Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline

Abstract

Chloroplasts possess bacterial-type systems for transcription and translation. On the basis of the identification of a Chlamydomonas reinhardtii gene encoding a RelA-SpoT homolog (RSH) that catalyzes the synthesis of guanosine tetra- or pentaphosphate [(p)ppGpp], we have previously suggested the operation of stringent control in the chloroplast genetic system. Although RSH genes have also been identified in several higher plants, the activities of the encoded enzymes and their mode of action in chloroplasts have remained uncharacterized. We have now characterized the intrinsic (p)ppGpp synthase activity of chloroplast extracts prepared from pea (Pisum sativum). Fractionation by ultracentrifugation suggested that the (p)ppGpp synthase activity of a translationally active chloroplast stromal extract was associated with 70S ribosomes. Furthermore, this enzymatic activity was inhibited by tetracycline, as was the peptide elongation activity of the extract. Structural comparisons between rRNA molecules of Escherichia coli and pea chloroplasts revealed the conservation of putative tetracycline-binding sites. These observations demonstrate the presence of a ribosome-associated (p)ppGpp synthase activity in the chloroplasts of a higher plant, further implicating (p)ppGpp in a genetic system of chloroplasts similar to that operative in bacteria.

Figure 1

Assay of the S-30 fractions of pea chloroplasts and E.coli cells for (p)ppGpp synthase activity. The S-30 fractions of pea chloroplasts (A, E, I) or of E.coli strains W3110 (B, F, J) or CF1678 (C, G, K) were incubated with [α-³²P]GTP (A, B, C), [γ-³²P]ATP (E, F, K) or [γ-³²P]GTP (I, J, K), after which ³²P-labeled nucleotides were separated by 2D-TLC on PEI-cellulose sheets and visualized by autoradiography. The origin and the positions of spots corresponding to ADP, ATP, GMP, GDP, GTP, ppGpp, pppGpp and orthophosphate are indicated. Data are representative of experiments performed with three independent chloroplast or bacterial extracts. As a negative control, the 2D-TLC profile of each nucleotide used for labeling is shown on the right end of each panel as ‘no extract’ (D, H, L).

Figure 2

Characterization of the labeled pppGpp-like nucleotide by nucleotide-hydrolyzing enzymes. The radioactive compounds corresponding to putative pppGpp were extracted from PEI-cellulose sheets and subjected to enzyme digestion analysis. Abbreviations for enzymes are indicated as: Myo, myokinase; AK, bacterial alkaline phosphatase; PDE-I, snake venom phosphodiesterase type I; T2, ribonuclease T2; and PDE-I + T2, combination of the phosphodiesterase and the ribonuclease. Enzyme reactions were performed as described in the Materials and Methods. After the digestion, ³²P-labeled compounds were separated by 1D-TLC as described previously (6) and visualized by autoradiography. Four detected nucleotide spots (a–d) are indicated in the picture with arrowheads. Pi, Gp₂, Gp₃ and Gp₄ indicate orthophosphate, guanosine diphosphate, guanosine triphosphate and guanosine tetraphosphate, respectively. These positions were determined by co-chromatography of similarly labeled nucleotides prepared from the E.coli W3110 extract (13).

Figure 3

Association of (p)ppGpp synthase activity with 70S ribosomes in pea chloroplasts. (A) Schematic representation of the procedure for fractionation of pea chloroplast and bacterial cell extracts. (B) Assay of the chloroplast and E.coli W3110 P-150(suc) fractions for (p)ppGpp synthase activity with [α-³²P]GTP as substrate. The chloroplast P-150, S-150, P-150(wash), and S-150(wash) fractions, and the reconstituted fraction consisting of chloroplast P-150(wash) and S-150(wash) were examined. The reaction mixtures were subjected to 2D-TLC and autoradiography. Data are representative of experiments performed with three independent chloroplast or bacterial extracts. Relative intensities of the pppGpp spots appeared from the chloroplast fractions were: P-150, 100; S-150, 2.6; P-150(suc), 4.6; P-150(wash), 17.2; S-150(wash), 5.8; P-150(wash) + S-150(wash), 49.9. Spot intensities were quantitated based on the densitometric analysis as described in Materials and Methods.

Figure 4

Inhibition of the peptide elongation activity of pea chloroplasts by tetracycline. The peptide elongation activity of the S-30 fractions of pea chloroplasts and E.coli W3110 cells was determined in the absence (closed circles) or in the presence (closed squares) of 500 μM tetracycline with a poly(U)-dependent in vitro translation assay (A and B), and the dose effect of tetracycline on the translation activity was examined in the presence of several concentrations (0, 50, 250 and 500 μM) of tetracycline (C and D). The reaction mixture contained 0.025 mCi/ml of l-[2,3,4,5,6-³H]phenylalanine, with a specific radioactivity of 108 Ci/mmol, and the incorporation of [³H]phenylalanine into poly-phenylalanine is indicated by disintegradations per minute (dpm). One dpm is equivalent to 8.34 ×10⁴ pmol phenylalanine per ml in the reaction. Data represent the incorporation of [³H]phenylalanine into TCA-precipitable material during the incubation of reaction mixtures for the indicated times. The activity of a reaction mixture without poly(U) (open circles) was also determined as a negative control. Results are representative of experiments performed with three independent chloroplast or bacterial extracts, and SDs are indicated by bars. The dose-effect analysis was performed by measurement of the incorporated tritium at 4 min after the start of the reaction.

Figure 5

Inhibition by tetracycline of the (p)ppGpp synthase activity of pea chloroplast extract. (A) The S-30 fractions of pea chloroplasts and E.coli W3110 cells were assayed for (p)ppGpp synthase activity with [α-³²P]GTP as substrate in the presence of tetracycline at concentrations of 0 μM (lanes 1 and 5), 50 μM (lanes 2 and 6), 250 μM (lanes 3 and 7) or 500 μM (lanes 4 and 8). Labeled nucleotides were separated by 1D-TLC on PEI-cellulose and visualized by autoradiography (12 h exposure). (B) Longer exposure (36 h) of the TLC plate shown in (A). (C and D) The intensities of the ppGpp spots obtained with the S-30 fraction of E.coli (C) or of the pppGpp spots obtained with the S-30 fraction of pea chloroplasts (D) were measured for the autoradiogram shown in (B). Data are expressed relative to the corresponding values for the incubations performed in the absence of tetracycline, and are representative of experiments performed with three independent chloroplast or bacterial extracts.