Studies on regulation of transcription and translation during differentiation of nicotians.... cultures

Abstract

The resumption of development and differentiation in callus cultures is brought about by phytohormones. To provide insight into some of the molecular events associated with this differentiation process, the present study investigates the control of gene expression at both transcriptional and translational levels during Nicotiana sanderae callus differentiation into shoots.

Anatomical studies conducted at various stages of shoot formation revealed well-defined phases of morphogenesis, including meristematic activity, bud formation, development of lignified xylem elements, and the emergence of leaves.

The contents of DNA, RNA, and protein, as well as their synthesis rates, were measured in both callus and differentiating cultures at various growth stages. DNA content was higher in the callus than in the differentiating tissue, possibly due to increased endoreduplication. However, RNA and protein contents were consistently higher in the differentiating tissue across all time points. The synthesis rates of these macromolecules varied: the differentiating tissue exhibited a higher protein synthesis rate after five days, while RNA synthesis increased marginally after 15 days of growth in the differentiation medium.

RNA polymerase activities in isolated nuclei were measured at different stages of growth and shoot differentiation. Total polymerase activity was higher in the differentiating tissue at early developmental stages. The contributions of RNA polymerase I and II to total activity were assessed through transcription assays using low (50 mM) and high (150 mM) concentrations of ammonium sulfate, with or without ?-amanitin. Template availability, measured using E. coli RNA polymerase, indicated a large excess of transcriptionally available template in callus nuclei compared to differentiating nuclei. When assessing the levels of free versus template-engaged polymerases (using poly d(AT) as a template), callus nuclei exhibited a major fraction of RNA polymerase activity in the free form (i.e., not engaged with DNA), whereas in differentiating tissue, most enzyme molecules were already template-engaged.

No significant differences were observed in the overall nature or type of total RNA transcribed in differentiating tissue, as determined by DNA/RNA filter hybridization. However, this does not exclude the possibility of differential synthesis of unique RNA species during shoot differentiation. Fractionation of pulse-labeled RNA from callus and differentiating tissues on polyacrylamide gels revealed the presence of novel RNA species synthesized during differentiation.

Gene expression regulation at the translational level was also examined. At early stages of organogenesis (24 hours), endogenous mRNA translation in vitro was enhanced. These endogenous mRNAs were capable of reinitiation in vitro, as evidenced by their sensitivity to inhibition by aurin tricarboxylic acid. However, polysomes from callus tissue were translated more efficiently than those from 24-hour-old differentiating tissue when tested in a wheat germ extract system. This may reflect a shift in the polysome profile toward smaller polysomes in response to differentiation signals, possibly due to preferential initiation of low molecular weight mRNAs or nonspecific reinitiation of polysome-associated mRNAs in callus but not in differentiating tissue.

To explain the apparent paradox between decreased polysome translation and increased endogenous mRNA translation in vitro in 24-hour-old differentiating tissue, the efficiency of supernatant factors in translating exogenously added mung bean polysomes was tested. The 3–100kDa fraction from differentiating tissue enhanced in vitro translation of heterologous polysomes more effectively than that from callus.

The amount of poly(A)+ RNA at various stages of differentiation was quantified. An increase in poly(A)+ RNA content was observed. When tested in a wheat germ system, the translatability of this poly(A)+ RNA was significantly higher than that from callus tissue. In vitro translation products, separated by polyacrylamide gel electrophoresis, revealed additional protein bands that may be specific to differentiation.

In summary, the onset of shoot differentiation in Nicotiana sanderae is characterized by:

(a) an increased rate of nuclear transcription by RNA polymerases I and II, due to greater engagement of RNA polymerases with DNA templates, and

(b) enhanced translational efficiency, likely resulting from increased polyadenylation and elevated activity of protein synthesis factors.