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The Role of Carbohydrate-Metabolizing Enzymes in Sugar Sensing and Differentiation in Sugar Beet Cell Lines 

Dubravko Pavoković1,2*, Osman Omarszad2, Stéphane Maury2,3, Claude Joseph2,3, Alain Delaunay2,3, Marijana Krsnik-Rasol1 and Daniel Hagège2


1Department of Biology, Faculty of Science, University of Zagreb, Horvatovac 102A,
HR–10000 Zagreb, Croatia
2University of Orléans, UFR-Faculty of Sciences, UPRES EA 1207, Laboratory for Biology of Ligneous Plants and Crop Cultures (LBLGC), Rue de Chartres, BP 6759, FR–45067 Orléans Cedex 2, France
3INRA, USC1328 Trees and Responses to Hydric and Environmental Constraints (ARCHE), FR–45067 Orléans Cedex 2, France

Article history:

Received April 20, 2011
Accepted September 23, 2011

Key words:

carbohydrate metabolism, differentiation, invertases, NMR, sucrose synthase

Summary:

Plant development is influenced by changes in the levels and types of sugars produced metabolically. The normal (N), habituated organogenic (HO) and habituated nonorganogenic (HNO) sugar beet cell lines originate from the same mother plant but exhibit distinct levels of morphogenesis and differentiation, and contain different levels of simple carbohydrates. We aim to elucidate whether differences in the abundance and activity of enzymes involved in carbohydrate metabolism and sugar sensing/signalling help explain the different carbohydrate profiles and differentiation states of the cell lines. Using 13C NMR spectroscopy to analyze cultures of the cell lines over 28 days, we found that N cells, accumulated sucrose; HO cells sucrose, glucose and fructose; and HNO cells glucose and fructose. Of three invertase isoforms, the activity of cell wall invertase (CWI) was highest in all the cell lines, and CWI activity was greatest in HNO line. The specific accumulation of intracellular carbohydrates during subculture correlated strongly with CWI activity but less so with the vacuolar and cytoplasmic invertase isoforms, or with sucrose synthase activity. Cell lines showed differences in how sugars regulated invertase and sucrose synthase activity. The role of sugar sensing in the regulation of CWI activity was investigated in the cell lines using glucose and sucrose, as well as carbohydrate analogues such as mannitol, 2-O-deoxyglucose and 3-O-methylglucose. Differences in the regulation of CWI activity by carbohydrates across the three cell lines suggest that CWI can be fine-tuned according to the specific carbohydrate requirements of each line during growth. Differences in sugar signalling pathways across the cell lines were explored using glucose and sucrose in the presence of inhibitors of protein kinases or phosphatases. Taken together, our findings suggest that specific regulation of CWI activity plays an important role in determining the intracellular carbohydrate levels of sugar beet cell lines, and possibly their differentiation state as well.


*Corresponding author:           dubravko@biol.pmf.hr
                                               ++385 1 460 6268
                                               ++385 1 460 6286

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