Light‐ and sucrose‐dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape (Brassica napus L.)

Abstract

Light‐dependent gene expression was analysed in photomixotrophic cell suspension cultures of rape (Brassica napus L.) growing in media containing either 2.0% or 0.6% sucrose. During growth in darkness phytochrome type I and NADPH‐protochlorophyllide oxidoreductase (Pchlide reductase) accumulated in both cell culture lines to a similar extent. Illumination with continuous white, blue or red light, but not with far‐red light, resulted in disappearance of both chromoproteins within 24 h in both cell cultures. Further analysis showed that the phytochrome system of rape cell cultures reacts in a similar way to that of re‐etiolated dicotyledonous plants, showing rapid Pfr destruction and rapid Pfr dark reversion. In contrast, the light‐dependent expression of genes encoding the major chlorophyll a‐ and b‐binding protein (CAB) and the re‐accumulation of chlorophyll were found to be strongly dependent on sucrose concentration in culture media. Whereas cells grown in darkness in medium containing 2.0% sucrose showed, after exposure to continuous white light, a very weak re‐induction of CAB mRNA, CAB protein and chlorophyll accumulation, the cells in medium containing 0.6% sucrose reacted very strongly. It was also possible to demonstrate that phytochrome (by high irradiance response, HIR, and by low fluence response, LF) and the blue/UV‐A receptor are involved in the light‐dependent gene expression of CAB. Similar to complete cells, protoplasts derived from the two different cell cultures showed an almost identical sucrose concentration‐dependent and light‐quality‐dependent regulation of CAB mRNA accumulation. As the dark‐grown photomixotrophic cells and protoplasts reflect some typical photoregulatory characteristics known from dark‐grown plants it is supposed that this system will be an excellent tool for studying biochemical and molecular biological aspects of light‐dependent signal transduction in cells of higher plants.

Sucrose Control of Phytochrome A Signaling in Arabidopsis

“…Metabolic repression of gene expression of light-regulated photosynthetic genes, such as CAB and RBCS genes, has been proposed to be a mechanism for the feedback inhibition of photosynthesis by carbohydrates (Sheen, 1990;Harter et al, 1993;Krapp et al, 1993;Jang and Sheen, 1994;Van Oosten et al, 1994). The analysis of severa1 sun mutants provided evidence for a close interaction between metabolic repression and light signaling in Arabidopsis seedlings.…”

“…Feedback inhibition of photosynthesis by sugars occurs at the leve1 of electron transport and enzyme activity (reviewed in Woodrow and Berry, 1988;Portis, 1992) and through repression of nuclear genes that encode proteins involved in photosynthesis. Metabolic repression of CAB and/or RBCS gene expression has been found in photomixotrophic cultures and protoplasts of rapeseed (Harter et al, 1993), autotrophic cell cultures of Chenopodium (Krapp et al, 1993), and intact Arabidopsis, potato, tobacco, and tomato plants (Cheng et al, 1992;Krapp et al, 1993;Van Oosten et al, 1994). By using a transient expression system for maize protoplasts, it has been shown that the activity of seven maize photosynthetic gene promoters is repressed by sugars (Sheen, 1990;Jang and Sheen, 1994).…”

Light-regulated modification and nuclear translocation of cytosolic G-box binding factors in parsley.

“…Cytosolic and nuclear probes were prepared for SDS-PAGE by denaturation in one volume of boiling (95%) SDS-sample buffer for 10 min. Total protein amounts were determined according to Harter et al (1993). PAGE, Protein Gel Blotting, Immunodetection, and Antisera Protein extracts were separated by SDS-PAGE according to Schagger and von Jagow (1987).…”

“…PAGE, Protein Gel Blotting, Immunodetection, and Antisera Protein extracts were separated by SDS-PAGE according to Schagger and von Jagow (1987). Protein gel blotting and immunodetection were performed as described by Harter et al (1993). Preimmune serum and antiserum raised against G-box binding factor 1 (GBF-1) of Arabidopsis were a gift from A.R.…”

“…Antiserum raised against phosphoenolpyruvate (PEP) carboxylase of maize was obtained from R. Chollet (University of Nebraska-Lincoln; Budde and Chollet, 1986). Before use in immunodetection, the antisera were diluted 1:l OOO in washing buffer as previously described (Harter et al, 1993).…”