Plastid protein synthesis is required for plant development in tobacco

Abstract

Chloroplasts fulfill important functions in cellular metabolism. The majority of plastid genome-encoded genes is involved in either photosynthesis or chloroplast gene expression. Whether or not plastid genes also can determine extraplastidic functions has remained controversial. We demonstrate here an essential role of plastid protein synthesis in tobacco leaf development. By using chloroplast transformation, we have developed an experimental system that produces recombination-based knockouts of chloroplast translation in a cell-line-specific manner. The resulting plants are chimeric and, in the presence of translational inhibitors, exhibit severe developmental abnormalities. In the absence of active plastid protein synthesis, leaf blade development is abolished because of an apparent arrest of cell division. This effect appears to be cell-autonomous in that adjacent sectors of cells with translating plastids are phenotypically normal but cannot complement for the absence of plastid translation in mutant sectors. Developmental abnormalities also are seen in flower morphology, indicating that the defects are not caused by inhibited expression of plastid photosynthesis genes. Taken together, our data point to an unexpected essential role of plastid genes and gene expression in plant development and cell division.

Fig. 1.

Recombination-induced knockout of plastid translation (RIKT). (A) Region of the tobacco plastid genome from which the chloroplast transformation vector pRB115 was derived. (B) Physical map of the plastid targeting region in transformation vector pRB115 (cloned as SacI/KpnI fragment into pBluescript). Note that the selectable marker gene aadA is flanked by only one region of homology with the plastid genome. Restriction sites lost because of ligation of heterologous ends are shown in parentheses. Sites from the pBluescript (pBS) polylinker are underlined. Prrn, promoter derived from the plastid rRNA operon; TpsbA, 3′ UTR derived from the psbA gene. (C) Cointegrate produced by plastid transformation with pRB115. A single homologous recombination event (indicated by the dotted arrow between the two maps in A and B) results in incorporation of the entire plasmid vector into the plastid genome. The cointegrate is unstable, and homologous recombination in the directly repeated psaA/psaB/rps14/trnfM region will recreate the wild-type genome and eliminate the aadA-resistance gene. In the presence of the translational inhibitor spectinomycin, cell lines that have completely lost the aadA as the result of such recombination events will no longer produce plastid-encoded proteins. pBS, pBluescript backbone (not drawn to scale).

Fig. 2.

Phenotype of tobacco plants with recombination-induced knockout of translation. (A) Segregating progeny of transplastomic RIKT plants. Variegated seedlings consist of both cells with translating and cells with nontranslating plastids whereas white seedlings have completely lost the spectinomycin-resistance gene by recombination and thus bleach out in the presence of the drug. (B) Close-up showing three seedlings with different degrees of variegation. (C) Phenotype of a RIKT plant on spectinomycin-containing synthetic medium. The arrow points to a leaf with half of the leaf blade missing. (D) Wild-type (left) and two RIKT (right) plants transferred from spectinomycin-containing synthetic medium to the soil. (E) The same plants after 3 weeks of growth in the soil. Note appearance of normal leaves in the RIKT plants in the absence of spectinomycin. (F) A RIKT plant transferred from spectinomycin-containing medium to drug-free medium. The lower leaves developed in the presence of the antibiotic (marked with filled arrows) whereas the upper leaves grew in the absence of spectinomycin (empty arrows) and gradually became more wild-type-like. (G) Reexposure of the same plant to spectinomycin-containing medium. Bleaching of leaf sectors in those leaves that developed in the absence of spectinomycin suggests that the these cell lines would have ceased to divide in the presence of the antibiotic and caused the phenotypic aberrations seen in leaves of RIKT plants. (H) Abnormal flower morphology in RIKT plants. The arrows point to missing sectors in the petals.

Fig. 3.

Spectrum of leaf phenotypes in RIKT plants. (Scale bar: 2 cm.) Upper left corner shows a wild-type leaf.

Fig. 4.

Southern blot confirming active excision of pRB115 in vivo. Total cellular DNA from the wild type and five independently generated transplastomic plants was electrophoresed in a 0.7% agarose gel with or without prior digestion with SalI and probed with the radiolabeled insert from pRB115. Analysis of the undigested samples reveals presence of the identical conformations of pRB115 in planta as in E. coli (oc, open circular conformation; lin, linear conformation; ccc, covalently closed circular conformation = supercoiled plasmid). Digestion with SalI produces a prominent band of 7.6 kb as expected for linearized pRB115. Note that little or no excision by recombination has occurred in the Nt-pRB115–10B (faint 7.6-kb band) and Nt-pRB115–12A plants (virtual absence of the 7.6-kb band), which correlates with the lack of a strong phenotype in these plants. The strongly hybridizing fragment of 23 kb present in both the wild-type and transplastomic lines originates from hybridization to the psaA/B region of the chloroplast genome. Minor bands are likely to originate from plasmid multimers (31) and/or promiscuous plastid DNA present in the nuclear genome (21, 32). M, molecular weight marker (sizes indicated in kb).

Fig. 5.

Recombinational loss of the aadA-resistance gene in bleached leaf sectors. Two green (G1 and G2) and two white (W1 and W2) leaf sectors from plants treated as shown in Fig. 2G were comparatively analyzed by DNA gel blot analyses. (A) Hybridization of undigested and SalI-digested DNA samples to an aadA-specific probe detects recombined-out pRB115 plasmids as well as aadA copies integrated into the plastid genome (compare with Fig. 4). (B) After digestion with XhoI or BamHI, hybridization to a psbA-specific probe detects identical fragments in green sectors, white sectors, and wild-type leaves (Wt). XhoI digestion produces a fragment of 8.5 kb; BamHI digestion generates a 4.8-kb fragment.