Shanghai Institute of Plant Physiology, Fonglin Road, Shanghai 200032, China
During the past decade we have been engaged in studies of some physiological and biochemical aspects of rice embryos (Oryza sativa L. subsp. japonica). In this paper we report the results obtained recently on the developmental changes in transcription activity.
Using 3H-uridine as a precursor, we measured the activity of total RNA synthesis in vivo in the embryos at four different stages. The results reflected the amount of newly synthesized RNA. In the differentiation stage (4-13 days after anthesis, DAA), the incorporation of 3H-uridine into total RNA tended to increase, while the maximal peak occurred on the 13 DAA, that is the transition point between stages of differentiation and maturation (Fig. 1). After 13 DAA, the activity began to drop until the completion of embryogenesis, except for a steady phase at 15-18 DAA.
Fig. 1. Incorporation of 3H-uridine into RNA of rice embryos during
embryogenesis. A. per 10 embryos; B. per 106 cells.
Rice embryos were manually dissected from fresh kernels and
incubated in 3H-uridine (20 uCi/ml) at 25 deg.C for 1 hr, and were
homogenized. The 5% TCA-insoluble fraction was transferred to nitrocellulose
microfilms, washed with TCA and dried under infrared lamp. The cpm was
measured. The experiments were triplicated, and the data were calibrated
with the control counterparts.
On the embryo cell basis, however, the activity per cell did not change significantly among all detected samples, suggesting that the increase in total transcription capacity of embryos could be attributed mainly to the increment of embryo cell number in these periods (Qin and Tang 1985).
It has been proven that alpha-amanitin, at the concentration of 1 mg/ml, inhibits specifically the activity of RNA polymerase II so as to stop mRNA synthesis without much effect on RNA polymerases I and III. Therefore, rice embryos from different developing levels were treated with alpha-amanitin (1 ug/ml). The incorporation of 3H-uridine was measured. As shown in Fig. 2, the inhibitory peaks occurred in the early differentiation stage (7 DAA) and mid-maturation stage (15-18 DAA), indicating the higher activity of RNA polymerase II and the more new-synthesized mRNA on the basis of total transcription activity. The weak inhibitory effect on the 13 DAA embryos, showing the less synthesis of mRNA, suggested that the high transcription activity in this period (Fig. 1) may be related to the synthesis of rRNA and tRNA.
Fig. 2. Effect of alpha-amanitin on incorporation of 3H-uridine into RNA of
rice embryos during embryogenesis.Inhibitory rate=1-cpm(alpha-amanitin
treatment)/cpm(control).
All operations were the same as for Fig. 1, except adding alpha-amanitin (1.0 ug/ml) to 3H-uridine solution.
In order to have an idea about the changes in mRNA species during seed development, we assayed to detect poly(A+)-RNA (assumed to be mRNA) by electrophoresis. As shown in Fig. 3, there were at least 10 detectable species of poly(A+)-RNA in the seed at differentiation stage, with a broad distribution of molecular weight (Fig. 3A). This may reflect the requirement for cell ditterentiation, proembryo organization and endosperm organogenesis. But in the early maturation stage, the species of poly(A+)-RNAs seemed to be somewhat decreased. Yet, there was some increment in high molecular weight (ca. 23s) fractions (Fig. 3B). This may be related to tremendous synthesis of storage proteins, especially glutelin at this time (Zhang and Tang 1985; Kim et al. 1986). There were much less species of poly(A+)-RNA at late-maturation stage, indicating, the completion of seed development (Fig. 3C).
According to the results described above, it can be concluded that, similarly as is observed in other plants (Allen et al. 1985); Fabijanski et al. 1985; Walling et al. 1986), the expression of rice seed genes involves a mechanism of
Fig. 3. Electropherograms of poly(A+)-RNAs from seeds at the differentiation
stage (A: 7-10 DAA), early maturation stage (B: 12-18 DAA) and late
maturation stage (C: 25 DAA).
Total RNA was extracted from seeds at a certain stage with phenol-chloroform-SDS procedure. By oligo(dT)-cellulose chromatography, poly(A+)-RNA was obtained. Agarose (1.5%) electrophoresis was done in phosphate buffer, with formaldehyde and formamide in gels. Ribosomal RNAs of E. coli was used as standard. The gels were scanned at 260 nm with Shimadzu 190 scanner.
developmental regulation which functions mainly at the transcription level.
References
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Fabijanski, S., G. J. Matlashewski and I. Altosaar, 1985. Characterization of developing oat seed mRNA: Evidence for many globulin mRNAs. Plant Mol. Biol. 4: 205-210.
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