Plant growth conditions and phenotypic analysis
Solanum tuberosum (potato) var. ‘Desirée’ and AtFtsZ1 overexpressing lines were grown in Magenta boxes containing MS Reg media (4.33 g/l MS basal salt mixture; 25 g/l sucrose; 100 mg/l myo-inositol; 170 mg/l sodium phosphate monobasic monohydrate; 440 mg/l calcium chloride dihydrate; 0.9 mg/l thiamine-HCl; 2 mg/l glycine; 0.5 mg/l nicotinic acid; 0.5 mg/l pyridoxine–HCl; 1 × MS vitamins; 3 g/l phytagel; pH 5.8). Transplastomic lines of both genotypes (wild-type and macro) were grown in selective MS rooting media60.
For determination of growth characteristics, apical shoots (same length and age) from both transgenic and wild-type plants were in vitro propagated. After 2 weeks, small plantlets with roots were transferred to soil, Pro-Mix BK25 (Griffin Greenhouse Supplies, Inc. Tewksbury, MA, USA), and grown to anthesis (9- and 10-weeks-old for wild-type and AtFtsZ1 lines, respectively) or full maturity (24-weeks-old for both genotypes). Pots of 3.8 or 11.4 l volume were used to grow plants at anthesis or full maturity, respectively. Plant height (cm), fresh and dry weight (g) of leaves and total biomass were measured. The total number, along with the dry and fresh weights (g) of tubers were also measured. For determination of dry weight, plant tissue was dried for 1 week at 50 °C. Both leaf and tuber areas were obtained by image analysis using ImageJ 1.41o software (National Institute of Health, Bethesda, MD, USA).
CO2 assimilation (A) values per unit of leaf area (µmol m−2 s−1) were obtained using a LI-6800 portable photosynthesis system (LI–COR Biosciences, Lincoln, NE, USA) at atmospheric CO2 concentrations (400 µmol mol air−1), constant irradiance (1000 µmol photons m−2 s−1), ambient temperature of 23 °C, a vapour pressure deficit (VPD leaf) of 0.8–1.2 kPa and a flow rate of 200 µmol s−1). The leaf chlorophyll content index (CCI) was obtained using a portable CCM-200 plus chlorophyll content meter (OPTI-SCIENCES Inc., Hudson, NH, USA).
Results were expressed as mean ± standard deviation (sd) of six and four biological replicates for each genotype (AtFtsZ1 independent lines (Macro 1 and 2) along with wild-type control) for the growth experiments at anthesis and full maturity, respectively. ANOVAs with post-hoc Tukey statistical analysis (IBM SPSS software) (p < 0.05) were performed to determine if differences were statistically significant among wild-type and AtFtsZ1 lines. Plants grown in vitro and in pots were subjected to congruent environmental conditions, 16/8 h of light and dark respectively, and the temperature was kept constant at 24 °C.
Construction of transformation vectors
The vector pAP202 containing the full-length cDNA of AtFtsZ1 was used to generated AtFtsZ1 overexpressing lines as published previously36. For the construction of pIR and pSSC, the tobacco (Nicotiana tabacum) plastome IR (trnI/trnA; 102,623–105,457 bp and 105,458–110,067 bp; GenBank: KU199713.1) and SSC (ndhG/ndhI; 119,184–120,988 bp and 120,989–126,029 bp; GenBank: KU199713.1) regions, respectively, were synthesized by GeneArt (Thermo Fisher Scientific, Waltham, MA, USA). The two homologous regions were modified including several SNPs to facilitate cloning. Before introducing the selection cassette between homologous arms, the trnI/trnA and ndhG/ndhI regions were cloned into pMK vector (Thermo Fisher Scientific, Waltham, MA, USA). A chloroplast selection cassette (Prrn-SD::aadA::5′UTR::GFP::psbA3′UTR) was PCR amplified from the pLD-PTD-GFP plasmid61 using 1Fw/1Rv primers and cloned into the PmeI site of trnI/trnA and ndhG/ndhI sites generating pIR and pSSC plasmids, respectively. A list of primers used in this work is shown in Supplementary Table S1.
Generation of transgenic lines
Potato AtFtsZ1 overexpressing lines (macro-chloroplast lines) were generated by Agrobacterium-mediated transformation. The vector pAP20236 was introduced into Agrobacterium tumefaciens LBA4404 by the freeze thaw method62. For each transformation, 20 internodes of ~ 1 cm-length from 1-month-old in vitro potato plants were used. Internodes were then transformed via Agrobacterium and transgenic plants were regenerated in selective media, as previously described63. Putative transgenic lines were screened by fluorescent microscopy for clearly-defined enlarged chloroplasts. Thereafter, selected lines were genetically characterized for construct integration and transgene expression.
The PDS-1000/He biolistics device (Bio-Rad, Hercules, CA, USA) was subsequently used to transform chloroplasts of both macro-chloroplast and wild-type potato plants64. Macro-chloroplast line 1 was used for chloroplast transformation. Per each transformation ~ 6 cm2 of leaf tissue from 1-month-old in vitro potato plants were used. Per each shoot 0.3 mg of gold-particles (0.6 µm in diameter) binding 1 µg of plasmid were used following the manufacturer protocol (Seashell Technology, La Jolla, CA, USA). Transplastomic plants of both genotypes (wild-type and macro-chloroplast) were then regenerated from transformed leaf material incubated in selective media as described previously60. The second round of transplastomic plants were obtained by applying the same protocol of tissue culture/selection/regeneration60. All lines were genetically characterized for the presence of the selection cassette integrated and GFP expression.
Total DNA extraction and PCR analysis
Total genomic-DNA preparations from leaves were obtained using ~ 50 mg of tissue and the CTAB-based procedure of extraction64. PCR (25 cycles) was performed in 25 µl reaction-volume by using 5 ng of total genomic DNA and the DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). For molecular characterization of AtFtsZ1 overexpressing lines, the pairs of primers 2Fw/2Rv and 3Fw/3Rv were used to check for nptII/AtFtsZ1 cassette integration and the internal control actin (GeneID-102593904), respectively. For genotyping of transplastomic lines, the two pairs of primers 4Fw/4Rv and 5Fw/5Rv were used to verify cassette integration in the trnI/trnA and ndhG/ndhI sites of plastome, respectively. The two pair of primers 6Fw/6Rv and 7Fw/7Rv were used to amplify full-length GFP (NCBI ID: AEX93343.1) and aadA (NCBI ID: AAR14532.1) genes, respectively. The primers 8Fw/8Rv were used to amplify an internal portion of rbcL (NCBI ID: 4099985).
Total RNA extraction and reverse transcriptase PCR
Total RNA extraction was performed using Tri-Reagent (Molecular Research Center, Inc, Cincinnati, OH, USA) according to manufacturer’s protocol. Per each extraction ~ 50 mg healthy leaf tissue was used. Total RNA preparations were cleaned and subjected to DNase treatment using the RNA Clean & Concentrator Kit (Zymogen, Irvine, CA, USA) according to manufacturer’s instruction. The cDNA synthesis was performed using the Super Script III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instruction. cDNA was quantified using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), and several serial cDNA dilutions were tested by PCR (as described before) for the presence of the transgene AtFtsZ, whereas the two internal controls rbcL (plastome) and ef1α (nuclear) were used for normalization. The pair of primers 9Fw/9Rv, 10Fw/10Rv and 11Fw/11Rv were used to amplify and internal fragment (~ 100 bp) of AtFtsZ (Tair ID: AT5G55280), rbcL (NCBI ID: 4,099,985) and ef1α (NCBI ID: NM_001288491.1), respectively.
Southern blot analysis
DNA probes for detection of pIR and pSSC constructs integrated into potato plastome (GenBank: NC_008096.2) were designed on IR (104,457–104,978 bp) and SSC (120,269–120,790 bp) regions, respectively. The PCR DIG Probe Synthesis Kit (Roche, Indianapolis, IN, USA) was used to synthesize digoxigenin(DIG)-sUTP-labelled IR and SSC DNA-probes using the pair of primers 12Fw/12Rv and 13Fw/13Rv, respectively. Total genomic DNA from leaf tissue was extracted using CTAB, as described above. After quantification, 1 µg of DNA for each pIR and pSSC sample was digested using KasI/HindIII and FspI/ScaI restriction enzymes, respectively. The DNA fragments were separated on 0.9% agarose gel, and after that, the gel was depurinated, denatured and transferred on a nylon membrane as described previously13. The membrane was then incubated with the DIG-labelled probe and detected using the anti- digoxigenin-AP Fab fragments detection kit (Roche Indianapolis, IN, USA) accordingly to the manufacturer’s protocol.
PCRs were performed in 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA), in a total volume of 15 µl per reaction using 1X PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 5 ng of total genomic DNA from leaves and 0.5 µM of each primer. Primers 20 bp-long with an annealing temperature of ~ 57 °C and able to amplify a ~ 100 bp-fragment were design using the online software Primer3 input v. 0.4.0 (Howard Hughes Medical Institute and by the National Institutes of Health)65. The primers 10Fw/10Rv and 14Fw/14Rv were use to detect the plastome gene rbcL (NCBI ID: 4099985) and the nuclear gene actin (GeneID-102593904), respectively (Supplementary Table S1). The QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) was used to perform real-time PCR, whereas the QuantStudio Real-Time PCR Software v1.1 (Thermo Fisher Scientific, Waltham, MA, USA) was used to aquire amplification data. The data were expressed as Log2(2−ΔCT) of rbcL vs actin. Results were expressed as mean ± standard deviation (sd) of 3 biological replicates and 8 technical replicates per biological replicate per each genotype. An ANOVA post-hoc Tukey statistical analysis (p < 0.05) was performed to determine statistically significant difference among means (IBM SPSS software).
The amount of GFP accumulated in leaf tissue of transplastomic lines was quantified by using the Fluorometric GFP Quantification Kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer’s protocol66. The results were expressed as mean ± standard deviation (ng GFP/mg fresh weight) of two indipendent experiments, resulting in a total of 2 biological replicates and 2 technical replicates per biological replicate for each independent transplastomic line. We analyzed 15 pIR and 8 pSSC lines for each genotype, normal and macro-chloroplast.
Confocal microscopy and chloroplast size determination
Leaf tissue from 3-week-old in vitro plants was imaged using an Olympus Fv1200 confocal microscope (Olympus, Center Valley, PA, USA). GFP was excited at 488 nm and detected at an emission wavelength of 509 nm. Chlorophyll autofluorescence was visualized using an excitation wavelength of 543 nm and an emission wavelength of 667 nm. Confocal images were acquired using the manufacturer’s Olympus FV10-ASW Viewer software Ver.4.2a (Olympus, Center Valley, PA). Confocal images were processed using the online software ImageJ 1.41o (National Institute of Health, Bethesda, MD, USA). The same ImageJ software was also used to estimate chloroplast size in both AtFtsZ1 overexpressing lines and wild-type controls. The results were expressed as mean ± standard deviation (sd) and the statistical analysis was performed using SPSS statistics 25 software (IBM).
Transmission electron microscope (TEM)
Leaf tissue from 3-week-old in vitro plants were chemically fixed in glutaraldehyde/ paraformaldehyde along with an osmium tetroxide solution and embedded in EMBed-821 resin (Electron Microscopy Sciences, Hatfield, PA, USA) as described previously67. Ultrathin sections (60–100 nm) of embedded tissue were cut using a diamond knife and a Leica EM UC7 ultramicrotome (Leica, Buffalo Grove, IL, USA). Ultrathin sections were post strained using aqueous solutions of uranyl acetate and lead citrate as described previously67. Images were obtained using a JEOL 1400 transmission electron microscope operating at 80 kV (JEOL, Peabody, MA, USA) equipped with a Gatan OneView camera (Gatan, Pleasanton, CA, USA). TEM micrographs were processed using the software ImageJ 1.41o (National Institute of Health, Bethesda, MD, USA).
Fluorescence imaging by the fluorescence-inducing laser projector (FILP)
Plant fluorescence patterns were characterized using the Fluorescence-Inducing Laser Projector (FILP), a custom-built instrument for imaging fluorescence in plants. FILP imaging was performed on 3-week-old transplastomic lines in pots43. GFP fluorescence in plants was acquired at 150 ms exposure time using 465 nm excitation and 525 nm emission notch (50 nm) filter. Standoff detection was performed at 3 m from the laser source. FILP images were processed using ImageJ 1.41o software (National Institute of Health, Bethesda, MD, USA).