High Yield Isolation and Rapid Recovery of Protoplasts From Suspension Cultures of Tomato (Lycopersicon esculentum)

Tewes, Annegret; Glund, Konrad; Walther, Reinhard; Reinbothe, H.

doi:10.1016/s0044-328x(84)80049-5

Cited by 55 publications

(16 citation statements)

References 16 publications

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“…Cell suspension cultures of tomato (Lycopersicon esculentum Mill cv Lukullus) were propagated as described previously (24). Cells grow logarithmically for about 3.5 d thereby increasing number of cells tenfold.…”

Section: Materials and Methods Plant Materialsmentioning

confidence: 99%

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Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Nürnberger¹,

Abel²,

Jost³

et al. 1990

Plant Physiol.

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108

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Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3'(2') monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an Mr of 22,000 (sodium dodecyl sulfatepolyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pi of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2',3'-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5' purine residues adjacent to the cleavage site.

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Section: Materials and Methods Plant Materialsmentioning

confidence: 99%

“…Cells grow logarithmically for about 3.5 d thereby increasing number of cells tenfold. Growth of the culture was followed by counting cells according to (24). In experiments carried out under conditions of phosphate starvation (referred to as -Pi'), KH2PO4 was omitted from the modified Murashige-Skoog medium (24).…”

Section: Materials and Methods Plant Materialsmentioning

confidence: 99%

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Nürnberger¹,

Abel²,

Jost³

et al. 1990

Plant Physiol.

Self Cite

108

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“…Tewes et al (14) mentioned that the shorter the incubation in enzyme solution, the more rapid and of f ective was recovery of protoplasts in culture.…”

Section: Resultsmentioning

confidence: 99%

Plant regeneration from mesophyll protoplasts of tomato cv. Ponderosa

Sakata

Nishio

Takayanagi

1987

Engei Gakkai zasshi

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“…Cells oftomato (Lycopersicon esculentum Mill cv Lukullus) were propagated in suspension culture (28). Protoplasts were prepared according to the method of Abel and Glund (1).…”

Section: Materials and Methods Cell Culture And Preparation Of Protopmentioning

confidence: 99%

“…In an attempt to elucidate the hypothesized (12,31) role of plant vacuoles in cellular RNA breakdown, we have shown in previous studies that isolated vacuoles (13,14) from suspension-cultured tomato cells (28) contain the bulk (60-80%) of the intracellular RNA-degrading activity (1). The vacuolar ribonucleolytic enzyme has been purified (2), extensively characterized (2, 3), and classified as RNase I according to the classification of Wilson (31).…”

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confidence: 99%

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

Abel

Blume²,

Glund³

1990

Plant Physiol.

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We have shown that highly purified vacuoles of suspensioncultured tomato (Lycoperskon esculentum) cells contain RNAoligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5-3H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment.About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3'-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes.Involvement in degradative pathways of the cellular turnover of macromolecules is thought to be one of the most prominent functions of vacuoles in plants (for a review, see refs. 5 and 6). This has mainly been concluded from the occurrence of a broad spectrum of acid hydrolases in plant vacuoles (24), rendering this organelle homologous to the lysosomes in animal cells (6).Until now, proteolysis is the best-characterized lytic process in that plant vacuoles have been found to participate. In most cases, intracellular proteases have been found to be located in the vacuole (see ref. 6). This organelle was shown to be directly involved in the breakdown of storage proteins during germination in seeds (22) and to be able to acquire and degrade intracellular proteins in suspension-cultured sycamore cells (8,9).In contrast to these studies, which support the role of vacuolar proteases in the continuous turnover of cellular proteins, little is as yet known about a possible nucleolytic function of plant vacuoles. Although vacuoles contain a set of potentially nucleolytically acting enzymes [nuclease, ribonuclease, acid phosphatase (24)], which could completely digest nucleic acid substrates down to nucleosides and phosphate, the biological significance of the individual vacuolar enzymes and of the vacuolar compartment per se in intracellular nucleolytic events remains to be proven.In

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High Yield Isolation and Rapid Recovery of Protoplasts From Suspension Cultures of Tomato (Lycopersicon esculentum)

Cited by 55 publications

References 16 publications

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Plant regeneration from mesophyll protoplasts of tomato cv. Ponderosa

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

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