted in scionswith an altered sucrose metabolism in either the sink or source organs. These scionswere subjected to biomass distribution, soluble sugar quantification and C4C]-radiolabelling experiments. The latter revealed an unaltered state of sugarpartitioning from the above-ground tissues of the Apo/GUS scions and a significantshift in sugar partitioning towards the roots of the GUS/Apo scions in comparison tothe control GUS/GUS scions. Phenotypic changes, opposite to those observed inApo-Inv lines expressing the heterologous invertase in both sink and source organs,could initially be observed in the GUS/Apo and Apo/GUS scions. However, nosignificant differences in phenotype or biomass distribution could be observedbetween the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting.This inconsistency between phenotype and sugar partitioning might beexplained by an increase in the respiration rate of the tissues as supported by thesoluble sugar content. These results highlight the complexity and adaptability ofsucrose metabolism and sugar partitioning. In addition, it confirms that sugarpartitioning can be modulated by sink-source interactions and emphasise theimportance of invertases in the regulation of sugar partitioning through its ability toalter sink strength.This study forms part of the rapidly expanding initiative to unravel the controlmechanisms of sugar partitioning. The results obtained in this study confirmed againthat the introduction and expression of a single heterologous gene in transgenicplants could provide significant insight into the regulation of this process. It wasshown here that the expression of sugar transporters is closely regulated by sugarlevels and therefore fulfils a vital function in sugar sensing and consequently theregulation of sugar partitioning. The data presented in this study also demonstratedthe intricate and flexible nature of the relationship that exists between sugarmetabolism, partitioning and growth phenomena.

[发布机构] Stellenbosch University