Cell-to-cell movement of plastids in plants

Abstract

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

Fig. 1.

Phenotypes of the graft partners and the G1 graft transfer plant. (A) Partner P1 is N. tabacum (2N = 48) with a nuclear gentamycin resistance transgene and wild-type N. tabacum plastids and mitochondria. Partner P2 has a wild-type N. sylvestris (2N = 24) nuclear genome, N. undulata plastids with aadA transgenes for spectinomycin selection and the aurea young leaf color phenotype (bar^au gene), and N. undulata mitochondria that confer cytoplasmic male sterility (CMS-92). Shown is also the G1 plant and its markers. (Black scale bar: 10 cm.) (B) Flower morphology of the P1 and P2 partners and G1 PGT plant. (White scale bar: 1 cm.)

Fig. 2.

Identification of plastid graft transfer events. (A) Grafted plant. Note that the P2 scion shown here is green because the expression of the bar^au gene is restricted to fast-growing tissue and is sensitive to environmental conditions. (B) Selection in cultures of 1–2-mm graft sections for gentamycin and spectinomycin resistance. On the left are stem sections from above (P2) and below (P1) the graft and on the right from the graft region. Note a green, proliferating callus that yielded the G4 PGT plants.

Fig. 3.

SSR markers confirm N. tabacum chromosomes in the G4 plant by testing each of the 24 chromosomes (numbered 1–24). Lanes are marked with s, G4, and t for the P2, G4, and P1 plants, respectively (see legend of Fig. 1). Some markers do not amplify the N. sylvestris template (13). White dots indicate the 200-bp fragment of the 20-bp molecular-mass ladder.

Fig. 4.

Identification of the source of mtDNA in the PGT plants. (A) Schematic representation of the tobacco mtDNA master circle with the position of polymorphic regions marked. Repeated regions are marked with boxes. (B) Mitochondrial DNA sequence polymorphisms. (C) Map position of polymorphic sites relative to the sequencing primers and gene features.

Fig. 5.

Identification of the N. undulata plastids in the PGT plants. (A) Identity plots of the plastid genomes of the transplastomic P2 partner carrying N. undulata ptDNA (u) with the aadA and bar^au transgenes (GenBank accession no. JN563930); the G1, G3, and G4 (G) PGT plants; and the P1 partner with N. tabacum ptDNA (t) (GenBank accession no. Z00044) aligned with the mVISTA program using a 500-bp sliding window. Shown above the map are the positions of the DNA probes (#1 through #6) and DNA polymorphisms (*1 through *7). (B) Plastid DNA sequence polymorphisms. For map position, see Fig. 5A. (C) DNA gel blot to identify RFLP markers in ptDNA. For probes see Fig. 5A.

Fig. 6.

Model for cell-to-cell movement of plastids via initial cytoplasmic connection in graft junctions. (A) Cells at graft junction reconnect by plasmodesmata. Arrows point to sites where opposite parts of the contact walls are synchronously thinned (11). These are future sites of plasmodesmata. Proplastids (ovals), mitochondria (small circles), and nuclei (large circles) are identified in scion and rootstock. Ns, N. sylvestris; Nt, N. tabacum; Nu, N. undulata. (B) Proplastid is transferred via initial cytoplasmic connection. (C) Transferred spectinomycin-resistant plastid takes over on selective medium. Note that the cells derive from the bottom cell in Fig. 6B.