Cloning of a polycistronic cDNA from tomato encoding gamma-glutamyl kinase and gamma-glutamyl phosphate reductase.

We isolated from a tomato cDNA library the tomPRO1 locus, which encodes gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1 sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1 locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified from E. coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1 locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.

Osmotin gene expression is posttranscriptionally regulated.

Accumulation of both osmotin mRNA and osmotin protein in tissues of tobacco (Nicotiana tabacum L. var Wisconsin 38) is subject to complex developmental control. Osmotin was found to be most abundant in tobacco roots and in tissues of the outer stem comprised primarily of epidermis, and it was less abundant in the corolla. It was a minor protein in other tissues and was undetectable in some tissues, including those of developing and mature seeds. The mRNA abundances did not always reflect the amount of protein accumulation because in some tissues high levels of mRNA but not protein were measured and vice versa. Accumulation of osmotin mRNA but not protein occurred in some plant tissues due to treatment with abscisic acid, wounding, and tobacco mosaic virus infection. Ethylene induced the accumulation of osmotin mRNA and, to a small extent, protein in seedlings, but was ineffective with cultured cells. Exposure of cultured cells and plants to NaCl caused high levels of both mRNA and the protein to accumulate. Thus, the accumulation of osmotin mRNA is controlled developmentally and by at least five hormonal or environmental signals. However, posttranscriptional processes can limit osmotin accumulation.

Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Delta-Pyrroline-5-Carboxylate Reductase.

Tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCl accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of Delta(1)-pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of Delta(1)-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCl-adapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and Delta(1)-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCl-dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties.

Stable NaCl Tolerance of Tobacco Cells Is Associated with Enhanced Accumulation of Osmotin.

Osmotin is a major protein which accumulates in tobacco cells (Nicotiana tabacum L. var Wisconsin 38) adapted to low water potentials. Quantitation of osmotin levels by immunoblots indicated that cells adapted to 428 millimolar NaCl contained 4 to 30 times the level of osmotin found in unadapted cells, depending on the stage of growth. Unadapted cells accumulated low levels of osmotin with apparent isoelectric points, (pl) of 7.8 and >8.2. Upon transfer of NaCl-adapted cells to medium without NaCl and subsequent growth for many cell generations, the amount of osmotin declined gradually to a level intermediate between that found in adapted and unadapted cells. NaCl-adapted cells grown in the absence of NaCl accumulated both pl forms; however, the form accumulated by cells adapted to NaCl (pl > 8.2) was most abundant. Adapted cells grown in the absence of NaCl exhibited absolute growth rates and NaCl tolerance levels which were intermediate to those of NaCl-adapted and unadapted cells. The association between osmotin accumulation and stable NaCl tolerance indicates that cells with a stable genetic change affecting the accumulation of osmotin are selected during prolonged exposure to high levels of NaCl. This stable alteration in gene expression probably affects salt tolerance.

Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl.

Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (+/-) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K(2)SO(4), Na(2)SO(4), sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Psipi) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Psipi differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Psipi difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na(+), K(+), Cl(-), free amino acids, or organic acids could not account for the Psipi differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na(+), K(+), and Cl(-) was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Psipi. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment.

Hormonal regulation of protein synthesis associated with salt tolerance in plant cells.

Cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) synthesize a predominant 26-kDa protein upon exposure to abscisic acid (ABA). ABA also accelerates the rate of adaptation of unadapted cells to NaCl stress. The ABA-induced 26-kDa protein is immunologically cross-reactive to, and produces a similar pattern of peptides after partial proteolysis as, the major 26-kDa protein associated with NaCl adaptation. Both have pI values of >8.2. The synthesis of the ABA-induced 26-kDa protein is transient unless the cells are simultaneously exposed to NaCl stress. There is an association between increased intracellular accumulation of ABA during cell growth and commencement of synthesis of the 26-kDa protein. ABA induces the synthesis of an immunologically cross-reactive 26-kDa protein in cultured cells of several plant species. In tobacco plants, synthesis of the 26-kDa protein could be detected in several tissues but the highest level of expression was seen in outer stem tissue. In root tissues, exogenous ABA greatly stimulated the synthesis of 26-kDa protein as compared to outer stem tissue and leaf. We suggest that ABA is involved in the normal induction of the synthesis of 26-kDa protein and that the presence of NaCl is necessary for the protein to accumulate.

Abscisic Acid accelerates adaptation of cultured tobacco cells to salt.

Adaptation of tobacco (Nicotiana tabacum L. var Wisconsin 38) cells to NaCl was accelerated by (+/-) abscisic acid (ABA). In medium with 10 grams per liter NaCl, ABA stimulated the growth of cells not grown in medium with NaCl (unadapted, S-0) with an increasing response from 10(-8) to 10(-4) molar. ABA (10(-5) molar) enhanced the growth of unadapted cells in medium with 6 to 22 grams per liter NaCl but did not increase the growth of cells previously adapted to either 10 (S-10) or 25 (S-25) grams per liter NaCl unless the cells were inoculated into medium with a level of NaCl higher than the level to which the cells were adapted. The growth of unadapted cells in medium with Na(2)SO(4) (85.5 millimolar), KCl (85.5 or 171 millimolar), K(2)SO(4) (85.5 millimolar) was also stimulated by ABA. ABA (10(-8)-10(-4) molar) did not accelerate the growth of unadapted cells exposed to water deficits induced by polyethylene glycol (molecular weight 8000) (5-20 grams per 100 milliliters), sorbitol (342 millimolar), mannitol (342 millimolar) or sucrose (342 millimolar). These results suggest that ABA is involved in adaptation of cells to salts, and is not effective in promoting adaptation to water deficits elicited by nonionic osmotic solutes.