Plants and growth conditions
SALK T-DNA insertion mutants were used in this study. Generation, formal identification, and submission of the mutant lines to public seed stocks was done by the SALK Institute Genomic Analysis Laboratory (SIGnAL) [24]. The four sets of confirmed homozygous lines with the NASC (Nottingham Arabidopsis Stock Centre) order numbers N27941, N27951, N27942, and N27952 comprised altogether 14,282 T-DNA mutants. All lines are available from NASC (http://arabidopsis.info). Fourteen seed pools of approx. 1000 mixed mutant lines each were prepared. To this end, 2–4 seeds per line were flipped out of every mutant stock tube by a bended needle forming a little hook. Thus, one seed pool contained approx. 3000 seeds, and in sum > 42,000 seeds were used in the screen. Fourteen plant trays (size: 0.6 m × 0.4 m × 0.06 m) with inlets (tray-sized, holes in the bottom to prevent water logging) were filled with soil (4 parts Floradur propagation substrate (Floragard) mixed with 1 part quartz sand). Seeds of every pool were evenly spread in each tray and vernalized for 2 days at 4 °C in the dark.
NO2 fumigations
The mutant screen was done in walk-in size chambers housing 4 air tight fumigation chambers (www.helmholtz-muenchen.de/eus/facilities/phytotron), which allowed simultaneous fumigation of 4 plant trays in parallel. The growth conditions were set to 250 μmol m− 2 s− 1 light intensity, 14 h light-10 h dark cycle, 23 °C/18 °C (day/night), and 70%/90% (day/night) relative humidity. The plants were grown for 2 weeks in the chambers, and mutants exhibiting chlorosis or lesions were removed before the NO2 treatments. NO2 was generated by the reaction of 15% NO with 100% O2 in mixing vessels containing Raschig glass rings. The NO2 concentrations were adjusted by regulating the NO flux rate. The concentrations of NO2 were monitored with an AC3 2 M chemiluminescent oxides of nitrogen analyzer (Environnement S.A.). Generally, the fumigations started in the morning at approx. 2 h after onset of the light period. Plants were fumigated with 10 ppm NO2 for 1 h and 2 days later with 30 ppm NO2. 10 ppm NO2 had no visible effect on Col-0 (mutant background) and most mutants. However, exposure to 30 ppm NO2 caused dead leaf areas or complete leaf collapse in most of the tested plants. Sensitive plants displayed symptoms already after 10 ppm NO2 whereas tolerant plants were hardly affected even by 30 ppm NO2.
Imaging
UV-induced fluorescence was detected using a hand-held UV lamp (Blak-Ray B-100AP; UVP). Plants were photographed with a Nikon DC300 digital camera.
Sampling of candidate mutants, and DNA extraction
Mutants showing NO2 phenotypes were sampled at 48 h after fumigation when symptoms became clearly visible due to bleaching of the dead leaf areas. Mutants that showed obvious phenotypes compared to the overall appearance of the fumigated seedlings were selected based on the assumption that the vast majority of mutants does not have an NO2 phenotype different from the Col-0 background line. NO2 sensitive and tolerant mutant seedlings without roots were sampled into polypropylene tubes containing 10 to 12 glass beads (1.7–2.0 mm, Roth) and were immediately frozen in liquid N2. The sampled seedlings were homogenized twice for 10 s in a Silamat S6 bead mill (Ivoclar Vivadent). DNA was extracted using the DNeasy Plant Mini kit (Qiagen) and quantified by Quant-iT Picogreen dsDNA (Life Technologies) according to the manufacturer’s instructions.
Adapter ligation-mediated PCR
Genes containing T-DNA insertions were identified by adapter ligation-mediated PCR following a published protocol [6]. In brief, genomic DNA from mutant plants was digested with the Ase1 restriction enzyme, and Ase adapters (long strand adapter 2 plus short strand of adapter Ase) were ligated to the cut sites. Fragments containing T-DNA were selectively amplified by LBa1/AP1 primer pairs binding T-DNA as well as adapter sequences. Subsequently, a nested PCR with LBb1/AP2 primers was run to selectively amplify T-DNA/gDNA junctions before sequencing. According to O’Malley et al. (2007) this step can be omitted when using Ase adapters but we found that the additional PCR enhances sequencing quality and success rate.
T-DNA-specific target enrichment and next generation sequencing
Genomic DNA from 59 (Pool I) or 65 (Pool II) plants was pooled to give two samples each containing 1 μg DNA. The DNA was precipitated by mixing with 1/10 sample volume of 3 M sodium acetate, pH 5.2, and 2 volumes of 100% cold ethanol. Samples were then incubated at − 20 °C for at least 20 min. After centrifugation at maximum speed the resulting pellet was washed once with 1 mL 70% ethanol, briefly air-dried, and finally resuspended in 100 μl of Tris-EDTA (TE) buffer. Two libraries were prepared from the DNA pools I and II using the GS-FLX+ Rapid Library (Roche) according to the manufacturer’s instructions, which involved shearing of the DNA, fragment end repair, and adapter ligation. Small fragments were removed by AMPure XP beads (Beckman), and the appropriate length distribution of DNA fragments between 500 and 1500 bp was confirmed by measurements with the Bioanalyzer 2100 (Agilent). Libraries I and II were amplified by ligation mediated PCR using adapter-specific Rapid-A and -B primers as described in the NimbleGen SeqCap EZ Library LR User’s Guide v2.0 [25], and DNA was quantified with the Quant-iT Picogreen dsDNA assay (Life Technologies).
DNA fragments containing T-DNA were isolated from the amplified libraries using T-DNA border-specific 70mer probes, the SeqCap EZ Hybridization and Wash Kit (Roche Nimblegen), and SeqCap EZ Developer Reagent (Roche Nimblegen). The following biotinylated 70mer probes designed to bind T-DNA left- and right border (LB/RB) sequences of the Agrobacterium tumefaciens vector pROK2 were employed for target enrichment: LB1: GAG CTG TTG GCT GGC TGG TGG CAG GAT ATA TTG TGG TGT AAA CAA ATT GAC GCT TAG ACA ACT TAA TAA C; LB2: TAT ATT GTG GTG TAA ACA AAT TGA CGC TTA GAC AAC TTA ATA ACA CAT TGC GGA CGT TTT TAA TGT ACT G; LB3: CAA CAG CTG ATT GCC CTT CAC CGC CTG GCC CTG AGA GAG TTG CAG CAA GCG GTC CAC GCT GGT TTG CCC C; RB1: CTC CGC TCA TGA TCA GAT TGT CGT TTC CCG CCT TCA GTT TAA ACT ATC AGT GTT TGA CAG GAT ATA TTG G; RB2: CAG TGT TTG ACA GGA TAT ATT GGC GGG TAA ACC TAA GAG AAA AGA GCG TTT ATT AGA ATA ATC GGA TAT T; RB3: GGG TAA ACC TAA GAG AAA AGA GCG TTT ATT AGA ATA ATC GGA TAT TTA AAA GGG CGT GAA AAG GTT TAT C.
Following the NimbleGen SeqCap EZ Library LR User’s Guide v2.0 a hybridization mixture was prepared including among others 1 μg amplified library DNA and the six different 70mer probes. The latter were adjusted to 3.75 × 106 molecules of each oligonucleotide in a total volume of 4.5 μl water, replacing the SeqCap EZ Library in the User’s Guide [7]. The hybridization mixture was incubated at 95 °C for 10 min and then at 47.5 °C for 40 h [7]. DNA fragments bound to the biotinylated probes were captured with Dynabeads M-270 streptavidin (Invitrogen) and amplified using 454 Rapid-A and -B primers. Target enrichment was verified by qPCR with the T-DNA right border-specific primers T-DNA_R_Rev CTG TGG TTG GCA TGC ACA TAC and T-DNA_R_For AGA TTG TCG TTT CCC GCC TT. Small fragments, primers, and primer dimers were removed with AMPure XP beads, the fragment length distribution was checked by Bioanalyzer 2100 (Agilent), and DNA concentration was determined with the Quant-iT Picogreen dsDNA assay (Life Technologies). Emulsion PCR, emulsion breaking, and sequencing using a 454 Genome Sequencer FLX instrument were performed as described in the NimbleGen SeqCap EZ Library LR User’s Guide v2.0 [26].
As Roche stopped its service for GS FLX+ sequencing in 2017, future studies will be performed using the Illumina MiSeq platform (Illumina). Here, the TruSeq DNA sample prep kit (Illumina) will be applied for sample preparation as described by the manufacturer. The read length of Illumina sequencing is in the range of 250 bases per read, if the paired end modus is used. Illumina sequencing has previously been reported to be applicable for targeted genome sequencing [7].
Bioinformatic analysis and T-DNA insertion line identification
Bioinformatic analysis was performed using the CLC Genomics Workbench 11 software (Qiagen). Two datasets of 207,785 reads (dataset 1) and 154,847 reads (dataset 2) were used for the identification of T-DNA insertion sites. To identify T-DNA-containing sequences all reads were blasted against the T-DNA insertion vector pBIN-pROK2 using the BLASTN function under default settings. By filtering the data for high-scoring segment pair (HSP) length ≥ 30 and ≤ 343 bp and an e- value ≤5,72*E− 11 unspecific hits and parts of “only T-DNA” hits were filtered out. Then the reads were further trimmed with the following settings in the CLC Genomic Workbench: read length > 40 bp, low quality limit of 0.05, max. 2 ambiguous bases. Finally, one nucleotide on the 5′ terminus and adapter sequences were cleaved off and vector sequences from the vector pBIN-pROK2 were labeled as trimmed and therefore ignored for the mapping. Afterwards the remaining reads were mapped against the Arabidopsis genome sequence. Sequence alignments were performed with the ApE-Plasmid Editor (http://jorgensen.biology.utah.edu/wayned/ape/).
Re-screen using ion leakage- and stomatal conductance measurements
Selected candidate mutants and WT plants were grown in pots at 65–85 μmol m− 2 s− 1 light intensity, 14 h light–10 h dark cycle, 20 °C/18 °C (day/night), and 65–68% relative humidity. Three-week-old plants were used for the ion leakage measurements whereas basal stomatal conductance was determined in 4-week-old plants. A fumigation chamber housing individual plants up to one tray of plants was used for re-screening of selected candidate mutants as described recently [10, 27]. Plants were fumigated for 40 min with 30 ppm NO2, which caused visible symptoms in 40–60% of the leaf area in WT plants. Immediately after fumigation 2 seedlings were collected into 30 mL of deionized water, and the background water conductivity (μS cm− 1) was determined using a conductivity meter (GLM 020A, Greisinger Electronic). After 24 h the sample conductivities were measured and the background values were subtracted. The resulting corrected sample conductivities were normalized to their respective conductivities measured after freezing and reheating to RT (i.e. 100% conductivity). Results are given as relative ion leakage. Basal stomatal conductance was measured in untreated plants using the Leaf Porometer Model SC-1 (Decagon Devices). The measurements took place in the growth chamber at 2 h – 4 h after start of the light period.