In previous work we have determined the primary structure of two of the five ribonucleases which are induced by phosphate starvation in cultured tomato cells. Here, we present the isolation and characterization of the cDNAs for the extracellular ribonuclease LE and the intracellular, but extravacuolar ribonuclease LX. Structural analysis of these cDNAs together with partial protein-sequencing of vacuolar ribonucleases LV1, LV2 and LV3 revealed a family of very similar ribonucleases. From these data we assume identify between ribonucleases LE and LV3 for which the targeting mechanism has to be shown. Furthermore, RNase LV1 and RNase LV2 might be posttranslational processing products of RNase LX which travel to the vacuoles after splitting off the putative ER retention signal present at RNase LX. Additionally, we show by northern blot analysis that phosphate starvation in plant cells leads to an increase in the steady-state level of this type of enzymes revealing close similarities of the plant response to a limited supply of inorganic phosphate with the PHO regulation in bacteria and fungi.

The primary structure of an intracellular ribonuclease (RNase LX) from cultured tomato (Lycopersicon esculentum) cells has been determined. Previous studies have shown that the protein is located inside the tomato cells but outside the vacuoles and that its synthesis is induced after depleting the cells for phosphate [Löffler, A., Abel, S., Jost, W., Beintema, J. J., Glund, K. (1992) Plant Physiol. 98, 1472-1478]. Sequence analysis was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the protein. RNase LX consists of 213 amino acids and has a molecular mass of 24300 Da and an isoelectric point of 5.33. The enzyme contains 10 half-cystines and there are no potential N-glycosylation sites detectable in the sequence. RNase LX, as compared to an extracellular tomato RNase (RNase LE), which is also phosphate regulated and the amino acid sequence of which was recently established [Jost, W., Bak, H., Glund, K., Terpstra, P. & Beintema, J. J. (1991) Eur. J. Biochem. 198, 1-6] has 60% of all amino acids identical and in identical positions, revealing a high degree of similarity between both proteins. In contrast to RNase LE, RNase LX has a C-terminal extension of nine amino acids. The C-terminal tetrapeptide HDEF may be a retention signal of the protein in the endoplasmic reticulum.

Although present in different organisms and conserved in their protein sequence, the biological functions of T2 ribonucleases (RNase) are generally unknown. Tomato (Lycopersicon esculentum) LX is a T2/S-like RNase and its expression is known to be associated with phosphate starvation, ethylene responses, and senescence and programmed cell death. In this study, LX function was investigated using antisense tomato plants in which the LX protein level was reduced. LX protein levels normally become elevated when leaves senesce and antisense inhibition of LX retarded the progression of senescence. Moreover, we observed a marked delay of leaf abscission in LX-deficient plants. This correlated with specific induction of LX protein in the tomato mature abscission zone tissue. LX RNase gene regulation and the consequences of antisense inhibition indicate that LX has an important functional role in both abscission and senescence.

Ribonuclease LX (RNaseLX) from tomato (Solanum lycopersicum L.) belongs to the RNase T2/S-RNase superfamily of plant endoribonucleases and this is a report on the characterization of the RNaseLX gene and its encoded protein as a member of the phosphate starvation response in tomato. RNaseLX gene sequences were cloned by a PCR-assisted approach. RNaseLX promoter sequences contained the conserved binding motif of the transcription factor PHR1 known to mediate phosphate starvation-dependent gene expression. The increase of RNaseLX transcript levels in roots during phosphate starvation correlated with high promoter activity in transgenic plants carrying a PromLX::uidA gene construct and pointed to transcriptional control of RNaseLX expression. Histochemical staining for beta-glucuronidase activity and immunodetection of RNaseLX protein revealed striking RNaseLX expression in main and lateral root tips of phosphate-starved transgenic plants, specifically in epidermal cells, as well as in lateral and adventitious root primordia. Induced RNaseLX expression in roots correlated with stimulated growth and elongation of primary and lateral roots during phosphate deprivation. Phosphate-starvation-induced RNaseLX transcript levels in roots were not modulated by auxin or ethylene. These data indicate that the role of intracellular RNaseLX in the phosphate starvation response is connected with specific RNA turnover processes at the root tip.