Regulation of RNA Synthesis by DNA-Dependent RNA Polymerases and RNases during Cold Acclimation in Winter and Spring Wheat

Abstract

Chromatin DNA-dependent RNA polymerases and RNases activities were measured in winter and spring varieties to understand the overall regulation of RNA synthesis during cold acclimation. We found that total RNA polymerase activities were significantly higher in chromatin isolated from winter wheat compared to the spring wheat during the acclimation period. This increase was parallel to the increase in protein and RNA contents during hardening. The ratio of RNA polymerase I to RNA polymerase II activity was higher than 2 in winter wheat after 30 days of hardening compared, to a ratio of 0.90 under the nonhardening conditions. The increase in activity and the ratio of polymerase I to polymerase II was maintained after the separation of the enzymes from the template, suggesting that RNA synthesis is regulated in part at the enzyme level. On the other hand, the chromatin associated RNase activity decreased in both varieties during acclimation, indicating a nonspecific inhibition caused by low temperature rather than a selective genetic response associated with cold acclimation.The enhancement of RNA and protein synthesis is one of the most important physiological changes in the hardy plant cell during the development of cold tolerance. It has been suggested that increased protein synthesis may result in the synthesis of cryoprotective substance(s) which increase membrane resistance to freezing and induce old hardiness (3,4,7,10,13,14,16,27,29). The mechanism by which cold hardening conditions regulate protein and RNA synthesis in hardy varieties is not fully known, but there is general agreement that frost hardening is associated with de novo protein synthesis. To understand the nature of this metabolic change, we studied the regulation of protein synthesis at the transcriptional level as a first step to elucidating the overall mechanism of regulation of cold hardiness. For this purpose, the functional activity of the chromatin was determined by measuring the change in DNA-dependent RNA polymerases and in RNase activities during hardening of two varieties of wheat differing in their degree of cold resistance. [3H]thymidine (20 Ci/mol) from New England Nuclear were used to label the RNA and DNA, respectively. The seedling was carefully removed with the root system intact and washed thoroughly before incubation in the nutrient solution containing the radioactive uridine or thymidine at a concentration of I ,uCi/ml. Gramicidin D, 6 Ag/ml (Calbiochem) was added to prevent bacterial growth during the experiment. MATERIALS AND METHODSExtraction of nucleic acids was carried out as described above and aliquots of RNA and DNA fractions were counted in a Nuclear Chicago ISOCAP 300 liquid scintillation spectrometer to determine incorporation of the radioactive precursor.Preparation of Chromatin. The tissues were homogenized in a Waring Blendor in 3 volumes per unit weight of: 0.5 M sucrose, 50 mM Tris-HCl (pH 8.0), 10 mM MgCI2, 4 mM ,B-mercaptoethanol, and 6 ,ug/ml gramicidin D. The homogenate was filtered through eigh...

Synthesis of Freezing Tolerance Proteins in Leaves, Crown, and Roots during Cold Acclimation of Wheat

“…tion, translation, or protein turnover rate during hardening. The increase in DNA-dependent RNA polymerases activity observed in our earlier study (14), may be a result of the activation by such regulatory factors during hardening. This regulation at the transcription level may be part of the genetic control responsible for the differential expression of specific freezing tolerance mRNAs and their products.…”

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures

“…Previous studies have demonstrated increased concentration of soluble proteins during hardening (1,26). Increases in soluble proteins having apparent mol wt of 30 and 25.5 kD in Brassica napus cv Goreczanski (12) and 240 and 115 kD in wheat (25) have been reported, while little change has been reported in alfalfa (5) and rye (1).…”

“…In the present study, rRNA was also observed to increase during hardening. These changes would facilitate the increased rate of protein synthesis during hardening reported by numerous workers (15,26). The …”

“…Cold acclimation in plants has been associated with increased RNA (I15, 21,26,28), while DNA contents may increase modestly (26) or may not increase (6,15). Increased DNA content has been suggested to represent increased copies ofrDNA (21,26), because there is increased rRNA content during cold acclimation (6,21). In the present study, rRNA was also observed to increase during hardening.…”

“…Sarhan and Chevrier (26) began examining genetic control of low temperature acclimation of winter wheat through a study of the regulation of protein synthesis at the transcriptional level. They quantitated the activities of RNases and RNA polymerases, DNA levels, RNA levels, and synthesis of DNA and RNA.…”