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Developmentaly specific protein and glycoprotein patterns in Mammillaria gracillis tissue culture

As plants with Crassulacean Acid Metabolism (CAM), cacti are highly affected by artificial environmental conditions in the tissue culture. In vitro propagated plants of Mammillaria gracillis Pfeiff. (Cactaceae) spontaneously produce callus. This habituated callus regenerates normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: wild strain B6S3 (tumour line TW) and the rooty mutant GV3101 (tumour line TR). Both tumour lines grow vigorously without expression of any morphogenic potential. The aim of this work was to obtain information about causes and conditions leading to growth of habituated callus and to the spontaneous regeneration in the callus culture in order to contribute to better understanding of cell differentiation and dedifferentiation mechanisms. Gene expression in cactus shoots, habituated callus, normal and hyperhydric regenerants as well as in both tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by 1-D and 2-D SDS-polyacrylamide gel electrophoresis and silver stained. Cellular and extracellular glycoproteins with D-mannose and D-glucose in their glycan component were detected on 1-D protein blots by concavalin A-peroxidase staining. For further characterisation of glycan component, biotin-labelled lectins RCA120, UEA I, MAA and SNA were used. SDS-PAGE of cellular proteins showed a few tissue-specific bands. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The 42 kDa cellular protein band was detected in all cactus tissues on the silver stained gels. The corresponding glycoprotein of 42 kDa, reacting with Con A, was highly expressed only in habituated callus, hyperhydric regenerants and both tumours. On protein blots sialic acid, which has not yet been found in plants, was detected in glycoproteins of all cactus tissues. HPAEC-PAD analysis confirmed the presence of the sialic acid in a glycan component of the 42 kDa protein from tumour TW. Patterns of proteins secreted in the nutrient medium of suspension-cultured cells showed differences between untransformed tissues (callus and hyperhydric regenerants) and tumours, while in extracts of shoots and normal regenerants no extracellular proteins were detected. The N-glycosylation of cellular and extracellular proteins can be related to the specific developmental stage of cactus tissue.

Cactaceae; plant tissue culture; glycoprotein pattern

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