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Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation

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Abstract

Normal (N), habituated nonorganogenic (HNO), and tumour (T) sugar beet cell lines were analysed in order to establish specific patterns of extracellular proteins and identify protein markers that might explain the distinct phenotypical characteristics. Electron microscopy showed that the ultrastructure of N cells corresponds to that of parenchyma cells, and that these cells contain plastids with large starch grains. HNO and T cells had enlarged, lobed nuclei with an increased number of nucleoli; the number of nuclei in HNO cells was greater than in T cells. The T plastids were elongated, with reduced thylakoids and abundant phytoferritin deposits, while HNO plastids were small and vacuolated, with an irregular, underdeveloped thylakoid system. The extracellular proteome of the cells was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Greater differences in protein expression were observed between the HNO and N lines than between the T and N lines. Sixteen of the most prominent bands differentially expressed among the cell lines were cut out from the gel and analyzed by mass spectrometry. Cell wall-modifying enzymes were identified, including a peroxidase whose expression was twofold higher in N and T tissue than in HNO tissue; pectinesterase, which was expressed at a level threefold lower in the T line than in the other cell lines; and xyloglucan endotransglucosylase, which was expressed at a level sixfold higher in HNO and T tissue. Three proteins belonged to the chitinase gene family and their expression was higher in HNO and T tissue than in N tissue. The differential expression of these proteins suggests that these play a role in cell line-specific cell wall composition and cell-to-cell adhesion.

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Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

BA:

6-benzyladenine

HNO:

Habituated nonorganogenic

MALDI:

Matrix-assisted laser desorption ionisation

N:

Normal

MS/MS:

Tandem mass spectrometry

PI4P 5-kinase:

Phosphatidylinositol-4-phosphate 5-kinase

SDS–PAGE:

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

T:

Tumour

TOF:

Time of flight

XTH:

Xyloglucan endotransglucosylase

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Acknowledgments

This work was supported by grant 119-1191196-1200 to MKR from the Ministry of Science, Education and Sports of the Republic of Croatia.

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Correspondence to Dubravko Pavoković.

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Pavoković, D., Poljuha, D., Horvatić, A. et al. Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation. Plant Cell Tiss Organ Cult 108, 111–119 (2012). https://doi.org/10.1007/s11240-011-0019-3

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