Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2017 Jul 24;175(1):186–193. doi: 10.1104/pp.17.00857

Efficient Plastid Transformation in Arabidopsis1,[OPEN]

Qiguo Yu a, Kerry Ann Lutz b, Pal Maliga a,c,2
PMCID: PMC5580780  PMID: 28739820

100-fold increased plastid transformation frequency is achieved in ACC2-defective Arabidopsis.

Abstract

Plastid transformation is routine in tobacco (Nicotiana tabacum) but 100-fold less frequent in Arabidopsis (Arabidopsis thaliana), preventing its use in plastid biology. A recent study revealed that null mutations in ACC2, encoding a plastid-targeted acetyl-coenzyme A carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC β-carboxylase subunit. We bombarded ACC2-defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance (aadA) gene and gfp, encoding the green fluorescence protein GFP. Spectinomycin-resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic messenger RNA, which would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin-hypersensitive Slavice and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Slavice and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops.

Plastids are semiautonomous plant organelles with thousands of copies of the approximately 155-kb genome localized in 10 to 100 plastids per cell. The plastid genome of higher plants encodes about 100 genes, the products of which assemble with approximately 3,000 nucleus-encoded proteins to form the plastid transcription and translation machinery and carry out complex metabolic functions, including photosynthesis and fatty acid and amino acid biosynthesis. Plastid transformation is routine only in tobacco (Nicotiana tabacum; Svab et al., 1990; Svab and Maliga, 1993), but reproducible protocols for plastid transformation also have been described in tomato (Solanum lycopersicum; Ruf et al., 2001), potato (Solanum tuberosum; Valkov et al., 2011), lettuce (Lactuca sativa; Kanamoto et al., 2006; Ruhlman et al., 2010), and soybean (Glycine max; Dufourmantel et al., 2004). Still, the technology is available in only a relatively small number of crops. Arabidopsis (Arabidopsis thaliana), the most widely used model plant, is one of the species that is recalcitrant to plastid transformation. In Arabidopsis, only two transplastomic events were identified in 201 samples (Sikdar et al., 1998), a sample size that would have yielded approximately 200 events in tobacco using the technology available in 1998. Until now, the reasons for the low efficiency in Arabidopsis were not understood.

Spectinomycin, the agent for the selection of transplastomic events, binds to the 16S rRNA, blocking translation on the prokaryotic type 70S plastid ribosomes (Wirmer and Westhof, 2006; Wilson, 2014) and inhibiting greening and shoot regeneration in tissue culture cells (Svab et al., 1990). When the plastid genome is transformed with the aadA gene encoding aminoglycoside-3′′-adenylyltransferase (AAD), the AAD protein modifies the antibiotic such that it no longer binds to the 16S rRNA and translation proceeds, enabling greening. Tobacco, when cultured on a spectinomycin medium, bleaches and proliferates at a slow rate due to the inhibition of plastid translation. Transplastomic tobacco cells are identified by the ability to green and regenerate shoots in spectinomycin-containing tissue culture medium. In contrast, Brassica napus, a close relative of Arabidopsis, bleaches but continues to proliferate as albino shoots on a spectinomycin medium in the absence of chloroplast ribosomes (Zubko and Day, 1998). Two major studies by Parker et al. (2014, 2016) revealed the existence of rare Arabidopsis accessions in which plastids are extremely sensitive to spectinomycin. Seeds of most accessions in the study germinated on spectinomycin and developed into small albino seedlings or rosettes, including RLD (Reduced Number of Long Days), the accession used in the 1998 plastid transformation experiment. However, seeds from hypersensitive accessions germinated but did not develop beyond the cotyledonary stage. Genetic analysis revealed that the spectinomycin sensitivity of hypersensitive accessions is due to mutations in the ACC2 nuclear gene. The ACC2 gene produces the homomeric acetyl-CoA-carboxylase (ACCase) that is imported into plastids and, in tissue culture, partially duplicates the function of heteromeric ACCase, one subunit of which is encoded in the plastid accD gene (Fig. 1A). When plastid translation is blocked by spectinomycin, no heteromeric ACCase is made, and the homomeric enzyme enables a limited amount of fatty acid biosynthesis to occur, thereby reducing the impact of the absence of heteromeric enzyme in culture, making spectinomycin selection inefficient. In the absence of a functional ACC2 gene, fatty acid biosynthesis is dependent on the availability of heteromeric ACCase enzyme, the β-carboxylase subunit of which is translated on plastid ribosomes (Fig. 1B).

Figure 1.

Figure 1.

Open in a new tab

Elimination of ACC2 function makes plastid transformation efficient in Arabidopsis. A, Heteromeric ACCase (hetACC) localizes in the chloroplast and is encoded by nuclear genes CAC1-A (At5g16390; Biotin Carboxyl Carrier Protein1 [BCCP-1]), CAC1-B (At5g15530; BCCP-2; not depicted), CAC2 (At5g35360; Biotin Carboxylase [BC]), CAC3 (At2g38040; α-subunit of Carboxyltransferase [α-CT]), and the plastid-encoded gene accD (AtCg00500; β-subunit of Carboxyltransferase [β-CT]). The homomeric ACC1 (At1g36160; homACCase) enzyme localizes in the cytoplasm, and the ACC2 (At1g36180; homACCase) enzyme is imported into the chloroplast via the TIC/TOC membrane protein complex. If translation of the plastid accD mRNA is blocked by spectinomycin, the nuclear homomeric ACC2 gene enables a limited amount of fatty acid biosynthesis, thereby reducing the impact of the absence of heteromeric enzyme in culture, making spectinomycin selection inefficient. B, In hypersensitive acc2 mutants, the absence of the homomeric ACCase makes the cells dependent on plastid translation to produce the heteromeric ACCase enzyme for fatty acid biosynthesis.

We hypothesized that inefficient plastid transformation in our original study was due to the tolerance of Arabidopsis to spectinomycin and that the transformation of hypersensitive mutants defective in ACC2 function should result in the efficient recovery of transplastomic clones. We report here that the efficiency of plastid transformation in the acc2 background is increased approximately 100-fold and comparable to that of tobacco, confirming our hypothesis.

RESULTS

Plastid Transformation with Vector pATV1 and Identification of Transplastomic Events

The plastid transformation vector pATV1 targets insertion upstream of the trnV gene in the inverted repeat region of the plastid genome (Fig. 2A). Vector pATV1 carries a dicistronic operon, in which the first open reading frame (ORF) encodes the aadA spectinomycin resistance gene and the second ORF encodes the green fluorescence protein GFP (Fig. 2A). Polycistronic mRNAs are not translated on the eukaryotic type 80S ribosomes in the cytoplasm; thus, the accumulation of GFP in chloroplasts in spectinomycin-resistant clones indicates plastid transformation.

Figure 2.

Figure 2.

Open in a new tab

Molecular characterization of the Sav-0 transplastomic clones. A, Map of the plastid genome with the integrated aadA-gfp dicistronic operon. The NruI-XbaI region is contained in the plastid transformation vector pATV1. P and T mark the positions of the PrrnLatpB promoter and the TpsbA terminator in the dicistronic vector. The black box at the aadA N terminus marks the atpB downstream box sequence (Kuroda and Maliga, 2001). The ribosome entry site is marked by black semiovals. The positions of the rrn16 and trnV plastid genes and relevant restriction enzyme sites are marked. Thick black and red lines indicate probes used for DNA and RNA gel-blot analyses, respectively. B, DNA gel blot using the rrn16 probe (A) indicates that the transplastomic Sav-0 calli and leaves are homoplastomic, carrying only the 4.7-kb EcoRI fragment and lacking the 2.7-kb wild-type fragment (wt). C, RNA gel-blot analyses using both the aadA and gfp probes (A) recognize the same 2-kb dicistronic mRNA.

Plastid transformation was carried out in the Columbia (Col-0) accession and the Col-0 ACC2 T-DNA insertion line acc2-1 (SALK_148966C), which was shown by Parker et al. (2014) to be hypersensitive to spectinomycin. We also evaluated plastid transformation efficiency in the Slavice (Sav-0) accession that was the most sensitive to spectinomycin in the study of Parker et al. (2014). The Sav-0 ACC2 gene carries 15 missense mutations; however, the hypersensitivity to spectinomycin is thought to be due to one mutation (G135E) that alters a conserved residue immediately preceding the biotin carboxylase domain (Parker et al., 2016). Plants were grown aseptically on Arabidopsis Revised Medium with 5% (w/v) Suc (ARM5 medium; Fig. 3A); leaves for plastid transformation were harvested from plants grown under aseptic conditions and placed on ARMI medium (see “Materials and Methods”). The leaf tissue was bombarded with gold particles coated with vector DNA. After 2 d, the leaves were stamped with a stack of razor blades, cut into 1-cm2 pieces, and transferred onto the same medium (ARMI) containing spectinomycin (100 mg L−1; Fig. 3B) to facilitate the preferential replication of plastids containing transformed ptDNA copies. The ARMI medium induces the division of leaf cells and the formation of colorless, embryogenic callus. After 7 to 10 d of selection on ARMI medium, spectinomycin selection was continued on the ARMIIr medium, which induces greening. Since spectinomycin prevents the greening of wild-type cells, only spectinomycin-resistant cells formed green calli. Visible green cell clusters appeared within 21 to 40 d on the selective ARMIIr medium (Fig. 3C). Illumination of plates with UV light revealed intense fluorescence of GFP in the green calli (Fig. 3D).

Figure 3.

Figure 3.

Open in a new tab

Identification of Arabidopsis transplastomic clones. A, Sterile Sav-0 plants grown in petri dishes (diameter, 10 cm) for 6 weeks. B, Two days after bombardment, the Sav-0 leaves are incised and transferred to selective spectinomycin (100 mg L−1) medium. C, Sav-0 leaves on selective medium 1 month after bombardment. Note the scanty callus formation and green cell cluster (arrow). D, The culture shown in C, illuminated with UV light. Note the green fluorescence indicating GFP accumulation in the green cell cluster. E, Sav-0 plant regenerated from transplastomic clone #6 in sterile culture. F, Culture illuminated with UV light. G, Sav-0 #3 seed progeny illuminated with UV light. Bar = 1 mm.

In the wild-type Col-0 sample (four bombarded plates), no transplastomic event was found. We obtained eight events on five bombarded plates using leaf tissue in the acc2-1 mutant background and four events on four bombarded plates in the Sav-0 accession (Table I). This transformation efficiency is comparable to the transformation efficiency obtained with current protocols in tobacco: four to five transplastomic events per bombardment (Maliga and Tungsuchat-Huang, 2014).

Table I. Identification of transplastomic events in Arabidopsis.

Au, Gold particles; Hepta, using the biolistic gun Hepta adaptor instead of a single flying disk; Tu, tungsten particles.

Plasmid Left/Right Arm Marker Gene Accession Tissue Gun No. of Plates No. of Transplastomic Events Reference
kb
pGS31A 1.1/0.9 Prrn:LrbcL:aadA:TpsbA RLD Leaf Single, Tu/1 μm 201 2 Sikdar et al. (1998)
pAAK176 1.7/0.8 Prrn:LrbcL:aadA:TpsbA RLD Leaf Hepta, Au/0.6 μm 10 0 Reported here
Ler Leaf Hepta, Au/0.6 μm 4 0 Reported here
pTT626 1.7/0.8 Prrn:Lcry9:aadA-gfp:TpsbA RLD Leaf Hepta, Au/0.6 μm 14 0 Reported here
pATV1 1.7/0.8 PrrnLatpB:aadA:Lcry9:gfp:TpsbA RLD Leaf Hepta, Au/1 μm 2 0 Reported here
Ler Leaf Hepta, Au/1 μm 1 0 Reported here
Col-0 Leaf Hepta, Au/0.6 μm 4 0 Reported here
Col-0 acc2-1 Leaf Hepta, Au/0.6 μm 5 8 Reported here
Sav-0 Leaf Hepta, Au/0.6 μm 4 4 Reported here
Open in a new tab

This is a significant advance, as high-frequency plastid transformation in Arabidopsis has been pursued since the publication of the original report (Sikdar et al., 1998). Since 2007, 26 plates of RLD and five plates of Landsberg erecta (Ler) leaf tissue were bombarded, none of which yielded a transplastomic event (Table I). In contrast, nine bombardments of leaves with the acc2 null background yielded 12 transplastomic clones. Even though the technology improved significantly since 1998, no transplastomic clones were obtained until ACC2-defective leaf tissue was used for bombardments (Table I), providing overwhelming support for the absence of ACC2 activity being critical for high-frequency plastid transformation in Arabidopsis.

Confocal Microscopy to Confirm Transplastomic Events

GFP is encoded in the second ORF; thus, GFP accumulation is expected only if the mRNA is translated in plastids on the prokaryotic type 70S ribosomes known to translate polycistronic mRNAs (Staub and Maliga, 1995). Thus, GFP accumulation was anticipated only if the gfp gene is expressed in chloroplasts.

Putative transplastomic lines were identified by green cell cluster formation and were confirmed as transplastomic events by detecting the localization of GFP to plastids by confocal microscopy (Fig. 4). Overlay of the GFP and chlorophyll channels indicates that the clones are heteroplastomic, carrying transformed and wild-type plastids in the same cells. A good example for mixed plastids is shown in the overlay of GFP and chlorophyll channels in Col-0 acc2-1 #3 in Figure 4. The chloroplasts were not well developed in most tissue culture cells. Chlorophyll was detected in only a localized region of plastids, in line with thylakoid biogenesis initiating from a localized center (Schottkowski et al., 2012). Good examples are overlays of Col-0 acc2-1 #5 and Sav-0 #1 in Figure 4.

Figure 4.

Figure 4.

Open in a new tab

GFP accumulates in chloroplasts. Shown are confocal images collected in the GFP, chlorophyll, and merged channels on a Leica TCS SP5II confocal microscope. Excitation wavelengths were at 488 and 568 nm, and detection was at 500 to 530 and 650 to 700 nm, respectively. Note the absence of GFP and chlorophyll in the wild-type Col-0 callus cells and mixed GFP-expressing transgenic and wild-type plastids in the Col-0 acc2-1 #1 and Sav-0 #6 lines. Note the absence of wild-type plastids in the leaves of Sav-0 #6 plants. Yellow color in the merged images indicates the colocalization of GFP and chlorophyll in plastids. Note that cells in the small green cell clusters are heteroplastomic. The only exception are cells in Sav-0 6 leaves, which are homoplastomic due to prolonged selection in tissue culture. Bars = 10 μm.

The heteroplastomic state detected in the cells of the green clusters was not maintained, and eventually, wild-type plastids (ptDNA) disappeared in the callus cells after continued cultivation on selective media. The homoplastomic state is confirmed by the uniform accumulation of GFP in the leaves of a Sav-0 #6 plant shown in Figure 4 and by DNA gel-blot analyses of calli shown in Figure 2B.

Regeneration of Transplastomic Sav-0 Plants and Transmission of GFP to Seed Progeny

After the bombardment of Col-0 and Sav-0 leaves, the selection of transplastomic events was carried out according to the published RLD protocol (Sikdar et al., 1998). However, when the transplastomic clones were transferred to the RLD shoot induction medium, the calli did not proliferate. Therefore, we transferred the transplastomic calli to media that were used successfully to regenerate plants from other accessions. We found that the two-step regeneration protocol described for shoot induction in the C24 background (Motte et al., 2013) triggered shoot regeneration in two surviving Sav-0 calli. Calli of Sav-0 transplastomic lines #3 and #6 were briefly (3 d) exposed to callus induction medium containing 0.5 mg L−1 2,4-dichlorophenoxyactetic acid (2,4-D) and 0.05 mg L−1 kinetin and then transferred to a shoot regeneration medium containing 0.15 mg L−1 indole acetic acid (IAA) and 1.6 mg L−1 phenyladenine. Phenyladenine is a potent compound for shoot regeneration through the inhibition of CYTOKININ OXIDASE/DEHYDROGENASE activity (Motte et al., 2013). Shoots from the calli developed in 45 to 60 d and flowered and formed siliques in sterile culture in 250-mL Erlenmeyer flasks (Fig. 3E). The plants glow intensely when illuminated with UV light, indicating high-level GFP accumulation (Fig. 3F). Confocal microscopy suggests the uniform transformation of plastid genomes in the leaves of regenerated Sav-0 #6 plants (Fig. 4) and was confirmed by molecular analyses (Fig. 2B).

The transplastomic shoots were transferred to larger 500-mL Erlenmeyer flasks containing ARM for seed set, where they continued to grow. It is noteworthy that the shoots did not have any roots or rosette leaves; thus, they could be best described as inflorescence cultures. The siliques harvested from the Sav-0 #6 plants were empty, while the Sav-0 #3 shoots produced six seeds. One transplastomic Sav-0 #3 seed germinated on spectinomycin. The cotyledons of this seedling fluoresced under UV light, indicating GFP accumulation (Fig. 3G).

Molecular Analysis of Transplastomic Arabidopsis Clones

DNA and RNA gel-blot analyses were carried out on the callus and shoots of Sav-0 transplastomic lines #3 and #6. Wild-type plastids present in the cells of the green clusters were gradually lost by the time DNA gel-blot analyses were carried out, confirming uniform transformation of the plastid genomes in both calli and shoots (Fig. 2B). RNA gel-blot analyses indicate the presence of a 2-kb dicistronic transcript detected by both the aadA and gfp probes (Fig. 2C).

DISCUSSION

Development of a Plastid Transformation Protocol in Arabidopsis

We report here approximately 100-fold enhanced plastid transformation efficiency per bombardment in the acc2 null background: eight events in five bombarded samples in the Col-0 acc2-1 line and four events in four bombarded samples in the Sav-0 background. The increase from one event per approximately 100 bombardments to one event per one bombardment is due in part to technological advances. However, the lack of success with the latest technology in a large number of bombarded samples (Table I) provides overwhelming evidence that the key to success was the choice of Arabidopsis lines lacking ACC2 activity.

The identification of transplastomic events in the RLD ecotype took 5 to 12 weeks in 1998 (Sikdar et al., 1998). The use of spectinomycin-sensitive acc2 knockout lines and the pATV1 dicistronic operon vector shortened the time period for the identification of transplastomic events to 3 to 5 weeks. Use of the acc2 knockout lines shortened scoring because the proliferation of nontransformed cells was efficiently inhibited by spectinomycin, enabling identification of the spectinomycin-resistant green cell clusters. Spectinomycin resistance may be due to the integration of aadA in the plastid genome, integration of aadA in the nuclear genome, and fortuitous expression from an upstream promoter or spontaneous mutations in the rrn16 gene (Svab and Maliga, 1993). GFP, encoded in the second ORF, is expressed only in the chloroplasts, enabling the rapid identification of transplastomic clones in a small number of heteroplastomic cells by confocal microscopy.

Once transplastomic clones are identified, the next major step is plant regeneration. There is diversity for shoot regeneration potential in Arabidopsis accessions. Col-0 is well known for its recalcitrance to shoot regeneration from cultured cells. Therefore, no attempt was made to regenerate shoots from the Col-0 transplastomic callus tissue. There is no information about the tissue culture properties of the Sav-0 accession. Our first attempts at Sav-0 shoot regeneration from the transplastomic clones proved successful, yielding flowering shoots in culture (Fig. 3E). However, the seeds, with one exception, failed to germinate. Seed viability was apparently compromised by somaclonal variation, accumulated genetic changes due to the tissue remaining in culture for close to 1 year (Bairu et al., 2011).

The first step toward obtaining a system that yields fertile transplastomic Arabidopsis will be obtaining ACC2 null mutations in regenerable accessions. Shoot regeneration protocols have been worked out from root (Márton and Browse, 1991) and leaf explants (Lutz et al., 2015) of the RLD accession and from protoplasts (Chupeau et al., 2013), leaf explants (Zhao et al., 2014), and inflorescence stem explants (Zhao et al., 2013) of the Wassilewskija accession. Thus, RLD and Wassilewskija will be our targets for ACC2 mutagenesis.

Plastid Transformation in Arabidopsis Provides a Template for Recalcitrant Crops

The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in all Arabidopsis accessions and in crops having a plastid-encoded accD gene and a plastid-targeted ACC2 enzyme. The Arabidopsis ACC2 enzyme has an N-terminal extension compared with ACC1 (Supplemental Fig. S1A). The N-terminal extension is a plastid-targeting sequence, as shown by subcellular localization of a GFP fusion protein (Babiychuk et al., 2011). The ACC1 and ACC2 genes are present in most Brassicaceae species, including Arabidopsis lyrata, Camelina sativa, Camelina rubella, Brassica oleracea, B. napus, and Brassica rapa. The homomeric ACC2 enzyme in these species has an N-terminal extension compared with ACC1 (Supplemental Fig. S1; Bryant et al., 2011). Thus, a targeted mutation in the ACC2 N-terminal extension should create a spectinomycin-hypersensitive variant. Plastid transformation has been achieved in cabbage (Brassica oleracea var capitata). Thus, knockout of ACC2 is apparently not necessary to obtain transplastomic events in this crop, at least in the two cultivars tested (Liu et al., 2007, 2008). Plastid transformation in cauliflower (Brassica oleracea var botrytis) has been obtained at a very low frequency (Nugent et al., 2006). Plastid transformation in B. napus also has been obtained, but no homoplastomic plants could be obtained (Hou et al., 2003; Cheng et al., 2010). Plastid transformation in Lesquerella fendleri, another oilseed crop in the Brassicaceae, was feasible but inefficient (Skarjinskaia et al., 2003). Deletion of ACC2 in the latter cases is expected to boost plastid transformation efficiency.

CONCLUSION

The experiments reported here establish that plastid transformation frequency is approximately 100-fold higher in ACC2 null mutants. A system to routinely obtain fertile transplastomic Arabidopsis requires ACC2 null mutants in a regenerable accession in which transplastomic plants can be rapidly obtained.

MATERIALS AND METHODS

Tissue Culture Media

The tissue culture media were adopted from Sikdar et al. (1998), originally described by Márton and Browse (1991). The culture media are based on Murashige and Skoog (MS) salts (Murashige and Skoog, 1962). ARM consists of MS salts, 3% (w/v) Suc, 0.8% (w/v) agar (A7921; Sigma), 200 mg of myoinositol, 0.1 mg of biotin (1 mL of 0.1 mg mL−1 stock), and 1 mL of vitamin solution (10 mg of vitamin B1, 1 mg of vitamin B6, 1 mg of nicotinic acid, and 1 mg of Gly per mL) per liter, pH 5.8. ARM5 medium consists of ARM supplemented with 5% (w/v) Suc. ARMI medium consists of ARM containing 3 mg of IAA, 0.6 mg of benzyladenine, 0.15 mg of 2,4-D, and 0.3 mg of isopentenyladenine per liter. ARMIIr medium consists of ARM supplemented with 0.2 mg L−1 naphthaleneacetic acid and 0.4 mg of isopentenyladenine per liter. The stocks of filter-sterilized plant hormones and antibiotics (100 mg L−1 spectinomycin HCl) were added to media cooled to 45°C after autoclaving.

Shoot regeneration in the transplastomic Sav-0 clones was obtained on an ARM containing 2,4-D (0.5 mg L−1), kinetin (0.05 mg L−1), and spectinomycin (100 mg L−1; 3 d) followed by incubation on an ARM containing IAA (0.15 mg L−1), phenyladenine (1.6 mg L−1), and spectinomycin (100 mg L−1; Motte et al., 2013). Seed was obtained by growing shoots on MS salt medium containing 3% (w/v) Suc and 0.8% (w/v) agar (A7921; Sigma), pH 5.8.

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) Sav-0 (CS28725) and Col-0 homozygous acc2-1 knockout line (SALK_148966C) seeds were obtained from the Arabidopsis Biological Resource Center. The Col-0 seeds were obtained from Juan Dong (Rutgers University). The RLD and Ler seeds were purchased form Lehle Seeds.

For surface sterilization, seeds (25 mg) were treated with 1.7% (w/v) sodium hypochlorite (5× diluted 8.5% (w/v) commercial bleach) in a 1.5-mL Eppendorf tube for 15 min with occasional mixing (vortex). The bleach was removed by pipetting and washed three times with sterile distilled water. Seeds were germinated on 50 mL of ARM5 medium in deep petri dishes (20 mm high and 10 cm in diameter). The plates were illuminated for 8 h using cool-white fluorescent tubes (2,000 lx). The seeds germinated after 10 to 15 d of incubation at 24°C. To grow plants with larger leaves, seedlings were transferred individually to ARM5 plates (four plants per deep petri dish). The plates were illuminated for 8 h with cool-white fluorescent bulbs (2,000 lx) and incubated at 21°C during the day and 18°C during the night. One- to 2-cm-long, dark green leaves were harvested for bombardment after incubation for an additional 5 to 6 weeks.

Plastid Transformation and Selection of Transplastomic Lines

The plastid transformation vector pATV1 reported here targets insertion in the inverted repeat region of the plastid genome upstream of the trnV gene (Fig. 2). The DNA sequence was deposited in GenBank under accession number MF461355. The pAAK176 and pTT626 plastid transformation vectors share the plastid-targeting region with vector pATV1. The pAAK176 vector carries the Prrn:LrbcL:aadA:TpsbA marker gene present in vector pHK34 (Kuroda and Maliga, 2001). The aadA gene is between two loxP sites, which facilitate the excision of the marker gene, leaving behind a loxP target site (Lutz et al., 2004). The pTT626 plastid transformation vector encodes the aadA-gfp fusion protein (Khan and Maliga, 1999) in a PrrnLcry/TpsbA cassette (Chakrabarti et al., 2006).

Plastid transformation in Arabidopsis was carried out using our 1998 protocol, as shown in Figure 3 (Sikdar et al., 1998). The leaves (each 10–20 mm) were harvested from aseptically grown plants and covered the surface of agar-solidified ARMI medium in a 10-cm petri dish. We used 100 to 120 leaves to cover the surface of the plate. The leaves were cultured for 4 d on ARMI medium and then bombarded with pATV1 vector DNA. Transforming DNA was coated on the surface of microscopic (0.6 μm) gold particles, then introduced into chloroplasts by the biolistic process (1,100 p.s.i.) using a helium-driven PDS1000/He biolistic gun equipped with the Hepta adaptor (Lutz et al., 2011). The plates were placed on the shelf at the lowest position for bombardment.

Following bombardment, the leaves were incubated for an additional 2 d on ARMI medium. After this time period, the leaves were stamped with a stack of 10 razor blades to create parallel incisions 1 mm apart. The stamped leaves were cut into smaller (1 cm2) pieces, transferred onto the same medium (ARMI) containing 100 mg L−1 spectinomycin, incubated at 28°C, and illuminated for 16 h with fluorescent tubes (CXL F025/741). After 8 to 10 d, the leaf strips were transferred onto selective ARMIIr medium containing 100 mg L−1 spectinomycin for the selection of spectinomycin-resistant clones. The leaf strips were transferred to a fresh selective ARMIIr medium every 2 weeks until putative transplastomic clones were identified as resistant green calli.

Confocal Microscopy to Detect GFP in Plastids

The subcellular localization of GFP fluorescence was determined by a Leica TCS SP5II confocal microscope. To detect GFP and chlorophyll fluorescence, excitation wavelengths were at 488 and 568 nm, and the detection filters were set to 500 to 530 and 650 to 700 nm, respectively.

DNA and RNA Gel-Blot Analyses

Total leaf DNA was prepared by the cetyltrimethylammonium bromide protocol (Tungsuchat-Huang and Maliga, 2012). DNA gel-blot analyses was carried out as described (Svab and Maliga, 1993). Total cellular DNA was digested with the EcoRI restriction enzyme. The DNA probe was the ApaI-SphI ptDNA fragment encoding the plastid rrn16 gene (Fig. 2).

Total cellular RNA was isolated from leaves frozen in liquid nitrogen using TRIzol (Ambion/Life Technologies) following the manufacturer’s protocol. RNA gel-blot analyses were carried out as described (Kuroda and Maliga, 2001). The probes were as follows: for aadA, a 0.8-kb NcoI-XbaI fragment isolated from plasmid pHC1 (Carrer et al., 1991); and for gfp, a fragment amplified from the gfp coding region using primers gfp-forward p1 (5′-TTTTCTGTCAGTGGAGAGGGTG-3′) and gfp-reverse p2 (5′-CCCAGCAGCTGTTACAAACT-3′; Fig. 2).

Alignment of Homomeric ACCases

The alignment of homomeric ACCases in the Brassicaceae family was carried out with MultAlin software (Corpet, 1988).

Accession Numbers

The DNA sequence of the pATV1 Arabidopsis plastid transformation vector was deposited in GenBank under accession number MF461355.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Arun K. Azhagiri and Tarini Tungsuchat Huang for the plastid transformation vectors pAAK176 and pTT626, respectively.

Glossary

RLD

Reduced Number of Long Days

ORF

open reading frame

Col-0

Columbia

Sav-0

Slavice

2,4-D

2,4-dichlorophenoxyactetic acid

MS

Murashige and Skoog

IAA

indole acetic acid

Footnotes

1

This research was supported in part by the NSF Eukaryotic Genetics Program (grant no. MCB-039958 to P.M.) and the Theresa Patnode Santmann Faculty Development Award in Bioscience to K.A.L. Q.Y. is the recipient of a Charles and Joanna Busch Predoctoral Fellowship.

[OPEN]

Articles can be viewed without a subscription.

References

  1. Babiychuk E, Vandepoele K, Wissing J, Garcia-Diaz M, De Rycke R, Akbari H, Joubès J, Beeckman T, Jänsch L, Frentzen M, et al. (2011) Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family. Proc Natl Acad Sci USA 108: 6674–6679 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bairu MW, Aremu AO, Van Staden J (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63: 147–173 [Google Scholar]
  3. Bryant N, Lloyd J, Sweeney C, Myouga F, Meinke D (2011) Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis. Plant Physiol 155: 1678–1689 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carrer H, Staub JM, Maliga P (1991) Gentamycin resistance in Nicotiana conferred by AAC(3)-I, a narrow substrate specificity acetyltransferase. Plant Mol Biol 17: 301–303 [DOI] [PubMed] [Google Scholar]
  5. Chakrabarti SK, Lutz KA, Lertwiriyawong B, Svab Z, Maliga P (2006) Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers resistance to potato tuber moth. Transgenic Res 15: 481–488 [DOI] [PubMed] [Google Scholar]
  6. Cheng L, Li HP, Qu B, Huang T, Tu JX, Fu TD, Liao YC (2010) Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons. Plant Cell Rep 29: 371–381 [DOI] [PubMed] [Google Scholar]
  7. Chupeau MC, Granier F, Pichon O, Renou JP, Gaudin V, Chupeau Y (2013) Characterization of the early events leading to totipotency in an Arabidopsis protoplast liquid culture by temporal transcript profiling. Plant Cell 25: 2444–2463 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Corpet F. (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16: 10881–10890 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dufourmantel N, Pelissier B, Garçon F, Peltier G, Ferullo JM, Tissot G (2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55: 479–489 [DOI] [PubMed] [Google Scholar]
  10. Hou BK, Zhou YH, Wan LH, Zhang ZL, Shen GF, Chen ZH, Hu ZM (2003) Chloroplast transformation in oilseed rape. Transgenic Res 12: 111–114 [DOI] [PubMed] [Google Scholar]
  11. Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M, Yokota A, Tomizawa K (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15: 205–217 [DOI] [PubMed] [Google Scholar]
  12. Khan MS, Maliga P (1999) Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat Biotechnol 17: 910–915 [DOI] [PubMed] [Google Scholar]
  13. Kuroda H, Maliga P (2001) Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol 125: 430–436 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Liu CW, Lin CC, Chen JJ, Tseng MJ (2007) Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant Cell Rep 26: 1733–1744 [DOI] [PubMed] [Google Scholar]
  15. Liu CW, Lin CC, Yiu JC, Chen JJ, Tseng MJ (2008) Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella. Theor Appl Genet 117: 75–88 [DOI] [PubMed] [Google Scholar]
  16. Lutz KA, Azhagiri A, Maliga P (2011) Transplastomics in Arabidopsis: progress towards developing an efficient method. In Jarvis RP, ed, Chloroplast Research in Arabidopsis. Springer Science+Business Media, New York, pp 133–147 [DOI] [PubMed] [Google Scholar]
  17. Lutz KA, Corneille S, Azhagiri AK, Svab Z, Maliga P (2004) A novel approach to plastid transformation utilizes the phiC31 phage integrase. Plant J 37: 906–913 [DOI] [PubMed] [Google Scholar]
  18. Lutz KA, Martin C, Khairzada S, Maliga P (2015) Steroid-inducible BABY BOOM system for development of fertile Arabidopsis thaliana plants after prolonged tissue culture. Plant Cell Rep 34: 1849–1856 [DOI] [PubMed] [Google Scholar]
  19. Maliga P, Tungsuchat-Huang T (2014) Plastid transformation in Nicotiana tabacum and Nicotiana sylvestris by biolistic DNA delivery to leaves. In Maliga P, ed, Chloroplast Biotechnology: Methods and Protocols. Springer Science+Business Media, New York, pp 147–163 [DOI] [PubMed] [Google Scholar]
  20. Márton L, Browse J (1991) Facile transformation of Arabidopsis. Plant Cell Rep 10: 235–239 [DOI] [PubMed] [Google Scholar]
  21. Motte H, Galuszka P, Spíchal L, Tarkowski P, Plíhal O, Šmehilová M, Jaworek P, Vereecke D, Werbrouck S, Geelen D (2013) Phenyl-adenine, identified in a LIGHT-DEPENDENT SHORT HYPOCOTYLS4-assisted chemical screen, is a potent compound for shoot regeneration through the inhibition of CYTOKININ OXIDASE/DEHYDROGENASE activity. Plant Physiol 161: 1229–1241 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473–497 [Google Scholar]
  23. Nugent GD, Coyne S, Nguyen TT, Kavanagh TA, Dix PJ (2006) Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake into protoplasts. Plant Sci 170: 135–142 [Google Scholar]
  24. Parker N, Wang Y, Meinke D (2014) Natural variation in sensitivity to a loss of chloroplast translation in Arabidopsis. Plant Physiol 166: 2013–2027 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Parker N, Wang Y, Meinke D (2016) Analysis of Arabidopsis accessions hypersensitive to a loss of chloroplast translation. Plant Physiol 172: 1862–1875 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ruf S, Hermann M, Berger IJ, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19: 870–875 [DOI] [PubMed] [Google Scholar]
  27. Ruhlman T, Verma D, Samson N, Daniell H (2010) The role of heterologous chloroplast sequence elements in transgene integration and expression. Plant Physiol 152: 2088–2104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schottkowski M, Peters M, Zhan Y, Rifai O, Zhang Y, Zerges W (2012) Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 109: 19286–19291 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sikdar SR, Serino G, Chaudhuri S, Maliga P (1998) Plastid transformation in Arabidopsis thaliana. Plant Cell Rep 18: 20–24 [Google Scholar]
  30. Skarjinskaia M, Svab Z, Maliga P (2003) Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea. Transgenic Res 12: 115–122 [DOI] [PubMed] [Google Scholar]
  31. Staub JM, Maliga P (1995) Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids. Plant J 7: 845–848 [DOI] [PubMed] [Google Scholar]
  32. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87: 8526–8530 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90: 913–917 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tungsuchat-Huang T, Maliga P (2012) Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants. Plant J 70: 717–725 [DOI] [PubMed] [Google Scholar]
  35. Valkov VT, Gargano D, Manna C, Formisano G, Dix PJ, Gray JC, Scotti N, Cardi T (2011) High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences. Transgenic Res 20: 137–151 [DOI] [PubMed] [Google Scholar]
  36. Wilson DN. (2014) Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat Rev Microbiol 12: 35–48 [DOI] [PubMed] [Google Scholar]
  37. Wirmer J, Westhof E (2006) Molecular contacts between antibiotics and the 30S ribosomal particle. Methods Enzymol 415: 180–202 [DOI] [PubMed] [Google Scholar]
  38. Zhao X, Liang G, Li X, Zhang X (2014) Hormones regulate in vitro organ regeneration from leaf-derived explants in Arabidopsis. Am J Plant Sci 5: 3535–3550 [Google Scholar]
  39. Zhao XY, Su YH, Zhang CL, Wang L, Li X, Zhang XS (2013) Differences in capacities of in vitro organ regeneration between two Arabidopsis ecotypes Wassilewskija and Columbia. Plant Cell Tissue Organ Cult 112: 65–74 [Google Scholar]
  40. Zubko MK, Day A (1998) Stable albinism induced without mutagenesis: a model for ribosome-free plastid inheritance. Plant J 15: 265–271 [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES