Conversion of Proplastids to Amyloplasts in Tobacco Cultured Cells Is Accompanied by Changes in the Transcriptional Activities of Plastid Genes

Abstract

When tobacco (Nicotiana tabacum L.) cultured cells at the stationary phase were transferred to culture medium that contained cytokinin (benzyladenine) instead of auxin (2,4-dichlorophenoxyacetic acid), proplastids in the BY-2 cells were converted to amyloplasts within 48 h. The data obtained from in vitro transcription assays using isolated plastid-nucleoids (nuclei) strongly suggested that amyloplast formation in BY-2 cells was accompanied by changes in the transcriptional activities of plastid genes.Plastids in higher plants have their own DNA, and they differentiate into various specialized organelles in an organspecific manner. It is of interest to determine how the expression of plastid genes is controlled during the process of plastid differentiation. The regulation of plastid gene expression during the biogenesis of chloroplasts has been extensively studied because of the importance of their principal function, photosynthesis. By contrast, the biogenesis of amyloplasts and the regulation of plastid gene expression during this process have not been examined in detail, even though amyloplasts are the sites of synthesis and storage of starch and are important constituents of the storage organs of higher plants. Studies of the processes and the mechanisms involved in the formation of amyloplasts are essential if we are to clarify the way in which plants turn on their storage functions.To study the process of amyloplast formation, we have established procedures for the induction of amyloplast formation in suspension-cultured tobacco (Nicotiana tabacum L.) cells (line BY-2). We have also analyzed the regulation of transcription of plastid genes in amyloplasts by an in vitro transcription assay using isolated plastid-nucleoids. The conditions for the culture of tobacco cell line BY-2, derived from Nicotiana tabacum L. cv Bright Yellow-2 (3), were described previously (14). For the induction of the transformation of proplastids to amyloplasts, auxin (2,4-D; 0.2 mg L-'), which was present in the conventional medium, was replaced by cytokinin (BA; 1 mg L-`), and 8.5 mL of a suspension of cells in stationary phase were transferred into 150 mL of the modified medium. The formation of amyloplasts was monitored by counting numbers of amyloplasts per cell under a light microscope. For measurement of the starch content of the cells, protoplasts prepared from 1 x 105 cells were treated with Sarkosyl (1%, w/v) and proteinase K (1 mg mLU-) at 300C for 1 h, and the hot-ethanol (80%, v/v) insoluble fraction was used for the extraction of starch. Starch was extracted with hot water and perchloric acid and quantified by the phenol-sulfuric acid method (1). Isolation of Proplastid-Nucleoids and AmyloplastNucleoidsProcedures for the isolation of proplastid-nucleoids (nuclei) were described previously (8). Amyloplast-nucleoids (nuclei) were isolated from BY-2 cells that had been cultured in the modified medium for 48 h. The cells were collected from 1.8 L of culture by sedimentation under gravity, and they were treated wi...

Disruption of Maize Kernel Growth and Development by Heat Stress (Role of Cytokinin/Abscisic Acid Balance)

“…It is well documented that cytokinins play a major role in promoting cell division (Letham, 1963;Nishinan and Syono, 1980;Summons et al, 1980), but there is no information on their effect on endosperm cell number in maize. Moreover, it has been shown that cytokinins can promote chloroplast biogenesis and development (Parthier, 1979;Parthier et al, 1982;Caers and Vendrig, 1986) and convert proplastids to amyloplasts in cultured tobacco cells (Sakai et al, 1992). However, their effect on amyloplast biogenesis in cereal grains has not been documented.…”

Proteome Analysis of the Rice Etioplast

“…These differences could be a consequence of the differentiation status of both plastid types. Although etioplasts differentiate into chloroplasts after illumination, BY-2 plastids remain non-photosynthetic in the light and can only differentiate into amyloplast-like organelles after hormone depletion (37). It is also possible that the BY-2 plastid metabolism reflects the long term adaptation of the BY-2 cell culture to the carbohydrate supply from the growth medium.…”

Retrograde signals arise from reciprocal crosstalk within plastids

“…This regulation is postulated to be accomplished by plastid-to-nucleus signaling, 1,2 although the overall signal transduction pathway(s) are not well characterized. By applying a specific differentiation system in tobacco Bright Yellow-2 (BY-2) cultured cells, 3,4 we recently reported that the regulatory system of nuclear gene expressions modulated by a plastid signal was also observed during differentiation of plastids into amyloplasts. 5 While retrograde signaling from plastids was previously speculated to consist of several independent pathways, we found inhibition of OGE and perturbation in the cellular content of one tetrapyrrole intermediate, heme, seemed to interact to regulate amyloplast differentiation.…”