Planta
Planta (1981)152:379-380
9 Springer-Verlag 1981
Short Communication
Enhanced expression of a distinct plastid DNA region in mustard seedlings by continuous far-red light Gerhard Link Biologisches Institut II, Universitfit Freiburg, Schfinzlestr.i, D-7800 Freiburg i. Br., Federal Republic of Germany
Abstract. Fragments of chloroplast D N A from mustard (Sinapis alba L.) seedlings, generated by the restriction endonuclease Eco RI, were used to assess the frequency of complementary sequences in mustard R N A by D N A / R N A hybridization. A pronounced increase in hybridization to a single D N A fragment was found with R N A from seedlings irradiated with continuous far-red light, compared to R N A from dark-grown seedlings. Key words: Chloroplast D N A
Light control - R N A
Sinapis.
In higher plants, genes for ribosomal RNAs, transfer RNAs, and messenger RNAs are located on chloroplast D N A (cpDNA) (Review by Bedbrook and Kolodner 1979). Several of these R N A species appear to be developmentally regulated since they were found to be more abundant in chloroplasts than in etioplasts (Harel and Bogorad 1973 ; Bedbrook et al. 1978). For a better understanding of the control mechanisms in light-induced chloroplast formation much attention has been given to the photoreceptor(s) involved. It is well documented that many aspects of plastid development including changes in ultrastructure and enzyme levels are controlled by phytochrome and do not depend on the presence of photosynthetic pigments (Mohr 1977; Schopfer 1977). However, little is known on phytochrome control at the level of plastid R N A synthesis. In previous reports it was shown by Thien and Schopfer (1975a, b) that in mustard seedlings, continuous far-red light operating through phytochrome (Hartmann 1966; Sch~fer 1975) promotes synthesis and accumulation of plastid ribosomal RNAs. Evidence is presented in this communication that the expression of a c p R N A region which does not code for r R N A is likewise enhanced by farred light.
Recently, mustard cpDNA has been purified and a restriction endonuclease map of the circular double-stranded molecule was constructed (Link et al. 1981).Positions of transcribed regions have been located on this map by gel transfer hybridization (Southern 1975) of cpDNA fragments with radioactive chloroplast RNA (G. Link, unpublished results). In this procedure the radioactivity bound to each DNA fragment reflects the amount of hybridized RNA and thus the steady-state concentration of RNA species transcribed from each fragment. By using RNA from various stages in plastid developmentthe transcriptional program of the organelle can be revealed. In the experiment shown in Fig. 1 c p D N A fragments which had been generated by restriction endonuclease Eco RI (track 1) were hybridized with R N A from mustard cotyledons, and hybrids were detected by autoradiography (tracks 2 and 3). Strong radioactive signals are due to hybridization with rRNAs (data not shown) which represent the most abundant plastid RNAs. Other D N A fragments which do not contain r R N A genes show comparatively weak hybridization with RNA, suggesting that the non-ribosomal RNAs are present in lower frequency. While many D N A fragments hybridize with similar intensity to RNAs from either dark-grown (track 2) or light-grown seedlings (track 3), one single fragment, El5, hybridizes intensely to R N A from lightgrown seedlings, but only weakly to R N A from darkgrown seedlings. In these experiments equal amounts of R N A were applied in both hybridization reactions. D N A fragmets which contain r R N A genes are hybridized equally despite the known increase in the amount of r R N A per cotyledon, following irradiation of seedlings with far-red light (Thien and Schopfer 1975 a). It is therefore concluded that the differential hybridization of El5 reflects a pronounced increase in the steady-state concentration of R N A transcribed from this fragment relative to plastid rRNAs. D N A fragment El5 is 1.9 kilobase pairs in size. A c p D N A sequence in Zea mays with a similar length was recently shown to code for a polypeptide of
0032-0935/81/0151/0379/$01.00
380
G. Link: Photoregulated plastid gene expression t h e size a n d l o c a t i o n o f t h e c o d i n g s e q u e n c e w i t h i n t h i s r e g i o n ( L i n k a n d B o g o r a d 1980). The author would like to thank Professor H. Mohr for his encouragement and for providing research facilities. This work is supported by the Deutsche Forschungsgemeinschaft (SFB 46/Habilitanden-Stipendium).
References
Fig. 1. Gel transfer hybridization of chloroplast DNA fragments with RNA from mustard cotyledons. Seedlings were grown in darkness or under standard far-red light (Mohr 1966) at 25~ for 72 h. RNA from cotyledons was purified (Link et al. 1978) and labelled in vitro with 32p to a specific radioactivity of 3.7.10 ~1 Bq g 1 (Bedbrook et al. 1978). Mustard cpDNA was digested with restriction endonuclease Eco RI (Yoshimori et al. 1972). In two parallel experiments DNA fragments were separated according to size (decreasing from top to bottom) by electrophoresis in 1.0% agarose gels, stained, and photographed (track 1). Fragments were then transferred to cellulose nitrate sheets (Southern 1975) and hybridized with 50 ng of RNA from either dark-grown seedlings (track 2) or seedlings irradiated with farred light (track 3) in 0.3 M NaC1 and 0.03 M sodium citrate at 60~ for 12h. The sheets were then washed three times in 0.15 M NaC1 and 0.015 M sodium citrate at 60 ~ C for 1 h each and exposed to X-ray film for 16 h. DNA fragments containing sequences for plastid rRNAs are marked (r). E15, cpDNA fragment number 15 with regard to size
34,500, t h e m R N A o f w h i c h is p r e s e n t i n a m u c h h i g h e r s t e a d y s t a t e c o n c e n t r a t i o n in c h l o r o p l a s t s t h a n in e t i o p l a s t s ( B e d b r o o k e t al. 1 9 7 8 ; B o g o r a d et al., 1980). E x p e r i m e n t s a r e i n p r o g r e s s , (1), t o c h a r a c t e r ize t h e g e n e p r o d u c t ( s ) o f E l 5 a n d , (2), t o d e t e r m i n e
Bedbrook, J.R., Link, G., Coen, D.M., Bogorad, L., Rich, A. (1978) Maize plastid gene expressed during photoregulated development. Proc. Natl. Acad. Sci. USA 75, 3060-3064 Bedbrook, J.R., Kolodner, R. (1979) The structure of chloroplast DNA. Annu. Rev. Plant Physiol. 30, 593-620 Bogorad, L., Jolly, S., Kidd, G.H., Link, G., McJntosh, L. (1980) Organization and transcription of maize chloroplast genes. In: Genome organization and expression in plants, pp. 291-304, Leaver, C.J., ed., Plenum Press, New York Harel, E., Bogorad, L. (1973) Effect of light on ribonucleic acid metabolism in greening maize leaves. Plant Physiol. 51, 10-16 Hartmann, K.M. (1966) A general hypothesis to interpret "high energy phenomena" of photomorphogenes~s on the basis of phytochrome. Photochem. Photobiol. 5, 469483 Link, G., Coen, D.M., Bogorad, L. (1978) Differential expression of the gene for the large subunit of ribulose bisphosphate carboxylase in maize leaf cell types, Cell 15, 725-731 Link, G., Bogorad, L. (1980) Sizes, locations, and directions of transcription of two genes on a cloned maize chloroplast DNA sequence. Proc. Natl. Acad. Sci. USA 77, 1832 1836 Link, G., Chambers, S.E., Thompson, J.A., Falk, H. (1981) Size and physical organization of chloroplast DNA from mustard seedlings. Mol. Gen. Genet. 181, 454457 Mohr, H. (1966) Untersuchungen zur phytochrominduzierten Photomorphogenese des Senfkeimlings (Sinapis alba L.). Z. Pflanzenphysiol. 54, 63-83 Mohr, H. (1977) Phytochrome and chloroplast development. Endeavor 1, 107-114 Schiller, E. (1975) A new approach to explain the "High Irradiance Response" of photomorphogenesis on the basis of phytochrome. J. Math. Biol. 2, 41-56 Schopfer, P. (1977) Phytochrome control of enzymes, Annu. Rev. Plant Physiol. 28, 223-252 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517 Thien, W., Schopfer, P. (1975 a) Control by phytochrome of cytoplasmic and plastid rRNA accumulation in cotyledons of mustard seedlings in the absence of photosynthesis. Plant Physiol. 56, 660-664 Thien, W., Schopfer, P. (1975b) Stimulation of precursor rRNA synthesis in the cotyledons of mustard seedlings by phytochrome. Planta 124, 215-217 Yoshimori, R., Roulland-Dussoix, D., Boyer, H.W. (1972) R factor-controlled restriction and modification of DNA: restriction mutants. J. Bacteriol. 112, 1275 1279
Received 9 March; accepted 3 April 1981
Planta (1981)152:379-380
9 Springer-Verlag 1981
Short Communication
Enhanced expression of a distinct plastid DNA region in mustard seedlings by continuous far-red light Gerhard Link Biologisches Institut II, Universitfit Freiburg, Schfinzlestr.i, D-7800 Freiburg i. Br., Federal Republic of Germany
Abstract. Fragments of chloroplast D N A from mustard (Sinapis alba L.) seedlings, generated by the restriction endonuclease Eco RI, were used to assess the frequency of complementary sequences in mustard R N A by D N A / R N A hybridization. A pronounced increase in hybridization to a single D N A fragment was found with R N A from seedlings irradiated with continuous far-red light, compared to R N A from dark-grown seedlings. Key words: Chloroplast D N A
Light control - R N A
Sinapis.
In higher plants, genes for ribosomal RNAs, transfer RNAs, and messenger RNAs are located on chloroplast D N A (cpDNA) (Review by Bedbrook and Kolodner 1979). Several of these R N A species appear to be developmentally regulated since they were found to be more abundant in chloroplasts than in etioplasts (Harel and Bogorad 1973 ; Bedbrook et al. 1978). For a better understanding of the control mechanisms in light-induced chloroplast formation much attention has been given to the photoreceptor(s) involved. It is well documented that many aspects of plastid development including changes in ultrastructure and enzyme levels are controlled by phytochrome and do not depend on the presence of photosynthetic pigments (Mohr 1977; Schopfer 1977). However, little is known on phytochrome control at the level of plastid R N A synthesis. In previous reports it was shown by Thien and Schopfer (1975a, b) that in mustard seedlings, continuous far-red light operating through phytochrome (Hartmann 1966; Sch~fer 1975) promotes synthesis and accumulation of plastid ribosomal RNAs. Evidence is presented in this communication that the expression of a c p R N A region which does not code for r R N A is likewise enhanced by farred light.
Recently, mustard cpDNA has been purified and a restriction endonuclease map of the circular double-stranded molecule was constructed (Link et al. 1981).Positions of transcribed regions have been located on this map by gel transfer hybridization (Southern 1975) of cpDNA fragments with radioactive chloroplast RNA (G. Link, unpublished results). In this procedure the radioactivity bound to each DNA fragment reflects the amount of hybridized RNA and thus the steady-state concentration of RNA species transcribed from each fragment. By using RNA from various stages in plastid developmentthe transcriptional program of the organelle can be revealed. In the experiment shown in Fig. 1 c p D N A fragments which had been generated by restriction endonuclease Eco RI (track 1) were hybridized with R N A from mustard cotyledons, and hybrids were detected by autoradiography (tracks 2 and 3). Strong radioactive signals are due to hybridization with rRNAs (data not shown) which represent the most abundant plastid RNAs. Other D N A fragments which do not contain r R N A genes show comparatively weak hybridization with RNA, suggesting that the non-ribosomal RNAs are present in lower frequency. While many D N A fragments hybridize with similar intensity to RNAs from either dark-grown (track 2) or light-grown seedlings (track 3), one single fragment, El5, hybridizes intensely to R N A from lightgrown seedlings, but only weakly to R N A from darkgrown seedlings. In these experiments equal amounts of R N A were applied in both hybridization reactions. D N A fragmets which contain r R N A genes are hybridized equally despite the known increase in the amount of r R N A per cotyledon, following irradiation of seedlings with far-red light (Thien and Schopfer 1975 a). It is therefore concluded that the differential hybridization of El5 reflects a pronounced increase in the steady-state concentration of R N A transcribed from this fragment relative to plastid rRNAs. D N A fragment El5 is 1.9 kilobase pairs in size. A c p D N A sequence in Zea mays with a similar length was recently shown to code for a polypeptide of
0032-0935/81/0151/0379/$01.00
380
G. Link: Photoregulated plastid gene expression t h e size a n d l o c a t i o n o f t h e c o d i n g s e q u e n c e w i t h i n t h i s r e g i o n ( L i n k a n d B o g o r a d 1980). The author would like to thank Professor H. Mohr for his encouragement and for providing research facilities. This work is supported by the Deutsche Forschungsgemeinschaft (SFB 46/Habilitanden-Stipendium).
References
Fig. 1. Gel transfer hybridization of chloroplast DNA fragments with RNA from mustard cotyledons. Seedlings were grown in darkness or under standard far-red light (Mohr 1966) at 25~ for 72 h. RNA from cotyledons was purified (Link et al. 1978) and labelled in vitro with 32p to a specific radioactivity of 3.7.10 ~1 Bq g 1 (Bedbrook et al. 1978). Mustard cpDNA was digested with restriction endonuclease Eco RI (Yoshimori et al. 1972). In two parallel experiments DNA fragments were separated according to size (decreasing from top to bottom) by electrophoresis in 1.0% agarose gels, stained, and photographed (track 1). Fragments were then transferred to cellulose nitrate sheets (Southern 1975) and hybridized with 50 ng of RNA from either dark-grown seedlings (track 2) or seedlings irradiated with farred light (track 3) in 0.3 M NaC1 and 0.03 M sodium citrate at 60~ for 12h. The sheets were then washed three times in 0.15 M NaC1 and 0.015 M sodium citrate at 60 ~ C for 1 h each and exposed to X-ray film for 16 h. DNA fragments containing sequences for plastid rRNAs are marked (r). E15, cpDNA fragment number 15 with regard to size
34,500, t h e m R N A o f w h i c h is p r e s e n t i n a m u c h h i g h e r s t e a d y s t a t e c o n c e n t r a t i o n in c h l o r o p l a s t s t h a n in e t i o p l a s t s ( B e d b r o o k e t al. 1 9 7 8 ; B o g o r a d et al., 1980). E x p e r i m e n t s a r e i n p r o g r e s s , (1), t o c h a r a c t e r ize t h e g e n e p r o d u c t ( s ) o f E l 5 a n d , (2), t o d e t e r m i n e
Bedbrook, J.R., Link, G., Coen, D.M., Bogorad, L., Rich, A. (1978) Maize plastid gene expressed during photoregulated development. Proc. Natl. Acad. Sci. USA 75, 3060-3064 Bedbrook, J.R., Kolodner, R. (1979) The structure of chloroplast DNA. Annu. Rev. Plant Physiol. 30, 593-620 Bogorad, L., Jolly, S., Kidd, G.H., Link, G., McJntosh, L. (1980) Organization and transcription of maize chloroplast genes. In: Genome organization and expression in plants, pp. 291-304, Leaver, C.J., ed., Plenum Press, New York Harel, E., Bogorad, L. (1973) Effect of light on ribonucleic acid metabolism in greening maize leaves. Plant Physiol. 51, 10-16 Hartmann, K.M. (1966) A general hypothesis to interpret "high energy phenomena" of photomorphogenes~s on the basis of phytochrome. Photochem. Photobiol. 5, 469483 Link, G., Coen, D.M., Bogorad, L. (1978) Differential expression of the gene for the large subunit of ribulose bisphosphate carboxylase in maize leaf cell types, Cell 15, 725-731 Link, G., Bogorad, L. (1980) Sizes, locations, and directions of transcription of two genes on a cloned maize chloroplast DNA sequence. Proc. Natl. Acad. Sci. USA 77, 1832 1836 Link, G., Chambers, S.E., Thompson, J.A., Falk, H. (1981) Size and physical organization of chloroplast DNA from mustard seedlings. Mol. Gen. Genet. 181, 454457 Mohr, H. (1966) Untersuchungen zur phytochrominduzierten Photomorphogenese des Senfkeimlings (Sinapis alba L.). Z. Pflanzenphysiol. 54, 63-83 Mohr, H. (1977) Phytochrome and chloroplast development. Endeavor 1, 107-114 Schiller, E. (1975) A new approach to explain the "High Irradiance Response" of photomorphogenesis on the basis of phytochrome. J. Math. Biol. 2, 41-56 Schopfer, P. (1977) Phytochrome control of enzymes, Annu. Rev. Plant Physiol. 28, 223-252 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517 Thien, W., Schopfer, P. (1975 a) Control by phytochrome of cytoplasmic and plastid rRNA accumulation in cotyledons of mustard seedlings in the absence of photosynthesis. Plant Physiol. 56, 660-664 Thien, W., Schopfer, P. (1975b) Stimulation of precursor rRNA synthesis in the cotyledons of mustard seedlings by phytochrome. Planta 124, 215-217 Yoshimori, R., Roulland-Dussoix, D., Boyer, H.W. (1972) R factor-controlled restriction and modification of DNA: restriction mutants. J. Bacteriol. 112, 1275 1279
Received 9 March; accepted 3 April 1981
