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

Abstract

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

Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana

“…An analogous function has been proposed for Pi-starvationinducible vacuolar ribonucleases. Vacuoles are equipped with an equivalent set of nucleolytic enzymes (LoÈ er et al 1992) and were shown to contain intermediates and end products of RNA degradation (Leinhos et al 1986;Abel et al 1990). Thus, remobilization of Pi from intra-and extracellular nucleic acid substrates to maintain constant cytoplasmic Pi concentrations is a likely function of a subset of Pi-starvation-inducible genes (Fig.…”

Characterization of three novel members of the Arabidopsis thaliana equilibrative nucleoside transporter (ENT) family

“…To obtain final evidence that the plasma membrane is the physiological destination, independent experiments using specific antisera will be needed. In purified tomato vacuoles, RNase activity as well as RNA oligonucleotides and uridine were detected [33,34]. Sequence analysis of AtENT2 and AtENT5 reported by the ARAMEMNON database (http://aramemnon.botanik.unikoeln.de/index.html) using the algorithm of the iPsort software [32] indicates a possible plastidic or mitochondrial localization respectively.…”

Suppression of LX Ribonuclease in Tomato Results in a Delay of Leaf Senescence and Abscission

“…The presence of vacuole-localized RNases and RNA (Boller and Kende, 1979;Glund, 1986, 1987;Abel et al, 1990) has led to the suggestion that vacuoles may participate in nucleic acid turnover. Secretion of RNases from the embryo and aleurone into the endosperm during germination (Barker et al, 1974;Rogers and Rogers, 1999) is thought to aid mobilization of stored RNA.…”