Phytochrome Control of the Expression of Two Nuclear Genes Encoding Chloroplast Proteins in Lemna gibba L. G-3

Abstract

Hybridization probes for two nuclear-coded chloroplast proteins of Lemna gibba L. G-3 have been constructed in order to investigate phyto-chrome regulation of specific sequences. The first probe is a cDNA clone encoding the small subunit of ribulose 1,5-bisphosphate carboxylase. This probe was isolated from a set of Lemna cDNA clones in the bacterial plasmid pBR322. The second probe is a subelone of a genomic clone encoding the light-harvesting chlorophyll a/b-protein. This clone was isolated from a set of genomic clones constructed in the lambda vector Charon 4 with L. gibba DNA fragments generated by partial EcoRl digestion. The identity of these clones was confirmed by in vitro translation of RNA which hybridized to the cloned DNA. Plants grown under continuous white light contain high concentrations of both RNA sequences; however, when these plants are put into darkness the concentration of these RNAs decreases rapidly relative to the total amount of RNA. Plants grown in the dark with intermittent red light (2 minutes/8 hours) and put into complete darkness for 8 days also contain lower concentrations of the sequences in the total RNA. One minute of red light after this dark period results in a rapid increase in the levels of RNA hybridizing to the probes. The effect of red light can be reversed by far-red light. These experiments demonstrate that phytochrome action can rapidly influence either the rates of transcription or the rates of degradation of these mRNAs. The expression of two nuclear genes encoding chloroplast proteins is under the control of light in Lemna gibba (55, 56, 59). These proteins are the SSU3 of RuBP carboxylase (17) and the light-harvesting Chl a/b-protein of the thylakoid membrane (52). Light affects the expression of these genes in Lemna by causing an increase in the amounts of the translatable mRNA encoding these polypeptides. The changes are accompanied by corresponding changes in the synthesis of the proteins in vivo (55, 59). This effect of light in Lemna is mediated by phytochrome (56). Correlated changes in translatable mRNA levels and the rates of in vivo synthesis of several polypeptides in response to light have also been reported in various other species of higher plants: these include a 32,000 D chloroplast membrane protein of Spirodela (35), maize (5), and mustard (31); phenylalanine ammonia lyase in cultured parsley cells (43); RuBP carboxylase in peas (6); phosphoenolpyruvate carboxylase in maize leaves (46); the Chl a/ b-protein and NADPH-Pchlide-oxidoreductase in barley (1, 41). SSU, small subunit; RuBP, ribulose 1,5-bisphosphate. Apel (1, 2) has shown that in etiolated barley the response of the two mRNAs to light is mediated by phytochrome. However, none of these studies unravel exactly which step(s) in the expression of these genes are affected by phytochrome. Phytochrome action might affect the transcription of specific mRNAs or posttranscrip-tional processing, packaging, or degradation of these mRNAs. For example, it has been suggested (18) that red light acts by mobilizing preexisting mRNA onto polysomes. The studies discussed above could not have detected mRNA sequestered in an untranslatable form. To study such questions, we have isolated molecular hybridization probes that can be used for the quanti-tation of the concentration of specific RNA sequences, coding for the SSU of RuBP carboxylase and the Chl a/b-protein. Both the SSU and the Chl a/b-protein are encoded by nuclear genes (1 1, 27, 29) and synthesized from polyadenylated (poly[A]) RNA on cytoplasmic ribosomes as precursor polypeptides which are transported into the chloroplasts (3, 12, 13, 16, 21, 55). In Lemna, it is not clear whether the two major polypeptides associated with the light-harvesting Chl a/b-complex (57) represent products of one or two genes, since antibodies against the two polypeptides immunoprecipitate a single band of 32,000 D from in vitro translation products (58). We have used poly(A) RNA from light-grown plants to prepare a cDNA library from which we have isolated the sequence for the SSU of RuBP carboxylase. We have isolated a hybridization probe for the Chl a/b-protein RNA from a library of genomic DNA of L. gibba cloned in the Charon 4 derivative of bacterio-phage lambda. We can now demonstrate that the content of these sequences is rapidly regulated by phytochrome action in L. gibba and we suggest that this regulation is occurring at the level of transcription. MATERIALS AND METHODS Growth of Plants. Lemna gibba L. G-3 was cultured aseptically in E medium with 1% sucrose in a growth chamber under constant white light at 27°C ± 2°C. Plants were also cultured in the dark with intermittent red light (2 min/8 h) in the same medium supplemented with 3 !LM kinetin at 270C + 2°C as described earlier (56). Plants were grown under four different red light regimes. RC-plants grown for 8 weeks under intermittent red light (2 min red light every 8 h); D-plants grown as RC-plants but put into darkness after 7 weeks for 8 d; 1'R-plants grown as D-plants but treated with 1 min of red light 2 h before harvesting; FR plants grown as 1 R-plants but instead of 119 min darkness after the 1 min red light pulse, the tissue was put into far-red light for 10 min followed by darkness for 109 min. After these light treatments, the tissue was harvested under green safe lights, washed with distilled H20 and frozen in liquid N2. Isolation of RNA. Lemna total RNA was isolated by phenol extraction and salt precipitation as previously described (19, 59). Poly(A)RNA was isolated using oligo (dT) cellulose as previously described (55). 717